Advances in Cancer Research, Volume 18

T VOLUME 18 Contributors to This Volume K. D. Bagshawe Charles Heidelberger R. W. Baldwin John H. Kersey Robert...

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T VOLUME 18

Contributors to This Volume

K.

D. Bagshawe

Charles Heidelberger

R. W. Baldwin

John H. Kersey

Robert A. Good

Fanny Schapira

Sen-itiroh Hakornori

Beatrice D. Spector

Yee Chu Toh

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLElN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

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

Consulting Editor

ALEXANDER HADDOW Chester Beatty Research Institute Institute of Cancer Research Royal Cancer Hospital London, England

Volume 78 ACADEMIC PRESS

New York and London

A Subridiory of Horcourt Brace Jovanovich, Publishers

1973

T PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITl'ED IN ANY FORM OR BY A N Y MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR A N Y INFORMATION STORAGE AND RETRIEVAL. SYSTEM, WlTHOUT PERMISSION IN WRITING FROM THE PUDLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York. New York 10003

United Kingdom Edition published by ACADEMIC PRESS. INC. (LONDON) LTD. . 24/28 Oval Road. London NW1

LIBRARY OF CONORESS CATALOO CARD NUMBER: 52-13360

PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS CONTRIBUTORS TO VOLUME18

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ix

Immunological Aspects of Chemical Carcinogenesis

R . W. BALDWIN I. Introduction . . . . . . . . . . . . . . I1. Expression of Tumor-Associated Antigens on Chemically Induced Tumors . . . . . . . . . . . . . I11. Isolation and Characterization of Tumor-Associated Antigens . . IV. Embryonic Antigen Expression in Chemically Induced Tumors . V . Deletion of Normal Tissue Antigens from Carcinogen-Induced Tumors VI . Immunosurveillance and Chemical Carcinogenesis . . . . . VII . Conclusions . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

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1

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3 21 30 35 41 65 67

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77 80

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lsozymes and Cancer

FANNY SCHAPIRA I. Introduction . . . . I1. Oxidoreductases . . . I11. Lyases . . . . . IV. Phosphotransferases . . V . Other "ransferases . . VI.Hydrolases . . . . VII . Summary and Conclusions Addendum . . . . References . . . .

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93

99 111 116 129 134 135

Pbsiological and Biochemical Reviews of Sex Differences and Carcinogenesis with Particular Reference to the liver

YEE CHU TOE I. Sex Differences in Phyeiological Functions I1. Sex Differences in Biochemical Functions . I11. Sex Differences in Carcinogenesis . . . IV. General Discussion . . . . . . References . . . . . . . .

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156 171 183 188 195

Immunodeficiency and Cancer

JOHN H. KERSEY. BEATRICE D. SPECTOI~. AND ROBERT A . GOOD

I. Introduction . . . . . . . . . . . . I1. Experimental Models Linking Immunodeficiency and Cancer V

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211 212

vi

CONTENTS

I11. Human Diseases References .

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217 225

Recent Observations Related to the Chemotherapy and Immunology of Gestational Choriocarcinoma

K . D. BACSHAWE

I. Introduction . . . . . . . . . . . . I1. General Aspects of Trophoblastic Tumors . . . . I11. Human Chorionic Gonadotropin as a Tumor Index Substance IV. Immunological Aspects of Choriocarcinoma . . . . References . . . . . . . . . . . .

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231 232 234 246 260

Glycolipids of Tumor Cell Membrane

SEN-ITIROH HAKOMORI

I. Introduction . . . . . . . . . . . . . . . I1. Structure and Organization of Glycosphingolipids in Membrane . . I11. Change of Glycolipids in Experimental Tumors and Transformed Cells in Vitro . . . . . . . . . . . . . . . . IV. Glycolipids of Human Tumors and Human Leukemic Cells . . . V . Cell Contact. Contact Inhibition. and Glycolipid Synthesis . . . VI . Change of Glycosyltransferases and Hydrolases . . . . . . VII . Reactivity of Cell Surface Glycolipids and Glycoproteins of Normal. Fetal. and Cancer Cells with Macromolecular Reagents . . . . VIII . The Significance of Membrane Glycolipid Changes in Regulation of Cell Growth and Intercellular Linkages : Some Comments and Speculations References . . . . . . . . . . . . . . .

265 269 277 253 2S6 290 293 302 308

Chemical Oncogenesis in Culture

CHARLES HEIDELBERCER

I. Introduction . . . . . . . . . . . . I1. Organ Cultures . . . . . . . . . . . I11. Hamster Embryo Cells . . . . . . . . . I V. Transformation of Fibroblastic Cells Derived from Mouse Ventral Prostate . . . . . . . . . . . V . 3T36ike Systems . . . . . . . . . . VI. Other Cell Systems . . . . . . . . . . VII . Liver Cell Systems . . . . . . . . . . VIII . Combined Effects of Chemicals and Oncogenic Viruses . I X . Metabolism of Polycyclic Aromatic Hydrocarbons . . . X . Binding to Cellular Macromolecules . . . . . . XI . Metabolic Activation of Polycyclic Hydrocarbons . . . XI1. Conclusions . . . . . . . . . . . . References . . . . . . . . . . . .

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317 315 323 333 337 338 339 341 343 346 349 355 360

CONTENTS

AUTHORINDEX.

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vii 367 401 405

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CONTRIBUTORS TO VOLUME 18 Numbers in parentheses refer to the pages on which the authors' contributions begin.

K. D. BAGSHAWE, Department of Medical Oncology, Charing Cross Hospital (Fulham), London, England (231)

R. W. BALDWIN, Cancer Research Campaign Laboratories, University of Nottingham, Nottinghum, England (1) ROBERT A. GOOD, Sloan-Kettering Institute for Cancer Research, New York, New York (211) SEN-ITIROH HAKOMORI, Department of Pathobiology, School of Public Health, and Department of Microbiology, School of Medicine, University of Washington,Seattle, Washington (265) CHARLES HEIDELBERGER, McArdle Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin (317) JOHNH. KERSEY, Departments of Pathology and Pediatrics, University of Minnesota, Minneapolis, Minnesota (211)

FANNYSCHAPIRA, Institut de Pathologie Mole'culaire, Paris, France (77) BEATRICE D. SPECTOR, Departments of Pathology and Pediatrics, University of Minnesota, Minneapolis, Minnesota (211) YEECHUToH,' Sub-Department of Endocrine Pathology, The University of Liverpool, The Liverpool Clinic, Liverpool, United Kingdom (155)

' Present address: Department of Physiology, Faculty of Medicine, University of Singapore, Singapore. ix

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N R. W. Baldwin Cancer Research Campaign laboratories, University of Nottingham, Nottingharn, England

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I. Introduction . . . . . . . . . . . . 11. Expression of Tumor-Associated Antigens on Chemically Induced Tumors . . . . . . . . . . . . . . A. Polycyclic Hydrocarbons . . . . . . . . . B. Aminoazo Dyes . . . . . . . . . . C. 2-Acetylaminofluorene . . . . . . . . . . . . D. Alkylnitrosamine . . . . . . . . . . E. Physical Agents . . . . . . . . . . F. Significance of Tumor Antigen Expression in Chemical . . . . . . . . . . . . . Carcinogenesis. 111. Isolation and Characterization of Tumor-Associated Antigens . . . A. Aminoazo Dye-Induced Rat Hepatomas . . . . . . . B. Diethylnitrosamine-Induced Guinea Pig Hepatomas . . . . C. Polycyclic Hydrocarbon-Induced Tumors . . . . . . D. Characterization of Membrane-Associated Tumor Antigens . . . IV. Embryonic Antigen Expression in Chemically Induced Tumors . . . V. Deletion of Normal Tissue Antigens from Carcinogen-Induced Tumors . VI. Immunosurveillance and Chemical Carcinogenesis . . . . . A. Cell Antigen Changes during Chemical Carcinogenesis . . . . B. Immunosuppression and Chemical Carcinogenesis . . . . . C. Cellular and Humoral Immune Responses to Tumor-Associated Antigens . . . . . . . . . . VII. Conclusions . . . . . . . . . . . . References . . . . . . . . . .

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I. Introduction

During the past two decades, the concept that tumors express antigens against which the host is capable of evoking an immune response has been firmly established. Contemporary research in tumor immunology can be considered to date from the studies of Foley (1953) demonstrating that destruction of subcutaneous grafts of 3-methylcholanthrene- (MC) induced mouse sarcomas in inbred (syngeneic) mice by ligation of their blood supply resulted in the development of immunity against subcutaneous challenges with the same tumor. The validity of these studies, as well as comparable findings with hlC-induced rat sarcomas (Baldwin, 1955), was critically analyzed by Prehn and Main (1957), who conclusively showed that induction of immunity to transplanted sarcoma 1

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R. W. BALDWIN

grafts was a tumor-specific phenomenon since immunized mice could still accept skin grafts from the tumor donor, precluding the involvement of an alloantigenic immune response. Subsequently, Klein e t al. (1960) established that tumor immune rejection reactions were evoked against MC-induced mouse sarcomas in the autochthonous host where the possibility that immunity could be accounted for by residual heterozygosis was excluded, and these findings led to a more general acceptance of the concept that host-mediated immune reactions play a role in neoplasia. Since these early reports, whose objective waS to demonstrate conclusively the reality of tumor rejection antigens, investigations have broadened to analyze the involvement of tumor-host interactions in the whole process of oncogenesis. Tumors induced by chemical carcinogens have figured in many of these studies and the object of this review is to consider the implications of the findings from these investigations in chemical carcinogenesis. Evidence shows that tumor rejection antigens are specific and permanent, or quasi-permanent, characteristics of cells transformed by chemical carcinogens. Chemically induced tumors also express antigens normally present in embryonic, but not adult, tissue which may be viewed as products of dedifferentiation processes induced by the carcinogen, and concurrently there is frequently a deletion of normal tissue antigens. Characterization of the antigen profiles of tumor cells in comparison with those of their normal counterparts thus provides a sophisticated method of analyzing cellular changes induced by Fhemical carcinogens. Even more importantly, many of the new antigens appearing in chemically induced tumors are specific components of the plasma membrane so that these immunological markers make possible studies of changes in cell surface architecture resulting from neoplastic transformation. Implicit in this approach is a need for a greater understanding of molecular mechanisms of control a t the cell membrane level (Changeux, 1969) especially with regard to the view that aberrations of the cell surface may be the critical changes in neoplasia (Markert, 1968; Burger and Noonan, 1970; Sela et al., 1970; Boyse, 1971). Since chemically induced tumors have neoantigens capable of evoking immune rejection reactions in the autochthonous host, this raises the question of the part, if any, played by immunosurveillance in chemical carcinogenesis, especially in view of the known immunosuppressive properties of many chemical carcinogens. For such control to be effective, the initial populations of transformed cells must express tumor distinctive antigens and the degree of immunogenicity b u s t be sufficient to provoke an effective tumor-immune response. This response will depend upon the accessibility of the transformed cell to the host’s immunological appa-

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

3

ratus and the integrity of this system a t the time cells arise. The objective of this review, therefore, will be to consider the degree of involvement of immune mechanisms in modifying tumor formation and growth during chemical carcinogenesis.

It. Expression of Tumor-Associated Antigens o n Chemically Induced Tumors

A. POLYCY CLIC HYDROCARBONS 1. Tumor Rejection

Decisive evidence of tumor-specific immunogenicity has been provided by tumor rejection studies in syngeneic hosts with tumors induced by several polycyclic hydrocarbons including 3-methylcholanthrene, benzo [ a ]pyrene, 7,12-dimethylbenz [ a ] anthracene, dibenz [a$] anthracene, and dibenzo [a,i]pyrene (Table I). Historically sarcomas induced by 3methylcholanthrene (MC) were the first to be examined for tumorspecific antigenicity, and there are several properties, including rapid development following a single injection of carcinogen and high frequency of tumors showing immunogenicity, indicating their suitability for immunological studies. Consequently there is a comprehensive body of knowledge on the immunology of MC-induced sarcomas, although by and large, other polycyclic hydrocarbon-induced tumors show comparable immunological characteristics. MC-induced sarcomas in several species, including rat, mouse, and guinea pig (Foley, 1953; Baldwin, 1955; Prehn and Main, 1957; Klein et al., 1960; Morton et al., 1965; Oettgen et aZ., 1968), have tumorassociated antigens so that prior exposure of compatible hosts to transplanted tumor cells in a manner which prevents progressive growth results in the development of a specific resistance against a subsequent inoculation of viable tumor cells which grow in control animals. This can be achieved by implanting tumor grafts treated with X- or 7-irradiation (10-15,000 It) so that tumor cells have a limited survival, or by surgical excision of a developing tumor mass. Comparable immune rejection reactions have been detected against MC-induced sarcomas in autochthonous hosts, and this was a significant finding in establishing the existence of tumor-specific antigens. For instance, immunity was detected against primary MC-induced mouse sarcomas following surgical amputation of hind limbs bearing a primary tumor and repeatedly treating the operated mouse with X-irradiated cells of the excised tumor, which was maintained by transplantation in syngeneic mice (Klein et al., 1960). Also suppression of growth of reimplanted biopsies of primary rat sar-

R. W. BALDWIN

4

TABLE I CARCINOGEN-INDUCED TUMORS SHOWN TO EXPRESS TUMOR REJECTIONANTIGENS Compound

Tumor type

Species Antigenic potency

Polycyclic hydrocurbom 3-Methylcholanthrene

Sarcoma

Mouse Rat Guinea pig Mouse

Skin papilloma/ carcinoma Mouselrat Bladder papilloma/ carcinoma Mammary carcinoma Rat Mouse I n uitro transformed Mouse mouse prostate cells Mouse Dibenz[a,h]anthracene Sarcoma Guinea pig Mouse 7,12-Dimethylbenz[a]anthracene Sarcoma Guinea pig Epithelioma Mouse Sarcoma Mouse/rat Benzo[a]pyrene Sarcoma Mouse Dibenzo[a,zlpyrene

Strong-inactive Strong Strong Weak -

Weak Weak Strong-weak Strong Strong Strong Strong Strong Strong Weak

Amirwazo Dyes 4-Dimethylaminoazobenzene (and 3’-methyl derivative) o-Aminoazotoluene

Hepatoma

Rat

Strong

Hepatoma

Mouse

Weak

Aromatic Amines 2-Acetylaminofluorene

Mammary carcinoma Rat Hepatoma Rat Ear duct carcinoma Rat

Inactive or weak Inactive or weak Inactive or weak

Hepatoma

Guinea pig Rat Pulmonary carcinoma Mouse I n vitro transformed Mouse mouse prostate cells

Strong Strong-weak Inactive Active

Pulmonary adenoma Plasma cell tumor Sarcoma Sarcoma Osteosarcoma

Weak or inactive Weak Weak Strong Weak

A lkylnitrosamines Diethylni trosamine Nitrosoguanidine Miscellaneous Urethan Mineral oil Plastic films Ultraviolet radiation Strontium (WSr)

Mouse Mouse Mouse Mouse Mouse

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

5

comas was obtained following ligation of the primary tumor (Takeda et al., 1966; Takeda, 1969) or its total excision (Mikulska et al., 1966). In general, however, the immune response was greater and more reproducible if some interval of time was allowed between surgical removal of the primary tumor and rechallenge. This is reflected by the lack of immunity observed against mouse sarcomas when challenge inocula were given immediately after surgical excision of the primary tumor (Stjernsward, 1968), and this was held to reflect anergy of the primary tumor host induced by the growing tumor and the residual immunosuppressive action of 3-methylcholanthrene. A more likely explanation is provided by studies (Hellstrom et al., 1970) showing that serum from mice bearing primary sarcomas contains a factor that blocks the lymphocyte-mediated reaction against tumor-associated antigens, but this humoral factor is rapidly (within 4 days) eliminated after tumor removal. Although MC-induced sarcomas are generally held to be highly immunogenic, there are quite large variations in antigenicity between tumors in different species and, more importantly, between individual tumors in a single species. The strain differences are illustrated by the high immunogenicity of sarcomas in the guinea pig (Oettgen et al., 1968), where immunized animals have been observed to reject challenges with up to 6 gm of viable tumor tissue, whereas the level of resistance elicited against rat and mouse sarcomas is much lower, producing protection against challenges with viable cell inocula (10"to los cells) or a t best small tumor grafts (Baldwin, 1955; Klein et al., 1960; Takeda, 1969). Antigenic variability of hlC-induced sarcomas arising within a single species has also been reported (Old et al., 1962; Johnson, 1968; Takeda, 1969; Prehn, 1969), and these studies demonstrated a correlation between decreased immunogenicity and increased latent induction periods. This suggested that immunoselection by the host may be a contributory factor to antigen loss, but Prehn (1970) has established that antigenic variability was not dependent upon temporal distribution in tumor induction since sublines selected from opposite poles of single primary MC-induced sarcomas showed different antigenic potentialities. It has also been shown that tumors induced by 3-BIG treatment of cells in vitro (Mondal et al., 1970, 1971 ; Embleton and Heidelberger, 1972) or in vivo in the immunologically protected confines of Millipore chambers (Basombrio and Prehn, 1972a,b) show antigenic variability. It would appear, therefore, that the high antigenicity of most hlC-induced sarcomas is acquired during neoplastic transformation. In contrast with the comprehensive studies on the immunogenicity of RIC sarcomas, other types of tumor induced by this carcinogen have received comparatively little attention. One sqiiamous cell carcinoma was

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shown to be highly immunogenic (Old et al., 1962), and there is also evidence that tumor-specific antigens can be detected on skin papillomas (Lap$, 1968, 1969) and bladder papillomas and carcinomas (I. Hellstrom and Hellstrom, 1972). Also mammary tumors induced by MC in mice (Prehn, 1962) and rats (Kim, 1970) express tumor-associated antigens, although their immunogenicity is generally much weaker than that observed in sarcomas. Surprisingly, mammary tumors which arise with extremely short latent periods in certain rat strains following oral administration of several polycyclic hydrocarbons, including 3-methylcholanthrene (Huggins et al., 1961), do not appear to have been the subject of any detailed immunological study. Tumor antigens have also been detected in cell culture lines derived from C,H mouse prostate and transformed in vitro with 3-methylcholanthrene (Mondal et al., 1970, 1971 ; Embleton and Heidelberger, 1972). Individual clones of transformed cells produced fibrosarcomas following injection into mice, and ligation of these tumors resulted in the development of immunity to rechallenge with cells of the same clone. The immunogenicity of these in vitro transformed cells was such that the maximum cell inoculum rejected was about lo5 tumor cells, which is comparable with that observed with some in vivoinduced tumors. These findings are of particular significance in that they provide a system for the analysis of tumor antigen expression in relation to the cellular macromolecule changes involved in chemical carcinogenesis. A significant feature of the tumor rejection antigens associated with MC-induced sarcomas in several species is that they are distinctive components of individual tumors, so that immunization against one tumor does not confer resistance to the host against histologically similar tumors even when induced by the same carcinogen (Prehn and Main, 1957; Klein et al., 1960; Old et al., 1962; Morton et al.,1965; Basombrio, 1970). Similarly, sarcomas induced with benzo [ a ]pyrene are antigenically distinct (Globerson and Feldman, 1964; Delorme and Alexander, 1964), and in one study it was established that two tumors originating in a single mouse each had characteristic antigens (Globerson and Feldman, 1964). Generally, however, the number of individual tumors examined has been rather restricted so that it has not been possible to clearly define the extent of this antigenic specificity. Other studies have suggested, alternatively, that MC-induced sarcomas may have cross-reacting antigens. For example, in early studies Prehn and Main (1957) described two pairs of mouse sarcomas showing cross-reactivity, and more recently Reiner and Southam (1967, 1969) reported that in addition to the individually distinctive tumor antigens, MC-induced sarcomas also express weaker common antigens. These were detected in mice immunized against several

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

7

sarcomas either simultaneously or sequentially which resisted challenge with tumors not included in the immunization schedule. One objection to this form of treatment is the possibility that a nonspecific stimulation may modify the growth of unrelated tumors. This is emphasized by comparable studies (Basombrio, 1970), where immunization against a single sarcoma occasionally produced resistance against other sarcomas, but this was not a reproducible event. Also, in studies analogous to those of Reiner and Southam (1969) , immunization of mice against several transplanted sarcomas by excision of discrete tumor masses did not consistently elicit resistance to tumors other than the one used for treatment. From this study, it was concluded that the resistance evoked against these sarcomas was directed against individual antigens expressed on each tumor, the cross-reactions being nonreproducible events. Contrary to the general consensus of opinion, Koldovsky and Svoboda (1963) reported a high frequency of antigenic cross-reactivity in MCinduced mouse sarcomas. Also, Takeda (1969) found that rat sarcomas which initially displayed individual antigenicities became cross-reacting after prolonged transplantation. This suggests that the anomalously high cross-reactivity may reflect infection with endemic virus, and experimental support for this suggestion is provided by studies showing that MC-induced mouse sarcomas which initially were not susceptible to polyoma virus induced transplantation resistance became sensitive after polyoma virus infection (Sjogren, 1964). Comparably, cross-reacting tumor-associated antigens have been detected in two of three MC-induced strain-2 guinea pig sarcomas (Holmes et al., 1971), whereas sarcomas induced in strain 13 guinea pigs have individually distinct antigens (Morton et al., 1965; Oettgen et al., 1968). Since virus-associated cavian leukemias have been detected only in strain 2 guinea pigs, it was postulated that cross-reactivity of the sarcomas in this strain could be explained by the presence of this leukemogenic virus or another contaminant virus. This remains unproved, however, since, although virus particles were found in two of the tumors, none was seen in the other sarcoma. Cross-reacting antigens have also been reported in MC-induced bladder tumors (I. Hellstrijm and Hellstrom, 1972), and in preliminary studies, it was reported that the appearance of MC-induced primary bladder papillomas in rats was delayed following immunization with bladder papilloma tissue before the carcinogen is applied (Taranger et al., 1972a). The nature of the antigens associated with MC-induced rat bladder tumors, which appear to be akin to those detected on human bladder tumors (Bubenik e t nl., 1970a,b), has yet to be defined. It may be relevant, however, that cellular damage induced by the implanted pellet is thought to be a contxibutory factor in bladder carcinogenesis.

8

R.

W.

BALDWIN

2. Cellular Immunity Tumor-associated antigens on polycyclic hydrocarbon-induced tumors, especially MC-induced sarcomas, have been more clearly characterized by analyses of the cellular and humoral immune reactions which they elicit. Cellular immunity is generally held to be of paramount importance in tumor rejection, and this is well established in studies showing the capacity of sensitized lymphocytes to adoptively transfer tumor immunity to normal syngeneic hosts (K. E. Hellstrom and Hellstrom, 1969). For instance, neutralization studies have shown that lymph node cells (Klein et al., 1960) and peritoneal exudate cells (Old et al., 1962) from mice immunized against syngeneic MC-induced sarcomas suppressed tumor growth when administered together with viable tumor cells to normal compatible hosts. Moreover the individual antigenicity of the tumors was revealed since peritoneal exudate cells only suppressed growth of the immunizing tumor. In vitro detection of cellular immune reactions was first reported by Rosenau and Morton (1966), who showed that spleen cells from immunized mice were cytotoxic for MC-induced mouse sarcoma cells in tissue culture, but the effects were not marked and the method of assay, involving the counting of surviving cell nuclei, has not found general acceptance. An alternative method of detecting cell-mediated immune reactions, developed by Hellstrom (1967), measures the capacity of sensitized lymphocytes to inhibit the capacity of tumor cells in tissue culture to form colonies, a technique which reflects the ability of the lymphocytes to both kill target cells and inhibit their growth. In this way, lymph node (LN) cells taken from mice between 3 and 8 days after surgical removal of a primary MC-induced sarcoma were shown to decrease the colonyforming capacity of sarcoma cells prepared from the excised tumor and explanted in vitro (HellstrBm et al., 1968). This suppression was significantly different from the background effect of L N cells from normal mice. The tumor specificity of the reaction was confirmed by showing that L N cells taken from mice exposed t o another MC-sarcoma only produced inhibitions comparable to that of L N cells from normal donors. Similarly, L N cells taken from rats immunized with irradiated grafts of MC-induced sarcomas inhibited the colony-forming capacity of plated tumor cells in tissue culture, and the specificity of these reactions was confirmed since LN cells were only inhibitory against cells of the immunizing tumor (Baldwin and Moore, 1971). In contrast, Taranger et al. (1972b) have reported that LN cells from mice bearing MC-induced bladder tumors destroyed culture bladder tumor cells derived not only from the lymphocyte donor, but also from other carcinogen-treated mice.

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

9

Cross-reactivity was detected between antigens associated on both bladder papillomas and carcinomas, although not with tumors arising in different tissues. Delayed hypersensitivity tests have similarly been applied for the detection of cellular immune reactions to tumor-associated antigens on MC-induced sarcomas. This procedure has proved to be particularly effective for demonstrating tumor immunity to guinea pig sarcomas induced by a number of polycyclic hydrocarbons including MC (Oettgen et al., 1968). These tumors are exceptional in that the tumor-specific antigen can readily be prepared in soluble form (Oettgen et al., 1968; Holmes et al., 1970), and these soluble components elicit delayed-type hypersensitivity reactions specific for each individual tumor. Moreover, these reactions may be demonstrated in vitro by assaying the capacity of the soluble antigens to inhibit migration of sensitized peritoneal macrophages and less reproducibly by the release of migration-inhibitory factors from immune lymph node cells (Bloom et al., 1969). Cutaneous delayed hypersensitivity reactions have also been observed in mice sensitized against MC-tumors either by surgical removal of growing tumors or immunization with disrupted tumor cells in Freund’s adjuvant (Halliday and Webb, 1969). These reactions were elicited using viable tumor cells, tumor extracts being ineffective, and the specificity of the test was confirmed by showing that reactions were only produced against cells of the immunizing tumor. This may explain why Wang (1968) was unable to detect individually distinctive immune reactions against benzo [ a ]pyrene-induced rat sarcomas since homogenized and sonicated tumor extracts were used for testing. In this case, the delayed hypersensitivity reactions appeared to be detecting a common antigenic component of the tumors since complete cross-reactivity was detected. The macrophage migration inhibition assay has also been used to detect tumor-specific immune reactions in vitro to MC-induced mouse sarcomas using both whole tumor cells and tumor extracts (Halliday and Webb, 1969; Halliday, 1971). 3. Humoral Antibody Passive transfer of immunity to MC-sarcomas with immune serum is not generally effective, since, depending upon conditions as yet not well defined, inhibition or enhancement of tumor growth may be obtained (Moller, 1964; Bubenik et al., 1965). One important factor is the relative concentration of tumor-specific antigen a t the cell surface (Linscott, 1970) so that while leukemic cells are susceptible to cytotoxic antibody both in vivo and in vitro (Old and Boyse, 1965; Pasternak, 1969), cells of solid tumors are less readily killed. More critical analyses of the in-

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R. W. BALDWIN

fluence of immune serum immunoglobulins on growth of MC-induced sarcomas (Bloom and Hildemann, 1970) reveal that the variable effects observed in earlier studies (Moller, 1964; Bubenik and Koldovsky, 1964; Bubenik et al., 1965) probably reflect responses to different immunoglobulins. These results implicate IgM antibody in resistance and IgG antibody in enhancement. This may reflect the effects of these immunoglobulin types a t the tumor cell surface since IgG is relatively inefficient for binding complement, but central inhibition of regulation of sensitized lymphocyte function by IgG antibodies is also probably involved (Bloom and Hildemann, 1970). Even though mouse sarcoma cells are not susceptible to cytotoxic antibodies in conventional assays (Old and Boyse, 1965), the activity of tumor-immune serum can be detected against cultured sarcoma cells This was first observed by Hellstrom et al. (1968), who showed that exposure of MC-sarcoma cells to sera from mice immunized by surgical excision of primary tumors, in the presence of added complement, inhibited their capacity to form colonies. These effects were significantly different from the response to normal serum, and sera from mice immunized against other sarcomas was ineffectual, again demonstrating the tumor specificity of the reactions. Complement-dependent tumor-specific cytotoxic antibody against MC-induced sarcomas has also been detected using the microassay technique of Takasugi and Klein (1970), in this case cytotoxicity being defined by the survival capacity of treated cells (Bloom, 1970; Bloom and Hildemann, 1970). Furthermore colony inhibition methods have been applied to demonstrate cytotoxic antibody in sera from rats immunized against irradiated grafts of rat sarcomas (Baldwin and Moore, 1971), and in this case the specificity of these reactions was identical to that detected by rejection of viable tumor cells and by membrane immunofluorescence assay of tumor-immune sera (Baldwin et al., 1971a,b). Although methods detecting interaction of labeled antibody with antigens localized in the cell membrane provide little information on the significance of these interactions in tumor rejection mechanisms, they allow more precise analysis of cell antigen expression. I n some cases, radioiodine-labeled antibody has been used to detect these interactions (Harder and McKhann, 1968), but undoubtedly the membrane immunofluorescence test provides the most versatile system. Early studies by Lejneva et al. (1965) on the interaction of mouse sarcoma cells with tumor-immune sera indicated the potential of the immunofluorescence method, although the validity of the tests was not fully confirmed in cross-reaction studies with different tumors. Subsequent to extensive analysis of rat hepatoma antigens (see Section II,B),

11

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

membrane immunofluorescence tests have been employed for a critical analysis of antigen expression on MC-induced rat sarcomas (Baldwin et al., 1971a,b). I n the first place it was 'established that sera produced by immunization of syngeneic rats with irradiated sarcoma cells showed significant membrane immunofluorescence reactions with cells of the immunizing tumor, this being visualized as point or segment fluorescence staining a t the cell surface membrane. In contrast to the reproducible reactions of each antiserum with cells of the immunizing sarcoma, only one of the sera showed any membrane staining with a wide range of other sarcomas (Table 11). Also, except in one case, tumor-specific antibody could only be absorbed from immune serum by cells of the immunizing tumor. Since in cross-tests, absorption with 100 times more cells than the numbers needed with the immunizing tumor generally failed to remove tumor-specific antibody, this also indicated that these sarcomas do not contain weak common antigens as well as more individual components. This was further emphasized by tests comparable to those described by Reiner and Southam (1967, 1969), in which attempts were made to detect cross-reacting antibody in rats immunized with pooled sarcomas (Baldwin et al., 1971b). Although serum from immunized rats reacted in membrane immunofluorescence tests with cells of the sarcomas used in the immunization pool, no reactions were detectable with other sarcomas. These findings are therefore in accord with the studies of TABLE I1

SPECIFICITY OF TUMOR ANTIGENS ASSOCIATEDWITH 3-METHYLCHOLANTHRENE-INDUCED

RAT SARCOMAS

Immune reactions with cells of

Cross-tested sarcoma

Immunizing sarcoma Assay methods Serum membrane immunofluorescence Fluorescence indices Antibody absorption by viable sarcoma cells* FI of absorbed serum with cells of immunizing sarcomac

Number positive

Number negative

Number positive

Number negative

8

0

1

120

0.49-0.74 6

0

0.34-0.57 0.00-0.22 1 35

0.00-0.16

-

0.00-0.07 0.35-0.68 ~

Derived from Baldwin et al. (1971e,b, 1972a). a 5 X 1W to 107 cells/ml of immunizing tumor: 108 celb/ml of cross-tested sarcoma. FI of unabsorbed sera ranged from 0.50 to 0.64. a

12

R. W. BALDWIN

Basombrio (1970) showing that immunization with pooled sarcomas does not produce immunity to unrelated sarcomas. B. AMINOAZO DYES Tumor-associated rejection antigens are expressed on hepatomas induced by 4-dimethylaminoazobenzene (DAB) in the rat (Gordon, 1965; Baldwin and Barker, 1967a) and by o-aminoazotoluene in the mouse (Riliiller, 1968). In general, the immunogenicity of rat hepatomas, ns reflected by the maximum tumor cell inoculum rejected by immunized syngeneic hosts (5 x 1 0 to lo6 cells), is comparable with that of polycyclic hydrocarbon-induced rat sarcomas (Baldwin, 1955; Baldwin and Pimm, 1971), and tumor antigen expression is a common feature (Fig. 1). The hepatoma-associated antigens are also individually distinct components as indicated by the specificity of the resistance elicited against transplanted hepatoma cells in syngeneic recipients (Baldwin and Barker, 1967a). This is further emphasized by experiments showing that four distinct hepatoma nodules arising in a single rat treated with 3'-methylDAB were antigenically distinct (Ishidate, 1970). The specificity of hepatoma-associated antigens was also revealed in adoptive transfer tests

n

DENA-induced hapotorno

IMMCMOGENIC

11

$

I

DAB-induced hepatorno AAF-in&cdmo

mory

.

&IDcoTcinoma

A A F - i r b c d ear duct cOrcinorno

0

U

n

2

4

1 12

20

40

30 LATENT

PERIOD OF

50

60

1

m

INDUCTION (WEEKS)

FIG.1. Immunogenicity of carcinogcn-induced rat tumors correlated with latent periods of induction. DEXA. dicthylnitrositmine; DAB, 4-tlimrthylaminoazobcnscnc ; AAF, 2-acetylaininofluorcnc.

13

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

showing that peritoneal exudate cells suppressed growth only of the immunizing tumor when transferred to normal compatible rats (Baldwin and Barker, 1967%; Ishidate, 1967). A more comprehensive and critical analysis of tumor antigen expression on DAB-intluced rat hepatomas has been provided by in vitro studies of the cellular and humoral immune responses to these tumors when transplanted into syngcncic hosts. Initially, membrane immunofluorescence tests with viable tumor cells in suspension established that a specific antibody reaction could be obtained with sera from syngeneic rats immunized against transplanted tumor cells to give significant fluorescence indices (defined as the proportion of unstained tumor cells in a sample treated with normal minus the proportion of unstained cells treated with immune serum divided by the former figure) (Baldwin and Barker, 196713; Baldwin et al., 1971a,b). Using a panel of antisera from rats immunized against six individual transplanted hepatomas, membrane immunofluorescence cross tests were then carried out against a range of up to 16 unrelated DAB-induced hepatomas (Table 111),but no positive reactions were obtained in a total of 67 cross tests (Baldwin et al., 1971a,b). The individual specificity of the DAB-rat hepatoma antigens was further confirmed by comparison of the capacity of intact tumor cells to absorb antibody from tumor-immune sera. Initial studies estabTABLE I11 SPECIFICITY OF TUMOR ANTIGENS ASSOCIATEDWITH AMINOAZO DYE-INDUCED RAT HEPATOMAS Immune reactions with cells of: Immunizing hepatoma Assay method Serum membrane immunofluorescenceO Fluorescence indices Inhibition of colony formationb (CI) by Serum CI% Lymph node Cells

Cross-tested hepatoma

Number positive

Number negative

Number positive

Number negative

16

0

0

67

0.30-0.74

-

-

0.00-0.22

0

3 9.3-10.8 0

69 0.0-6.5 67 0.0-22.0

7 29.2-61.7 7 40.1-79.0

0

-

-

a Derived from Baldwin et al. (1971a,b). Fluorescence indices of 0.30 or greater represent significant reactions. Derived from Baldwin and Embleton (1971a).

14

R. W. BALDWIN

lished conditions under which antibody could be absorbed from a serum with cells of the immunizing tumor. It was then established that treatment of further aliquots of the serum with 10-fold excesses of other hepatomas caused only a slight nonspecific lowering of the fluorescence index of the serum (Baldwin et al., 1971a). In this way it was established that DAB-induced hepatomas were devoid of any minor cross-reacting tumor-specific antigen which may have been masked by a more prominent individually distinct tumor antigen. The individual specificities of DAB-induced rat hepatoma antigens was further elaborated in studies showing that serum from tumor-immune rats was only cytotoxic for cells of the immunizing tumor, this being demonstrated by assay of complement-dependent inhibition of colony formation of cultured hepatoma cells (Baldwin and Embleton, 1971a). Whereas colony inhibitions of between 29 and 62% were obtained when sera were tested against cells of the immunizing tumor, cross tests with up to 12 other hepatomas failed to demonstrate any cross-reactivity (Table 111).The colony inhibition technique was also used to explore the specificity of lymphocyte-mediated immune reactions of the rat hepatomas and comparably, these reactions were shown to be highly specific. As with the serum studies, significant inhibition of colony formation was consistently obtained when lymph node cells were tested against cells of the immunizing hepatoma, but were totally ineffective for cells of other hepatomas (Baldwin and Embleton, 1971a). It was concluded from these comprehensive surveys that the tumor antigens associated with DAB-induced rat hepatomas are highly characteristic membrane components. It is interesting to compare these findings obtained in syngeneic rats with earlier reports on abnormal antigens in rat hepatomas as detected by immunodiffusion analyses of antisera prepared in rabbits against subcellular tumor fractions (Baldwin, 1965; Baldwin and Barker, 1 9 6 7 ~ ) . These studies established that antigens could be identified in soluble cell sap and microsomal fractions of DAB-induced rat hepatomas which were not present in normal adult liver. Certain of these tumor antigens, however, were later found to cross-react with extracts of other adult tissues such as lung and kidney or with newborn rat serum (Baldwin and Barker, 1967~).This throws doubt on the tumor specificity of the other supposedly tumor antigens, especially since they proved to be common to several hepatomas. Also, the antigens detected by these immunochemical procedures cannot be defined in relation to the host without corroborative studies in syngeneic systems and so may have little relevance to immune reactions in aminoazo dye carcinogenesis. Nevertheless these antigens, which may be viewed as time or tissue displaced components, may also reflect cellular derangements induced by the carcinogen.

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENEXSIS

15

c. 2-ACFXYLAMINOFLUOlWNE 2-Acetylaminofluorene (AAF) -induced rat tumors behave anomalously in not showing consistent or marked immunogenicity. For example, tumor transplantation immunity could only be elicited against two of eleven mammary carcinomas in syngeneic rats immunized either by excision of growing tumor or by repeated implantation of irradiated tumor cells (Baldwin and Embleton, 196%). Similarly only three of ten AAFinduced hepatomas and one out of three ear duct carcinomas elicited tumor rejection responses to transplanted tumor cells and, where demonstrable, resistance was weak as reflected by the maximum number of tumor cells rejected by immunized rats (Baldwin and Embleton, 1971b). Tumor-specific antibody, again, which only reacted with cells of the immunizing tumor, was detectable by membrane immunofluorescence tests in the examples where weak tumor rejection reactions were obtained, but not in any others (Baldwin and Embleton, 1969a, 1971b). It is therefore unlikely that the lack of resistance observed following immunization with the majority of AAF-induced tumors reflects an inappropriate immune response eliciting circulating antibody with enhancing activity and only a minor stimulation of the cellular reaction. AAF-induced tumors, therefore, must be considered as not being significantly immunogenic, although of course the practical limitations of the tumor rejection test make it impossible to decide absolutely that the tumors do or do not express weak immunogenicity. Why this should be has yet to be adequately resolved, but the low frequency of antigenic tumors occurring in rats treated with AAF can be correlated with their relatively long latent induction periods (Baldwin and Embleton, 1971b). It is clear, for instance, that DAB-induced and DENA-hepatomas and M,C-induced sarcomas in the rat arise relatively rapidly (Fig. l ) , and in these situations, a high proportion of the tumors express tumor-associated antigens which render the cells immunogenic. On the other hand, AAF carcinogenesis produces tumors in which the latent induction periods are much more variable, but in most cases they are significantly greater than that observed with aminoazo dyes or polycyclic hydrocarbons. I n these tumors the frequency and “strength” of tumor antigen expression is proportionately lower. Contrary to the findings in tests in syngeneic animals, rat hepatomas induced by AAF have been shown to have antigens not present in normal rat liver by their reactions with heterologous antisera against subcellular tumor fractions. In earlier reports these antigens were detected by in vivo or in vitro binding of purified radioiodine labeled rabbit antihepatoma microsome antibody (Isojima et al., 1966). Comparable results have also

16

R. W. BALDWIN

been reported in studies on the immunofluorescence staining of primary and cultured hepatoma cells by antitumor microsome antibody (Isojima et al., 1969). These reactions were visualized by intracellular staining of acetone-fixed hepatoma cells, and so while thcy show a difference in antigen content between AAF-induced hepatoma and normal liver cells, the studies do not indicatc whether they reflect qualitative or quantitative changes. Also the tumor specificity of the hepatoma reactions must be questioned since, while not present in normal liver, these antigens may be expressed in other adult or embryonic tissues. Again, therefore, thc relevance of thesc changes in relation to tumor-host interactions cannot be established without supporting evidence from syngeneic systems.

D. ALKYLNITROSAMINE Diethylnitrosamine (DENA)-induced hepatomas in strain 2 guinea pigs are particularly immunogenic, and immunity can be induced by excision of transplanted tumor or by intradermal or intramuscular injection of subliminal doses of tumor cells such that progressive growth is prevented (Zbar et al., 1969). The immune response to intradermal inoculations of viable tumor cclls, where temporary growth occurred to form a papule which then regressed, was sufficiently pronounced even to induce rejection of the same hepatoma simultaneously implanted intramuscularly where normally progressive growth would have resulted (Wepsic et at., 1970). These guinea pig hepatomas mostly have characteristic individual antigens, although threc out of eight examples were found to share a t least one antigen (Zhar et al., 1969). I n some examples, also, hepatomas initially showing individually specific antigens, acquired cross-reactivity with a long-transplanted tumor line, but the characteristics of these common antigens have yet to be defined. It is interesting t o note that MC-induced sarcomas in strain 2 guinea pigs have also been reported to have cross-reacting tumor rejection antigens (Holmes et al., 1971), but other studies (Morton et al., 1965; Oettgen et al., 1968) showed that the antigcns associated with sarcomas in strain 13 guinea pigs were individually distinct. An added complication is the finding that viruslike particles are present in a strain 2 guinea pig leukemia (Opler, 1967; Nadel et al., 1967), so that the possible influence of viral agents on antigenic cross-reactivity of chemically induced guinea pig tumors needs to be further explored (Holmes et al., 1971). Cellular immunity is particularly pronounced in guinea pigs immunized against DENA-induced hepatomas, and it was possible to adoptively transfer tumor immunity, measured either by suppression of tumor growth or delayed cutaneous hypcrsensitivity reactions, with peri-

IMMUNOLOGICAL ASPECTS O F CHEMICAL CARCINOGENESIS

17

toneal exudate cells from sensitized donors (Kronman et al., 1969; Wepsic et al., 1970; Zbar et al., 1970). These cellular immune reactions were also detected in vitro by assaying the capacity of lymphocytes from peritoneal exudate cell preparations to inhibit tumor cell DNA synthesis, measured by incorporation of tritiated thymidine (Oppenheim et al., 1970). In contrast, a variety of assays including immunofluorescence staining and passive transfer tests have failed to detect any hurnoral antibody response in immunized guinea pigs. Diethylnitrosamine-induced hepatomas in the rat are also immunogenic as defined by the capacity of irradiated tumor cells to elicit rejection reactions against transplanted tumor cells in syngeneic recipients (Baldwin and Embleton, 1971b). In vitro studies of the immune response to these tumors have been little explored so far, but weakly positive membrane immunofluorescence reactions were obtained in tests with one hepatoma (Baldwin and Embleton, 1971b). This contrasts with the highly reproducible serum antibody responses to hepatomas induced by aminoazo dyes (Baldwin and Barker, 1967b; Baldwin et al., 1971a,b), but interestingly corresponds to the lack of circulating antibody detected against guinea pig hepatomas (Oppenheim et al., 1970). The antigenic profiles of DENA-induced rat hepatomas compared with normal tissues have been analyzed by immunodiffusion techniques using heterologous antisera prepared against tumor homogenates (Garisoain and Arcos, 1970). These showed the presence of an antigen in hepatoma extracts which, while absent from normal liver, was still detectable in embryonic and newborn livcr. Again, this antigen, like that detected in both rat and mouse hepatomas induced with aminoazo dyes, is probably an intracellular component so that its relation to the cell surface expressed tumor antigens which represent new components relative t o the adult host remains in question. The only other tumors induced with diethylnitrosamine which so far have been characterized immunologically are pulmonary adenocarcinomas in mice (Pasternak et aZ., 1966). With these, there was no conclusive evidence for tumor immunogenicity since tumor rejection reactions could not be regularly demonstrated in immunized animals even when challenge inocula as low as 2 X lo4viable tumor cells were used. There is now available a host of related alkylnitrosamines with particular tissue specificities, such as the induction of adenocarcinomas of the small intestine and colon by N-nitrosomethylurea (Magee and Barnes, 1967). Also there is a substantial body of evidence on the nature of the interaction between nitrosamine metabolites and cellular macromolecules (Magee and Barnes, 1967), and it is surprising that these systems have not yet been fully explored immunologically. A study of nitros-

18

R. W. BALDWIN

amine-induced tumors, for example, should provide an insight into the possible relationship between carcinogen-tissue interaction and tumor antigen expression. Cell lines transformed in vitro by N-nitrosomethylurea (Sanders and Burford, 1967) also provide another model for studying the possible significance of tumor antigen expression in neoplastic transformation. I n this context, studies with one transformed line from C,H mouse prostate cells treated with nitrosoguanidine (Emblcton and Heidelberger, 1972) have detected characteristic ncw cell surface antigens capable of eliciting immune responses in syngeneic mice.

E. PHYSICAL AGENTS Experimental tumors induced by physical agents also have tumorassociated rejection antigens. These include murine sarcomas induced by UV irradiation (von Graffi at al., 1964, 1965) or by implantation of inert membrane films made of plastic materials, such as cellophane (Klein e t al., 1963), polyvinylchloride, and silicone (Horn et al., 1965), or Millipore filter (Prehn, 1963a). Methods used for eliciting immunity to plastic film-induced tumors transplanted into syngeneic mice were similar to those employed with hydrocarbon-induced sarcomas, but in general the degree of resistance demonstrated was weak. Immunized mice only rejected low numbers of tumor cells (lo3 to lo4), and there were proportionately more examples where resistance to transplanted tumor cells could not be detected. Many of these sarcomas have long-latent induction periods ; for example, cellophane film-induced sarcomas mostly arose between 13 and 20 months (Klein et al., 1963). Compared with the high immunogenicity of rapidly MC-induced murine sarcomas (Klein e t al., 1960), this suggested an inverse relationship between tumor antigen expression and latent induction period. On the contrary, however, UVinduced murine sarcomas, are highly immunogenic (von Graffi et al., 1964, 1965), but also have relatively long latent induction periods. Plasma cell tumors induced in BALBJc mice given intraperitoneal injections of mineral oil may also be considered in this category, and with these, immunity has been induced against transplanted tumor cells in syngeneic hosts using tumor cells attenuated with sodium iodoacetate (Lespinats, 1969). This was accompanied by the formation of tumorspecific antibody detectable by immunofluorescence methods (Lespinats, 1970). Specificity tests revealed that the immune serum initially reacted only with cells of the immunizing tumor, but prolonged immunization resulted in the appearance of cross-reacting antibodies. Interestingly, plasma cell tumors induced by mineral oil contain type A viruslike particles (Dalton et al., 1961 ; Kuff et d., 1968), and the response to tumor immunization is very similar to that reported with strain 2 guinea pig

IMMUNOLOGICAL ASPECTS O F CHEMICAL CARCINOGENESIS

19

sarcomas, whcre it has bcen postulatcd that antigcnic cross-reactivity may be explained by the presence of a common viral agent (Holmes et al., 1971).

F. SIGNIFICANCE OF TUMOR ANTIGENEXPRESSION IN CHEMICAL CARCINOGENESIS Pcrhaps the most significant featurc of thc tumor-rejection antigens associated with chemically induced tumors is their great diversity. In comparing tumor antigenicity with the expression of histocompatibility antigens and the genetic control of antibody production, Burnet (1970) postulated that a wide range of antigens may exist in normal cells. This, however, would only be manifested when the antigenic pattern of the cell line from which the tumor arose is made available in adequate concentration by the monoclonal proliferation of the tumor. This concept would appear untenable, since it has been established that tumors arising following 3-methylcholanthrene treatment in vitro of a cloned cell line of mouse prostate cells each possess individually distinct tumor rejection antigens (Embleton and Heidelberger, 1972). Similarly, characteristic antigenic specificities have been detected in tumors induced in vivo by treatment of cloned BALB/c 3T3 cells with 3-methylcholanthrene in Millipore chambers (Basombrio and Prehn, 1972a,b). An alternative concept is that the carcinogen induces a mutationlike heritable alteration resulting in the expression of new antigens a t the ccll surfacc. On this basis, it is necessary to postulate that there are a number of sites which, when randomly modified by direct or indirect carcinogen interaction, produce changes in the genome of the cell leading to the expression of new surface antigens. This hypothesis must also take into account the appearance of nonantigenic as well as antigenic tumors after exposure to potent carcinogens, such as 3-methylcholanthrene (Prehn, 1970), 2-acetylaminofluorene (Baldwin and Embleton, 1969a, 1971b), and urethan (Prehn, 1962). One possibility initially proposed by Prehn (1967), that the appearance of weak or nonantigenic tumors reflects the influence of immunological surveillance, is borne out to some extent by correlations between the immunogenicity of a tumor and its latent induction period (Fig. 1). That is, rapidly arising tumors are frequently significantly more immunogenic than those developing more slowly. It is apparent, however, that antigenic variability occurs in tumors with comparable latent induction periods (Prehn, 1970). Also, tumors having significant immunogenicities generally retain this property through many generations of transplantation in immunologically competent hosts, indicating that the expression of tumor antigen is a stable characteristic property. hlore significantly, tumors induced by

20

R.

W.

BALDWIN

3-methylcholanthrene in cells contained within the immunologically protected confines of a Millipore chamber (Basombrio and Prehn, 1972a,b) or in in vitro culture conditions (Rlondal et al., 1970, 1971; Embleton and Heidelberger, 1972), where immunosurveillance is positively excluded, also exhibit antigenic variability. Furthermore, in both systems a number of the MC-induced tumors lacked detectable immunogenicity. Comparably, cells showing spontaneous transformation in these systems were generally deficient in tumor-rejection antigens, and this is consistent with the known infrequency of antigenicity of spontaneously arising tumors (Prehn and Main, 1957; Hammond e t al., 1967; Baldwin and Embleton, 1969b). From these considerations, the expression of new cell surface antigens would appear not to be a necessary product of malignant transformation, possibly because certain sites of interaction with carcinogen within the cell genome do not code for plasma membrane-expressed protein. Accordingly, it may be postulated that a chemical carcinogen acts randomly to produce changes in the genome of the cell which may be expressed as a malignant transformation and also give rise to new cell surface membrane antigens, although these two events are not necessarily interdependent. If it is accepted that the antigenic characteristics are imparted to cells during neoplastic transformation by chemical carcinogens and are not modified during tumor growth and progression, this hypothesis must account for the general lack of immunogenicity of AAF-induced rat tumors, particularly hepatomas (Baldwin and Embleton, 1969a, 1971b), in comparison with DAB-induced hepatomas which almost always express tumor-associated antigens (Baldwin and Barker, 1967a ; Baldwin et al., 1971a,b). Both these carcinogens undergo similar metabolic activation in vivo involving N-hydroxylation and esterification to provide ultimate carcinogens (E. C. Miller and Miller, 1969; J. A. Miller and Miller, 1969). The close correspondence in the mode of action of these carcinogens is further reflected by the comparable sarcomogenic activities in the rat of the synthetic esters, N-benzoyloxy-AAF (E. C. Miller and Miller, 1969; J. A. Miller and Miller, 1969) and N-benzoyloxy-4-methylaminoazobenzene (RIAB) (Poirier et al., 1967; E. C. Miller and Miller, 1969; J. A. Miller and Miller, 1969). Analogously, these esters attack the transforming DNA for Bacillus subtilis, giving similar mutagenic indices (Maher et al., 1968). The ultimate carcinogenic metabolites of AAF and DAB also show overall similarities in the patterns of interaction with target cell macromolecules including protein, DNA, and RNA (E. C. Miller and Miller, 1969; J. A. Miller and Miller, 1969). Both hepatocarcinogens interact with soluble liver proteins belonging to a small group of relatively basic proteins, electrophoretically defined as h, proteins (Sorof

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

21

et al., 1963, 1969; Sorof, 1969). However, more precise studies of molecular size profiles determined by gel filtration methods (Sorof et al., 1970) have detected differences in the proteins involved in binding different carcinogens. The principal conjugates of AAF in rat liver are h2 proteins with molecular weights of approximately 150,000, whereas the h, proteins binding 3’-methyl DAB have molecular weights between 60,000 and 80,000. So far no distinctivc differences are apparent in the binding of metabolites of AAF and DAB with either DNA or RNA, covalent linkage to position 8 of guanine being a preferential site of attack (E. C. Miller and Miller, 1969; J. A. Miller and Miller, 1969; Weinstein et al., 1971). However, considerable refinements are needed in studying the significance of carcinogen-nucleic acid interactions since as yet there is no clear indication of which nucleic acid bases are critical for carcinogenesis (Magee, 1971). From these considerations, it is conceivable that the variations observed in the expression of tumor rejection antigens in AAF- and DAB-induced hepatomas may reflect differential sites of carcinogen attack, either directly or indirectly, in the DNA of the cell genome, the implication being that AAF-induced modifications do not code for cellsurface membrane components. Howcver, more precise analyses of tumor antigen expression are needed to correlate with the much more sophisticated analyses of carcinogen tissue interactions. It would be important to know, for example, whether sarcomas induced by the N-benzoyloxy esters of AAF and DAB also differ immunologically. A further advance would be the analysis of tumor antigen expression on cells transformed in vitro by different chemical carcinogens. Tissue culture cell systems may also be used to investigate the related problem of whether carcinogen-treated nontransformed cells express neoantigens. 111. Isolation and Characterization of Tumor-Associated Antigens

Tumor-associated rejection antigens are almost certainly located within the cell surface membrane; it is difficult to conceive how intracellular antigens could operate to produce rejection reactions, because the tumor cell membrane is largely impermeable to antibody and although the mode of action of sensitized lymphocytes is still not yet clearly dcfincd, the general opinion is that cell-cell interactions are involved a t least in the primary interaction (Perlmann and Holm, 1969). The importance of cell membrane-associated antigens has been more directly demonstrated by in vitro studies of the cytotoxicity of immcne serum or sensitized lymphocytes for culture tumor cells (K. E. Hellstrom and Hellstrom, 1969; Perlmann and Holm, 1969). Tumor antigens detected by membrane immunofluorescence tests with viable tumor cells in suspension are also, by definition, exposed a t the cell surface, and although there is

22

R. W. BALDWIN

no direct evidence that these are thc same as the tumor rejection antigens, this seems likely in view of their identical specificities. Tumor rejection antigens may thus be viewed as weak alloantigens analogous to the cell membrane-associated H-2 antigens in mice and the HL-A system of humans (Amos, 1969; Kissmeyer-Nielson and Thorsby, 1970; Reisfeld and Kahan, 1971). Conscquently, the rapid advances in mcthodology for alloantigen isolation and chemical characterization (Reisfeld and Knhan, 1970, 1971) are relevant to the study of tumor antigens. Another important factor has been the development of reliable in vitro methods for measuring tumor antigen, since early attempts to define the antigenicity of isolated tumor cell fractions by their capacity to elicit tumor immunity in compatible hosts gave inconsistent or, more often, totally negative results. Serological methods involving antibody interaction, such as membrane immunofluorescence, are particularly useful, but present assay methods are still relatively insensitive and laborious and need refinement. Measurement of binding of radioisotope-labeled antibody may prove applicable for this purpose, providing that the problems of nonspecific antibody uptake by dead cells can be controlled. Alternatively inhibition of lymphocyte or serum-mediated cytotoxic reactions for plated tumor cells may provide a useful assay, especially since the development of microtest systems (Takasugi and Klein, 1970) and the use of radioisotope target cells (Cohen e t al., 1971). Despite these methodological limitations, it has now been possible in a number of tumor systems to isolate subcellular fractions retaining tumor specific antigen so that more precise chemical characterization becomes feasible. A. AMINOAZO DYE-INDUCED RAT HEPATOMAS Using procedures designed initially for the isolation of plasma membrane-associated mouse H-2 antigens (Ozer and Wallach, 1967) membrane fractions prepared from transplanted DAB-induced hepatomas following cell rupture by intracytoplasmic cavitation with nitrogen gas were found to retain tumor-specific antigen (Baldwin and Moore, 1968, 1969a). I n these studies, the antigen content of membrane fractions was measured by their capacity to specifically absorb antibody from tumorimmune sera prepared by immunizing syngeneic rats against intact tumor cells. Also, since antibody absorption was measured by the loss of reactivity of sera with intact tumor cells, the tests measured antigen expressed on the surface of hepatoma cells. In this way it was established that isolated membrane fractions retained tumor-specific antigens, whereas none was detected in the soluble cell extracts. However, the yield of tumor antigen relative to that expressed on intact hepatoma cells was generally less than lo%,whereas almost total recovery of rat alloantigen

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

23

was obtained from thcsc hcpatoma cells using identical methods (Baldwin and Moorc, 1969b). Initial attempts to fractionate hepatoma membrane preparations by differential centrifugation showed that the antigen was widely distributed among particles of various sizes (Baldwin and Moore, 1969a). This is because cell rupture following nitrogen pressure homogcnization results in a high dcgrcc of membrane fragmentation yielding particles widely differing in sizc. Controlled mechanical cell rupture in a Potter-Elvchjem hornogenizcr under conditions which allow the integrity of nuclei to be maintained has since been shown to yield much more defined cell or tissue homogenates for membrane fractionation (Baldwin and Price, 1973). With one hepatoma (D23), fractionation of the nuclear sediment (loo0 g pellet) of these homogenates using rate-dependent centrifugation in an A-XI1 zonal rotor or employing a flotation technique through sucrose solutions of density 1.17 in a B-XIV zonal rotor yielded membrane preparations with an approximately 10-fold increase in antigenic activity compared with that of total subcellular membrane fractions. This is illustrated in Fig. 2 in which the antigenic activity of various subcellular fractions of hepatoma D23 isolated by a B-XIV zonal centrifugation procedure is compared by their capacity to absorb tumor-specific antibody. After the isolation of antigenically active membrane fractions from hepatoma D23 cells, it was established that tumor-specific antigen could 0.7 7 0.6 Ly X

f Ly

0.5 0.4

U

0.3 # U

:0.2

5

0.1

Y

0.0

0

20

40 60 80 100 MG P R O T E I N PER ML

120 140 160 ANTISERUM

180

200

FIG.2. Tumor-spcrific antigen activity of plasma membrane fractions from rat hrpatoma D23. Tumor-specifir antigen was assaycd by thc capacity of individual membrane fractions to absorb antibody from tumor-immune serum, this being measured by the reduction of the serum fluorescence index when tested on hepatoma D23 cells. V-V, Total subcellular membrane sedimented from hepatoma D23 homogenates a t 78,000g for 30 minutes; 0-0, extranuclear membrane fraction sedimented a t 78,000g for 30 minutes from the 1OOOg supernatant of hepatoma 1000 g pellet; 0-0, B-zonal membrane isolated by flotahomogenates. 0-0, tion through sucrose solution ( d 1.17) by centrifugation in a B-WV zonal rotor (R. W. Baldwin and M. R. Price, unpublished observations).

24

R. W. BALDWIN

be solubilized by limited papain digestion (Baldwin and Glaves, 1972a). DEAE-cellulose chromatography of solubilized membrane preparations yielded a chromatographically discrete component (Fig. 3) retaining antigenic activity assayed by its capacity to block the interaction of tumorimmune sera with viable target hepatoma D23 cells. Polyacrylamide gel electrophoresis of this component showed, however, that it was still heterogeneous containing several proteins. Further purification by sucrose gradient centrifugation followed by preparative polyacrylamide gel electrophoresis has yielded a major antigenic fraction with a molecular weight, 55,000 (Baldwin et al., 1 9 7 3 ~ )Other . large proteins (up to 22 S) have also been isolated as pure or partially pure components retaining tumor-specific antigen activity, although these represent only a small proportion of the total antigen recovered. Their significance as reflecting the true state of the antigenic determinant at the cell surface or possibly as discrete aggregates of smaller protein subunits is not known. Isolated plasma membrane fractions from rat hepatomas as well as papain-solubilized preparations are immunogenic in syngeneic hosts eliciting tumor-specific antibody directed specifically against cell membraneassociated tumor antigens (Baldwin and Moore, 1969a; Baldwin and Glaves, 1972a). These humoral antibody responses are detectable by membrane immunofluorescence staining and consistent with the data obtained following immunization with intact tumor cells, the response is specifically directed against cells of the immunizing tumor membrane. However, immunization with isolated plasma membrane fractions does

;iI

0

10

20

30

40

z N

4

Ti 50

60

70

FIQ.3. DEAE-cellulose chromatography of papain+olubilized plasma membrane from rat hepatoma D23 (Baldwin and Glaves, 1972a)..

IMMUNOLOGICAL ASPECTS O F CHEMICAL CARCINOGENESIS

25

not result in the development of tumor immunity (Baldwin and Moore, 1971). Analysis of this form of immunization indicated that the most consistent response was the production of humoral antibody which a t least in vitro was cytotoxic for cells of the immunizing hepatoma as measured by inhibition of colony formation by plated tumor cells (Baldwin et al., 1973a). Additionally, however, membrane-immune sera showed high levels of blocking antibody in that they markedly inhibited the lymph node cell-mediated inhibition of colony formation by plated hepatoma cells. This marked humoral antibody response together with the much weaker and less consistent cell mediated reaction probably accounts for the lack of tumor immunity elicited by immunization with hepatoma membrane fractions. A further consequence of membrane immunization, again most likely due to thc predominant humoral antibody 'response, is that treated animals become unresponsive to immunoprotective immunization with intact hepatoma cells.

B. DIETHYLNITROSAMINE-INDUCED GUINEAPIGHEPATOMAS Human histocompatibility antigens have recently been solubilized by extraction with hypertonic solutions of potassium chloride (Reisfeld and Kahan, 1970; Reisfeld et al., 1971), and this procedure has been applied by Meltzer et al. (1971) for the extraction of tumor-specific antigen from diethylnitrosamine-induced guinea pig hepatoma cell lines. Under optimal conditions, 3 M KC1 extraction led to a recovery of 1540% of the antigenic activity present on viable hepatoma cells as defined by the capacity of cells or isolated extracts to elicit delayed hypersensitivity reactions. Significantly the soluble extracts showed the same individual specificities as the intact hepatomas so that, for example, soluble antigen from hepatoma line 1 cells elicited delayed skin reactions in guinea pigs immunized to line 1 tumor but not in line 10 immune animals, Although antigen extraction initially led to substantial loss of activity, isolated extracts were stable and partial purification of the antigen has been achieved following ammonium sulfate precipitation and gel filtration chromatography on Sephadex G-200. The antigenic fraction eluted in the included volume of the gel confirming solubility and suggesting a molecular weight of 75,000 to 150,000.

C. POLYCYCLIC HYDROCARBON-INDUCED TUMORS Plasma membrane can be readily separated from ascitic mouse lymphomas induced in DBAJ2 mice with 7,12-dimethylbenz [ a ]anthracene so that relatively stable cell ghosts can be isolated following gentle mechanical homogenization (Wolf and Avis, 1970). These cell ghost preparations as well as large plasma mcmbranc fragments isolated by A-XI1

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zonal ultracentrifugation of tumor homogenate are significantly immunogenic, as little as 50 pg membrane protein per mouse inducing resistance against subsequent challenge with 3 X lo3 lymphoma cells. However, cross-reactions occurred in rejection tests with a spontaneous C,H mammary tumor and 2 of 4 DBAJ2 leukemias, and it was suggested that the new antigen expressed on the lymphomas may be virus dependent. Surface membrane ghosts from a long-transplanted 3-methylcholanthrene (MC)-induced murine sarcoma (Meth A) were also found to be immunogenic and although less active, disrupted membrane also induced resistance in a proportion of treated mice (McCollester, 1970). The specificity of Meth A membrane immunization was not evaluated, however, and Halliday and Webb (1969) previously reported that sonicated MCinduced sarcoma cells did not elicit delayed hypersensitivity responses. Solubilized extracts of MC-induced mouse sarcomas have also been reported to retain tumor-specific immunogenicity as measured by rejection of viable tumor cells in immunized mice (Pilch, 1968). I n this study mice immunized either with cell-free saline extracts (7000 g supernatants) or fluorocarbon tumor extracts were shown to reject challenges with viable sarcoma cells, but treatment afforded no protection against challenge with an unrelated spontaneously arising mammary adenocarcinoma. The specificity of the immune response was not evaluated by cross-challenges with other MC-induced sarcomas which have different tumor-rejection antigens, although it was established that no tumor immunity was produced following immunization with extracts of pooled normal mouse tissues. Under the conditions of antigen isolation used in this study, the cell-free extract will contain large amounts of membrane material and also fluorocarbon extraction undoubtedly solubilizes (disperses) membrane lipoprotein so that these studies may be measuring weak immune reactions t o membrane-associated antigens. An earlier, but unconfirmed, study reported that immunity to MC-induced rat sarcomas could be obtained following immunization with crude lipopolysaccharide extracts (Matsumoto, 1965). These findings have not been confirmed (Baldwin and Barker, unpublished observations), but it has been established that cell surface-expressed, tumor-specific antigen can be isolated in membrane fractions of MC-induced rat sarcomas after cell rupture by intracytoplasmic cavitation with nitrogen gas (Baldwin and Pimm, unpublished observations). I n these studies, the sarcoma-specific antigen was determined by its capacity to specifically absorb antibody from serum taken from tumor-immune rats. Since this antibody is detected by membrane immunofluorescence staining with viable sarcoma cells, this defines the surface localization of the tumor antigen. Also, cross-absorption tests

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

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with a series of MC-induced sarcomas indicated that membrane preparations from individual sarcomas only reacted with antibody directed specifically against the tumor. Sarcoma membrane fractions were also immunogenic in syngeneic rats producing antibody detectable by membrane immunofluorescence reactions with cells of the immunizing sarcomas, but as with the rat hepatoma studies (Baldwin and Moore, 1969a), there was no concomitant development of tumor immunity even though challenges were given with threshold doses of viable sarcoma cells. There is substantial evidence indicating that tumor-rejection antigens associated with MC-induced guinea pig sarcomas can be isolated in subcellular fractions following cell rupture by mechanical methods (Oettgen et al., 1968; Holmes et al., 1970). I n this case, antigen was separated as a soluble component, but this may reflect a particular lability of guinea pig cell membrane since alloantigens in this species can, atypically, be isolated in a soluble form (Kahan et al., 1969). Sarcomas induced by M C as well as DMBA in strain 13 guinea pigs were shown to release soluble tumor-specific antigen after tumor homogenization, this being defined as the fraction not sedimented a t 100,OOOg for 60 minutes and not retained on filtration through 0 . 2 , ~pore size M4llipore filter (Oettgen et al., 1968). These soluble extracts were immunogenic in syngeneic guinea pigs producing rejection of viable cells of the immunizing sarcoma. The sarcoma extracts also elicited delayed cutaneous hypersensitivity reactions in guinea pigs immunized either with intact tumor cells or isolated membrane. Significantly, both these parameters of tumor immunity were highly specific, tumor extracts only reacting with guinea pigs immunized against the homologous tumor. Comparably, Holmes et al. (1970) isolated a ‘‘soluble” antigen from MC-induced sarcomas in strain 2 guinea pigs following tumor cell rupture by ultrasonication, but in this case, the conditions of centrifugation (130,000 g for 15 minutes) does not exclude the presence of small membrane particles in the isolated fraction. Nevertheless the tumor extracts were immunogenic producing a degree of tumor resistance comparable with that achieved using irradiated tumor cells. Further studies of the solubilized antigen, both by its capacity to elicit tumor immunity and the production of delayed cutaneous hypersensitivity reactions in tumor-immunized guinea pigs, provided more conclusive proof for the soluble nature of the active component, since it was not sedimented following centrifugation a t 130,OOOg for 60 minutes. Fractionation of DEAE-cellulose yielded an active component, but this was shown by discontinuous polyacrylamide gel electrophoresis to contain a t least 13 components so that characterization of the antigen has not yet been achieved.

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D. CHARACTERIZATION OF MEMBRANE-ASSOCIATED TUMOR ANTIGENS The degree of sophistication of biochemical studies with isolated tumor associated antigens still does not approach that attained in studies on human and mouse alloantigens, undoubtedly one of the major limiting factors is the insensitivity of techniques for assaying tumor antigens. This has already been emphasized in studies on rat hepatoma antigens where antibody absorption methods based on immunofluorescence assays are inadequate for the analysis of the limited amounts of material provided by chromatographic fractionation of solubiliaed membrane. In this case, however, radioimmunoassay procedures offer the possibility of approaching the sensitivity of the cytotoxicity-inhibition tests with 51Crlabeled target lymphoid cells which have been widely applied for studying alloantigen purification (Sanderson, 1964; Wigzell, 1965). Even with the limited evidence on tumor antigen characteristics, it is evident that these antigens appear chemically similar to the cell membrane alloantigens in that closely similar methods are applicable for the isolation of both antigen classes. This is particularly apparent in the case of mouse TL (thymus-leukemia) antigen which occupies a special position in being expressed on thymus cells in some mouse strains (designated TL ) and also on leukemic cells in TL-negative strains of mice which normally never express this antigen (Boyse et al., 1968a; Boyse and Old, 1969). Solubilization of lipoprotein from a radiation-induced leukemia by enzymatic degradation allows chromatographic separation of H-2 antigens into a series of identifiable components each containing just one or only a few of the many specificities determined by the H-2 locus (Davies et al., 1969). This fractionation excludes all other antigens tested for except T L and these appear as superimposed peaks within the family of H-2 peaks (Davies et al., 1969). Furthermore TL and H-2 antigens are related by their location on the cell surface (Boyse et al., 1968b). Biochemical characterization of alloantigen specificities is now a t an advanced stage of development (Reisfeld and Kahan, 1970). In the mouse H-2 system, individual antigens have been isolated from papain-solubiliaed membrane fractions having molecular weights in the region of 65,000, and it has been established that the alloantigenic specificities are defined by glycoprotein molecules containing 8 5 4 0 % protein, about 3 4 % neutral sugar, 1 4 % glucosamine, and 1-2% sialic acid (Shimada and Nathenson, 1969; Yamane and Nathenson, 1970a,b). Precise characterization of the haptenic configuration conferring the individual H-2 antigenic specificities is still unresolved although their susceptibility to protein denaturants and prolonged proteolytic enzyme digestion implies that protein configurations are important. Moreover comparison of the

+

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

29

peptide composition of reactive glycoprotein fragments of two H-2 alloantigens (H-2b and H-2d) indicated that whereas most of the peptides were identical, three peptides of H-2b and four peptides of H-2d were distinctive. This implies that whereas most of the protein structure in the two alloantigens were similar, differences were discernible and it was suggested that protein structure may pIay a central role in determining alloantigen activity (Shimada et al., 1970). Furthermore, guinea pig transplantation antigens (MW 15,000) isolated by low-frequency sound homogenization, have been reported to contain a t most one carbohydrate residue per molecule and HL-A antigens isolated in this way (MW 34,600) contained at most two residues of carbohydrate per molecule (Reisfeld and Kahan, 1971). Sialic acid appears not to be essential for H-2 activity since digestion of neuraminidase-treated antigen with p-galactosidase and fi-N-acetylglucosaminidase removed about 70% of the galactose and 25% of the N-acetylglucosamine without any loss of antigenic activity (Muramatsu and Nathenson, 1971a). Moreover, analyses of the carbohydrate moiety from glycoproteins carrying H-2b and H-2d specificities indicated that each contained two chains of approximately 12-15 carbohydrate residues. The chains recovered from each antigenic species as glycopeptides after pronase digestion appeared identical so that differences in carbohydrate structure were not thought to account for antigenic specificities (Muramatsu and Nathenson, 1970, 1971b). On the contrary, Sanderson et al. (1971) have reported that human HL-A antigens isolated as soluble products by papain digestion of spleen cell membranes are glycoproteins containing some 8--15% carbohydrate. In this case, prolonged Pronase digestion yielded glycopeptides of molecular weight about 8 O W l O , O o O , and chemical analyses of these fractions suggested that carbohydrate is concerned with antigenic specificity. These results were viewed as indicating that the cell genome directly determines a particular amino acid sequence serving as a specific receptor for the antigenic determinant, this being glycoprotein. Characterization of cell surface-associated tumor antigens is still a t a primitive stage of development, but at least the methods developed for alloantigen isolation now make this feasible. For instance H-2d alloantigens isolated from Meth-A tumor cell membrane preparations were similar to those prepared from DBA-2 normal mouse spleen cells in respect to their serological profiles as well as chemical properties defined by amino acid and carbohydrate composition, molecular weight, and chromatographic properties (Muramatsu and Nathenson, 1971b ; Yamane and Nathenson, 1970). With aminoazo dye-induced rat hepatomas there is evidence suggest-

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ing that protein, carbohydrate and lipid moieties are all involved in the arrangement of the tumor-specific antigen in plasma membrane. This has been established in studies on the influence of various enzymatic modifications of either intact hepatoma cells or isolated membrane fractions on their capacity to absorb tumor-specific antibody (Baldwin et al., 1 9 7 1 ~ ) I. n this way it was established that membrane-associated tumor antigen could be degradedJreleased from plasma membrane by treatment with papain, lipase, and P-glucosidase. No antigen loss was observed following treatment with P-galactosidase or a-glucosidase indicating the involvement of glucopyranoside P-(1 - 4) linkages. Also there was no loss of tumor-specific antigen by neuraminidase treatment suggesting that sialic acid is not important. A more precise characterization of the chemical species involved in tumor antigen is now possible since a single antigenically active component has been isolated from papain-solubilized hepatoma membrane, and preliminary studies indicate that this fraction is predominantly protein with trace amounts of carbohydrate (Baldwin et al., unpublished observations). Since these aminoazo dye-induced r a t hepatomas are known to express individually distinct antigens (see Section II,B), it should be possible to elucidate the configurational changes accounting for this specificity in a manner analogous to that now being attempted with alloantigens. IV. Embryonic Antigen Expression in Chemically Induced Tumors

The tumor antigens detected in many carcinogen-induced tumors have been defined by their capacity to elicit specific immune reactions either in the autochthonous host against primary growths or in syngeneic hosts against transplanted tumor cells. This excludes the possibility that tumor antigens are normal tissue antigens inappropriately expressed with regard to organ specificity (space relationship) unless related to antigens of tissues normally in privileged sites (e.g., lens protein). It is conceivable, however, that tumor antigens are cell components normally expressed during embryonic differentiation but repressed in adult life (time-displaced antigens). I n this case, the tumor antigens may be viewed as resulting from gene derepression, which may be either directly or indirectly attributed to the action of the chemical carcinogen. Embryonic antigens have now been shown to be associated with several experimental tumor types. Hepatomas induced by o-aminoazotoluene in mice (Abelev et al., 1963; Abelev, 1968, 1971) and 4-dimethylaminoazobenzene or diethylnitrosamine in rats (Baldwin and Barker, 1967c ; Stanislawski-Birencwajg et al., 1966, 1967; Garisoain and Arcos, 1970) synthesize a-fetoproteins normally present in embryonic, but not adult, normal serum. These antigens are detectable by immunodiffusion

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methods in soluble fractions from hcpatoma homogenates and also are present in the serum of tumor-bcaring hosts, so that they may be viewed as secretory products of the tumor ccll (Abelev, 1968, 1971; Uriel, 1969). Therefore, a t least as far as DAB-induced rat hepatomas are concerned, these antigens differ from the tumor rejection antigens which are firmly associated with plasma membrane (Baldwin and Moore, 1969a ; Baldwin et al., 1971b; Baldwin and Glaves, 1972a). However, evidence of embryonic antigens associated with the surface membrane of tumor cells has been obtained in several tumor systems. Hamster cells transformed by SV40 have been shown to cross-react with antigens on fetal hamster cells so that immunization with irradiated (5000 R) 9- to 12-day-old fetal tissue produced immunity against transplanted tumor (Coggin et al., 1970, 1971). This was accompanied by the production of cytostatic antibody as measured by the inhibition of growth of SV40 hamster tumor cells in diffusion chambers (Coggin and Ambrose, 1969). It was further shown that adenovirus 31 tumorigenesis could be interrupted by a single injection of fetal hamster cells if administered to neonatally infected hamsters 3 weeks after birth and before the appearance of the tumors (Coggin et al., 1971). SV40 oncogenesis could be similarly interrupted, especially in male hamsters, and it was reported that the effect could also be achieved using irradiated (5000 R) human embryonic kidney cells (Ambrose et al., 1971a). Since it was earlier shown that immunization of hamsters with irradiated mouse embryo cells produced cytostatic antibody and protection against transplanted SV40-induced tumor cells (Coggin et al., 1970), these results were interpreted as indicating that SV40 oncogenesis results in the reactivation of specific fetal genes, which may be immunologically similar in cells of widely different origin. The occurrcnce of embryonic antigens on SV40 transformed cells is further emphasized by studies showing that sera from multipregnant hamsters reacted in immunofluorescence tests with antigens on the surface of these tumor cells (Duff and Rapp, 1970). Also, Baranska et al. (1970) reported that serum obtained from guinea pigs immunized with unfertilized C57BL/6 mouse eggs was cytotoxic for SV40-transformed 3T3 cells. Antibody could be absorbed with SV40 transformed cells, but the antiegg serum was not cytotoxic for 3T3 cells or lymph node cells. These findings have been interpreted as indicating that the tumor-specific transplantation antigens associated with SV40-induced tumors are in fact embryonic antigens (Ambrose et al., 1971a; Coggin et al., 1971), but the extension of this concept to other tumor systems is unproved. Thus, while there is also evidence of fetal antigen expression on BALBJc spleen cells infected with Rauscher leukemia virus (Hanna et al., 1971), fetal cell immunization has been reported to be without influence on the development of virus-induced mammary tumors (Blair, 1970) or to evoke trans-

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plantation resistance to polyoma tumor cells (Ting, 1968; Kit et al., 1969). In these studies, however, nonirradiated fetal cells were used for immunization, and it was suggested (Coggin et al., 1970) that the negative results may be explained on the basis of rapid maturation of the fetal cell in the adult environment. It was subsequently shown that implantation of nonirradiated fetal hamster cells which produced embryomas did not elicit any transplantation immunity to SV40-induced tumors (Coggin et al., 1971). Surgical removal of the embryomas resulted in the development of cytotoxic antibody and the “cured” hamsters then rejected challenge with SV40 tumor cells. Because tumors induced by individual viruses generally express crossreacting tumor rejection antigens (Deichman, 1969; Pasternak, 1969), it cannot readily be distinguished whether there are both new virus-coded products and retrogenically activated fetal gene antigens (Ambrose et al., 1971a). I n the case of chemically induced tumors, however, where individual tumors generally have characteristically distinct tumor rejection antigens, this analysis of antigen expression on neoplastic cells is more feasible. I n the studies of Duff and Rapp (1970) and Baranska et al. (1970), the embryonic antigens expressed on SV40-induced tumors were not conclusively demonstrable on either DMBA- or MC-induced tumor cells. More conclusive evidence that MC-induced murine sarcomas have embryonic antigens associated with the cell surface is provided by the finding that colony formation of tumor cells in culture was significantly inhibited by exposure to lymph node cells from multiparous mice (Brawn, 1970). These observations were confirmed in studies with MC-induced rat sarcomas (,Baldwin e t al., 1971b, 1972a), where it was established the serum or lymph node cells were cytotoxic for cultured tumor cells as measured by the microcytotoxicity test (Takasugi and Klein, 1970). Also, embryonic antigens on rat sarcoma cells could be detected by membrane immunofluorescence reactions with serum from multiparous rats. In comparable studies it was also established that DAB-induced rat hepatomas have embryonic antigen expressed a t the cell surface. Multiparous rat sera were cytotoxic for hepatoma cells as measured by complementdependent inhibition of colony formation or direct cytotoxicity for plated tumor cells, and gave positive membrane immunofluorescence staining. Comparably, lymph node cells from multiparous rats reacted with embryonic antigens on hepatoma cells producing significant inhibition of colony formation by plated target tumor cells (Baldwin et al., 1971b, 1972a). These observations raise the possibility that the tumor-specific antigens detected on chemically induced tumors are in fact embryonic antigens, as already implied from studies with SV40 induced tumors (Coggin

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et al., 1970, 1971; Ambrose et al., 1971a). As already discussed, a significant feature of the antigens associated with carcinogen-induced tumors is their great diversity as reflected by the individually characteristic immune response elicited by each tumor. In order to account for this, therefore, it would be necessary to postulate that neoplastic transformation by chemical carcinogens leads to the reactivation of a whole array of fetal antigens, each thmor having a distinct characteristic component. While such a concept is not impossible, it is not in accord with the postulate (Ambrose et al., 1971a) that viral oncogenesis involves activation of specific fetal genes which may code for immunologically similar products in cells of widely different origin. More conclusive proof that, a t least in MC-induced rat sarcomas and DAB-induced hepatomas, the embryonic antigens differ from the tumor-specific antigens has been provided by analyses of the specificities of the embryonic antigens. First, it was established that individual sera from multiparous rats reacted in membrane immunofluorescence tests with a range of both hepatomas and sarcomas. Second, it was found that absorption of these sera with cells of one target tumor removed antibody reacting with both the absorbing tumor and also other unrelated tumors (Baldwin et al., 1971b, 1972a). In this way it was established that embryonic antigens are common to the two tumor types of different histological origin and induced by different carcinogens. This contrasts with extensive published data showing that antibody from tumor-immune sera could be absorbed only by cells of the immunizing tumor (Baldwin et al., 1971a,b), which reflects the individual specificity of the rejection antigens associated with these tumors. From these studies it has been postulated that the embryonic antigens expressed on chemically induced rat hepatomas and sarcomas are not specific products induced by the carcinogen involved, but reflect a more general response to malignant transformation. A similar conclusion has been drawn by Ting et al. (1972), who reported that embryonic and tumor-specific antigens on polyoma and SV40-induced mouse tumors could be distinguished by differential absorption of antisera produced in syngeneic mice against tumor or embryo cells. Antibody in tumor-immune sera could be removed by absorption with syngeneic, allogeneic, or xenogeneic tumor cells transformed by the same virus, this being measured by reduction of the uptake of 1261-labeledantibody by viable target tumor cells in suspension. Antibody was not removed, however, following serum absorption with nonpolyoma or non-SV40-induced tumors. In contrast, antisera prepared by immunization of male mice (C3H/HeN) with X-irradiated syngeneic tissue from 7- to 14-day-old embryos reacted with various tumor cell lines, but not normal spleen or thymus cells. The reactivity of these sera with polyoma or SV40-induced

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tumor cells could be removed by absorption with various types of tumor cells including those not induced by polyoma or SV40 (e.g., leukemias induced by Rauscher and Gross viruses and by DMBA) and by embryonic tissues. Although the characteristics of the embryonic antigens associated with rat hepatomas and sarcomas have yet to be defined, there is evidence to suggest that they may be present in the soluble cytoplasmic proteins of tumor cells. This is implied by the positive immunofluorescence reactions obtained following treatment of embryo cells with antisera from syngeneic rats immunized with soluble extracts of rat sarcomas, whereas no tumor-specific antibody is detectable in these sera (Baldwin and Glaves, unpublished observations). These findings distinguish the embryonic antigens from the tumor-specific antigens which are known not to be present in intracellular fractions from hepatomas and sarcomas (Baldwin and Moore, 1969a ; Baldwin and Pimm, unpublished observations, suggesting that the embryonic antigens detected a t the cell surface of the tumor cells may only show transient expression. This may explain why in these tumors the fetal component appears to play little role in tumor rejection reactions, which are initiated by the individually characteristic antigens intimately associated with the cell membrane (Baldwin and Moore, 1969a; Baldwin et al., 1971b; Baldwin and Glaves, 1972a). This is further supported by the finding that immunization with irradiated (5000 R) 12-day-old rat embryo cells or excision of growing embryomas only produces a weak and inconsistent tumor rejection response in syngeneic rats challenged with a transplanted rat hepatoma, whereas the hepatoma cells are highly immunogenic (Baldwin, Glaves, and Vose, unpublished findings). Nevertheless, there is evidence with other tumor systems, e.g., SV40 and adenovirus 31-induced hamster tumors (Coggin e t al., 1971), that embryonic antigen may contribute more significantly to tumor rejection responses. This may reflect the concentration and degree of association of embryonic antigens a t the cell surface membrane of the tumor cell and could explain, for example, the reports of cross-reactivity between MC-induced rat and mouse bladder tumors (I. Hellstrom and Hellstrom, 1972; Taranger et al., 1972a,b). Another factor which may be important in this respect is the reIative contribution of cell mediated and humoral immune responses to tumor rejection. Thus humoral cytotoxic antibody elicited against embryonic antigens has been reported to be closely associated with the immunity elicited against SV40 hamster tumor cells (Coggin et al., 1970, 1971), and it has been postulated that humoral factors are of prime importance in SV40 tumor immunity (Ambrose et al., 1971b). On the contrary, immune rejection of chemically induced rat hepatomas and sarcomas is

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

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primarily mediated by sensitized lymphoid cells (Baldwin and Barker, 1967a; Baldwin and Embleton, 1971a; Baldwin and Moore, 1971) and humoral antibody is either ineffective or enhances tumor growth (Baldwin and Barker, 1967b ; Baldwin and Pimm, unpublished observations). V. Deletion of Normal Tissue Antigens from Carcinogen-Induced Tumors

Since the demonstration of covalent binding of hepatocarcinogenic aminoazo dyes to rat liver proteins (Miller and Miller, 1947), binding of metabolites to target tissue proteins has been reported with almost all classes of chemical carcinogens (Arcos and Arcos, 1969; E. C. Miller and Miller, 1969; Heidelberger, 1970). The binding of many chemical carcinogens to DNA and RNA has also been observed and a central problem of chemical carcinogenesis is the interpretation of the significance of these interactions in neoplastic transformation. Subsequent to the demonstration of the protein interactions of carcinogenic aminoazo dyes and 2-acetylaminofluorene, it was established that the liver proteins involved in these metabolic interactions were markedly diminished in concentration or even undetectable in the resulting hepatomas. These findings were interpreted as reflecting critical cellular deletions associated with neoplastic transformation and therefore it is not surprising that immunological methods have been used to define so-called “antigen deletions” in neoplastic cells. Since initially much of the biochemical evidence for protein deletions in tumors was obtained in studies with carcinogen-induced hepatomas, these have been extensively investigated by immunological methods. One of the earliest demonstrations of antigen deletion following malignant change was reported by Weiler (1952, 1956a, 1959) showing, by complement fixation methods, that organ-specific rat liver antigen was absent from carcinogen-induced hepatomas. These observations were confirmed and extended by Nairn et al. (1960), who showed, using immunofluorescence tests with tissue cryostat sections, that absorption of rabbit antinormal rat liver microsome antisera with DABinduced hepatoma tissue did not entirely abolish its reactivity with normal liver. Immunofluorescence tests with cryostat sections have also been used to demonstrate loss of normal microsomal antigens from 2acetylaminofluorene-induced primary rat hepatomas (Hiramoto et al., 1961) and transplanted mouse hepatomas originally induced with oaminoazotoluene were similarly shown to be totally or partially deficient in a liver-specific antigen (Engelhardt et al., 1963). Additionally, Fischer and Weiler (1962) used fluorescein-labeled antiserum against guinea pig complement to detect loss of normal liver antigens from a diethylnitrosamine-induced guinea pig hepatoma. The conclusion from these studies is that pronounced qualitative modification of normal liver antigen ex-

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pression occurs in carcinogen-induced hepatomas. This approach for studying antigen expression in tumors is limited, however, since it is not possible to define the antigens involved and the qualitative nature of the tests limits their value in determining the degree of antigen modification. More precise definition of the normal liver antigens deleted from aminoazo dye-induced hepatomas has been sought using immunodiffusion techniques. In this way, Abelev (1965) demonstrated that several of the seven organ-specific antigens detected in mouse liver parenchymal cells were deleted or greatly reduced in concentration in o-aminoazotoluene-induced hepatomas. There was considerable variation in the deletion patterns from individual hepatomas, no single antigen being deleted in all cases. Comparison of the antigenic composition of primary hepatomas with first generation transplants also indicated the variability of antigen deletions, but in all cases transplanted tumors revealed further antigen losses (Guelstein and Khramkova, 1965). The subsequent antigen deletions observed on tumor transplantation may reflect selection of distinct clones from the original tumor and is comparable with other reports (Prehn, 1970) indicating that sublines of 3-methylcholanthrene-induced mouse sarcomas expressing variable tumor-specific antigenicity can be established by transplantation techniques. Many primary hepatomas, however, contain normal liver elements (Reuber, 1966) so that the more marked deletions observed in transplanted hepatomas may simply reflect the elimination of normal tissue contamination. Immunodiffusion and immunoelectrophoresis methods have similarly been applied to characterize antigen deletions from DAB-induced rat hepatomas (Baldwin, 1964). At least eight antigens localized in the soluble cytoplasmic fraction of normal liver were not detectable in primary hepatomas. These antigens were still present, however, in liver taken from the primary hepatoma-bearing hos& indicating that antigen loss did not result from a nonspecific effect during carcinogen feeding. Also, antibody reacting with normal liver antigen could not be removed by preabsorption of antisera with hepatoma extracts, demonstrating that the observed deletions probably reflected qualitative loss of normal liver antigens from hepatoma. Using these methods, it was further established that a t least two major antigen components detectable in sodium deoxycholate-solubilized fractions of normal liver microsomes were deleted from primary DAB-induced rat hepatomas. These major antigens could be more precisely defined by immunoelectrophoresis showing that a t least ten normal microsoma1 antigens were not detectable in hepatoma microsome extracts (Baldwin, 1964). Deletion of liver antigen from DABinduced hepatomas has been reported also by Kalnins and Stich (1963). I n this case cellular localization of the deleted antigens was not defined,

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although it was found that individual hepatomas differed in the number of liver antigens deleted. Deckers (1964) also defined five organ-specific antigens in normal liver distributed between various cell organelles and some of these were deleted from primary DAB-induced hepatomas. More extensive deletions were observed when the hepatomas were transplanted into syngeneic recipients so that all but one of the normal liver antigens was lost. Comparable with the extensive studies on aminoazo dye-induced hepatomas, loss of antigens located in normal liver microsomes (Kitagawa et al., 1966a; Baldwin and Barker, 1967d) and soluble cytoplasmic proteins (Baldwin and Barker, 1967d) has been detected in hepatomas induced by diethylnitrosamine and 2-acetylaminofluorene. Again, the normal liver antigen profile showed a more pronounced simplification following tumor transplantation, and it was noted that similar deletions were obtained in hepatomas induced by all three hepatocarcinogens. The complexity of the deletions of normal liver antigens associated with various cell organelles in carcinogen-induced hepatomas presents a major difficulty in interpreting the significance of these changes in carcinogenesis. In this context, the term antigen refers to the fact that the components are antigenic in heterologous hosts such as rabbits and there is no evidence that they have immunological properties in the autochthonous host. On this basis, antigen changes may simply reflect the pronounced modifications of various liver enzymes which are known to occur during aminoazo dye carcinogenesis (hlorris, 1965; Poirier and Pitot, 1970). Because of these criticisms, other studies have examined the deletion from tumors of cell proteins known to be involved in carcinogen binding during the early stages of carcinogenesis, the objective being to ascertain whether these two events are causally related. Immunodiffusion studies of solubilized microsome fractions from 2-acetylaminofluorene (AAF)-treated liver showed that antibody directed toward the 2-aeofluorenyl group reacted with several microsomal components, indicating the presence of bound carcinogen metabolites. This anti-2-azofluorenyl antibody was prepared by immunizing rabbits against synthetic conjugates prepared by coupling hemocyanin with diaeotized 2-aminofluorene. The reactivity of the purified and lZ5I-labeled 2-azofluorenyl antibody with solubilized microsomal fractions of normal liver and hepatoma was then compared by radioimmunoelectrophoresis. By this means, it was established that several microsomal components which bind carcinogen were not present in liver from rats fed for long periods on AAF diet or in the hepatoma which subsequently developed (Kitagawa et al., 1966a). Subsequently it was established by immunofluorescence staining of liver sections that 2-azofluorenyl-antibody reacted with cytoplasmic com-

38

R. W. BALDWIN

ponents and also there was predominant staining of the cell boundary and perinuclear zone in liver cells taken from rats fed or injected with AAF (Tanigaki e t al., 1967). I n contrast hepatoma nodules in AAF-fed rats often showed little staining even though closely associated hepatic cells in the sections still showed strong binding. I n comparable studies (Baldwin e t al., 1960), it was established that fluorescein-labeled antiaminoazo-dye antibody prepared against a conjugate of bovine serum albumin and DAB reacted with protein-bound carcinogen during the early stage of azo dye carcinogenesis, whereas the binding was markedly diminished in hepatoma cells. These results may be interpreted as reflecting deletion of the liver cell components which bind carcinogen metabolites. Alternatively, the lack of carcinogen binding detected by low uptake of anticarcinogen antibody may reflect the absence of the required enzyme systems needed to convert carcinogen to the reactive metabolites. An alternative approach was adopted in studies on the distribution of a basic azo dye-binding protein in normal rat tissues and DAB-induced hepatomas (Baldwin et al., 1968). In this case, a basic azo dye-binding protein was isolated from normal rat liver using methods initially developed by Ketterer e t al. (1967) and rabbit antisera were prepared against this highly purified protein. These antisera were then used in immunodiffusion tests to detect basic azo dye-binding protein in primary and transplanted hepatomas induced by a variety of hepatocarcinogens. I n most cases, the basic protein was still detectable in primary hepatomas, although very much reduced in concentration when compared with normal liver. On transplantation into syngeneic rats, most of the tumors were found to be deficient in the protein so that it is possible that the low levels detected in primary hepatomas may be due to contamination with normal tissue elements. The data did not allow a clear distinction to be made between this and the alternative effect of tumor selection on transplantation. Nevertheless, these studies provided a direct correlation between carcinogen-protein interaction during the early stages of aminoazo dye carcinogenesis and the eventual deletion of the protein in the neopIastic cell. Modification of the cell surface membrane has been widely implicated in carcinogenesis (Emmelot and Benedetti, 1966; Wallach, 1968), and it has been postulated (Sachs, 1965) that the changes in cell regulatory mechanisms produced by transformation of normal cells by carcinogenic agents can be ascribed to a change in the cell surface membrane. Whether these changes result from intracellular derangement rather than independent phenomena at the cell surface does not detract from their importance in carcinogenesis and tumor progression, if one views these

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

39

events as a release from a normal homeostatic control in which cell surface events are all important. As already described, loss of cell surface components in carcinogen-induced hepatomas was suggested by studies (Baldwin et aE., 1960; Tanigaki et aE., 1967) showing that tumor cells failed to bind carcinogen as measured by uptake of anticarcinogen antibody. More direct evidence of antigenic modification of the cell surface is provided by the studies of Beloshapkina and Khramkova (1967) in which rabbit antisera against normal mouse liver cell ghosts was absorbed with suspensions of ascitic hepatoma cells. Immunofluorescence tests were then used to show that, while absorbed sera did not react with viable hepatoma cells in suspension, positive staining was still obtained with cryostat sections of normal liver. Although these reactions with liver cells were located a t the cell periphery, this does not necessarily indicate that the antigens are expressed on the outer cell membrane. Similarly, although it has been possible to detect deletion of normal liver plasma membrane components from transplanted rat hepatomas by comparative immunodiffusion studies using antisera prepared against highly purified membrane fractions (Baldwin, 1964; Baldwin and Glaves, unpublished observations), this does not preclude the possibility that the membrane antigens are intracellular components or a t least localized on inner membrane structures. A more critical appraisal of normal liver plasma membrane changes during aminoazo dye carcinogenesis has been provided in studies with a range of DAB-induced hepatomas transplanted in syngeneic rats (Baldwin and Glaves, 197213). Membrane immunofluorescence methods with rabbit antinormal rat liver membrane antiserum were used to detect liver antigens expressed on normal parenchymal liver and hepatoma cells in suspension, an important factor being that the test system specifically localized components a t the cell surface membrane. Deletion of cell surface liver antigens was then demonstrated by showing that absorption of an antiliver membrane antiserum with hepatoma cells until it no longer reacted with the absorbing tumor did not abolish reactivity toward liver cells. The normal liver antigens which were deleted from hepatoma cells have not yet been characterized, but they are liver-specific components since reacting antibody could not be absorbed with other normal rat tissue, and possibly may be viewed as differentiation antigens (Boyse and Old, 1969; Schlesinger, 1970; Burnet, 1971). Each hepatoma line studied was shown to be deficient in a characteristic normal liver antigen, and this would seem to preclude the idea that the deleted components have any specific growth-controlling properties unless it is further postulated that a multiplicity of such substances are expressed on normal liver cells. Since, however, the hepatomas have previously been shown to ex-

40

R. W. BALDWIN

press individually distinctive tumor antigens (Baldwin and Barker, 1967a; Baldwin et al., 1971a,b), these studies indicate a parallelism between the loss of individual plasma membrane-associated liver antigens and the concomitant gain of tumor-specific antigen. This correlation suggests that both events may be causalry related, reflecting a specific interaction in carcinogenesis, and that the appearance of tumor-specific antigen results in a replacement or displacement of normal liver cellsurface components. While deletion of normal tissue antigens from tumor has been most extensively studied with carcinogen-induced hepatomas, similar observations have been reported with other experimentaI tumor systems. Deletion of kidney-specific antigen from stilbestrol-induced renal carcinomas was detected by means of complement fixation and immunofluorescence methods (Weiler, 1956b; Nairn e t al., 1960), and loss of soluble kidney antigens has been reported in transplanted renal tumors originally induced by X-irradiation (Deckers, 1964). Loss of normal muscle antigens from MC-induced rat sarcomas has been observed (Fel and Tsikarishvili, 1964) and MC-induced murine squamous cell carcinomas are deficient in normal epidermal antigens as measured by lack of immunofluorescence staining of tumor cryostat sections with antiepidermis antiserum (Carruthers and Baumler, 1965). Further studies indicated that antigen deletions were more pronounced in deeper parts of the tumors, and a t the invading edge there was almost total depletion of normal epidermal antigen, suggesting that antigen deletion may be a faotor in local invasion by malignant cells (Muller and Sutherland, 1971). A more precise characterization of the epidermal components which are deleted from carcinogen-induced tumors could be obtained by comparison of the cell membrane antigens on viable epidermal and tumor cells in suspension, and it would be particularly interesting to ascertain whether squamous cell carcinoma cells are deficient in epidermal chalones (Bullough, 1965; Bullough and Laurence, 1968). This approach has been adopted in showing a reciprocal relationship between normal H-2 antigen and tumorspecific antigen expression on MC-induced mouse sarcomas (Haywood and McKhann, 1971). Quantitative absorption of monospecific H-2 antisera by cells of five MC-induced sarcomas was used to study the variation of individual H-2 antigenic determinants. When these results were compared with the tumor-specific immunogenicity, determined from the dose of tumor cells needed to kill 50% of preimmunized mice, a precise inverse relationship was obtained. That is, the most immunogenic tumor showed the greatest loss of H-2 antigens and the least antigenic tumor possessed the greatest amount of H-2. However, all the H-2 antigens on a single tumor were altered to the same degree so that tumor antigen

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

41

expression could not be accounted for by replacement of a particular H-2 determinant. In comparable studies, loss of H-2 antigens has been demonstrated in TL( + ) leukemia (Boyse and Old, 1969), Moloney Iymphomas (Fenyo et al., 1968$, and polyoma tumors (Ting and Herberman, 1971), and HL-A antigen loss has been reported in a human lymphoma (Seigler et al., 1971). These observations, together with the findings on deletion of plasma membrane-associated liver-specific antigen from DAB-induced rat hepatomas (Baldwin and Glaves, 1972b) may be interpreted as reflecting distortions of normal cell membrane architecture following neoplastic transformation. Within the limited evidence available, however, it cannot be established whether these deletions reflect actual loss of normal antigens, resulting from replacement by tumor-specific or other neoantigen components, or reflect reorientation of normal cell surface antigen, perhaps by the introduction of neoantigens. Such interpretations have relevance to the concepts that cell surface disorganization, arising either dependently or independently of changes in the genome, is a critical event in chemical carcinogenesis (Markert, 1968; Sachs, 1965) so that precise mapping of cell surface antigens using immuno-electron microscopy (Boyse et al., 1968b; Boyse and Old, 1969; Aoki e t al., 1969) is desirable. VI. lmmunosurveillance and Chemical Carcinogenesis

Immunological reactions in the autochthonous host to antigens associated with chemically-induced tumors are well documented (see Section 11). This was revealed in studies showing suppression of growth of reimplanted biopsies of 3-methylcholanthrene-induced rat sarcomas following ligation of the primary tumor (Takeda et al., 1966; Takeda, 1969) or its total excision (Mikulska et al., 1966). Comparable transplantation studies with DMBA-induced hamster sarcomas has demonstrated “concomitant immunity,” whereby tumor-bearing animals rejected a second inoculum of the same tumor but not unrelated tumors (Lausch and Rapp, 1969, 1971). The existence of tumor-specific immune reactivity in primary tumor-bearers has also been detected by in vitro assay of lymphocytemediated inhibition of colony formation of plated tumor cells in MCinduced sarcomas (Hellstrom et al., 1970) and aminoazo dye-induced rat hepatomas (Bddwin et al., 1973b). Consequently, immunosurveillance mechanisms may be viewed as limiting factors in chemical carcinogenesis by both restricting the establishment of transformed cells and influencing the growth of established tumors. For effective surveillance, however, several criteria must be satisfied. In the first place, tumor-associated antigens must be expressed in the early population of tumor cells since it is well established that even under optimal

42

R. W. BALDWIN

conditions tumor-immune reactions can only effectively eliminate small numbers of tumor cells. Important factors in this respect are the accessibility of the transformed cells to the host’s immunological apparatus and the functional integrity of this system a t the time transformed cells arise. A further important point is that the immune response elicited by the host must lead to a significant tumor rejection reaction and not result in the enhancement of tumor growth. DURING CHEMICAL CARCINOGENESIS A. CELLANTIGENCHANGES

Many chemical carcinogens interact covalently with cell macromolecules during the early stages of carcinogenesis so that new antigenic specificities are produced through the introduction of these haptenic groups. For instance, the maximum level of protein-bound metabolites of DAB in rat liver is known to occur after carcinogen feeding for around 4 weeks (Miller and Miller, 1947), and a t this time, new antigenic components are detectable in the soluble fraction from liver homogenates (Baldwin, 1962). These abnormal components also reacted in immunodiffusion tests with antisera prepared against synthetic conjugates of DAB linked to bovine serum albumin (BSA) through the carbamido linkage (Baldwin et al., 1960; Green et al., 1967). No reactions were detected against antisera prepared against BSA or conjugates with other carcinogens, an observation indicating that the abnormal antigens contained bound aminoazo dye metabolites. Comparably, carcinogen-binding antigenic components have been demonstrated in cytoplasmic fractions of liver from mice treated with o-aminoazotoluene (Korosteleva, 1957, 1965; Muller, 1967). Microsome fractions of liver from rats treated with AAF also contain components which fix lZ5I-labeled antibody prepared against protein conjugated with diazotized 2-aminofluorene, indicating the presence of bound carcinogen metabolites (Kitagawa et al., 1966a). Generally, however, these carcinogen-modified antigens are intracellular components, and it is questionable whether they play any part in immunosurveillance reactions to carcinogen-modified cells because of their inaccessibility to antibody or sensitized lymphocytes. Additionally, however, there is suggestive evidence that carcinogen-binding modifies components in cell surface membranes. Thus rabbit antisera against microsome fractions of liver from rats treated with 3’-methyl-DAB reacted in immunofluorescence tests with antigens localized on the surface of carcinogen-treated, but not normal liver cells (Green and Ghose, 1964). Also antisera against 2-azofluorene-protein conjugates reacted with liver cells taken from AAF-treated rats (Kitagawa et al., 1966b). However, the significance of modifications resulting from carcinogen binding at the cell surface in terms of immunological reactions in the autochthonous host have not yet been explored.

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

43

Evidence for immunological reactions against carcinogen-treated tissue in the autochthonous host or in syngeneic hosts against transplanted tissue is less substantial. Skin treated with 7,12-dimethylbenz [ a ] anthracene was shown to have new antigenic specificities causing its rejection when grafted to syngeneic mice (Math&,1967), but it was not established that this resulted from an immunological reaction to new antigens in the skin graft. Short-term treatment with polycyclic hydrocarbons can nonspecifically damage skin (Silberberg e t al., 1948; LappB, 1968), and their cytotoxicity to normal mammalian cells in vitro is well established (Huberman and Sachs, 1966; Diamond et al., 1967; Alfred e t al., 1969). Consequently, rejection of DMBA-treated skin may reflect cytotoxicity of the carcinogen rather than host immunological reactions to new antigens. This point has subsequently been investigated by Basombrio and Prehn (1972b), who distinguished between the toxic effect of DMBA and the presence of neoantigens by transplanting carcinogen-treated skin into normal mice immunodepressed by injections of cyclophosphamide or by thymectomy followed by 450 R of X-irradiation. These experiments indicated that immunosuppression did not increase the survival of DMBAtreated skin grafts, and therefore it was concluded that the skin rejection seen in normal mice reflects DMBA toxicity. More conclusive evidence for tumor antigen expression in premalignant lesions is provided by studies on MC-induced mouse skin papillomas (Lap@, 1968, 1969), where the accessibility of the carcinogen-treated tissue and the feasibility of skin-grafting techniques allowed the detection of minor antigenic changes. In this way it was established that the number of papillomas developing in skin initially treated with MC and then grafted to syngeneic mice was correlated with the immune competence of the graft recipients (Lap& 1968). Decreasing immunological competence by sublethal X-irradiation accelerated papilloma appearance and increased their incidence. Conversely, immune stimulation with bacterial adjuvants [methanol-extracted residues (MER) of Bacillus CalmetteGudrin] delayed papilloma appearance and decreased their incidence. The characteristics of the neoantigens associated with MC-induced papillomas have still to be determined although transplantation tests established that each papilloma expressed individually distinct rejection antigens (Lap$, 1969). Moreover, the antigens on individual papillomas were reported to persist through progression to malignancy, being detectable on skin carcinomas derived from individual papilloma lines. Further studies designed to investigate the influence of immunological environment on papilloma progression and regression (Lapp6 and Prehn, 1969) again established that a greater number of papillomas initially developed in MC-treated mouse skin transplanted to syngeneic mice when they were immunosuppressed (thymectomized and X-irradiated)

44

R. W. BALDWIN

compared with normal control or immunostimulated mice. Moreover, the dynamics of papilloma regression were consistent with the immunological reactivity of each group (Fig. 4 ) . Thus the greatest proportion of regressing papillomas (34J48; 71%) was observed in the most immunologically competent group, and the most immunosuppressed group (thymectomized and X-irradiated) showed the lowest proportion of regressing papillomas (13/43 ; 30%). Histological assessment of papillomas in MC-treated mouse skin transplanted into syngeneic mice of varying degrees of immunocompetence provided further evidence for the immunological mediation of papilloma regression (Lapp6, 1971a). Focal microscopic lesions exhibiting mononuclear cell infiltration were commonly found only in immunologically competent mice, and these were taken to be papilloma regression sites. Sublethally irradiated or antilymphocytc serum-treated mice developed macroscopic papillomas without the appearance of these microscopic lesions. The influence of immunocompetence on papilloma progression was also analyzed by means of the transphnted mouse skin system (Lapp6 and Prehn, 1969). Consistent with the data on papilloma regression, the time of appearance of squamous cell carcinomas was shorter, and the

...'...... -................" .....A (30/43) ~

50

(00

60

200

Doys after grafting

FIQ. 4. Persistence of methylcholanthrene-induced papillomas in mouse skin grafted to syngeneic recipients with various degrees of immunocompetence (Lapp6 , controls; 0. * * .O, methanol-extracted residues of Baand Prehn, 1969). X-x cillus Calmette-Gukrin (0.25 or 0.60 mg) ; A-A, sham thymectomized and X-irradiated (450 R) ; A- - - -A, thymectomized and X-irradiated (450 R).

IMMUNOLOGICAL ASPECTS O F CHEMICAL CARCINOGENESIS

45

final incidence greater, in immunosuppressed syngeneic mice receiving grafts of MC-treated skin. In order to determine an index of the rate of progression, the populations of papillomas were sampled a t fixed time intervals, and the numbers of new malignancies were expressed as a fraction of the total papillomas which had persisted during that time interval. These calculations showed that the percentage of papillomas progressing to skin carcinomas over five 40-day periods following transplantation of MC-treated skin to mice with either diminished or enhanced immunological competence was remarkably uniform (9-17%/40-day papilloma units). These findings are consistent with the hypothesis that papilloma antigenicity as reflected by susceptibility to regression, is unrelated to malignant progression. These studies establish that early populations of cells transformed by 3-methylcholanthrene have new antigenic specificities with the capacity to provide immunological rejection reactions. However, such effects have been detected against transplanted tissues, and undoubtedly this manipulation may enhance the response to weak antigens. For instance, premalignant MC-induced mammary hyperplastic nodules which are immunogenic on transplantation into syngeneic mice arouse litle reaction while in situ in the mammary pad (Prehn, 1967), and it has been claimed (Blair and Moretti, 1967) that the mammary pad is an immunologically privileged site. In order to account for the lack of immunological response to skin papillomas in situ it was also postulated that epidermis may be immunologically unresponsive (Lapp5, 1968), but this can hardly be so in view of skin reactivity to a variety of hypersensitizing agents. AND CHEMICAL CARCINOGENESIS B. IMMUNOSUPPRESSION

If immunosurveillance is held to be important in the control of neoplasia, it follows that factors causing defects in this system, especially thymus-dependent (T) immune responsiveness, will facilitate tumor development. Indeed it is well established in viral oncogenesis that thymectomy at or near birth leads to an increased tumor incidence (Vandeputte et al., 1963; Law, 1965; Allison et al., 1967; Allison and Taylor, 1967; Allison, 1970). Similarly selective immunosuppression with antilymphocyte or antithymocyte serum markedly enhances the response to oncogenic viruses (Allison and Law, 1968; Law et al., 1968; Vandeputte, 1969). The de now development of malignant disease in recipients of transplanted organs undergoing prolonged immunosuppressive therapy (Doll and Kinlen, 1970; Starzl et al., 1970; Schneck and Penn, 1971) affords further evidence that host restraint factors play some role in neoplasia, although in these situations the etiologic factor involved is unknown.

46

R.

W.

BALDWIN

1. Thymectomy

The influence of immunosuppression on chemical carcinogenesis is less clear-cut than in viral oncogenesis. After Miller’s report (1961) that neonatal thymectomy significantly reduced host immunocompetence, it was claimed that thymectomized mice developed more skin tumors when treated with benzo[a]pyrene (Miller et al., 1963). Contrary results were reported by Yasuhira (1969), who failed to observe any marked influence of thymectomy on the induction of skin papillomas or carcinomas following repeated skin painting with MC. This study also failed to show any difference in skin tumorigenesis in intact or newborn thymectomized mice following initiation with an oral dose of urethan and promotion by skin-painting croton oil. There are similar discrepancies in the reports on the influence of thymectomy on the carcinogenic response to subcutaneous injections of polycyclic hydrocarbons. Grant and Miller (1965) reported that neonatal thymectomy of C57BL mice rendered them more susceptible a t the young adult stage to a subcutaneous dose (200 pg) of MC. This was manifested by a reduction of latent induction period in thymectomized mice so that, at 14 weeks, 78% had developed sarcomas compared with 42% of sham-thymectomized control mice. However, the final incidence of sarcomas in both groups was similar, approaching loo%, and there was no difference in the growth rates of the tumors evaluated from the time taken to develop from small (5 mm) nodules to a mean diameter of 15 mm. An enhanced response to MC in inbred AKR and outbred CF1 mice, but not inbred C,H mice following neonatal thymectomy was also reported by Nomoto and Takeya (1969). In the sensitive strains, tumors arose more rapidly whereas in AKR mice the final tumor incidence was increased from 70% to 100%. However, in other studies (Balner and Dersjant, 1966; Johnson, 1968) neonatal thymectomy of C57BL mice did not markedly enhance susceptibility t o M C carcinogenesis, and the incidence and growth rates of tumors were not significantly different from those in controls. Also, although thymectomy a t 3 days of birth was reported to shorten latent induction periods (Johnson, 1968) , this was not observed in the previous study (Balner and Dersjant, 1966), where thymectomy was carried out a t 24 hours and mice so treated had proven immunoincompetence as evidenced by their failure to reject skin allografts. In fact, the most consistent feature arising from these studies was the increased immunogenicities of the sarcomas arising in thymectomized mice. This was qualitatively revealed in the work of Balner and Dersjant (1966), who showed that resistance could be induced in syngeneic mice against transplants of 9/10 tumors arising in thymec-

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

47

tomized mice, whereas only 5/10 tumors from control, sham-thymectomized mice had comparable immunogenicities. A more precise analysis of tumor immunogenicity was attempted by Johnson (1968), who compared the immune response elicited against tumors arising in thymectomized and nonthymectomized mice by the rejection of a standard challenge dose of tumor cells (lo3) in normal syngeneic mice immunized by tumor excision. This study indicated significantly increased immunogenicities in tumors arising from a dose of 250 pg of MC in thymectomized mice compared with sham-operated controls. When a 4-fold larger dose of carcinogen was used, however, tumor immunogenicity was not enhanced in thymectomized mice. This suggests that the variable influence of thymectomy on MC-carcinogenesis may be due to the immunosuppression induced by the carcinogen which is known to be dose dependent (Stjernsward, 1969). An increased sensitivity of neonatally thymectomized mice to chemical carcinogenesis has also been reported in studies on lung adenoma formation in mice treated with 7,12-dimethylbenz [ a ]anthracene or urethan (Ribacchi and Giraldo, 1965; Trainin et al., 1967; Lapp6 and Prehn, 1970). Moreover, neonatally thymectomized Swiss mice inoculated repeatedly with competent lymphoid cells from the same random-bred strain were more susceptible to the tumorigenic effect of urethan than nongrafted thymectomized resistance (Trainin and Linker-Israeli, 1969) . This was attributed to a further loss of immunocompetence in these animals since they exhibited impairment of the homograft response, a decrease in circulating lymphocytes and a depressed hemagglutinin response. It was also reported that restoration of immunologic competence of thymectomized mice by repeated thymus implantation reduced the response to urethan, the number of lung adenomas per mouse was similar to that of intact controls (Trainin and Linker-Israeli, 1970). 2. Anti1ymphocy te Serum

The influence of immunocompetence on chemical carcinogenesis has also been analyzed using selective immunosuppression by antilymphocyte serum (ALS) . An increased response to subcutaneous MC carcinogenesis in mice has been reported (Balner and Dersjant, 1969; Cerilli and Treat, 1969; Rabbat and Jeejeebhoy, 1970) although the main effect was a shortening of the latent induction period for sarcoma development, and the final tumor incidence in ALS-treated mice were not significantly different from those in controls. I n contrast, Fisher et al. (1970) were unable to detect any significant influence of ALS treatment in MC-treated C57BL mice as measured by sarcoma incidence or latent period of tumor induction. Also, the immunogenicities of sarcoma arising in ALS-treated

TABLE IV EFFECT OF ANTITRYMOCYTE SERUM ON MOUSE SKINTUMORIGENESIS~ Squamous carcinoma

_____

Skin papilloma incidence after

Group

Treatment of host

1

INITIATION 7 Days 3 Days ATS X 2 DMBA

2

NRSX2

3 Days

7 Days

DMBA 7 days

DMBA

3

4

DMBA

PROMOTION Earlg phaue 7 Days 6 Weeks ATS croton oil

+

24 Weeks Croton oil 7 Days 6 Weeks

30 Weeks CrotonoilX60 30 weeks Croton oil X 60 30 weeks Crotonoil X 60

10

15

20

25

30

35

40

45

Weeks

Weeks

Weeks

Weeks

Weeks

Weeks

(%)

(%I

(%)

(%I

(%I

Weeks (%)

Weeks (%)

(%)

Average latent Incidence period (%) (days)

F

? td LF c1

85 (3.3)

100 (8.5)

100 (14)

81 (2.5)

100 (8.7)

71 (3.7)

100 (21)

-

-

-

23

203

100 100 (13.1) (18.4)

100 (19.5)

-

-

-

0

-

100 (7 5 )

100

100 (16.4)

100 (18)

-

-

-

13

217

55 (2.4)

100

100 (92)

100

100 (17)

-

-

100 (17.5)

33

217

(5)

(12)

100 (17)

(12)

5

6

NRS

DMRA

DMBA

+ croton oil

24 Weeks Croton oil 8 Days 30 Weeks Croton oil

L d e phase 13 Weeks Croton oil 14 Weeks Croton oil ATS 7Days 13 Weeks Croton oil 14 Weeks Croton 08 NRS 7 Days 27 Weeks Croton oil 7 Days

7

DMBA

+

8

DMBA

+

9

DMBA

After Croton oil treatment 30 Weeks Croton oil

7 Days 10

DMBA

64 (2.1)

82 (3 ) (13 weeks) 72 (3 )

100

100 (7.1)

100

-

-

-

(42 weeks) 100

11

DMBA

7 Days 12

0

DMBA

7 weeks ATS X 7 30 Weeks Croton oil 7 weeks AT8 X 7 30 Weeks

Croton oil

Derivad ftom Haran-Ghera and Lurie (1971).

161

100 (10.5)

16

20 1

-

33

245

(14)

66 (2.5)

-

100 (7 )

-

9

196

78 (3 )

-

91 (7)

-

4

259

100

41

280

14

315

14

340

-

100 (15.6)

7 Days

10

(10)

100 (9.5)

(10)

-

-

100

100

(12.7)

(11.7)

100 (9.3)

50

R. W. BALDWIN

mice were not markedly diff e m i t from those developing in untreated control mice. A more extensive analysis of ALS treatment on MC-carcinogenesis was reported by Wagner and Haughton (1971) using several strains of mice, but again there was no evidence of an enhanced carcinogenic response. In this study, ALS was administered throughout the course of tumor development, and the efficacy of the immunosuppression was established by the impairment of the capacity of treated mice to reject a n allogeneic tumor. In one experiment, ALS treatment was initiated a t the time of MC treatment (dose 250 pg), but no difference was seen between the number of tumors or the rate of tumor development in treated and control mice. Also, treatment with ALS starting 50 days after M C injection, a t a time when malignant cell clones would be expected to appear, had no effect on primary development or metastasis formation. Studies to delineate the influence of antithymocyte serum (ATS) -induced immunosuppression at various stages of skin carcinogenesis by 7,12-dimethylbenz [ a ]anthracene (DMBA) in SWR female mice have also been reported (Haran-Ghera and Lurie, 1971). The immunosuppressive effect of ATS treatment was indicated by the acceptance of allogeneic tumor grafts in treated mice. Even so, short-term immune impairment before topical application of DMBA did not modify its initiating effect, the subsequent response to promotion with croton oil skin painting, measured from tumor incidence, latent period, and degree of progression to malignancy, being comparable in normal control and ATS-treated mice (groups 1-2, Table IV). ATS treatment either early (weeks 1-6; group 4) or late (weeks 13-27, group 7) during the promotion phase with croton oil also did not markedly influence the papilloma response to DMBA. This is indicated by the data summarized in Table IV showing the comparable tumor incidence, latent induction periods, and numbers of papillomasjmouse in the immunosuppressed ATS-treated mice (groups 4 and 7) compared with either untreated controls (groups 6 and 9) or mice treated with normal rabbit serum (groups 5 and 8 ) . In control mice treated during the promotion phase with croton oil or croton oil and normal rabbit serum (groups 5, 6, 8, and 9 ) , the rate of papilloma regression within 15 weeks after croton oil treatment was discontinued (week 30) was 3 0 4 2 % (based on the number of papillomas present at the end of the promotion treatment). I n contrast, no such papilloma regression was observed in mice treated with ATS either early or late during the promotion phase (groups 4 and 7). However, ATS treatment initiated following the completion of the promotion phase with croton oil (30 weeks; group 10) did not change the rate of papilloma regression, 38% of the papillomas disappearing over a period of 15 weeks. This compares with regression rates of 40% and 46%, respectively, over

IMMUNOLOGICAL ASPECTS OF CHEMICAL CARCINOGENESIS

51

the same period in micc trcatcd with normal rabbit serum (group 11) or left untreated (group 12) after the croton oil phase. There was, however, a significant difference in the incidence of skin carcinomas in ATS-treated mice (41%) compared with that in controls (14%). The conclusion from these studies, therefore, is that growth and regression of DMBA-induced skin papillomas is not markedly influenced by the immune status of the host and consequently such events are not mediated by immune reactions. A further point from these findings was that ATS treatment could not replace the promotion stage with croton oil. Therefore it was argued that, even though croton oil was immunosuppressive (Bluestein and Green, 1970), this function played little part in its promotional function. Comparably, Lapp6 (1971b) found that immunosuppression with ALS retarded, but did not prevent, the eventual appearance of skin papillomas developing in MC-treated skin a t the site of wound-stimulated epithelial proliferation. There were no significant differences in the final skin tumor incidences in AM-immunosuppressed mice compared with either normal mice or mice immunostimulated with MER fraction of BCG. It was possible, however, to discern significant differences in the rate of papilloma development, especially those arising within 30 days, this being greatest in ALS-treated mice. This was more evident when the distribution of the “early-arising” papillomas was correlated with the capacity of the mice to reject skin allografts. I n this case significantly more tumors were found in weak allograft responders (rejection scores greater than 19 days) than in strong allograft responders and most of the tumor-bearing mice had received ALS treatment. The findings of Haran-Ghera and Lurie (1971) present a contradictory pattern when compared with other studies (Lapp6 and Prehn, 1969) showing a relationship between immunological competence and the regression of MC-induced skin papillomas when transplanted to syngeneic recipients. It should be emphasized, however, that this transplantation manipulation may serve to call attention to the tumor rejection antigens detectable on these papillomas (Lapp6, 1968) and the findings of HaranGhera and Lurie (1971) question the contribution of this immunemediated rejection in situ. It is noteworthy, that transplantation of subcutaneous tissue from the site of injection of benz[a]anthracene or dihenzo [ailpyrene accelerates carcinogenesis (Homburger and Treger, 1970). A further point of controversy relates to the significance of host immune reactivity ‘in the progression of skin papillomas to malignancy. From analyses of the numbers of papillomas becoming malignant in syngeneic grafts of MC-initiated skin on mice with varying degrees of immunocompetence, it was concluded that this was a random event

52

R. W. BALDWIN

(Lapp6 and Prehn, 1969). A further corollary of these findings was that papilloma antigenicity, as reflected by susceptibility to regression, is unrelated to malignant progression. On the contrary, treatment with ATS a t the same time as croton oil skin painting, conditions which prevented regression of papillomas, was found to increase the incidence of skin carcinomas (Haran-Ghera and Lurie, 1971) suggesting that immunological factors may contribute to this process. 3. Immunosuppression by Chemical Carcinogens

The immunosuppressive action of several chemical carcinogens is well established, but whether, as initially postulated (Prehn, 1963b), this activity plays a significant role in chemical carcinogenesis is questionable. Early studies (Malmgren et al., 1952) established that 3-methylcholanthrene and dibenz [ a ]anthracene inhibited humoral antibody response in mice to sheep erythrocytes, but the doses were far greater than those necessary for tumor induction. The immunosuppressive action of these chemicals a t carcinogenic doses has since been substantiated in studies showing that a single low dose exposure to carcinogen prior to antigen led to depressed levels of antibody-forming cells as determined by the hemolytic plaque technique (Stjernsward, 1965, 1966a, 1968). With MC, a single exposure prior to antigen administration resulted in prolonged immunosuppression for a period of several months. This effect was dose dependent in the range 0.1 mg to 1.0 mg of carcinogen, but greater doses of up to 5.0 mg had little further effect (Stjernsward, 1967). Repeated exposure to small doses of carcinogen led to a cumulative immunosuppressive effect, and this was obtained following administration either by subcutaneous injection or skin painting (Stjernsward, 1967, 1969). Other studies also established that MC acts as an immunosuppressant only in mice which are susceptible to its carcinogenic action. Consequently, I strain mice in which the sarcoma incidence following a single dose (0.1 mg) of carcinogen was only 11% responded as well as control mice to sheep erythrocytes (Stutman, 1969). C3HJf mice, on the other hand, in which the same dose of RlC induced sarcomas in all treated mice, showed a marked immunosuppressive response to the carcinogen. Contrary findings on the immunosuppressive influence of MC have been reported by Ball (1970), who observed that doses of between 60 and 120 pg injected into newborn CFWJD mice did not depress the immunological response to sheep erythrocytes a t 28-63 days of age. Nevertheless, mice so treated developed both thymic lymphomas and sarcomas. 3-hlethylcholanthrene also exerts a suppressive effect on cell-mediated immune reactivity. This was originally established in studies showing that subcutaneously administered carcinogen increased the survival of

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male skin grafts to female mice (Prehn, 1963b) or enhanced the takes of allogeneic tumor (Rubin, 1964). However, this immunosuppression was not sufficient to significantly influence rejection of skin allografts (Linder, 1962; Stjernsward, 1965). In these situations, skin graft survival was only prolonged at the time primary tumors appeared, and so tumor-associated effects may have been contributory factors. Further, more conclusive evidence of the influence of MC treatment on cell-mediated immune responses was provided, however, in studies (Prehn, 1963b) showing that growth of first transplant generation sarcomas was enhanced in treated mice. Moreover, the degree of this effect was proportional to the immuno,genicity of individual sarcomas. Confirmatory studies were reported by Stjernsward (196613) with 75 individual primary sarcomas where the growth of first-generation transplants in syngeneic recipients was tested by inoculating various tumor cell doses between los and lo6 cells. At the Iowest cell dose (lo3), 32/39 tumors grew in MC-treated mice compared with 17/40 in untreated controls. A significant difference in tumor takes was also observed a t a cell dose of lo4 cells, but challenge inocula greater than this were not influenced. This indicated that the MC-induced immunosuppression is not particularly marked and therefore may not be discernible when tests involve alloantigen responses. Even so, Fox and Bock (1967) reported that repeated MC skin painting allowed progressive growth of the allogeneic 6C3HED lymphoma in DBA/2 mice. Consistent with the MC studies, several other carcinogenic polycyclic hydrocarbons have been reported to exert immunosuppressive effects. Assaying the capacity to suppress antibody forming cells in the hemolytic plaque assay Stjernsward (1966a, 1969) established that the potent carcinogens benzo[n] pyrene and 7,12-dimethylbenz[a] anthracene were jmmunosuppressive whereas noncarcinogenic hydrocarbons like anthracene and benzo [el pyrene were inactive. Contrary findings have been reported by Ball (1970), who was unable to demonstrate any substantial suppression of the humoral antibody response to sheep erythrocytes in adult mice injected intramuscularly with DMBA (1 mg). Also, in contrast to earlier findings on the immunosuppression induced by neonatal injection of DMBA (6-30 pg) (Ball et al., 1966; Ball and Dawson, 1969), carcinogenic doses (60-120 pg) of benzo[ujpyrene and MC given a t birth did not result in any detectable immunosuppression (Ball, 1970). Interference with cellular rather than humoral immune responses is more likely to be a significant factor in modifying carcinogenesis. This has been measured in one study (Rubin, 1964) by assaying the effect of repeated skin painting on the capacity of DBA/2 mice to reject grafts of the allogeneic 6C3HED lymphosarcoma, and these tests again estab-

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lished a correlation between carcinogenic and immunosuppressive activities. In this series of tests, potent carcinogens such as 7,12-dimethylbenz [a] anthracene, dibenz [a$] anthracene; benzo [a]pyrene, and 10-methylbenz [ a ]anthracene produced marked immunosuppression leading to enhanced growth of the allogeneic tumor in 75--100% of the treated mice. Solvents used for skin painting, such as benzene and acetone as well as a series of noncarcinogenic hydrocarbons (anthracene, pyrene, 1’-, 2‘-, 3’-methylbenz [ a ] anthracene) , produced no effect on host immunocompetence, so that the 6C3HED tumor was rejected. Comparably this tumor rejection system has been used to show that the carcinogens 7,g-dimethylbenz [aJ acridine and 4-nitroquinoline-l-oxide were immunosuppressive (Fox and Bock, 1967). In general, therefore, there is a good correlation between the immunosuppressive action of polycyclic hydrocarbons measured either by their effect on humoral or cell-mediated immune reactions and carcinogenic potential. Immunosuppression may also be viewed as a component of urethan carcinogenesis. Repeated treatment with urethan produce a depressant effect on humoral antibody response in mice (Malmgren e t al., 1952; Haran-Ghera and Peled, 1967; Parmiani e t al., 1969, 1971). Although impairment of antibody formation was found to correlate with the yield of leukemia (Parmiani e t aE., 1969), the doses needed to produce significant immunosuppression were far greater than those used for urethan carcinogenesis and, moreover, did not seem to influence cellular immunity as measured by rejection of tumor allografts (Rubin, 1964). Further studies (Parmiani, 1970) also failed to detect urethan-induced suppression of cellular immunity when analyzed by rejection of mouse skin grafts differing a t the strong H-2 locus. When, however, the response was measured by the rejection of C57BL/6 male skin grafts on C57BLJ6 femaIe mice, a significant increase in graft survival was noted in urethan-treated mice (Lapp6 and SteinmulIer, 1970; Parmiani, 1970). There was also a significant increase in takes of an antigenic MC-induced sarcoma in female mice treated with urethan, although no modification of growth of 8 nonantigenic sarcoma was noted (Parmiani, 1970). Whereas large doses of urethan (120-150 mg) were necessary to induce cellular immunosuppression in adult BALB/c strain mice, newborns were much more susceptible, and a single dose of 1.0 mg in 4-day-old mice, prolonged skin allograft survival (Lapp6 and Steinmuller, 1970). However, the immune depression in neonates may be due to the damaging effect of urethan on the thymus, thus being comparable to the known effect of neonatal thymectomy on urethan carcinogenesis (Trainin et al., 1967; Trainin and Linker-Israeli, 1969). The possibility that depression of cell-mediated immunity facilitates

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the carcinogenic response to urethan in the lungs of highly susceptible BALB/c mice has been examined by correlating the incidence of lung adenoma formation with the immunocompetence of the mice as assessed by survival of DBA/2 skin allografts (Lapp6 and Prehn, 1970). These studies established that lung adenomas were found preferentially in urethan-treated mice in which the allograft survival times were increased. Thymectomy is known to increase the incidence of urethan-induced lung adenomas (Trainin et al., 1967; Trainin and Linker-Israeli, 19691, and similar studies on urethan-treated thymectomized BALB/c mice showed that male mice with increased allograft survival times developed the largest tumors. These findings are similar to the correlation between MCinduced skin papilloma formation and the extent of immunosuppression by ALS (Lapp6 and Prehn, 1969; Lapp6, 1971a) and were interpreted as demonstrating that urethan directly facilitates adenoma development by interfering with immunological surveillance (Lapp6 and Prehn, 1970). Few other chemical carcinogens have been studied in such detail for immunosuppressive activities, but even so, there is a general correlation of this property with carcinogenic activity. Malmgren et al. (1952) reported that 4-dimethylaminoazobenzene (DAB) markedly reduced the hemolysin titers of mice sensitized by sheep erythrocytes. This correlation is not very convincing, however, since relatively large doses of DAB (500-1000 mg/kg body weight) were used, and mice are not particularly susceptible to DAB carcinogenesis. The immunosuppressive action of DAB in rats has since becn confirmed (Baldwin and Glaves, unpublished observations) in studies showing that a single intraperitoneal dose (250 mg/kg body weight) depressed the response to sheep erythrocytes, as assayed by reduction in the number of hemolytic plaquc-forming spleen cells. This was also reflected in the reduction of hemolytic and hemagglutinin antibody levels in serum throughout the period of the primary immune response. The immun,osuppression induced by DAB was also dose dependent, so that doses greater than 100 mg/kg depressed the numbers of hemolytic plaque-forming cells following immunization with sheep erythrocytes, whereas low doses (of the order of 40 mg/kg) stimulated the response. This may be a reflection of two contrasting effects of DAB, high doses producing immunodepression while low doses allow the expression of a reticuloendothelial stimulating action (Bismarck et al., 1966). Perhaps more significantly, analysis of the effects of continuous carcinogen feeding in the low protein diet used for hepatoma induction revealed a progressive increase in immunocompetence. Hence, while rats fed on carcinogen diet, for 30 days showed a 10% depression in the number of hemolytic plaque-forming cells by 130 days of carcinogen feeding a 52% deprcssion was obtained. These effects were obtained in comparison

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with controls fed the low protein diet thus excluding this as the cause of the immunodepression. Finally, in structure-activity studies it was established that hepatocarcinogens including DAB, 3’-methyl-DAB, and 4’fluoro-DAB were immunosuppressive whereas noncarcinogenic 2’5’dichloro-DAB and 2-methyl-DAB were stimulatory. This effect, however, was evident only in assays of the reduction in numbers of hemolytic plaque-forming spleen cells, the levels of hemolytic and hemagglutinin antibodies hardly being affected a t any interval throughout the primary response to sheep erythrocytes. Furthermore, DAB treatment has not been found to modify the rejection of skin allografts or to reduce the level of tumor-specific antibody elicited by immunization with hepatoma cells. Several other carcinogens including N-nitrosomethylurea (Parmiani et al., 1971) and 2-acetylaminofluorene (Baldwin and Glaves, unpublished observations) have been found to be immunosuppressive, based on their capacity to inhibit the antibody response to sheep erythrocytes. In addition, Kripke and Weiss (1970) reported that mineral oil which induces plasma cell tumors in BALB/c mice depressed immunological responsiveness in this strain of mice as measured by the humoral antibody response to bacteriophage T2 and by spleen weight assay for graft-versushost reactions. The effects on humoral antibody response were not observed in C57BL mice which are refractory to mineral oil carcinogenesis. AND HUMORAL IMMUNE RESPONSES TO TUMORC. CELLULAR ASSOCIATED ANTIGENS

For immunosurveillance mechanisms to inhibit growth of carcinogeninduced malignant cell clones, the immune response elicited against tumor-associated antigens must be of sufficient magnitude to prevent “sneaking through” as postulated by Old and Boyse (1964), whereby immune cytotoxic or cytostatic reactions are judged to be incapable of controlling a rapidly dividing tumor cell population. This will depend first on the magnitude of the immune response, which in turn will be regulated by the degree of antigenic dissimilarity between the neoplastic cell and the host and its exposure to the host’s immunological apparatus. Second, for effective control of tumor cell populations, the autochthonous host must be capable of eliciting immune responses sufficiently rapidly in order to limit growth of dividing tumor cells. As already indicated in Section 11, the amount of tumor antigen expressed on chemically induced tumor cells, even in the most favorable examples, such as MC-induced sarcomas, is limited when compared with alloantigen expression. This is reflected by the finding that, even under optimal conditions of immunization, there is a limit t o the number of

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transplanted tumor cells (often no more than lo6) rejected by immunized hosts. Moreover, the immunogenicity of tumor cells in the autochthonous host may be further restricted by the masking of tumor antigens by coating substances which may be tumor cell products or nonspecifically associated host material. Currie and Bagshawe (1969) showed that the immunogenicity of MC-induced mouse sarcomas is increased following incubation with neuraminidase. This effect was most marked following intraperitoneal injection of tumor cells into syngeneic mice, where untreated tumor cells developed progressively to kill the mice. Incubation of tumor cells with purified Vibrio cholera neuraminidase prevented tumor development and subsequent challenges of the mice with trocar fragments developed much slower than in controls. It is interesting, however, that one of the five sarcomas which lacked immunogenicity was not affected by neuraminidase treatment, suggesting that antigen masking is unlikely to account for the lack of immunogenicity of other chemically induced tumors, such as AAF-induced hepatomas and mammary carcinomas (Baldwin and Embleton, 1969a, 1971b). The interpretation of these studies was that neuraminidase treatment removes cell surface sialic acid and thereby facilitates recognition of tumor-associated antigens. This interpretation was also suggested by Simmons et al. (1971a,b), who showed that neuraminidase treatment of MC-induced murine sarcoma cells enhanced their immunogenicity to such an extent that they could be used to induce immunological suppression of established tumor grafts. Neuraminidase treatment has also been reported to increase the immunogenicity of allogeneic tumors, such as the Landschiitz tumor (Currie and Bagshawe, 1968) and TA3 cells (Sanford, 1967), while controlled exposure of leukemia L1210 cells results in loss of growth potential (Bekesi et al., 1971). The effect of neuraminidase on L1210 cells was correlated with the amount of N-acetylneuraminic acid released on incubation and treated cells were still highly viable as determined by dye exclusion tests. Mice injected with neuraminidase-treated cells were shown subsequently to reject a challenge with 105 L1210 cells, which in the system used was about 100,000 times the 50% lethal tumor dose. Sialic acid is known to be intimately associated with the cell surface of many tumor types (Weiss, 1967; Winzler, 1970), and plasma membrane-associated sialoglycopeptides have been isolated from rat hepatoma cells (Walborg et al., 1969; Smith and Walborg, 1972). As yet, however, the effectiveness of neuraminidase treatment on tumor immunogenicity has not been generally explored. With DAB-induced rat hepatomas, neuraminidase treatment does not lead tcr a marked increase in the amount of tumor antigen exposed at the cell surface assayed by comparing the capacity of treated and untreated hepatoma cells to absorb tumor-specific antibody

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(Baldwin et al., 1 9 7 1 ~ )However, . the mode of action of neuraminidase treatment in increasing tumor immunogenicity is still not resolved, and it may well be that removal of surface sialic acids facilitates cell-cell contact phenomena involved in the initiation of a cellular immune response to tumor-associated antigens, rather than producing an overall increase in tumor antigen exposure. Since sensitized lymphocytes are primarily responsible for the rejection of neoplastic cells (K. E. Hellstrom and Hellstrijm, 1969), it is evident that for immunosurveillance to be effective, tumor-associated antigens must evoke an effective cell-mediated response. The alternative production of humoral antibody contributes little to tumor rejection except perhaps in the case of lymphoid tumors which are susceptible to cytotoxic antibody (Old and Boyse, 1965; Wahren, 1968; Herberman and Oren, 1971). Moreover, humoral antibody may even lead to enhancement of tumor growth as demonstrated in studies with R1C-induced mouse and rat sarcomas (Moller, 1964; Bubenik and Koldovsky, 1964; Bloom and Hildemann, 1970; Baldwin and Pimm, unpublished observations), and DAB-induced rat hepatomas (Baldwin and Barker, 1967b) showing that tumor-immune serum sometimes enhanced tumor growth in syngeneic recipients. Generally, however, the effects were not very great when compared with the enhancement induced by serum in allogeneic tumor systems (Kaliss, 1958; Takasugi and Hildemann, 1969a,b; Takasugi and Klein, 1971). Also enhancement by passively transferred tumor-immune serum is not conclusive proof that such effects are operative against tumors in the autochthonous host. This is emphasized by studies (Baldwin and Pimm, unpublished observations) on the influence of serum on the growth of MC-induced rat sarcomas transplanted into syngeneic recipients, where tumor-bearer serum was not enhancing. Furthermore, the response to serum from tumor-free donors, whose subcutaneous tumors had been surgically removed, and repeatedly immunized rats, was quite variable, ranging from complete inhibition to enhancement of tumor growth. The role of humoral antibody in immunity to transplanted MCinduced mouse sarcomas has been more fully explored (Bloom and Hildemann, 1970) to show that in this system only IgG antibody was enhancing. Antibody of the 7 s class obtained from resistant or actively enhanced hosts regularly produced enhancement of tumor growth in previously untreated recipients. Also, IgG fractions obtained from antibody-enhanced hosts were very effective in vivo even when there was too little IgG to be detected in vitro by their cytotoxicity for plated tumor cells. IgM antibody, on the other hand, was cytotoxic in vitro toward mouse sarcbma cells, and passively administered fractions significantly inhibited tumor growth. From these findings it was postulated that pro-

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duction of passive enhancement as opposed to resistance following administration of whole tumor-specific antisera was determined by whether IgG or IgM was the predominating molecular class of antibody produced. This competitive action of 7 S and 19 S immunoglobulins could therefore account for the opposite effects of passively transferred tumor-specific antisera reported in other studies. 1. Tumor-Immune Reactions in the Tuntor-Bearing Host

The view that a tumor-bearing host elicits a significant cellular immune response against its autochthonous tumor is supported by reports of “concomitant” immunity, where tumor-bearing animals were able to reject a second challenge with the same tumor, but not one which was immunologically different. For example, Lausch and Rapp (1969) observed significant levels of resistance against a subsequent tumor challenge in hamsters bearing syngeneic transplants of a 7,12-dimethylbenz [ a ]anthracene-induced sarcoma. Animals bearing the DMBA-induced sarcoma were not protected against challenge with hamster tumors transformed by simian papovavirus SV40. Concomitant immunity has also been observed in mice bearing syngeneic transplants of MC-induced sarcomas (Bard et al., 1969; Deckers et al., 1971; Vaage, 1971, 1972; Belehradek et al., 1972). It is notable, however, that comparable studies have not been able to demonstrate a similar concomitant immunity in animals bearing primary sarcomas induced in mice with MC (Stjernsward, 1968) or with benzo[a]pyrene in rats (Mikulska et al., 1966). A number of factors can be considered to account for this discrepancy including immunosuppression by residual carcinogen, while the transplantation technique may enhance the tumor-immune response in the tumorgraft recipient, as already discussed in relation to studies on the rejection of mouse skin papillomas (see Section V1,A). Another factor undoubtedly is the size of the initial tumor a t the time tests are carried out for concomitant immunity, and in the preliminary studies reported by Belehradek et al. (1972) mice bearing 10-day-old, but not 30-day-old1 sarcoma grafts were able to reject a second challenge with the same tumor. The view that the tumor-bearing host is producing an immunological reaction against tumor-associated antigens which is affected by the presence of a tumor mass is supported by an earlier report (Old et al., 1962) showing that spleen cells from mice bearing syngeneic grafts of MCinduced sarcomas were unable to adoptively transfer resistance when tested immediately after removal, but when spleen cell recipients were challenged after 7 days, tumor resistance was observed. These findings were confirmed in several studies (Mikulska et al., 1966; Stjernsward, 1968; Bard and Pilch, 1969), and Deckers et al. (1971) reported a com-

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prehensive analysis of the requirements for adoptive transfer of immunity by spleen cells from mice bearing MC-induced sarcomas. This showed that the capacity of spleen cells from tumor-bearers to transfer tumor immunity developed 7-10 days after tumor implantation (10 tumor cells) subcutaneously, but this also required an interval of a t least 7 days between spleen cell transfer and tumor challenge. More direct evidence that a tumor-immune response is influenced by a growing tumor is provided by studies of Alexander and his collaborators (Dclormc et al., 1969; Alexander et al., 1969; Alexander and Hall, 1970) on the lymphoid cell populations in the thoracic duct lymph during growth of syngeneic transplants of benzo [ a ]pyrene-induced rat sarcomas. Initially, immunoblasts are released in response to antigenic stimulation by the transplanted tumor, but this response soon decreases in spite of continued stimulation of the regional lymph nodes by the progressively growing tumor. Immediately after tumor excision, however, immunoblasts are again released into the thoracic duct lymph. There are also several reports indicating that reintroduction of tumor may lead to the abrogation in vivo of tumor-specific immunity. This was demonstrated in studies (Wepsic e t al., 1971) showing that immunity to an intradermal challenge with guinea pig hepatomas, conferred by intracardial transfer of peritoneal cells from specifically immune donors, was abrogated by intraperitoneal injection of viable or X-irradiated tumor cells. This abrogation could be achieved when lo9 irradiated hepatoma cells were administered, whereas lower doses of hepatoma cells were less effective. It was also shown that injection of irradiated tumor cells in admixture with sensitized peritoneal exudate cells prevented the development of resistance to intradermal tumor challenge. I n similar studies Vaage (1971) showed that mice challenged with viable MC-induced sarcoma cells after complete surgical removal of transplants of the same tumor rejected this challenge. When, however, the first sensitizing tumor was left in situ until 4 days after the challenge injection of tumor cells, the immune resistance of the host was impaired. A similar impairment of tumor rejection was also obtained so that a dose of X-irradiated (8000R) tumor tissue, ( 5 X to 5 X lo-? gm) implanted a t the time of tumor excision abrogating resistance to tumor challenge 4 days later. The dynamics of this effect have been explored in greater depth (Vaage, 1972) to show that the depression of host resistance by injection of X-irradiated (10,000 R ) murine sarcoma cells was most effective during the week after challenge in mice sensitized to the tumor by surgical removal of a subcutaneous tumor graft. This depression was specific and was not impaired by the presence of a growing implant or by injecting irradiated cells of an antigenically unrelated syngeneic mammary carcinoma. Injection of graded doses of killed tumor

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cells about the time of tumor challenge showed that the depression was dose related. Doses from 1.0 to 10 pg of irradiated sarcoma tissue did not affect the level of resistance, but three injections of fibrosarcoma tissue in doses ranging from 50 pg to 50 mg each produced significant impairment. 2. In Vitro Studies Cellular immune responses to tumor-associated antigens in rats and micc bearing primary or transplanted MC-induced sarcoma have been demonstrated in. vitro using colony inhibition methods (Hellstrom et al., 1968, 1970; Baldwin and Moore, 1971; Belehradek et al., 1972; Stutman, 1972). Comparable studies have shown the presence of cytotoxic lymphocytes in rats bearing DAB-induced primary and transplanted rat hepatomas (Baldwin et al., 1973b) and rats and mice with MC-induced bladder papillomas and carcinomas (I. Hellstrom and Hellstrom, 1972 ; Taranger et al., 1972a,b). There is also a growing body of evidence from studies with virus-induced tumors as well as human tumors (reviewed by K. E. HellstrGm and Hellstrom, 1969, 1970, 1972) showing that lymphocytes from tumor-bearing hosts are specifically cytotoxic. These observations led to the suggestion that the serum of tumor-bearing hosts may contain circulating factors which interfere with cell-mediated immunity, these being primarily conccrncd in tumor-rejection (K. E. Hellstrom and Hellstrom, 1970) . Evidence supporting this concept was provided initially in studies with Moloney virus-induced sarcomas (I. Hellstrom and Hellstrom, 1969) showing that serum from tumor-bearing mice blocked the inhibitory effect of sensitized lymph node cells. This effect was detected in vitro with the colony inhibition technique by demonstrating that prior treatment of plated tumor cells with decomplemented serum from mice with progressively growing Moloney sarcomas protected them from the cytotoxic action of sensitized lymph node cells. Serum from mice with spontaneous mammary carcinomas or MC-induced sarcomas showed no such “blocking” activity. I n a similar fashion it has been established that sera from rats and micc with progressively growing MC-induced primary or transplanted sarcomas (Hellstrom et al., 1968, 1970; Baldwin and Embleton, unpublished observations ; Stutman, 1972) and aminoazo dye-induced hepatomas (Baldwin et al., 197313) specifically protect cultured tumor cells from the inhibitory effect of lymph node cells sensitized against the tumor-associated rejection antigens. An important question concerning the role of serum blocking factors is whether they appear before tumors begin to grow progressively ; i.e. : Can they influence small populations of tumor cells? Evidence on this point is still not very substantial, but Hellstrom et al. (1970) observed blocking activity in

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serum 7 days after mice were inoculated with 5 X lo7 viable MC-sarcoma cells, at a time when tumors were not palpable. Similarly, blocking activity was detectable in serum a t the time when primary polyoma tumors first became palpable, and in some instances this activity preceded visible or palpable growths (Sjogren and Bansal, 1971; Sjogren and Borum, 1971). The appearance of blocking activity in serum is thus an early phenomenon in tumor development. Several findings suggest that one of the blocking factors in serum from tumor-bearing hosts involves tumor-specific antibody. First, the specificity of serum blocking is generally similar to that of the antibody response elicited against the tumor-associated antigens. For instance, sera from rats bearing transplants of DAB-induced hepatomas only block lymph node cell-mediated inhibition of colony formation by cells of the homologous tumor (Baldwin et ul., 1973b), and this is consistent with the specificity of the cell-mediated and humoral antibody responses detected by the colony inhibition method against these tumors (Baldwin and Embleton, 1971a). Second, fractionation of Moloney sarcoma-bearer mouse serum by Sephadex G-200 chromatography (I. Hellstrom and Hellstrijm, 1969) and rat hepatoma-bearer serum by either G-200 chromatography or density gradient centrifugation (Baldwin and Robins, unpublished observations) indicating that blocking factors separated with 7 S immunoglobulins. Furthermore, the blocking activity of Moloney sarcoma-bearer serum could be neutralized by heterologous anti-mouse 7 S immunoglobulins or by absorption with Moloney sarcoma cells. Blocking substances were similarly absorbed from the serum of rats bearing a transplanted hepatoma (D23) with viable hepstoma D23 cells, and, as in the tests with Moloney sarcomas, the blocking active material could be eluted with pH 3 buffer (Baldwin and Robins, unpublished observations). By means of comparable methods, immunoglobulin-containing eluates have been obtained from intact cells and subcellular membrane fractions from primary benzo [ u ]pyrene-induced mouse sarcoma and from spontaneous mammary carcinomas (Ran and Witz, 1970; Witz, 1971). The eluted immunoglobulin was characterized by immunodiffusion predominantly as IgG2, and preliminary studies indicated that these fractions enhanced tumor growth (Ran and Witz, 1972). Serum blocking activity is rapidly lost, however, in animals rendered tumor-free by surgical removal of subcutaneously growing tumor (Hellstrom et al., 1970; Baldwin et uE., 1973b). For example, serum taken 5 days after excision of syngeneic grafts of DAELinduced rat hepatomas no longer blocked lymph node cell cytotoxicity, and this was accompanied by the appearance of complement-dependent cytotoxic antibody. Serum blocking activity has been observed to be

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rapidly lost following regression of Shop6 papillomas (Hellstrom et al., 1969) or Moloney sarcomas (I. Hellstrom and Hellstriim, 1969), and similar effects have been detected in the serum of patients who have become clinically symptom free (Hellstriim et d., 1971a; Sjogren et d., 1972). These observations are difficult to reconcile with the view that the serum factor abrogating cell-mediated immunity is circulating antibody unless it is further postulated that this antibody class has an especially short half-life. Furthermore, it has also been demonstrated that the in vitro blocking activity in serum of tumor-bearing animals or patients can be neutralized by the addition of appropriate amounts of serum from tumor-free individuals (I. Hellstrom and Hellstrom, 1970; Bansal and Sjogren, 1971; Hellstrom et al., 1971b) which is not compatible with the concept of antibody as the blocking species. One explanation of the above findings is that the factor in tumorbearer serum which blocks target tumor cells from lymphocyte-mediated cytotoxicity is antigen-antibody complex. This concept is supported by the finding that the blocking factor in serum from mice bearing Moloney virus or MC-induced sarcomas can be absorbed onto the respective tumor cells, and, after elution with glycine buffer, (pH 3.1), separated into high and low molecular weight components which individually lack activity (Sjogren et al., 1971). Fractionation was achieved by membrane ultrafiltration first through a filter retaining molecules above approximately 100,000 molecular weight (fraction Elm) which includes immunoglobulins. The filtrate was subsequently passed through a second membrane retaining molecules with a molecular weight above 10,OOO (fraction E10). Both fractions were then adjusted to pH 7.4, concentrated to the original serum volume for testing for the capacity to block tumor cells against sensitized lymphocytes. These tests showed that individual fractions were devoid of blocking activity while activity was restored when they were recombined. Blocking factors have also been eluted in a similar fashion from human tumors (Sjogren et al., 1972), and again high and low molecular weight fractions individually failed to block the cytotoxic action of specifically sensitized patient’s lymphocytes, but a 1:1 mixture of the two was blocking. I n tests where the two fractions were added separately to plated tumor cells, treatment with the “low molecular weight” followed by the “high molecular weight” fractions did not produce abrogation of lymphocyte cytotoxicity. If the sequence was reversed, treating tumor cells with the “high molecular weight” component first, then blocking occurred. From these observations, it has been suggested that the blocking factor interacting with target tumor cells may be antigen-antibody complex which following dissociation a t low pH, can be separated into an antibody containing fraction (E100) and an antigen-rich fraction (E10).

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Direct proof that tumor-specific antigen-antibody immune complexes can block lymphocyte-mediated cytotoxicity in vitro for cultured tumor cells has been obtained in tests with a transplanted rat hepatoma D23 (Baldwin et al., 1972b). As already discussed, serum from tumor-bearing rats blocks the action of sensitized lymph node cells against plated hepatoma cells. Following surgical removal of subcutaneous tumor grafts, blocking activity is rapidly (3 days) lost, this being accompanied by the appearance of complement-dependent cytotoxic antibody in the “postexcision” serum (Baldwin et al., 1973b). It was then possible to demonstrate that tumor-specific antigen-antibody complexes formed by the addition of solubilized membrane antigen from hepatoma D23 cells (Baldwin and Glaves, 1972a; cf. p. 23) to LLpost-excision”serum resulted in the appearance of blocking activity (Baldwin et al., 197213). This effect was dependent upon the relative amounts of antigen and antibody in the system so that a t low ratios no significant blocking was observed, presumably because the amount of immune complex was inadequate. With increasing antigen to antibody ratios, blocking activity was detected, and then in regions of antigen excess, this activity again disappeared. Comparably, addition of soluble hepatoma D23 antigen to tumor-bearer serum which initially displayed blocking action resulted in the loss of this activity. The hepatoma D23 antigen preparation used in these studies was prepared by papain solubilization of tumor membrane and contained proteins predominantly of molecular weight 55,000. It is possible, however, that the tumor antigen bound to antibody in tumor-bearer serum may be of much lower molecular size, since this presumably is liberated as a consequence of tumor cell degeneration. This would also seem to be indicated by the data of Sjogren e t al. (1971) on the membrane filtration characteristics of the small molecular weight components separated following low pH dissociation of immune complexes from tumor-bearer sera, and is consistent with the isolation of blocking substances with 7 S components (I. Hellstrom and Hellstrom, 1969; Baldwin and Robins, unpublished observations). I n the above studies, blocking activity was evaluated by incubating plated tumor cells with serum which was removed before exposure of the target cells to sensitized lymphocytes. Probably a more important mode of blocking is by direct interaction with sensitized lymphocytes, and this may be effected by both circulating tumor antigen and antigen-antibody complexes. This was indicated in the experiments of Sjogren et at. (1971) showing that the low molecular weight fraction from tumor-bearer serum (E10) was itself blocking if added to plated Moloney sarcoma cells and left in the system after addition of sensitized lymphocytes. I n another system, Brawn (1971) has demonstrated that the cytotoxic action of

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lymphocytes sensitized to H-2 antigens may be abrogated by exposure to sera containing soluble H-2 antigens of the target cell specificity. This hypothesis is also consistent with the in vivo data (Wepsic et al., 1971 ; Vaage, 1971, 1972) showing that tumor-specific immune rejection reactions can be abrogated by injection of tumor-antigen extracts. The view that circulating factors, which may be tumor-specific antigen or antigen-antibody complexes, play a role (dominant?) in counteracting homeostatic control of tumor growth is further supported by experiments showing that injection of sera from rats bearing progressively growing polyoma tumors or low pH eluates of serum-treated polyoma tumor enhances growth of syngeneic polyoma tumor transplants (Bansal et al., 1972). Development and growth of primary Moloney sarcomas in mice was also enhanced by pretreatment of tumor-bearing hosts with serum from mice bearing progressively growing Moloney sarcomas (Pierce, 1971). Conversely, growth of polyoma tumors can be retarded or completely suppressed by inoculation of rats with “unblocking” sera which counteract the in vitro lymphocyte-blocking activity of tumor-bearer serum (Bansal and Sjogren, 1972). These so-called “unblocking sera” were prepared from rats or rabbits primed by inoculation with Bacillus Calmette-GuBrin, followed by the injection of neuraminidase and mitomycin-treated polyoma tumor cells. When inoculated into rats which had previously received a syngeneic graft of a polyoma tumor, the unblocking sera inhibited (or counteracted) the appearance of blocking antibody and led, in a number of instances, to complete regression of progressively growing tumor. Inoculation of unblocking sera from either rats or rabbits in combination with splenectomy also led to the retardation of tumor growth (in two cases complete regression) of primary polyoma virusinduced kidney sarcomas. The specific role of circulating tumor antigen or tumor antigen-antibody complexes in modifying lymphocyte cytotoxicity for tumor cells is now being actively investigated. It is likely that in addition to tumorspecific antigen, other tumor-associated antigens such as embryonic antigens and their immune complexes will all contribute to the antagonistic humoral responses which operate against the lymphocyte-mediated tumor-rejection reactions. VII. Conclusions

Immunological responses to neoantigens expressed on preneoplastic and neoplastic cells can influence either positively or negatively the course of chemical carcinogenesis. It may be that tumor-rejection reactions mediated by sensitized lymphocytes are much commoner events than was hitherto believed, and this would be in accord with the concept that im-

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munosurveillance is a controlling factor in neoplasia (Burnet, 1971). On the other hand, there are antagonistic immune responses which limit lymphocyte reactivity, including direct “blocking” by humoral factors, such as circulating tumor antigen as well as antibody and antigen-antibody complexes. The interplay between cellular and humoral immune reactions to tumor-associated antigens is still poorly understood, but circulating lymphocyte-blocking factors seem to be important in preventing the rejection of rapidly growing tumors. Whether such effects operate against chemically induced tumors and a t what stage in carcinogenesis they may appear has still to be investigated. Another important question, which has not been adequately evaluated, is the contribution of depressed immunological competence in chemical carcinogenesis. It is evident that immunosuppression either by thymectomy or treatment with antilymphocyte serum modifies the response to chemical carcinogens far less than in viral oncogenesis. This may be because the degree of neoantigen expression on chemically induced tumors is frequently lower than that of virus-induced tumors where virus-associated antigens a t the tumor cell surface contribute appreciably to immunological recognition. It has been suggested (Prehn, 1971) that weak immunological reactions may even favor tumor growth and a closer analysis of the cellular and humoral immune responses to tumor-associated antigens in animals displaying various degrees of immunological competence would seem desirable. The notion that immunosuppression by a chemical carcinogen contributes to its carcinogenic action cannot be substantiated on the evidence presented, which generally has relied upon evaluation of depressed humoral antibody responses to protein antigens or xenogeneic erythrocytes or enhanced growth of allogeneic tumors, antigenic stimuli which are not really comparable with tumor-associated antigens. Here, also, a more critical appraisal of immune deficiencies will be needed to establish whether carcinogen-induced immunosuppression has any real influence in chemical carcinogenesis. Recognition must be given to the considerable body of evidence showing that aberrations of cell surface architecture occur in many, if not all, chemically induced tumors. This may result in the appearance of new cell surface antigens, some of which appear to be specific components of the tumor cell while others arc products of re-expressed fetal genes. I n some cases, the appearance of neoantigcns may also be accompanied by the deletion (real or masking) of cell-surface antigens usually expressed on normal cell types. These findings represent only a beginning in the study of tumor cell surface components, but already more precise biochemical characterizations of tumor-associated antigens is possible. This should lead to the development of refined immunological assays for study-

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ing the biosynthesis of tumor-associated antigens, while immunoelectron microscopy should provide accurate methods for localizing antigens within discrete regions of the cell surface. These developments will therefore make possible the analysis of cell surface changes during chemical carcinogenesis, using in vitro systems a t a level consistent with that employed for the analysis of specific interactions of carcinogen metabolites with cellular macromolecules and may lead to the interpretation of metabolic events in relation to tumor antigen expression a t the cell surface.

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ISOZYMES AND CANCER Fanny Schapira Institut de Pathologie Molhculaire. Pork. France

I. Introduction . . . . . . . . I1. Oxidoreductascs . . . . . . . A . Lactic Dehydrogenase . . . . . B . Malate Dehydrogenase . . . . C. Isocitrate Dehydrogenase . . . . D . Alcohol Dehydrogenase . . . . E. Aldehyde Dchydrogenasc . . . . I11. Lyases . . . . . . . . . A. Aldolase . . . . . . . . B. Serine Dehydratase . . . . . IV . Phosphotransferases . . . . . . A . Hexokinase . . . . . . . B . Pyruvate Kinase . . . . . . C. Adenylate Kinase . . . . . . D . Phosphofructokinase . . . . . E. Uridine Kinase . . . . . . F. Thymidine Kinase . . . . . . G . Carbamyl Phosphate Synthetase . . V. Other Transferases . . . . . . A . Aspartate Aminotransferase . . . B . Branched-Chain Amino Acid Trnnsferases C . Glycogen Synthetase . . . . . D . Phosphorylases . . . . . . E . DNA Polymerase . . . . . . F. Transfer RNA Methylases . . . . VI . Hydrolases . . . . . . . . . . . . . . . A . Introduction B. Alkaline Phosphatases . . . . . C . Acid Phosphatases . . . . . . D. Fructose-1, 6-Diphosphatase . . . E. Glutarninase . . . . . . . I? . Esterases . . . . . . . . VII . Summary and Conclusions . . . . Addendum . . . . . . . . References . . . . . . . .

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I. Introduction

Our first purpose is to give a definition of the isozymes . This question has been controversial for a long time . I n 1964, a subcommittee of the 77

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International Union of Biochemistry recommended : “Multiple enzyme forms in a single species, catalyzing essentially the same reaction, but differing in various ways should be called ‘isozymes,’ or ‘isoenzymes.”’ Nevertheless this definition was too extensive, and new recommendations appeared in 1971 from the I U P A G I U B Commission of Biochemical Nomenclature. We shall quote them here according to the papers published in several journals. 1. The term “multiple forms of the enzymes” defines every protein possessing the same enzyme activity and occurring naturally in a single species. 2. The term “isoenzyme” or “isozyme” should apply only to those multiple forms of enzymes arising from genetically determined differences in primary structure. It follows from that definition that proteins conjugated with other groups (for example, phosphorylases a and b ) , polymers of a single subunit (for example glutamate dehydrogenase) , and the allosteric modifications of enzymes are not isoenzymes. But this definition does not exclude, for example, acid and alkaline phosphatases, although these phosphatases are generally considered to be separate enzymes. The forms derived from different organs are sometimes called “heteroenzymes.” For the enzymes occurring in different forms in the mitochondria and in the supernatant of the same cell, the term “isoenzyme” is convenient. According to this nomenclature, genetic variants (for example, variants of glucose-6-phosphate dehydrogenase) are to be distinguished from isoenzymes: we shall call them “alleles” or “allelic variants,” and “isoenzymes,” the other molecular forms synthesized on different loci. Finally, the clearest case is that of heteropolymers of two or more polypeptide chains, noncovalently bound, the first and the best known example of which is lactic dehydrogenase. Obviously this definition includes also the homopolymers susceptible to hybridization: for example, the pure homopolymer muscle-lactic dehydrogenase and the pure homopolymer heart lactic dehydrogenase. Structure studies have shown that an increasing number of enzymes with multiple molecular forms are recognized as oligomers susceptible to hybridizing each other. The Nomenclature Committee of 1964 recommended the numbering of individual isoenzymes on the basis of their electrophoretic mobility, the number 1 being assigned to the form having the highest mobility toward the anode. The Committee of 1971 added that isozymes should not be labeled on the basis of tissue distribution. Instead of setting forth general data concerning techniques of isozymic separation and physicochemical and immunological properties of isoenzymes, we shall give detailed information on lactic dehydrogenase, taken

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as the best example of an enzyme with multiple molecular forms (for general methods, see King, 1965 ; Bodansky and Schwartz, 1967; Wilkinson, 1970a). According to the preceding definition, isoenzymes are generally oligomeric in structure, and isozymic modifications are related to the replacement of one or several subunits of one type by subunits of another type. Consequently, isozymic changes lie a t the molecular level; that is why knowledge of isoenzymes has renewed the study of enzymatic modifications in cancer. As recalled by Bergel (1961), three main hypothesis may account for enzymatic alterations in cancer: 1. Warburg’s hypothesis: The discovery of Cori and Cori (1925a,b) and of Warburg (1930, 1956) that tumors possess a high rate of glycolysis cannot be presented here in detail. For this question, and its relation to the impairment of respiration, the Pasteur effect, and the Crabtree effect in tumors, see the review and discussion of Weinhouse (1955, 1956). We shall recall only that this increase of glycolysis is found in fast growing tumors, but not in “minimal deviation hepatomas” (Aisenberg and Morris, 1961 ; Elwood et al., 1963). 2. The deletion hypothesis: This theory was advanced by Miller and Miller (1953), and then developed by Potter (1958, 1964). It postulates that, during malignancy, the cell loses certain essential constituents. For the Millers it appeared to be a loss of proteins caused by azo dye carcinogens, and Potter emphasized a deletion of enzymes involved in catabolic reactions. But many well differentiated hepatomas did not exhibit such a deletion. 3. Greenstein’s hypothesis : Greenstein (1956) considered that the enzyme complement of tumors tended to resemble each other, so that cancer tissue more closely resembles another tumor than its tissue of origin. The link with Warburg’s theory could be that cancer cells have lost some enzymes of respiratory cellular metabolism. On the other hand, Greenstein pointed out certain analogies between the metabolism of hepatomas and fetal liver. Greenstein’s theory was criticized: Bottomley et al. (1963) particularly gave many examples of tumors with divergence of enzyme patterns (see also Pitot, 1966). More recently, the impairment of control mechanisms was stressed. The lack of hormonal induction of tryptophan oxygenase in hepatomas, even well differentiated (Auerbach and Waisman, 1958; Pitot, 1963) has been pointed out in particular. We shall come back, in relation to isoenzymic changes, to the hypothesis of an impairment of control mechanisms in cancer. For detailed reviews of biochemical aspects of malignancy see Knox (1967), Shonk and Boxer (1967), and Wenner (1967).

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II. Oxidoreductases

A. LACTICDEHYDROGENASE 1. Generalities The most intensively studied enzyme with multiple molecular forms in cancer is lactic dehydrogenase (LDH) (L-lactate :NAD oxidoreductase, EC 1.1.1.27). We shall begin by recalling the main data on L D H isozymes. Without giving a history of the discovery of multiple forms of LDH, it may be recalled that Meister (1950a,b) observed two components by Tiselius electrophoresis of crystalline beef heart LDH. Both possessed enzymatic activity as shown by Neilands (1952). Wieland and Pfleiderer (1957) extensively investigated many tissues of different origins, and found that L D H can be resolved by electrophoresis on paper or in starch block into five proteins. The heterogeneity of serum L D H was first discovered by Vesell and Bearn (1957). The starch gel electrophoresis technique of Smithies (1955) coupled with histochemical procedures, allowed Hunter and Markert (1957) to perform a good and simple resolution. This technique was first applied to the study of esterases, and then t o L D H (Markert and Mgller, 1959) according t o a modified procedure of tetrazolium staining (Nachlas et al., 1957). From the work of Appella and Markert (1961), and Markert (1963), it is known that each isozyme is a tetramer, which may be dissociated by urea or guanidine into four subunits, the molecular weight of each of which is 35,000. Moreover, crystalline L D H of each type, muscle (abbreviation M or A) and heart (abbreviation H or B ) , was prepared and extensively purified by column chromatography and electrophoresis. The two types were mixed in equal proportions in 1 M NaCl and frozen. Resolution of the mixture by electrophoresis on starch gel generated five isozymes in the proportions of 1 :4: 6 :4 : 1, which correspond to the random association of subunits in tetramers. Fondy et al. (1964) isolated and crystallized the hybrid H,M2 from chicken liver, and found its fingerprint pattern to be virtually identical to that of a 1: 1 mixture of H, and M,. Amino acid analysis of the hybrid shows that it is intermediate between the pure types. There are consequently five isozymes, corresponding to the pure tetramers, M, and H,, and to the three hybrids, M,H,, M,H,, and M,H,. Isozymes from human tissues were also purified with analogous results (Wachsmuth and Pfleiderer, 1963). Hybridization in vitro is generally possible between diff erent species (Chilson et al., 1965 ; Saito, 1972), but in some species, hybridization does

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not occur, either i n vivo or i n vitro (Salthe et al., 1965). The synthesis of the two types is dependent on two different genes, as shown by the discovery of human erythrocyte variants, either of M type, or of H type: (Boyer et al., 1963; Kraus and. Neely, 1964; Nanee et al., 1963; Vesell, 1965a). The results of Shaw and Barto (1963) crossing a mouse strain having mutant L D H with a normal strain and observing the expected isozymes in offspring, have also confirmed this theory. Moreover, structural loci of M-LDH and H-LDH are not linked in humans (Ruddle et al., 1970; Shows, 1972; Boone et al., 1972). The two types posses an amino acid and peptide composition very different from each other. On the contrary, the tryptic peptides of LDH from rat heart and pig heart, for example, are very similar (Wieland and Pfleiderer, 1961). Pesce et al. (1964) find the same similarities between the M type from ox and from chicken. It is possible to prepare specific antisera against each type. Nisselbaum and Bodansky (1961) , Avrameas and Rajewsky (1964), Fondy et al. (1964), and many other authors have found that heart type and muscle type are immunologically different without cross reaction between the two types. Rajewski et al. (1964) showed that hybrids react together with isozymes 1 and 5. Moreover, this strict type specificity is in contrast with a poor species specificity. Several authors found more than five bands in mouse tissues (Fritz and Jacobson, 1965; Allen, 1961 ; Koen and Shaw, 1965). Even 15 active fractions might be found which appear to follow a binomial distribution (Costello and Kaplan, 1963). Some analogous subbands have been demonstrated also in chick embryo by Croisille (1964, 1967) and in fetal guinea pig by Fieldhouse and Masters (1968), and, as we shall see, in some cancerous tissues. But these results are not perfectly explained in spite of the findings of Dudman (1969) , who has shown the role of thiols, and of the extensive work of Houssais (1966) and of Koen (1967), who pointed out the role of aggregates and the role of aging i n vitro. It is possible to compare the subbands with the “X” band found by Blanco and Zinkham (1963) and Zinkham et al. (1963) in adult human testis. This “sixth isoenzyme” migrates between LDH, and LDH,. For Stamhaugh and Buckley (1967) , the X band does not involve a third gene for its synthesis. This assumption is based on the findings of Stegink and Vestling (1966) , who suggested that each subunit contains two polypeptide chains: an enzymatically active chain, and another one that would determine the charge. Hybridization might occur between these different chains and would explain subbands and the X, isozyme, but this hypothesis is not yet confirmed, and Goldberg (1971) was able to prove the immunochemical specificity of LDH “X” (for discussion, see Biserte, 1970).

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In summary, it is generally accepted that animal tissues contain mainly five LDH isozymes. Each isozyme is a tetramer; there are five isozymes, numbered from 1 (H,) to 5 (M,). Electrophoretic separation may be mainly performed on cellulwe acetate [first described by Wieland and Pfleiderer (1957)l ; on agar gel [technique extensively studied by Wieme (1965) and Wieme et al. (1962)], on acrylamide gel (Davis, 1964) ; or on starch gel, as recalled earlier. After electrophoresis, specific coloration is performed according to Markert and MPler (1959). For more detailed techniques, see Dreyfus and Schapira (1967) and Wilkinson (1970a). Chromatographic separation was described by Hess and Walter (1960, 1961). It is also possible to separate LDH isozymes according to several kinetic properties. Each isozyme responds differently to changes in pyruvate concentration (Plagemann et al., 1960). At fixed pH, each isozyme exhibits a different substrate concentration optimum. The greater the electrophoretic mobility of an isozyme, the lower its pyruvate concentration optimum. Kaplan e t al. (1960) carried out separate reactions a t high and low concentrations of substrate, and calculated the activity ratio: ( lo4 M pyruvate) : (3.3 X 10-~M pyruvate) . Cahn e t al. (1962), Kaplan and Cahn (1962), and also Markert and Ursprung (1962) have shown that tissues such as skeletal muscle, where type M is predominant (and consequently where the LDH allows the rapid conversion of pyruvate into lactate), exhibit mainly anaerobic glycolysis. These authors point out that heart, kidney, and brain, which are tissues with high rates of cellular respiration, possess an LDH which is almost entirely of H type. The inhibition of LDH by accumulation of its substrate pyruvate would direct the carbohydrate metabolism toward the citric acid cycle. For example, Wilson et al. (1963) found good correlation between the degree of substrate inhibition by LDH of pectoral muscle of birds and their flight habits. This hypothesis of the team of Kaplan is the basis of the well known theory of a shift toward an M-type LDH in cancerous tissues. Actually, there is much experimental evidence for a role of anaerobiosis in the synthesis of M subunits. In culture, a constant shift toward a predominant M-type pattern was found by several authors (Philip and Vesell, 1962; Vesell e t al., 1962; Blanco et al., 1967; Delain, 1972), and it was shown that this shift from H, to M, can be retarded under high pressure of 0, (Cahn, 1963). Moreover, Goodfriend et at. (1966) have shown, in cultures of monkey heart cells, that oxygen represses the biosynthesis of the M subunits. Low oxygen tensions, on the contrary, increase the synthesis of LDH, isozyme ; the percentage of M subunits reaches the maximum at 2.5% oxygen.

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Actinomycin D suppresses this synthesis when added before exposure to anoxia. Oxygen would activate a repressor. The effect of anaerobic environment on LDH isozymes is similar for HeLa cells, as shown by Cribbs and Kline (1971). These facts were confirmed, but the interpretation of the physiological role of M subunits is not universally accepted. For Vesell and Pool (1966)) levels of pyruvate high enough to significantly inhibit LDH of heart type do not occur in vivo. The point a t issue is whether or not this inhibition, which occurs a t 25”C, persists a t 37°C and in vivo (Everse et al., 1970). Stambaugh and Post (1966) demonstrated the differential effects of substrate concentration and temperature on the activity of H, and M, isoenzymes, as well as on purified enzymes, and on homogenates of rabbit tissues. And in human tissues, Latner et al. (1966a) found that homogenates of human liver, containing predominantly the M, isoenzyme, are inhibited significantly less than homogenates of heart, a t 37°C as well as a t 25°C. Vesell (1965b) has also focused attention on the different behavior of whole homogenates and of purified isozymes. Moreover Vesell’s group has shown that the extent of substrate inhibition of both types depends on enzyme concentration. At physiological high enzyme concentrations, no inhibition by pyruvate occurs (Wuntch et al., 1970). It is also appropriate to point out that liver, in mammals, is an aerobic tissue; nevertheless it contains almost exclusively muscle-type LDH. On the contrary, mature erythrocytes, platelets, and lenses which are anaerobic in metabolism, exhibit only traces of LDH 5 (the M, isozyme) (Vesell, 1965c; Hulk, 1971). In any case, Fritz (1965) has pointed out that muscle LDH is a regulatory enzyme with allosteric properties. Other properties allow one to differentiate between the two types. Kaplan and Ciotti (1961) showed that the rate of reaction differs according to the type, when NADH is replaced by some analogs. Wrdblewski and Gregory (1961) distinguished isozymes according to thermostability, muscle type being more thermolabile than heart type. For clinical purposes, measurement of the rate of reduction of a-ketobutyrate (2-oxobutanoate) and the determination of the activity ratio hydroxybutyrate dehydrogenase : lactic dehydrogenase. (SHBDH/SLDH) is very useful; the H-type LDH reduces the second substrate faster than does the M type (Rosalki and Wilkinson, 1960; Plummer et al., 1963). The so-called “a-hydroxybutyrate dehydrogenase” is very probably identical to heart LDH (Balinsky, 1966). The differential effect of inhibitors was studied especially by Plummer et al. (1963) and Emery (19671, who developed a technique based on preferential inhibition of LDH, by urea. Among the methods of separation, the most important

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are the chromatography techniques, particularly with DEAE-Sephadex (Richterich et al., 1963). The distribution of L D H isozymes varies according to the tissue, the species, and the age, Almost all animal species possess the two types, and it was possible to describe a phylogenetic relationship on the basis of their distribution (Kaplan et al., 1960; Kaplan and Ciotti, 1961 ; Thoai and Roche, 1968). We have seen the tissue distribution, and we have recalled that in mammals, skeletal muscles are generally of type M (although red muscles x e of H type (Blanchaer and Van Wijhe, 1962). Liver, except in ruminants, is also of type M, while heart, brain, and the renal cortex are of type H. The LDH hybrids appear to be the most abundant isoenzymes in most other tissues. An important factor of variation in the distribution of isoenzymes lies in their ontogenic evolution. Alarkert and Mprller (1959) and then Flexner et al. (1960) have given evidence for differences between the number of components of LDH during the maturation of several animal tissues. The ontogenic evolution was first described for human tissues by Pfleiderer and Wachsmuth (1961) and by Dreyfus e t al. (1962), who showed that fetal muscle is of type H, or mixed, while the adult muscle is mainly of type M. But this evolution varies according t o the species, and it seems difficult to describe a general fetal pattern. Pfleiderer and Wachsmuth (1961) found in the early stage of life, the distribution pattern in human tissues would show a maximum in band 3. This “undifferentiated pattern” was also described by Vill6e (1966), while Hinks and Masters (1966), and Fieldhouse and Masters (1966) showed that there is no evolution in one direction only. I n chicken and in rabbit (Dawson et al., 1964; Dawson and Kaplan, 1965), there is a fetal pattern of muscle, as in the human; but not in rat or mouse. I n any case it is important to point out that fetal liver in most mammalian species contain more H subunits than does adult liver (Fine et al., 1963). Generally speaking, the isozymic pattern is very constant for a species and a tissue (Rlarkert and Sladen, 1966). Nevertheless, some hormonal factors could be demonstrated; Goodfriend and Kaplan (1964) found that the administration of estradiol to immature female rats and rabbits resulted in an increase of the hl, isozymes of the uterus; estradiol would inactivate a repressor. The same increase is seen during gestation in women (Richterich and Burger, 1963) and in rabbits (Biron, 1964). Variations of isozymic pattern of the reproductive tract during the menstrual cycle or under the influence of steroid hormones, were studied by several authors (Fottrell et al., 1969) ; (Galbraith et al., 1970; Patterson and Masters, 1970; Georgiev et al., 1970). Nevertheless, the stability

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of the isoenzymic pattern in adult tissues contrasts with the extensive changes that occur during embryonic life. 2. Lactic Dehydrogenase Isozymes in Cancer

a. Isozymes in Blood. i. Serum. Hill and Levi (1954) showed that serum L D H activity was increased in some neoplastic diseases. Wr6blewski and La Due (1955) found that it was elevated during chronic and acute leukemia. Afterward, this elevation was found in the ascitic fluid of mice or rats bearing sarcoma or Ehrlich tumor (Hill, 1956) and in serum of mice with transplantable leukemia (Friend and Wrbblewski, 1956; Hill and Jordan, 1957). I n mice with transplantable tumors, a close correlation was observed between the level of L D H and the growth or the regression of the tumor (Riley and Wrbblewski, 1960), but in man, the elevation was very inconstantly found, as pointed out by Zimmerman and Weinstein (1956). On the other hand, one must keep in mind the findings of Warburg et al. (1924; Warburg, 1930) and of Cori and Cori (1925a,b) showing that tumors in vitro and in vivo used more glucose and produced more lactate than do normal tissues. It was actually confirmed that, in many tumors and in malignant tissues in culture, the L D H activity of the tumor and of the fluid medium is higher than that of the tissue of origin (Wrbblewski, 1958; Shonk et al., 1965). But Weinhouse (1955, 1956) has pointed that not only is there no experimental basis for the assumption that oxidative metabolism is impaired in cancer, but also, some normal tissues glycolyze as highly as many tumors. Also, Weber e t al. (1961) and Elwood et al. (1963) have proved that, although the glycolytic rate is increased in the rapidly growing tumors, it is similar to that found in normal liver in many slowgrowing hepatomas. Riley and Wr6blewski (1960) described virus-infected mice with a considerable and permanent incrcase of serum L D H ; in these infected mice, i t was the slowest-migrating fraction of serum L D H which was increased (Riley 1963, 1968). But the normal pattern of plasma L D H in mouse is discussed. Warnock (1964) and Riley (1968) found only two isozymes. On the contrary, Plagemann et al. (1962) reported that four isozymes may be separated ; and Vesell and Brody (1964) reported seven. Because many mouse tissues are predominantly of the M type (Fountain et al., 1970), the source of the elevation remained unknown, until Mahy and Rowson (1965) showed that the clearance of M type is impaired, after virus infection, while the clearance of the H-type isoenzyme remains unchanged. Blockade of the reticuloendothelial system causes an enzymatic pattern similar to that found in infected mice.

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Since the beginning of the isoeyme studies, the distribution of isoeymes in human serum in normal and pathological states has been investigated. Vesell and Bearn (1957, 1958) localized LDH activity in several fractions of serum. They showed that in myocardial infarction there is an elevation of the electrophoretically fastest migrating isoeymes, while in serum of patients with myelogenous leukemia, the intermediary isozyme was increased. By various methods, fractionation of serum was improved by Wieme (1959), Wieme and Van Maercke (1961), Wr6blewski and Gregory (1960), Hill (1958),Hess and Walter (1960), Rosalki and Wilkinson (1960), and Starkweather et al. (1966b), and the presence of five LDH fractions in normal human serum was demonstrated. In normal human serum the most anodic isoeymes are preponderant although muscle and liver (the main tissues susceptible of being the source of the circulating enzyme) possess an LDH of M type, with predominant cathodic isoeymes. The reason was brought up by Boyd (1966), who demonstrated that the sheep plasma clearance of M-type LDH is faster than the clearance of the H type. If it is the same for man, this fact will perhaps explain why the study of LDH isozymes often appeared deceptive, although according to Vesell (1961), alterations of the serum LDH pattern correlate with the pattern of the damaged tissue. The knowledge of LDH isozymes was applied to clinical medicine, particularly for the diagnosis of myocardial infarction (Wieme, 1959; Wrbblewski and Gregory, 1960) and in liver injuries (Hess and Walter, 1960; Wieme and Van Maercke, 1961; for reviews, see Latner and Skillen, 1968; Wilkinson, 1970a) ; but in clinical cancerology results appeared inconstant. Richterich et al. (1961, 1963), and Von Zuppinger et al. (1962) found an increase of the slowest fractions, migrating as a-globulins in malignant effusions, but not in serum; this increase contrasts with the isoenzyme composition in transudates, which is similar to that of the serum. Guttler (1967) has confirmed these data. The estimation of HBDH:LDH activity ratio allowed Elliott and Wilkinson (1963) to find an increase of M subunits in sera of some patients with primary carcinoma of liver; but usually the ratio was normal except in cases of carcinoma with liver metastases (Wilkinson, 1970b). Many further studies were performed on the alterations of isoenzymic pattern of sera in malignancy, but results were often divergent. Starkweather et al. (1966a) found an abnormal isoeneyme pattern in 39 sera on 48 patients with lung cancer, but these abnormalities varied according to the stage of the disease, the effect of therapy, and other unknown factors. Wieme (1959) found an “indifferent” pattern in sera of patients with leukemia and carcinomas, and Cenciotti and Mariotti (1964) in sera of rats with experimental tumors. Wieme et al. (1968) reported that,

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after cytostatic treatment of bronchogenic carcinoma, the progressive return of LDH, to a normal value is of good prognostic value. The increase of LDH, is often transient, and when observed a t the beginning of treatment, it seems to be a good sign, and its normalization has always preceded radiological improvement. Zondag (1965) and Zondag and Klein (1968) studied 474 patients with malignancy. I n 204 patients having a primary tumor without metastases, only 65 had abnormal LDH isozymes; this anomaly was generally the increase of fractions 2-3 or 4. When metastases were clinically proved, the percentage of anomaly was almost doubled. The same abnormal pattern was very often encountered in Hodgkin’s disease and in lymphosarcoma. In a special group of 15 tumors originating in the testis or ovary, 13 (87%) showed abnormalities, but it was the fast-moving fractions (LDH 1 and 2) which were raised. More recently, Seck et al. (1970) studied the isoenzymic distribution in serum of African patients with primary cancer of liver. They found a strong increase of LDH 5 in 35 out of 37 cases with presence of a-fetoprotein. The proportion of LDH 5 was 33% instead of 9%. These interesting results are in striking contrast with the results found in other types of cancer; the association with some degree of liver cytolysis (as shown by the frequent increase of serum transaminases) may explain the release of M subunits from liver. Nevertheless, Garbe et al. (1971) also found a slight increase of LDH 4 and 5 in sera of patients with cancers, at various locations. It seems of special interest that several authors have found supplementary bands of LDH isoenzymes in serum of patients with cancer. Fujimoto et al. (1968) and Wilkinson (1970b) reported a case of carcinoma of the esophagus with metastases in the liver. The serum LDH was greatly raised, and the high hydroxybutyrate dehydrogenase activity proved the increase of H subunits. Moreover, electrophoresis showed extra bands between LDH, and LDH, and between LDH, and LDH,. One sample of the liver tumor showed abnormally high electrophoretic mobilities of the LDH isoenaymes: the abnormalities in the serum isoenayme pattern consequently might reflect the synthesis by the tumor of an abnormal type of subunit more positively charged than the normal ones. However, no definitive explanation may be given now. This observation may be compared to that of Beautyman (1962) and to experiments of Thkret and LalCgerie (1967) on transplantable carcinomas of rats. These authors found many additional “subbands” in fraction 5, in serum, and in tumors; the LDH activity was also very high both in serum and in tumor tissues. We shall come back to this question of supernumerary bands, described also by Latner (1964) and by Lundh (1967). In summary, the constancy and the significance of the elevation of

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serum LDH in malignancy remains conflicting. It seems that in leukemia, the elevation of serum LDH activity would be more constant than in other malignant states (Bierman et al., 1957), especially in myelocytic leukemia (Magill et al., 1959). Many studies were devoted to the pattern of serum and of white and red cells in leukemia; but results were inconclusive. Elliott and Wilkinson (1963) reported that the activity ratio 2-HBDH:LDH, which reflects the relative amount of H subunits, is generally normal in serum of patients with chronic or acute leukemia, but is abnormal in sera of myelomatosis: low in /?-myeloma, high in y-myeloma. Zondag and Klein (1968) indicate the most anodic or the most cathodic isozyme, or all 5 isozymes may be increased. Obviously, it is principally the LDH pattern of leukemic leukocytes which gave rise to the most work. The main difficulty was the heterogeneity of the cell population. ii. Blood cells. Different normal cells display very different patterns. In the normal granulocytes, most of the LDH activity lies in the cathodic fractions. According to Rabinowitz and Dietz (1967), the H : M ratio in granulocytes is equal to 38/62. On the contrary, this ratio is equal to 67/32 in normal lymphocytes. Consequently, these authors pointed out that meaningful comparisons cannot be made with mixed cell populations. Another possibility for divergent results comes from the storage ( H and M LDH have different sensitivity to cold storage) and from various methodologies used. We shall try to draw general conclusions from several articles on this subject: Andersen et al. (1964), Bottomley et al. (1966), Hul6 (1967), Malaskova and Holeysovska (1969), Kamiya (1970). The most extensive studies were performed by Dioguardi and Agostoni (1965), by Dioguardi et al. (1962-1963, 1965, 1966), and by Rabinowitz and Dietz (1967). In lymphatic leukemia and lymphosarcoma cell leukemia, bands 2 and 3 are predominant, as in normal lymphocytes; isozyme 1 is somewhat diminished and isozymes 3 and 4 are increased. But these differences are slight, and the leukemic and normal lymphocytes resemble each other more closely than do normal mixed cells. I n chronic and acute myeloid leukemia, the third fraction predominates while it is the fifth fraction which is the most relatively abundant in normal granulocytes. Myeloblasts, the most immature cells, are lowest in LDH 5 and highest in LDH 1. It seems, consequently, that there is no correlation, in leukemia, between malignancy and an increase of M subunits in white cells. In red cells of patients with chronic or acute leukemia, an increase of the fifth isoenzyme is frequent (Starkweather et al., 1965; Zittoun et al., 1970), but there is a correlation between the LDH pattern of red cells and their age. Vesell and Beam (1962a,b) have shown that the

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nucleated cells possess the five isoenzymes whereas normal human erythrocytes display only anodic isozymes on electrophoresis. Moreover, Rosa and Schapira (1964) separated older and younger mature red cells by osmotic lysis and showed that the youngest erythrocytes may be resolved into four isozymes, while there are only two isozymes in the oldest ones. The M subunits in the red cells seem to have a shorter half-life than the H subunits. Starkweather et al. (1965) have extended these findings, showing that in hematologic diseases, especially Hodgkin’s disease and multiple myeloma, the change in LDH pattern of red cells reflects the dynamic state of erythropoiesis. The relative increase of LDH 1 is an index of an older cell population; and conversely, increases in LDH 5 indicate hyperactive erythroid tissue. I n platelets, a similar isoenzyme modification was found (Knudsen, 1971). b. Isozymes in Organs and Tissues. The tissue modifications of the isozymic pattern in malignant diseases have given rise to considerable interest. Nisselbaum and Bodansky (1963) showed that purified liver and hepatoma human LDH displayed the same kinetic and immunological properties, purification of these forms giving the same isozymes in both cases. Before purification, many authors were able to find a modification of the isozymic LDH partition in several tumors, generally with an increase of the M subunits; this increase would be characteristic of the malignant tissue. This assertion was first made by Pfleiderer and Wachsmuth (1961) on several tumors from different organs. But for Starkweather and Schoch (1962) the LDH of tumors has lost the heterogeneity of normal tissue. Studying human neoplasms from lungs and testes, liver metastases and leukemias, they found that in all cases most of the activity appeared in fraction 3. Bar et al. (1963) and Hoch-Ligeti et al. (1965) also found a frequent increase of the third isoenzyme. Richterich et al. (1962, 1963), found, as did Pfleiderer and Wachsmuth (1961) , that malignant neoplasms of cervix uteri, lymph nodes, kidney, lung, stomach, and colon show an absolute and relative increase in the electrophoretically slow migrating fractions. Clausen and Gerhardt (1963) proposed a general law that malignant changes are associated in all organs with a considerable increase of the slowest migrating LDH isozymes. This change is most readily detectable in brain tissue, where there is a normal predominance of the fastest bands. The lowering of the H : M ratio below the normal limit of 1.5 would be constant in m.alignant g l i o m (Gerhardt et al., 1967); Nagy et al. (1971) confirmed these data and studied the cellular distribution of LDH isoenzymes in brain tumors. The shift of LDH toward a predominance of M type was described by Goldman and Kaplan (1963)

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for several human tumors. With Hall, these authors (Goldman et al., 1964) performed an extensive study on LDH in malignant human neoplasms compared with benign tumors and normal controls. The normal and tumoral tissues examined were stomach, bowel, lung, thyroid, kidney, pancreas, ovary, prostate, breast, brain, and uterus; the authors found an absolute increase of M LDH in the tumors, not merely a shift toward an M pattern. I n contrast, the benign tumors of thyroid, ovary, and uterus possessed the same type of LDH as the normal tissue of origin. Poznanska-Lumde et al. (1966) compared the LDH pattern of a lymph node tumor with that of a normal lymph node, and found a marked shift toward the slow isoenzymes; they also studied neoplasms of the digestive tract and confirmed previous findings of other authors by their personal technique, using 2-oxobutyrate as substrate. Yasin and Bergel (1965) studied the distribution of LDH in normal and malignant stomach and also found a consistent increase in the proportions of M subunits in carcinoma; Leese (1965) reached the same result by microelectrophoretic techniques, allowing a study of the histologic distribution. The findings of Nissen and Bohn (1965) on carcinoma of stomach, colon, kidney, lung, and prostate were similar. Stanislawski-Birencwaj g and Loisillier (1965) compared mastitis, fibroadenoma, and carcinoma of the mammary gland. They found that in cancerous gland, the relative activity of LDH 5 is six times higher than in mastitis or fibroadenoma. They also found a significant difference between the LDH epithelioma and the LDH of peritumoral region tissue. Okabe et al. (1968) found a consistent shift toward the muscle-type isozymes in uterine neoplasms, while isozymes from uterine myoma were indistinguishable from the normal tissue of origin. Ishihara (1968) found that M, isoenzyme to be increased a t the expense of the H, isoenzyme in carcinoma of cervix and ovary. This deviation of the isoenzymatic distribution in the tumors of the female genital tract, was also described by Stagg and Whyley (1968). According to Latner et al. (1966b) and Langvad (1968), this shift toward the slower migrating zones may be seen even where the microscopic evidence indicated no abnormality. We also noted that carcinoma of thyroid (Omi and Anan, 1970) and of prostate (Oliver et al., 1970) have an M-type pattern. It seems, consequently, that the shift toward an M-type pattern is frequently encountered in cancerous organs mainly of the genital or digestive tract. In experimental tumors, analogous results were obtained (see, e.g., Hershey et al., 1966, in rat breast cancer). Also, in human digestive tumors maintained in organ culture, Burtin et al. (1971) found only isoenzyme 5. Despite the build-up of evidence for the deviation of LDH toward the M type in many tumors, its interpretation is not clear. We have earlier indicated that it is necessary to keep in mind reservations about the

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metabolic significance of the L D H isozyme type. While the majority of cancer tissues have a predominance of M subunits, this is not the case for all cancers. We point out the work of Kline and Clayton (1964) and of Johnson and Kampschmidt (1965). Kline and Clayton studied the L D H isozymes during development of azo dye tumors in rat livers. While livers of normal rats exhibit only LDH 5, livers of rats fed hepatocarcinogen contained LDH 4 and even sometimes LDH 3. Johnson and Kampschmidt, studying rats with transplanted tumors, also saw that dietary 3’-MeDAB produces the appearance of two or three supplementary isozymes. Rosado et al. (1969) also found that there was not always a preponderance of M form in tumors. The growth rate or the degree of differentiation did not seem to influence the relative proportion of H and M subunits. These authors pointed out that the physiological significance of the M type of liver L D H in rodents is not clear. They concluded that there is not a causal relationship between the high glycolysis of tumor cells, their LDH activities, and their subunit composition. Schapira and de N6chaud (1968) and Schapira and Josipowica (1970) proposed a tentative explanation. They found a shift toward an H-type pattern in primary (slow growing) or transplantable ascitic (fast growing) r a t hepatomas. Studying the pattern of rat fetal liver, they pointed out that the fetal liver contains more H subunits than the adult liver. Their theory of a fetal pattern in cancerous tissues will be revealed with the aldolase isoaymes. They also found supplementary abnormal subbands, in some of these hepatomas, analogous findings to those of Latner (1964) and of ThQret and LalQgerie (1967) in various tumors. It is interesting to point out that Schapira and de NQchaud found supplementary bands in rat fetal liver, and we recall that chick embryo and fetal guinea pig also contain additional subbands (Croisille, 1964; Fieldhouse and Masters, 1968; Soetens et al., 1965) also noted supernumerary fractions in two brain tumors. Recently Schapira et al. (1972) described in a human rhabdomyosarcoma a shift toward a heart pattern, and compared it to the pattern of the human fetal muscle. In conclusion, the shift toward an M type in cancerous tissues is frequently but not constantly encountered, and the relation between the isozymic modifications and an increase of glycolysis is doubtful. We shall come back to other possible interpretations of alterations in the abnormal distribution of the L D H isoenzymes in malignancy.

B. MALATEDEHYDROGENASE “Simple malate dehydrogenase” (Kun, 1963) (L-malate :NAD oxidoreductase, EC 1.1.1.37) exists in a t lcast two forms: onc mitochondria1

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and the other cytoplasmic (Thorne, 1960). Kinetic and immunologic properties of both forms are different: upon electrophoresis, the supernatant form migrates to the anode and the mitochondrial form migrates to the cathode. Moreover, this last form is more susceptible to oxidation of sulfhydryl groups (Skilleter et al., 1970). Both forms of NAD-malate dehydrogenase (MDH) from mammalian tissues seem to be different proteins. Electrophoresis on starch gel of mitochondrial enzyme allowed Thorne et al. (1963) to separate up to six bands with similar properties, while the cytoplasmic enzyme gave only one band, the properties of which were different. Rabinowitz and Dietz (1967) using leukocyte extracts separated two bands by acrylamide gel electrophoresis and showed that, in chronic and acute leukemia, the fastest band diminished to the benefit of the slowest one. In normal breast tissue, the supernatant form is the most abundant, and the mitochondrial form predominates in breast carcinoma (Hershey et al., 1966). C. ISOCITRATE DEHYDROGENASE There are two isocitrate dehydrogenases with a different cellular localization: one with NAD as coenzyme (threo-D,-isocitrate :NAD oxidoreductase, decarboxylating, EC 1.1.1.41) and the other with NADP (threoD,-isocitrate :NADP oxidoreductase, decarboxylating EC 1.1.1.42). The NAD-linked enzyme is exclusively mitochondrial while the NADP-linked enzyme is found both in mitochondria and in supernatant (for review of properties and distribution, see Criss, 1971). Lowenstein and Smith (1962) demonstrated immunological differences between intra- and extramitochondrial enzymes of rat liver. Hawtrey (1962) showed that mitochondria of rat hepatomas induced by 3'-MeDAB possess both NAD- and NADP-linked isocitrate dehydrogenases and that activity of NAD-linked enzyme was increased. McLean and Brown (1966) found in these hepatomas that NADP-linked isocitrate dehydrogenase was lowered in the cell supernatant. Stein et al. (1967a,b) identified in mitochondria of Ehrlich ascites carcinomas an NAD-specific isocitrate dehydrogenase which was very active, and had regulatory properties. The predominance of the NAD-linked enzyme was seen only in tumor and in brain mitochondria.

D. ALCOHOL DEHYDROGENASE Recently, Bertolotti and Weiss (1972a) found that electrophoretic analysis of alcohol dehydrogenase (alcohol :NAD oxidoreductase, EC 1.1.1.1) in rat hcpatoma cells revealed two bands: one corresponding to normal liver enzyme, and another with faster migration toward the anode.

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They found a band with the same mobility in rat stomach and also in rat fetal liver.

E. ALDEHYDE DEHYDROGENASE A1dehyde:NAD oxidoreductase (EC 1.2.1.3) activity is much increased in rat hepatoma, and several bands are obtained by electrophoresis on polyacrylamide gel, instead of a single band from normal liver (Feinstein and Cameron, 1972).

Ill. Lyases

A. ALDOLASE 1. Generalities We shall restrict this review to class I aldolases. The aldolases of bacteria, yeast, fungi, and blue-green algae (class 11) possess very different properties: in particular they require a divalent metal ion as coenzyme (Rutter, 1965). Fructose-l-6-diphosphate~-glyceraldehyde-3phosphate-lyase (EC 4.1.2.13) and ketose-l-phosphate aldehyde-lyase (EC 4.1.2.7) would be better called, respectively, aldolase A and aldolase B, because each acts on the two substrates, fructose 1,6-diphosphate (FDP) and fructose l-phosphate (F-1-P), although a t different rates (Hers and Jacques, 1953; Hers, 1957; Schapira 1961a,b). Moreover, a third type of aldolase exists in higher animals, aldolase C , which was more recently isolated in brain (Penhoet et al., 1966; Foxwell et al., 1968; Lebherz and Rutter, 1969). The three types have some common properties: molecular weight about 160,000 and ability to react with dihydroxyacetone phosphate to form a Schiff base intermediate (Morse and Horecker, 1968a). A three-subunit model was first proposed for the aldolase molecule, based on the results of acid or urea dissociation (Stellwagen and Schachman, 1962; Deal et al., 1963) and on the characterization of three active sites (Ginsburg and Mehler, 1966). There are three C-terminal tyrosines (Drechsler et al., 1959; Kowalski and Boyer, 1960; Rutter et al., 1961; Winstead and Wold, 1964) and three N-terminal prolines (Sine and Ham, 1967). Chan et al. (1967) proposed a three-subunit model with nonidentical chains; but the sedimentation studies of aldolase dissolved in 6 M guanidine hydrochloride led Kawahara and Tanford (1966) to propose a four-subunit model. The obtaining by Penhoct et al. (1966) of five-membered sets, contain-

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ing three hybrid forms, by dissociation and recombination in vitro of two types of aldolases (A-B) (A-C or B-C) strengthened the hypothesis of a tetrameric molecule. Subsequent work gave further evidence for this structure (Morse et aZ., 1967; Woodfin, 1967; Lai, 1968; Lai and Chen, 1968; Meighen and Schachman, 1970; Eagles et al., 1969). Adelman et al. (1968a) gave an explanation for the failure to detect four substratebinding sites. This tetrameric structure is the active form of the enzyme, as shown by Masters and Wurzor (1971) ; although Chan and Mawer (1972) presented evidence for aldolase activity of monomers, subunit interactions confer stability to the tetrameric form. However, the subunits of the tetrameric molecules are not identical. Susor et al. (1970) resolved the crystalline muscle aldolase into five components by electrofocusing, and proposed that they were the result of a random assembly of two types of subunits which they called a and /I. There would be a deamidation in vivo of an asparaginyl residue during the beginning of life, explaining the age-dependent change in the ratio a:/3 found in rabbit muscle aldolase (Koida et al., 1969; Lai and Horecker, 1970). Aldolase A contains 32 cysteine residues per mole of enzyme (Lai, 1968; Steinman and Richards, 1970; Lai et aE., 1971), some of which play a role in the catalysis (Swenson and Boyer, 1957). Several functional sulfhydryl groups were detected a t the active center (Lai et al., 1968) ; liver aldolase also contains cysteine a t the active center (Horecker, 1970). Aldolases A and B have several distinctive properties ; the most striking one is the relatively high activity of aldolase B toward the second substrate, fructose l-phosphate, which allows liver aldolase to metabolize fructose. The aldolase activity ratio FDP:F-1-P of aldolase B is 1.0, whereas it is higher than 50 for aldolase A. The purification of bovine liver aldolase by Peanasky and Lardy (1958) and then of r a t and rabbit liver aldolase by Rajkumar et al. (1966) have shown that there was only one enzyme in liver with activities toward both substrates. Muscle and liver aldolase are two distinct proteins; the differences displayed toward carboxypeptidase action are associated with the function of the carboxy-terminal residues (Blostein and Rutter, 1963). The enzymatic lesion of hereditary fructose intolerance, which appears to be due to a structural mutation of aldolase B, also indicates that the two types, A and B, are coded by different genes (Hers and Joassin, 1961; Nordmann et al., 1968). Spolter et al. (1965), extensively studied the two types A and B, and showed that muscle aldolasc, but not liver aldolase, is competitively inhibited by ATP. However, many characteristics are the same for the two types. Tetrameric structure, first demonstrated for aldolase A, was also proved for aldolase B (Gracy et al., 1969); and Gurtler and Leuthardt (1970) even found two types of subunits in liver

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aldolase which were analogous to two subunits, a and p, of muscle aldolase. Aldolase C also is a tetramer, as shown by electron micrographs of the three types (Penhoet e t al., 1967, 1969b). The kinetic properties of aldolase C appear to be intermediate between those of aldolase A and B, the aldolase activity ratio FDP:F-1-P being between 5 and 10, according to the species. The main distinctive property of aldolase C is its negative charge, which allows its easy separation by electrophoresis (Foxwell e t al., 1968). Despite their common structure, and the similarity of their active sites (with a lysine residue), the three types have significantly different amino acid compositions and are immunologically distinct. Blostein and Rutter (1963) differentiated liver and muscle aldolase by immunochemical methods. Further studies confirmed that, from an immunological point of view, there is a strict type specificity, but a feeble species specificity. Antimuscle mammalian aldolase antiserum cross-reacts completely with other mammalian aldolases, but not with muscle avian aldolases (Ikehara e t al., 196913). Antibodies prepared against pure aldolase of each type (A4,B,, or C,) also react with hybrids (Penhoet and Rutter, 1971). We shall not review in detail the methods used for isolation and purification of aldolases A, B, and C (Taylor e t al., 1948; Rensing et al., 1967; Morse and Horecker, 1968b; Penhoet e t al., 1969a), nor the techniques for estimation of aldolase activities and isozymic separation (Sibley and Lehninger, 1949a; Schapira, 1960; Blostein and Rutter, 1963; Yenhoet e t al., 1966). The three forms are widely distributed among the tissues of higher animals. Although every species has its own characteristic tissue distribution, nevertheless aldolase A is almost unique in skeletal muscle of all species; aldolase B is always present in liver, kidney, and intestine; and aldolase C is always found in brain. Aldolase A, however, appears to be present in all tissues, even in liver (but in feeble amount in normal adult liver). Aldolase B is restricted to liver (where it is almost unique in mammals), kidney (Wolf and Leuthardt, 1957), and intestine (Schapira, 1961a). Aldolase C has a broad tissue distribution in birds and in ruminants (Herskovits e t al., 1967; Sheedy and Masters, 1969) ; but in several mammals, it is found principally in brain, where it is hybridized with aldolase A (Penhoet et al., 1966). Aldolase C, also exists in some other tissues, namely lens, gonads, spleen, and blood cells (Baron e t al., 1968; Dikow and Genowa, 1969a,b; Hatzfeld and Schapira, 1969; Rutter, 1969; Kochman e t al., 1971). Hybrids of alklolases A and B, or A and C, are found in several tissues where five isozymes are found: three hybrids, and the two pure forms. Recently, hybrids between B and C have been detected also in vivo (Lebherz, 1972).

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The study of ontogenic evolution of the isoenzymic aldolase distribution is of special interest for the modifications in cancerous tissues: Hers and Joassin (1961) and Schapira et al. (1962) pointed out that the substrate specificity of aldolase in adult liver was different from that in fetal liver. Nordmann and Schapira (1966, 1967a,b) showed that fetal liver produced an aldolase with the same properties as muscle aldolase. Furthermore, Schapira et al. (1970, 1971) and Schapira and Nordmann (1969) gave kinetic, electrophoretic, and immunological evidence of the presence, not only of aldolase A, but also of aldolase C in fetal liver. The ontogenic evolution of aldolase in other tissues was less extensively studied. Nevertheless, it was shown that fetal muscle of some mammals contains aldolase C (Schapira et al., 1968), as do also heart, spleen, and lung (Masters, 1968). More generally, all tissues display an ontogenic evolution, except perhaps for brain, for which the transition from aldolase A to hybrids A-C is discussed (Masters, 1968; Kochman et al., 1971; Lebherz and Rutter, 1969). 2. Aldolase Isozymes in Cancer

Warburg and Christian (1943) demonstrated the elevation of aldolase activity in serum of tumor-bearing rats. Sibley and Lehninger (1949a,b), using a more precise method of determination, showed that some patients with cancer exhibited abnormally high serum aldolase activity. Sibley (1958) demonstrated in cancerous rats that the tumor itself was the source of the increased aldolase in serum. Baker and Govan (1953) demonstrated a frequent elevation of serum aldolase activity in patients with prostatic cancer, and its diagnostic and prognostic significance. It is possible to distinguish between muscular and hepatic origin of a hyperaldolasemia according to the FDP:F-1-P ratio, which is related to the two types of aldolase (Schapira, 1961). The aldolase activity ratio is between 5 and 10 in normal adult serum; it is raised above 30 in some muscular diseases, and lowered to nearly 1.0 in hepatitis and in liver glycogen storage diseases (Schapira et al., 1957; Schapira, 1 9 6 1 ~ ) . Schapira and Payet (1960) showed that there is a hyperaldolasemia in the course of primary liver cancer; and that the serum aldolase is not the liver type (the FDP:F-1-P ratio being about 3). They advanced the hypothesis that the serum aldolase of these patients came from the tumor itself, and consequently that the tumor aldolase was not of liver type. Actually, in the cancerous region, they found a higher FDP:F-1-P ratio than in the normal region. The substrate specificity of hepatoma aldolase is different from the normal one, and may be compared to the substrate specificity of the fetal liver: in human fetal liver, the aldolase activity ratio is not 1.0, but

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between 2.0 and 3.0 (Schapira et al., 1962). In a more extensive study, the mean aldolase activity ratio in human hepatomas was found to be 5.5, instead of 1.02 in normal adult liver; and a ratio as high as 30 was found in the experimental ascitic hepatoma of Zajdela. In cancer, the biosynthesis of adult aldolase (and probably also that of some other differentiated enzymes) appears to be repressed, while the embryonic form appears to be derepressed (Schapira et al., 1963).On the other hand, Rutter et al. (1963) found that growing populations of cells contain primarily aldolase A. The aldolase activity ratio was 9 in Novikoff hepatoma, and 110 in Morris 3159 hepatoma. Ashmore e t al. (1963) described in several experimental hepatomas a change from liver-type to muscle-type fructose metabolism; but did not study the enzymatic pattern. The hypothesis that, in hepatoma, aldolase B was partially replaced by aldolase A was proved by the action of ATP (Nordmann and Schapira, 1966; Sugimura et aZ., 1966) and principally by the action of antialdolase A antiserum (Nordmann and Schapira, 1967a,b). By electrophoresis, these authors visualized in rat hepatomas induced by 3‘-MeDAB, an increase of aldolase A (normally present in feeble amount in normal liver) a t the expense of aldolase B. The electrophoretic pattern of fetal liver aldolase was similar to the cancerous aldolase pattern. These findings were confirmed and extended by several other authors. Adelman et al. (1967) contrasted well differentiated hepatomas with an aldolase activity ratio of between 1 and 5 , and poorly differentiated hepatomas with a ratio of about 50. Pietruszko and Baron (1967) using another method, found a similar electrophoretic pattern. Matsushima et al. (1968) obtained electrophoretic evidence for the presence of hybridaldolases A-B in Yoshida ascites hepatoma. Tsunematsu and Shiraishi (1969) and Yandov and Dikow (1970) confirmed and extended the previous data on serum aldolase in patients with primary liver cancer. It is important to point out that Brox et al. (1969) and Gracy et al. (1970) crystallized aldolase from a Novikoff ascites hepatoma. They compared the properties of this cancerous aldolase with those of aldolases of normal rat liver and muscle. Substrate specificity, amino acid composition, tryptic fingerprints and effect of digestion with carboxypeptidase, were identical for muscle and tumor aldolases (and different for normal liver aldolase). Analogous findings were made by Ikehara et al. (1970) on aldolase of a primary hepatoma induced by 3‘-MeDAB. The increase of aldolase A corresponds to an “absolute” increaSe of its amount, as proved by Nordmann (1969) using immunological methods. The liver aldolase of tumorbearing rats is normal (Suzuki et al., 1969). The increase in the level of aldolase A appears in the early stage of hepatocarcinogenesis induced by

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3'-MeDAB and is fixed irreversibly; the isozymic modification is not produced by another azo dye noncarcinogen, 2-MeDAB (Endo et al., 1970). I n order to demonstrate that the increase of aldolase A in hepatoma is related to its predominance in fetal liver, aldolase modifications in other tissues were searched. In mouse spleen the normal ratio of the two aldolase activities was found to be about 9 whereas in cancerous spleen a decrease of this ratio to 4.5 was observed. The isozymic modification was apparently the contrary of that found in cancerous liver; but in both cases, it is the specific, adult type which is lowered-type B in hepatoma, type A in spleen reticulosarcoma-at the expense of the fetal type (Schapira and Tran Ba LOC, 1965; Schapira, 1966). Consequently the increase of aldolase A seems to be due to the derepression of fetal enzymes, and it is not specific for cancerous tissues. We have reviewed some properties of the third type of aldolase, the more recently discovered aldolase C. Schapira and Josipowicz (1970) characterized it by electrophoresis on starch gel in rat fetal liver, and in Zajdela ascites hepatoma. Sugimura et al. (1970) in a strain of Yoshida ascites heptoma showed the presence of a hybrid A,C by electrophoresis on cellulose acetate, and by DEAE-cellulose column chromatography, and Schapira et al. (1970) demonstrated the presence of the three AC hybrids and of the pure tetramer C, in solid, fast growing hepatomas. They compared the pattern of this cancerous aldolase to the fetal one. The presence of aldolase C both in fetal liver and in fast growing hepatomas was definitely proved ,by the action of the specific antialdolase C antiserum (Schapira et al., 1971). Isozymic modifications of aldolase in some other organs were investigated. Sat0 et al. studied the aldolase isozyme pattern in human and experimental brain tumors. The relative amounts of aldolase A and C appear to vary according to the origin of the tumor; aldolase A predominated in meningiomas, while the aldolase pattern of gliomas was the same as in normal brain (Sato et al., 1970, 1971). Ikehara et al. (1969a) found an increase of aldolase A activity in liver of patients with extrahepatic tumors, but the exact significance of these findings and similar ones of Pandov and Dikow (1970) is not clear. Kawabe et al. (1969) purified and crystallized aldolase from a transplantable rat sarcoma and showed that it was identical to aldolase A of normal rat muscle. Schapira et al. (1972) found A-C hybrids in two human rhabdomyosarcomas, and compared this pattern to that of fetal muscle. I n summary, the modifications of aldolase isozymes in hepatoma represent a t the present time the first and the most striking example of the resurgence of fetal molecular forms of enzymes in cancer.

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B. SERINE DEHYDRATASE L-Serine hydro-lyase, deaminating (EC 4.2.1.13) was isolated from rat liver by Selim and Greenberg (1959, 1960), who showed that the preparation displayed a cystathionine-synthesizing activity and an Lthreonine dehydratase activity ; purification was performed by Nagablushanam and Greenberg (1965), and cystathione synthetase activity was further separated (Brown et al., 1966). This enzyme appeared to be inducible by diet and hormones (Suda, 1967). Inoue and Pitot (1970) showed that the crystalline enzyme, the molecular weight of which is 65,000, may be separated into two components by chromatography on DEAE-cellulose and acrylamide gel electrophoresis. The relative amount of the two forms vary under different conditions. The synthesis of the more electropositive form (I) is regulated by glucagon, while the other form (11) is regulated by cortisone. In adrenalectomized rats, all enzyme is in form 11, as with glucagon. With a 90% protein diet, both forms are in approximately the same amount. Moreover, in Morris hepatoma 7800, form I1 decreased to the benefit of form I, and adrenalectomy reduced only partly the relative amount of this form. Synthesis of both forms occurs independently.

IV. Phosphotransferases

A. HEXOKINASE 1. Generalities

The existence of a glucose phosphorylating enzyme in mammalian liver was first postulated by Slein et al. (1950). Several laboratory studies later established that liver contains two enzymes that can phosphorylate glucose, designated as ATP :~-hexose-6-phosphotransferase (EC 2.7.1.1) and ATP :~-glucose-6-phosphotransferase (EC 2.7.1.2). The main advances in this field were performed by the group of Weinhouse (Di Pietro et al., 1962; Lin et al., 1962; Sharma et al., 1963, 1964) and the group of Sols (Vifiuela et al., 1963; Sols et al., 1964) and of Walker (1962, 1963; Walker and Rao, 1963). According to the terminology of Sols, “glucokinase” was distinguished from “hexokinase” mainly on the basis of its different affinity for glucose. Glucokinase is found in liver only, and its K, for glucose is about 10 mM. In contrast, the K , for glucose of hexokinase was found to be about 0.01 mM (Sols et al., 1964) ; moreover hexokinase is inhibited by glucose 6-phosphate. Glucokinase activity decreases in fasting, and virtually

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disappears in diabetic animals, as first shown by D i Pietro and Weinhouse (1960). It reappears on refeeding glucose and by insulin administration, and this reappearance is due to the true synthesis of a new protein (Sharma et al., 1963, 1964). More recently, the presence of three forms of hexokinase with a low K , for glucose was demonstrated in rat liver, and all four enzymatic forms are now generally called “hexokinases” (Schimke and Grossbard, 1968). Gonzalez et al. (1964) separated four fractions by DEAE-cellulose column chromatography. Katzen et al. (1965) using electrophoresis on starch gel showed the presence of four bands in rat liver. Katzen and Schimke (1965) designated the four bands as types I-IV, in order of increasing mobility; Type IV or “high K , glucokinase,” could be detected only in liver, where the other three types were present. The distribution of these three types varies according to the tissue and the age (Katzen and Schimke, 1965; Grossbard and Schimke, 1966; Katzen, 1967). Types I and I1 are widely distributed, especially in intestine and heart. Skeletal muscle contains predominantly type 11. In adult fat pads, the three types are found with predominance of type I (Hanson and Fromm, 1967) ; brain and kidney hexokinase activities consist largely of type I. However, Pilkis and Hansen (1968) and Shatton et al. (1969) showed that kidney also contains type IV. In liver and in kidney, type IV may be resolved into two electrophoretically distinct components which have approximately the same Michaelis constants for glucose but are immunologically distinct. Type I1 also exists in two forms, but only one form may be detected in fat pad by electrophoresis when 8-mercaptoethanol is incorporated in the staining solution (Katzen et al., 1968). I n testes and epididymal fat pad, a new and apparently unique form of hexokinase was characterized by Katzen (1967). This form is absent from immature epididymal fat pad. The electrophoretic migration of the “sperm type” in starch gel is slower than migration of the other types; its Michaelis constant for glucose is about 100 JAM.Type I11 has the lowest K , ( 5 p M ) . It is inhibited by a high glucose concentration. This high concentration must be added to the staining mixture, after starch gel electrophoresis, for the revelation of type IV. Thermostability and the action of inactivating agents also allows a distinction between the four forms. I n human red cells, Eaton et al. (1966) found seven bands with hexokinase activity, but Holmes et al. (1967) characterized only type I and type I11 hexokinases in adult blood. In hemolyzates of newborn infants, and in adults with persistence of fetal hemoglobin, these authors found type I1 hexokinase. But with a more sensitive method, Kaplan and Beutler (1968) were able to show that this hexokinase of cord blood is

a

a supplementary isoenzyme; this band was also found, although in lesser amount, in adult blood. No type I1 hexokinase was found in cord blood, and Kaplan and Beutler (1969) consider that hexokinase type I is duplicated in red cells. From the very start, Walker (1962) showed that glucokinase is present only in mature adult liver. But more recently, one of the two bands of type IV was found in rat fetal liver by Shatton et al. (1969). Type I11 hexokinase appears between day 15 and day 16 of rat fetal life (Sigman et al., 1972). Immunological methods can distinguish the four types. Moreover, Pilkis and Hansen (1968) have shown that antiserum prepared against the faster band of type IV did not inhibit the slower band; the significance of these findings is not clear. Katzen and Schimke (1965) and Walters and McLean (1966) pointed out that all tissues known to be highly sensitive to insulin are rich in type I1 hexokinase (for example, muscle, heart, and epididymal fat pad and lactating mammary gland) ; the fastest band of hexokinase I1 is considerably diminished in tissues of diabetic animals, and it is restored after insulin administration (Katzen, 1967). It has been suggested that there is a thiol-disulfide interchange between the hormone, and a form of the enzyme (Katzen, 1966). Brain and kidney are insensitive to insulin action and contain largely type I hexokinase. We shall not review the relationship between the multiple forms of hexokinase and insuIin action (see Katzen, 1967) ; it is possible that there is a cellular compartmentation of the different forms (Katzen et al., 1970). Rose and Warms (1967) raised the question of the possible role for the regulation of carbohydrate metabolism of mitochondria1 hexokinase ; and Berthillier et al. (1970) showed that there are glucokinases bound to the microsomal fraction. Grossbard et al. (1966) found that the pattern of tissue distribution of hexokinase isozymes is similar in several mammals. In all species studied, the most rapidly migrating form was that which had a high K , for glucose. Brown et al. (1967) characterized four bands in human liver, from I to IV, of which band IV was a “high K,,, glucokinase.” Moreover, they found a supplementary band migrating toward the cathode a t pH 7.4 and with a low Km for glucose. I n heteroploid cell cultures originating in human liver, Katzen et al. (1965) found only two bands with hexokinase activity, but no “high Km glucokinase.” The molecular weight of glucokinase is about 48,000 (Pilkis and Krahl, 1966) whereas that of the three hexokinase types is about 96,000. However, hexokinases are not simply dimers of glucokinase because antiserum against glucokinase fails to inhibit hexokinase activity (Pilkis et al., 1968). At the present time, the most likely hypothesis is that the hexokinase molecule would be an oligomer with dissimilar subunits, such

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as lactic dehydrogenase and aldolasc (Pilkis, 1972). It must be recalled that Kenkare and Colowick (1965) have shown that yeast hexokinase is a tetrameric molecule, the subunits of which can be dissociated and reassociated. Methods of measuring hexokinase activity were described by Crane and Sols (1955) and by Di Pietro and Weinhouse (1960). For the separation of multiple molecular forms, electrophoresis was performed principally on starch gel: to the staining mixture containing glucose (at high or low concentration) ATP, MgCl,, NADP, and glucose-6-phosphate dehydrogenase were added. The substitution of a fluorimetric procedure for the tetrazolium-formazan system of Katzen and Schimke (1965) gave to Kaplan and Beutler (1968) a better isozymic separation. 2. Hexokinase Isozymes in Cancer The studies of isozymic hexokinase modifications in cancer began with the findings of Weinhouse et al. (1963), who showed the lack of glucokinase activity in Morris 5123 hepatoma. These authors were able to correct the glycolysis impairment in this tumor and in several other minimal deviation hepatomas by adding an excess of yeast hexokinase. On the contrary, the Dunning LC 18 tumor, and the Novikoff tumor had I very high glycolysis and were not affected by hexokinase addition. Moreover, Weinhouse et al. pointed out that in these fast growing hepatomas with high hexokinase activity, the K , for glucose was low. This work was subsequently developed ; distinguishing phosphotransferases according to their K , for glucose, Elwood et a2. (1963), Sharma et al. (1964), and Manjeshwar et al. (1965) found that all tumors induced by 3’-MeDAB had lower glucokinase activities, even those which were hepatocarcinomas. On the contrary, hexokinase was generally high. I n transplanted hepatic fast-growing tumors, a high total hexokinase activity was found, contrasting with the low (or even absent) glucokinase activity. The only tumor which had maintained a high glucokinase activity was the slow growing 7787 Morris hepatoma. Whether or not these modifications are related to the type of cells remains questionable. Extensive studies showed that the absence of glucokinase and the increase of hexokinases in liver cancer could not be correlated with their histological type; that is to say, it was not restricted to bile duct cells in transplanted and in primary hepatomas (Sharma et al., 1965). However, the glucokinase level closely followed the degree of differentiation of hepatomas. Farina et al. (1968) pointed out that in the more rapidly growing hepatomas, many enzymes, involved in liver function (for example, glucokinase, aldolase B, and pyruvate kinase liver type) were reduced or lost; and, moreover, that there is an increase of enzymes present in low amount in normal liver (for example, hexokinase). These

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data on the modifications of the hexokinase: glucokinase ratio were confirmed by several authors (McLean and Brown, 1966). Sugimura et al. (1966) found only two molecular species of hexokinases (with K, 10-5M and 10-4M) in ascites hepatoma, instead of three species and a glucokinase in normal liver. Lea et al. (1969) compared these modifications in regenerating liver and in hepatomas with a similar growth rate; they found a lowering of glucokinase and an elevation of hexokinase 48 hours after partial hepatectomy, but these changes are much less marked than in hepatomas. Patterns obtained by chromatography on DEAE-cellulose confirmed these data. Shatton et al. (1969) particularly stressed the comparison with the fetal pattern. I n 20-day-old rat fetal liver, isozymes I, 11, and I11 are preponderant although a very feeble isozyme IV band is present. The reversal toward a fetal pattern of hexokinase isozymes was compared to the fetal pattern of aldolase isozymes. Sat0 et al. (1969) pointed out that in fetal liver type I1 is preponderant as in hepatomas; but these authors, differing with Farina et al. (1968), found an analogous pattern in regenerating liver. From the studies of Lea et al. (1970), it appears that the question of isozymic modifications in regenerating liver remains controversial; but it seems that the decrease in growth rate in hepatomas is not accompanied by the restoration of glucokinase activity; and also that there is not an exact correlation in regenerating liver, between the decrease in “high K , glucokinase” and the increase in “low K, hexokinases.” Knox et al. (1970b) pointed out that, although fetal and regenerating liver grow as fast as the fastest hepatomas, their hexokinase concentration (per gram or per cell) is much lower than in these tumors. Some data on cellular distribution have appeared more recently: Saito and Sat0 (1971) found that a great part of hexokinase activity in Yoshida ascites hepatoma is bound to mitochondria, whereas in normal rat liver hexokinase is principally in the supernatant fraction. This mitochondrial activity is predominantly of type I (in contrast with the finding of predominance of type I1 in hepatomas). On the contrary Bhatty and Hickie (1971) found that the hexokinase distribution in slow growing hepatomas is similar to that of liver, that is to say predominant in the soluble fraction. Recently an analogous isozymic modification has been described in another cancerous tissue: Kikuchi et al. (1972) showed that uterine carcinomas contain type I1 hexokinase, while normal uterus contains only type I. As pointed out by Weinhouse et al. (1972a), poorly differentiated tumors have high hexokinase levels, and consequently a high glycolytic activity. Weinhouse compared this high hexokinase activity with the high activity of the nonhepatic-type pyruvate kinase isozymes

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in fast-growing hepatomas. We shall come back to these modifications and to their general significance. B. PYRUVATE KINASE

1. Generalities Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) catalyzes the transfer of a phosphoryl group from phosphoenol pyruvate to ADP; the reaction is activated by K+ ions. It is widely distributed in lower and in higher animals. Von Fellenberg et al. (1963) described a method for the electrophoretic separation and visualization of pyruvate kinase on agar gel. They showed that the migration of pyruvate kinase of skeletal and heart muscle and of brain rat extracts was different from that of liver extracts ; kidney pyruvate kinase showed an intermediary migration between muscle and liver pyruvate kinase. Cytoplasmic and mitochondria1 pyruvate kinases had an identical electrophoretic migration. Tanaka et al. (1965) presented evidence for the presence of two types of pyruvate kinase (PK) in rat liver, which they called type M and type L. Both types were separated by electrophoresis on starch block; type L is lowered in diabetic and in fasting animals and is restored by insulin treatment; in contrast, M type is almost insensitive to hormonal and dietary regulation. Tanaka et al. (1965) showed that type L is restricted to liver and kidney, while type M is distributed in muscle, brain, and heart, and is present also in liver and kidney. By purifying rat muscle pyruvate kinase and injecting it into rabbits, these authors were able to obtain an antiserum, neutralizing the M type, but not the L type. They also showed that in an ascitic hepatoma, AH 130, the pyruvate kinase was not L type, but M type; we shall come back to these findings. Bigley et al. (1968) using chromatographic, electrophoretic, and immunological methods, separated three isoeymes, which they called I, 11, and 111, from anode to cathode on starch gel electrophoresis a t pH 8.6. Isozyme I, the most anodic, is found in liver, where it is predominant, and in erythrocytes; isozyme 111, which remains near the origin, is found in skeletal and heart muscle, in brain, and in leukocytes. A minute amount of isozyme I11 is also found in liver and in kidney; the main kidney form, isozyme 11, has an intermediate mobility. An antiserum prepared against partially purified red cell pyruvate kinase almost completely inactivated isozyme I, partially isozyme 11,and not a t all isozyme 111. Chromatography on DEAE-cellulose and fractional precipitation with ammonium sulfate also separated these isozymes. In rat tissues also, three forms of pyruvate kinase were described

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(Susor and Rutter, 1968) : antiserum to muscle-type pyruvate kinase was active against skeletal muscle, heart, lung, spleen, intestine, testis, brain, and kidney enzymes, although the electrophoretic migration of muscle and heart enzyme was different ,from that of others. Brain and kidney exhibited two bands ; and electrophoresis of liver extract resulted in two bands, a main isoayme (liver type) with a fast anodic migration, and a minor cationic isoeyme. Liver-type pyruvate kinase exhibits regulatory properties. Hess et (21. (1966) had reported that yeast pyruvate kinase is stimulated by fructose diphosphate (FDP) and that the affinity curve for phosphoenol pyruvate (PEP) was sigmoidal. Taylor and Bailey (1967) and Tanaka et al. (1967) demonstrated the same phenomena for liver pyruvate kinase ; FDP acts as an allosteric activator a t a very low concentration. Moreover, liver-type is inhibited by ATP, by Cu2+,and by several amino acids. In contrast, the muscle and brain pyruvate kinase is inhibited only by phenylalanine (Passeron et al., 1967; Weber and Lea, 1967; Weber, 1969; Rozengurt et al., 1970; De AsGa et al., 1971). Weber et ul. (1967) and Llorente et ul. (1970) emphasized the importance of the regulatory properties of pyruvate kinase for the control of glycolysis and gluconeogenesis. The two types of liver pyruvate kinase have been distinguished according to their kinetic, electrophoretic, and immunological properties (Bailey et al., 1968; De AsGa et uZ., 1971; Hess and Kutzbach, 1971; Marshall and Walker, 1972). The two types were designated as type L (or LA) and type M (or LB). The M type of liver shows no evidence of cooperative interactions with phosphoenol pyruvate, nor activation by fructose diphosphate. Nevertheless, it is not identical to type RI from muscle because it is inhibited by several amino acids. As in the case of muscle pyruvate kinase, the activity of the M type liver pyruvate kinase is unaffected by dietary modifications (Marshall and Walkcr, 1972). The problem of the different molecular forms of liver is very complex. Criss (1969) separated four forms by isoelectrofocusing, with isoelectric points of 5.5, 5.75, 6.00, and 6.75; in skeletal muscle, only one isozymc was found, with an isoelectric point of 6.75. This microheterogeneity was also found by Hess and Kutzbach (1971) in pig liver, and by Susor et al. (1970) in rabbit muscle; and Schapira and Gregori (1971), by electrophoresis on starch gel, distinguished three isozymes in rat fetal liver. Marshall and Walker (1972) showed that the M type has a high activity in rat fetal liver, while the activity of L type is low. The M type decreases progressively during fetal life, at the profit of L type. Pyruvate kinase of adipose tissue is of type L or type M according to

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the species (Leveille, 1969). Nevertheless the tissue distribution of the different types is very similar in higher animals. Steimetz and Deal (1966) found that rabbit muscle pyruvate kinase is a tetramer and advanced the hypothesis that it would consist of two identical protomers, each protomer consisting of two nonidentical polypeptide chains. The tetrameric structure was confirmed by Cottam et al. (1969) and by X-ray crystallographic studies of Mc Pherson and Rich (1972). Interest has been focused on erythrocyte pyruvate kinase since the discovery of inherited deficiencies, with hemolytic anemia (Valentine et al., 1961; Tanaka et al., 1962). By purifying the enzyme, Cartier et al. (1968a,b) were able to distinguish two forms, one with allosteric kinetics and another with regular Michaelis-Menten kinetics: the two forms appeared to be interconvertible. These results were confirmed and extended (Ibsen et al., 1971; Blume et al., 1971; Boivin et al., 1972). Jacobson and Black (1971) demonstrated that one form is equivalent to the T form of Monod et al. (1965) while the other one corresponds to the R form; binding of either PEP or F D P converts the T form into the R form. The leukocyte pyruvate kinase is different from the erythrocyte enzyme (Campos et al., 1965) and is similar to the muscle type (Bigley et aE., 1968). Two interconvertible forms of pyruvate kinase were also found in epididymal fat pad; the fastest migrating form has sigmoid kinetics with PEP as substrate and is activated by FDP, while the slowest one is insensitive to F D P (Pogson, 1968). The electrophoretic migration of erythrocyte pyruvate kinase appears to be nonidentical to that of liver enzyme. Nevertheless, Bigley and Koler (1968) have described a patient with a deficiency of both red cell and liver pyruvate kinase. Kinetic, chromatographic, electrophoretic, and immunological methods were used to distinguish the different types of pyruvate kinase. Activity is generally measured according to Bucher and Pfleiderer (1955), by coupling the reaction with the lactic dehydrogenase system. Electrophoresis, first used by von Fellenberg et al. (1963) was improved by Susor and Rutter (1971). We should like to point out that many problems have not yet been solved, particularly concerning the number of different molecular forms of pyruvate kinase and the relationships between them. 2. Pyruvate Kinuse Isozymes in Cancer

Tanaka et al. (1965) were the first to show modifications of pyruvate kinase isozymes in cancer; they showed that in ascitic hepatoma AH 130, most of the enzyme is of type M. Lo et al. (1968a,b) and Farina et al. (1968) confirmed these findings, comparing pyruvate kinase in rat hepatoma and in normal rat liver.

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Pyruvate kinase activity attained high values in poorly differentiated tumors, while activity of well differentiated hepatomas was lower than that of normal liver. By chromatography on DEAE-cellulose, Lo et al. (1968a,b) separated two forms of the enzyme; form I was not adsorbed; form I1 was adsorbed and then eluted; this last form corresponded to the liver type. Isozyme I1 predominated in normal liver, and in highly differentiated hepatoma. On the contrary in Novikoff hepatoma, and in other poorly differentiated tumors, nearly all the enzyme was isozyme I. However, Taylor et al. (1969) showed that the pyruvate kinase of a fast-growing hepatoma (3924-A) was different not only from the liver enzyme, but also from the muscle enzyme. The authors compared the kinetics, thermostability, the action of FDP and of several effectors in liver, in muscle, and in rapidly growing hepatoma pyruvate kinases. Generally speaking, tumor pyruvate kinase resembled muscle enzyme and did not have regulatory properties. But the inhibition by alanine, ATP, and copper was similar to that of the liver type. Moreover, FDP stabilized the hepatoma pyruvate kinase, not the muscle enzyme. By electrophoresis, tumor enzyme migrated in an intermediate position, and its great instability also distinguished it from both muscle and liver types. Nevertheless, hepatoma pyruvate kinase was not immunologically distinct from muscle pyruvate kinase. Criss (1969) was able to provide new data by isoelectrofocusing. I n the well differentiated Morris hepatoma 9633, the four normal forms were greatly diminished, and a new form with an isoelectric point of 7.28 was revealed. This new form was predominant in the poorly differentiated Novikoff hepatoma. This isozyme was not found in adult or fetal liver, nor in diabetic, fasted, or regenerating liver. From the results of Taylor et al. (1969) and of Criss (1969), it would appear that a new pyruvate kinase isozyme in hepatoma exists principally in poorly differentiated hepatoma. More recently, Weinhouse et al. (1972a) reported that this fifth form differs kinetically from the preponderant muscle and liver forms. Schapira and Gregori (1971) showed that pyruvate kinase isozyme of a poorly differentiated hepatoma (Reuber H 178) resembles placental pyruvate kinase from the point of view of its electrophoretic migration and the action of fructose diphosphate. Moreover, one of the three bands found in fetal liver (day 16) had the same migration as the tumor enzyme. Consequently, it appears that many problems remain ; the partial purification of pyruvate kinase from Yoshida ascites tumor cells performed by Neri et al. (1970) is not yet sufficient to solve them. Another problem was raised by the findings of Suda (1967), who studied the changes of pyruvate kinase isozymes in the liver of Walker tumor-bearing rats. Suda reported that the M type was predominant in these livers;

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parabiotic techniques (Suda et al., 1966) gave results suggesting a “messenger” coming from the cancer cells, flowing into the blood, and modulating the pattern of several enzymes. Obviously, these interesting findings need subsequent development and confirmation. CampadelliFiume et al. (1970) did not find a shift toward the M type in liver of rats bearing the Morris hepatoma 5123, the host liver isozyme remaining subject to dietary regulation. But Yanagi et al. (1971) confirmed the increase in muscle-type pyruvate kinase activity in the liver of tumorbearing animals. They studied mice with Ehrlich ascites tumor, and found that, the isoelectric resolution of pyruvate kinase from livers of tumorbearing mice showed the increase of minor peaks normally found in liver. Work on pyruvate kinase in cancerous tissues was performed almost entirely on hepatomas, but recently, Farron et al. (1972) found that in rhabdomyosarcoma N 5104 carried in Fischer rats the pyruvate kinase pattern was abnormal. Its electrophoretic migration toward the anode on cellulose acetate strips was faster, and was similar to that of fetal muscle pyruvate kinase (day 16). Fetal and cancerous enzymes had the same electrophoretic mobilities as the M-type pyruvate kinase of adult and fetal liver. I n conclusion, many problems remain concerning the relationship of different pyruvate kinase isozymes, and their possible hybridization in vivo; but it seems that in cancerous tissues there is a modification of the isozymic distribution, with an increase or the appearance of forms normally present in other tissues of the same organism, or in the same tissue during fetal life. C. ADENYLATE KINASE

ATP: AMP phosphotransferase (EC 2.7.4.3) was first described in muscle and named “myokinase” and was later found in liver and in crythrocytes ; more recently adenylate kinase was recognized in many animal tissues (for review and general properties, see Noda, 1962). I n human erythrocytes, Fildes and Harris (1966) described a procedure to detect the molecular forms of the enzyme after starch gel electrophoresis. They found two common alleles, each of which may be resolved in two isozymic bands: consequently, three bands are seen by electrophoresis of red cells of heterozygotes. I n rat liver, adenylate kinase was first found in mitochondria (Heldt and Schwalbach, 1967). Adelman et al. (1968a,b,c) showed that there is a high activity in the soluble portion of the cytoplasm, and that this activity is subject to dietary and insulin control, while the mitochondria1 enzyme remains unaffected. Klethi and Mandel (1968) showed by electrophoresis, followed by specific coloration, that there is a specific tissue polymorphism of adenylate kinase, muscle, heart, brain, and erythrocytes displaying a different

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pattern from liver. Khoo and Russell (1972) studied the adenylate kinase isozymes by immunochemical methods in rabbit and human tissues. Two types can also be distinguished, from the degree of inhibition by antimuscle adenylate kinase antiserum: a muscle type, present also in brain and in erythrocytes; and a liver type, present also in testis and in splecn. The electrophoretic pattern and thc inhibition by AgNO, confirmed this distinction. On the other hand, Criss et al. (1970a) separated four forms of thc enzyme by isoelectric focusing in rat liver supernatant; their isoelectric points were: 5.9 (isozyme I ) , 7.0 (isozyme 11),7.6 (isozyme 111), and 8.2 (isozyme IV). The major isozyme was isozyme 111; its activity increased in fasted rats and decrcascd upon refeeding. Criss also studied the subcellular localization of these four forms, and found that isozyme I11 was located in the outer compartment of mitochondria. From the molecular weight, it seemed that isozyme I11 existed as monomer, dimer, or trimer in rapid equilibrium (Criss, 1970; Criss et al., 1970a). I n fetal liver, adenylate kinase activity is low and is distributed in both compartments (Hommes et al., 1971). Criss et al. (1970b) applied their method of isoelectric focusing to the hepatoma adenylate kinase, and compared the elution pattern with that of normal liver. In highly differentiated hepatomas, the pattern was very similar, but in Novikoff hepatoma, they noted a sharp decrease in isozyme 111. The pattern was intermediate in Morris hepatoma 9633 (the differentiation of which was also intermediate). Criss e t al. pointed out that it was the form responsive to diet which was the most diminished in poorly differentiated hepatomas.

D. PHOSPHOFRUCTOKINASE Phosphofructokinase (PFK) (ATP: ~-fructosc-6-phosphate l-phosphotransferase, EC 2.7.1.11) exists in multimolecular forms in mammalian tissues. The enzyme, the molecular weight of which is about 360,000, is composed of several subunits. Lnyacr et al. (19691, who purified the human muscle and the red cell enzymes showed that their properties are different: Erythrocytc PFK migrates faster on electrophoresis and is more inhibited by ATP and less by citrate than muscle PFK. According to Layzcr and Conway (1970) there cxist in human tissues four different isozymes: a muscle, a red cell, and two platelet forms of PFK. Muscle and erythrocyte isoenzymes are immunologically related, whereas the isoenzymes in platelets are unrelated to the muscle enzyme. I n glycogenosis with muscle PFK deficiency, the enzyme level decreases only partially in red blood cells (Tarui et al., 1965) and is normal in leukocytes.

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I n the rat, Taylor and Bew (1970) distinguished two forms by chromatography on DEAE-cellulose. Muscle contained only one form, whereas liver contained preponderantly another form (“liver type”), but also muscle type. Recently, Tsai and Kemp (1972) were able to dissociate and to hybridize in witro rabbit muscle and liver P F K ; cellulose acetate electrophoresis revealed five isoenzymes. Consequently it seems that rabbit muscle and liver P F K were tetramers. On the other hand, Tanaka et al. (1971) separated from rat tissues four peaks with P F K activity by column chromatography numbered in order of elution. Type I was present in muscle, heart, and brain; type I1 in brain, spleen, kidney, and testis; type I11 in spleen, stomach, kidney, and testis; and type I V only in liver. I n ascites hepatoma, Tanaka et al. found not only type IV, but also types I1 and I11 in equal amounts. Immunochemical methods, using antiserum against type I (muscle type), confirmed the distribution of the three main types (types I1 and I11 were not distinguished). Sumi and Ui (1972) distinguished P F K of Ehrlich ascites tumor cells from P F K of other mouse tissues by their behavior toward inhibitors. I n man, Meienhofer et al. (1972) recently found that the PFK of leukemic white cells differs from the P F K of normal white cells; immunological studies showed that the leukemia enzyme was similar to the muscle enzyme. It is possible to assume that in the rat and human as in the rabbit, isoenzymes of phosphofructokinase are heteromers susceptible to hybridization, and that in some cancerous tissues there is a decrease of specific subunits, with appearance of subunits of another type.

E. URIDINEKINASE Two forms of ATP: uridine 5’-phosphotransferase (EC 2.7.1.48) were separated in rat liver by Krystal and Webb (1971). The form with a molecular weight of 120,000 predominated in normal adult r a t liver and in regenerating liver, but a form with a low molecular weight was predominant in fetal liver and was abundant in Novikoff ascites hepatoma.

F. THYMIDINE KINASE Multiple forms of ATP: thymidine 5’-phosphotransferase (EC 2.7.1.21) may be differentiated according to their sensitivity to thymidine 5’-triphosphate (TTP) and deoxycytidine 5’-triphosphate (dCTP) . Bresnick et al. (1964) showed that in normal liver dCTP and TTP inhibited thymidine kinase to about the same extent. I n contrast, dCTP did not inhibit the enzymes of fetal or regenerating liver whereas TTP had a pronounced effect. I n “minimal deviation hepatomas” thymidine kinase reacted toward dlCTP and TTP as adult liver, but in contrast,

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Novikoff and Dunning hepatomas were resistant to dCTP, and sensitive to TTP. Recently, Jones e t al. (1972) partly purified thymidine kinase from human fetal liver and spleen. The pH, thermostability, electrophoretic pattern, and molecular weight, as well as the sensitivity to dCTP were different from that of the adult enzyme. This “fetal form” was found in several cancer tissues, but not in benign tumors; and in fibroblasts transformed by SV,, virus, thymidine kinase changed from the adult to fetal form.

G. CARBAMYL PHOSPHATE SYNTHETASE ATP: carbamate phosphotransferase (EC 2.7.2.5) forms carbamyl phosphate from COz, ATP, and ammonia or glutamine. Carbamyl phosphate is a precursor in urea and pyrimidine synthesis. Carbamyl phosphate synthetase is present in liver and intestine of rat (Hall e t al., 1960; Jones e t al., 1961) and also in hematopoietic mouse spleen (Tatibana and Ito, 1967). There are two forms of carbamyl phosphate synthetase: one utilizes ammonia (type I) and the other one utilizing glutamine (type 11) (Hager and Jones, 1965). Hager and Jones (1967a,b) have shown that adult rat liver contains principally form I (in mitochondria), while fetal liver contains form I1 (in the supernatant) ; the latter type utilizing glutamine is replaced before birth by type I utilizing ammonia. These authors were able to characterize type I1 as the form in Ehrlich ascites carcinoma. Mayfield e t al. (1967), and Yip and Knox (1970) also found that the carbamyl phosphate synthetase of several hepatomas was of type 11, as in fetal liver. Moreover, they found a correlation between the growth rate of the tumors and the increase of carbamyl phosphate type 11. V. Other Transferases

A. ASPARTATE AMINOTRANSFERASE Two forms of L-aspartate :2-oxoglutarate aminotransferase (EC 2.6.1.1) (glutamic-oxaloacetic transaminase) were found in animal tissues (Fleisher e t al., 1960). In rat liver, Boyd (1961) showed that one form was principally mitochondrial, and the other was in the supernatant. Both enzymes differ in substrate affinities, in pH optima, and in electrophoretic mobility (Bodansky et al., 1966). Inhibitors of the two forms are different (Michuda and Martinez-Carrion, 1970). Morino e t al. (1963, 1964) crystallized both isozymes from beef liver and showed that they displayed distinct physicochemical and immunological properties. Otani and Morris (1965) separated the two forms by electrophoresis

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on starch block, followed by elution and measurement of transaminase activity. All types of hepatomas studied possessed the two isozymic components, a “cationic” and an “anionic” one; but the percentage of anionic isozyme seemed higher. Sheid et al. (1965) found that both aspartate aminotransferases were lowered in fast-growing hepatomas ; but generally the supernatant fraction isozyme was relatively higher in hepatomas than in normal adult liver. Nisselbaum and Bodansky (1969) purified the two transaminases from rat liver; and they prepared an antiserum active against the anionic but not against the cationic isozyme. These immunological methods allowed them to compare the transaminases of normal rat liver and of hepatomas, but results varied with the type of tumor. The most striking result was with Morris hepatoma 5123 D, where the percentage of anionic isozyme was considerably increased; the serum transaminase activity (which comes from the supernatant fraction) was forty times that in normal rats. Sheid and Roth (1965) showed that, whereas aspartate aminotransferase was inducible by cortisone in normal and in regenerating liver, it was not inducible in embryonic liver or in hepatomas. We recall the results of Reynolds et al. (1971), who studied the induction of tyrosine aminotransferase by glucocorticoids and glucagon and by high protein diet. They found that the inducibility in hepatomas may be compared to the inducibility in neonatal livers. Nevertheless, in some hepatomas, tyrosine transaminase was very responsive to hormones, while inducibility of glutamic-oxaloacetic transaminase was lost. Aspartate aminotransferase isozymes (GOT) were also studied in senim of patients with neoplastic diseases. Bodansky et al. (1966) showed that the serum GOT level was often raised, and that electrophoresis of serum resolved transaminase activity into two zones instead of only one in normal serum. A mitochondrial, cationic component was found in addition to the normal anionic supernatant component. However, this pattern is not specific: Schmidt et al. (1967) showed the same appearance of the mitochondrial GOT in the serum of patients with acute hepatitis.

B. BRANCHED-CHAIN AMINOACIDTRANSFERASES Two types of transaminases with 2-oxoglutarate for branched-chain amino acids (leucine, and also valine and isoleucine) (EC 2.6.1.e) were isolated from rat liver and purified by Aki et al. (1968). Type I predominates in the supernatant while type I1 predominates in the mitochondria; but the two types were found in both fractions. While type I was active for the three branched-chain amino acids, type I1 was active only for leucine. Type I is widely distributed in various rat tissues, while type I1 is found only in liver. From pig heart, Taylor and Jenkins (1966)

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isolated and purified type I. An antiserum prepared against this type also inhibited type I from rat liver, but not type 11. Ichihara and Takahashi (1968) showed that only type I is found in fetal rat liver. Type I1 appears after birth an’d is inducible by cortisone and by diet (Ichihara et al., 1967). I n pig brain, Aki et al. (1969) found another type (type 111) slightly different from type I. Ogawa et al. (1970; Ogawa and Ichihara, 1972) studied the distribution of the three types in rat tissues; type 111 similar to pig brain enzyme was found only in brain; type I1 seemed specific for liver; type I was widely distributed, and was present in skeletal muscle, heart, kidney, and spleen and also in normal liver. Principally, Ogawa et al. showed that in Yoshida ascites hepatoma (AH 130) type I1 had disappeared, and instead these authors characterized type I11 and type I, in the supernatant fraction. hloreover they purified this type I11 from the ascites tumor, and showed that its kinetic, electrophoretic and immunological properties were identical to those of a component of rat brain transaminase. In primary tumors induced by feeding 3’-MeDAB, type I11 appeared also, while type I1 disappeared; type I1 also tended to decrease in nontumorous parts of the liver. Consequently, branched-chain amino acid transaminases provide a new example of a qualitative alteration of the isozymic pattern in cancer cells. It must be stressed that normal adult liver contains no type 111; but this type may be found in another tissue of the same species, the brain. Moreover, in fetal liver, the activity of type I is high, while that of type I1 is very low. Hepatoma seems to reproduce the same pattern ; nevertheless, Ogawa and Ichihara (1972) have not found type I11 in fetal liver.

C. GLYCOGEN SYNTHETASE UDPglucose: glycogen a-4-glucosyltransferase (EC 2.4.1.11) exists in two forms in mammalian muscle and liver. One form depends for activity on glucose 6-phosphat6 (D form) and the other is independent (I form) (Leloir and Cardini, 1962; Villar-Palasi et al., 1966). The two forms have not yet been completely separated. D and I forms are interconvertible, not only in muscle, but also in liver, presumably by phosphorylation and dephosphorylation. Glucose and cyclic-AMP are antagonistic effectors (Hers et al., 1970). Thermal stability and sensitivity to sulfhydryl inhibitors differ in muscle and in liver (Steiner et al., 1965). In the pmsence of physiological concentrations of inorganic phosphate, liver glycogen synthetase D is almost inactive, even with high levels of glucose 6-phosphate (De Wulf et al., 1968; De Wulf and Hers, 1968). K. Sat0 et al. (1972a,b) have partly purified the D and I forms of

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muscle and liver glycogen synthetases, and also of two ascites hepatomas. The effect of maleate was similar to that of inorganic phosphate, and in Tris-maleate buffer, the D form in liver is active only a t pH levels above 8.0, even in the presence of glucose 6-phosphate. I n contrast, muscle glycogen synthetase activity is almost independent of p H between 7.0 and 9.0. Moreover, these authors found that hepatoma glycogen synthetases displayed the same pH activity curves of the D form as the muscle enzyme, in the presence of glucose 6-phosphate. The apparent K,, value for UDPglucose of the D and I forms was identical for the muscle and the cancer enzyme, and different from that of liver glycogen synthetase, in which the apparent I<,,, is 20 times greater for the D form than for the I form. K. Sat0 et al. (1972a,b) pointed out that although the two ascites hepatomas studied (AH 66 F and AH 130) differed markedly in glycogen content, nevertheless their glycogen synthetases were both of the same muscle type. Glycogen synthetase yields another example of a liver type of enzyme replaced by a muscle type in hepatoma.

D. PHOSPHORYLASES a-1,4-Glucan:orthophosphate glucosyltransferase (EC 2.4.1.1) plays an important part in the regulation of glycogen metabolism. I n mammalian muscle, phosphorylase exists in two interconvertible forms : phosphorylase a, active; and phosphorylase b, inactive. Phosphorylase b is converted into active phosphorylase a by a phosphorylase kinase, and the reverse reaction is catalyzed by phosphorylase phosphatase. AMP activates both forms of the enzyme in muscle, but in liver phosphorylase b is almost insensitive to AMP. Liver and muscle enzymes are synthesized by two different genes, as shown by studies of enzymatic deficiencies: muscular glycogenosis, or McArdle disease, is due to a lack of muscle phosphorylase activity (Schmid and Mahler, 1959), and some forms of hepatic glycogenosis are accompanied by a partial deficiency in liver phosphorylase (Hers, 1959). Almost all the liver phosphorylase activity is found in the particulate fraction, but in ascites hepatomas, an important part has been found in the supernatant by Takeda et al. (1967). Moreover, these authors found that the tumor enzyme was much more activated by AMP than the liver enzyme. Nigam (1967) made similar observations on Novikoff hepatoma. Lea et al. (1972) pointed out that, even in the most rapidly growing hepatomas, the activation of phosphorylase by AMP was very inferior to the activation of muscle phosphorylase, although it is more important than that .of normal liver enzyme. Moreover the effect of cystcine would be different, and would not support the hypothesis of the replacement of a liver type enzyme by a muscle type one.

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However K. Sato et al. (1972~)recently found by kinetic methods and by isoelectric focusing that phosphorylases of poorly differentiated hepatomas possess properties of the muscle enzyme. E. DNA POLYMERASE

DNA nuclcotidyltransferase (dcoxynucleoside triphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7.) was first purified by Kornberg et al. (1959) from extracts of Escherichia coli; it was shown that the composition of the DNA formed is determined by the nature of the “primer.” Partially degraded and heated DNA is a more efficient “primer” than undenatured DNA (Imsande and Handler, 1961). DNA polymerase purified from calf thymus required denatured DNA (Yoneda and Bollum, 1965). Ove et al. (1969, 1970) showed that rat hepatomas have increased DNA polymerase activity when compared with normal liver. Two peaks of enzyme activity were separated on Sephadex column chromato,graphy. I n normal and regenerating rat liver, there was a predominance of peak I1 containing fractions acting with native DNA as primer. I n transplantable hepatomas, peak I was preponderant in proportion to the tumor growth rate ; it contained enzyme preferentially using denatured DNA. It is interesting to point out that, during regeneration, the increased marked DNA polymerase activity resembled that of normal liver and differed from that of hepatomas. Ove et al. (1970) also found that DNA polymcrasc of rat fetal liver preferentially used denatured DNA. Peak I activity was very high 6 days before birth (even higher than in hepatomas) and then decreased and was barely detectable a t birth, when its level was comparable to those in minimal deviation hepatomas. Peak I1 was lower than peak I in fetal liver. The findings of Iwamura et al. (1968) were similar. I n regenerating liver, results were divergent (de Recondo et al., 1964; Mantsavinos and Munson, 1966; Ono and Umehara, 1968). However, Roychondhury and Bloch (1969) showed that preferential utilization of denatured-DNA as primer by DNA polymerase from Ehrlich ascites cells depended on the stage of purification. Consequently, the exact importance of the differences between the DNA polymerases of adult, fetal, regenerating, and cancerous liver is not yet known.

F. TRANSFER RNA METHYLASES The tRNA methylases are a group of enzymes catalyzing the transfer of the methyl group from S-adenosyl-L-methionine to accepting sites on tRNA. Differences in extent of methylation between different tissues may reflect differences in their tRNA methylases (Riddick and Gallo, 1970). All these enzymes are species specific. Srinivasan and Borek (1964) were the first to suggest that tRNA methylases could be important in car-

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cinogenesis; in fact, increased tRNA methylase activity was found in several experimental and human tumors, first by Bergquist and Matthews (1962) and then by Tsutui et al. (1966). Baguley and Staehelin (1968) and Hacker and Mandel (1969) , and in transformed cell cultures (Gallagher et al., 1971) (for discussion, see Magee, 1971). Riddick and Gallo (1970) demonstrated a correlation between the growth rate of the tumors and the increase of tRNA methylase activity. Moreover, they showed that this activity seemed to be related to the percentage of immature cells. Fetal liver of mouse and rabbit displays high levels of tRNA methylase activity; as does rat fetal brain (Simon et al., 1967). On the other hand, Nau et al. (1969) showed that there is a different substrate specificity for the tRNA methylases of mouse liver and of mouse myeloma. Kerr (1970, 1971) demonstrated that normal tissues have an inhibitor of tRNA methylases and that the high molecular weight component of this inhibitor is absent in embryonic and tumor tissues. Consequently, it is possible that differences between normal methylases and fetal and tumor methylases are not really related to isozymic differences. Nevertheless, more recently Buch et al. (1972) showed that nicotinamide and its analogs were able to inhibit tRNA methylase activity in several tumors and were without effect on the tRNA methylase activity of normal tissues.

VI.

Hydrolases

A. INTRODUCTION Alkaline and acid phosphatases (orthophosphoric monoester phosphohydrolases; respectively, EC 3.1.3.1 and EC 3.1.3.2) are generally considered to be separate enzymes. We shall discuss the isozymes of both alkaline and acid phosphatases. The mammalian enzymes are often called “nonspecific phosphatases” to distinguish them from enzymes with more definite substrates (for example, glucose-6-phosphatase) . Since the work of Robison (1923) on bone phosphatase, a considerable number of publications was devoted to the tissue phosphatases (for review, see Roche, 1950).

B. ALKALINE PHOSPHATASES 1. Generalities

It was recognized for many years that “nonspecific” alkaline phosphatases are widely distributed in animal tissues. These hydrolases may act on many substrates; their pH optimum is between 9.2 and 9.6. Com-

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parison between activities toward different substrates allow phosphatases from different tissues to be distinguished. P-glycerophosphate was first used as substrate a t pH 7.4 by Kay (1930) and a t pH 8.6 by Bodansky (1933). Phenyl phosphate was introduced by King and Armstrong (1934) and p-nitrophenyl phosphate by Ohmori (1937) and King and Delory (1939). Phenolphthalein phosphate and p- and a-naphthyl phosphate (Babson and Read, 1959), are also frequently used. Ahmed and King (1960a,b) demonstrated that purified alkaline phosphatase of human placenta hydrolyzes 8-glycerophosphate and phenyl phosphate a t nearly the same rate, whereas the kidney and intestinal enzymes are much more active toward phenyl phosphate than 8-glycerophosphate. Nisselbaum et al. (1961) compared the activity of several human tissues with P-glycerophosphate and with several other substrates. For example, the relative activity of the enzyme from bone, liver, kidney, and spleen toward p-nitrophenyl phosphate was about twice that of the intestinal phosphatase. Conversely, the latter was more active against adenosine 5’-phosphate. Moss et al. (1967) showed that liver and intestinal phosphatases act also on pyrophosphates ; the pyrophosphatase activity of human placental enzyme was demonstrated by Sussman and Laga (1968). The action of effectors was extensively studied. According to Bodansky (1930, 1937), there is inhibition by several amino acids and bile salts; and Mg2+ is a potent activator (Ahmed et al., 1959), except for placental phosphatase. EDTA inactivates the phosphatases, except the placental enzymes (Conyers et al., 1966), and zinc and cobalt appear to be essential for activity (Engstrom, 1961a,b), especially for placental phosphatases (Harkness, 1968b). Fishman et al. (1962) studied 130 compounds, listed many inhibitors, and demonstrated distinctions between intestinal and liver alkaline phosphatases. L- ( + ) -Phenylalanine inhibits the intestinal enzyme stereo-specifically and uncompetitively. Electrophoretic methods has provided other ways of separating multiple forms of phosphatases. Moss and King (1962) submitted extracts of human bone, liver, kidney, and intestine to starch gel electrophoresis, and then recovered by elution the separated fractions. They found several fractions in each tissue; the major bands of liver and bone migrated more slowly than bands of kidney and intestine. Moreover the K, for P-naphthyl phosphate was lower in liver than in the other tissues. Minor bands of phosphatase activity displayed the same K, values, pH optimum, and thermostability as the major bands of the same tissue. Intestinal phosphatases have been resolved into two components by DEAEcellulose column chromatography : which differ in thermostability (Moss, 1964).

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The visualization of zones of phosphatase activity after starch gel electrophoresis allowed a better distinction between different tissue enzymes (Estborn, 1959; Boyer, 1961; Hodson e t a)., 1962). At pH 8.6, human liver is resolved in two bands, the major band of which is slightly faster than that of bone extract. Intestine and kidney show three bandsa minor, slow-moving band and two major bands, one similar to that of bone, and another faster (see Moss, 1970). Incubation with neuraminidase before electrophoresis accentuated the differences in migration between the different types (Robinson and Pierce, 1964) ; neuraminidase was almost without effect on purified intestinal alkaline phosphatase, while it retarded the two bands of liver and bone extracts (Moss et al., 1966). The effect was the same with crude and purified extracts of placental phosphatase. Preincubation with neuraminidase also converts the electrophoretic pattern of human kidney alkaline phosphatase into a single zone. Immunochemical methods provide important data. Schlamowitz (1953) prepared an antiserum against alkaline phosphatase from dog intestine, and showed the presence of two phosphatase components in the enzyme preparation. Later Schlamowitz (1958) also prepared specific antiserum against human intestinal and bone phosphatases, and showed the absence of any cross reactions between the two types of antibodies. These antisera also displayed a high degree of species specificity. Boyer (1961) prepared an antihuman placental phosphatase antiserum and an antihuman bone phosphatase antiserum in rabbit. Four classes of human phosphatases have been distinguished by these various methods : bone and kidney, liver, intestine, and placenta (Sussman et al., 1968; Moss, 1970). According to some authors, there is only one type of alkaline phosphatase within a single tissue, and the heterogeneity observed by submitting a tissue extract to electrophoresis might result from artifacts (Moss, 1969). Nisselbaum et al. (1961) showed the influence of the mode of preparation of intestine and kidney phosphatases on their chromatographic behavior. I n part, the heterogeneity of several phosphatases might be due to a variable content of sialic acid residues (Moss et al., 1966; Bettane et al., 1972). L. Fishman et al. (1971) separated by precipitation with ammonium sulfate two fractions in human liver alkaline phosphatase, with different electrophoretic properties. Fishman e t al. (1972) found analogous results with human intestinal alkaline phosphatases. It must be pointed out that, as for many enzymatic molecular forms, alkaline phosphatase from one tissue more closely resembles the phosphatase of another species originating from the ,same tissue than it resembles another

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tissue from the same species (Moss, 1969). Ontogeny of alkaline phosphatase has been studied by Moog et al. (see 1969 review) in the small intestine of both mice and rats. They showed that duodenum phosphatase is heterogeneous a t all stages, and that there is a “cross reacting material” during the first stages. It seems that there is an inactive precursor, perhaps activated by cortical hormones. The importance of the study of properties of placental phosphatase for cancerology must be emphasized. Ahmed and King (1960a,b) purified the enzyme. Harkness (1968a,b) found its molecular weight to be 125,000. Ghosh (1969) focused his work on the multiple molecular forms of human placental phosphatase. Two major forms were separated on Sephadex G-200; although they differ in molecular weight, their kinetic reactions were identical, so that form B might be a molecular aggregate. Form A was obtained in a homogeneous condition and the amino acid composition of this sialoglycoenzyme was determined. Ghosh (1969) compared the properties of human placental and intestinal alkaline phosphatases. Both are inhibited to the same extent by L-phenylalanine, and by sulfhydryl reagents, but optimum pH was more alkaline for placental enzyme, and K , was much higher. Principally, placental alkaline phosphatase is neuraminidase sensitive, whereas intestinal phosphatase is resistant. Neuraminidase treatment slows the migration of intestinal phosphatase, but not that of placental phosphatases; that this effect is due to the release of sialic acid from the molecule has been shown by Ghosh et al. (1967). A very important property of human placental phosphatase is its thermostability (Moss and King, 1962) (Moss, 1970) and its resistance to denaturation by urea. Placental alkaline phosphatases of other species are not so thermostable. Placental phosphatase is completely stable a t 55”C, while half-inactivation of intestinal phosphatase occurs in 60 minutes. Both enzymes are antigenically distinct, but partially cross react. We shall not discuss in detail the genetic polymorphism of placental phosphatase. It was first described by Boyer (1961), who showed that in serum of pregnant women, two zones with alkaline phosphatase activity are observed by starch gel electrophoresis. These fast anodic bands appear between week 15 and week 28 of pregnancy, and are of placental origin; they represent about half of the total serum alkaline phosphatase activity of pregnant women (Posen et al., 1969). Depending on the subjects, one or another or both components are present; this presence is under genetic control and three phenotypes may be distinguished. Robson and Harris (1965) were able to distinguish six common phenotypes, and to show that they are determined by three alleles a t a single locus. More recently, Beratis et al. (1970) separated six components whose molecular size and charge were different, by chromatographic fractionation. Kitchener et al.

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(1965) showed that the fetal serum phosphatase is not of placental origin. In placenta, it is possible to distinguish not only allelic variants, but also several forms in the same tissue. Lo and Kellen (1971) lshowed that removal of zinc by chelation dissociated placental alkaline phosphatase into two subcomponents, onc of which is active in the absence of zinc and magnesium ions. The subcomponent with smaller molecular weight did not react with the specific antiserum prepared in rabbits against purified human placental alkaline phosphatase. It seems, consequently, that the human placental enzyme does exist in true multiple molecular forms. We recall that alkaline phosphatase from E. coli is a dimeric molecule (Schlesinger, 1965, 1967; Schlesinger and Barrett, 1965) and that EDTA inhibits dimerization. The most important differential properties of placental phosphatases are summarized in Table I. Serum alkaline phosphatase has been extensively studied for several decades, and the problem of its tissue origin has raised many discussions (for reviews, see Sarles, 1954; Wilkinson, 1970a) and is not completely solved. Schlamovitz and Bodansky (1959) proposed immunological arguments for a bone origin of the serum enzyme, but electrophoretic studies brought evidence for a predominant liver origin of serum alkaline phosphatase in normal adults (Hodson et al., 1962). Results vary according t o the authors and the electrophoretic method used. It seems that the fastest band is constant and is of liver origin. Two slower bands are seen; one probably from bone, and another from intestine, as shown by the inhibiting action of L-phenylalanine and the resistance to the action of neuraminidase (Robinson and Pierce, 1964). By electrophoresis on starch gel a slow band is occasionally found; its presence depends on the A B 0

TABLE I PROPERTIES OF PLACENTAL ALKALINEPHOSPH ATASFS Property Thermosensitivity Effect of Lphenylalanine Effect of neuraminidase Immunochemical properties

Remarks Stability when heated 30 minutes at 56°C (in contrast to the three other types) Stereospecific inhibition of the uneompetitive type (different from bone and liver type; similar to intestinal type) Electrophoretic mobility retarded (different from the intestinal type; similar to the other types) Partial cross reaction with intestinal type; antigenically distinct from other types

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blood groups and on the A B H secretor or nonsecretor status, and also on diet (Arfors et al., 1963) (Langman et al., 1966). Yong (1966) showed that in children serum alkaline phosphatase originates not only in liver, but also in intestine. Moreover, it is known that bone phosphatase is an important component of the serum enzyme in children as shown by the combination of study of thcrmostability with clectrophoretic methods (Warnock, 1966). In pathological sera, hyperphosphatasemia comes from multiple sources, originating in liver, bone and intestine. But the problem is not entirely solved; for example in Paget’s disease, elevation of alkaline phosphatase is probably due to the liver enzyme (Sussman, 1970) as shown by immunological methods. In hypophosphatasemia, serum alkaline phosphatase values of patients and of their parents are below normal (Fraser et al., 1955). Much diversity of results is due to multiplicity of methods of measuring alkaline phosphatase activity and of differentiating its isozymes. The multiplicity of possible substrates and various inhibitors, may also contribute to the diversity of the results of the electrophoretic techniques (see Wieme, 1965; Wilkinson, 1970a). 2. Alkaline Phosphatase Isozymes in Cancer

For many years, serum alkaline phosphatase was investigated in the course of cancer of several tissues, bone included. Its activity is increased principally in primary bone tumors, osteogenic sarcoma, and in metastasis to bone, principally carcinoma of prostate or parathyroid, with osteoblastic metastatic lesions. Serum alkaline phosphatase is also raised in liver metastases. However, various methods were used and it is not easy to apply them to the problem of the tissue origin of this elevated serum activity (for review, see Schwartz et al., 1969). Staining techniques after starch gel electrophoresis allowed Latner ( 1965) to demonstrate different mobilities for liver and bone alkaline phosphatases. Schlamowitz and Bodansky (1959) showed that antiserum against bone phosphatase precipitates serum phosphatase of patients with bone metastases. Fennelly et al. (1969) found that in the hyperphosphatasemia of liver origin, thermost,ability at 56OC was greater than in controls or in patients with bone diseases; by incubating during 20 minutes, Fennelly et al. (1969) found a more marked thermostability in cancer patients with hepatic metastases than in nonmalignant hepatic diseases. Urea inhibition gave similar although less striking results. However, the findings of Sussman (1970) on the liver origin of hyperphosphatasemia in Paget’s disease shows the complexity of the problem of tissue origin. A most important discovery concerning alkaline phosphatase isoenzymes in cancer was made by Fishman et al. (1968a,b). These authors

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characterized an alkaline phosphatase which was undistinguishable from placental enzyme in serum and tumor of a patient with bronchogenic carcinoma. This type of phosphatase was named “Regan” after the name of the patient and was extensively investigated (W. H. Fishman et al., 1968a,b, 1971; Fishman, 1969; Stolbach et al., 1969). Both enzymes were sensitive to L-phenylalanine, unaffected by heating during 16 minutes a t 55°C and 5 minutes a t 65”C, and were very active against phenyl phosphate a t pH 10.7. Electrophoresis on starch gel, using sodium a-naphthyl phosphate a t this pH, and performed on heated serum, revealed a “placental band.” Action of neuraminidase, K , optimum pH, and inhibition by p-hydroxymercuribenzoate were similar. Immunological techniques brought definitive evidence. Antisera to placental and Regan isoenzymes were identical when tested by the Ouchterlony double diffusion technique ; the titration of the antigens gave the same loss in activity for both isoenzymes titrated against both antisera. Moreover, by adding antibodies to placental or Regan isoenzymes before electrophoresis, these isoenzymes failed to migrate and consequently can be easily recognized. Fishman was able to demonstrate that Regan isozyme is identical to the variant “B” of placental isozyme. It has been found in the serum of patients with cancer of various organs: lungs, gastrointestinal tract, genital tract. The exact frequency of its occurrence in cancer is not yet determined, but seems to be about 10%. Ovarian and pancreatic carcinoma have the highest incidence. During therapy, Regan isoenzyme progressively disappears (Stolbach et al., 1969) ; but Nathanson and Fishman (1971) found the Regan isoenzyme in the serum of some patients without cancer-principally during the course of ulcerative colitis, familial polyposis of the colon, and some types of cirrhosis. These findings are explainable on the basis that the appearance of this isoenzyme is due to a nonspecific derepression of the genome. W. H. Fishman et al. (1971) consider the Regan isoenzyme as a carcinoplacental antigen, by analogy with the carcinoembryonic antigens. Moreover, they pointed out that, although the fetus does not produce this isoenzyme, the placenta is of embryonic origin. Other authors have made similar findings. Elson and Cox (1969) compared the placental alkaline phosphatase with the enzyme produced by HeLa cells, after partial purification. Molecular weight and kinetic properties were similar. Electrophoresis showed a triple-band pattern for the HeLa isozyme as well as for the placental isozyme with the phenotype “FS,” although with slower migration. But immunodiffusion of both phosphatases using an antibody against HeLa cell enzyme showed that there are additional antigenic sites on tk;is last form. From the electrophoretic pattern, Elson and Cox asserted that both alkaline phosphatases are dimeric enzymes. Beckman

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et al. (1970) confirmed these findings, but identified the electrophoretic pattern of HeLa cell phosphatase with the phenotype S (the most common one among Negroes). Tan and Aw (1971) found some differences in thermostability between the two purified enzymes. It must be noted that the alkaline phosphatase activity of HeLa cells is inducible by glucocorticoids (Griffin and Cox, 1966). On the other hand, Warnock and Reisman (1969) have described an alkaline phosphatase, the properties of which resembled those of the placental form. It was found in extracts of tumors in 8 out of 10 patients with hepatocellular cancer; and the sera of 3 of 5 patients showed the same isozyme. It seems likely that the isozyme described by Warnock and Reisman, and that described by Fishman are of the same molecular species. However, recently Higashino et al. (1972) found in patients with hepatocellular carcinoma, a variant alkaline phosphatase, the properties of which were slightly different from those of placental isoenzyme. Research on experimental tumors is not very abundant. In virus-induced leukemia of mice, there is a marked increase in the alkaline phosphatase activity of liver, with a change in its properties: the proportion of Mg2+independent phosphatase becomes much greater (Cory and Whitford, 1971). Neumann et al. (1971) found the rate of hydrolysis of p-nitrophenylphosphate is increased in thymus of mice with lymphoid leukemia. Recently, Bettane et al. (1972) pointed out the multiplicity of the alkaline phosphatase patterns in the microsomal fraction of murine plasmacytomas. They had an antigenic relationship with the liver and spleen enzymes, but whether these types are different from the normal type or not was not specified. In conclusion, the study of alkaline phosphatase isozymes may be useful for the diagnosis of bone or liver cancers. From a practical as well as a theoretical point of view, major interest resides in the placental type found in cancerous sera and tissues.

C. ACIDPHOSPHATASES 1. Generalities These phosphomonoesterases are almost as widespread as alkaline phosphatases. Their pH optimum varies from 5.0 to 6.0 according to their origin, and also the substrate. Substrates for alkaline phosphatases are also substrates for acid phosphatases: P-glycerophosphate, phenylphosphate, p-nitrophenyl phosphate, phenol phthalein phosphate, and a- or P-naphthyl phosphate (Babson et al., 1959; King, 1965) depending on the tissue origin. Acid phosphatases are found in most tissues, and in blood plasma. Abul-Fad1 and King (1947, 1948, 1949) first differentiated acid phosphatases of red cells and of prostate by their behavior toward

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some inhibitors. Formaldehyde inhibits the activity of red cell enzyme, but not that of prostate, whereas tartrate has the reverse effect. I n fact, the development of studies with various methods has stressed the complexity of the problem. The widespread distribution of these hydrolases, and the lack of specificity of their substrates, renders the classification of their molecular species very difficult; moreover, there are not only isoenzymatic forms, but also allelic forms. Methods used to distinguish them were chromatography on DEAE-cellulose columns (Moore and Angeletti, 1961) and on Sephadex (Di Pietro and Zengerle, 1967; Hcinrikson, 1969) ; electrophoresis on starch gel (Estborn, 1959; Hopkinson et al., 1963; Lundin and Allison, 1966) and on acrylamide gel (Allen and Gockerman, 1964). Specific staining after electrophoresis was performed using a- or p-naphthy1 phosphate, and diazonium salts as coupling reagents. These techniques were combined with the use of different substrates; in a single tissue, several bands with phosphatase activity may be found, and each band displays different activity and affinity for the different substrates. Thermal inactivation, and principally action of inhibitors (fluoride, tartrate, formaldehyde and Cuz+) were commonly used to distinguish the different forms. Acid phosphatases are principally lysosomal enzymes (de Duve e t al., 1955) but may be found also in other cellular fractions (Neil and Horner, 1964) with different properties (Nelson, 1966). Treatment of homogenates by Triton X-100 increases markedly the activity and results in the appearance of supplementary bands (Allen and Gockerman, 1964). Several acid phosphatases with different substrate specificities may have different cellular distributions (Vaes and Jacques, 1965). On the other hand, the heterogeneity within one tissue seems to be related to a cellular heterogeneity, as shown by the study of acid phosphatase isozymes in leukocytes (Li et al., 1970). A great number of bands, up to 17, have been detected by starch gel electrophoresis in some human and animal tissues (Sur et al., 1962; Lundin and Allison, 1966). Beckman and Beckman (1967) characterized four components (A,B,C,D) in different human organs, and also in human cell cultures, component C seeming specific for the placenta, where component A is lacking. The component A is found most frequently in kidney and probably contains sialic acid. All four components are inhibited by L- ( f ) -tartrate, and all react with antiserum to liver acid phosphatase. The relationship between these forms is not perfectly clear. Some variants have been found for placental acid phosphatase, but genetically determined variants were principally described in red cells. Four forms also were separated in rat testicular tissue (Vanha-Pertulla, 1971), with very different substrate specificities ;

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but form IV was tartrate-resistant and seems similar to the erythrocyte enzyme. Hopkinson et al. (1963) and Harris et al. (1968) recognized six phenotypes, determined by three alleles. The erythrocyte acid phosphatases are distinct from prostatic acid phosphatase: erythrocyte enzyme is inhibited by formaldehyde and not by L-tartrate, and the reverse is true for prostatic enzyme. Moreover, the erythrocyte phosphatase is inhibited below p H 4.8, but is slightly activated a t a more alkaline pH (Abul-Fad1 and King, 1948). Prostatic acid phosphatase is also “copper-resistant.” These properties may be reflected in serum, and the determination of tartrate-labile prostatic acid phosphatase in serum is now a routine test (King and Jegatheesan, 1959). But the source of acid phosphatase normally present in serum is unknown; Zucker and Borelli (1958) suggest it comes from platelets. De Grouchy (1958) described a technique of revealing acid phosphatase activity after electrophoresis and found only one zone of acid phosphatase activity in normal sera. But, Dubbs et al. (1960) later characterized three bands. Rozenszajn et al. (1968) showed that the three bands separated in serum by disc electrophoresis on polyacrylamide gel correspond to the bands found in spleen, liver, and lymph nodes. In prostate, two additional isoenzymes were found (migrating after albumin). Inhibition tests with formaldehyde and with L-tartrate and use of various substrates allow the recognition of the tissue origin of phosphatasemia (see King, 1965). 2. Acid Phosphatase Isozyrnes in Cancer

Gutman and Gutman (1938) showed that serum acid phosphatase activity is increased in patients with carcinoma of the prostate with metastases. These findings were later made specific: elevation was found in 282 of 349 cases with bone metastases, and only in 42 of 218 without metastases (Bodansky, 1960). The importance of distinguishing the prostatic origin of an hyperphosphatasemia is now well recognized. Angeletti et al. (1961) fractionated acid phosphatase of normal serum into two components by chromatography on DEAE-cellulose columns. I n serum from patients with prostatic carcinoma, the second fraction was more abundant ; it corresponded to the localization of acid phosphatase in prostatic chromatograms. Action of tartrate confirmed the origin of this second peak. Moreover Angeletti et al. (1961) showed that the enzymatic distribution in serum of patients with benign hyperplasia of the prostate was similar to that of normal serum. By starch gel electrophoresis, a supplementary band was characterized in serum of patients with prostatic carcinoma (Grundig et al., 1965). Rozenszajn et al. (1968)

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also, sporadically found this heat-labile band, but Goldberg et al. (1966) found only a single zone. The prostatic enzyme is formaldehyde-stable, copper-resistant, and tartrate-labile. P-Glycerophosphate is a good substrate for this phosphatase (Schwartz et al., 1969), especially to distinguish elevated activity due to prostatic carcinoma from that due to Gaucher’s disease. Nevertheless, a-naphthyl phosphate was used by Babson et al. (1959), who estimated the ratio for the activities of prostatic to erythrocytic acid phosphatase with different substrates. In the course of carcinoma of prostate with metastasis, the level of serum enzyme does not reflect exactly the state of disease (Schwartz e t al., 1963). In cancerous tissues, isozymes of acid phosphatase were less extensively studied. Moore and Angeletti (1961) compared the distribution of acid phosphatase peaks obtained by chromatography on DEAE-cellulose of normal rat liver and those with hepatoma, and of normal mouse epithelium and those with squamous cell carcinoma. In both cases, tumors had a greater proportion of the basic peak. Li et al. (1970) studied acid phosphatase in normal and leukemic human leukocytes and separated isozymes by acrylamide gel electrophoresis and by ion exchange chromatography. In normal leukocytes, four bands with acid phosphatase activity were recognized. In chronic lymphocytic leukemia, there was a great predominance of the band with intermediate mobility ; the predominant band had a slightly different mobility in acute leukemia from blast cells ; and in leukemic reticuloendotheliosis, another isoenzyme appeared, which was tartrate-resistant (Yam et al., 1971). The tumor cells of spleen and bone marrow were submitted to histochemical investigation and showed strong tartrate-resistant acid phosphatase activity (Katayama et al., 1972). On the other hand, acid phosphatase activity in lymphosarcoma was weak and tartrate-labile. The authors believe the different properties of acid phosphatase may represent an approach to differentiate leukemic reticuloendotheliosis and lymphosarcoma. A tartrate-resistant isoenzyme was also found in lymph nodes of a patient with Hodgkin’s disease (Katayama et al., 1972). The ectopic production of this isoenzyme is not yet explained and may be of great interest. Maggi and Franks (1968), studying some mouse tumors, showed that well differentiated breast adenocarcinomas, and slow-growing hepatomas possess a molybdate insensitive acid phosphatase, as do normal adult mice. On the contrary, in undifferentiated fast-growing tumors, a high molybdate-sensitive acid phosphatase activity was found, as in the tissues of animals 1-3 weeks old (Maggi et al., 1967). This review has shown that many problems concerning isoenzymes of acid phosphatase remain. It would be necessary to purify the different

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fractions with acid phosphatase activity in order to understand their physiological and pathological significance.

D. F'RUCTOSE-1 ,6-DIPHOSPHATASE 1. Generalities A specific hexose diphosphatase (~-frutose-1,6-diphosphatel-phosphohydrolase, EC 3.1.3.11) was discovered in rabbit liver by Gomori (1943), and its essential role in gluconeogenesis was shown. This enzyme, termed fructose diphosphatase (FDPase) , was purified from rabbit liver; its molecular weight (Pontremoli et al., 1965) is about 127,000, and it is easily dissociated into two subunits by exposure to pH 2.0. The enzyme seems to be composed of four nonidentical polypeptide chains (Pontremoli et al., 1966; Sia et al., 1969). Adenosine 5'-monophosphate (AMP) is a noncompetitive allosteric inhibitor (Taketa and Pogell, 1965). FDPase activity of rat liver is under hormonal control (Weber et al., 1965). FDPase occurs mainly in liver and kidney, but significant FDPase activity may be found in skeletal muscle (Salas et al., 1964), where it is also inhibited by AMP (Opie and Newsholme, 1967). Immunological studies provided evidence for the identity of liver and kidney enzymes, while muscle FDPase appeared to be a distinct enzyme (Enser et al., 1969) ; some biochemical properties also are different (Fernando et al., 1969). I n fetal liver, the regulatory properties of FDPase are very similar to those of adult liver FDPase (Wallace and Newsholme, 1967). 2. Fructose Diphosphatase Isozymes in Cancer Sat0 and Tsuiki (1968) found a relatively high FDPase activity in extracts of mouse Ehrlich ascites carcinoma, and they partially purified the tumor enzyme. They studied the inhibition of FDPase activity in mouse tissues by AMP, and showed that the tumor enzyme displayed, just as did the muscle enzyme, a very high sensitivity to AMP, 50% AMP for muscle or tumor FDPase inhibition requiring only 1.0-2.0 activity and about 130 p.M AMP for mouse liver FDPase (at p H 7.4). These observations were extended to rat tissues and hepatomas (Sato and Tsuiki, 1969). Two types of FDPase were recognized from the sensitivity to AMP inhibition: one type, with Ki for AMP in a range of 100-200 f l exists in liver and kidney: this Iiver-type is sensitive to hormonal control; another type, with a very low Ki,was present in the muscle of several species, and was found (with low activity) in rapidly growing hepatomas. In slow growing hepatomas, FDPase activity was liver type. Consequently, although fetal liver FDPase seems to be similar to the adult liver enzyme, fructose-1,6-diphosphataseoffers another

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example of isozyme modifications in hepatomas characterized by the replacement of the liver-type enzyme by a muscle type.

E. GLUTAMINASE 1. Generalities Errera (1949) showed that two different enzyme systems catalyze the deamidation of glutamine in rat liver: glutaminase I is bound to insoluble particles, and hydrolyzes glutamine more actively in the presence of phosphate; glutaminase I1 resides in the soluble fraction and is activated by pyruvate (L-glutamine amidohydrolase, EC 3.5.1.2). Kvamme et al. (1965) and Katunuma et al. (1966) found that this enzyme exists in two forms in kidney; one form requires phosphate (Phosphate-dependent glutaminase: “PDG”) ; the other is activated in vitro by maleate (phosphate-independent glutaminase “PIG”). Phosphate-independent glutaminase is found in various tissues (kidney, liver, brain, and placenta) and is activated in v i m by N-acetylamino acids (Katumuma et al., 1967). The pH optimum and the thermostability of both isozymes varied from tissue to tissue. Katunuma et al. (1968) found that kinetics of affinity of liver PIG for glutamine was allosteric and becomes normal in the presence of N-acetylglutamate. Horowitz and Knox (1968) compared the properties of phosphate-independent glutaminase in adult rat liver and in kidney. They showed that both enzymes differ in pH optima, affinity for glutamine and for phosphate, and action of effectors. Liver type (glutaminase L) with a great affinity for phosphate was found only in adult and regenerating liver. Kidney type (glutaminase K) was found in many tissues, mainly kidney, brain, small intestine and also fetal liver. The two isozymes are mitochondrial. I n kidney as in liver, glutaminase activity without phosphate was very low when glutaminases were purified. It appears to be the endogenous phosphate which acts as activator in crude preparations. I n spite of these uncertainties, the different authors have shown that fetal liver contains a glutaminase which is similar to kidney enzyme: it is resistant to heat and to p-chloromercurihenzoate action but immunological properties are identical. 2. Glutaminase Isozymes and Cancer Katunuma et al. (1968) pointed out that while regenerating liver contains only liver-type glutaminase, AH-130 hepatoma also contains the kidney-type, phosphate-independent glutaminase. Horowitz and Knox (1968) also found kidney type glutaminase in Novikoff and LC 18 hepatoma and in fetal liver; but these authors found activities were phosphate-dependent. I n a more extensive study, Linder-Horowitz et al.

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(1969) compared the type of glutaminase and the growth rate for several transplantable hepatomas. I n all hepatomas, glutaminase was for the most part phosphate dependent and was of the kidney type, except for the slowest-growing tumor. Type was recognized from the K, for phosphate and the p H optimum. But Katunuma et al. (1969) found no direct correlation between the kidney type and the degree of dedifferentiation. Knox et al. (1970a) also studied mammary carcinomas. They found a high correlation between their growth rate and the glutaminase concentration and they pointed out that glutaminase activity was undetectable in normal mammary gland. They concluded that this presence marked a qualitative difference between normal and neoplastic tissue.

F. ESTERASES Multiple forms of esterases have not been fully characterized and have generally wide substrate specificities. Their classification remains controversial. For differences between cholinesterases (EC 3.1.1.8), arylesterases (EC 3.1.1.2), and aliesterases (EC 3.1.1.1), see Augustinsson (1961). Few experiments were devoted to esterase modifications in cancerous tissues. Yoshimura (1971) compared the pattern after electrophoresis in polyacrylamide gel in normal mouse skin, and in skin carcinoma. During carcinogenesis, the slowest migrating component “Ia” increases, a t the expense of the two others (I1and 111).It seems that band I a is an arylesterase. Bartholomew et al. (1969) conlpared diploid and SV40 transformed human lung cell cultures. They found that a cathodic isoenzyme of the diploid line, resistant to eserine, was very diminished in the transformed cell lines. However, Tyndall e t al. (1971) comparing the electrophoretic patterns of normal, leukemic and fetal mouse thymus and spleen, showed that the esterases of the prealbumin region were those which were decreased in leukemic and fetal tissues, a t the benefit of the esterases of the post albumin zone. VII. Summary and Conclusions

Before concluding, we present a table summarizing the main distinctive isozymic modifications occurring in serum of cancerous patients (Table 11). It is not possible to prepare a similar table with tissue isozymic modifications because of the considerable variations occurring with the tissue origin of the tumor. This review is necessarily inc‘omplete. The body of data on the isozymic modifications in cancer is continually increasing. Moreover, we have noted that the definition itself was revised (and probably will be revised again) so that some authors have described modifications in

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TABLE I1 ISOZYMIC MODIFICATIONS IN SERVM OF CANCEROUS PATIENTS Enzyme

Origin of cancer

Isozymic modifications in serum

Increase of isozymes 3 to 5 Increase of FDP activity with ratio FDP/F-1-P about 3 Bone (primary and Thermosensitive, stable to L-phenylalrlr nine. Retarded at electrophoresis by bone metastasis) neuraminidase Intermediate for thennostability, stable Liver (metastasis) to L-phenylalanine. Retarded at electrophoresis by neuraminidase Various organs (prin- Thermostable; inhibited by L-phenylcipally ovaries and alanine, urea, and EDTA. Retarded pancreas) a t electrophoresis by neuraminidase. Inhibited by antiserum antiplacental alkaline phosphatase Prostate (with Activity increased with 0-glycerophosmetastasis) phate or a-naphthylphosphate as substrates. Tartrate labile, formaldehyde reaistant

Lactic dehydrogenase Various organs Aldolase Hepatoma Alkaline phosphatase

Acid phosphatase

cancer of enzymatic forms which are perhaps not true isoenzymes. Nevertheless, we shall try to draw some general conclusions, taking into account the most recent reviews on this subject. The first idea which arises is that there is in fact a correlation between the growth rate of the tumors, and the degree of isozymic abnormalities. Weber and Lea (1967) advanced the “molecular correlation concept,” which included more generally the antagonistic pattern of enzymes involved in the synthesis and in the catabolism of carbohydrate and nucleic acid metabolisms; but also the degree of isozymic changes. But it seems that this concept is not sufficient to explain what kind of isozymic changes occur. If we recall one of the main hypotheses on the enzymatic changes in cancer, we see that Warburg’s theory was not well sustained by the modifications of molecular forms found in cancerous tissues. The shift toward a muscle-type of lactic dehydrogenase isozymes is not always a shift toward an increased glycolysis; and in some instances, cancerous tissues may exhibit a shift toward the heart type. Moreover, many enzymes with modified molecular forms in cancer, are without direct relationship to glycolytic metabolism. The “deletion hypothesis” is not directly concerned with the question of isozymes. It may be noted that, if some isoiymes disappear almost completely, or even completely, during

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the cancerous process, some others appear, which were not present in the normal organ. Greenstein’s conception, that all neoplastic tissues tend to have the same enzymatic pattern is not entirely confirmed by the isozymic study ; we have seen that, on the contrary, different patterns may be found in various tissues. For example, lactic dehydrogenase shows a shift to an M type in cancers of digestive tract and in many other cancers, but an increase of the H subunits is found in some hepatomas, and in leukemic leukocytes. Aldolase “A” is increased in hepatomas, but decreased in reticulosarcoma. Pierce (1970) put forward the idea that what has been interpreted as dedifferentiation “is in reality an abortive attempt a t differentiation by the neoplastic stem cells.” Nevertheless, Greenstein’s idea that some degree of “dedifferentiation” occurs in cancer is verified to a certain extent by the study of isozymes, in the sense that there are often the most differentiated molecular forms which disappear from cancerous tissues. For example, as pointed out by Weinhouse (1970, 1972) , in rapidly growing hepatomas, there is replacement of several liver-type isozymes by nonhepatic types. Weinhouse noted the loss of such hepatic “marker” isozymes as glucokinase, aldolase B, pyruvate kinase type L, and also adenylate kinase 111. It is also possible to add the type I1 of branched-chain amino acid transaminases. Still more important is the appearance in cancerous organs (especially in liver) of molecular forms totally or almost totally absent from the normal adult organ. Two kinds of abnormal isozymes may be recognized. 1. Resurgence of Fetal Forms of Enzymes We cite principally the resurgence, in fast growing hepatomas, of aldolases A and C, the kidney type of glutaminases, and type I1 of hexokinases. Also, alcohol dehydrogenase and principally phosphorylases may be added. Alkaline phosphatase, lactic dehydrogenase, pyruvate kinase, uridine, and thymidine kinases offer other somewhat less demonstrative examples. But mainly, the comparison has to be made with the presence of fetal antigens in some cancer cells. Abelev (1963, 1965) and his group (Abelev et al., 1963) showed that serum of mice with hepatomas contained an embryo-specific a-globulin. These findings were extended to man (Uriel et al., 1967; Tatarinov, 1964) and this protein was called a-fetoprotein. a-Fetoprotein is produced in hepatocellular carcinoma, and there is evidence from in vivo and in vitro experiments for its synthesis by the tumor itself (Goussev et al., 1971; Rioche et al., 1970). It seems that liver perivascular parenchyma cells are the site of this synthesis (Abelev, 1968), but only a few cells of the tumor (Goussev et al., 1971) are in-

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volved. These data are especially interesting for comparison with the synthesis of fetal-type enzymes in hepatomas (for review on a-fetoprotein see Abelev (1971a ; Masseyeff, 1972). Another fetal antigen was also found, occurring in gastrointestinal tumors, called carcinoembryonic antigen (Gold and Freedman, 1965 ; Von Kleist and Burtin, 1969). It must also be recalled that, fetal liver is generally deficient in the same enzymes as are fast-growing hepatomas (for detailed review, see Knox, 1972). 2. Resurgence of Molecular Forms of Enzgmes Present in Other Tissues We recall that “muscle type” of several enzymes appear in hepatomas: for example this type of fructose diphosphatase, glycogen synthetase, phosphofructokinase isozymes I1 and 111, normally found in several organs but not in liver, may be found in some hepatomas. It must be noted that the “fetal type” in liver is often an “adult type” in other tissues: for example muscle and brain for aldolases, kidney for glutaminase, brain for branched-chain amino acid transaminases. All enzymatic molecular forms which appear abnormally in cmcerous tissues have always been found in some tissues of the same organism-either in another organ, or in the same organ during fetal life. A truly specific cancer form of enzyme has apparently not yet been found. This fact is especially striking because the properties of the molecular forms of enzymes are generally species specific. The molecular form abnormally found in cancerous tissue always has the characteristics of its species. These data give important arguments for an epigenetic change in cancer. 3. Discussion

The notion of “fetal antigens” is, in fact, very general, and the “Regan isoenzyme” may also be considered as of fetal origin. .On the other hand, the molecular forms of enzymes possess antigenic properties, and consequently their fetaI forms which are recurrent in cancer are also “fetal antigens’’ from this point of view. We recall more generally the “neoantigens” of the cancer cells (Math6 et aZ., 1968). Alexander (1972) suggested that the ‘lonco-foetal antigens” may only be “one aspect of a more general phenomenon, namely the synthesis of aberrant proteins of a wide variety”; the production of ACTH by some lung carcinomas, and more generally the “para endocrine cancer syndromes,” are well known (Waldenstrom, 1970; Goodall, 1969). Katunuma et aZ. (1972) have recently shown that the mode of amino nitrogen excretion in rat ascites hepatonia was more similar to that of chicken than to that of rat normal liver. Katunuma et al. (1972) concluded that “abnormal gene expressions

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may exist in some hepatomas as arising not only from ontogenic aspect but also from phylogenic aspect.” It must also be stressed that the resurgence of isozymes, or antigens, or other proteins is not a specific phenomenon in cancer. Using a high-sensitivity immunoautoradiography technique, Abelev (1971a,b) was able to find a-fetoprotein in the sera of patients with viral hepatitis. Other “transitory cell antigens,” especially a P-fetoprotein in man and an a,-fetoprotein in rat, can reappear in adult organisms in some nonneoplastic, as well as in neoplastic, states (Uriel, 1971). Obviously liver regeneration is one of these states. From the start of the studies on fetal antigens in cancer, it was shown that a-fetoprotein synthesis may be induced by partial hepatectomy (Abelev, 1965), and we have reported several examples of isozymic modifications in regenerating liver. Nevertheless, it is important to recall that there is no parallelism between cellular multiplication and the production of fetal proteins. For example, the resurgence of aldolase “C” in some hepatomas is not seen in regenerating liver (Weber and Schapira, 1972), and the production of a-fetoprotein always remains very weak (Stanislawski-Birencwajg, 1965). Indeed, cell proliferation is not sufficient to account for the synthesis of recurrent proteins. I n very different processes-for example, in muscular diseases-it is possible to find a “fetal-type” pattern of several enzymes, similar to that found in rhabdomyosarcoma (Schapira et al., 1966, 1968, 1972). Schapira et al. had set forth in 1963 the hypothesis that the synthesis of the adult forms of enzymes was more or less repressed in cancer, while that of embryonic forms would be derepressed. It was added that it would be the most specific and preponderant molecular form which would be repressed, to the benefit of the less specific one (Schapira, 1966). Potter (1969, 1970) expressed an analogous idea, although more restrictive, by his formula “oncogeny as blocked ontogeny.” Ontogeny is “a time sequence of switching certain genes from of to on” and switching others from on to of, for transcription and translation. I n oncogeny (blocked ontogeny), the switch on and off of certain genes would be reversed; cells would have a resistance toward various normal feedback controls. Matsushima et al. (1968) expressed this conception as a “disdifferentiation.” Uriel (1969) supposed that “retrodiff erentiation” is a physiological or pathological potentiality of the mature cell, as differentiation is a physiological potentiality of the stem cell. But, as pointed out by Quelin et al. (1972) for the synthesis of a-fetoprotein, it would be possible that these fetal forms are synthetized by a few stem cells (subsisting in the adult tissue), not by the differentiated cells. More generally, Markert (1968) postulatcs that cancer is a disease of

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differentiation. S. Sat0 et al. (1972) have recently found that it is possible by repeated transplantations, to “reactivate” hexokinlase type I11 in a slowly growing substrain derived from a fast-growing tumor without this isozyme. To these very interesting findings, it is possible to compare the work of Peterson and Weiss (1972). These authors have shown that the mouse fibroblast genome can be induced to produce mouse albumin by hybridization with rat hepatoma cells. By the same technique of hybridization, Weiss and Chaplain (1971) and Bertolotti and Weiss (1972b) have shown that when specific chromosomes are lost, some differentiated functions (such as tyrosine aminotransferase inducibility and synthesis of aldolase B) may reappear. These different experiments clearly demonstrate that cells retain genetic information for their nonexpressed functions. There would be “a disordered mechanism” of gene activation in cancer (Weinhouse et al., 1972a). Repression of genes which code for liver-type isozymes seems to precede derepression of genes which code for other isozymes. But the concepts of “repression” and “derepression” probably do not apply perfectly to eukaryotes. As stressed by Dreyfus (1969), the existence of regulator genes is not demonstrated in higher animals, and there is no real proof of a negative type of control mechanism analogous to the control mechanisms discovered in microorganisms by Jacob and Monod (1961). As suggested by Weinhouse (1970) the translational phase rather than the transcriptional phase might be involved. Pierce (1970) among several hypotheses on the role of viral DNA in malignancy, advanced that viral DNA may cause synthesis of a product, the effect of which would be mediated via the cytoplasm, and would create an imbalance of cytoplasmic controls, secondarily altering nuclear expression. It is obviously impossible to draw definite conclusions from this increasing accumulation of information and discussions, and we would simply stress that, if the knowledge of isozymic forms has a t first brought very useful data for clinical purposes, it now appears that it may bring a new approach to the understanding of carcinogenesis a t the molecular level. Addendum Imamura and Tanaka (1972) have classified rat tissues into three groups according to the distribution of their pyruvate kinase isosymes. Muscle and brain are of M1 type, while liver posseases L type as the major component. Spme adult tissues, such as spleen, lung, ovary, and testis, and principally fetal tissues, contain M2 type, which is also the minor component of the adult liver. M2 type is the main component of several hepatomas, and also of regenerating liver. Chromatography on DEAE cellulose results in three separate peaks in rat fetal liver, the major one of which resembles the form of poorly differentiated hepatomas (Weinhouse, lW2b). Sudn ct nl. (1972) showed that pyruvate kinnse M2

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type was increased in liver of tumor-bearing animals, and that this increase was due to a “de novo” synthesis of the enzyme. K. Sat0 et al. (1973)) extending their previous findings (1972~))showed that phosphorylase isozymes may be separated in rat timues by isoelectric focusing. Muscle phosphorylase b has an isoelectric point at 6.1, while liver phosphorylase focuses at 5.9. The Novikoff hepatoma phosphorylase, like the phosphorylase of 2 l d a y fetal liver, has an isoelectric point a t 5.6. Lack of activation by sulfate ions also allows us to distinguish Novikoff hepatoma and fetal liver forms from the adult liver form. Antiserum antimuscle phosphorylase inactivates only partly hepatoma phosphorylasc; tumor isosyme differed both from muscle and from liver isozyme. The same authors, studying glycogen synthetases, brought new evidence for the replacement of liver type by muscle type in poorly differentiated hepatomas, particularly by comparing active and inactive forms of the enzyme.

ACKNOWLEDGMENTS The author wishes to acknowledge support by a grant from the “Ligue Nationale Francaise contre le Cancer.‘’ Thc valuable assistance of Miss I r b e Jczioiski is decply appreciated.

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PHYSIOLOGICAL AND BIOCHEMICAL REVIEWS OF SEX DIFFERENCES AND CARCINOGENESIS WITH PARTICULAR REFERENCE TO THE LIVER Yee Chu Tohl Sub-Department of Endocrine Pathology, The University of Liverpool, The Liverpool Clinic, Liverpool, United Kingdom

I. Sex Differences in Physiological Functions

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A. Hypothalamus B. Adenohypophysis C. Adrenal Gland D. ThyroidGIand. E. Growth Hormone . . . . . F. Miscellaneous . . . . . . 11. Sex Differences in Biochemical Functions A. Enzymes . . . . . . . B. Proteins . . . . . . . C. Lipids . . . . . . . . D. Nucleic Acids . . . . . . 111. Sex Differences in Carcinogenesis . . A. Hepatic Tumors . . . . . B. Extrahepatic Tumors . . . . IV. General Discussion References . . . . . . .

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Cancer is a broad biological problem. It concerns not only pathology, but also physiology, biochemistry, genetics, immunology, and virology. It is hoped that experimental work on animals will lead us to understand the causes and growth of the disease and ultimately to control human neoplasia. One of the problems is the curious distribution of tumors between the two sexes, and the reason for it is not clear. In the case of the primary and secondary sex organs, this difference is not surprising in view of the great differences in anatomical structures, physiological functions, and susceptibilities to tumor development between men and women. Therefore, the higher incidence of the ovary and uterine cancer as compared with the testis and prostate cancer could be attributed to the greater cyclic stimulation of these reproductive organs in females than in males. As to the breast, which is a rudimentary accessory organ in men, Present address: Department of Physiology, Faculty of Medicine, University of Singapore, Singapore. 155

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the higher incidence of breast cancer in women is therefore not obscure. But it is not a t all obvious why in most other organs men should develop cancers more frequently than women (the evidence for this generalization and a discussion of exceptions, will be given later). This problem must be solved if a full understanding of the process of carcinogenesis is to be achieved. The subsequent sections of this review attempt to give a general view on sex differences, and their relationship to carcinogenesis, particularly in the liver, will be discussed later. 1. Sex Differences in Physiological Functions

The functions of the body are controlled and regulated by the nervous system together with the hormonal or endocrine system. The hypothalamic-hypophysial-gonadalsystem matures in different ways between males and females, and the hormonal imbalance produced early in the undifferential period of reproductive development could permanently not only alter the normal reproductive neuroendocrine relationship, but also affect the functions of adrenal and thyroid, sexual behavior, skin transplantation, metabolizing systems in the liver, and sensitivity toward carcinogens.

A. HYPOTHALAMUS The sex difference in the hypothalamus and perhaps other parts of the brain could easily be observed and measured by differences in the control of pituitary-target functions, such as the different sexual pattern of gonadotropic secretion and its effect on various components of sexual behavior, the sex difference in adrenal cortical secretion and its response to ACTH, the difference in thyroid function between males and females, and the sex difference in the growth and its relationship to somatotropic hormone-all of which will be described below in detail. But the most direct evidence concerning sex differences in the hypothalamus is the anatomical, biochemical, and pharmacological differences in the hypothalamus itself. 1. Physioanatornical Sex Differences of the Hypothalamus Changes in nuclear size as an indication of the functional activity of the hypothalamic neuron (Leveque, 1953; Edstrom, 1957; Hyddn, 1959) following hormonal alteration either by castration or administration of sex hormones have been used as a criterion to study the interkelationship between endocrine glands and the hypothalamus (Szent6gothai et al., 1968). Ifft (1964) considered that a n increase in the nuclear size was an indication of increased activity of the hypothalamic neuron. I n fact, the heavier weight and greater cross-sectional overall area of the brain of

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male rats compared with that of females have been found to be primarily due to the difference in cell size instead of the difference in the number of neurons (Pfaff, 1966). a. Sex Differences. In prepubertal rats, the nuclear volumes in the medial preoptic area of the hypothalamus were greater in males than in females (Docke and Smollich, 1968). Hyyppa (1969), however, did not find such a sex difference in the ontogenetic development of rat hypothalamic nuclei. Therefore, the sexual differentiation of hypothalamic neurons must take place during the neonatal period. I n fact, the results of castration of male rats on the first day of life showed that the brains of males and females had a similar morphology (i.e., nuclear size) a t this time (Pfaff, 1966; Dorner and Staudt, 1968, 1969). On the other hand, if testes were removed after the critical sexual differentiation period of the hypothalamus (14 days of life in rats), a masculine characteristic of nuclear size was still retained (Dorner and Staudt, 1968, 1969). b. Estrogenization. Administration of estrogen caused the cellular degeneration of supraoptic and paraventricular nuclei of the rat hypothalamus (Garcia and Ferreira, 1951, 1953). Lisk and Newlon (1963) reported that nucleoli of arcuate and mammillary neurons were significantly smaller when estradiol was implanted into those areas of the hypothalamus of male and female rats. Neonatal injections of estrone to female rats caused a decrease of nuclear size in various parts of the hypothalamus (Arai and Kusama, 1967, 1968; Arai, 1969), and the nuclei were atrophied. This neurosecretory degeneration was found to be in proportion to the quantities of estrogen administered (Garcia and Ferreira, 1953). c. Androgenization. Androgen treatment in adult intact male rats, however, did not cause significant enlargement of nuclear size in the preoptic anterior hypothalamus (Dorner and Staudt, 1968), but it did increase the nuclear volume of preoptic and ventromedial nuclei of male rats when castrated on day 1 but not on day 14 of life, and this structural change of neonatal castrated rats could be prevented by a single injection of testosterone on day 3 of life (Dorner and Staudt, 1968, 1969). The effect of androgen in adult female rats when spayed postpubertally was greater than in similarly treated males. Docke and Smollich (1968) also reported that neonatal administration of testosterone to female rats whether ovariectomized or not, significantly increased the nuclear size in the medial preoptic nucleus of the hypothalamus. 2. Physiobiochemical Sex Differences of the Hypothalamus

The secretions of adenohypophysial hormones are controlled by the polypeptide substances released from the neurosecretory cells of the hypothalamus and travel by means of the portal blood vessels to the

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anterior pituitary causing the secretion of the corresponding tropic hormone, that in turn stimulates its target endocrine gland to release another hormone. These polypeptide substances are called releasing factors (Harris and Reed, 1966; McCann and Porter, 1969). Changes of the hypothalamic oxidative metabolism would influence the synthesis of releasing factors, because the oxidative metabolism in the central nervous system is one of the main sources of high-energy compounds involved in the peptide-synthesizing system. In fact, similar cyclic changes of the oxygen uptake and the activity of succinic dehydrogenase were found in the hypothalamus, hypophysis, uterus, and ovary of female rats with the lowest value during diestrus, and the highest one during estrus (Moguilevsky and Malinow, 1964; Moguilevsky et al., 1964b). In prepubertal rats, males had a higher oxygen uptake compared with females in the anterior hypothalamic area (Moguilevsky et al., 1969b) whereas in adult animals, proestrous and estrous females showed a greater value of oxygen consumption than that of males in anterior as well as in posterior hypothalamic regions (Moguilevsky et al., 1968). There were no sex differences of oxygen uptake in the middle hypothalamus which was also the only part of hypothalamus that did not show cyclic changes of oxygen consumption (Moguilevsky, 1965). Gonadectomy of adult male rats seduced oxygen uptake in anterior and posterior hypothalamus (Moguilevsky, 1966; Moguilevsky et al., 1966) and administration of testosterone restored the level (Gordan and Elliott, 1947; Eisenberg et al., 1949; Moguilevsky, 1966; Moguilevsky et al., 1966). Moguilevsky et al. (1969a) also demonstrated that castration decreased and injection of testosterone recovered the cytochrome oxidase activity in the hypothalamus of adult male rats. The hypothalamic oxidative metabolism increased during sexual maturation in male rats and prepubertal castration stopped the rise (Moguilevsky et al., 1964a). Neonatal injection of testosterone to female rats produced a similar level of oxygen uptake to that of males (Moguilevsky and Rubinstein, 1967; Moguilevsky et al., 196913; Libertun et al., 1969). Orchiectomy at birth decreased oxygen consumption to that of female rats whereas ovariectomy had no effect (Moguilevsky et al., 1969b). Estradiol and progesterone when added in vitro to the estrous-female rat hypothalamus reduced thc uptake (Moguilevsky and Malinow, 1964). 3. Physiopharmacological Sex Differences of the Hypothalamus

The hypothalamus is the final common pathway of various levels of the central nervous system that could participate in the control of internal hormonal secretions through the hypothalamic-hypophysial system. Evidence has increased to support the belief that adrenergic as well as

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cholinergic mechanisms in the hypothalamus are involved in the control of gonadotropic hormonal secretions (Markee e t al., 1947, 1948, 1952; Everett, 1964; Kobayashi e t al., 1966; Lippmann e t al., 1967; Kordon e t al., 1968; Kordon, 1969; Hamon e t al., 1970). The hypothalamus contains considerable amounts of biogenic amines, such as norepinephrine, dopamine, serotonin, and acetylcholine. I n male rats, castration increased the anterior hypothalamic content of norepinephrine and its rate of synthesis, while decreasing dopamine (Donoso el al., 1967, 1969; Anton-Tay and Wurtman, 1968) that acted as a precursor of norepinephrine in the brain (Udenfriend and ZaltzmanNirenberg, 1963). However, Hyyppa and Valavaara (1970) showed no significant effect of castration on the hypothalamic content of norepinephrine and serotonin. On the other hand, castration was shown to increase dopamine content in the hypothalamus, and administration of testosterone lowered the level to normal (Hyyppa and Lorentz, 1969). I n female rats, ovariectomy caused a rise of norepinephrine and its turnover in the anterior hypothalamus (Stefano et al., 1965; Donoso and Stefano, 1967 ; Anton-Tay e t al., 1969 ; Anton-Tay and Wurtman, 1968) but Sandler (1968) was not able to detect any changes. Kobayashi e t al. (1964, 1966) reported a temporary decrease of monoamine oxidase activity in the anterior hypothalamus of female rats a t day 14 after castration, which was followed by an increase. Neonatal administration of testosterone to intact female rats did not show any differences in the level of norepinephrine in the anterior hypothalamus from that of normal estrous rats but was significantly increased in those ovariectomized and androgenized female rats (Donoso and Cukier, 1968). On the other hand, estrogen and progesterone treatment in castrated as well as in intact female rats decreased the anterior hypothalamic content of norepinephrinc and increased its precursor, dopamine (Donoso and Stefano, 1967; Donoso and Cukier, 1968). A reduction of monoamine oxidase and choline acetylase activities was also observed in the posterior hypothalamus of castrated female rats treated with estradiol (Kobayashi e t al., 1966).

B. ADENOHYPOPI~YSIS The pituitary gland that once was called the master of endocrine glands, now becomes the servant of the hypothalamus, because its functions have been discovered to be controlled by the central nervous system. Apparently, the sex difference of the hypothalamic activity would certainly affect its control over the pituitary secretions. One of the obvious sex differenccs is the presence in the female and

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the absence in the male of estrous or menstrual cycle. This female reproductive cycle was first shown by Smith and Engle (1929) to be correlated with the gonadotropic secretions from the guinea pig anterior pituitary. I n the male there is as yet no evidence that this type of cyclic changes occur in the level of gonadotropic sbcretion. Since the gonadotropic hormones, including luteotropic hormone appear to be identical in both sexes, the only difference is the pattern of its secretion, being cyclic in the female and acyclic in the male. I n normal females, the secretion of FSH by the pituitary stimulates the growth of ovarian follicles and the secretion of estrogen from the ovary. A high titer of estrogen in the circulation inhibits (through the hypothalamic-hypophysialsystem) the release of FSH and stimulates the secretion of LH that causes ovulation and the formation of corpora lutea. A rise of progesterone in the blood stream inhibits the secretion of LH. Therefore, a cyclic pattern of gonadotropic secretion is established. Since ovulation apparently requires the abrupt release of LH, the formation of corpora lutea in the tested ovarian graft is taken as a criterion of gonadotropic secretory pattern (Pfeiffer, 1936; Harris, 1964; Gorski and Wagner, 1965). The presence of corpora lutea in such an ovarian graft is considered as evidence of cyclic secretion of gonadotropic hormones, and the absence as evidence of acyclic or masculine mode of gonadotropic secretion. The formation of corpora lutea as an indicator of cyclic release of gonadotropin correlated with the estrous cycle has also been proved in neonatal castrated male rats with both ovarian and vaginal tissues transplanted side by side under abdominal skin (Yazaki, 1960). The sex difference in the pattern of adenohypophysial gonadotropic secretion, however, is not genetically determined. Pfeiffer (1935, 1936), who investigated the effects of gonadectomy and gonadal transplantation in newborn rats on the secretory pattern of gonadotropic hormones when the animals became adult, had clearly demonstrated that the “sex type” of the anterior pituitary was not genetically determined, but was secondary and dependent upon the presence of differentiated sex glands at birth. The action of sex hormones in the neonate, on the other hand, is not directly on the hypophysis, for the adult pituitary, when transplanted beneath the hypothalamus of hypophysectomized female rats, was found to be functioning normally in maintaining estrous cycles of female rats regardless of whether male or female donors were used (Greep, 1936; Harris and Jacobsohn, 1952; Martinez and Bittner, 1956). Segal and Johnson (1959) also showed that transplantation of neonatally androgenized female rat pituitary into the sella turcica of untreated-hypophysectomized females restored normal cycles and ovulation. On t,he other hand, transplantation of the normal pituitary tissue into hypophysec-

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tomized-androgen-sterilized female rats failed to maintain cycles. In addition, the adenohypophysis of neonatally androgenized female rats was able to function normally and caused ovulation by electrical stimulation of various regions of the hypothalamus (Barraclough and Gorski, 1961; Gorski and Barraclough, 1963). Moszkowska and Kordon (1961) showed a sex difference in the hypothalamic influence on gonadotropic secretion by a combined implantation of hypothalamus and hypophysial tissue to either male or female rats. Obviously, the sex difference in the pattern of pituitary gonadotropic function does not reside within the hypophysial tissue itself, but depends upon the sexual differentiation of the hypothalamic controlling mechanism during neonatal life. Since the neonatal gonad plays a leading role in sexual differentiation, it is possible that the presence of testes and the absence of ovaries induces a “male type” of brain or the absence of testes and the presence of ovaries develops a “female type” of brain. Gorski and Wagner (1965), using corpora lutea as an indicator, beautifully demonstrated that after neonatal gonadectomy of male or female rats, which at the same time either received ovarian transplants or not, corpora lutea were present in the postpubertal-tested ovarian grafts. Thus, the presence .or absence of ovaries seem to have no effect on the sexual differentiation of the hypothalamic gonadotropin regulating mechanism in newborn rats. On the other hand, when testes were grafted a t the same time to the neonatal gonadectomized male or female rats, no corpora lutea were present (Pfeiffer, 1935, 1936), and this suppression by transplanted testes on the development of a cyclic control of the hypothalamic pituitary system was only effective within 3 days after birth (Yazaki, 1960; Gorski, 1966). Testicular hyper- or hypofunction produced by neonatal administration of human chorionic gonadotropin to rats caused permanent changes of hypothalamic-hypophysial-gonadalfunction whereas ovarian hyper- or hypofunction during this critical period produced no effect on reproductive function (Dorner et al., 1969). Therefore, it seems highly probable that the hypothalamus in the rat a t birth is undifferentiated and potentially cyclic. The presence of testes induces a male type of brain in both genetic male and female rats, and its absence results in the development of a female type of brain. A similar situation was also found in the sexual differentiation of the embryo in which testes was required for the determination of the male phenotype, whereas ovaries seem to play a minor part in the differentiation of the female type (Raynaud and Frilley, 1947; Jost, 1970; Jost and Picon, 1970). Cytogenetically, the mammalian Y chromosome is also a strong male determinator for the differentiation of the undifferentiated gonad into a testis (Mittwoch and Delhanty, 1969; Mittwoch, 1970). On the contrary, ovaries are formed in human embryo

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and fetuses even with only a single X chromosome (Singh and Carr, 1966). The neonatal testes, essential for the sexual differentiation were indeed found to be able to produce androgen in rats (Wells and Fralick, 1951; Niemi and Ikonen, 1961, 1962; Nourmura et al., 1966; Resko et al., 1968), mice, pigs, calfs, goats (Moore, 1939 ; Albert, 1961 ; Burns, 1961), bulls (Hooker, 1944; Santamarina and Reece, 1957; Abdel-Raouf, 1960), rabbits (Lipsett and Tullner, 1964), and men (Gillman, 1948; Sniffen, 1950; De la Balze et al., 1960; Segal, 1964; Huhtaniemi e t al., 1970). Acevedo e t al. (1961, 1963), Bloch et al. (1962), Bloch (1964, 1967), Noumura e t al. (1966), Steinberger and Ficher (1968), and Resko (1970) showed that rats, armadillos, guinea pigs, dogs, monkeys, and human fetal or neonatal testes have the capacity to convert progesterone to androgen whereas the same week-old fetal ovaries of the same species appeared to be quiescent. The androgen secreted by fetal testes, however, does not succeed in completing the process of sex differentiation of the hypothalamus during intrauterine life, for castration of male rats on the first day of life produces a female type of brain. This might be because the amount of androgen secreted by the fetal testes is not sufficient to have a permanent effect on the hypothalamus prenatally. The minimal dosage of androgen required to masculinize the hypothalamus is 10 pg when given to 4- or 5-day-old rats (see below). In newborn male rats the plasma testosterone concentration is 0.08 pg/lOO ml of plasma (Lloyd and Weisz, 1967). Although this is about 2-3 times the level present in the adult female rat during proestrus, it is not enough to produce a permanent androgenic effect a t this age. Another possibility is the poor fetal circulation in which the androgen is rapidly metabolized before it has sufficient effective concentration on reaching the hypothalamus. It is also possible that the maternal progesterone protects the fetal hypothalamus from being masculinized by the fetal androgen (see below). A fourth possibility is that the fetal hypothalamus is still insensitive to it. The major androgen present in the early postnatal period of developing rats was found to be testosterone (Resko e t al., 1968). Gonadotropic hormone also has been detected in the fetal pituitary gland of pigs (Smith and Dortzbach, 1929), horses (Hellbaum, 1935), sheep (Mauleon and De Reviers, 1969), and men (Siegmund and Mahnert, 1928). Jost and his colleagues (Jost, 1948; Jost and Colonge, 1949; Jost and Yicon, 1970), using rats, reported that when fetal testes were grafted into the seminal vesticles of castrated adults they stimulated the adult sexual structures, but if the adult rat was hypophysectomized no such androgenic activities were observed and the grafted fetal testes were atrophied. On the other hand, if gonadotropic

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hormone was administered to such a hypophysectoniized host, the grafted fetal testes recovered their testicular functions. A similar result was also found in the male fetus which when decapitated showed underdeveloped reproductive organs indicating insufficiency of testicular secretions, but these abnormalities were completely restored when the gonadotropin was given to the rat fetus a t the same time as decapitation (Jost, 1951). Moreover, administration of gonadotropic hormone to the newborn rat caused a significant increase in testicular weight (Yaginuma et al., 1969). All this evidence suggests that the pituitary-testicular axis already functions during the perinatal period and that pituitary hormones are required for the stimulation of the testes to secrete enough testosterone, which later plays an important role in sexual differentiation. If the testosterone secreted by the testes is important for the masculine differentiation of the hypothalamus during the neonatal period, it should be possible to cause such a differentiation by injecting the exogenous steroids instead of gonadal transplantation. The animal with masculine differentiation of the hypothalamic-hypophysial system after neonatal administration of steroids showed ovulatory failure, polycystic ovaries without corpora lutea, and constant estrous vaginal cornification (Harris, 1964). The sensitivity of the brain to exogenous hormones depends upon the age of the treated animals and the dosage administered. However, there is no clear-cut definition. A single neonatal injection of testosterone that resulted in a permanent anovulatory sterility was effective within a critical period from birth to about day 5 or 6 in rats (Barraclough, 1961; Swanson and van der Werff ten Bosch, 1964b; Gorski, 1968), day 10 in mice (Barraclough and Leathem, 1954; Barraclough, 1955, 1967), day 3 or 4 in hamsters (Alleva et al., 1969), and probably prenatal ages in guinea pigs (Goy et al., 1964), monkeys (Young et al., 1964), cows and pigs (Zimbelman, 1964). The minimal effective dosage of androgen required for the masculinization of the hypothalamus is 10 pg when given to 4- or 5-day old rats (Gorski, 1966; Barraclough, 1967). The larger the dose of androgen, the sooner the anovulatory syndrome develops (Swanson and van der Werff ten Bosch, 1964a). In rats, the dose of androgen which is sufficient to cause a tonic pattern of gonadotropic secretion when administered postnatally, however, has no effect when given prenatally to the pregnant doe or into the amniotic sac. Although the vagina was affected, the ovaries appeared to be normal, with corpora lutea present (Forsberg, 1960; Cagnoni et al., 1964; Swanson and van der Werff ten Bosch, 1964b, 1965). On the contrary, such masculine effects were observed when androgen was administered directly to the fetus or indirectly with relatively high doses to the gravid rats (Swanson and van der Werff ten Bosch, 1965).

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Thus, the failure to induce a tonic-gonadotropic pattern of the brainpituitary complex in the first place seems due to the poor placental transmission of sufficient steroid from the maternal to the fetal body. Another explanation is the protective effect of progesterone secreted by the pregnant rat. Kincl and Maqueo (1965) showed that progesterone injected concomitantly with androgen to the 5-day-old female rat prevented the sterilizing effect of the androgen. The constant vaginal estrous rat resulting from neonatal treatment with androgen could also be changed by cyclic administration of progesterone (Boros et al., 1969). However, Lloyd and Weisz (1967) reported that this protective effect of progesterone was only temporary, for a typical anovulatory polycystic ovary and persistent vaginal estrus developed later in life a t about 1W 300 days of age. As to the precise site in the hypothalamus for the action of neonatally injected sex hormones, Barraclough and Gorski (1961) first proposed a double controlling system of the gonadotropic secretion: the tonic center of arcuate-ventromedial region and the cyclic center of anterior-hypothalamic-preoptic area. In normal female rats, the tonic center of the hypothalamus causes the pituitary to release an amount of gonadotropin just sufficient to maintain estrogen secretion from the ovaries but not enough to initiate ovulation, whereas the cyclic neural center controls ovulation by triggering the ovulatory release of gonadotropic hormone from the hypophysis. At birth the anterior-hypothalamic-preoptic area is either undifferentiated or potentially cyclic. I n the presence of the endogenous or exogenous steroids the preoptic hypothalamus loses its cyclic activity, and therefore a masculine tonic pattern of gonadotropic secretion develops (Gorski and Barraclough, 1963 ; Gorski and Wagner, 1965; Gorski, 1966; Barraclough, 1967). The degree of androgenization of the hypothalamus is also dosage-dependent, for the ovulation could be induced by hypothalamic electrical stimulation in rats treated with 10 pg of androgen (Gorski and Barraclough, 1963) but not in rats treated with 1.25 mg of androgen (Barraclough and Gorski, 1961). I n male rats, if endogenous and exogenous androgen is important during the critical phase for the differentiation of the pituitary-diencephalic system into a male type, then it should be possible to inhibit this differentiation by simultaneously injecting antiandrogenic steroid and therefore allow a female type of brain to develop in genetically male rats. Hamada et al. (1963) and Neumann et aE. (1966,1970) induced a vagina in male rats by administration of an antiandrogen, cyproterone acetate, to the pregnant rats during the second half of gestation and continuously for the first 3 postnatal weeks. Cyclic changes in the vaginal mucosa were also observed in these feminized male rats following castration and

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ovarian transplantation (Neumann and Elger, 1966). Larger corpora lutea were present in the grafted ovary of the treated animal as compared with the control. I n female rats, concurrent administration of cyproterone acetate with testosterone propionate also prevented the neonatal androgen-induced masculinization (Wollman and Hamilton, 1967). The antiandrogen action, however, is not due to increased inactivation of androgen in the liver (Wollman and Hamilton, 1966), but by a competitive action on androgen receptors within the target organ, presumably the central nervous system (Junkmann and Neumann, 1964). If the action of antiandrogen is on the brain, then by using tranquilizing or anesthetic agents one should be able to produce a similar result. Indeed, reserpine (Kikuyama, 1961; Kawashima, 1964; Arai and Gorski, 1968a), chlorpromazine (Kikuyama, 1962; Arai and Gorski, 1968a), and pentobarbital or phenobarbital (Arai and Gorski, 1968a,b) given simultaneously with androgen to neonatal rats inhibited the development of an androgenized neural center. The masculinizing action of androgen on the brain was shown to have taken place within 12 hours of neonatal injection, for administration of an antiandrogen agent after this period had no inhibitory effect (Arai and Gorski, 1968b; Kobayashi and Gorski, 1970). Since in neonatal gonadectomized male and female rats, whether receiving a t the same time an ovarian transplant or not, a cyclic sexual pattern of gonadotropic secretion developed (Gorski and Wagner, 1965) ; similar administration of exogenous estrogen instead of ovarian transplants would not be expected to have any effect on the sexual differentiation of the hypothalamo-gonadotropin-regulating mechanism. I n fact, however, estrogen when injected shortly after birth to either intact or gonadectomized male and female rats produced an acyclic pattern of pituitary gonadotropic secretion (Greene and Burrill, 1941; Turner, 1941; Gorski, 1963a; Arai, 1964; Kincl et al., 1965). It is possible that cyclic pattern of gonadotropic regulation develops in the absence of any sex steroids in the circulating blood, but there is some evidence that the fetal or neonatal ovary could possibly secrete estrogen or progesterone. Roberts and Warren (1964) demonstrated the capabilities of the bovine fetal ovary to metabolize progesterone in vitro in the same way as the adult ovary (Sweat et al., 1960). A similar result was also found in 15- and 19-week-old human fetal ovary (Bloch, 1964). Tillinger et al. (1957) even detected a small amount of estradiol in the urine of human newborn babies. It may be that, in fact, the perinatal ovary, like the perinatal testis, does have some degree of activity, though the ovary becomes functional a t a later stage than the testis (Clark, 1935; Maekawa, 1953). If the detected estrogen in the fetal or neonatal life comes from the mother, why does this maternal estrogen not cause masculinization? It does not

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seem, therefore, that the secretion of the fetal or neonatal ovary necessarily had any effect on sexual differentiation; this conflicts with respect to the effect of exogenous estrogen. The minimal dosage of estrogen required to modify the female pattern of cyclic release of gonadotropin when injected to 5-day-old female rats is 5 pg of estradiol benzoate (Gorski, 1963b). This could not be considered a physiological dose, because 7-littermate ovaries or a large piece of adult ovary when transplanted to 2-dayold castrated male rats did not interfere with the development of a female type of gonadotropic secretion (Gorski, 1966). Therefore, the administration of 5 pg or more of estrogen should be considered to have a pharmacological effect. In fact, 0.5 pg, but not 1 pg, of estradiol benzoate when given to 5-day-old female rats produced no effect a t all on the development of a cyclic gonadotropic release (Gorski, 196310). Apparently, the cyclic pattern of gonadotropic regulation develops in the absence of androgen during the perinatal period. Similarly, the masculine effect of neonatal administration of other steroids or synthetic compounds, such as deoxycorticosterone acetate (Selye and Friedman, 1940; Takasugi, 1953), human chorionic gonadotropin (Bradbury and Gaensbauer, 1939), stilbestrol (Hale, 1944; Ladosky, 1967), hexestrol (Gorski, 1963a), “anabolic steroids” (Jacobsohn, 1964), and cholesterol (Takasugi, 1953) should be regarded as a pharmacological effect. Progesterone, as mentioned before, when injected simultaneously with androgen to the pregnant rats had a protective effect from masculinization of her off spring. However, when progesterone was administered alone to newborn female rats (Takasugi, 1953; Gorski, 1963a) , or when progesterone was given orally to pregnant women (Wilkins, 1959, 1960), it produced a masculine syndrome in the infants. On the other hand, Revesz et al. (1960) showed that progesterone when given to pregnant female rats did not androgenize her offspring, but Gar-methyl-l7~-acetoxyprogesteronedid produce pseudohermaphrodism. However, Lyster et al. (1959) did not find any androgenic effect due to this compound. Recently, by the use of ovarian ascorbic acid depletion and ovarian weight augmentation assay methods, the actual LH and FSH content of the anterior pituitary was measured in both male and female rats from the neonatal period through sexual maturity (Moore, 1965-1966 ; Matsuyama et al., 1966; Lloyd and Weisz, 1967; Lisk, 1967, 1968; Kragt and Ganong, 1968; Labhsetwar, 1969, 1970; Fawke and Brown, 1970). In infancy, there was a peak of gonadotropic concentration (pg/gm of pituitary) that occurred before puberty, followed by a nadir a t the time of vagina opening or sexual maturation. The prepubertal peak of FSH appeared earlier than that of LH (Labhsetwar, 1970). The peak level of both LH and FSH in the female was significantly higher than in the malc

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a t this age. The situation was completely reversed following sexual maturation, in which the adult males had a greater pituitary gonadotropic content than adult female rats (Ramirez and McCann, 1963). The sexual differentiation of the hypothalamus not only has its effect on the gonadotropic secretion but also affects other adenohypophysial tropic releases. Details of sex differences of ACTH and TSH will be described in connection with adrenal and thyroid functions. Growth hormone will also be described in order. C. ADRENAL GLAND In most species (with the exception of hibernating rodents such as golden hamsters) the adrenal weights of males were smaller than those of females (Deane, 1962). In rats, this sex difference was not observed until they were approximately 50 days old and reached a maximum a t about 70 days, from then a sex difference persisted until old age (Baca and Chiodi, 1965). Ovariectomy of the pregnant rat resulted in atrophy of the fetal adrenal, and administration of estradiol, but not of testosterone, prevented it (Eguchi, 1962). Gonadectomy increased the adrenal weight in the male and decreased in the female rat whereas injection of testosterone lowered, and estradiol restored, the level (Kitay, 1963 ; Bernstein, 1964). The sex difference in adrenal weight is consistent with the sex difference in the amount of corticosterone present. The heavier is the gland, the higher is the adrenal content of corticosterone (Halberg and H a w , 1960; Critchlow et al., 1963; Nandi et al., 1967). The circulating level of corticosterone was found to be higher, and its biological half-life shorter, in females than in male rats (Kitay, 1961; Glenister and Yates, 1961; Critchlow et aZ., 1963). Sex differences have also been described in the rats of metabolism of corticosterone and cortisone by rat liver tissue in vitro (Troop, 1959; Urquhart et al., 1959; Hagen and Troop, 1960; Glenister and Yates, 1961; Kitay, 1961). Female rats demonstrated a higher and more persistently increased plasma level of corticosterone after stress than did male animals (IGtay, 1961) ; this finding suggested that the greater secretion of corticosterone observed in female rats might be due to the greater adrenal sensitivity to stimulation by ACTH. In fact, the concentration of ACTH in the blood has been reported to bc higher in the female than in the male (Barrett, 1960), and estrogen ad1967). ministration doubled its level (Fonzo et d., A similar sex difference was also found in the activity of the corticosteroid-binding (Gala and Westphal, 1964, 1965a), which first appeared a t 30 days of age and reached a maximum level a t 48 days when the female rat had approximatcly twice the binding activity of the malc

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(Gala and Westphal, 1965b; Keller et al., 1966). Castration and estradiol injection increased activity in the male whereas testosterone treatment lowered it in the female (Gala and Westphal, 1965b, 1966b). Hypophysectomy, even with the administration of estradiol, failed to produce any significant increased activity, indicating that the effect of sex hormones might probably mediate through the pituitary gland rather than have a direct action on the binding activity (Gala and Westphal, 1966a). In addition, sex differences were also observed in the ascorbic acid content (Baca and Chiodi, 1965), phosphorylase activity (Cohen and Fagundes, 1966), lipid content (Whitehead, 1935), D P N H oxidation (Allan, 1959), succinoxidase activity, and glucose oxidation (Kalant and Sellers, 1954) of the adrenal gland.

D. THYROID GLAND The activity of the thyroid gland seems to be affected by the secretions of the gonad because many clinical and experimental works showed a higher incidence of thyroid gland diseases in females than in males. Higher radioiodine uptakes in the thyroid gland of women have also been reported (Quimby et al., 1950; Krokowski et al., 1964). I n mice, females showed a significantly higher uptake of 1311 than males (Schreiberova and Kapitola, 1969). A cyclic change in radioactive iodine uptake by the thyroid gland has also been observed, with the highest value during estrus in rats (Feldman, 1956; Brown-Grant, 1962a; Kennedy et al., 1964) and in guinea pigs (Brown-Grant, 1962b) or during proestrus in mice (Boccabella and Alger, 1961). Ovariectomy decreased the thyroid: serum (T: S) radioiodine concentration ratio (Boccabella and Alger, 1964) whereas castration produced no significant alteration (Feldman, 1956). Estrogen treatment increased radioactive iodine uptake both in intact and in gonadectomized male and female rats (Noach, 1955; Feldman, 1956; Florsheim, 1958; Boccabella and Alger, 1964). However, a sex difference of T : S ratio in response to estrogen injection still persisted in gonadectomized rats (Boccabella and Alger, 1964). Hypophysectomy markedly reduced the thyroidal radioiodine uptake (Yamada et al., 1966), and administration of estradiol to hypophysectomized or hypophysectomized-ovariectomized rats increased but could not restore the level of the uptake (Boccabella and Alger, 1964; Yamada et al., 1966). A sex difference in the incorporation of T,-1311 into the brain has also been noted in the rat (Ford et al., 1964). The thyroxine secretion rate (TSR) was greater in female than in male rats (Gregerman and Crowder, 1963). Gonadectomy caused a significant reduction of TSR, and the fall could be restored by treatment with estradiol in the female and testosterone in the male (Moon and

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Turner, 1960; Kumaresan and Turner, 1967). Neonatal administration of testosterone to female rats showed a significant decline of TSR (Kennedy et al., 1964; Kumaresan and Turner, 1966). A sex difference of the thyroid-stimulating-hormone (TSH) in plasma and pituitary has also been claimed (D’Angelo, 1968). Castration of male rats decreased the TSH secretion whereas administration of testosterone prevented the damage (Van Rees et al., 1965). I n female rats ovariectomy also reduced TSH levels in serum and pituitary (D’Angelo, 1966; D’Angelo and Hughes, 1967). A profound sex difference of TSH secretion still remained in the response to estrogen treatment in intact rats; females showed a progressive decline in TSH content of plasma and pituitary whereas males produced no significant alteration in the plasma TSH level and a slight drop in the TSH content of the pituitary (Amesbury et al., 1965; D’Angelo, 1968). In addition to the difference in the thyroid-pituitary functions between males and females, a sex difference in the renal retention of thyroxine has also beeh reported in mice (Kamei et al., 1965).

E. GROWTHHORMONE The influence of sex hormones on growth has long been suspected because females are generally smaller than males. Observation of rats in the laboratory showed a significantly greater growth pattern (body weight and length) in the male than in the female, and these sex differences were not revealed until they were 21 days old (Grunt, 1964b). Castration of adult male rats decreased the growth while ovariectomy of adult females produced significantly greater length and heavier body weight (Frendenberger and Billeter, 1935; Rubinstein et al., 1939a ; Grunt, 1964a). However, there were no significant differences between male and female rats when gonadectomy was performed on the first day of life (Grunt, 196413). I n mice, a similar sex difference of body weight was noted and a single injection of goldthioglucose which produced hypothalamic lesions also showed a sex difference relative to subsequent obesity (Schindler and Liebelt, 1967). On the other hand, a completely different picture was found in hamsters; the adult female was heavier and longer than the male, And castration before or after puberty increased growth in the male but not in the female (Swanson, 1967a,b, 1970). Direct measurement of growth hormone content in the pituitary either by tibia1 epiphysial bioassay or radioimmunoassay also showed a higher concentration in the male than in the female rat; ovariectomy increased the level whereas orchiectomy produced no alteration ; estrogen treatment lowered the level and testosterone administration enhanced the

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concentration (Jones e t al., 1965; Birge e t al., 1967; Josimovich e t al., 1967; MacLeod et al., 1969). In contrast, the female rat showed a greater concentration of growth hormone content in the plasma (Schalch and Reichlin, 1966; Quabbe, 1970). I n mice, however, the pituitary growth hormone content was reported to be higher in the female than in the male of C3HJHe but not C57BL/6 strain (Yanai and Nagasawa, 1968). Sex differences and the effect of sex hormones on osseous growth have also been described in humans and in animals (Gardner and Pfeiffer, 1943;Roche, 1968; Coleman, 1969).

F. MISCELLANE~US Isologous transplantations of skin from males were frequently rejected by female recipients, whereas male to male, female to female, or female to male skin grafts were almost always successful in mice (Eichwald and Silmser, 1955; Eichwald e t al., 1957; Short and Sobey, 1957). However, the strength of rejection varied from strain to strain of mice, being strongest in C57BL and weakest in A/Jax strain mice (Eichwald e t al., 1958). This sex influence in transplantation was also observed in tissues other than the skin, such as thymus (Hirsch, 1957), lymph nodcs (Feldman, 1958), lung, salivary gland, blood, spleen, and liver (Eichwald et al., 1958). Rejection of male grafts by females of the same strain indicates that the graft carries a male-specific antigen which is absent in the female. By using mice of exceptional chromosomal constitution, XO female and XXY male, Celada and Welshons (1963) reported that this sex-linked antigen was not determined by a histocompatibility gene located on the single X chromosome normally found in a male of C3H and C57BL strains of mice. Before a definite conclusion could be drawn that the Y chromosome is the cause, they suggested that the male antigen might be just one of the consequences of sexual differentiation or a product of any histocompatibility locus whose expression is male sex dependent, for such is the case in the mouse sera whose gene controlling the titer of late-acting component complements is secondary and depends on the level of androgen (Urbach and Cinader, 1966; Churchill e t al., 1967), and it is also true that the gene controlling the Hi-agglutinogen in chickens depends upon the level of estrogen (Scheinberg and Reckel, 1961, 1962). In fact, neonatal orchidectomy interrupted the sexual differentiation of C57BL strain of mice significantly inhibits the development of the expression of the male antigen in their skin (Vojtiskova and Polackova, 1966; Polackova and Vojtiskova, 1968). On the other hand, male antigen could be induced in cells lacking the Y chromosome by exposing the newborn female mouse skin in a male environment (Engelstein, 1967; Polackova, 1969). Apparently, both male and female mice posscss the gcnctic

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information necessary for the formation of male antigen. The fact that this antigen normally appears only in the male may be only secondarily dependent on the Y chromosome, and the presence of a male-hormonal environment may be of primary importance in determining the activity of the “male” histocompatibility locus. Moreover, sex differences and the effect of sex hormones on the skin grafts in the histocompatibility gene located on the autosomal loci including H-1, H-2, H-3, H-4, H-7, H-8, H-9, H-10, H-11, H-12, and H-13 (Graff e t al., 1966; Galton, 1967; Kongshavn and Bliss, 1970) and the immune response (Kaliss, 1957; Batchelor and Chapman, 1965) have also been demonstrated. A sex difference in the red blood cell count has been observed in all species of animals commonly used in the laboratory as well as in men and the count, in general, was greater in the male than in the female (Adams and Shevket, 1929; Rosahn e t al., 1934; Riddle and Braucher, 1934; Kamenoff, 1937; Lewis, 1941 ; Korenchevsky and Hall, 1944). Allan and Alexander (1968) reported a higher leukocyte count in men than in women. Differences in blood level of ascorbic acid between men and women have also been noted (Dodds, 1969). For details of the effect of gonadectomy and the administration of sex hormones on the blood, refer to reviews of Grant and Root (1952) and Zemplenyi (1968). The action of steroids on other nonsexual organs, including the heart, kidneys, and liver tissues, and on the general growth of the body has also been investigated, and it was found that the male sex hormone exerted a stimulating effect whereas the effect of female sex hormone was predominantly depressing (Korenchevsky and Ross, 1940; Korenchevsky and Hall, 1941; Korenchevsky e t al., 1941). The sex difference and the effect of hormones on the liver functions will be described in more detail in the following section. II. Sex Differences in Biochemical Functions

Physiological sex differences presuppose biochemical ones, in seeking to explain the differences between males and females in the underlying molecular basic processes of life. Each type of cell in the organism has its own characteristic morphology as viewed under the microscope. This specific morphology is due to the unique biochemistry of enzymes, lipids, proteins, and other macro- and micromolecular structures, The possible role of the mechanism of action of carcinogens on these molecular and cellular levels has been revealed recently by the results of the covalent binding of residues of chemical carcinogens with DNA, RNA, and protein and the way they were metabolized by the enzymes. Although numerous reports have shown sex-linked differences of metabolism in

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various types of tissues, an extensive review will be given here with particular reference to the liver. A. ENZYMES Many anabolic and catabolic processes fundamental to all the living organisms and vital for the synthesis of normal and abnormal tissuecomponents depend on the catalytic effect of enzymes. It seems that nearly every alteration in the physiological conditions of an animal produces changes in the amount of enzymes. Sex differences in the activity of various enzymes were reviewed by Knox et al. (1956) and Barzilai (1965).Those concerned with the metabolism of carcinogens,steroids, and drugs will be described. 1. Carcinogen-Metabolizing Enzymes

The aromatic amine and amide carcinogens required biochemical transformation before they can exert their carcinogenic effect at the site distant from the area of application. This suggests that a metabolic process is essential for the manifestation of their carcinogenic properties. According to Miller (1970), there are at least two steps of activation of these carcinogens :a. N-Hydroxylation. For example, one of the carcinogens, N-2-fluorenylacetamide (FAA), is hydroxylated on the nitrogen leading to the N-Hydroxy-N-2-fluorenylacetamide(N-OH-FAA) , a reaction which proceeds via the endoplasmic reticulum or the microsomal fraction of the liver (Miller et al., 1960; Miller and Miller, 1969). This N-hydroxy derivative when tested on the liver of rats was found to be more powerful carcinogen than its parent compound (Miller et al., 1961). After a single intraperitoneal injection of W-labeled N-OH-FAA, adult male rats excreted less of a dose in the urine than did adult females and the urine of males contained more sulfate conjugates and less of the glucuronic acid conjugates than that of females (Weisburger et al., 1964; Miller et al., 1970). Castration of adult male rats increased the excretion of N-O-FAA whereas administration of testosterone prevented the rise (Lotlikar et al., 1964). b. Esterijkation. Evidence shows that the final reactive form of carcinogenic amines and amides comes from the enzymatic esterification of the N-hydroxy derivatives (Miller and Miller, 1969). It is believed that the sulfuric acid ester of N-OH-FAA may be an ultimate carcinogen, at least in hepatocarcinogenesis, and that the hepatic enzyme which generates this ester is sulfotransferase (Miller, 1970). Sex differences of this enzyme activity have also been found in the liver of mature rats; in the male, it is 5-fold higher than in the female, but this difference is not

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seen in weanling rats (De Baun et al., 1968, 1970). Gonadectomy of weanling male and female rats produced no significant alteration in hepatic N-OH-FAA sulfotransferase activities. Administration of testosterone to normal or ovariectomized female rats significantly increased the sulfotransferase activities whereas it has no effect in intact or castrated male rats. On the other hand, estradiol reduced the level of hepatic sulfotransferase activity in both normal and gonadectomized male and female rats (De Baun et al., 1970). Hypophysectomy or thyroidectomy significantly decreased liver sulfotransferase activity whereas adrenalectomy or combined adrenalectomy-castration of male rats produced little effect on the response (De Baun et al., 1970). Each of these endocrine alterations on the effect of sulfotransferase activity also caused a similar change in rat liver in respect to susceptibility to the carcinogenicity of N-OH-FAA. Sex differences in other metabolic enzymes were also noted. c. S-Adenosylmethionine Synthetase. This enzyme catalyzes the formation of S-adenosylmethionine from ATP (adenosine 5-triphosphate) and L-methionine in the presence of reduced glutathione and magnesium (Cantoni, 1951). Male rats had a lower level of this enzyme in their liver than the females (Natori, 1963; Sheid and Bilik, 1968). In male rats, castration increased the enzyme activity, and subsequent injection of androgen brought down the level to that of the normal intact males (Natori, 1963; Pan et al., 1968). In female rats, ovariectomy resulted in a decrease of enzyme activity whereas administration of estrogen restored the level. Estradiol when given to the intact male rats also mised the level of the enzyme to that of the female (Pan et al., 1968). Recently, it was found that mammalian fetal liver and various neoplastic tissues contained only a small amount of this enzyme, in contrast with the high level in normal adult rat liver (Hancock, 1966, 1967). Sheid and Bilik (1968) even showed the nonexistence of this enzyme in transplantable hepatomas. Obviously, the higher level of this enzyme in the female rat liver is consistent with its resistance to the hepatocarcinogenesis. d. L-Asparaginase. This enzyme has been used for the treatment of cancer. The amount of L-asparaginase in the liver of fetal or newborn rats and mice and in the serum of newborn guinea pigs was found to be very low compared with that in the adult animals (Tower et al., 1963; Bonetti et al., 1969 ; Patterson and Orr, 1969). Its activity was decreased in regenerating liver and was almost absent in transplantable hepatoma (Bonetti et al., 1969). The enzyme had also an inhibitory effect on mitosis (Becker and Broome, 1967) and RNA polymerase activity (Becker and Broome, 1969) of regenerating liver. Along with its antitumor activity was the higher enzyme activity in the female than in the male, and gonadectomy decreased, and administration of estradiol in-

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creased, its activity in the liver of both male and female rats (Bonetti et al., 1969). e. p-Glucuronidase. An increase of P-glucuronidase concentration in the liver of carcinogen-treated rats has been reported (Hoch-Ligeti et al., 1966). Glucuronide synthesis in vitro of rat liver was much higher in males than in females and castration decreased the formation whereas administration of testosterone restored it (Kunzel and Muller-Oerlinghausen, 1969). On the other hand, urinary excretion of N-GIO-FAA (glucuronide of N-OH-FAA) was found to be lower in male than in female rats (Miller et al., 1970), suggesting that probably a higher concentration of N-GIO-FAA remains in the liver available for the carcinogenesis that resulted in a higher incidence of tumors in males than in females. Further investigations are required. f. Catalase. Liver catalase has been used as an important character in the study of the relationship between host and tumor, for a depression of its activity almost invariably occurs in all animals with growing tumors; the degree of depression is proportional to the size of the tumor; removal of the tumor restores the level and the enzyme activity is not affected by rapidly growing nonmalignant tissue such as an embryo (Kampschmidt, 1965). A sex difference in the liver and kidney catalase has also been found in nearly all strains of mice and rats with a higher level in males than in females (Adams, 1950, 1951, 1956). Castration decreased the liver and kidney catalase levels and administration of testosterone to female rats and castrated males increased the enzyme activity (Adarns, 1956). Hypophysectomy caused an increased liver catalase activity in normal rats (Gaebler and Mathies, 1951; Utsugi, 1960a), and it also prevented a decrease in tumor-bearing rats and mice (Utsugi, 1960a,b). Administration of thyroxine reduced the liver enzyme level (Yamamoto et al., 1959; Girkin and Kampschmidt, 1960) whereas thyroidectomy enhanced the activity (Zeckwer et al., 1954). g. Metabolism of AfEatoxin B,. Aflatoxin B,, an extremely effective hepatocarcinogen in the rat when fed either as naturally occurring contaminant in peanut meal or as mixed aflatoxin (Butler and Barnes, 1963; Butler et al., 1969) was metabolized slower in the male than in the female rat (Purchase and Steyn, 1969). The difference in the susceptibility of males and females to the aflatoxin could be a result of the difference in the ability of male and female rat liver to detoxify the carcinogen, for, there were no differences in the ability of absorption from the stomach (Purchase and Steyn, 1969). 2. Drug-Metabolizing Enzymes

A vast number of foreign compounds are metabolized in the liver by the microsomal enzyme system. It can be divided into two processes.

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a. Hydroxylation. Sex differences in the hydroxylation of hexobarbital (Quinn et al., 1958; Kato and Gillette, 1965a,b) and aniline (Page and Vesell, 1969) have been shown to be more active in the liver microsomes of male than of female rats, but there were no clear-cut sex differences in mice and rabbits (Kato et al., 1970a). However, Noordhoek and Rumke (1969) claimed that hexobarbital was hydroxylated faster in the 9OOg liver supernatant from female than from male mice. The higher metabolism of hexobarbital in male rats was due to the higher activity of NADPN oxidase, NADPN-cytochrome c reductase and the content of cytochrome P-450 in the microsomal electron transport system (Kato, 1967; Kato and Takahashi, 1968; Kato et al., 1968a, 1970a). Schenkman et al. (1967), however, found that the sex difference in the rate of the metabolism of hexobarbital by liver microsomes was due to the differences in the apparent disassociation constant (K,) and the maximal velocity (V,) instead of a difference in the content of cytochrome P-450. The metabolism of hexobarbital by liver microsomes of male rats was impaired by castration (Kato and Gillette, 196513) and the damage could be restored by subsequent testosterone treatment (Booth and Gillette, 1962). Administration of testosterone to female rats also enhanced the activity of liver microsomal enzymes to metabolize hexobarbital (Quinn et al., 1958; Booth and Gillette, 1962). Conversely, estradiol in adult male rats reduced the activity of these enzymes. Hypophysectomy in early postnatal life or in the adult inhibited the activity of the hexobarbital metabolizing enzymes in the liver of male rats (Yarn and Du Bois, 1967; Nair and Bau, 1967). Recently, bilateral electrolytic lesions in the posterior hypothalamus, but not in other parts of the brain, was shown to have a similar inhibitory effect on the liver microsomal enzyme system as metabolized hexobarbital in male rats, indicating the role of the hypothalamic-hypophysial system in the regulating of enzyme activity in the liver (Nair et al., 1970). b. N-Demethylation. Significant differences in N-demethylations of ethylmorphine (Davies et al., 1969; Page and Vesell, 1968) and aminopyrine (Kato and Gillette, 1965b; Kato et al., 1968a,b) between two sexes were also found in the liver microsomes with a higher level in the male than in the female. Again, no differences were observed in mice and rabbits (Kato et al., 1968a). These sex differences of N-demethylations could be explained by the differences in the amount of enzyme (V,), the , the rate of cytoapparent affinity of the enzyme for substracts ( K m ) and chrome P-450reduction (Davies et al., 1968; Gigon et al., 1968). 3. Steroid-Metabolizing Enzymes Changes in steroid metabolism in the liver and their possible association with the clinical symptoms of the patient or the experimental dis-

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eases of the animal have been received increasing attention during the past decades. a. Metabolism of Androgen. It has been reported that the major pathway for the metabolism of testosterone in the liver microsomes of male rats was via the formation of polar metabolites (ultraviolet absorbing fraction consisting of hydroxylated compounds) whereas in females it was metabolized through the formation of nonpolar products (A4reduced metabolites with no ultraviolet light absorption) (Jacobson and Kuntzman, 1969; Kato et al., 1970b). Castration of male rats decreased the in vitro metabolism of the testosterone and the fall could be completely restored by the subsequent administration of testosterone or methyltestosterone. On the other hand, simultaneous injection of estradiol or diethylstilbestrol blocked the action of androgen on the hydroxylation of testosterone (Kato et al., 1969). The activity of hepatic steroid sulfatase was found to be higher in males than in females of various strains of rats (Roy, 1958; Burstein and Westort, 1967; Burstein and Zucker, 1967). However, there were no significant sex differences of the enzyme activity in fetal or newborn rats (Pulkkinen, 1961). On the other hand, Lewis (1968) reported that female rats excreted greater amounts of the administered androsterone, epiandrosterone, dehydroepiandrosterone, or testosterone as sulfuric acid conjugates than did male rats. Castration lowered hepatic steroid sulfatase activity in male rats whereas giving testosterone restored the level. The enzyme activity also decreased in hypophysectomized rats. Administration of growth hormone to the hypophysectomized rats caused a significant increase of the enzyme level (Burstein, 1968). I n addition, sex differences and the effect of sex hormones on 3phydroxysteroid dehydrogenase activity in rat liver have also been demonstrated (Rubin and Strecker, 1961; Rubin et al., 1963). b. Metabolism of Estrogen. The rate of the metabolism of estrone and estradiol-17P in the liver microsome was shown to be greater in males than in female rats. Gonadectomy reduced estrogen metabolism both in males and in female rats and the drop could be recovered by giving testosterone to males and estradiol-17p to females (Lehmann and Breuer, 1969). Further information on the metabolism of estrogens in the liver refer to reviews of Barzilai (1965) and Adlercreutz (1970). c. Metabolism of Adrenocortical Steroids. Sex differences have been described in the rate of the metabolism of corticosterone and cortisone by rat liver tissue in vitro. Liver homogenates from adult male rats reduced the 17,21-dihydroxy-20-ketoneside chain of cortisone a t a more rapid rate than did similar preparations from female rats (Troop, 1958, 1959; Hagen and Troop, 1960). On the other hand, female rat livers metabo-

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lized the ring A of cortisone and corticosterone faster than did the male (Yates et al., 1958; Forchielli et al., 1958; Troop, 1958; Kitay, 1961). Orchiectomy decreased side-chain reduction and increased the rate of ring A metabolism of cortisone ; administration of testosterone prevented these changes. On the other hand, estradiol given to male rats caused a drop of side-chain reduction. Oophorectomy slightly elevated cortisone side-chain reduction but had no effect on ring A metabolism; testosterone treatment increased side-chain metabolism and depressed ring A activity while estradiol produced a reverse picture (Troop, 1959; Hagen and Troop, 1960). These sex differences, however, could only be observed after day 30 of life in rats (Denef and De Moor, 1966; Schriefers, 1967). Castration of male rats on day 24 of life reduced but did not completely prevent the masculinizing metabolism of cortisone (i.e., lowered ring A metabolism and increased side-chain reduction) in rat liver homogenates. However, if testes were removed within 10-17 hours after birth, the pattern of cortisol metabolism when tested in the adult no longer differed from that of neonatally castrated female rats. On the other hand, there were no differences whether ovariectomy was performed early in life or later, suggesting that probably the liver, like the hypothalamus, is undifferentiated at birth and potentially “feminine”; the presence of testes induces a “male type” of liver enzyme systems first evident after puberty, and its absence develops into a “female type” of liver (De Moor and Denef, 1968). A single neonatal injection of testosterone to female rats, ovariectomized on the first day of life, developed and maintained during adult life a masculinizing steroid metabolism in the liver whereas testosterone given during adult life induced only a temporary male type of steroid metabolism. In male rats, neonatal administration of testosterone completely prevented the development of the neonatal castration-induced female type of liver enzymes that metabolized the steroids (Denef and De Moor, 1968). In contrast, a single subcutaneous injection of estradiol to normal male rats on day 5 but not on day 15 of life delayed the sexual differentiation of cortisol metabolism. However, when estradiol was given t o male rats on the first day of life, a female type of cortisol metabolism emerged (Denef and De Moor, 1969).

B. PROTEINS Protein is the fundamental component of all living organisms. Changes of metabolism as a result of dysfunction lead to a qualitative as well as quantitative alteration of the protein and its urinary excretion. It is generally believed that carcinogenic agents induce cancer through interactions with tissue components. Extensive investigations were, there-

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fore, focused on chemical carcinogens binding with proteins and nucleic acids. 1. Sex-Associated Proteins

A sex-associated protein component in extracts from livers of male rats and absence from those of females was first reported by Bond (1960) and subsequently confirmed by Barzilai and Pincus (1965). This sex-linked protein component, however, was not present in the liver of immature male rats (Barzilai and Pincus, 1965). Castration of male rats reduced the amount of sex-associated protein fraction in the liver extracts and administration of testosterone restored the level, whereas estradiol caused a further decrease of the protein concentration. In intact male rats, testosterone slightly depressed while estradiol significantly decreased the amount of sex-associated protein in the liver extracts. On the other hand, sex-linked protein component appeared in intact as well as in the ovariectomized female rats after testosterone treatment (Bond, 1961, 1962; Barzilai and Pincus, 1965). The absence of the sex-associated protein component in immature male rats and its presence in testosteronetreated females, indicates that it is not a sex-chromosome-linked protein but a sex-hormone-dependent protein component. 2. Sex-Associated Urinary Proteins

It has been known for many years that both mice and rats normally excrete a large quantity of protein in their urine, the male has a higher level of proteinuria than the female and there are no differences in prepubertal animals (Bell, 1933; Shih, 1935; Wicks, 1941; Sellers et al., 1950; Roy and Neuhaus, 1967). The major urinary component of the sex-associated protein was found to be the prealbumin in the mouse (Thung, 1956, 1962; Bao-Linh et al., 1964) and a,-globulin in the rat (Bao-Linh et al., 1965; Roy and Neuhaus, 1966a). A higher concentration of the sex-related urinary protein in males than in females could also be found in the serum, though a t a much lower level than in the urine, and also in the extracts of rodent kidney and liver (Rumke and Thung, 1964; Roy and Neuhaus, 1966b). It is now clear that the sex-dependent urinary protein originated from the liver, in which it was released to the plasma, and was excreted by the kidneys into the urine (Finlayson et al., 1965; Roy and Neuhaus, 196613; Rumke et al., 1970). Whether this sex-related protein synthesized in the liver and excreted in the urine is the same as the sex-associated protein component discovered by Bond (see Section II,B,l), we still do not know. In male rats or mice, castration before puberty produced more severe damage of urinary protein excretion than that in adult life; giving testosterone to them raised the level to that of the normal excretion; the

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level declined after withdrawal, whereas administration of estradiol to normal males completely abolished the male-specific urinary protein excretion. In female rodents, ovariectomy did not affect the quantity of protein excretion but was significantly increased after testosterone treatment; withdrawal of the hormone reduced the excretion (Sellers et al., 1950; Finlayson et al., 1963; Roy and Neuhaus, 1967). However, administration of testosterone to spayed and hypophysectomized female rats completely failed to produce a response, an observation suggesting that the increased effcct of testosterone on the sex-linked urinary protein excretion might be mediated through the pituitary (Kumar et al., 1969). 3. Carcinogen-Binding Proteins I n 1964, Weisburger et al. reported that more radioactivity of 14Clabeled N-OH-FAA was bound to the liver protein in males than in female rats. A higher level of hepatic protein-bound methionyl-FAA (a metabolite of the carcinogen) in the liver of male, as compared with female rats, was also observed after intraperitoneal or stomach tube administration of N-OH-FAA. However, this sex difference of protein binding of fluorenyl carcinogen was not seen in immature rats. Gonadectomy of male and female rats yielded no significant alteration. Testosterone when given to either intact or ovariectomized female rats caused 50% increase in the protein-bound methionyl-FAA derivatives in the liver whereas estradiol treatment of normal or gonadectomized male and female rats reduced the binding to 1&20% of those in the controls. Adrenalectomy of male rats did not affect hepatic protein binding to the carcinogen, but thyroidectomy and hypophysectomy significantly decreased the binding (De Baun et al., 1970). Hypophysectomy also reduced the amount of amino acid incorporation into the liver microsomes, and treatment of hypophysectomized rats with growth hormone or a combination with triiodothyronine restored the level (Korner, 1965 ; Tata and Williams-Ashman, 1967).

C. LIPIDS Lipid constructs a higher proportion of cellular membranes. For example, the hepatic microsomal membranes consisting of fragments of the endoplasmic reticulum are mainly phospholipids (Favarger, 1963). Changes of microsomal membranes during chemical carcinogenesis were reported in the rat liver and in actual hepatomas (Arcos and Arcos, 1958). The alteration of membrane permeability may allow the entry of more carcinogen or its metabolites into the cell. Moreover, many enzymes, such as cytochrome c oxidase, DPNH-cytochrome c reductase, etc., contain predominantly phospholipid (Marinetti et al., 1958;

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Fleischer et al., 1961). In fact, phospholipid makes up about 80% of the liver lipid (Ostwald et al., 1965). Female rats tend to have a higher body fat content than male rats, guinea pigs, and mice (Boycott and Damant, 1908). Loeb and Burr (1947) also reported that female rats stored more fat than males when fed on an essentially fatty acid-deficient diet. On the other hand, when rats were fed a fat-free diet, males showed more severe dermal symptoms than did females (Ostwald et al., 1965), and the amount of essential fatty acid required for the male was greater than for the female (Greenberg et al., 1950; Pudelkewicz et al., 1968). A significantly higher proportion of stearic acid in the adipose tissue of men than of women has also been reported (Insull and Bartsch, 1967; Jagannathan, 1969). Therefore, a sex difference in lipid metabolism exists. As far as the liver is concerned, male rats had about 60-70 mg more phospholipid than did females during the period of rapid growth on an essentially fatty acid-deficient diet (Ostwald et al., 1965). The higher content of phospholipid in the liver of males was due to the higher proportion of palmitic acid and eicosatrienoic acid. On the other hand, females maintained a greater proportion of stearic acid as compared with males (Okey et al., 1961; Ostwald et al., 1965). Morin (1965) showed a decrease of stearic acid in the choline-containing phospholipids, particularly in the lecithins of the liver microsomes of male rats when fed on a fat-free diet containing a carcinogen, 4-dimethylaminoazobenzene. There are two metabolically different types of lecithins in rat liver, one with predominantly palmitic acid and linoleic acid that incorporated 32Pmore rapidly than did the other type, which contain mainly stearic acid and arachidonic acid (Collins, 1960; Harris and Robinson, 1960). A greater incorporation of 32Pinto the lecithin (Holtzman and Gillette, 1968) and phosphatidylethanolamine (Holtzman et al., 1970) of liver microsomal phospholipid in male rats than in females has also been reported. Lee et al. (1962) demonstrated that the uptake of 32Pwas significantly greater in the liver phospholipid of tumor-bearing mice than of normal ones. An increase of 32Pincorporation into the liver phospholipid was also found to be correlated with mitotic activity in rats (Levin et al., 1955, 1956, 1958). Neonatal administration of testosterone significantly increased the uptake of 32Pinto the hepatic microsomal phospholipid of intact and ovariectomized females as well as in the castrated male rats whereas estradiol produced no effect (Toh, 1971a). Hypophysectomy decreased 32P incorporation into liver phospholipid of rats (Scott, 1968). Tata and Williams-Ashman (1967) also reported a marked reduction in the amount of phospholipids in all three rough microsomal subfractions of rat liver after hypophysectomy, and the fall could be

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restored by a combined trcatmeiit of hypophysectomized rats with growth hormone and 3,3,5-iodo-~-thyronine. Growth hormone alone was also able to restore hepatic lipid metabolism of hypophysectomized rats, and the increases were mainly in the lecithin and cephalin fractions (Liberti et al., 1970). Sex differences were also reported in other lipid metabolism (Ostwald et al., 1965; Cardoso et al., 1968; Christiansen et al., 1969; Hay and Gray, 1970).

D. NUCLEICACIDS The nucleus contains most of the genetic materials encoded in the strands of DNA and directs specific cellular functions, such as protein synthesis via RNA. I n seeking the explanation of complete or a t least partial hereditary loss of controls of growth that characterizes neoplasms, attention has been focused on the role of the nucleic acids in carcinogenesis. It has been shown that normal cells could be transformed into neoplastic cells by exposing them to nucleic acids of malignant cells. Thus, administration of RNA or DNA isolated from hepatoma (De Carvalho and Rand, 1961; Cantarow et al., 1966), parotid gland tumor (Hays and Carr, 1962), kidney tumor (Jones, 1964) and leukemic tissue (Latarjet et al., 1958; Bielka and Graffi, 1959) produced tumors in rats and mice. Hormonal effect, on nucleic acid metabolism has been widely investigated in various kinds of tissues; for detail, refer to reviews of Tata (1966,1967). 1. Ribonucleic Acid ( R N A )

A sex difference in RNA binding to carcinogen has been reported, in which male rats had a higher specific radioactivity than female rats in liver transfer and ribosomal RNA after a single injection of FAA-9-14C or N-OH-FAA-9-I4C (Irving et al., 1970; Irving and Veazey, 1971). It also appeared that in male rats 72% of the fluorene residues was bound to ribosomal RNA, and in females only 33% was bound. Recently, Fink et al. (1970) and Agarwal and Weinstein (1970) reported that this covalent attachment of the fluorene residues to the transfer RNA modified their amino acid acceptance capacities, codon recognition, and ribosomal binding. The in vivo incorporation of uracil-2-'*C (Cantarow et al., 1958), glycine-2-14C (Kodama and Kodama, 1970), and cytidine-5-8H (Fujii and Villee, 1968) into the liver RNA of rats were also markedly increased after the administration of testosterone. Kidson and Kirby (1964) observed an increased synthesis of messenger-RNA in the liver of both male and female rats after the administration of testosterone,

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whereas stilbestrol produced no significant effect in males but increased mRNA synthesis in females. Castration of males rats decreased the specific activity of DNA-dependent RNA polymerase in the liver nuclei, and administration of testosterone to the castrated rats returned it to normal (Widnell and Tata, 1966). Villee and Fujii (1968) showed that administration of RNA isolated from the liver of adult male rats into the seminal vesicle stimulated growth and protein synthesis, but was not as effective as the RNA extracted from seminal vesicle itself. On the other hand, RNA isolated from the liver of adult female rats produced no effect. A single injection of testosterone into neonatal female rats caused a higher uptake of 32Pinto the liver nuclear RNA, while a single administration of estradiol into male neonates lowered the rate of incorporation (Toh, 1 9 7 1 ~ ) . Hypophysectomy or thyroidectomy caused a marked reduction in the amount of radioactivity fSzPor I4C) incorporated into RNA (Tata and Widnell, 1966; Tata and Williams-Ashman, 1967; Gupta and Talwar, 1968), DNA-dependent RNA polymerase (Widnell and Tata, 1966; Tata and Widnell, 1966), and polyribosomes (Staehelin, 1965; Tata and Widnell, 1966; Garren et aZ., 1967) of rat livers. Treatment of growth hormone, thyroid hormone or a combination of growth hormone and thyroid hormone stimulated their synthesis in the liver of these hypophysectomized or thyroidectomized rats. 2. Deoxyribonucleic Acid ( D N A ) Males had a higher DNA content than females in the liver of rats fed 3’-methyl-4-dimethylaminoazobenzene(Li et al., 1965). A higher activity of DNA in cancerous livers than in normal ones has also been reported (Troll et al., 1968). Ove et al. (1969) showed that the increase of DNA synthesis in rat hepatoma was characterized by a rise in key anabolic enzyme activity, the DNA polymerase and a fall in catabolic enzyme activity, the deoxyribonuclease. Changes in DNA content per nucleus parallel the degree of ploidy of chromosomes, and it is generally accepted that the nucleic of the parenchymal cells of mammalian liver have DNA contents in the ratio of diploid (2N), tetraploid (4AT),octaploid ( 8 N ) , . . . (Helweg-Larsen, 1952; Epstein and Gatens, 1967). These various levels of ploidy also correspond in ratio with the cell volume (Doljanski, 1960; Epstein, 1967), nuclear volume (Swartz, 1956 ; Epstein and Gatens, 1967), nucleolar volume (Shea and Leblond, 1966; Nadal, 1967), and nuclear histone level (Alfert and Geschwind, 1953) of hepatocytes. Swartz et al. (1960) found a higher percentage of octaploid nuclei in the liver of male than of female rats. The development of octaploid nuclei, however, was asso-

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ciated with sexual maturation, because neonatal castration of male rats produced a more significant inhibition of growth and polyploidization of liver cells than did castration of males at weaning (Swartz et al., 1960; Swartz, 1962). On the other hand, Swartz and Sams (1961) did not show any significant effect on liver polyploidization when intact newborn rats were given very large amounts of testosterone (500 pg, 6 times a week) or estrone (80 pg, 3 times a week) for 80 days. These authors interpreted the failure as a dosage phenomenon, for large quantities of testosterone might actually inhibit body growth (Rubinstein et al., 193933) while small doses of testosterone produced a significant change in growth rate (Rubinstein and Solmon, 1941). In fact, giving a single subcutaneous injection of 500 pg of testosterone propionate within 24 hours after birth markedly increased the liver nuclear volume of female rats whereas 250 pg of estradiol caused a drop in both males and females (Toh, 1971b). Thyroidectomy prevented the formation of polyploid nuclei and thyroidectomy plus castration showed as many polyploid nuclei in the liver as the thyroidectomized intact male rats, indicating that the increased production of octaploid nuclei during sexual maturation was not due to the direct action of testosterone on liver polyploidization (Carri&e, 1962). Treatment of thyroidectomized rats with thyroxine led to an increase of polyploid nuclei in rat liver. Hypophysectomy interrupted the progress of polyploidization in the liver nuclei and administration of sex hormones to the hypophysectomized-castrated rats did not alter the ploidy distribution (Hoffmann and Swartz, 1962). Administration of thyroxine to the hypophysectomized rats also failed to produce any effect whereas when growth hormone was given the number of polyploid nuclei in the liver of hypophysectomized rats increased ; administration of growth hormone to the thyroidectomized rats also increased the formation of polyploid nuclei (CarriBre, 1962). Apparently, growth hormone is the main important anterior pituitary hormone responsible for the general polyploidization of the liver nuclei (Helweg-Larson, 1952; Leuchtenberger e t al., 1954; Di Stefano e t al., 1955; Di Stefano and Diermeier, 1956; Geschwind e t al., 1958) and the effects of sex steroids and thyroid hormone are mediated via the pituitary rather than by direct action on the liver (Hoffmann and Swartz, 1962; CarriBre, 1962). Ill. Sex Differences in Carcinogenesis

Men always have a higher incidence of cancer than women, apart from cancer of the reproductive organs and a few other exceptions (Shimkin, 1965; Ashley, 1969a,b). A similar situation of sex differences in the incidence of tumors is also found in animals fed various carcinogens,

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particularly the sex difference in the incidence of liver tumors, which will be described in more detail in the following section. A. HEPATIC TUMORS The incidence of primary liver cancer has been reported to be higher in men than in women (Bonne, 1935; Hall and Sun, 1951; Yen and Cowdry, 1954; Chung and Ch’en, 1957; Barrera and Dalmacio-Cruz, 1958; Marsden, 1958; Berman, 1958; Higginson and Oettle, 1960; Muir, 1961; Higginson, 1963, 1970). This sex difference is not limited to the high-incidence areas of Africa and Asia-for example, in Singapore the ratio men:women is 7.2-but is also found in the lower-incidence areas of America and Europe-for example, in Liverpool, the ratio is 5.0 (Doll et al., 1966). Sex differences in the incidence of primary liver cancer were also found in animals, particularly rodents. Thus, male rats (Kirby, 1947; Rumsfeld et al., 1951; Morris and Firminger, 1956; Sidransky et al., 1961; Bielschowsky, 1961) or mice (Leathem, 1951 ; Kirschbaum, 1957; Heston and Vlahakis, 1968; Hancock and Dickie, 1969; Biancifiori, 1970) have a greater susceptibility to hepatocarcinogenesis than females, whether spontaneous or chemical carcinogen induced. This sex difference in liver tumor incidence still remained even when a single dose of carcinogen was administered subcutaneously or by stomach tube to newborn animals (Toth, 1968; Epstein and Mantel, 1968; Porta and Terracini, 1969 ; Vesselinovitch, 1969). The difference, however, was dependent upon the dose of carcinogen given to the animal and the duration of the experiment, for, when given a higher dose or a longer period of treatment, the female achieved as high an incidence of liver tumors as the male (Stasney et al., 1947; Bielschowsky, 1961 ; Biancifiori, 1970). Castration of adult male rats decreased liver tumor incidence, and administration of testosterone restored the susceptibility (Firminger and Reuber, 1961 ; Morris and Firminger, 1956). Giving testosterone to intact male rats also intensified tumor development (Stasney et al., 1947; Firminger and Reuber, 1961). On the other hand, male rats receiving diethylstilbestrol had a lower liver tumor incidence (Reubner and Firminger, 1962). Castrated male rats treated with diethylstilbestrol also showed more resistance (Morris and Firminger, 1956). By contrast, progesterone increased the incidence of heptocellular carcinoma in intact and castrated male rats (Reuber and Firminger, 1962). In female rats, ovariectomy produced no differences from those of the intact group (Morris and Firminger, 1956) . However, administration of testosterone accelerated the development of neoplastic hepatic lesions in both intact and spayed female rats (Firminger and Reuber, 1961). Diethylstilbestrol,

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when given to female rats failed to have any effect on liver tumor incidence (Reuber and Firminger, 1962), but Stasney et al. (1947) reported that estradiol dipropionate did cause some effect on liver tumor formation. Progesterone also enhanced the incidence of hepatocarcinoma in castrated female rats but did not affect the incidence in intact females (Reuber and Firminger, 1962). Neonatal injection of testosterone also has been reported to increase liver tumor incidence in female rats while estradiol given a t birth to male rats decreased hepatic tumor incidence (Weisburger et al., 1968). Recently, Toh (1972) showed that subcutaneous implantation of testosterone pellets produced N-2-fluorenylacetamide-induced hepatic tumors in neonatally castrated male and female rats whereas intrasplenic implantation of testosterone exerted no effect. Adrenalectomy has been reported to decrease or completely block carcinogenesis (Perry, 1961; Reuber, 1965a). Administration of cortisone to either adrenalectomized or intact male and female rats produced no appreciable effect in altering the hepatocarcinogenesis (Firminger and Reuber, 1961; Reuber, 1969) but deoxycorticosterone acetate had a striking protective effect against hepatocarcinomas both in adrenalectomized and in intact rats (Symeonidis et al., 1954; Firminger and Reuber, 1961; Reuber, 1965a). Thyroidectomy completely inhibited the development of hepatocellular carcinomas (Bielschowsky and Hall, 1953; Goodall, 1965). Treatment of thyroidectomized rats with thyroid powder (Reuber, 1965b) or iodide (Goodall, 1965) restored the carcinogenic effect in rat livers. Although thyroidectomy prior to the administration of carcinogen prevented hepatoma formation, thyroidectomy performed after 14 weeks (Bielschowsky and Hall, 1953) or 23 weeks (Reuber, 1969) of carcinogen feeding offered no protection. Hypophysectomy completely blocked carcinogenesis in rat liver with every chemical carcinogens except dimethylnitrosamine (Skoryna, 1955; Griffin et al., 1953; Goodall and Butler, 1966, 1969; Lee and Goodall, 1968; Weisburger, 1968). Hypophysectomy of rats 14 weeks (Skoryna, 1955) but not 23 weeks (Reuber, 1969) after the initiation of carcinogenesis also totally prevented the conversion of N-2-fluorenylacetamideinduced hyperplastic hepatic cells into malignant tumors. Administration of growth hormone to the hypophysectomized rats restored the hepatic carcinogenic activity of azo dye (Robertson et at., 1954; Griffin et al., 1955). Adrenocorticotropin, thyrotropin, and gonadotropin were also able to produce some effect (Robertson et al., 1954). On the other hand, O’Neal et al. (1958) claimed that administration of ACTH did not produce any signs of N-2-fluorenyldiacetamide action in the liver of hypophysectomized rats. Thyrotropic hormone when given to the hypo-

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physectomized rats was also reported to exert no azo-dye carcinogenic effect a t all (Griffin et al., 1955). Implantation of the pituitary tumor MtTJF4 as a source of hypophysial hormones significantly shortened the latency period and increased the incidence of N-OH-FAA-induced hepatomas in male and female rats (Weisburger and Yamamoto, 1964). The relevance of the influence of pituitary, thyroid, and adrenal hormones to that of androgen in liver carcinogenesis will be discussed later.

B. EXTRAHEPATIC TUMORS Sex also plays a significant role in carcinogenesis other than in the liver. Thus, a higher incidence of skin cancer (except melanoma) in men than in women has also been reported (Carmichael, 1961; Carmichael and Silverstone, 1961). I n rats, 3-methylcholanthrene (Gruenstein et al., 1966) or N,N-2,7-fluorenylenebis-2,2,2-trifluoroacetamide (Morris et al., 1963) induced cutaneous neoplasma were found to be male specific. I n mice, males were more susceptible than females to skin carcinogenesis initiated by a small dose of 7-12-dimethylbenz [alanthracene and promoted by croton oil (Bates, 1968). I n hamsters, skin tumors were induced in males to a greater extent than in females by 9,10-dimethyl-1,2benzanthracene (Shubik et al.,1960). It is also a well established fact that men develop lung cancer more frequently than women, and the male to female sex ratio ranges from 1.5:1 (Lee and Ts’o, 1963) to 9.2:l (Shinton, 1963). A large number of statistical studies have been published showing that there was a casual relationship between cigarette smoking and bronchogenic carcinoma in the human. I n view of the different external social environment between two sexes, a higher frequency of lung cancer seems to be due to a higher consumption of the “carcinogen,” i.e., cigarette smoking in men than in women, but in an identical animal experimental condition, after exposure to the similar inhalation of cigarette smoke, male mice still developed a higher incidence of lung tumors than females (Leuchtenberger et al., 1963). Moreover, a greater incidence of lung cancer was found in men than in women among non-cigarette smokers (Haenszel and Taeuber, 1964). Mori-Chavez (1962) also reported that the incidence of spontaneous pulmonary tumors was higher in male mice than in female mice. On the other hand, among surgically treated patients with localized cancer of the lung, the survival rate was substantially higher in women than in men (Connelly et al., 1966). There are many factors that could determine resistance or susceptibility of the respiratory tract to cigarette smoke. Haenszel and Taeuber (1964) suggested that the sex difference in the incidence of lung cancer was probably due to a steroidal factor rather than to the amount of intake carcinogen.

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A greater incidence of salivary gland tumors in men than in women was found in Malaya and Singapore (Loke, 1967; Tock e t al., 1970), but the opposite was observed in Uganda (Davies et al., 1964). Ashley (1969b) and Evans and Cruickshank (1970), however, did not find significant sex differences in the incidence of salivary gland tumors in the white population in either the United States or Europe. A preponderance in the incidence of salivary gland tumors in males was also found in rats given 9,10-dimethyl-1,2-benzanthracene(Cherry and Glucksmann, 1965). Castration decreased the incidence of salivary gland tumors; administration of testosterone increased and accelerated the induction while stilbestrol inhibited and retarded rat tumor formation (Glucksmann and Cherry, 1966). Cancer of the lip, buccal cavity, pharynx, and esophagus were found to be more common in men than in women (Millar, 1961; Cohen and Hoe, 1961 ; Shanta and Krishnamurthi, 1963). A male-predominant nasopharyngeal carcinoma was reported in Asians and Africans (Muir, 1962; Linsell, 1964; Buell, 1965 ; Loke, 1966; Shanmugaratnam, 1971). Mortality from cancer of the stomach was remarkably high in men of Chile, Japan, and Iceland (Sigurjonsson, 1966). The incidence of rectal cancer was higher in men than in women, but this sex difference was reversed in the case of cancer of large intestine (Ederer e t al., 1961; Ashley, 1970). A greater incidence of spontaneous tumors of the gastrointestinal tract was also reported in male than in female hamsters, and castration significantly decreased the incidence (Fortner, 1961). Cancer of the pancreas was seen to be significantly more prevalent in men than in women (Muir, 1961). Spontaneous pancreatic tumors were also found predominantly in male rats (Rowlatt and Roe, 1967) and in male hamsters (Fortner, 1961). A sex difference in the incidence of estrogen-induced kidney tumors has also been reported in hamsters. Thus, prolonged treatment of estrogen led to the induction of malignant renal tumors in the male, but rarely in the female hamster (Kirkman and Bacon, 1950; Kirkman and Wurster, 1957). However, there was no fundamental genetic difference between the kidneys of male and that of female hamsters with respect to their susceptibility to the tumorigenic properties of estrogen, because tumors still developed in the subpannicular transplanted kidney fragment from newborn female hamster into estrogen-treated male host. Renal tumors might also be induced by estrogen treatment in immature or ovariectomized adult female hamsters. Neonatal administration of testosterone to female hamsters produced kidney tumors in adult life after continuous estrogen treatment. Tumors could also be produced in the kidney of adult female hamsters by starting estrogen treatment dur-

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ing metestrus, indicating that it was the high lcvel of progestcrone that normally prevented the induction of kidney tumors in estrogen-treated female hamsters (Kirkman, 1959). From clinical statistical observation, the incidence of bladder cancer was more common in men than in women (Ashley, 1969b). Armstrong and Bonser (1947) and Foulds (1947) also found a greater susceptibility to the induction of bladder tumors by hr-2-fluorenylacetamide in males than in female mice of RIII, IF, and White Label strains. In contrast, Clayson and Bonser (1965) oberved a higher incidence of bladder tumors in females than in male AbXIF mice after oral administration of 1,4ethylsulfonylnaphthalene- l-sulfonamide. Pituitary tumors in mice have been reported to have a greater incidence in males than in females (Jacobs and Huseby, 1967). In rats, males developed a higher percentage of mammo-somatotropic and gonadotropic pituitary tumors than females (Yokoro et al., 1964; Griesbach, 1967). Stocks (1969) observed a greater susceptibility of men to pituitary and adrenal diseases than of women except between 35 and 55 years of age. Sex is also an important factor in leukemogenesis. Male preponderance in this type of cancer has been reported in Japan (Nishiyama, 1970), the United States, and England and Wales (Tivey, 1954; Fraumeni and Wagoner, 1964; Stark and Oleinick, 1966). The influence of gonads on leukemogenesis in the mouse, however, varies from strain to strain. Jenkins et al. (1966) and Jenkins and Upton (1963) showed that male mice of the RF strain were more susceptible to leukemia induction by cell-free filtrates or by radiation than were females, and estradiol treatment prolonged the latency and lowered the incidence of myeloid lcukemia. On the other hand, the incidence of spontaneous and methylcholanthrene-induced leukemia was found to be greater in females than in males of C58 and DBAJ2 strains of mice (Liebelt and Liebelt, 1962). In contrast to the above male predominance in the incidence of cancer, women tended to be more susceptible to thyroid cancer than men (Meyer, 1962; Stocks, 1969). Spontaneous tumors of the thyroid gland in mice were also found to be higher in females than in males (Jones et al., 1966). Stocks (1969) showed a greater incidence of malignant melanoma of the skin in women than in men. In hamsters (Lipkin, 1970), transplanted melanoma grew faster in the female, while the opposite occurred in mice (White, 1959). IV. General Discussion

The striking difference in the incidence of cancer between males and females is of great interest, because this may lead to the elucidation of some factors possibly relevant to the etiology of carcinogenesis. Such

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neoplasms often arise in tissues not usually considered to be either primary or secondary sex organs, yet make their presence known to be greatly influenced by the secretion of the gonad. Liver is a very good example, and a vast knowledge of morphology, biochemistry, pharmacology, and physiology has accumulated on it under normal and pathological conditions. At first sight, the sex difference in the incidence of cancer might be thought to be due to the different environments in which the two sexes live and work. In modern western countries, men have a greater exposure to the industrial potentially carcinogenic substances, but a higher incidence of liver cancer in men is still found in those underdeveloped countries. Moreover, the incidence rate is lower in Europe and North America than in Africa and Asia. The higher incidence of liver cancer in indigenes of oriental countries may be attributable to helminthic infections of the liver, but Bonne (1935) did not find this to be the case in Indonesia, and other investigators (e.g., Chung and Ch’en, 1957; Barrera and DalmacioCruz, 1958) consider this to be unlikely. Alcoholism frequently induces liver damage, and sometimes even cirrhosis or hepatoma. Social factors that result in higher consumption of alcohol by men than by women might explain predominance in the incidence of liver cancer in males, but this is not the case in the Far East; for example, the Malay people consume very little, if any, alcohol (Bonne, 1935). The very strongly spiced food, particularly of Malays and Indians, has been suspected to contain certain carcinogenic substances. Men and women consume the same food, so why should men have a greater incidence of liver cancer than women? Furthermore, the diets of African people do not usually contain spices, yet liver incidence is very high in South African Bantu (Berman, 1958). In contrast, Mexicans eat much spiced food, but the incidence of primary liver cancer is relatively low in Central and South America (Higginson, 1963, 1970). Perhaps, men eat more than women, and therefore a higher incidence of liver cancer in men is due to greater uptake of “carcinogens” in the food. However, Rumsfeld et al. (1951) reported that the sex difference in liver tumor incidence of rats was not influenced by the amount of intake of food or carcinogen. Moreover, there were no differences between male and female rats in the absorption of carcinogen from the stomach (Purchase and Steyn, 1969). Although malnutrition has been considered to be one of the causes of cirrhosis, and perhaps also of primary liver cancer in Africa and certain parts of Asia, it would not explain the differences in the incidence of liver cancer between men and women who suffered from the same deficiency. On the other hand, malnutrition also occurs in the Caribbean and Central and South America, where liver cancer is not prevalent (Higginson, 1963,1970). Furthermore, protein and

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caloric deficiencies in most animal experimental conditions decrease rather than increase tumor incidence (Ross and Bras, 1965; Tannenbaum and Silverston, 1953). If all the factors of the external environment discussed above are responsible for the higher incidence of primary liver cancer in men, then in identical animal experimental conditions, there should not be any difference in the incidence of hepatocellular carcinoma between the two sexes; however, as discussed in Section 111, a similar pattern of sex difference in the incidence of hepatic tumor is still found in mice and rats fed similar amounts of various chemical carcinogens. A sex difference in the incidence of cancer is also found in prepubertal children whose environment does not differ very much (Bigelow and Wright, 1953). In mice and rats also, administration of a small dose of chemical carcinogens shortly after birth produced a greater response in males than in females (see Section 111).Apparently, the difference in the external environments between males and females would not of itself account for the sex difference in the incidence of cancer. It has been suggested by Ashley (1969a,b, 1970) that a lower frequency of cancer in the female is due to her greater immunological POtential and that the gene concerned with immunity is located on the X chromosome, rather than on the Y chromosome of the male. As discussed in Section I,F, it has been proved that the gene which controls the development of a sex-linked antigen of the skin graft rejection is located neither on the X chromosome (Celada and Welshons, 1963) nor on the Y chromosome (Engelstein, 1967; Polackova, 1969). The important role of the genetic factor in carcinogenesis is very complicated. Clear-cut evidence for its direct participation has not yet been demonstrated, and it can only be considered under the most stringent conditions. The genetic control of tumorigenesis was reported to reside largely in the end organ (Heston and Dunn, 1951; Trentin and Gardner, 1958; Dux and Riluhlbock, 1966). If the genetic influence is not effective through systemic mechanism but is localized in the organ, and the gene controlling the occurrence of tumors has its primary action in the tissue, then transplantation of an organ to its opposite sex should retain its sex-chromosomal controlling mechanism over its susceptibility toward carcinogen. But the contrary is found, for example, in the kidney: when a piece of it was transplanted from a newborn female hamster into a male host, there was no fundamental genetic difference between the kidney of males and that of females with regard to susceptibility to the tumorigenic properties of estrogen (Kirkman, 1959; see also Section 111). On the other hand, neonatal administration of testosterone to female hamster induced a “male type” susceptibility of the female kidneys toward “carcinogen” (Kirk-

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man, 1959). Therefore, by analogy, it could be argued that the difference in the incidence of cancer in the nonsexual organs between males and females is not primarily determined by the X or Y chromosome. Taking adrenocortical steroids metabolizing enzymes as a criterion, the liver, like the hypothalamus and skin graft (see Section I), is undifferentiated a t birth and potentially “feminine.” The presence of a male sex hormone induces a “male type” of hepatic enzyme system and its absence results in the development of a “female type” of liver (De Moor and Denef, 1968; see also Section 11).If the “sex of the liver” is not determined by the X or Y chromosome, but by its hormonal environment during the neonatal life, then alteration of its endocrine status a t birth should be able to change its sensitivity toward carcinogen. Indeed, administration of testosterone to newborn female rats increased the incidence of N-hydroxy-2-fluorenylacetamide-inducedhepatic tumors, whereas estradiol given to newborn male rats decreased the subsequent incidence of tumors (Weisburger et al., 1968). Obviously, hormones play a very important part in the incidence of cancer between males and females. As it appears that sex hormones play a leading role in the sex difference of hepatocarcinogenesis, it could possibly be either the presence of the testosterone that induces a higher incidence of hepatic tumors or the presence of the estrogen that protects the liver against tumor development. Reuber and Firminger (1962)clearly demonstrated that in gonadectomized male and female rats, whether given diethylstilbestrol or not, there were no differences in liver tumor incidence. Morris and Firminger (1956) even showed a similar resistance among intact females, ovariectomized females, castrated males and castrated male rats treated with diethylstilbestrol. Administration of diethylstilbestrol to the intact male rats decreased the liver tumor incidence as compared with the controls (Reuber and Firminger, 1962),perhaps owing to its antiandrogen properties (Lerner, 1964). Thus, estrogen seems to have very little effect on hepatocarcinogenesis. On the other hand, when testosterone was given to the gonadectomized or intact male and female rats, it greatly increased the incidence of hepatic carcinoma (Morris and Firminger, 1956; Firminger and Reuber, 1961). Apparently, therefore, the presence of testosterone intensified liver tumorigenesis. If testosterone is responsible for the higher incidence of the liver cancer in male rats, then the question arises whether testosterone has a direct or an indirect effect via other endocrine glands on liver carcinogenesis, because castration only reduced the tumor incidence but was unable to protect against tumor development (Rumsfeld et al., 1951; Morris and Firminger, 1956; Firminger and Reuber, 1961; Reuber and Firminger, 1962),whereas thyroidectomy or hypophysectomy completely

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inhibited the liver tumor formation (Bielschowsky and Hall, 1953; Griffin et al., 1953; Skoryna, 1955; Goodall, 1965; Weisburger, 1968). Recently,

Toh (1972) implanted testosterone pellets either into the subcutaneous tissue or into the spleen showed that testosterone was not effective directly in promoting hepatic carcinogenesis, but was effective indirectly, perhaps through some other endocrine glands. The possible mechanism of the action of testosterone on liver tumorigenesis is indicated in Fig. 1. Adrenalectomy had been shown to decrease or completely block liver tumor development (Perry, 1961), but administration of testosterone to the adrenalectomized rats increased tumor incidence (Firminger and Reuber, 1961). Therefore, the possibility of involving the adrenal gland in the effect of testosterone on hepatocarcinogenesis seems unlikely. Furthermore, administration of cortisone produced no appreciable effect (Firminger and Reuber, 1961; Reuber, 1969), and deoxycorticosterone acetate even decreased liver tumor incidence (Symeonidis et al., 1954; Firminger and Reuber, 1961; Reuber, 1965a). Thyroidectomy completely suppressed liver tumor formation in rats fed either X-2-fluorenylacetamide or 2-fluorenamine (Bielschowsky and Hall, 1953; Goodall, 1965, 1966). Administration of testosterone to thyroidectomized rats also failed to have any carcinogenic effect (Reuber, 1965b, 1966) suggesting that the effect of testosterone might be mediated via the thyroid gland. On the other hand, hypophysectomy had also been reported to inhibit totally the hepatic tumorigenicity of several powerful carcinogens of azo dye, aflatoxin, and aminofluorene groups (Griffin et al., 1953; Skoryna, 1955; Goodall and Butler, 1966, 1969; Weisburger, 1968) ; testosterone was also ineffective in restoring the actvity (Robertson et al., 1953, 1954; Griffin et al., 1955). This does not mean, however, that the effect of testosterone was directly mediated through the pituitary, because even in the presence of the hypophysis, administration of testosterone to thyroidectomized rats did not exert any neoplastic effect (Reuber, 1965b). On the other hand, testosterone was effective when

FIQ.1. The possible mechanism of action of testosterone on liver carcinogenesis.

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given simultaneously with thyroid hormone to thyroidectomized or thyroidectomized-castrated rats (Reuber, 1966). Therefore, it seems likely that the effect of testosterone on liver carcinogensis might be mediated via the thyroid secretion rather than the pituitary. Thus, the problem arises whether the thyroid hormone has a direct effect on hepatocarcinogenesis. Administration of thyroid hormone in the drinking water to hypophysectomized rats failed to produce any signs of hepatic carcinogenic effect (Bielschowsky et al., 1962). Moreover, hypophysectomy of rats 14 weeks (Skoryna, 1955), but not 23 weeks (Reuber, 1969), after the initiation of carcinogenesis completely prevented the conversion of hyperplastic hepatic cells into malignant tumors whereas thyroidectomy after 14 weeks (Bielschowsky and Hall, 1953) or 23 weeks (Reuber, 1969) of carcinogen feeding failed to have a total inhibition of tumor development. Even after 23 weeks of carcinogen administration, the degree of protection against liver tumor formation was still greater in hypophysectomized rats than in thyroidectomized ones (Reuber, 1969).Therefore, thyroid hormone seems unlikely to have a direct effect on the liver tumor formation, although it may exert an indirect effect. Bielschowsky and Hall (1953) reported that the failure to develop liver tumors in thyroidectomized rats was due to the absence or decreased number of pituitary acidophils and therefore decreased the level of growth hormone secretion that would normally be required for the tumor development. A good correlation between prompt acidophil degranulation and the low level of growth hormone has already been shown (Schooley et al., 1966). Recently, Suzuki and Shibasaki (1970)reported that thyroidectomy decreased the amino acid incorporation into the pituitary growth hormone, and administration of thyroxine to the thyroidectomized rats restored the synthesis. I n fact, a hypophysis-mediated action of thyroxine on the body growth was clearly demonstrated in rats by Eartly and Leblond (1954)and Scow (1959). If the growth hormone is the main important anterior pituitary hormone responsible for liver tumor induction by chemical carcinogens, then an increase in the level of growth hormone should be able to increase tumor formation. Indeed, given growth hormone, the thyroidectomized rats developed N-2-fluorenylacetamide-induced hepatoma (Bielschowsky, 1958). Growth hormone also increased liver tumor incidence in both castrated male rats and ovariectomized female rats (Reuber, 1968). I n hypophysectomized rats, growth hormone was able to restore the activity of azo dye in liver tumor formation; however, other tropic hormones, such as ACTH, TSH, and gonadotropin, were also able to produce some effect (Robertson et al., 1954;Griffin et al., 1955). From the above discussion, i t seems likely that the effect of testos-

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terone on hepatic carcinogenesis may be mediated via the thyroid gland, which in turn acts on the pituitary-a mechanism similar to that of liver nuclear polyploidization (see Section II,D,2).I n fact, Reuber (1969) recently demonstrated that the degree of protection against tumor development due to the alteration of hormonal status in male rats of previously formed hyperplastic hepatic nodules was of a similar order (percentages of hepatic carcinoma formation : untreated control 10J12 = 83%, castration 9/13 = 69%, thyroidectomy 7/14 = 50%, and hypophysectomy 3/12 = 25%). In view of the neuroendocrinological effect on mammary tumor induction in rats (Welsch e t al., 1970) and mice (Montemurro and Toh, 1968) , a possible participation of the hypothalamus due to its control over the pituitary secretions in the liver carcinogenesis should not be neglected. Sex differences in the physiological functions of the hypothalamichypophysial system has already been described a t the beginning of this review, and the testosterone certainly plays a very important role in the sexual differentiation of the hypothalamus. Evidence showing the hypothalamic effect on the hepatocarcinogenesis is scanty or absent, but there are several ways in which the hypothalamus could be involved. Testosterone might act through the hypothalamus, which in turn acts on the pituitary to increase its secretion, presumably growth hormone, and therefore increase liver tumor incidence, but this mechanism would not be able to account for the complete inhibition of hepatic tumorigenesis after thyroidectomy (Bielschowsky and Hall, 1953 ; Goodall, 1965, 1966). It may be that testosterone increases the thyrotropic releasing factor from the hypothalamus, therefore increases pituitary TSH secretion. A higher level of TSH would certainly enhance thyroid hormone secretion, which in turn acts on the pituitary to increase growth hormone release. Alternatively, testosterone may act directly on the thyroid, which then causes an increase of somatotropic releasing factor from the hypothalamus, therefore increasing pituitary growth hormone secretion. However, investigations are required to ascertain the role of the hypothalamus in the formation and growth of liver tumors, before a precise mechanism of the action of testosterone can be discussed. It is generally believed that the action of hormones in hepatic carcinogenesis is to modify the metabolic activation of chemical carcinogens and therefore controls the entire process. Biochemical differences between males and females in DNA, RNA, enzyme activity, protein component, and lipid metabolism have already been described in Section 11. If the effect of testosterone is mediated through the thyroid, the pituitary, and possibly the hypothalamus, then a physiological effect on liver tumor induction as a result of the alteration of these mediators should be correlated with a similar effect on the biochemical metabolism in the liver.

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Indeed, thyroidectomy or hypophysectomy had been reported to decrease the carcinogen-metabolizing enzyme activity (De Baun et al., 1970), protein binding to the carcinogen (De Baun et al., 1970), phospholipid metabolism (Cornatzer et nl., 1953; Scott, 1968), RNA turnover (Tata and Williams-Ashman, 1967 ; Gupta and Talwar, 1968), and DNA polyploidization (CarriBre, 1962; Hoffmann and Swartz, 1962) of the rat liver. Hypothalamic lesions also lowered the level of hepatic enzyme systems that metabolized p-dimethylaminoazobenzene (Shimazu, 1965) and hexobarbital (Nair et al., 1970). Administration of growth hormone increased all these biochemical parameters in the liver of rats (Geschwind et al., 1950; Carrihre, 1962; Korner, 1965; Tata and WilliamsAshman, 1967; Gupta and Talwar, 1968; Liberti et al., 1970). Electrical stimulation of the hypothalamus also elevated the activity of tryptophan pyrrolase of rabbit liver (Shimazu, 1962, 1964). Recently, the possible role of tRNA in the regulation of protein synthesis has been greatly emphasized (Martin, 1969; Sueoka and Kano-Sueoka, 1970), and Fink et al. (1970) and Agarwal and Weinstein (1970) even showed that covalent attachment of the carcinogen, N-2-fluorenylacetamide residues to the tRNA modified its amino acid acceptance capacities, coden recognition, and ribosomal binding. A significantly higher level of binding of FAA and N-OH-FAA to the tRNA in males than in females has also been reported in the rat liver (Irving et al., 1970; Irving and Veazey, 1971). Jackson et al. (1970) demonstrated that administration of growth hormone to the hypophysectomized rats produced two additional new species of tRNA which were not present in the liver of the control hypophysectomized animals. It is concluded that environmental factors may act like a “carcinogen” to induce cancer, the higher incidence of cancer in males than in females may be secondarily dependent on the sex chromosome in its role as a carrier of genetic messages leading to “maleness,” and the sex difference of hormonal pattern may very well be of primary importance in determining the metabolic activation of the “carcinogen” (available environmental potentially carcinogenic substances) and therefore seems likely to be the most important factor responsible for the curious distribution of cancer in nonsexual organs between men and women. ACKNOWLEDGMENT This work was supported by grants from the North-West Cancer Research fund.

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IMMUNODEFICIENCY AND CANCER1 John

H. Kersey, Beatrice D. Spector,

and Robert A. Good

Deporfments of Pathology and Pediatrics, University of Minnesota, Minneopolis, Minnesota and Sloon-Kettering Institute for Cancer Research, New York, New York

I. Introduction . . . . . . . . . . . . . 11. Experimental Models Linking Immunodeficiency and Cancer . A. Experimental Immunodeficiency and Neoplasms Induced by DNA Viruses B. Experimental Immunodeficiency and Neoplasms Induced by RNA Viruses . . . . . . . . . . . . C. Virus- and Chemical-Induced Immunodeficiency . . . D. Genetic Factors in Host Resistance to Tumors . . . . E. Graft versus Host Models of Immunodeficiency . . . . 111. Human Diseases . . . . . . . . . . . . A. Disorders with Abnormalities of Humoral (B-Cell) Immunity B. Human Disorders with Abnormalities of Cell-Mediated (T-Cell-Dependent) Immunity . . . . . . . . C. Other Genetic Human Disorders with High Risk of Cancer . D. Drug-Induced Immunodeficiency . . . . . . . E. Virus Isolation from Immunodeficient Individuals . . . References . . . . . . . . . . . . .

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

211 212 212 213 214 215 216 217 217 219 222 224 224 225

I. Introduction

Evidence indicating a relationship between the immunologic system and malignancy arose partially from a hypothesis presented by Thomas (1959). This hypothesis suggested that a major role of the immune system (and especially the cell-mediated system) may be in host defense against malignant cells. The postulate was later expanded by the suggestion that development of malignant cells is a frequent event and under normal conditions these cells are destroyed by immunologic control or surveillance mechanisms (Good, 1970; Burnet, 1971). Early discussions of immune surveillance suggested that immunodeficiency would be associated with earlier and more frequent cancers, especially of the lymphoreticular system, in experimental animals and man. Subsequent studies relating immunodeficiency and cancer are the subject of this review. 'Supported in part by grants from the American Cancer Society, the National Foundation-March of Dimes, and a contract from the Special Virus Cancer Program (NIH-71-2261). 211

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II. Experimental Models linking Immunodeficiency and Cancer

A. EXPERIMENTAL IMMUNODEFICIENCY AND NEOPLASMS INDUCED BY DNA VIRUSES Experiments relating the immune surveillance hypothesis were first reported by Vandeputte et al. (1963), who demonstrated that neonatally thymectomized rats inoculated with polyoma virus developed significantly more soft tissue sarcomas, especially in kidney and bone, than did nonthymectomized controls. Delay of virus inoculation until 2-3 weeks of age resulted in the development of cerebral hemangiomas but no sarcomas. Using the hamster model, Defendi et al. (1964) reported increased oncogenicity of polyoma virus in thymectomized animals. Later, Law and associates (Law, 1966) reported that certain strains of mice with greater immunologic competence (e.g., C57/B1) were more resistant to polyoma virus-induced tumors than less immunocompetent (e.g., C3H) strains. Allison and Taylor (1967) found that neonatally thymectomized rats of two strains inoculated with another DNA virus, simian virus 40 (SV40), developed more tumors after a shorter latent period than did nonthymectomized control rats. I n these studies, skin tumors induced with a chemical, 7,12-dimethylbenzanthracene,were not affected by neonatal thymectomy. More recent studies of experimental immunodeficiency and DNA virus-induced tumors have utilized antilymphocyte serum (ALS) . Data reported by Allison et al. (1967) indicated that tumors could be readily induced by adenovirus in susceptible (CBA) mice using either ALS or neonatal thymectomy. Angistein and co-workers (1965) demonstrated that antithymic cell sera increased the size of Sarcoma 180 implants and fostered growth of transplantable mammary adenocarcinoma. Allison and Law (1968) reported additional experiments using C3H mice in which neonatal ALS treatment and polyoma virus inoculation resulted in frequent development of tumors of parotid gland, mammary gland, and hair follicles. Under the same experimental conditions C57/B 1 mice developed frequent parotid tumors but no mammary or hair follicle tumors. These and other experiments demonstrated that ALS treatment is often more effective than neonatal thymectomy in enhancing the development of polyoma-induced neoplasms. Interesting observations were made by Gaugas and co-workers (1969), who reported that thymectomized and ALS-treated mice developed frequent polyoma-type tumors when treated with Mycobacterium lepra. These investigations indicate that experimental immunodeficiency enhances tumor induction by several DNA viruses, including SV40,

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adenovirus, and polyoma virus. Direct evidence of new antigenicity of cells transformed by DNA viruses was reported by Sjiigren (1964) and Habel (1965) in transplantation studies. Subsequent studies have indicated that tumors induced by DNA viruses generally are highly immunogenic despite the absence of any significant viral replication. Lymphocytes that are cytotoxic for tumor cells are often demonstrable during the latency period before evidence of tumor growth as well as throughout the course of the tumor (Sjogren and Borum, 1971). Of importance are studies of Vandeputte and De Somer (1965), Allison et al. (1967), and Law (1969), which demonstrated that antiviral antibody titers were the same in untreated and immunosuppressed animals and that immune lymphoid cells but not immune sera could reduce tumor frequency in immunodeficient animals. These data provide additional support for the postulate that immunodeficiency results in increased tumor growth because of decreased destruction of transformed cells rather than through effects on viral antibody or viral replication.

B. EXPERIMENTAL IMMUNODEFICIENCY INDUCED BY RNA VIRUSES

AND NEOPLASMS

Early work by McEndy, Boon, and Furth (1944) demonstrated that thymectomy often results in a decrease in mouse leukemogenesis. They showed that thymectomy of young adults of a high leukemic strain, AKR, drastically reduced the incidence of lymphoid leukemia. The viral etiology of AKR lymphoma was largely established by Gross (1957), who demonstrated that AKR lymphoma could be transmitted by cell-free extracts. The thymic dependency of Gross passage A lymphoma has been a matter of great interest. Law and Potter (1956) and Csrnes et al. (1956) reported that leukemia arising in thymectomized animals was not of donor origin when genetically identifiable thymic grafts were used to reconstitute capacity to develop malignancy. On the other hand, evidence provided by Kaplan and Brown (1954) and Kaplan e t al. (1956), Siegler and Rich (1963), and Goodman and Block (1963) established that in a t least certain strains of mice the first histologic changes of lymphoma occur focally within the thymus, extending throughout one of the thymic lobes and finally to the peripheral lymphoid tissue. Of interest were observations of Peterson et al. (1963) and Dent et al. (1965) that Gross passage A virus itself is immunosuppressive. These data suggest a complex interrelationship between virus, which is both immunosuppressive and oncogenic, and an organ of major immunologic import which may also be the target of leukemogenic influences. Additional understanding of these relationships came from studies of

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A.

GOOD

Allison and Law (1968), who clearly demonstrated that Moloney virusinduced leukemias and sarcomas are markedly increased by ALS while thymectomy significantly reduced the incidence of leukemias, as previously reported. These data suggest that intact immunologic defenses are of major importance in controlling RNA virus-induced leukemias and lymphomas in the mouse. “Spontaneous” neoplasms develop in mice with frequencies that vary between strains. A recent study of the incidence of ‘(spontaneous” neoplasms in neonatally thymectomized (C57JB1 X A) F, mice by Cornelius (1971) indicated that thymectomy markedly increased the incidence of “spontaneous” (possibly virally induced) lymphoreticular tumors, especially reticulum cell sarcomas. Early studies in the chicken indicated that thymectomy had no effect on the development of virally induced visceral lymphomatosis (Peterson et al., 1964a). Of special interest, however, was the observation that bursectomy prevented the development of virus-induced lymphomatosis when performed at 1 and 28 days of age. These and later studies (Cooper e t al., 1966a) indicated that the lymphomas regularly originate in one or many bursa1 follicles, and thus appear to be of B (bursal-dependent) origin. In contrast, thymectomy increases the incidence and decreases the latency period of soft tissue sarcomas induced by the Carr-Zilber strain of Rous sarcoma virus (RSV) in the chicken (Radzichovskaja, 1967). SjSgren and Jonsson (1970) further provided direct evidence for the presence of cytotoxic lymphocytes in chickens bearing Schmidt-Ruppin RSV virus-induced sarcoma. Recent observations by McArthur et al. (1972) indicated that bursectomy did not influence growth of Bryan strain Rous virus-induced sarcomas. Bursectomy markedly depresses antibody formation, while maintaining cell-mediated immunity intact. These data, therefore, suggest that antibody-mediated “blocking” factors cannot be held responsible for the growth of these tumors despite an intact cell-mediated immune response.

IMMUNODEFICIENCY C. VIRUS-AND CHEMICAL-INDUCED Numerous investigators have reported that tumor viruses may act as immunosuppressants. Early studies of immune response in animals inoculated with tumor viruses were reported by Old et al. (1960). They were able to demonstrate immunosuppressive effects of Friend leukemia virus, but not with Moloney virus. Peterson et al. (1963) found that C3H mice given Gross virus A as neonates had a significant reduction in bacteriophage neutralizing antibody in the preleukemic period. Cellular immune responses were also depressed as manifest by decreased ability to reject

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skin grafts (Dent e t al., 1965). Similarly, chickens infectcd with avian leukosis virus demonstrated decreased ability to produce antibody to bovine serum albulin and to certain bacteriophages in the preleukemic stage (Peterson et al., 1966). Furth et al. (1966) reported that the agglutinin response to sheep erythrocytes was depressed in preleukemic Gross passage A virus-infected rats. Cremer et al. (1966) found rats inoculated with Moloney virus to be deficient in production of sheep red cell antibody in both preleukemic and leukemic phases of the disease. The mechanisms of immunosuppression in these studies are not completely clear but could relate to antigenic competition (Adler, 1964) or to a block in stem cell differentiation (Siege1 and Morton, 1966). The end result would be a depression in immune surveillance mechanisms permitting uninhibited expansion of transformed clones. I n the early 1950’s, Malmgren et al. (1952) demonstrated that chemical carcinogens could also be immunosuppressive. Prehn (1963) further demonstrated that 3-methylcholanthrene could interfere with an immune reaction in the doses commonly used. Stutman (1969) found that I strain mice, which are relatively resistant to tumor development after subcutaneous injection of 3-methylcholanthrene, are also resistant to the immunosuppressive effects of the drug. In an extensive review of the influence of neonatal thymectomy and ALS treatment on chemical carcinogen-induced malignancy, Makela (1973) showed that these immunosuppressive influences often shortened incubation periods of chemical carcinogen-induced malignancies and increased the incidence of tumor initiated by chemical agents.

D. GENETICFACTORS IN HOSTRESISTANCE TO TUMORS Genetic differences in susceptibility to tumor induction are well documented in mouse systems. Susceptibility to naturally occurring or Friend virus-induced mouse leukemias, for example, is controlled by several genes (Lilly, 1972a). Genetic influences affect transformation of target cells as well as in the immunologic response to viral antigens and virally transformed cells. Splenic transformation with Friend virus is controlled by the FV-2 gene which is transmitted as a dominant trait (Lilly, 1970). By contrast, FV-2 is not of major importance in cell transformation with Gross passage A virus. Another gene, FV-I, determines host susceptibility to the helper component of Friend virus and other murine leukemia viruses but is not linked to H - 2 or FV-2 (Lilly, 1970; Pincus et al., 1971). The immune response to murine leukemia viruses is largely controlled by the Rgv-1 (Resistance-Gross Virus-1) gene. This gene, which has been mapped within the H - 2 region, influences the immune response by a t least two possible mechanisms. First, there exists in the same H - 2 subregion,

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the Ir-1 (Immune response-1) gene which regulates the immune response to a variety of natural and synthetic antigens (McDevitt et al., 1972). Second, the expression of a surface antigen of Friend virus-infected spleen (FMR) cells is controlled by a gene in the H-6 region (Lilly, 1972b). In susceptible mice, e.g., BALB/c, the concentration of F M R surface antigen falls progressively from the second week to the fourth week of infection, while antigen remains a t high levels in the more resistant, e.g., BALB/B, mice. These genetically determined differences may be the result of antigenic modulation, as is seen in thymic leukemia (TL) antigens (Boyse et al., 1967), and may result in a form of escape from the immune response. The Rgv-1 gene influences host response to virtually all RNA viruses of mice, including Friend virus (Lilly, 1968), Gross virus (Lilly et al., 1964), BALB-Tennant leukemia virus (Tennant and Snell, 1968), radiation leukemia virus (Kaplan, 1967), and mammary tumor virus (Muheback and Dux, 1971). With each of these RNA viruses, H - b and H-dd alleles are associated with susceptibility and H-db with resistance to neoplastic disease. Recently, Tennant presented additional evidence that host rejection of leukemic cells is a major mechanism of resistance to leukemia induction by BALB/Tennant leukemic virus, and that host rejection of leukemic cells is closely linked to histocompatibility antigens (Tennant, 1972).

E. GRAFTVERSUS HOST MODELSOF IMMUNODEFICIENCY Experimental graft versus host models generally utilize immunocompetent cells from parental mouse strains which are given to F, hybrid strain recipients. Lymphoid proliferation develops shortly after inoculation, followed by progressive lymphoid depletion and immunodeficiency (Howard and Woodruff, 1961; Blaese and Good, 1964; Kersey et al., 1971; Gatti et al., 1973). In certain mouse strain combinations, a large percentage of animals with chronic graft versus host reactions develop lymphoma (Walford and Hildemann, 1965; Schwartz and Beldotti, 1965; Gleichmann et al., 1972). Development of lymphoma in such experimental systems requires a major histocompatibility ( H - 8 ) difference between donor and recipient, immunocompetent donor lymphocytes, and generally high leukemia-lymphoma risk in one of the F, hybrid parents. One interesting GVH combination involves inoculation of low leukemia risk C57BL/6 donor cells into (C57BL/6 X NZB)F, hybrid recipients resulting in high lymphoma rate. High leukemia risk NZB donor cells into the same F, combination results in fewer lymphomas (Gleichmann et al., 1972). Of considerable interest are recent experiments demonstrating that the development of GVH-induced lymphomas is associated with murine

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leukemia virus activation (Hirsch et al., 1970). Similarly, blast formation in vitro in the mixed leukocyte culture system is often associated with leukemia virus activation (Hirsch et al., 1972). Finally, cell-free extracts from GVH-induced lymphomas will induce lymphomas when injected into newborn mice (Armstrong et al., 1972). Ill. Human Diseases

Data linking immunodeficiency and cancer in humans was first reported in the 1950’s and originated from several lines of evidence. Schier and his colleagues (1956) had observed that patients with Hodgkin’s disease were often unable to develop normal delayed hypersensitivity response. Observations in Minnesota confirmed and extended these findings to include studies of skin allograft survival in which significant prolongation was noted in Hodgkin’s patients and in two patients with congenital sex-linked agammaglobulinemia and lymphatic leukemia (Kelly et al., 1958; Good et al., 1962). These investigations further demonstrated that immunodeficiency became more severe with progression of the Hodgkin’s disease. At approximately the same time, data were presented demonstrating that immunodeficiency is often associated with chronic lymphatic leukemia (Brehm and Morton, 1955). I n subsequent years the number of reports linking immunodeficiency and malignancy has increased a t a remarkable rate (Gatti and Good, 1971; Good, 1972a; Waldmann et al., 1972) permitting more in depth analysis of the role of the immunologic. apparatus in relation to malignancy. To facilitate the handling of data, an immunodeficiency-Cancer Registry was established in Minnesota through which we now communicate regularly with all individuals involved in the investigation of malignancy and immunodeficiency. To date, a total of 114 persons with primary immunodeficiency disease have been reported to have developed malignancy (Kersey et al., 1973). A second large group of individuals (now totaling 94) have developed cancer while receiving immunosuppression drugs (Penn and Starzel, 1972). A third and even larger group of individuals are those whose immunologic abnormalities have become apparent only after development of malignancy. Secondary immunodeficiency has been reported in patients with widespread cancer of many types since the early work of Kelly, Good, and Varco (1958) and Southham (1961). A. DISORDERS WITH ABNORMALITIES OF HUMORAL (B-CELL) IMMUNITY Current data suggest that humoral immunity has origins in the bursa of Fabricius in the chicken and probably in the bone marrow or gutassociated lymphoid tissue of mammals (Cooper et al., 1966b; Good,

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J. H. KERSEY, B. D. SPECTOR, AND R. A. GOOD

197213). Cells that are antigen sensitive in these organs are termed B cells and have immunoglobulin surface receptors (Pernis et al., 1970). After antigenic stimulation B cells differentiate into plasma cells, the immunoglobulin-secreting cells. Abnormalities of the B-cell system are exemplified by sex-linked (Bruton’s) agammaglobulinemia, a disorder associated with inordinate susceptibility to infection with encapsulated pathogenic bacteria, absence of immunoglobulin-secreting plasma cells, and usually low or absent B lymphocytes. Lymph nodes of these patients are generally lacking in germinal centers and lymphocytes in cortical regions as well as deficiency of lymphocytes and plasma cells in medullary cords (Good, 1972a). By contrast, deep cortical regions are normally constituted and contain abundant lymphocytes. Of the 50-60 patients who have been studied with Bruton’s agammaglobulinemia, 5 have developed malignancy, and of these all have been leukemias or lymphomas. Two individuals developed acute lymphatic leukemia and one each a malignant lymphoma with leukemic expression, subacute monomyelocytic leukemia, and thymoma with leukemia (Page et al., 1963; Riesman et al., 1964; Gatti and Good, 1971) (Table I). Of interest is the observation that none of these were epithelial tumors, mesenchymal tumors, or nervous system tumors. It is possible that the absence of solid tumors in these individuals is due to lack of humoral antibodies which may be responsible for “blocking” of cell-mediated immune responses (Sjogren et al., 1971). A much larger group of patients will be necessary to test this hypothesis. AlterTABLE I TYPES OF MALIGNANCY REPORTED IN PRIMARY IMMUNODEFICIENCY DISEASES LymphoEpithelial reticular Wiskott-Aldrich syndrome Congenital sex-linked immunodeficiency Severe combined system immunodeficiency Ataxia-telangiectasia Common variable immunodeficiency IgA deficiency

0.

17

Leukemia 2 (9%)

Mesenchymal

Nervous system Total

1(5%)

1(5%)

21

0

1(16%;b) 4 (67%)

0

1(16%)

6

0

4 (80%)

1(20%)

0

0

5

5 (11%) 29 (64%) 13 (39%) 13 (39%)

7 (17%) 5 (15%)

2 (4%) 1 (3%)

2 (4%) 1 (3%)

0

0

0

3 (75%)

1(25%)

21 (18%) 65 (57%) 19 (17.5%) 4 (3.5%) 5 (4%) 0

b

Number of individuals reported to January, 1973. Percent malignancy type for each primary immunodeficiency disease.

45 33 4 114

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natively, the high rate of leukemia and lymphoma may be related to chronic antigenic stimulation with lymphoid proliferation plus the absence of normal feedback mechanisms (Schwartz, 1972). Isolated IgA deficiency involves the B-cell system and in certain individuals may result in no symptoms whereas in others this absence results in autoimmune phenomena, sinopulmonary infections, and/or gastrointestinal disorders. Current evidence indicates that the IgA system is involved primarily in local host defense, especially in the respiratory and gastrointestinal tracts (Tomasi and Bienenstock, 1969). IgA is a locally produced 7 s immunoglobulin which enters the vascular system or becomes complexed to a “secretory” piece, and enters the gastrointestinal and respiratory tracts. Thus, it is of some interest that an individual with isolated IgA deficiency developed gastric carcinoma (Fraser and Rankin, 1970), and another developed pulmonary carcinoma (Amman and Hong, 1971), suggesting that surveillance mechanisms in gastric and pulmonary environments may be deficient in persons with this syndrome. Two additional individuals with pulmonary carcinomas were found to have isolated IgA deficiency ; however, the possibility of a secondary immunodeficiency could not be excluded (Amman and Hong, 1971) (Table I). IgA deficiency is also regularly associated with ataxia-telangiectasia, a disorder involving also the IgE system, as well as cell-mediated (thymic-derived or T-cell) system. These patients have an inordinate number of neoplasms of many types, as will be discussed below, but of interest in this context are two gastric adenocarcinomas which developed in sisters aged 19 and 21 (Haerer et al., 1969).

B. HUMANDISORDERS WITH ABNORMALITIES OF CELL-MEDIATED (T-CELL-DEPENDENT) IMMUNITY Experimental data indicate that cell-mediated immune responses are largely dependent on cells which are influenced by or derived from the thymus. These thymus-dependent (T cell) cell populations appear to play a major role in the immune response to tumor cells, foreign (allogeneic) cells, and to facultative intracellular bacterial pathogens, fungi, and viruses (Good, 1972a). Islated deficiency of the T-cell system is present in the pharyngeal pouch (Di George) syndrome. These individuals do not develop thymus and parathyroids because of developmental defects of the 3rd and 4th pharyngeal pouch. Delayed skin responses to virus antigens are markedly depressed as is response to phytohemagglutinin (PHA) in vitro. The B-cell system, including immunoglobulin synthesis, remains intact, and circulating B lymphocytes are markedly increased in both relative and absolute number (GajlPeczalska et al., 1972). The few individuals described with this syndrome

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J. H. KERSEY, B. D. SPECTOR, AND R. A. GOOD

have not survived beyond infancy, and none thus far have developed malignancy. Other disorders with defective T-cell responses are generally associated with abnormal B-cell responses as well. Ataxia-telangiectasia involves the skin, blood vessels, and central nervous system and abnormalities of cell-mediated and humoral (especially IgA and IgE) responses. An estimated 450 individuals have been reported with this syndrome (Lambrecht and Snoijink, 1971) ; most have died in childhood of severe infections. However, a total of 45 have been reported to have developed cancer since the early reports of Boder and Sedgwick (1963), more recently reviewed by Boder and Sedgwick (1972). Included are 5 epithelial malignancies( 11% of total), 29 lymphoreticular malignancies (64%), 7 leukemias (17%), 2 mesenchymal malignancies (4%), and 2 nervous system tumors (4%) (Table I). Epithelial malignancies included gastric adenocarcinoma, ovarian dysgerminoma, and basal cell carcinoma (Levin and Perlov, 1971; Reed et al., 1966; Dunn et al., 1964; Haerer et al., 1969). Lymphoreticular tumors have included Hodgkin’s disease, lymphosarcoma, and reticulum cell sarcoma (Harley et al., 1957; Boder and Sedgwick, 1963; Peterson et nl., 1964b; Murphy, 1965; Rosenthal et nl., 1965; Fois, 1966; Smelby, 1966; Miller, 1967; Dugois et al., 1967; Gotoff et al., 1967; Aguilar et al., 1968; Morgan et al., 1968; Castaigne et al., 1969; Amman et al., 1969; Feigen et al., 1970; Levin and Perlov, 1971; Waldmann et al., 1972). Leukemias were undifferentiated or acute lymphoblastic in type (Hecht et al., 1966; Fois, 1966; Lampert, 1969; Taleb et al., 1969; Levin and Perlov, 1971). Mesenchymal malignancies were undifferentiated sarcomas or bone sarcoma (Boder and Sedgwick, 1963 ; Perez-Soler and Espadaler, 1964). One glioma and one medulloblastoma were reported (Young et al., 1964; Shuster et al., 1966). Wiskott-Aldrich syndrome is characterized by eczema, low platelets, and abnormalities of both T- and B-cell immune systems, manifest as abnormal delayed allergy response and low immunoglobulins. IgM catabolism is enhanced in this disorder, and isohemagglutin titers are low or absent (Blaese et al., 1971). Children with Wiskott-Aldrich syndrome usually die of severe infections or cerebral hemorrhage a t an early age. Twenty-one of this individuals have developed malignancies. Seventeen (81% of total) wre lymphoreticular, 2 (9%) leukemias, 1 (5%) mesenchymal, and 1 (5%) nervous system malignancy (Table I ) . Lymphoreticular tumors were most frequently reported to be malignant reticuloendotheliosis, or occasionally malignant lymphoma (Coleman et al., 1961 ; Kildeberg, 1961; Pearson et al., 1966; ten Bensel et al., 1966; Chaptal et al., 1966; Radl et al., 1967; Cooper et al., 1968; Huber,’1968; Brand and Marinkovich, 1969; Waldmann et al., 1972). One leukemia was an

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acute myelogenous form, and another was an acute undifferentiated type (ten Bensel et al., 1966; Gatti and Good, 1971). Multiple leiomyosarcomas were found in the one patient with a mesenchymal tumor, and astrocytoma was reported in the individual with a brain tumor (Amiet, 1963; Gatti and Good, 1971). Another disorder of infancy associated with severe disturbances of both T- and B-cell systems is severe combined system immunodeficiency. These children usually succumb to severe infections within the first year or two of life. Four of these individuals have developed lymphoreticular malignancies, including lymphomas and Hodgkin’s disease (Goldman et al., 1967; Lamvik and Moe, 1969; Tung et al., 1969; von Bermuth et al., 1970), and one developed acute leukemia (Kadowaki et al., 1965). Common variable (“late-onset”) primary immunodeficiency, previously often referred to as acquired agammaglobulinemia, is found in individuals who frequently have only mild symptoms until adulthood, when frequent infections or malignancy suggest the diagnosis. Most individuals with this disorder have both B- and T-cell abnormalities, although a broad spectrum of abnormalities has been reported. These immunodeficiencies are variable from time to time in the same patient and from patient to patient in the same family. The varying degree of abnormalities in this group has resulted in difficulty in determining the total number of affected individuals. These persons must be carefully evaluated in an attempt to distinguish them from the large group of individuals who have developed immunodeficiencies following the onset of malignancy. Reported tumors in common variable immunodeficiency include 13 (39% of total) epithelial tumors, 13 (39%) lymphoreticular tumors, 5 (15%) leukemias, 1 (376) nervous system tumor, and 1 (3%) mesenchymal malignancy (Table I). Epithelial tumors were gastric carcinomas, colon carcinomas, breast carcinomas, bladder carcinoma and cervical carcinomas (Gafni et al., 1960; Huiaenga et al., 1961; Hermans et al., 1966; Medical Research Council, 1970). Lymphoreticular tumors included lymphomas and malignant reticuloendotheliosis (Brehm and Morton, 1955; Fudenberg and Solomon, 1961; Pelkonen et al., 1963; Green et al., 1966; Schrub et al., 1967; Hudson and Wilson, 1968; Freeman et d . , 1970; Douglas et al., 1970; Medical Research Council, 1970; Waldmann et al., 1972). Leukemias were chronic lymphatic and acute lymphatic (Medical Research Council, 1970). One liposarcoma (Waldmann et al., 1972) and one medulloblastoma were found (Medical Research Council, 1970). A final form of immunodeficiency, the exact basis of which is not yet clear, is the Chediak-Higashi anomaly. These patients have gigantism of single membrane-bound intracellular particles, including phagosomes

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J. H. RERSEY, B. D. SPECTOR, AND R. A. GOOD

and lysosomes. These patients suffer from frequent infections. If the patients do not die of infection and live long enough, they all succumb to malignancies, particularly of the lymphoreticular system (Page et al., 1962; Windhorst et al., 1966). Reported malignancies by age in patients with primary immunodeficiency syndromes are distributed as would be expected from the age distribution of each disease (Fig. 1 ) . Ataxia-telangiectasia, for example, develops in infants and children, and thus most malignancies in this group have occurred before the age of 20. Similarly, tumors in sex-linked (Bruton’s) agammaglobulinemia, common variable immunodeficiency, Wiskott-Aldrich syndrome, and severe combined system immunodeficiency occurred largely before the age of 20. By contrast, the largest number of tumors in patients with common variable immunodeficiency developed in the 40-60 age group.

C. OTHERGENETIC HUMAN DISORDERS WITH HIGHRISK OF CANCER Certain inherited disorders which are associated with a high risk of cancer have skin fibroblasts which are abnormally susceptible to transformation in vitro with the tumor virus, simian virus 40. Most of these disorders have associated chromosomal abnormalities. Patients with Fanconi’s anemia, for example, have a high risk of acute leukemia and skin fibroblasts which are transformed 20 times more readily than those from normal individuals (Todaro et al., 1966). Klinefelter’s syndrome (XXY) is associated with high leukemia risk and high transformation susceptibility (Mukerjee et al., 1970), as is familial myelogenous leukemia (Snyder et al., 1970). Fanconi’s anemia, Klinefelter’s syndrome and familial myelogenous leukemia are without significant immunologic

-

Number of Known Cases of Cancer by Age; 0 70 years

FIG.1. Age of onset of malignancy in individuals with primary immunodeficiency disorders.

IMMUNODEFICIENCY AND CANCER

223

defects and thus appear to represent defects of target cell or intracellular surveillance (Kersey and Good, 1972). Such target cell defects may allow increased frequency of transformation by viruses, chemicals, irradiation, and for lymphoid cells by chronic antigenic stimulation, as depicted in Fig. 2 (defect 1).Our hypothesis suggests that even normal immunologic surveillance is inadequate to control malignant clones, which develop frequently in individuals with such defective target cells. Down’s syndrome (G trisomy) is associated with a high risk of leukemia (Krivit and Good, 1956) and increased fibroblast susceptibility to SV40 transformation in vitro (Todaro and Martin, 1967). Additionally, these individuals frequently have immunologic abnormalities of both T- and B-cell immunity (Sutnick et al., 1971), and thus Down’s syndrome may represent abnormality of both target cell (intracellular) surveillance and immunologic (intercellular) surveillance (defects 1, 2, and 3 in Fig. 2). Individuals with primary immunodeficiency diseases sometimes have chromosomal defects (Riesman et al., 1964; Hecht et al., 1966). However, recent experiments in our laboratories indicated that patients with primary immunodeficiencies are generally free of target cell defects which are detected in the SV40 transformation assay (Kersey et al., 1972). These data support the hypothesis that individuals with primary im-

Chemicals lrrodiat ion Cell mediated Immune response

Humaral Immune respmse

individual

FIG.2. High risk of cancer in certain individuals is probably related to defects of surveillance mechanisms: defects may be intracellular, i.e., of the target cell (I), or intercellular, i.e., cell-mediated immune responses (21, or of humoral immune responses (3).

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J. €KERSEY, I. B. D. SPECTOR, AND R. A. GOOD

munodeficiency syndromes have normal target cells and abnormalities of intercellular (immunologic) surveillance as depicted in the upper portion of Fig. 2 (defects 2 and 3 ) . The graphic representation in Fig. 2 indicates that the cell-mediated immune response and the humoral immune response are responsible for control of cancer at the level of the transformed cell. Individuals with one or both of these deficiencies would thus be unable to destroy one or a few transformed cells. By contrast, individuals with abnormal target cells (e.g., Fanconi’s anemia) may develop cancer in spite of normal immunologic surveillance. Finally, individuals with abnormalities of target cells and immunologic surveillance (e.g., Down’s syndrome) should be a t a very high risk for development of cancer.

D. DRUG-INDUCED IMMUNODEFICIENCY In recent years, convincing evidence linking drug-induced immunosuppression and cancer has been reported in recipients of organ transplants. In some instances tumors became evident after patients were given organs from donors with cancer; more often neoplasms have arisen de novo sometime after organ transplantation, and a total of 94 has now been reported (McKhann, 1969; Penn and Starel, 1973). Of interest are the large number of lymphoreticular malignancies of the brain which have developed in these patients (Schneck and Penn, 1971). Additionally, immunosuppressed organ transplant recipients appear to be a t high risk for development of malignant skin tumors (Walder et al., 1971).

E. VIRUS ISOLATION FROM IMMUNODEFICIENT INDIVIDUALS Immunosuppressed organ transplant recipients develop frequent virus infections, especially with cytomegalovirus and other herpes viruses (Rifkind et al., 1967; Craighead et al., 1967). These virus infections may be associated with graft rejection episodes (Lopez et al., 1973). Recently, Gardner and co-workers (1971) demonstrated a new virus isolate from an immunosuppressed patient. The virus, which they termed B.K., appeared to be a member of the papovavirus group. B.K. virus was smaller than wart virus, grew more slowly than SV40, and was serologically different from SV40. Progressive multifocal leukoencephalopathy (PML) is a chronic degenerative disease of the brain often associated with primary or secondary immunodeficiency. Padgett et al. (1971) found a papovalike virus from one of these patients. Very similar to B.K. virus, this virus was smaller than wart virus and did not appear to cross-react with polyoma or SV40 in immunofluorescent tests. More recently, Weiner et al. (1972) ,

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in independent studies, isolated papovavirus from the brains of two individuals with PML. In contrast to previous isolates, serologic studies utilizing fluorescent and neutralizing antibodies demonstrated a close relationship between the new virus isolates and SV40. Additional data reported by Lopez et al. (1972) indicate that individuals with primary immunodeficiency disorders have an unusual frequency of viral infections and are unable to eliminate live virus vaccines, e.g., polio. These reports indicating new and frequent viruses in immunodeficient individuals, suggest that considerable efforts are warranted in an attempt to isolate new viruses from these people, especially if they are bearing a tumor. The relationship of these findings to the concepts of immunosurveillance discussed above deserve continued attention and reflection. Surely, the accumulating evidence from work with primary and secondary immunodeficiencies continues to reveal the intimate association of the immunologic functions and malignant adaptation. However, the data to date do not totally support the concepts of immunosurveillance as originally postulated. There seems to be no question that primary immunodeficiencies focused on defects of humoral mechanisms favor development of leukemias and lymphomas over other malignancies. Indeed, each form of primary immunodeficiency seems to have a tendency to express its own constellation of malignancies. I n the aggregate, all forms of primary and secondary immunodeficiency tend to favor development of lymphoreticular cancers. It seems quite possible that the increased susceptibility to malignancy in immunodeficiencies reflects a susceptibility to etiologic agents which can operate more effectively in the absence of immunocomponent mechanisms. Regardless of the basis of increased frequency of cancer in patients with immunodeficiency, this intimate association deserves continuous study and detailed analysis. These efforts are currently in progress. REFERENCES Adler, F. L. (1964). Progr. Allergy 8,4157. Aguilar, M. J., Kamoshita, S., Landing, B. H., Boder, E., and Sedgwick, R. P. (1968). J . Neuropathol. Exp. Nezaol. 27, 659-676. Allison, A. C., and Law, L. W. (1968). Proc. SOC.Exp. Biol. Med. 127, 207. Allison, A. C., and Taylor, R. B. (1967). Cancer Res. 27, 703-707. Allison, A. C., Berman, L. D., and Levy, R. H. (1967). Nature (London), 215, 185187.

Amiet, A. (1963). Ann. Paediat. 201, 315-335. Amman, A. J., and Hong, R,.(1971). Medicine (Baltimore) 50, 223-236. Amman, A. J., Good, R. A., Bier, D., and F’udenberg, H. H. (1969). Pediatrics 44, 672-676.

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RECENT OBSERVATIONS RELATED TO THE CHEMOTHERAPY AND IMMUNOLOGY OF GESTATIONAL CHORIOCARCINOMA K. D. Bagshawe Deportment of Medical Oncology, Choring Cross Hospital (Fulhom) , London, England

I. Introduction . . . . . . . . . . . . . . 11. General Aspects of Trophoblastic Tumors . . . . . . . 111. Human Chorionic Gonadotropin as a Tumor Index Substance . . A. General Characteristics . . . . . . . . . . . B. Detection Methods . . . . . . . . . . . . C. Diagnosis and Monitoring of Tumor Mass . . . . . . D. Immunochemical Detection of Brain Metastases . . . . . E. HCG Assays in the Follow-up of Patients after Hydatidiform Mole IV. Immunological Aspectsof Choriocarcinoma . . . . . . . A. ABO Blood Groups in Trophoblastic Neoplasia . . . . . . B. H G A Antigens . . . . . . . . . . . . C. HL-A Antibodies . . . . . . . . . . . . D. Cellular Reaction to Choriocarcinoma . . . . . . . E. Rejection of Husband Skin Grafts . . . . . . . . F. Choriocarcinoma and Fetomaternal Immunity . . . . . G. Immune Responses in Relation to Cytotoxic Therapy for Choriocarcinoma . . . . . . . . . . . . References . . . . . . . . . . . . . .

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I. Introduction

The tumors of the human trophoblast form a spectrum of increasing malignancy ranging from benign hydatidifonn mole through invasive mole to choriocarcinoma. They are unique tumors in three respects: 1. They are natural allografts since the trophoblast is a fetal tissue and the tumors generally occur only in the maternal host. It follows that they are distinguished from the host by the genetic contributions of the male parent. 2. Trophoblastic tumors are also endocrinologically active, and synthesis of human chorionic gonadotropin (HCG) provides an indicator of tumor activity and of the effect of exogenous agents upon them. Studies on HCG provide a model for the information which might be gained from measurements of other tumor-associated antigens. 3. Many widely disseminated choriocarcinomas are curable by chem231

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otherapy. However, they are not all curable by present methods; some show drug resistance initially and others become resistant during therapy. Defining the basis of curability and noncurability of choriocarcinoma could have implications for a much wider range of tumors. In order to provide perspective for studies of specific topics related to choriocarcinoma, some general aspects of these tumors will be summarized. I f . General Aspects of Trophoblastic Tumors

Choriocarcinoma can arise from any term delivery, abortion, or hydatidiform mole. I n European populations, the incidence of mole is between 1 :1000 and 1 :2000 known pregnancies, but the risk of choriocarcinoma after mole is about 1000 times as high as that after normal term delivery. The risk of choriocarcinoma ranges from about 0.003% following normal term deliveries to about 3% for hydatidiform mole. Precise figures for world incidence cannot be given owing to large geographical differences which arise mainly from the greater frequency of hydatidiform mole in various parts of Southeast Asia, Africa, and the Middle East (Bagshawe, 1969 ; Park, 1971). Careful and scholarly studies of the histology and general pathology of trophoblastic tumors have recently been presented by Park (1971) and Ober et al. (1971). The characteristic lesion of choriocarcinoma is a purplish node in which a vascularized rim of tumor cells envelops an avascular central zone composed of necrotic debris and blood. Typically, areas of cytotrophoblast tend to be surrounded by syncytiotrophoblast, and villous structures with stroma are absent. Where choriocarcinoma has been found in an otherwise normal placenta, normal villi are present adjacent to the tumor (Brewer and Gerbie, 1966), and occasionally a few villi may persist where choriocarcinoma is removed within a few weeks of evacuating a mole. But generally persistence of villi typifies invasive mole and confers a more favorable prognosis. The multinucleated syncytial giant cells, in which mitoses are very rare, are formed by fusion of cytotrophoblast cells (Carter, 1964). The tumor frequently invades vessels so that hemorrhagic destruction of adjacent structures is prominent. Grossly dilated vessels in the periphery of tumor masses are readily demonstrated by arteriography. Local infiltration occurs, and tumor masses may form in the tissues adjacent to the uterus. In about 10% of choriocarcinomas, hysterectomy or autopsy fails to reveal any tumor in the uterus. Dissemination is hematogenous, particularly to the lungs, vagina, central nervous system, kidneys, gastrointestinal tract, skin, spleen, and liver. Lymphatic spread appears to be quite rare, and proof of lymph node involvement has been obtained in less than 1% of cases in the writer’s series.

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Invasive mole is a neoplastic proliferation of the trophoblastic elements of a hydatidiform mole in association with persisting villi. Its behavior is bizarre. Invasion occurs locally in the uterus. Blood vessels may be penetrated and a few small distant blood-borne metastases may form, particularly in the vagina and lungs, and rarely reach the brain. Marked vascular changes, which are not readily distinguished from those of choriocarcinoma, occur in the pelvis (Borrell et al., 1966; Brewis and Bagshawe, 1968). Invasive mole may cause severe uterine hemorrhage or perforate the uterus, but it frequently undergoes spontaneous degeneration. It can be effectively eliminated by hysterectomy or chemotherapy. I n practice, it is not always possible to obtain tissue which would allow a histological diagnosis of choriocarcinoma or invasive mole to be made without major surgery. Since invasive mole is virtually always curable by chemotherapy and choriocarcinoma is usually curable, i t is often desirable to avoid hysterectomy. Where i t is not possible to determine whether a tumor is invasive mole or choriocarcinoma, terms such as “post-mole trophoblastic neoplasm” or “malignant trophoblast disease” or “clinical choriomatosis” are frequently used. The problem does not arise after a term pregnancy nor after a nonmolar abortion since any persistent proliferation of trophoblast after nonmolar pregnancies seems always to be choriocarcinoma. It has been widely observed that the response of trophoblastic neoplasms to cytotoxic agents is influenced by the “age” of the tumor, that is, the latent period between the end of the antecedent pregnancy and the time of diagnosis and treatment (Ross et al., 1965; Bagshawe, 1969; Lewis, 1969; Kolstad et al., 1972). Tumors treated within 3 months of a term pregnancy or nonmolar abortion, or within 6 months of a hydatidiform mole have generally proved highly amenable to treatment even when widespread metastatic dissemination has occurred. Tumors treated later may show resistance, and the incidence of resistance tends to increase with the duration of the latent interval (Bagshawe et al., 1969). Low dose chemotherapy given soon after the antecedent pregnancy has, however, sometimes been associated with late resistance to a variety of cytostatic agents. The normal trophoblast has an extensive repertoire for the synthesis of peptides and steroids, and the pattern of activity changes during the course of pregnancy (Bagshawe, 1969; Biswas and Hindocha, 1972). Choriocarcinoma consistently expresses the peptide-synthesizing patterns of early trophoblast by being active in the production of HCG but relatively inactive in the production of human chorionic somatomammotropic hormone (human placental lactogen) and the placental form of alkaline phosphatase (Jacoby and Bagshawe, 1972). It may, however,

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differ from early normal trophoblast in that human chorionic thyrotropic hormone (HCT) is probably produced only by a minority of choriocarcinomas (Ode11 e t al., 1963). Ill. Human Chorionic Gonadotropin as a Tumor Index Substance

A. GENERAL CHARACTERISTICS Trophoblast was identified as the source of HCG by growing placental tissue in ectopic sites (Kido, 1937) and in tissue culture (Gey e t al., 1938). Later studies using immunofluorescent localization techniques have shown that both cytotrophoblast and syncytiotrophoblast synthesize HCG. The concentration of HCG in placental tissue changes during pregnancy and is a t a peak between weeks 8 and 10, when values of about 600 I U HCG per gram of placental tissue were found (Diczfalusy, 1953; Diczfalusy e t al., 1958). At term, values have fallen to 2-8 IU/g (Loraine and Matthew, 1953). HCG is a glycoprotein which dissociates in lOM urea into two nonidentical subunits. The a subunit which has a molecular weight of 18,OOO is similar to, and possibly identical with, the subunits of the pituitary luteinizing hormone (Morgan and Canfield, 1971; Bahl, 1969; Bell e t al., 1969). The P subunit has a molecular weight of about 30,000, and this is also similar to the ,8 subunit of LH, a finding which is consistent with the similarities between LH and HCG in their biological and immunological characteristics (Hartree e t al., 1971). However, differences between L H and HCB p subunits have been identified and have been used in the development of specific radioimmunoassay for HCG. Experience with P-HCG assay so far is limited, but it appears to offer the most practicable means for discrimination between HCG and L H so far devised. Current experience is largely based on assays for the intact HCG molecule, and elsewhere in this paper the assay methods referred to do not discriminate between HCG and LH. Sialic acid forms an important part of the carbohydrate component of HCG, and progressive desialylation of purified preparations is accompanied by reduction in electrophoretic mobility and biological potency (Whitten, 1948; van Hell e t al., 1968; van Hall et al., 1971). Desialylation has little effect, however, on the immunoreactivity of the molecule, which is remarkably stable in both plasma and urine. Plasma and urine concentrations of HCG tend to be similar. The renal clearance rate of HCG is about 1 ml/min with a range of 0.5-2.0 ml/min (Gastineau e t al., 1949; ,Johnson e t al., 1950; Bagshawe, 1969). This relatively low clearance rate for a stable substance tends to smooth

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out changes in plasma concentration and urine excretion rate as a reflection of changes in tumor secretion rate.

B . DETECTION METHODS Methods for detecting human chorionic gonadotropin (HCG) were applied in the diagnosis of trophoblastic tumor during the 1930’s, but it was only with the introduction of effective chemotherapy in the form of methotrexate that semiquantitative measurements of HCG in the urine came into regular use to monitor the course of the disease. The mouse uterine weight method was widely used up to about 1964 but since then, radioimmunoassays for HCG (Wilde et al., 1965, 1967; Midgley, 1966) have proved advantageous. Assays can be performed on urine, plasma, or spinal fluid, and, for most purposes, incubations of 2-16 hours provide high sensitivity, precision, and reproducibility. The detectable concentrations of HCG in plasma and urine of patients with trophoblastic tumors cover a 1O7-foldrange. Urinary excretion rates vary between about 5 I U of HCG and lo7 IU per 24 hours. The limit of detection for small amounts of HCG is imposed not by the sensitivity of radioimmunoassay methods, but by the cross-reaction between HCG and LH. The relationships between HCG and LH and assay sensitivity are shown in Fig. 1. HCG can be said to be present in body fluids when an estimate for HCG LH exceeds the physiological range of LH. This physiological range is equivalent to 5-50 I U of HCG for most of the menstrual cycle. The amount of LH in plasma and urine varies with the endocrine status of the subject; for full use to be made of HCG estimations by radioimmunoassay, familiarity with pituitary and ovarian physiology is necesssary. In brief, there is a cyclical variation in LH production during the menstrual cycle with the mid-cycle peak 5-7 times higher than the values found in the follicular and luteal phases. Deficient ovarian steroidogenesis is accompanied by values up to 5-10 times those of the follicular phase whereas administration of 17P-hydroxyestrogens suppresses both mid-cycle peaks and the high levels of LH associated with ovarian deficiency (Bagshawe et al., 1966; Franchimont, 1972).

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C. DIAGNOSIS AND MONITORING OF TUMOR MASS If measurement of a soluble tumor product is to be used as an index of tumor extent or activity, then the limitations of the relationship between quantity of product and tumor size need to be defined. Clinically, the requirement is for an index related to the number of tumor cells possessing mitotic potential rather than to tumor volume, since the contribution of necrotic tissue, blood, and other elements to tumor volume is generally irrelevant to the therapeutic problem. Sequential measure-

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ments of an index substance related to viable cell mass provide an index of tumor growth or regression rate. Ideally, we would measure the rate at which the tumor product is secreted by the tumor, but although this can be done for various substances, present procedures are laborious and do not lend themselves to repetition. In the case of HCG, plasma concentrations and urine excretion rate can be measured frequently, and the information obtained provides a reliable guide to the course of the disease. Corrections can be applied for the low clearance rate and for its slow equilibration throughout body fluids, but in practice, the main limitation on the information obtained is the sensitivity limit imposed by the crossreaction between HCG and LH. The “doubling time” for HCG excretion in patients with active choriocarcinoma falls in the range 30 hours to 15 days. I n early normal pregnancy, the “doubling time” is in the range 30-40 hours, and this may be related to a cell “doubling time” as short as 22 hours (Hamilton et al., 1962; Bagshawe, 1959). A broad correlation has long been noted between the amount of HCG excreted by a patient before treatment and the total size of her tumor masses. It has also been firmly established that if gonadotropin excretion is reduced to the normal L H range and remains normal for some time before treatment is discontinued, then the chances are good that the tumor will not recur. In the author’s series of more than 200 cases, there have been no relapses more than 12 months after discontinuation of chemotherapy. An important question then is whether, between these limits, there is a useful correlation between the amount of HCG produced and the amount of viable tumor. I n studies of choriocarcinoma cells grown in tissue culture, HCG production during log phase growth was found to be in the range lV4I U of HCG/cell per day, but during periods when cell growth was not apparent, lower values were recorded (Currie and Bagshawe, 1967). A value of I U of HCG per day was consistently recorded by Pattillo and Gey (1968) using the BeWo line of choriocarcinoma cells which has been carried through 350 generations in tissue culture. Estimates based on in vivo production of HCG have been made where solitary tumor masses of the order of 2-10 mms were removed surgically and where gonadotropin excretion fell to normal values subsequently, Estimates were made of the number of tumor cells by visual counting, and hormone production per cell was calculated. In 3 patients in the author’s series, values of approximately 5 X lC5 IU cell per day were obtained. The range of values from these crude methods is wide, to IU/ cell, but it is consistent with other estimates of cell numbers. The upper range of HCG production in vivo, about lo7 IU/day, is consistent with a total tumor cell population of 1011to 10l2 cells. Similar estimates have

238

R. D. BAGSHAWE

been made for other tumor cell populations in patients with advanced neoplasia (Collins et al., 1956). There is no evidence that HCG production per unit mass of tumor changes greatly with time in the individual patient. The possibility that drugs impair protein synthesis without causing cell death has to be recognized, but all our evidence has indicated that a major reduction in gonadotropin production was virtually always accompanied by other evidence of tumor cell death whenever other corroborative evidence was available. Between individuals, differences in HCG production per unit mass of tumor no doubt occur, but probably they do not exceed a 10-fold range on a scale with an overall magnitude loll to lo1*. Since the normal LH range in premenopausal subjects is equivalent to 5-40 I U of HCG during most of the menstrual cycle, i t follows that HCG values allow tumors to be monitored down to about lo6 cells. Several patients who have had chemotherapy for choriocarcinoma have then relapsed while having frequent HCG assays. Gonadotropin values in the range 50-200 I U of HCG per day in such patients have sometimes led to the detection and surgical removal of uterine tumors less than 10 mm3 in total volume provided there were clues to the anatomical location of the residual tumor. It therefore seems highly probable that there is a substantial degree of proportionality between the observed HCG values and the number of viable tumor cells. Chemotherapy is generally given as a series of courses of about 6 7 days in duration with rest phases of about 7 days intervening (Ross, 1966 ; Bagshawe, 1969). During chemotherapy, the pattern of change in HCG excretion or plasma concentration provides information which is valuable in clinical management. The first exposure to an effective cytotoxic agent is frequently followed by an increase in HCG values lasting 1-5 days. The mechanism of this rise is not known. One possibility is that interference with DNA synthesis in some way results in an increased rate of synthesis of HCG. Alternatively, and more probably, the rapid destruction of large numbers of replicating cells might result in the escape of HCG preformed within the cells. A fall in HCG values follows the start of a course of drugs after some 2-6 days and may continue for 5-18 days. The rate of fall varies greatly among patients and gives some indication of the sensitivity of the tumor ; the greater the rate, the more sensitive the tumor (Fig. 2). A first course of therapy lasting 4 7 days causes the HCG Concentration to fall by 75 to 99.9% of the initial values. If no further treatment is given during the following 23 weeks, HCG values level off and then start to increase again with doubling rates in the range 1.5-5 days. In the individual patient, successive courses of treatment with the same cytotoxic agent or combination of agents and with intervening rest

GESTATIONAL CHORIOCARCINOMA

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phases tend to produce fractional reductions in HCG values. This is best seen with tumors responding a t a moderate rate (Case A, Fig. 2). With each course of therapy, an increase of HCG production can be seen to occur before the corresponding fall. This may be a composite effect resulting from (1) tumor regrowth during the rest phase (2) the smoothing effect of the low renal clearance rate, and (3) the increased release of HCG from tumor cells during cytotoxic therapy as already discussed. The result is a stepwise reduction in HCG values which, when plotted on a log scale, is seen to be consistent with the concept of constant fractional cell kill as advanced by Skipper e t al. (1964). When a choriocarcinoma is very sensitive to the cytotoxic agents, this stepwise reduction in HCG values is less clearly seen (Case B, Fig. 2) because of the smoothing effect of the low renal clearance rate. On the other hand, with a resistant tumor, a reduction in HCG values still occurs, but the rate of reduction is reduced, the magnitude of the reduction is relatively small and it is equaled or exceeded by the increase which occurs in the succeeding rest phase. The general pattern of progression which is seen in a tumor that is initially sensitive but later becomes resistant is shown in Fig. 3. Where a tumor is potentially curable by chemotherapy, one problem is to decide how long to continue therapy. In general, treatment of choriocarcinoma must be continued for some time after normal values have been obtained. If treatment is stopped too soon, the tumor recurs and retreatment tends to be more difficult. Instances have been described where chemotherapy has not been conURINE HCG lU/DAY

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FIG.2. Human choricnic gonadotropin (HCG)excretion rates in two patients with sensitive tumors showing different rates of response.

240

K. D. BAGSHAWE CHEMOTHERAPY

FIG.3. Human chorionic gonadotropin (HCG) excretion in a patient whose tumor was resistant to the initial drug regimen and responded to the second regimen with a 3 log fall in HCG values and resolution of pulmonary metastases. Despite continued therapy with a wide range of agents, the tumor began to grow again and progressed to death. The lower line indicates the general pattern of HCG values seen in such resistant cases in diagrammatic form. tinued beyond the time of obtaining one or two negative “pregnancy tests,” and remission has been reported. Although there is the possibility that immune mechanisms are responsible for eliminating residual tumor in such cases, experience in the present series suggests that this is likely to occur only with invasive mole and that it does not occur, or is very rare, in choriocarcinuma. Yet to continue treatment beyond the period necessary for the individual patient is not only expensive and time-consuming but may also be dangerous. It is clear that treatment should continue so long as HCG can be detected, but just how long to continue therapy after gonadotropin values have fallen to the normal LH range remains a substantial problem. When gonadotropin values are in the normal LH range, it can be concluded only that the tumor cell population is about los cells or less. I n cases where the HCG values have fallen more or less exponentially, some indication of the minimum time required to reach “zero” cells can be obtained by extrapolation of the HCG production curve down to the theoretical 1-cell level (Bagshawe, 1969). This is illustrated diagrammatically in Fig. 4.

241

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The limitations of extrapolating data in this way are obviously considerable. Any slowing of the rate of response after normal LH values have been reached would tend to make it take longer to achieve total elimination of the tumor, and it has been found useful to take other in-

242

K. D. BAGSHAWE

formation into account when deciding on the termination of therapy. For instance, it has proved advantageous to continue treatment beyond the “extrapolation zero” if the tumor had a long latent period before starting treatment, or if there were large metastases (>5 cm diameter), or if there were metastases in the central nervous system. Depending on these variables, treatment has been continued for periods of 1-5 months after normal LH values were reached. Even so, relapses have not been completely eliminated. Where a patient has relapsed after treatment, the recurrence has been detected by HCG assay before any other symptoms or signs were present (Fig. 5 ) . The occurrence of drug resistance is clearly indicated by HCG values (Figs. 3 and 5 ) . I n some instances, resistance to a drug regimen has been detected and other agents were substituted successfully before symptoms or clinical signs developed (Figs. 5 and 6 ) . Thus one of the most striking applications of HCG measurement as a tumor index is its ability to detect tumors a t a subclinical stage, to indicate the pattern of drug resistance and sensitivity, and then to enable treatment to be carried out successfully.

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243

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D. IMMUNOCHEMICAL DETECTION OF BRAIN METASTASES As a consequence of the blood-brain barrier, many substances released into body fluids are found in much lower concentration in cerebrospinal fluid than in plasma. This is true of HCG and in patients who have trophoblastic tumors without evidence of metastases in the central nervous system, the spinal fluid concentration is about one two-hundredth of that in the plasma (Bagshawe et al., 1968). When metastases are present in the central nervous system, the relative concentration of HCG in the spinal fluid is increased (Tashima et al., 1965). Plasma :spinal fluid ratios in the presence of intracranial metastases have ranged from 75: 1 to 1 :2 (Rushworth et nl., 1968). The extent of the tumor within and without the nervous system clearly influences the ratio observed. Ratios close to 1:1 have generally indicated tumor confined, or largely confined, to the central nervous system. The slow diffusion of negatively charged peptides between the spinal fluid and the blood results in a slow equilibration following primary changes in concentration in one or other compartment. Thus, if there is a rapid fall in the plasma concentrations of HCG, this will not be fully reflected in the spinal fluid concentration for several days, so that if the plasma :spinal fluid ratio is determined during this time, the ratio will be disturbed and it may fall below 100: 1.

244

K. D. BAGSHAWE

When the plasma level of HCG is relatively low, the concentration of HCG in the spinal fluid may be close to or below the level of detection of the radioimmunoassay. Concentration of the HCG in the spinal fluid has been achieved by various methods in this laboratory, but freezedrying has proved to be the most satisfactory and permits a 5 to 10-fold concentration to be achieved, the limit being imposed by the volume of spinal fluid which can be safely removed. HCG detection in the spinal fluid has generally proved to be more sensitive than clinical examination, scintillation scanning, electroencephalography, and arteriography in the detection of choriocarcinoma metastases in the brain. There have been a few instances, however, when early clinical signs have preceded an alteration in plasma :spinal fluid ratio, presumably depending on the anatomical relationship between the metastases and the spinal fluid. The p1asma:spinal fluid ratio provides a means of monitoring the response of the brain metastases to therapy more or less independently of disease elsewhere in the body. Intrathecal methotrexate combined with systemic chemotherapy has proved to be capable of eradicating intracranial metastases which have caused hemiplegia, blindness, and coma. Full recovery from extensive neurological damage has occurred in 8 of 10 such patients; 2 cases had some residual neurological deficit. However, where intracranial metastases develop a t a late stage in treatment, eradication has proved more difficult. Radiotherapy alone was successful in 5 of 21 patients (Brace, 1968). I n such cases, the best results have been obtained in the author’s series when systemic and intrathecal chemotherapy was combined with radiotherapy. Any patient showing signs of drug resistance needs to have plasma: spinal fluid ratios determined regularly. I n a significant number of resistant cases, tumor growth has continued in the nervous system when it has apparently been eliminated elsewhere. The blood-brain barrier which affects the diffusion of cytotoxic drugs is assumed to be responsible for this “sanctuary” effect in a fashion comparable to that of intracranial disease in leukemia.

E. HCG ASSAYSIN THE FOLLOW-UP OF PATIENTS AFTER HYDATIDIFORM MOLE I n contrast to the trophoblast of nonmolar pregnancy, trophoblastic fragments of mole tend to remain active long after the uterus has been evacuated. However, in only about 2 3 % of cases does choriocarcinoma develop ; in most of the remainder, the trophoblast dies out spontaneously. In others, invasive mole is found. It has been shown that the prognosis of patients who develop chorio-

GESTATIONAL CHORIOCARCINOMA

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carcinoma following hydatidiform mole depends on early recognition of the tumor and that, in turn, this is determined largely by the effectiveness of the follow-up methods (Bagshawe et al., 1969). The suitability of radioimmunoassay to large-scale operations and the fact that small aliquots from timed urine collections can be posted by patients to a central laboratory now facilitates his process of follow-up. A national scale screening laboratory follow-up service has been introduced in the United Kingdom for this purpose, and this is perhaps the first large-scale biochemical screening service for cancer to be introduced. Each patient has about 20 tests carried out over a 2-year follow-up period entailing a total of some 20,000 tests annually in the United Kingdom, but such a number is now well within the capacity of single automated radioimmunoassay apparatus (Butler et al., 1971 ; Bagshawe, 1973). In countries where systematic follow-up studies after hydatidiform mole are not possible, prophylactic chemotherapy has been tried (Apelo and Cunanan, 1964; Kaku, 1966; Manahan et aE., 1967; Koga and Maeda, 1968). Unfortunately, this has not been successful. Deaths have occurred from drug toxicity, and in one study, prophylactic chemotherapy increased the overall mortality 5-fold (Ratnam et al., 1971). In any case, the practice of giving agents known to be teratogenic (Powell and Ekert, 1971) to a relatively large number of patients when 95% of them would have no sequelae without treatment is questionable. It may be reasonable for a woman who has had chemotherapy for choriocarcinoma to have further pregnancies, but this scarcely warrants the liberal use of cytotoxic agents in hydatidiform mole. Chromosome studies on five children conceived after successful treatment for choriocarcinoma had a higher incidence of chromosome breaks than controls although the difference was not statistically significant (Cohen et al., 1971). Some centers, notably in the United States, have adopted the policy of giving chemotherapy to all patients still excreting HCG 6-8 weeks after evacuation of a mole (Brewer et al., 1964, 1968; Hammond and Parker, 1970; Goldstein, 1972). This has the virtue of being an easy rule to follow, and it reduces the number of patients given chemotherapy to about 20.40% of all mole patients. It can be criticized, however, on the grounds that this still results in giving chemotherapy to about four to eight times as many women as require it and that more stringent criteria might well be applied. A chemotherapeutic success rate of 100% is less impressive when it is recognized that 75% of the patients would recover without treatment. A follow-up study of 380 mole patients over a 12-year period has led to the following conclusions, HCG excretion rates persisting at a high level (>25,000 IU/day) several weeks after evacuation may call for

246

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prompt therapy to avoid uterine perforation, or hemorrhage, or because of metastatic disease. Cases with persisting HCG activity detected by radioimmunoassay require clinical and radiographic examinations monthly. Anemia may result from gastrointestinal tract metastases as well as from uterine bleeding. Treatment is indicated also if there is histological evidence of choriocarcinoma from uterine curettings, or vaginal biopsy, or if there are pulmonary metastases and HCG values are not falling spontaneously. Further, if there is stiIl HCG excretion detectable by radioimmunoassay 5-6 months after evacuation of the mole, then treatment should not be delayed further. Patients with levels of HCG detcctable by radioimmunoassay may have negative “pregnancy tests” for months or years and then develop overt choriocarcinoma with a high risk of drug resistance. Using these indications for treatment, not more than about 5% of mole patients have required therapy, and none of the others have developed choriocarcinoma during the period of observation. It is evident that the indications for treating patients with chemotherapy after mole vary substantially between different series. I n order to make valid comparisons of the effectiveness of various drug regimens used in different centers, it is essential to know the criteria for therapy and to subdivide the clinical material according to (a) the nature of the antecedent pregnancy (b) the available histological evidence, and (c) the interval between the end of the antecedent pregnancy and the start of treatment. The value of HCG estimations by radioimmunoassay in screening for choriocarcinoma, in diagnosis, in the monitoring of therapy, and in the follow-up care of patients can hardly be exaggerated. They have undoubtedly made a major contribution to the curability of this tumor. The need for comparable index substances for other tumors is evident. IV. Immunological Aspects of Choriocarcinoma

It is well known that when experimental tumors are transplanted t o allogeneic recipients, tumor rejection tends to occur. Metastases from a bronchial carcinoma transferred in an allografted kidney have regressed when immunosuppressive therapy was discontinued (Wilson et al., 1968). The fact that choriocarcinoma is a “successful” malignant allograft or hemiallograft is therefore surprising. However, it is also evident that mammalian evolution has rested securely on the success of the fetal allograft. It would not be possible to review here the complex factors which are known to be involved in the nonrejection of the fetal allograft, and these were recently reviewed in detail by Beer and Billingham (1971). Numerous studies indicate that the trophoblast, which forms the fetal component of the fetomaternal interface, has properties crucial to the survival of the fetus. Thus, the failure of the human immune system

GESTATIONAL CHORIOCARCINOMA

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to reject choriocarcinoma can be associated with a general failure to reject antigenic tumors on the one hand and with failure to reject the fetus in utero on the other. This link between tumor-host and fetomaternal relationships may have more than superficial significance. Several important issues can be defined with respect to our present knowledge of choriocarcinoma. 1. Do the rare conceptions which result in choriocarcinoma fail to carry the genetic potential for histocompatibility differences to distinguish them from the host? In other words, is there some degree of accidental histocompatibility matching of the mating couple? 2. If the conception is distinct from the host by virtue of strong histocompatibility antigens, is this antigenicity expressed on the tumor fully, partially, or not a t all? 3. If the tumor expresses paternally derived histocompatibility antigens, are the immune responses of the host muted in some way and does pregnancy play a part in this? 4. Is there direct evidence of an immune reaction to choriocarcinoma and, if so, can the antigens concerned be identified? A. ABO BLOODGROUPSIN TROPHOBLASTIC NEOPLASIA The antigens of the ABO system are known to be widely distributed and powerful transplantation antigens. Attempts to identify ABH group substances on choriocarcinoma cells in this laboratory have not, so far, been successful. Blood group A antigen was reported to have been detected on early human trophoblast (Gross, 1966), but this finding has yet to be confirmed. Szulman (1972) has recently reported failure to demonstrate H, A, and B antigens on trophoblast, and only basic H structure was found on placental vascular endothelium whereas the full antigenic complement was found on the endothelium and epithelia of the fetus proper according to its ABO blood group. Ideally, one would look for the presence of these and other antigens directly on the tumor tissue, but this is rarely possible and indirect methods are therefore used. The parental blood groups can be studied and, where choriocarcinoma follows a term live birth, the child can be assumed to have the same genetic information as the tumor. Various workers have examined the blood groups of patients with choriocarcinoma and mole. The first report of ABO blood groups distribution in patients with trophoblast tumors was that of Schmidt and Hertz (1961) , who found no disturbance in the groups of 28 patients selected for chemotherapy nor in 20 of their husbands. The cases were not analyzed according to histological findings nor to antecedent pregnancy. Scott (1962) analyzed data for 46 patients with histologically confirmed choriocarcinoma in the records of the Albert Mathiew Chorion-

248

K. D. BAGSHAWE

epithelioma Registry of the American Association of Obstetricians and Gynecologists. He found a shift from group 0 toward A, B, AB when compared with the expected distribution for the United States based on the data of Levine (1958), but it was not statistically significant. The data were interpreted as being consistent with the concept that women in group 0 are less likely to have a compatible fetus and therefore more likely to reject an early choriocarcinoma. Llewellyn-Jones (1965) presented data for 140 Chinese patients with trophoblastic tumors in Malaya also showing a shift away from group 0 toward group AB and group A. In 82 cases where the blood group of the husband was also recorded, the conceptus would have been ABO compatible with the mother in 51 cases and there was a 50% chance of compatibility in the other 31 cases. Further data for patients in Singapore have recently been provided by Dawood e t aE. (1971). These authors found no significant shift in the ABO blood group distribution in 351 patients with hydatidiform mole. In 87 cases of choriocarcinoma, there was a significant increase in the incidence of blood group A and a significant decrease in that of blood group B. No significant differences were observed between Chinese, Malays, Indians, or other races. The availability of data for blood group distribution for different geographical locations within Britain (Mourant et al., 1958; Kope6, 1970) facilitated the analysis of evidence from 260 patients who had various forms of trophoblastic tumor between 1957 and 1970 (Bagshawe e t al., 1971). The series included 69 nontreated patients who had hydatidiform moles which underwent spontaneous regression without sequelae. Estimates of the expected ABO frequencies were calculated individually for each patient according to the geographical location of her home or, in 23 cases, the patient’s, country of origin. The husbands’ ABO blood groups were also obtained in 139 instances. The number of patients in groups B and AB were insufficient for conclusions to be drawn. The principal findings for groups A and 0 are summarized in Table I. An excess risk associated with group A compared with group 0 was found in each subdivision of the patients. This was greatest for those with choriocarcinoma following live birth, where group A patients had more than twice the risk of group 0. In the data for the husbands, the relative risk was reversed being greater for group 0 than group A. The relative risk of 1.5 for all tumor classes is statistically significant ( P = 0.04) and was highest for patients with choriocarcinoma after mole. The right-hand column of Table I shows how the A/O excess in pa-

TABLE I RELATIVE RISKSFOR GROUPA AND 0 PATZENTS IN RELATION TO HUSBANDS’ GROUPS AND IN RELATION TO MATING FREQUENCY~ Relative risk Mating pairs

Tumor class

I I1 I11 IV V VI VII VIII IX

All cases Treated cases Not treated (hydat.idiform mole) Treated after nonmolar pregnancy Treated after molar pregnancy Choriocarcinoma after term live birth Choriocarcinoma after nonmolar abortion Choriocarcinoma after mole Invasive mole or no morphological diagnosis after mole

data taken from Bagshawe et al. (1971).

Patient For group A women compared with group 0

1 . 3 ( P = 0.06) 1 . 4 ( P = 0.06) 1.2 1 . 6 ( P = 0.08) 1.2 2 . 2 ( P = 0.05) 1.2 1.2 1.3

A X A matinga Husband compared with For group 0 husbands compared 0 women with A or 0 husbands with group A 1 . 5 ( P = 0.04) 2 . 0 ( P = 0.005) 0.9 2 . 2 ( P = 0.06) 1.9 ( P = 0.06) 1.8 5.4 4.7 ( P = 0.01) 1.1

A X 0 matings compared with 0 women wit,h A or 0 husbands

0.4

2 . 4 ( P = 0.001)

0.5 0.4 0.4 0.7 0.00 0.7

3.8 ( P = 0.001) 1.8 5 . 0 ( P = 0.001) 1.4 1.7 1.9

250

K. D. BAGSHAWE

tients combined with the O/A excess in the husbands resulting in highly significant differences between treated and nontreated classes. The excess risk of group A women is shown to depend on the group of the husband. Indeed, the risk for a group A woman with a group A husband is reduced to 0.4 compared with a group 0 woman. However, for a group A woman with a group 0 husband, the risk is increased to 2.4, a difference which is highly significant ( P = 0.001). The deficient risk for A X A matings and excess risk for A X 0 matings is evident in all treated classes. On the other hand, there is an excess of like matings (A X A + 0 X 0 > A X 0 0 X A) in the nontreated class. I n 24 instances where choriocarcinoma followed a term live birth, the ABO blood group of the child from that pregnancy was also determined. The expected number of incompatible children in a sample of this size was 3.9; the observed number was 2. In both instances where the child was incompatible and in 2 further cases of Rh incompatibility, extensive metastatic spread occurred early in the course of the disease. It may be significant that all four patients remitted promptly with chemotherapy and the remissions have been sustained. Although the number of AB patients in the series was not significantly increased, it was observed that these patients seemed to respond poorly to chemotherapy and only 2 of 8 achieved sustained remissions whereas 80% of the whole series of treated patients have had sustained remissions. A similar prognostic effect for group AB patients has been reported by Dawood et al. (1971). The dominant feature of the ABO data is the excess risk associated with A 9 X O d matings compared with A 9 X Admatings. Since both types of matings can produce only conceptions which are ABO compatible with the mother, it is clear that the mechanism cannot be accounted for simply in terms of ABO compatibility. A surprising finding was that the choriocarcinoma-associated children born from A X 0 matings were equally divided between group 0 and group A. This suggests that the powerful effect of blood groups on the development of choriocarcinoma is inexplicably influenced by genetic factors reflected in the ABO group of the husband but not in the ABO group of the conceptus. Obviously, the question arises whether this effect occurs a t or even before fertilization, but a t present, we can only speculate.

+

B. HL-A ANTIGENS Studies of HL-A antigens on the leukocytes of patients with trophoblastic tumors and of their husbands were reported soon after specific antisera became available. Robinson et al. (1963) reported two patients

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who were compatible with their respective husbands, and this was advanced in support of the hypothesis that genetic similarity between patient and husband favored growth of the tumor But as more leukocyte antigens were defined, so incompatibilities between patient and husband were recognized (Robinson et al., 1967). IvBskovA et al. (1968) and Amiel and Lebovici (1970) also reported small series. Mogensen and Kissmeyer-Neilsen (1968, 1969, 1971) have studied patients, their husbands and, where available, their children. In their most recent publications, these authors had studied 20 patients with choriocarcinoma, 16 with invasive mole, 30 of their husbands and 41 of their children, but none from pregnancies leading to choriocarcinoma. They concluded that generalized disease occurs when the matings are such that the conceptus could be compatible with the host a t one or both HL-A loci, whereas patient-husband incompatibility a t both loci favors localized disease. They have also suggested that HL-A typing has prognostic significance and that where incompatibility is pronounced, only small doses of chemotherapeutic agents need be given. Quite contrary conclusions have been reached after study of a larger series of cases in Britain (Lawler et al., 1971). I n 19 Caucasian families in which choriocarcinoma followed a term live birth, 14 children were incompatible with the mother for two paternally derived antigens, one belonging to each locus, and 4 children were compatible only for a first locus antigen. Only one child was antigenically compatible with the mother. There were examples of incompatibility for all the antigens tested. In comparable single-child families in the United Kingdom, 24% of families would be expected to have a child who inherited from the father one antigen present in the mother and 2% of children would be expected to be antigenically compatible with the mother (Lawler, 1971, 1973). The one child who was HL-A compatible with the mother was ABO incompatible. It seems clear that conceptions associated with subsequent development of choriocarcinoma are neither more nor less compatible with the patients than the children of normal mothers. In a study of 15 such families (Lewis and Terasaki, 1971), two instances of HL-A compatibility between child and mother were found. The mating patterns for HL-A antigens were studied on the basis of phenotypes in 75 Caucasian patients and their husbands (Klouda et al., 1972a). This included 41 patients with histologically confirmed choriocarcinoma and 34 with trophoblastic neoplasia not histologically confirmed, following mole. For each locus, each husband/wife pair was scored four times and the observed number of matings for each possible pair was compared with the number that would be expected. Their analysis showed that HL-A mating was at random for both first and second loci.

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There was no assortment leading to a tendency for like to mate with like. The risk of a woman getting a trophoblastic neoplasm is not influenced by her choice of mate with respect to the HL-A system. This series was also studied for an influence from the HL-A system on prognosis. Among 73 patients who had completed treatment, there were 4 deaths in 30 patients who had no antigens in common with their husbands and 8 deaths among 43 having one or two antigens in common. No correlation was found between the degree of compatibility and the duration of therapy necessary to obtain a remission in the survivors nor with the extent of metastases. If there is any effect from HL-A factors on prognosis, it is a slight one, and there would seem to be no basis whatsoever for modifying treatment regimen according to the HL-A antigens on the cells of the patient and her husband.

C. HL-A ANTIBODIES The presence of antibodies to the husband’s histocompatibility antigens in the serum of patients with trophoblastic tumors is not significant in itself since a single pregnancy is known to evoke such antibodies in some women and they are also found following blood transfusion. However, under certain circumstances antibodies may be significant. The occurrence and specificities of HL-A antibodies in patients with trophoblastic tumors have been reported. Math’&et al. (1964) found agglutinating activity against the husband’s cells in five cases and in one instance, these were found after a single mole pregnancy followed by choriocarcinoma. Mogensen et al. (1969) reported that lymphocytotoxic antibodies were present in four of their cases, and in three instances were monospecific. I n a study of 97 patients (Klouda et al., 1972b), cytotoxic antibodies were found against a panel of lymphocytes in 44 instances. Some of the antibodies were monospecific, but serial examinations revealed that the specificities and titers changed with time as treatment proceeded, the weaker antibodies disappearing first. Antibody production in many instances was attributable to pregnancy, but it was often boosted by blood transfusion. No correlation was found between the presence of antibodies and prognosis. In three instances in this series, there was strong evidence that a single molar pregnancy followed by a trophoblastic tumor had immunized the patient against HL-A antigens on the husband’s cells (Lawler et al., 1972). Such cases are notable because in molar pregnancy, in contrast to normal pregnancy, the transplacental passage of leukocytes as a source of immunizing cells is unlikely. The possibility that immunization results from contact between maternal cells and the stroma of the hydatidiform villi rather than trophoblast cannot be excluded. The fact that high anti-

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body titers are found during pcrsistencc of the trophoblastic neoplasm and decline following its eradication suggests this tissue as the source of continued antigenic stimulation. Attempts to demonstrate HL-A antigens by direct immunofluorescent techniques on trophoblast and choriocarcinoma cells have so far been less than conclusive. Loke et al. (1971) found evidence for the presence of HL;AP when molar trophoblast was grown in vitro. Unfortunately, it is difficult to be sure in such studies that all the cells are trophoblastic in origin. The relevant evidence from antibody studies is therefore fragmentary, but it would seem that, as long as choriocarcinoma is active, HL-A antibodies can often be found in variable titer, but that after regression has occurred, the titers decline. This would seem to suggest that, with the reservations already stated, the corresponding HL-A antigens are present on choriocarcinoma cells but not necessarily as numerous as on other tissues.

D. CELLULAR REACTION TO CHORIOCARCINOMA It has sometimes been said that there is no lymphocytic cellular response to the invading tissue of the normal placenta (Hertig, 1968). But Park (1971) has emphasized that this is true only during the first 2 weeks or so of nidation and that a predominantly lymphocytic infiltration may be seen in close association with trophoblast throughout most of human pregnancy. Similarly, various authors have commented on the absence of cellular reaction to choriocarcinoma (Hackett and Beech, 1961 ; Porter, 1966; Iliya et al., 1967). Although there are cases where few leukocytes are to be found, this is not true in the majority of instances examined here. Elston (1969) analyzed histological material from primary and metastatic growths in 38 patients in this series followed for periods of 5 to 13 years. The cellular reaction found in 34 instances was scored as “nil” in 3, “slight” in 15, “moderate” in 11, and “marked” in 5. The cells were mainly lymphocytes and histiocytes with some plasma cells; many of the large mononuclear cells had pyroninophilic cytoplasm. Polymorphs were present adjacent to necrotic areas and hemorrhage. To see whether the cellular reaction had any effect on prognosis, the “nil” and “slight” groups were pooled as “mild,” the “moderate” and “marked” groups were pooled as “severe.” Of 18 patients with mild cellular reactions, 12 died from their disease whereas only 2 of 11 died where the reaction was “severe.” Park (1971) has analyzed his data along similar lines and stated that his findings are essentially in agreement with those of Elston. Ober et al. (1971) also described lymphocytic infiltration around some chorio-

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carcinomas. In a more recent analysis, Elston and Bagshawe (1973) have compared the influence of the cellular reaction on prognosis with other factors that might influence prognosis, but no additional correlations were found. This was surprising in view of the observation that, for the series as a whole, the time interval between the end of the antecedent pregnancy and the start of chemotherapy shows a strong correlation with prognosis. The series available for histological examination was selected principally according to the need of certain patients for major surgery such as hysterectomy or thoracotomy in addition to chemotherapy. The series examined histologically is therefore not representative of the drugtreated series as a whole, but representative of the less amenable forms of the disease. It has to be emphasized that some patients with little or no mononuclear cell reaction respond well and that some who have a good reaction ultimately prove resistant, but the degree of correlation on present evidence is consistent with the concept that a marked host response to the tumor is more favorable prognostically than a feeble one. It seems reasonable to interpret the histological evidence as indicating a host immune response to antigens on the tumor cells. But there is, of course, no indication of the nature of the antigenic stimulus. Studies to determine whether there is a correlation between antigenic disparity a t HL-A loci and the degree of cellular response have yet to be reported. The possibility remains that tissue-specific or tumor-specific antigens may be involved. Apart from the histological evidence that a lymphocytic reaction occurs both in the normal placental bed and around many instances of choriocarcinoma, evidence for cellular immunity to paternal antigens as a result of pregnancy in mice was found by Soren (1967) using the spleen assay technique of Simonsen. Also, the reactivity of lymphocytes from wife/husband pairs has been studied in two-way cultures. Lewis et al. (1966) found that most pregnant women show a specific lack of responsiveness to their husbands’ cells compared with unrelated donor cells in mixed leukocyte cultures, but that patients with trophoblastic neoplasms appeared not to share this unresponsiveness. Halbrecht and Komlos (1968) found an increased blastic transformation in wifebusband leukocyte cultures in cases of abortion and hydatidiform mole. Ohama and Kadotani (1971) reported a lower level of reactivity in fertile couples than in sterile couples and those with aborted pregnancies. The lymphocytes of a patient with choriocarcinoma following term delivery responded to stimulation in a one-way reaction with cells from the infant (Rudolph and Thomas, 1971), but one-way mixed lymphocyte reactions have not yet been reported from any large series of patients.

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E. REJECTION OF HUSBAND SKINGRAFTS The immunoreactivity of choriocarcinoma patients has also been studied with skin grafts from their husbands. Robinson et al. (1967) and Math6 et al. (1964) each reported two patients showing prolonged acceptance of skin grafts from their husbands. On the other hand, Li (1967) reported accelerated rejection of similar grafts on two patients. Bagshawe and Golding (1970) reported a study of 94 skin grafts on 21 patients with choriocarcinoma which had proved resistant to chemotherapy. The mean rejection time for first grafts from the husband was 18.9 days (range 1 6 22 days) compared with a mean rejection time of 9.4 days (range 8-11 days) for grafts from unrelated donors. Thus, although these patients may have been immunosuppressed as a result of their preceding chemotherapy, a marked difference was observed between the rejection time of husband skin grafts and those of unrelated donors. A series of grafts from the husbands at intervals of 18-30 days resulted in a shortening of the mean rejection time, but after a series of 7 grafts (8 cases), it was still 11.1 days. Graft rejection times under 10 days were exceptional in these patients. Four patients received either intradermal BCG or husband’s leukocytes in Freund’s complete adjuvant between the first and second skin grafting. I n these patients, the second graft was rejected more rapidly (range 5-9 days). The tolerance to husband skin grafts appeared to be abrogated more readily after nonspecific immunization than after repeated “specific” stimulation with husband’s skin. In one instance, a skin graft from the child isogenic with the tumor was also transferred to the mother. The rejection time for the child’s skin was 42 days, for the husband’s 20 days and for an unrelated donor graft, 9 days. While it may be highly significant that patients with choriocarcinoma tolerate skin grafts from their husband substantially longer than grafts from unrelated donors, a full evaluation in relation to malignant disease is not possible because of the lack of data for the effect of pregnancy itself on the rejection of husband’s skin grafts. It has been shown that in mice, multiparity in interstrain matings induces prolonged acceptance of male strain tissues (Breyere and Barrett, 1960; Prehn, 1960; Porter and Breyere, 1964; Kaliss and Dagg, 1964). Using male strain tumor tissue as the challenge graft, Sazama and Breyere (1969) and Currie (1969) found that detectable levels of tolerance were induced by days 12 and 9 of pregnancy, respectively. Thus exposure to strong fetal antigens under the physiological conditions of pregnancy and the pathological circumstances of trophoblastic

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tumors results in prolonged acceptance of the tissues of the specific mating male.

F. CHORIOCARCINOMA AND FETOMATERNAL IMMUNITY Although the studies with the ABO blood groups indicate the clear operation of genetic factors in the causation of choriocarcinoma, the immunological implications are by no means clear, and the data for HL-A antigens are remarkable for the apparent lack of effect of these antigens. Genetically, choriocarcinoma is distinct from the host, but we cannot conclude that there is a corresponding degree of antigenic distinction expressed by choriocarcinoma cells. To pursue this point further, it is necessary to refer again to the normal fetomaternal relationship. Failure to reject the trophoblast was attributed to its apparent deficit of transplantation antigens (Simmons and Russell, 1963; Haskova, 1961). However, Kirby e t al. (1964) suggested that periplacental fibrinoid material, a mucoprotein rich in sialic acid, provided a continuous electron dense fetomaternal interface with characteristics appropriate to an immunological barrier. Currie et al. (1968) found that when they injected ectoplacental cones, consisting of trophoblast which is free or almost free of other cells, from A2G mice into adult male CBA mice, a subsequent skin graft from A2G mice on the CBA recipients evoked a first set response. But if the ectoplacental cone cells were desialylated by pretreatment with neuraminidase before injection, then a subsequent male strain graft evoked a second set response implying that the sialic acid in some way masked strain-specific transplantation antigens on trophoblast. This suggested that the glycocalyx of the cell was involved in antigen masking, or that sialic acid groups resulted in steric hindrance, or that desialation produced conformational changes in the cell periphery with an associated increase in immunogenicity . In vivo studies of the action of maternal lymphocytcs on normal human trophoblast (Douthwaite and Urbach, 1971) and of malignant trophoblast (Currie and Bagshawe, 1967) revealed that cytotoxicity was greatly increased by prior trypsinization of the trophoblast. On the other hand, Simmons e t al. (1971) using a similar but not identical model to that of Currie et al. (1968) concluded that neuraminidase did not unmask histocompatibility antigens on trophoblast, although like Currie and Bagshawe (1968, 1969) and Sanford (1967), Simmons and Rios (1971) found that neuraminidase did increase the immunogenicity of various typcs of malignant cell.

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The balance of evidence seems to indicate that normal trophoblast is, relative to other tissues, deficient in histocompatibility antigens but not in species-specific antigens. What part, if any, is played by sialoproteins in the masking of antigens on trophoblast remains controversial. The interaction of cellular and humoral immune responses has not been studied in choriocarcinoma, but again, evidence from normal pregnancy may be relevant. In a study with A2G female mice mated with CBA males, Currie (1969) detected a significant impairment of restraint to the growth of a fibrosarcoma induced in CBA mice by methylcholanthrene as early as the ninth day of the first pregnancy. Moreover, he was able to show that the ability of virgin A2G females to restrain the growth of grafts of this tumor was impaired by repeated injections of serum from A2G females which were multiparous by CBA males. This finding was consistent with the observations of immunological enhancement as originally suggested by Kaliss and Dagg (1964). Further evidence that immunological enhancement might be involved in the specific weakening of maternal responses to male antigens has been provided by Hellstrom e t al. (1969). The8e investigators mated BALB/C female mice with C3H males and, using their colony inhibition technique, found that maternal lymph node cells inhibited the growth of C3H fetal target cells in vitro. Further, they found that sera from pregnant mice abrogate this effect, presumptively as a result of blocking antibodies. It is not possible at the present time to assign relative orders of importance to the various mechanisms that appear to be involved in the success of the fetal allograft. It seems probable, but is not yet certain, that both normal and malignant trophoblast display antigens inherited from the male parent, but that they may be deficiently expressed compared with other fetal tissues. It may be that both sialic acid-containing prosthetic groups and blocking factors participate in antigen masking. It seems likely from the evidence so far reviewed that a t least some of the complex reactions that facilitate the escape of the fetus from immunological rejection also favor the growth of choriocarcinomas. Nevertheless, evidence from cellular reactions to the tumor make it improbable that the abrogation mechanisms are fully effective, and it seems reasonable to expect that some restraint is imposed by immunological mechanisms. We have no means of knowing whether such restraint is greater than that imposed on a wide range of other tumors. The fact that testicular choriocarcinoma grows in lymph nodes and rarely responds fully to cytotoxic therapy may be supposed to indicate the greater strength of the response to the gestational variety of choriocarcinoma. The argument, however, carries little weight since both testicular and primary

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ovarian choriocarcinomas treated a t an early stage in dissemination respond well to chemotherapy and may also be curable. G. IMMUNE REXPONSES IN RELATION TO CYTOTOXIC THERAPY FOR CHORIOCARCINOMA A further line of investigation with choriocarcinoma patients has been to determine their immunological status before and during chemotherapy. Indices of static, cellular, and humoral immunity have been measured in serial observations (Golding, 1972). Immunoglobulin levels proved to be in the normal range generally, although in some instances small increases occurred in IgG, IgA, and IgM values during therapy. For the study of humoral and cellular immunity, the 30 patients studied were divided into two groups. Group 1 consisted of patients with relatively early disease following hydatidiform mole who achieved sustained remissions with a methotrexate/folinic acid regimen (Bagshawe, 1969), and in no case did the duration of treatment exceed 5 months. All these patients had normal primary cutaneous reactions to dinitrochlorobenzene (DNCB). During therapy, the DNCB response was partially inhibited in five instances, and this impairment was still detectable 6 weeks after completion of therapy in two patients. Twelve patients were initially tuberculin positive, and a good correlation was observed between the tuberculin skin reaction and the in vivo leukocyte migration index (Sgiborg and Bendixen, 1967). On completion of therapy, only eight were positive, and 6 weeks later, one was still unresponsive. Lymphocyte transformation with YHA became impaired during the early stages of therapy, but soon after completion of therapy, 9/14 patients had values in excess of their initial responses. Humoral responses to salmonella Tennessee “H” antigen were normal in only three of the seven subjects studied in this group. There was marked impairment of primary antibody responses in two patients and in the secondary responses in five instances. In general, however, the impairment of immune responses in this group were relatively slight. Group 2 consisted of thirteen patients with more advanced disease following mole, abortion, or term pregnancy. These patients were treated initially with the same methotrexate/folinic acid regimen as group 1, but because of incomplete remission, they subsequently received treatment with actinomycin D, and some patients also received these agents in various combinations with 6-azauridine, cyclophosphamide, vincristine, and 6-mercaptopurine. Five of these patients died, two remain under treatment, six are in remission after treatment lasting 4-12 months.

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Of the five patients who died, two had low immunoglobulin levels and severely impaired cellular and humoral responses before receiving any treatment. Neither patient responded to the initial or subsequent therapy, a finding which is exceptional in gestational choriocarcinoma. The other three fatal cases had good initial cellular responses, but two of them had impaired humoral responses on admission and their cellular responses became impaired during therapy. The third died from pulmonary embolism. The combination drug therapy suppressed cell-mediated immune responses, and the longer it was continued, the more severe this effect became. The four patients showing the least immunosuppression in this group were numbered among the six who went into remission. It has to be recognized that these indices of immunity may reflect host responses to tumor antigens inadequately. Indeed, the fact that these nonspecific indices were often normal a t the outset, when husband skin graft rejection is known to be impaired, indicates their limitations. Serial, one-way mixed lympocyte reactions between patient and husband, would provide additional information, but such studies have been possible so far only on a limited scale. Even so, the present evidence suggests that indices of cellular immunity have some prognostic value and that poor responses, either initially or induced by chemotherapy, are associated with worsening of the prognosis. These findings need to be confirmed in more extensive studies of choriocarcinoma. They suggest that the immunosuppressive actions of cytotoxic agents may be an important self-limiting component which will have to be taken into account. Should the cytoreductive action observed clinically as “drug sensitivity’’ depend on a supplementary action from the immune system, then reducing that supplementary action could presumably contribute to “drug resistance.” Immunologically mediated cell losses are only one of several routes of “cell loss” from choriocarcinoma (Bagshawe, 1968). Cells are lost from the cytotrophoblast cell population by differentiation to syncytium and probably from nutritional causes, and these contributions are added to immunologically mediated cell deaths. Theoretical analyses of cytotoxic drug action based on growth rate of the tumor cell populations may have taken too little account of the contribution from cell losses by various routes. It has therefore been suggested that the curability of choriocarcinoma depends on a high rate of spontaneous cell loss (Bagshawe, 1970). The magnitude of the contribution from immunological mechanism is not yet known, but the fact that poor immune responses are frequently associated with drug resistance and advancing tumor growth suggests that

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they are significant. It remains to be seen whether measures to avoid specific immunosuppression can further reduce the incidence of drug resistance.

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Manahan, C. P., Abad, R., and Lopez, A. M. (1967). Zn “Choriocarcinoma” (J. F. Holland and M. M. Hreshchyshyn, eds.), pp. 72-75. Springer-Verlag, Berlin and New York. Mathk, G., Dausset, J., Hervet, IE., Amiel, J. L., Columbani, J., and Brute, G. (1964). J . Nut. Cancer Znst. 33, 193-208. Midgley, A. R. (1966). Endocrinology 79, 10-18. Mogensen, B., and Kissmeyer-Nielsen, F. (1968). Lancet i, 721-724. Mogensen, B., and Kissmeyer-Nielsen, F. (1969). Dan. Med. Bull. 16, 243. Mogensen, B., and Kissmeyer-Nielsen, F. (1971). Transplant. Proc. 3, 1267-1269. Mogensen, B., Kissmeyer-Nielsen, F., and Hauge, M. (1969). Transplant. Pruc. 1, 76-79.

Morgan, F. J., and Canfield, R. E. (1971). Endocrinology 88, 1045-1053. Mourant, A. E., Kopec, A. C., and Domaniewska-Sobczak, K. (1958). “The ABO Blood Groups.” Blackwell, Oxford. Ober, W. B., Edgcombe, J. H., and Price, E. B. (1971). Ann. N. Y . Acad. Sci. 172, 299-426. Odell, W. D., Bates, R. W., Rivlin, R. S., Lipsett, M. B., and Hertz, R. (1963). J. Clin. Endocrinol. Metab. 23,658-664. Ohama, K., and Kadotani, T. (1971). Amer. 3. Obstet. Gynecol. 109, 477479. Park, W. W. (1971). “Choriocarcinoma. A Study of its Pathology.” Heinemann, London. Pattillo, R. A., and Gey, G. 0. (1968). Cancer Res. 28, 1231-1236. Porter, J. B., and Breyere, E. J. (1964). Transplantation 2,246-250. Porter, K. A. (1966). I n “Recent Advances in Pathology” (C. V. Harrison, ed.), 8th Ed., pp. 242-319. Churchill, London. Powell, H. R., and Ekert, H. (1971). Med. J. Aust. 2, 1076-1077. Prehn, R. T. (1960). J. Nut. Cancer Znst. 25,883486. Ratnam, S. S., Teoh, E. S., and Dawood, M. Y. (1971). Amer. J. Obstet. Gynecol. 111, 1021-1027.

Robinson, E., Shulman, J., Ben-Hur, N., Zuckerman, H., and Neuman, Z. (1963). Lancet i, 300302.

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GLYCOLIPIDS OF TUMOR CELL MEMBRANE1 Sen-itiroh Ha komori Deportment of Pathobiology, School of Public Health, and Deportment of Microbiology, School of Medicine, University of Washington, Seattle, Washington

I. Introduction . . . . . . . . . . . . . , 11. Structure and Organization of Glycosphingolipids in Membrane . . 111. Change of Glycolipids in Experimental Tumors and Transformed Cells in V i t ~ o . . . . . . . . . . . . . . IV. Glycolipids of Human Tumors and Human Leukemic Cells . . , A. Blood Group Glycolipids or Glycoproteins . . . . . B. Ganglioside . . . . . . . . . . . . . C. Formman Glycolipid . . . . . . . . . . . . D. Human Leukemic Cells . . . . . . . . . . . E. Serum Factor Reactive to Lactosylceramide . . . . . . V. Cell Contact, Contact Inhibition, and Glycolipid Synthesis . . . VI. Change of Glycosyltrmsferases and Hydrolases . . . . . . VII. Reactivity of Cell Surface Glycolipids and Glycoproteins of Normal, Fetal, and Cancer Cells with Macromolecular Reagents . . . . A. Reactivity with Glycosylhydrolases . . . . . . . . B. Reactivity with Galactose Oxidase and External Labeling Pattern of Glycolipids and Glycoproteins . . . . . . . . , C, Reactivity with Phytoagglutinin (Lectin) . . . . . . . D. Reactivity with Agglutinin Derived from Animal Tissue and Cells . E. Reactivity with Antiglycolipid Antisera . . . . . . . VIII. The Significance of Membrane Glycolipid Changes in Regulation of Cell Growth and Intercellular Linkages: Some Comments and Speculations . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

.

.

265 269 277 283 283 285 285 286 286 286 290 293 293 294 295 300

300 302

308

I. Introduction

Tumor cells, whether transformed by viruses or by chemicals, exhibit various biological abnormalities related to the function of cell surface membranes, as listed in Table I. These aberrant surface functions, whether just one or several occur, are the common denominator of tumor cells. Some processes could be different manifestations of common molecular changes of membanes, which are listed in Table 11. 'This work was supported by grants from the U. S. Public Health Service (CA 10909 and CA 12710), and from the American Cancer Society Grant 3C-9-C. 265

266

SEN-ITIROH HAKOMORI

TABLE I ABERRANT BIOLOGICAL PROPERTIES OF TUMOR CELLSURFACE MEMBRANES ~~

Phenomena Enhanced transport of sugars, amino acids, and nutrients

~

Teleologic consequences

References

Increased intracellular concentration of nutrients that accelerate cell growth

Paul et al. (1966); Foster and Pardee (1969); Hatanakrt and Hanafuss (1970); Hatanaka et al. (1970); Sefton and Rubin (1971); Isselbacher (1972); Kalckar et al. (1973); G. S. Martin et al. (1971) Loss of contact inhibition Uncontrolled cell growth Abercmmbie and Ambrose of cell movement and by cell contact (1962); Todaro and Green cell division (1963) Decreased surface adhe- Infiltrative and meta&man (1960); Campbell and siveness static properties Edwards (1970); Edwards et al. (1971); Vicker and Edwards (1972) Loss of intercellular com- Independency and auton- hewenstein (1968, 1973) munication omy of tumor cells Presence of tumor specific Unknown (uncontrolled Foley (1953); Prehn and Main surface antigens cell division?) (1957); Klein (1959); Sjogren (1965)

During the past few years, evidence has pointed toward the relation of certain surface properties, such as contact inhibitability, intercellular linkages, and antigenicity of cells, with the structure and function of membrane-bound glycolipid or glycoprotein and their related enzymes. Other functions, such as sugar transport and its changes upon malignancy, are not necessarily related to membrane-bound complex carbohydrates, although the possibility cannot be denied that they could be indirectly related, as neuraminidase treatment of cells can enhance sugar transport (see Section VII) . Molecular changes of cell membrane associated with malignant transformation are itemized in Table 11, although none of these changes have been successfully correlated with the characteristic biological properties of tumor cells. It has, however, become increasingly apparent that some of these molecular changes are essential steps in the process of malignant transformation of cells, as these molecular changes of membranes, such as hematoside synthesis, can be reversed when the biological properties of tumor cells transformed by a temperature-sensitive mutant of oncogenic viruses, were reversed to a normal state a t nonpermissive temperature (see Section 111). The molecular change of membrane as expressed by reactivity to phytoagglutinin or by ganglioside synthesis was found to be normalized when the properties of

267

GLYCOLIPIDS OF TUMOR CELL MEMBRANE

TABLE I1 MOLECULAR CHANGES O F CELL SURFACE MEMBRANES ASSOCIATED WITH MALIGNANT TRANSFORMATION Chemical, physical changes ~

References ~

1. Increased negative surface charge demonstrated by cell-electrophoresisand histochemical staining 2. Change of total sialic acid, amino sugar, and neutral sugar content in membranes 3. Change of glycolipids a. Simplifications of glycolipid pattern (incomplete synthesis)

b. Accumulation of some glycolipid (which, in some cases, result from blocked synthesis) c. Blocked enzyme activities for glycolipid synthesis

~~

Abercrombie and Ambrose (1962); Forrester et al. (1962); Defendi . and Gasic (1963) Shen and Ginsburg (1968); Gulp et al. (1971); Perdue et al. (1971, 1972) Hakomori and Murakami (1968); Hakomori et al. (1968); Mora et at. (1969); Brady et al. (1969); Brady and Mora (1970) Hakomori and Murakami (1968); Siddiqui and Hakomori (1970); Brady et al. (1969); Cheema et al. (1970); Yogeeswaran et al. (1972); Diringer et al. (1972) Cumar et al. (1970); Den et al. (1971); Kijimoto and Hakomori (1971)

4. Change of glycoproteins

Wu et al. (1969); Meezan et at. (1969); Grimes (1970); Perdue et teins, especially lack of terminal sialic al. (1971, 1972); Saito et al. (1972); acid and decreased activity of sialyltransferase Grimes (1973) b. Enhanced fucose-labelingpattern Buck et al. (1970, 1971a,b) c. Increased level of a specific sialyltransferase Warren et al. (1972a,b); Bosman et al. (1968) d. Enhanced glycosylhydrolase Bosman (1969) 5. Change of sulfated mucopolysaccharides a. Heparitin sulfate and chondroitin sulfates Makita and Shimojo (1972); Saito and Ueman (1971) Hakkinen et al. (1968) b. Immunological change of glycoprotein sulfate 6. Lack of “contact extension” or “contact Hakomori (1970); Robbins and Macpherson (1971); Sakiyama et accumulation” of glycolipids al. (1972); Kijimoto and Hakomori (1972); Critchley and Macpherson (1972) 7. Change of reactivity of surface carbohydrate residues; reactivities with: Hakomori et al. (1968); Hakomori a. Antiglycolipid antibodies and lack of “contact masking” and Kijimoto (1972) a. Incomplete synthesis of some glycopro-

(Continued)

268

SEN-ITIROH HAKOMORI

TABLE I1 (Continued) Chemical, physical changes

References ~~~~

b. Phytoagglutinins:wheat germ agglutinin, Aub et al. (1963); Burger and Goldconcanavslin A, Ricinus communis, soyberg (1967); Burger (1969); Inbar bean, Wistariajloribunda and Sachs (1969); Nicolson (1971, 1972); Lis et al. (1970); Takeuchi et al. (1972); Tomita et al. (1970, 1972) c. Egg agglutinin Kawauchi et al. (1972) d. Invertebrate proteins Uhlenbruck and Sehrbundt (1968); Prokop et al. (1968) 8. Change of external labeling pattern Gahmberg and Hakomori (1973) 9. Tumor specific “proteolipid” Skipski et al. (1972)

the “flat revertant” were compared with those of the original population of transformed cells (see Sections 111 and IV,C) , The philosophy behind the recent development of cell surface biology is that plasma membrane is a regulatory organelle for cellular metabolism and replication, as well as for intercellular recognition, and that uncontrolled growth of cancer cells occurs under “epigenetic” regulatory dysfunction of cell surface membranes, rather than from somatic mutation. Although significant progress has been made concerning chemical properties and structure of plasma membrane, our knowledge about the function of plasma membrane in regulating cell metabolism, replication, and intercellular interaction is still very premature and fragmentary. Practically nothing is known about the relation of rhemical composition to structure and function of plasma membrane, and unfortunately, the observed chemical changes of plasma membrane, if any, cannot a t this time be correlated with the functional changes of plasma membrane, although this relation is extremely important for understanding various phases of cell biology. Some speculation and comment will be made about this possible relationship in Section VIII. This article is aimed a t a review and discussion of the structural, organizational, and functional changes of membrane glycolipids in association with malignant transformation of cells. Related data on glycoprotein, protein, and other lipids will also be treated subsidiarily. An extensive review on glycoprotcins, glycolipids, and mucopolysaccharides of the cell periphery has been made by Kraemer (1971, 1972), and relatively concise reviews of the same topic were made by Winzler (1970), Ginsburg and Kobata (1971), and Heath (1971). Reviews of our own work on glycolipid have also been published (Hakomori, 1971).

GLYCOLIPIDS OF TUMOR CELL MEMBRANE

269

II. Structure and Organization of Glycosphingolipids in Membrane

Glycosphingolipid consists of two hydrocarbon chains of ceramide and one Carbohydrate residue with great structural variety. The lipids listed in Table I11 have been isolated and characterized from animal cells and tissues. Three groups of these glycolipids can be classified according to structure of the carbohydrate backbone : ganglio-series, having gangliotetraose (Gal/31+3GalNAc/31+,4Gal/31+4Glc),globo-series having globo-tetraose or globo-neotctraosc (GalNAc~l+3Gala1+4or3Gal/31-+ 4Glc), and lactoseries having lacto-tetraose or lacto-neo-tetraose (Gal/31+ 3or4GlcNAc/31~3Gal/31+4Glc). Ganglio-series is always substituted with sialosyl groups, thus yielding a variety of gangliosides ; lacto-series have sialosyl or fucosyl residues attached, giving an extremely wide variety of compounds; globo-series has no known derivatives. Recently, a new type of highly complex glycolipid with branching structures has been isolated (see last line of Table 111).This includes ceramide deca- to tetrakaidecasaccharides, which were characterized by the presence of two different carbohydrate chains joined to a branching structure a t the galactosyl residue of lactosylceramide. Thus, a glycolipid having multispecific functions can be provided (Hakomori e t al., 1972). According to the systematic studies of Yamakawa and his colleagues, ganglioside, hematoside, and neutral glycolipids are present exclusively in ghosts (plasma membrane) of erythrocytes (Yamakawa and Suzuki, 1951, 1952; Yamakawa e t al., 1960; Yamakawa, 1966). Isolated plasma membranes of liver cells ( n o d and Gray, 1968), L cells (Weinstein e t al., 1970), BHK cells (Renkonen et al., 1970), MDBK bovine kidney cells, monkey kidney MK, HAK cells, hamster BHK and NIL cells (Klenk and Choppin, 1970), and mouse 3T3 cells and their various transformants (Yogeeswaran e t al., 1972) have enriched concentrations of ganglioside and neutral glycolipids as compared to total cells or other membranes. As both Renkonen et al. (1970) and Klenk and Choppin (1970) described, plasma membranes are enriched in all gangliosides present in whole cells, and therefore, no essential qualitative difference exists between total glycolipid pattern of whole cells and that of plasma membrane. This figure differs from membrane analysis of L cells carried out by Weinstein e t al. (1970), which indicated that only a part of hematoside and disialoganglioside was present in plasma membranes. Yogeeswaran et a1 (1972) recently observed that the ganglioside pattern of plasma membrane of 3T3 cells and various clonal isolates of viral transformants was the same as that of total cells, although all gangliosides were not as greatly enriched as described by Klcnk and Choppin and by Renkonen e t al. In our unpublished studies of rat liver cells (Siddiqui and Hakomori), even

TABLE I11 GLYCOSWINOOLIPIDS OF ANIMALCELLS 1. Cerebrosides, dihexosylmamide and sulfatide G@ 1+ lCer

Glcpl+lCer Gal,91+4Glcpl+ lCer (CDH)

H2SOo+3Galfll+4Glcfll-+l Cer

GalNAcp1-+3Gald-+4Galpl+4Glc+Cer Globoside (“cytolipin K”) GalNAcal+3Galal+3Galpl-+4Glc+Cer

Isolation: Thudicum (1874; also see Tierfelder and Klenk, 1930); Thudicum (1904); structure: Carter pt al. (1950) Occurrence, Gaucher spleen: Klenk (1941); liver, spleen, serum: Svennerholm (1963) Occurrence: Klenk and Rennkampf (1942); as human tumor hapten (“cytolipin H”): Rapport et at. (1959); identity of of cytolipin H with ceramide dihexoside of bovine kidney: Rapport el al. (1960); Isol. from erythrocytes: Yamakawa et al. (1960); kidney, Makita and Yamakawa (1962); structure: Yamakawa et al. (1962) Occurrence and isolation: B l i (1933); structure: Yamakawa et al. (1962); Stoffyn and Stoffyn (1963) Occurrence and isolation: MArtensson (1966); structure: Stoffyn d al. (1968) In mouse kidney showing sex hormone response : Gray (1971); in Fabry kidney: Sweeley and Klionsky (1963); structure: Handa d ol. (1971); Li et al. (1972) In Fabry kidney: Sweeley and Klionsky (1963); erythrocyte: Vance and Sweeley (1967); structure: Hakomori et al. (1971b); Li and Li (1971); Handa et al. (1971) Occurrence: Klenk and Lauenstein (1951); Yamakawa and Suruki (1952); structure: Yamakawa et al. (1965); Hakomori et al. (1971h) Occurrence and isolation: Rapport ct al. (1967); structure: Laine d al. (1972); Siddiqui et al. (1972) Occurrence: Brunius (1936); Yamakawa et al. (1961); isolation: Makita et al. (1966);structure: Siddiqui and Hakomori (1971)

N J

3

?

U

3

1 P %

sE

Hemdoside series (GMJ). NGNA2-+3Galp1+4Glc-+Cer

NANA248NAN A2+3Gal@1-+4Glc+Cer NGNA2+3Galp1+4Glc-+Cer

r

AcO Ganglioseries NANA2-13Gal-tCer GalNacp1-+4Cral@1-+4Glc-+Cer Gal~Ac@1-+4~1~1-+4Glc-+Cer 3

r

2 NANA (GM2)" Gall-t3GalNac~1+4Gal~1+4Glc+Cer

T

Occurrence: Yamakawa and Suzuki (1951); structure: Klenk and Padberg (1962) Occurrence: Svennerholm (1963); Klenk and Heuer (1960); st,ructure: Pun, (1969); Handa and Yamakawa (1964) Isolation and structure: Handa and Handa (1965); Handa and Yamakawa (1964) Isolation and structure: Pun, (1969) Isolation and structure: Hakomori and Saito (1969)

Isolation and structure: Siddiqui and McCluer (1968) Isolation and structure from guinea pig erythrocytes: Yamakawa et a2. (1960); structure: Yamakawa (1966); TaySachs' brain, Makita and Yamakawa (1963) TaySachs' ganglioside (GM2)-occurrence and isolation: Klenk (1939); structure: Makita and Yamakawa (1963); Ledeen and Salsman (1965) Occurrence and isolation: Klenk (1942); structure: Kuhn and Wiegandt (1963); Svennerholm (1963)

2

NANA (GMl). Gall -+3GalNac@1-+4Ga&5 l-+4Glc-+Cer 3 3

t

2 NANA

r

Occurrence and isolation: Klenk (1942); structure: Kuhn and Wiegandt (1963); Svennerholm (1963); Johnson and McCluer (1963)

2

NANA

(GD1a)"

(Continued)

TABLE 111 (Continued) N

Gall-+3GalNAcpl+4Ga~1+4Glc+Cer 3

Isolation and structure: Kuhn and Wiegandt (1963)

-3

N

r

NANA8+2NANA

Gall+3GalNAc~l+4Galpl-+4Glc~Cer 3

3

2

2

T

(GDlb)” Isolation and structure: Kuhn and Wiegandt (1963)

T

NANA

NANA 3

u,

r

2 NANA (GTi). Gal1+3GalNAcl+4Gall+4Glc+Cer

r

T NANA

N.4NA

(NANA)

NANA

T

7 Y

Isolation and structure of fish brain: Ishizuka et al. (1970); Wiegandt (1972); Ishizuka and Wiegandt (1972)

t

t

NANA

(tetrasialo or pentasialo-ganglioside)

5 . llLmto-SeTies”

Galfil+4GlcNAcp1-+3Galpl-+4Glc+Cer (“paragloboside”) Fucnl+2CTalfi1+4GlcNAcfi1~3Galfi l+4Glc-+Cer (H1-glycolipid)

Galp1+3GlcNAcfi1-+3Ga1fil+4Glc+Cer 4

r1

n

Fuc (Lea-active glycolipid)

Isolation and structure (human erythrocytes) : Siddiqui and Hakomori (1973); Wiegandt (1972) Isolation and structure (human erythrocytes): Stellner et al. (1973b) Isolation and structure (from adenocarcinonia): Hakomori and Jeanloz (1970)

3

5 P 2!

5E

Gal@l-+4GlcNAc@1-+3Gal~1-t4Glc-+Cer 3

Isolation and structure (from adenocarcinoma): Yang and Hakomori (1971)

t

1 ff

Fuc (X hapten) Galal--t3Gal,81+3GlcN~c@1~3Gal~1-,4Glc-tCer 2

Tsolation and structure (from Fabry’s pancreas): Wherrett and Hakomori (1973)

t

1 Fuc (B-glycolipid of human) Galal-t3Galp 1+4GlcNAc@1+3Gd~1+4Glc-*Cer (B-glycolipid of rabbit)

Gall--t30r4GlcNAc~l--tSGal~1-+4Glc--tCer

T

Isolation and structure (rabbit erythrocyte): Eto el al. (1968); Stellner et al. (1973a) Isolation and structure: Hakomori and Andrews (1970)

T

Fuc Fuc (Leb glycolipid) GalNAc~l+3Gal@l-t4GlcNAc~l--t3Gal~l--t4Glc--tCer 2

Isolation and structure (human ery-throcytes): Hakomori et aZ. (1972a)

t

1 Fuc (A-glycolipid)

NANA2+3Gal~l--t4GlcNAc~l-t3Gal~1+4Glc-tCer NGNA2-13Gallgl+4GlcNAc~l+3G~~l~4Glc~Cer Gd+GlcNAc~l+3Gall+4or3GlcNAc~1+3Gall+4Glc+Cer

t

Isolation and structure: Wiegandt (1970); Siddiqui and Hakomori (1973) Isolation and structure: Wiegandt (1970) Isolation and structure: Hakomori and Andrews (1970)

t

Fuc Fuc

(Continued)

274 SEN-ITIROH HAKOMORI

GLYCOLIPIDS OF TUMOR CELL MEMBRANE

275

ceramide was present a t much higher concentrations in plasma membrane than in other membrane fractions. It is generally accepted that most cellular glycosphingolipids are located on the plasma membrane, and therefore, gangliosides and hematosides can be used as “plasma membrane markers” in the same way that sodium/potassium-dependent ATPase or histocompatibility antigens have been used as “plasma membrane markers.” As yet there has been no positive evidence that glycosphingolipids are exposed on normal cell surfaces. To the contrary, more negative evidence has been presented; that is, (1) reactions of normal cells to antiglycolipid antisera have been weak or nonexistent (Hakomori, 1969; Hakomori et aZ., 1968), and (2) only a few glycolipids have been hydrolyzed by glycosylhydrolases on the cell membrane, although isolated glycolipids were easily hydrolyzed by the same enzymes (see Section VI1,A). Recently, a radioactive external labeling procedure for cell surface galactose and galactosamine has been developed, showing that globoside and ceramide trihexoside of intact erythrocytes can be labeled and exposed to the external environment (Gahmberg and Hakomori, 1973). Morphology of membrane glycoproteins and their mode of association with membranes have been elucidated by Marchesi about “glycophorin” of the erythrocyte membrane; i.e., multispecific carbohydrate chains are attached to hydrophilic peripheral regions of the peptide and to a hydrophobic proximal region associated with the lipid bilayer (Marchesi et al., 1972). The prototype of such a model was proposed by Morawiecki (1964) and by Winzler (1970). Probably a similar structural feature can be found in glycoproteins of other animal cell surfaces. Figure 1 demonstrates an idealized version of glycoprotein-glycolipid associations within the membrane. These associations are considered to be present a t the locations of “plasma membrane particles” (Pinto da Silva and Branton, 1970). For example, blood group A-antigenic sites were shown to be located in the “plasma membrane particles” (Pinto de Silva et al., 1971). Much evidence is currently being presented to show that membranes are composed of a mosaic structure in which globular molecules of integral proteins alternate with sections of phospholipid bilayer in the cross section of membrane. In some cases, the integral globular molecule either contains glycoproteins or strongly associates with specific lipid components. These mosaic components are in a fluid dynamic equilibrium (“fluid mosaic model’’ of Singer and Nicolson, 1972). The fluid dynamic state of membrane antigens was well described (Edidin, 1972), and extreme lability of “plasma membrane particles” with regard to their location has been demonstrated (Pinto da Silva and Branton, 1970; Nicolson, 1972; Singer and Nicolson, 1972). Glycoprotein and glycolipid inlaid in

276

SEN-ITIROH HAKOMORI

the lipid bilayer of membranes could thus be easily mobilized by a change of membrane dynamics, which determine the various regulatory functions of cells. Location and mobility of “plasma membrane particles” and/or of glycoprotein and glycolipid on membranes could be closely related to activity of microtubules, an idea resulting from analysis of the agglutination process (Berlin, 1972) (see Section VI1,C). Glycolipids have never been considered in any model presented thus far, including the “fluid mosaic model.” They could either be distributed among phospholipid bilayers or located in the plasma membrane particles. External labeling of glycolipid by galactose oxidase is now possible, and the results have allowed some speculation on arrangement of glycolipids on surface membranes, as seen in Fig. l (Gahmberg and Hakomori,

a

b

FIG.1. An idealized version of molecular arrangement of glycolipids and glycoproteins in membranes of normal (a) and fetal (b) erythrocytes: black ball, GalNAc or GlcNAc; white ball, Gal; dotted ball, Glc; dotted square, sialic acid; coiled or stretched lines, peptides ; G.O., galactose oxidase ; I.G., immunoglobulin for antiglycolipid (e.g., antigloboside). This figure illustrates three points: (1) glycolipids are directly embedded on the lipid bilayer through two hydrocarbon chains (ceramide) , and glycoproteins are associated with the lipid bilayer through hydrophobic peptide regions according to Marchesi’s “glycophorin” model (Marchesi et al., 1972) ; a major carbohydrate structure bound to lipid (glycolipid) is “globoside” with a terminal GalNAc+Gal +Gal+R, whereas a major carbohydrate structure bound to protein (glycoprotein) is sialyl-+Gal+GalNAc+R, although a few globosidic structures can also be found in glycoprotein (evidence proposed by the reaction of glycoprotein with antigloboside antibody : Watanabe and Hakomori, unpublished observations). This indicates that there is an essential difference in carbohydrate structures between glycoprotein and glycolipid within the same cell surface, although a common structure can be shared b e b e e n glycolipid and glycoproteins. Therefore, antiglycolipid antibody should be primarily directed to the glycolipid seated on the lipid bilayer; (3) possibly coincident to the loci of “plasma membrane particles” (Pinto da Silva et ul., 1971), glyco-

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1973). Change of organization of glycoproteins and glycolipids upon malignant transformation will be discussed in Section VII. I l l . Change of Glycolipids in Experimental Tumors and Transformed Cells in Vifro

Lipid extract of human tumor tissue reacts preferentially with certain rabbit antisera directed against human tumor tissue suspension, as indicated by thc complement fixation test. This classical observation, supported by the voluminous data of Witebsky (1929) and of Hirszfeld and associates (1929), was reinvestigated 25 years later by Kobayashi (1956) and by Rapport et al. (1959). Kobayashi followed the lipid hapten fraction of human gastric cancer tissue, taking complement fixation using antisera absorbed by normal gastric tissue. He finally obtained a “water soluble” lipid resembling the so-called “blood group lipid antigens.” Rapport, in his initial studies, recognized a specific lipid fraction able to react with rabbit antisera directed against the “mitochondria1 fraction” of various human tumors and of transplantable human epidermoid carcinoma (Rapport et al., 1959). The purified lipid hapten by chromatography was indistinguishable from ceramide dihexoside of bovine spleen previously isolated by Klenk and Rennkampf (1942) ; the complement fixation reaction between the lipid hapten and antihuman epidermoid carcinoma was effectively inhibited by lactose, and therefore, the structure lactosylceramide was assigned to this hapten previously named “cytolipin H” (Rapport et al., 1960). A definitive structural determination of this ceramide dihexoside was made by methylation (Yamakawa et al., 1962). Many other lipid haptens from various tissues have been assigned as cytolipins, designated by the initial letter of the original tissue, such as cytolipin G, K, R, P, etc. Lipid hapten of human gastroprotein and proteins are located close to each other on the lipid bilayer and form “bushes” of glycoprotein ; glycolipids can be located among these “bushes.” Galactose oxidase can penetrate between such “bushes” to label glycolipids, but other larger molecules like immunoglobulin cannot easily react with the glycolipids of normal erythrocytes as shown in Fig. la. In contrast, fewer glycoproteins and proteins w‘cw labeled on fetal erythrocytes, as seen in Fig. lb, and therefore, not only galactose oxidase but also immunoglobulin can react with globoside on fetal cell surfaces. Information obtained by external sugar labeling is important to illustrate subtle differences of surface structure. Only one possibility is shown in this figure. Studies on surface labeling of glycolipid and glycoprotein in other cells (3T3, BHK, NIL cells) showed that they must have similar spatial arrangements, as seen in this figure, but each cell type had a definitely different structure of carbohydrate, order, and spacing. Specificities of cell surfaces are therefore determined by (1) primary structure of carbohydrate on lipid and proteins, (2) order of arrangement, and (3) spacing of glycolipid and glycoproteins.

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intestinal tract tumors was named cytolipin G (Rapport and Graf, 1961), and that of lymphosarcoma (Murphy-Sturm) of Wistar rats was named cytolipin R (Rapport et al., 1967). The structure of cytolipin R was recently determined by Laine et al. (1972) (see Table 111). Although neither Kobayashi’s nor Rapport’s work has been critically studied by other investigators, the data indicate that glycolipids of tumor cells or tissue could be different from that of normal tissue either by chemical structure or by organization in membrane. It is clear, however, that neither “cytolipin H” nor “cytolipin R” are specific products of human cancer or rat sarcoma, because according to chemical analysis, these glycosphingolipids can be found in normal tissue as well. Nevertheless, it is possible that due to the change of membrane organization in tumor cells, these glycolipids could be recognized by the immunological machinery as “foreign” (Rapport and Graf, 1969; also see Section VI1,E). Change of glycolipid by malignant transformation has been clearly observed in tissue culture cells transformed by virus and/or chemicals. The first observation was made with BHK C13/21 cells transformed with polyoma virus and with spontaneously transformed clones. The level of hematoside was drastically reduced in virally transformed cells (BHKpy), but was only slightly reduced in spontaneously transformed cells, whereby the level of lactosylceramide was increased. Thus, a reciprocal relation was found between the quantities of hematoside and lactosylceramide (Hakomori and Murakami, 1968). This observation was further extended using BHK cells transformed with Rous sarcoma virus and mouse 3T3 cells transformed with polyoma and SV40 viruses. In all these cases, a decrease of hematoside was consistently observed, and the quantities of hematoside precursors, i.e., lactosylceramide or glucosylceramide, remained constant, or were slightly elevated (Hakomori et al., 1968). Although the level of hematoside decreased in transformed cells, the reactivity of hematoside on cell surfaces to antihematoside antisera increased to a great extent. The level of reactivity, measured by the release of 51Cr by immunolysis in the presence of complement, was found to be similar to trypsinized normal BHK or 3T3 cells (Hakomori et al., 1968). This finding was reminiscent of our previous observation that globoside residue of adult human erythrocytes were reactive with antigloboside only after trypsin treatment (Koscielak et al., 1968) and that fetal erythrocytes showed high reactivity with antigloboside antisera (Hakomori, 1969). This suggested that “cryptic” carbohydrate loci become “exposed” by transformation although the exact mechanism is not known at the present time (see Section VI1,E). Mora et al. (1969) observed a dramatic decrease of disialoganglioside (GDla) in tumorigenic 3T3 and ALN cells (both clones of mouse

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embryo) transformed with SV40 or polyoma virus, but hematoside level was not reported to be altered. Such a change was observed only in transformed cells carrying the virus genome, and was correlated with increased saturation density in culture and with higher incidence of rejection in the immunologically competent syngeneic host (Mora et al., 1969). Low enzyme activity for synthesis of GM2 ganglioside (UDP-Nacetylgalactosamine :hematoside-N-acetylgalactosaminyltransferase) was observed in virally transformed, but not in spontaneously transformed 3T3 or ALN cells. Repression of this particular enzyme was, therefore, correlated to the presence of a virogenome (Cumar et al., 1970). The absence of higher ganglioside homologs (GDla) and repressed activity of UDP-N-acetylgalactosamine :hematoside-N-acetyl-galactosaminyltransferase were found to be reversible, according to changes in growth properties in tissue culture. The “flat revertants,” derived from virally transformed cells and showing phenotypically normal growth behavior (Pollack et al., 1968), had higher ganglioside and restored activity of the sbove-mentioned enzyme (Mora et al., 1971), It is suspected, therefore, that reverted, normal phenotypic growth properties in culture are closely associated with normalization of the ganglioside pattern, and of enzymatic activity for synthesis of gangliosides, in spite of the continuing presence of a virogenome. As an example of chemically transformed cells, glycolipids of rat hepatocytes and Morris hepatoma cell lines were compared, showing a decrease in disialoganglioside (GDla) and an increase in monosialohematoside (GM3) and in monosialoganglioside (GM1) (see Table 111) (Brady et al., 1969). Thus, the change of ganglioside pattern associated with chemical carcinogenesis is very similar to the change associated with viral transformation, as observed in 3T3, BHK, and ALN cell systems, i.e., blocked synthesis of higher glycolipids and accumulation of a precursor (Brady et al., 1969). Gangliosides of Morris hepatoma and normal liver tissue have been compared independently by two laboratories : Siddiqui and Hakomori (1970) compared gangliosides of 5123 and 7800 Morris tumors to rapidly growing baby rat livers and adult rat livers, while Cheema et al. (1970) compared gangliosides of 5123 and 7777 Morris hepatoma to adult rat livers. I n all observed cases, the presence of trisialoganglioside (GT1) in either rapidly growing baby rat liver or in nongrowing normal rat liver and its complete absence in Morris hepatoma tissue was observed. Marked accumulation of disialoganglioside (GDla) and monosialoganglioside (GM1) was also observed. It is interesting that total lipid-bound sialic acid increased greatly in Morris hepatoma while it decreased greatly in transformed BHK cells. Blocked synthesis of GT1 ganglioside occurred in the former cells, which accom-

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panied accumulation of GM1 and G D l a ganglioside, thus giving an enormously high value of lipid-bound sialic acid. In contrast, blocked ganglioside synthesis in the latter case occurred with hematoside (GM3), which led to a decreased level of total sialic acid. The glycolipid patterns of normal adult and neonatal livers were similar and were characterized by the presence of trisialoganglioside, which was absent in Morris hepatoma, and by larger proportions of hematoside (both N-acetyl and N-glycolylhema‘toside) than in Morris hepatoma. It is noteworthy that the qualitative pattern of glycolipid in tissue does not simply reflect rapidity of cell growth, but is more closely related t o the intricate properties of cell membrane. The altered pattern of glycolipid in all three lines of Morris hepatoma resembled each other, however. The level of accumulated disialoganglioside depended on the degree of malignancy; i.e., the most rapidly growing 5123 showed the highest level and the most slowly growing 7800 showed the lowest level (Siddiqui and Hakomori, 1970). A considerable discrepancy between glycolipid changes of 3T3 cells transformed by SV40 virus was found by two groups of investigators: whereas in one study the level of hematoside was reported to be low in some 3T3 cells transformed with SV40 or polyoma viruses (Hakomori e t al., 1968), in the other study the level of hematoside was found to be greater, and a dramatic decrease of disialoganglioside (GDla) was observed in other transformed 3T3 cells (Brady and Mora, 1970). The results of Brady and Mora (1970) were essentially confirmed by Yogeeswaran e t al. (1972), while those of Hakomori e t al. (1968) were confirmed by Diringer e t al. (1972). A comparative study of various 3T3 cells with regard to ganglioside pattern has been undertaken in the author’s laboratory. These “3T3 cells” with a more fibroblastic appearance and showing less contact inhibition had only a trace quantity of disialoganglioside, whereas more typical “3T3” cells showing cobblestone appearance a t confluency demonstrated a high degree of contact inhibitability and contained a greater quantity of disialoganglioside (Hakomori, unpublished observations). I n the latter cells, the level of disialoganglioside greatly increased on cell-to-cell contact. It is possible that the differences observed in the two laboratories as “transformation-dependent” changes of 3T3 glycolipid are due to differences in glycolipid pattern of progenitor cells. Similarly, considerable variation in glycolipids has been observed in BHK cells originating from Stoker’s laboratory in Glasgow, and glycolipid variation seems to depend on cell history. BHK C13/21 cells with low passage and showing relatively low cell saturation density (about 8 X lo4 to 10i/cm2) had fairly large quantities of ceramide trihexoside

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(CTH). This glycolipid was usually absent in most BHK C13/21 cells with higher passage numbers and with higher cell saturation density. A dramatic decrease of ceramide trihexoside was observed in py-transformants of BHK cells that contained CTH, while decrease of hematoside in these cells was less pronounced than in cells without ceramide trihexoside (Hakomori, 1970). Thus, clonal variation of glycolipids and transformants was noticed, and such variation, which depends on glycolipid patterns of clones (“clonal variants”), has recently become more apparent and has been well documented by Sakiyama et ul. (1972) on various clones of NIL cells and their transformants, and by Yogeeswaran et al. (1972) on Balb mouse cell transformants. More recently, Diringer et al. (1972) found a consistent decrease of hematoside in 3T3 cells transformed with DNA viruses and a large increase of hematoside in cells transformed with RNA virus (murine-leukemic virus). The critical question, however, concerns the majority of nontransformed cell populations which may not represent the real progenitor of transformed cells, as only a small population of cells having higher susceptibility for transformation can be transformed by chemicals or virus, and observed changes in glycolipid may not reflect the actual changes due to transformation. I n other words, progenitor cells of transformants may not be identical with average cells with average glycolipid pattern. Another critical question is whether glycolipid change could be a secondary phenomenon and may not be essential for transformation. With these questions in mind, it becomes highly desirable to use a system which allows 100% transformation of cell populations. Chick embryonic fibroblasts transformed by high concentrations of Rous sarcoma virus inoculated with DEAE-dextran showed a rapid and complete transformation, ie., transformation occurred within 48 hours (Hanafusa, 1969). Transformation-dependent changes of glycolipids can be followed on a timecourse basis using this system. This study revealed a remarkable decrease of monosialo- and disialohematoside during the early stages of transformation (Hakomori et ul., 1971a). Decrease of disialohematoside was more sensitively recorded than monosialohematoside, and amounts of higher ganglioside did not change. The levels of lactosylceramide and glucosylceramide, precursors of hematoside, increased only slightly after complete transformation. In regard to the second question raised above, glycolipid profiles of chick embryonic fibroblasts transformed by temperature-sensitive mutants of Rous sarcoma virus will give a more definitive answer to the relation between glycolipid synthesis and expression of malignancy by surface membranes. Both chemical quaritity and synthesis of hematoside greatly decreased when chick embryonic fibroblasts were infected with

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temperature-sensitive mutants of Rous sarcoma virus and left a t permissive temperature (35°C). The chemical level and synthesis rate of hematoside reverted to normal a t 41" when the integrated virogenome was released, and also cell morphology and behavior became normal. Hematoside synthesis in membranes was perfectly reversible, indicating that the process of transformation is temperature-sensitive, and thus it is apparent that synthesis of glycolipid in membrane is indeed transformation-dependent (Wyke, Hakomori, and Vogt, unpublished observations). Warren et al. (1972b) recently reported that synthesis of hematoside and gangliosides in chick embryonic fibroblasts infected and transformed by temperature-sensitive mutants (T5) of Rous sarcoma virus was not restored to normal level a t non-permissive temperatures (41"C) , although morphology and growth behavior did return to a normal state. They regard glycolipid changes, therefore, as a nonessential process for transformation. This contrasts with the result of Wyke et al. (unpublished observations). However, no chemical quantities of glycolipids were determined in the experiments of Warren et al. (1972b). Although the general direction of glycolipid changes on malignant transformation is toward both simplification and incompletion of the carbohydrate chain, other changes directed toward increase or elongation of carbohydrate chain of glycolipids have also been noticed. Hakomori et al. (1971a) described the appearance of higher gangliosides on chick embryonic fibroblasts transformed with Rous sarcoma virus. In spite of this, the major change was a decrease or disappearance of disialohematoside, monosialoganglioside, and hematoside (see Fig. 1, columns 5 and 6, from Hakomori et al., 1971a). Recently, Yogeeswaran et al. (1972) found two different clonal isolates of 3T3 cells transformed by SV40 virus (3T3SV-479) and one clone of polyoma-transformed 3T3 clones (3T3-py-6) showing loss of disialoganglioside (GDla),in agreement with the results of Brady and Mora (1970). According to the figure of their chromatogram, considerable decrease of hematoside was present in the other two clonal isolates of SV40-transformed 3T3 cells in which GDla ganglioside was greatly elevated, although this was not discussed in their paper. This figure shows similarities t o the ganglioside changes observed in Morris hepatoma, i.e., decreased hematoside (GM3) level, increased GM1 and G D l a ganglioside, and absence of GT1 ganglioside, which is interpreted as blocked synthesis of GT1 ganglioside, although hematoside synthesis was blocked concurrently. More recently, Diringer et al. (1972) described a severalfold increase of hematoside in 3T3 clones transformed with Friend-murine leukemic virus, whereas the level of G D l a ganglioside remained constant. Splenomegaly with leukemic lesion was observed when susceptible mice were infected with Friend murine leukemic virus. Glycolipids of such a

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leukemic spleen were compared to that of normal, uninfected spleen, revealing a remarkable decrease of higher ganglioside and an accompanying increase of ceramide trihexoside and lactoside (Nishimura et al., 1972). I n our own studies using mouse fibroblasts transformed with SR virus A-31, a most remarkable change was observed in neutral glycolipid, rather than in ganglioside ; i.e., ceramide trihexoside was completely deleted, although the level of hematoside was lowered only slightly (Saito, Hakomori, and Vogt, unpublished observations) . IV. Glycolipids of Human Tumors and Human leukemic Cells

A difficulty in studying glycolipid changes in human tumors is the lack of suitable control tissue or cells, and the progenitor cells of human tissue are, in most cases, unknown. Another difficulty in studying human tumors is that they vary in morphology, chemical and antigenic properties, even within the same category of tumor. The pattern of glycolipids also varies greatly. A. BLOOD GROUPGLYCOLIPIDS OR GLYCOPROTEINS Deletion of blood group A and B antigenic activity in glycoprotein and in glycolipid of human adenocarcinoma has been known for two decades (Ohuti, 1949; Masamune et al., 1952, 1958; Iseki et al., 1962; Davidsohn et al., 1966; Kawasaki, 1958; Masamune and Hakomori, 1960; Kay and Wallace, 1961; Masamune and Shiozima, 1951), although the activities of H-antigen and Lewis antigens were less impaired and decreased to a slight degree, or were rather enhanced (Kawasaki, 1958; Iseki et al., 1962). A diminished reactivity or a total deletion of blood group antigen was observed even a t the premalignant lesions of cervical epithelial tissue (dysplasia, or atypia) and of oral epithelial cells (Davidsohn et al., 1969; Dabelsteen and Fulling, 1971). Blood group glycolipids of a few cases of human tumors have been reinvestigated and compared to normal gastrointestinal mucosa, and deletion of A and B glycolipid has been confirmed in several cases of human adenocarcinoma (Hakomori et al., 1967; Hakomori and Jeanloz, 1970). However, accumulation of an unusual quantity of fucose-containing glycolipid was noticed in some adenocarcinoma, which had Lea specificity in tumors of blood group host Leb (Hakomori and Jeanloz, 1964, 1970; Hakomori et al., 1967). Accumulation of glycolipids showing positional isomers of Lea called “X-hapten” have been isolated and characterized in some human adenocarcinoma (Yang and Hakomori, 1971). In some cases of adenocarcinoma, Le” glycolipid was present irrespective of blood group Lea status of the host (Hakomori and Andrews, 1970). It is difficult, however, to generalize these findings to all human adeno-

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carcinoma, as only a limited number of cases were analyzed, since the analysis was difficult and time-consuming. Accumulation of Lea-glycolipid was correlated with blocked synthesis of Leb glycolipid, and accumulation of HLeb-like glycolipids was correlated to blocked synthesis of A and B antigen. In earlier studies, fucose-containing glycolipid was found to inhibit cytoagglutination of human adenocarcinoma, caused by wheat germ phytoagglutinin. Agglutination was also inhibited by blood group A substance, but not by blood group B or H substance (Hakomori et al., 1967). A similar finding that hemagglutination caused by wheat germ hemagglutinin was inhibited by blood group A substance was reported by Uhlenbruck et al. (1968). It is assumed that a structure similar to wheat germ phytoagglutinin receptor sites is present in blood group A glycoprotein as one of the heterogeneous oligosaccharide chains. It is extremely interesting that synthesis of such a hapten site is correlated with gene A but not with gene B or 0. Some of the structures included in fucose and glucosamine-containing glycolipids of human tumor must share this structure with unknown oligosaccharide chain present in blood group substance A. It is suspected that the higher incidence of gastric cancer in blood group A people is due to early recognition of tumor cells in blood group B and 0 individuals (Hakomori et al., 1967). More recently, Hakkinen reported ('nee-A antigen” appeared in tumors of blood group B or 0 host. Although the chemical nature of this antigen is unknown, it cross-reacts with anti-A but not with anti-B or anti-0, and the higher incidence of gastric cancer in A individuals was attributed to this A-like antigen in tumors of 0 and B (Hakkinen, 1970). Such an incompatible blood group antigen in special kinds of human adenocarcinoma is of great interest in view of the fact that recognition of tumors could be different according to blood type of the host. I n relation to this finding, it is interesting to note that there is a degree of cross-reactivity between “carcinoembryonic antigen (Gold) ” (CEA, Gold and Friedman, 1965a,b) and blood group substance A, and that anti-A antibodies combined with CEA, but not with anti-B and anti-H antibodies (Gold et al., 1972). Most recently, a relation of blood group antigen to CEA was strongly suggested by Simmons and Pearlmann (1973). It is assumed that CEA as an incomplete blood group antigen and the degradation product of a blood group antigen reacts with anti-CEA antibody. Two immunodominant groups in CEA have been identified as N-acetylgalactosaminyl peptide (CEA-I) and as blood group I antigen complex (CEA-11) , respectively. Both of them are found in the internal part of the blood group carbohydrate chain. The I-antigen complex has been associated with the structure, +Gal/?l+30r4GlcNAc/?l +4Galp+R, which is the internal carbohydrate chain of blood group

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substances (Feizi et al., 1971). Although the major antigen site (CEA111) has not been identified, it was suspected to be the GlcNAcpl+GGal structure present on the inside of the blood group antigen. Thus, it has been greatIy suspected that CEA is an incomplete blood group substance of blood group ABH antigen, and that the deficient antigens have tumorspecific activity due to unmasking of a sequence that is cryptic in the normal structure (Simmons and Pearlmann, 1973). This view coincides with the “incompletion model” proposed by Hakomori and Murakami (1968).

B. GANGLIOSIDE Several cases of human cecal carcinoma were analyzed and their ganglioside pattern compared with that of normal colon tissue. I n all cases, the presence of GM2-gangliosidc (Tay-Sachs ganglioside) was noticed. The concentrations of GM2 ganglioside in normal colon tissue was very low. This finding was rather consistent with the accumulation of glycolipid due to blocked ganglioside synthesis (Kawauchi and Hakomori, unpublished observations) , Ganglioside of human brain tumor was studied by Kostic and Buchheit (1970). Comparison of different types of intracranial tumors-cerebellar gliomas, cerebral gliomas, and meningiomas-were compared with normal human brain tissue. Trisialoganglioside (GT1: G1) and disialoganglioside (GDlb :G2) decreased, whereas monosialoganglioside (GM1: G4) and hematoside increased, indicating again that blocked synthesis might occur in brain tumor, Seifert (1966) and Seifert and Uhlenbruck (1965) demonstrated the presence of a large amount of an additional ganglioside in brain tumors, which was present in only trace amounts in normal brain. The glycolipid was characterized as disialohematoside (NANA2+8NANA2+3Galpl+4Glc-+Cer) and was one of the accumulating gangliosides in human brain tumor.

C. FORSSMAN GLYCOLIPID Although Forssman antigen of experimental tumors, both in vivo and in vitro, has been extensively studied (Stern and Davidsohn, 1956; Fogel and Sachs, 1964), very little is known about Forssman glycolipid of human tumors. Since humans are regarded as Forssman-negative animals, no Forssman synthesis has been expected in human tissue. Recently, however, Kawanami reported the appearance of synthesis of Forssman haptcn glycolipid in one human tumor case (Kawanami, 1972). Carbohydrate composition and structure of the isolated and purified antigen was identical with that reported by Siddiqui and Hakomori (1971). The relation of this antigen t o “neo-A antigen,” as described before, will be of great intercst, as exprcssion of Forssman antigen in animals seems

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to be of a “carcinoembryonic” nature (Noonan and Burger, 1971). It is possible that Forssman synthesis in human cancer could also be expressed retrogenetically .

D. HUMAN LEUKEMIC CELLS Glycolipid of human leukemic cells as compared to normal leukocytes presents a difficult problem, as the progenitor of proliferous populations of leukemic cells cannot be assessed, and a sufficient amount of progenitor cells is difficult to obtain for analysis. Hildebrand et al. (1972), however, compared glycolipids of various types of leukemic cells and of nonleukemic human leukocytes. There was a significant decrease of higher ganglioside and a slight increase of lower ganglioside. Neutral glycolipids were also examined. There was a significant increase of glucosylceramide in various leukemic leukocytes as compared to nonleukemic leukocytes, although variation between different leukocytes was quite high (Hildebrand et al., 1971). Agglutinability of leukemic leukocytes by wheat germ agglutinin or by concanavalin A did not show a significant difference from normal leukocytes (Aub et al., 1965b). Kraus and Black (1972) compared agglutinability of various human leukemic leukocytes and normal leukocytes with Con A, wheat germ agglutinin, lentil agglutinin, and soybean agglutinin. Unlike tissue culture cells and their transformants, there was little difference in agglutinability demonstrated between normal and leukemic leukocytes by any phytoagglutinin. Con A and soybean aggiutinin barely agglutinated any human leukemic cells, except for two cases of normal lymphocytes. I n contrast, wheat germ agglutinin showed a strong reaction, irrespective of whether the leukocytes were normal or malignant.

E. SERUM FACTOR REACTIVE TO LACTOSYLCERAMIDE Serum of patients with cancer or of pregnant women gave a specific precipitin reaction with lactosylceramide or gave cytoagglutination inhibitable by lactosylceramide and a specific “T-globulin” of patients’ sera was isolated (Tal and Halperin, 1970; Tal et al., 1964; Tal, 1965). The findings are compatible with the results of Rapport (see Section 111) that “cytolipin H” (lactosylceramide) is a human tumor specific hapten, but are incompatible with findings of investigators that the serum factor reactive to lactosylceramide or to lactosylsphingosylprotein was found in normal serum as well (Brady, 1964; Taketomi, 1969). V. Cell Contact, Contact Inhibition, and Glycolipid Synthesis

One of the distinctive characteristics of eukaryocytes which is absent in prokaryocytes is the remarkable change of cell division and cell

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movement on cell-to-cell contact (“contact inhibition”) . Although this phenomenon is observable under specific culture conditions, the difference in the effect of cell contact on growth behavior and cell saturation density observed between transformed and nontransformed cells is quite obvious. This is regarded as an important surface-mediated control mechanism for cell division of eukaryocytes. Loss of contact inhibition has been described as a common property of tumor cells expressed in vitro (Abercrombie and Ambrose, 1962). This is an impressive rationale for the belief that tumor cells have lost control of cell division through surface membranes, although some doubt has been aroused in that “contact inhibition” can be an artificial mechanism inducible and/or reducible by concentrations of serum factors or of “nutrients” in cell cultures (Holley and Kiernan, 1968; Todaro e t al., 1965; Jainhill and Todaro, 1970; Holley, 1972). Holley (1972) postulated that the essential difference between normal and transformed cells is in transport properties of membrane for nutrients, as the concentrations of intracellular nutrients determine cell growth behavior, rather than special membrane changes for specific intercellular signals. This postulation is based on the enhanced sugar transport in transformed cells (see Table 11) and on the probable absence of specific signals between cells when they contact. Romans and Colby (1973) argued that the apparent enhancement of sugar uptake observed in 3T3 cells transformed with SV40 viruses is not due to the transport process of membrane itself, but to enhanced phosphorylation of the sugar by intracellular kinase. Enhanced sugar uptake is therefore not due to membrane property changes. Specific signals between cells when they contact may exist, as a remarkable change of glycolipid synthesis in membrane has been observed on cell-to-cell contact (Hakomori, 1970; Robbins and Macpherson, 1971). Cell contact is necessary for the enhanced chemical quantity and synthesis of glycolipid; “sparse quiescent cells” had the same glycolipid pattern as “sparse growing cells,” but glycolipid pattern is different in “confluent cells,” as observed in 3T3 cells (Hakomori, unpublished observations) and in NIL cells (Critchley and Macpherson, 1973). We cannot, however, ignore the fact that a remarkable change of glycolipid synthesis in membranes occurred on cell-to-cell contact. Further comments should be waived a t this time, because we have no extensive data about glycolipids of various types of “quiescent sparse” cells. Shen and Ginsburg (1968) observed that total carbohydrate content of HeLa cells grown as monolayer was greater than the same HeLa cells grown as suspension culture, when less chance of cell-to-cell contact occurs. Wu e t al. (1969) described the remarkable increase of a glycopeptide separated from a trypsinate supernatant of confluent 3T3 cells, as

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compared to sparsely growing cells. Well contact-inhibited BHK cells contained CTH, in contrast to less contact-inhibited BHK cells, which had no CTH. I n fact, in the contact-inhibited phase, BHK cells synthesized more CTH (Galal+4Galpl+4Glc+Cer) than in the sparsely growing phase, while concentrations of CDH (Galpl+4Glc+Cer) decreased in the contact-inhibited phase (Hakomori, 1970). Hematoside of well contact-inhibited BHK cells also showed an elevated level in the confluent phase. Monosialoganglioside (GM1) , disialohematoside, and monosialohematoside of 8166 human fibroblasts showed a similar increase on cell-to-cell contact. It is noteworthy that all these contactsensitive glycolipids dramatically decreased on malignant transformation, while transformed cells showed no variation in glycolipid levels on cellto-eel1 contact (Hakomori, 1970). Another study on chick embryonic fibroblasts also showed an increase of hematoside and disialohematoside a t the confluent phase and disappearance of these glycolipids with RSVtransformation (Hakomori e t al., 1971a). A similar glycolipid change on cell-to-cell contact was reported independently by Robbins, Macpherson, and their colleagues (Robbins and Macpherson, 1971; Sakiyama et al., 1972; Critchley and Macpherson, 1973). I n NIL cells CTH (Galal+4Galp1+4Glo,Cer), globoside (GalNAc/3l+3Galal+4Gal~l+4Glc+Cer), and Forssman glycolipid (GalNAoal+3GalNAc~1+3Galal+4Gal~l+4Glc+Cer) increased severalfold on cell-to-cell contact, and all these glycolipids were deleted by transformation with polyoma virus or with hamster sarcoma virus (Sakiyama et al., 1972). They also observed differences in contact inhibitability, cell saturation density, and tumorigenicity between clonal isolates of various NIL cells; e.g., one clone (2C1) showed a great increase in various neutral glycolipids as mentioned above, but had higher saturation density; another clone ( l C l ) , showing lower saturation density, did not have more neutral glycolipids with a longer carbohydrate chain, but showed a remarkable increase of hematoside on cell-to-cell contact, and this clone showed the lowest saturation density. Thus, “no relation was found between complexity of glycolipid and contact inhibitability of cells” (Sakiyama et al., 1972). This remark should be inappropriate, as globo-series and hematoside-series (see Table 111) should have different functions and cannot be compared on an equal basis. A conceptual model of extension response is shown in Fig. 2. A significant correlation was found between tumorigenicity and contact-dependent increase of glycolipid. The 1C1 clone without higher neutral glycolipid and which showed a remarkable increase of hematoside on cell contact was the least tumorigenic. In general, cells showing no density-dependent glycolipid changes had higher tumorigenicity, although an exception was reported. This ex-

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NORMAL CELLS : Extension Response +

@-(+

CER Low density (Growing)

tl -CER

High density (Confluent I

TRANSFORMED CELLS : Extension Response

-

WCER m

C

E

R Low density

tl

WCER W

C

E

R High density

FIG.2. A model explaining “glycosyl extension” on cell-to-cell contact of nontransformed cells and its absence in transformed malignant cells. Either sialyl residue (“NA”) or a-galactosyl residue (“Gal”) can be added onto hematoside Qr lactosylceramide (Gal+Glc+Cer> when cells are con(NA+Gal+Glc+Cer) fluent. Confluent cells therefore have more extended carbohydrates than cells without contact. This phenomenon is called “extension response” or “contact accumulation” and is virtually absent in transformed cells, where the carbohydrate chain is left as “incomplete.”

ceptional clonal isolate having neutral glycolipid showed a remarkable change of glycolipid level on cell contact, although it had high tumorigenicity (Sakiyama and Robbins, 1972). It is possible that change of glycolipid containing sialic acid is more related to contact inhibitability than is neutral glycolipid. Enzymatic background of contact accumulation will be discussed in the next section. It is unknown whether structural changes in glycoprotein occur on cell-to-cell contact. If cell-to-cell contact offers a specific signal on cell surfaces which can induce contact inhibition, the mechanism of intercellular association or intercellular linkage would be of great importance. It is possible that the higher the intercellular linkages, the greater the contact inhibitability of cells. Malignant cells in general exhibit much reduced intercellular linkage. Substantial progress was made‘ by recognition of glycosyltransferase on cell surfaces and the possible role of such glycosyltransferases on cell surfaces for interlinkages between cells (Roseman, 1971; Roth et al., 1971; Roth and White, 1972). A theory provided by Roseman is that a glycosyltransferase on one cell surface binds to its acceptor molecule on another cell surfacc, and an enzyme on one cell glycosylates the substrate of the counterpart cell. The phenomena “contact inhibition” and “contact

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accumulation” (Hakomori, 1970; Robbins and Macpherson, 1971; Sakiyama et al., 1972) essentially support this view. Also, the concept has been supported by Bosman’s study on the mechanism of platelet aggregation (Bosman, 1972a). Cell contact-dependent enhancement of Forssman glycolipid synthesis and of hematoside synthesis was studied in greater detail according to the degree of cell contact, i.e., correlation between the degree of synthesis of these glycolipids and cell population density. It was noticeable that synthesis of Forssman glycolipid in NIL E cells was 3-4 times enhanced a t a relatively early stage of cell contact but reduced in a later stage; similarly, hematoside synthesis also increased 4 times in NIL K cells a t the early stage of cell contact (Kijimoto and Hakomori, 1972). Critchley and Macpherson (1973) observed that the glycolipid pattern of NIL2 cells, whose growth was inhibited by low serum concentration or lack of glutamine (“sparse quiescent”), is obviously different from that of cells whose growth was inhibited by cell-to-cell contact a t high cell density, but is rather similar to that of “sparse growing cells.” A similar finding was obtained with 3T3 cells (Hakomori, unpublished results). These data indicate that “contact extension” and “contact accumulation” indeed depend on cell contact. It has also been suggested that the phenomena may be one step in a series of membrane changes which induce contact inhibition, but may not be a result of contact inhibition. Membrane changes associated with cell contact may be found not only in glycolipids but also in other molecular species; it is extremely noteworthy that a large increase in phosphatidycholine was found in confluent 3T3 cells, and no density-dependent change of phospholipid was observed in transformed cells (Cunningham, 1972). VI. Change of Glycosyltransferases and Hydrolases

Cumar et al. (1970) first described repression of enzyme activity of UDP-N-acetylgalactosamine :hematoside N-acetylgalactosaminyltransferase in mouse fibroblasts (3T3, Balb, NAL) transformed with various DNA viruses. Den et al. (1971) described a remarkably low activity of CMP-sialic acid :lactosylceramide :sialyltransferase in polyoma transformed BHK cells as compared to normal BHK cells. Enzyme activity was not only quantitatively lowered but also qualitatively altered; is., the enzyme activity of normal cells was enhanced after mixing with phosphatidyl glycerol and cardiolipin, while that of transformed cells was not enhanced by the addition of a particular phospholipid. The enzymes described by Cumar et al. (1970) and also by Den et al. (1971) did not show cell contact-dependent changes. Fishman et al. (1972) studied activities of various sugar nucleotide transferases for synthesis of ganglio-

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sides of mouse 3T3 cells. Neither P-galactosyl, N-acetylgalactosaminyl, nor sialytransferase showed enhancement of their activities a t higher cell population densities. This is in contrast to the results of Kijimoto and Hakomori (1971), which showed that a-galactosyltransferase for synthesis of ceramide trihexoside was enhanced a t higher cell population density. The enzyme activity of UDP-galactose :lactosylceramide-a-galactosyltransferase (for synthesis of ceramide trihexoside) was remarkably enhanced when cell population densities increased, while that of UDPgalactose :glucosylceramide-P-galactosyltransferase (for synthesis of lactosylceramide) remained unchanged or increased slightly when cell population density increased. In transformed NIL or BHK cells the activity of a-galactosyltransferase was reduced to a barely detectable level, while a-galactosidase activity increased. Thus, both synthetic and degradative enzymes cooperated to lower the level of ceramide trihexoside. P-Galactosyltransferase (for synthesis of lactosylceramide) was either unchanged or increased slightly in transformed cells (Kijimoto and Hakomori, 1971). It is interesting to note that enhanced a-galactosidase activity in sparsely growing cells was about the same as in transformed cells (Kijimoto and Hakomori, 1971). Bosman (1969) observed a great increase of various glycosylhydrolases in transformed cells; the same authors (Bosman et al., 1968) previously described enhanced activities of various glycosyltransferases in transformed 3T3 cells, i.e., a fucosyltransferase, a galactosyltransferase, and an N-acetylgalactosaminyltransferase. The sialyltransferase activities using desialyzed bovine submaxillary mucin and fetuin as acceptors from 3T3 cells and Balb/C cells were compared against SV40 transformed cells. A remarkable decrease of enzyme activities (one-third to one-half) was observed in transformed cells (Grimes, 1970). Glycosyltransferase activity was further extensively studied by Grimes (1973) in 3T3 cells (Balb cells) transformed with SV40 polyoma virus and hamster sarcoma virus (RNA virus). Sialyl transferase activities for desialylated submaxillary mucin and fetuin were all reduced except in polyoma transformed 3T3 cells. Fucosyl and galactosyl transferases were also reduced in those transformed cell lines, as compared t o the levels in normal cells. “Flat revertants” derived from SV40-transformed cells showed increased levels of sialyl transferase activity close to the level of normal 3T3 cells. A comparable finding was recently presented by Saito et al. (1972), who showed a much lower level of sialyltransferase activity in an extract of Yoshida’s hepatoma cells as compared to normal liver cells. Recently, Warren et al. (1972a) observed a great increase in activity of a special sialyl transferase whose acceptor structure contained a fucosyl residue and which was eluted slightly faster than the corresponding

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component of normal cells. This observation was based on a previous finding that a fucose-containing glycopeptide of transformed cells was eluted slightly faster than a similar glycopeptide of normal cells from a Sephadex G-50 column. The concentrations of such a glycopeptide were higher in actively growing cells than in slowly growing cells (Buck et aZ., l970,1971a,b). Sakiyama and Burge (1972) studied glycopeptide patterns of both normal and 3T3 or Balb cells; the patterns of normal and transformed cells were very similar except that a large peak observed in normal cells was absent in transformed cells. They observed a phenomenon similar to what Buck et al. (1970, 1971a,b) described, but regarded as insignificant because the difference was too small. The “early eluting” glycopeptide of Buck et al. was present on the surface of T5-transformed chick embryonic fibroblasts only when they were grown a t a permissive temperature (35”), and it disappeared when cells were normalized a t nonpermissive temperatures (Warren e t al., 1972b). The “early eluting” material was identified as having an extra sialic acid, and therefore a specific sialyltransferase was present when cells were actively growing or transformed (Warren et al., 1972a). The higher sialyltransferase activity of transformed cells is reminiscent of a previous report of Bosman et al. that many glycosyltransferases are enhanced in transformed 3T3 cells (1968). More recently, various virally transformed 3T3 cells showed 1.5 to 1.6 times the sialyltransferase activity than nontransformed 3T3 cells showed, if whole cells were desialyzed, trypsinized, homogenized with detergent and used as an acceptor (Bosman, 1972b). Although a number of studies on glycosyltransferase activities of normal and transformed cells have been published, the significance of this approach is rather limited a t present, as properties of glycosyltransferases and factors affecting their activities in vitro are unknown. Usually determination is made by incubating broken cells or particulate fractions with high concentrations of detergents, bivalent cations, deglycosylated substrate, and radioactive sugar nucleotide. Obviously, the activity of transferases, expressed by radioactivity incorporation into substrate, does not reflect physiological conditions or quantity of enzymes present in the cell membrane. The conditions described by Caputto and his associates, designed for physiological activity of membrane-bound glycosylhydrolases, would be more suitable for comparison of glycosyltransferases (Arce et al., 1971; Maccioni et aZ., 1972). Localization of glycosyltransferases in cells has received much attention: while major localization of glycosyltransferases should be in the Golgi apparatus, and progressive glycosylation of glycoprotein during transport of protein via the endoplasmic reticulum to the cell periphery

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has been postulated (for a review, see Kraemer, 1972). Possible localization of glycosyltransferases in cell surface membranes will be particularly interesting for understanding the mechanism of cell-to-cell interaction. The data of Roth et al. (1971) and Roth and White (1972) showed the presence of glycosyltransferase in cell membrane, which supports Roseman’s theory for the mechanism of intercellular interaction. Recently, the presence of a particular neuraminidase in mouse cell plasma membrane was described (Schengrund et al., 1972), and the role played by such an enzyme in intercellular association is most probable, but is yet to be determined. VII. Reactivity of Cell Surface Glycolipids and Glycoproteinr of Normal, Fetal, and Cancer Cells with Macromolecular Reagents

A. REACTIVITY WITH GLYCOSYLHYDROLASES Various glycosidases can hydrolyze surface-oriented carbohydrate residues to a limited degree. The effect of neuraminidase has been best studied in relation to the effect of neuraminidase on electrophoretic mobility of cells (for review, see Winder, 1970) and transplantability of tumor cells (Sanford, 1967). Numerous studies on surface property changes induced by neuraminidase are available, but studies on chemical changes of surfaces induced by neuraminidase are few, and some are rather controversial. Treatment of cat erythrocytes with neuraminidase hydrolyzed disialohematoside and sialyllacto-N-neotetraosylceramide (see Table 111), but hematoside was not hydrolyzed (Wintzer and Uhlenbruck, 1967). Neuraminic acid of hematoside in L cells was not hydrolyzed (Weinstein et al., 1970), while that of hematoside and GM1ganglioside in 3T3 cells was hydrolyzed (Yogeeswaran e t al., 1972). N-Acetylgalactosaminyl residue of globoside in human erythrocytes was not hydrolyzed when the intact human erythrocytes was treated with jack bean P-N-acetylgalactosaminidase, whereas globoside extracted from membranes was easily hydrolyzed by the same enzyme (Siddiqui and Hakomori, unpublished observations). Similarly, ceramide trihexoside (CTH) of human erythrocytes and of NIL cells was not hydrolyzed by a-galactosidase of figs, although the same glycolipid was hydrolyzed by the same enzyme in vitro (Siddiqui et al., unpublished study). After cells were treated with pronase, hydrolyzability of globoside increased slightly. No systematic studies have been made on whether change of hydrolyzability of glycolipid or glycoprotein occurs on malignant transformation. A limitation of this method is, however, that a steric effect of hydrolases is too large to pick up the subtle structural difference of cell surfaces. However, it is interesting to note that mouse TA3 tumor cells treated

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with neuraminidase reduced their transplantability remarkably, and this phenomenon was considered to be due to enhanced immunogenicity, as originally observed by Sanford (1967). This finding was successfully applied by Simmons and his associates (1971a,b) for immunotherapy of various tumors ; i.e., augmented immunogenicity was expressed in various experimental tumors treated with commercial Vibrio cholerae neuraminidase. The chemical basis of these phenomena is not known. It is well known that commercial neuraminidases contain significant activities of phospholipase and proteases, and thus immunological changes of cells treated with these enzymes would be hard to interpret (Kraemer, 1968). WITH GALACTOSE OXIDASEAND EXTERNAL LABELING B. REACTIVITY PATTERN OF GLYCOLIPIDS AND GLYCOPROTEINS

Treatment of erythrocytes with galactose oxidase (EC 1.1.3.9) followed by reduction with tritiated sodium borohydride (NaBsHH,)a t pH 7.4 allowed the labeling of galactosyl and N-acetylgalactosaminyl residues on external surfaces of cells with tritium t3H) (Steck, 1972; Gahmberg and Hakomori, 1973). Labeling patterns and specific activities of galactose and galactosamine in glycolipids and glycoproteins were determined after separation with gel electrophoresis and thin-layer chromatography. The labeling patterns of normal adult cells differed greatly from fetal cells, and were significantly altered when cell surfaces were modified by proteases and neuraminidase. The results of analysis indicated that: (1) the carbohydrate moieties of two glycolipids (globoside and ceramide trihexoside) and a t least three glycoproteins (molecular weight 9.5, 8.2, and 6.4 X lo4) were exposed to the external environment, but not ceramide dihexoside, ceramide monohexoside, or other glycoproteins with higher molecular weights; (2) the specific activities of galactosamine in glycolipid and of galactose in glycoprotein increased after protease treatment, although total activity of glycoprotein did not change; (3) labeling of glycoprotein was greatly enhanced by neuraminidase treatment, while that of glycolipid was enhanced to a lesser degree; (4) the “relative exposures” of glycoprotein and glycolipid differed greatly between normal and fetal erythrocyte surfaces. Glycoproteins of fetal cells had a very low label as compared to glycolipid. The labeling patterns of 3T3 cells, NIL cells, and their viral transformants have been compared with different cell population densities and with trypsin and EDTA treatment. Label activities for glycolipids and glycoproteins were characteristic for different cell types and differed greatly from one cell line to the other. Nontransformed cells had a much greater label for glycolipids after treatment with EDTA, and the label for ceramide trihexoside greatly diminished when cells became confluent.

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Such great variation in labeling patterns dcpends on cell contact and on EDTA treatment and was not, in general, observed in transformed cells. Normal 3T3 and NIL cells had a greatly enhanced label of glycolipid a t the confluent phase as compared to growing phase. These labeling patterns are coincident with the results of cell reactivity with antiglycolipid antisera (see Section VI1,E). Newly labeled peaks appeared after the cells were treated with neuraminidase ; qualitatively different peaks were demonstrated in virally transformed cell surfaces after neuraminidase treatment (Gahmberg and Hakomori, unpublished observations).

C. REACTIVITY WITH PHYTOAGGLUTININ (LECTIN) Since Aub et al. (1963, 1965a,b) described enhanced agglutination of ascites tumor cells by heated wheat germ lipase (containing phytoagglutinin), extensive studies using various normal and transformed cells with various phytoagglutinins have been carried out by a number of investigators (for review, see Burger, 1971b; Sharon and Lis, 1972; Osawa, 1972). Aub’s agglutination was studied with human tumor suspension, in comparison with cell suspension of parenchymatous organs. I n this case, the glycolipid fraction containing fucose and glucosamine prepared from human tumors was found to inhibit the agglutination (Hakomori et al., 1967; see also Section IV). The wheat germ phytoagglutinin was concurrently purified and identified to be a glycoprotein ; the agglutination was found to be effectively inhibited by N-acetylglucosamine and N-acetylchitobiose (Burger and Goldberg, 1967). The agglutinin-binding receptor complex was isolated from the neoplastic cell surface by hypotonic shock (Burger, 1968). Enhanced agglutination of tumor cells by wheat germ phytoagglutinin was inhibited by a “ganglioside fraction” obtained from polyoma-transformed BHK cells, but the same fraction derived from normal BHK cells was not as effective. Moreover, glycolipids derived from normal cells treated with periodate, sodium borohydride, and acid hydrolysis (Smith degradation) were able to inhibit malignant cytoagglutination by wheat germ agglutinin (Hakomori and Murakami, 1968). It was assumed, therefore, that malignant cells should contain more incomplete glycolipid which could have N-acetylglucosamine a t the end. The terminal of this malignant glycolipid could have been masked by another sugar residue in normal cells. A subsequent study by Burger showed, however, that normal cells can be agglutinated after treatment with a dilute solution of trypsin (0.005%), and therefore, enhanced reactivity of transformed cells to wheat germ phytoagglutinin was ascribable to the exposed reactive site rather than to creation of reactive sites on cell surfaces (Burger, 1969). EDTA-treated normal cells did not show increased reactivity.

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Independent from Burger’s observation, the idea of “cryptic” reactive sites was developed during observations on reactivities of cells to invertebrate agglutinins and that the surface glycolipids to their antisera, i.e., the reactivity to the invertebrate agglutinin, was greatly enhanced by proteolysis of cells (Uhlenbruck et al., 1968) ; the major glycolipid sites such as globoside and hematoside of normal erythrocytes were “cryptic” (Koscielak et al., 1968) while those of fetal cells were ‘(exposed” (Hakomori, 1969). Concurrently, hematoside groups in normal 3T3 and BHK cell surfaces were demonstrated to be “cryptic” as compared to their transformants (Hakomori et al., 1968). This finding will be discussed under subsection E. Also, specific antigenic sites of mouse tumor cells were demonstrated to be an “exposed” group of “cryptic” sites in normal cells which were also “exposed” by proteolysis (Hayry and Defendi, 1970). These antisera are heterogeneous and difficult to obtain, in contrast to phytoagglutinins, which are generally obtainable with high purity and are easy to handle. Extensive studies on cell surface properties using phytoagglutinin have therefore been rapidly developed. Burger’s finding was essentially confirmed by investigators a t the Weiamann Institute in Israel using concanavalin A (jack bean) (Inbar and Sachs, 1969) and an agglutinin of soybean (Lis et al., 1970), and by Osawa and his colleagues in Tokyo (Tomita et al., 1970, 1972). Consequently, agglutinabilities of various experimental tumor cells of mouse and rats and of normal erythrocytes using sixteen kinds of known phytoagglutinins were examined (Tomita et al., 1970). Also, agglutinability of 3T3, SV3T3, and their trypsin-treated cells were compared using fourteen kinds of agglutinin ; a remarkable difference in agglutinability between normal and transformed 3T3 cells was recorded by Ricinus communk, Wistaria floribunda, Pisum sativum, Solanum tuberosum, and Phaseolus vulgaris, in addition to wheat germ and concanavalin A (Tomita et al., 1972). Burger and Noonan (1970) demonstrated that contact inhibitability of transformed 3T3 cells was restored when the cells were treated with trypsinized “monovalent” concanavalin (Con A). This phenomenon was interpreted to mean that trypsinized “monovalent Con A” can bind to tumor-specific sites so that tumor cells are covered. The resulting covered cells showed the same surface status as normal 3T3 cells, thus restoring contact inhibition. On the other hand, a remarkable cytotoxicity of Con A to hamster and mouse cells was demonstrated in vitro as well as in vivo (Shoham et al., 1970). Restoration of contact inhibition in transformed 3T3 cells by “monovalent Con A” should be reinvestigated in view of a plausible objection that trypsin treatment of Con A may not result in monovalent Con A, but still retains cytotoxic bivalent properties (T.

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Osawa, personal communication). A slower rate of growth observed in the presence of “monovalent” lectin could be ascribable to modified cytotoxicity. However, Noonan and Burger’s phenomenon is now compatible with this reviewer’s observation that cell growth rate was slowed by some purified anti-glycolipid antibody without detectable cytotoxicity (Laine and Hakomori, unpublished observations). Friberg et al. (1971) observed that Con A inhibited tumor cell migration from a capillary into a planchette filled with growth medium. In the earlier studies of Inbar and Sachs (1969), they demonstrated increased absorption capability of tumor cells to 83Ni-labeled concanavalin A. However, their finding was sharply contradicted by subsequent studies of Cline and Livingston (1971) and of Ozanne and Sambrook (1971) using 1251-labeledwheat germ agglutinin and Con A. Polyoma or SV40-transformed 3T3 cells can absorb about the same labeled activity of phytoagglutinin as normal 3T3 cells do. These findings also contradict the observation of Fox et al. (1971) that cell surface carbohydrates were more exposed in transformed cells, as evidenced by persistent labeling with fluorescein-coupled wheat germ agglutinins, irrespective of cell cycle. Increased binding of fluorescent Con A a t the mitotic cell surface was described by Shoham and Sachs (1972), and H2 and viral surface antigens are expressed most strongly a t the S-phase (Cikes and Friberg, 1971). Thus, the cell-cycle dependent “exposure” of cell surface antigens has been supported. The exposure of wheat germ binding sites preceded change of nuclear DNA synthesis, and were observed on abortive transformation (Shepard et al., 1971). Binding activity of erythrocytes with soybean agglutinin was unchanged after treatment of erythrocytes with trypsin (Gordon e t al., 1972). According to the new “fluid mosaic” model of biomembrane (Singer and Nicolson, 1972), the location of carbohydrate sites on cell membranes may correspond to the sites of “plasma membrane particles” (Pinto da Silva and Branton, 1970), which are in a dynamic equilibrium state with other membrane components. Distribution of these loci on plasma membranes can be greatly affected by a subtle change in the dynamic equilibrium between plasma membrane components, i.e., such a change can be caused by change of pH, nutrients, and by modification of cell surface by enzymes (see Edidin, 1972). The mechanism of enhanced agglutination observed in transformed cells was put forth by Nicolson (1971), who observed the distribution of phytoagglutinin reactive sites on cell surfaces elucidated by new techniques of electron microscopy using ferritin-bound phytoagglutinin, revealing a remarkable topological difference between normal and transformed cells. Transformed tumor cells showed a clustering of reactive

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sites, in contrast to homogeneous distribution of reactive sites on normal cell surfaces (Nicolson and Singer, 1971; Nicolson, 1971). Clustering of agglutinin reactive sites was also induced by treatment with proteases, which was similar to the process associated with transformation. I n this study, topological rearrangement of reactive sites was shown to be temperature sensitive (Nicolson, 1972) . Although reactivity of cells with isotope-labeled wheat germ agglutinin or with concanavalin A did not show increased isotope uptake in transformed cells, it did show a remarkable increase of isotope uptake labeled to some other agglutinin paralleling the increase of agglutinability. Reactivity with labeled phytoagglutinins, Ricinus communis (which is directed to /3-galactose residue) and Wistaria floribunda (which is directed to p-N-acetylgalactosamine residue), was greatly increased by transformation, as tested with C31 cells transformed with bovine adenovirus type I11 (Takeuchi et al., 1972). Similarly, lymphoma cells had five times as much binding activity for lZ5I-C0nA as compared to lymphoid cells (W. T. Martin et al., 1971). Cell density-dependent decrease of surface reactive sites of bovine C31 cells has been demonstrated, as determined by the isotope-labeled Wistaria floribunda and Ricinus communis (Takeuchi et al., 1972). Recently, further details of the mechanism of agglutination have been studied. Binding of phytoagglutinin to the cell surface receptor occurred even a t 0°C. The second-step reaction, the process of agglutination, was found to be temperature sensitive and only occurred a t 25°C (Inbar, 1971). Agglutination of leukocytes by Con A was inhibited by colchicine and vinblastin as well as a t low temperatures (Berlin, 1972). These conditions have been known to induce depolymerization and inhibition of polymerization of microtubular proteins (Behnke and Forer, 1967). Thus, great interest has been aroused by the possibility that rearrangement of cell surface reactive sites for phytoagglutinins can be associated with the activity of microtubules, the organelle which controls self-assembly and motility of cells (for reviews, see Inoue and Sato, 1967; Tilney, 1968; Kennedy, 1969; Ishikawa, 1971). It is assumed that binding of phytoagglutinins to cell surface receptor sites does not require energy, but agglutination is an energy-dependent process for rearrangement of glycoprotein and glycolipid in the cell membrane; the process of rearrangement is closely associated with the reactivity of microtubules. An essential change in agglutinability of cells associated with malignant transformation is therefore ascribable to change of fluid dynamicity of membrane and to microtubular activity. Either “fluid dynamicity” or microtubule activity of membrane could be caused by

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chemical changc of membrane glycolipid or phospholipid, as already discussed in Sections IV and VI. However, agglutination of malignant or transformed cells may not always be more enhanced than the corresponding normal cells. In the original paper of Aub et al. (1965b), it was shown that agglutination of leukocytes of chronic lymphocytic leukemia was consistently less than the control normal leukocytes. A study of Kraus and Black (1972) showed that wheat germ agglutinin strongly agglutinated both normal and leukemic leukocytes to the same degree, whereas Con A and soybean agglutinin did not agglutinate normal and malignant leukocytes, i.e., no differential agglutination between normal and leukemic leukocytes has been demonstrated by lectins. Moore and Temin (1971) compared agglutination with wheat germ agglutinin and Con A in chicken cells infected with Rous sarcoma virus, avian leucosis virus (RAV) ; rat and mouse cells infected with murine sarcoma virus or murine leukemic virus. With the exception of rat cells converted by B77 virus, all chicken and mouse cells converted by RNA tumor viruses did not show marked increases in agglutinability. However, Kapeller and Doljanski (1972) described increased agglutinability in chick embryonic fibroblasts transformed by Rous sarcoma virus and by Con A and wheat germ agglutinin. More recently, Salzberg and Green (1972) tried to correlate agglutinability with replication of infected RNA viruses on the host cells; transformed rat, mouse, and cat cells that replicate sarcoma-leukemia virus complex are agglutinated by Con A, but mouse and human cells that replicate leukemic component alone are not agglutinated. The inability of various phytoagglutinins to distinguish agglutinability of normal human leukocytes and leukemic leukocytes has already been mentioned (see Section IV,D) . It is plausible, therefore, that agglutinability changes by phytoagglutinin may not be a universal phenomenon associated with transformation. It is noteworthy that Moscona (1971) reported an interesting difference in binding capability of chick embryonic cells of some organs with concanavalin A as compared to adult cells from the same organs. No such differences were found for the same cells using wheat germ agglutinin. Noonan and Burger (1971) have shown that mouse embryo cells derived from thymus, bone marrow, and spleen strongly agglutinated with wheat germ agglutinin, while newborn cells of those organs did not. Similar but less pronounced differences in agglutinability were shown by Con A in spleen cells of embryo and of newborn. Stronger agglutinability and hemolysis of fetal erythrocytes demonstrated by antigloboside will be described later (Hakomori, 1969; see Section VI1,E).

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D. REACTIVITY WITH AGGLUTININ DERIVED FROM ANIMALTISSUE AND CELLS Proteins and glycoproteins of some snail glands also showed hemagglutination (Prokop et al., 1965). Helix pomatia had agglutinins (“antiHp”) showing two specificities toward both a-N-acetylgalactosamine and P-N-acetylgalactosamine, and Cepala nemoralis had an agglutinin (antiACN) showing specificity to P-N-acetylgalactosamine (Schnitzler et al., 1969). Of particular interest, albumin glands of Helix hortensis and Achatia granulata contained an agglutinin directed to a “receptor” which can be destroyed by neuraminidase (Uhlenbruck and Pardoe, 1969; Pardoe et al., 1970). The “anti-Hp” showed a most remarkable agglutination of ascites tumor cells (Prokop et al., 1968), hepatoma cells (Schnitzler et al., 1968), and HeLa cells (Uhlenbruck and Sehrbundt, 1968). However, strict comparison of agglutination between these tumor cells and comparable normal cells has not been carried out. Recently, Kawauchi et al. (1972) found that various frogs’ eggs had a cell surface coat showing distinctively different reactivities to normal and transformed cells. Eggs of Ram japonica contained a basic protein which was easily dissociated from the egg cell surface by homogenization of the cells with physiological saline and which was strongly reactive to many different kinds of in vitro transformed cells and transplantable tumors. Normal progenitor cells and normal erythrocytes were not agglutinated. Further studies are expected in view of the idea that because egg is ontogenetically immature, it might contain some material showing affinity to ontogenetically immature tumor cells.

E. REACTIVITY WITH ANTIGLYCOLIPID ANTISERA Antisera against a few glycosphingolipids were prepared by injecting glycolipid (e.g., glohoside) with bovine serum albumin into rabbits. The antiserum against globoside was purified by absorption with BSA, and the antibodies were used for detection of globoside residues on cell surfaces (Koscielak et al., 1968). Although globoside is the major glycolipid component of human erythrocyte membrane, the reactivity of human erythrocytes to antigloboside was unexpectedly low. However, after treatment of erythrocytes with trypsin, the reactivities were enhanced to a great extent (from a titer of 1 :10 to 1 :1,000) (Koscielak et al., 1968). Not only were the agglutination titers enhanced, but also hemolysis titer and absorption capability of erythrocytes to antigloboside were enhanced. The results were interpreted to mean that “cryptic globoside” was “exposed” by trypsin digestion. Subsequent comparative studies on fetal and adult erythrocytes indicated that fetal cells have a much higher reactivity than

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adult erythrocytes, although cord erythrocytes have the same reactivity as adult erythrocytes (Hakomori, 1969). A remarkably high reactivity (1:500 to 1:2,000)was frequently observed in fetal erythrocytes up to the 3-month gestation period. Reactivity of fetal erythrocytes was about the same as trypsinized human erythrocytes. Treatment of adult erythrocytes with neuraminidase also results in greatly enhanced reactivity against antigloboside antisera. Antilactosylceramide showed no distinctive difference in reactivity between fetal and normal erythrocytes. Antihematoside antiserum also showed enhanced reactivity with fetal erythrocytes. The results of these studies suggest that globoside or hematoside groupings of fetal erythrocytes are “exposed” to the same degree as trypsinized adult erythrocytes. The reactivities of various normal cells, 3T3, and BHK cells with antihematoside antiserum and with antilactosylceramide antiserum were compared with the reactivities of virally transformed fibroblastic cells. Transformed cells showed significantly enhanced reactivities with antihematoside antiserum as compared to norma1 progenitor cells. However, no enhanced reactivity was observed when the reactivities were compared with antilactosylceramide antibody. Normal cells treated with trypsin showed about the same level of reactivity as transformed cells. These reactivity differences observed with antihematoside resembled the difference of agglutinability demonstrated by phytohemagglutinins. Further studies using NIL cells and purified anti-Forssman glycolipid antibodies labeled with formaldehyde-14C indicate that the reactivity of Forssman glycolipid on cell surfaces (measured with isotope fixation) greatly decreased when NIL-2 cells were confluent. However, since the synthesis of Forssman antigen was very much reduced in transformed NIL cells, the reactivity of transformed cells with anti-Forssman antibody also greatly decreased. NIL cells treated with ethylenediaminetetraacetic acid, however, showed rather increased reactivity with antiForssman antibodies, in concordance with enhanced synthesis of this glycolipid antigen when the cells were confluent (Hakomori and Kijimoto, 1972). Therefore, most of the Forssman glycolipid in NIL cells became nonreactive when the cells were confluent. EDTA may either remove or alter the material that masks Forssman antigen, or the membrane could be rearranged when the cells are detached. Another possibility is that the antigen is blocked by a complementary receptor site of counterpart, cells (either a cell surface enzyme or an unknown protein). In either case, the antigen is expressed by EDTA and becomes cryptic in the confluent state. The results of external labeling recently developed showed a similar tendency for some ceramide tetrasaccharides in 3T3 and BHK cells (see Section VI1,C; Gahmbcrg and Hakomori, 1973). A similar phenomenon

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could be found for cell surface hematoside, as the chemical quantity, immunogenic reactivity, and metabolic activity of this glycolipid are variable on cell-to-cell contact and on malignant transformation (Hakomori et al., 1968; Hakomori, 1970; Kijimoto and Hakomori, 1972). Reactivities of Forssman antigen in BHK cells are quite different from NIL cells; i.e., they are normally “cryptic” and are “exposed” by malignant transformation (Makita and Seyama, 1971 ; Burger, 1971a). Appearance of Forssman antigen in BHK cells during many passages or by transformation has been known for many years (Fogel and Sachs, 1964; O’Neill, 1968; Robertson and Black, 1969). Classical immunological studies carried out by Rapport and his associates (Rapport and Graf, 1961, 1969) may suggest that the change of glycolipid organization on cell surfaces can result in enhanced antigenicity of cellular glycolipids (cytolipins) ; a critical reinvestigation of this possibility with new ideas and techniques is encouraged. Glycosphingolipid covalently coupled to aminoethyl Sepharose has been developed for affinity chromatography (Laine and Hakomori, 1973). Further studies on quantitative determination and topological distribution of glycolipid in membrane is expected. Carbohydrate structures distributed on glycolipids and on glycoprotein may vary greatly from one cell to another. This information is now available by external surface labeling combined with immunoprecipitin with antiglycolipid antisera. It is now apparent that globoside structures are mainly present in the glycolipid fraction of human erythrocyte membrane, and only a very small amount of globoside structures are found in glycoproteins. Such information is important and will eventually be made known for various cell types. VIII. The Significance of Membrane Glycolipid Changes in Regulation of Cell Growth and lntercellular linkages: Some Comments and Speculations

Two kinds of membrane changes which involve glycoprotein and glycolipids have been established to be most closely associated with malignant transformation of cells: 1. Enhanced agglutinability of cells by some agglutinins; this is probably caused by altered dynamic motility of glycoproteins or glycolipid reactive sites on cell surfaces, which could be defined by the altered activity of cell surface microtubules and organization of the lipid bilayer, protein, and glycoprotein. 2. Blocked synthesis of ganglioside, neutral glycolipids, and possibly of some glycoprotein carbohydrates: this could be related to the lack of glycosyl extension or accumulation on cell contact (“contact extension”

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or “contact accumulation”) in transformed cells. Although blocked synthesis of glycolipids has been correlatcd with repressed activity of a specific glycosyltransferase (e.g., CMP-sialic acid :lactosylceramide sialyltransferase in BHK cells; UDP-GalNAc :hematoside GalNAc transferase in 3T3SV cells ; UDP-Gal :lactosylceramide a-Gal transferase in NILpy cells) , the possibility of regulation through activities of glycosylhydrolases bound to surface membranes (e.g., Schengrund et al., 1972) remains to be elucidated. Both these phenomena have been tested in many cases of transformation. Enhanced agglutinability has been observed in many cases of transformed cells with DNA virus, but evidence with RNA virus is limited. Blocked synthesis of glycolipid and related changes of glycoprotein have been found in many transformed cells tested thus far, and the change was reversed when malignancy was reverted (see Section 111). Besides these two changes, accumulation of some glycolipids in transformed cells has been often observed. This may occur as a result of blocked synthesis or as a result of blocked degradation; for example, accumulation of G D l a ganglioside in Morris hepatoma, in some clonal isolates of SV40 transformed 3T3 (Yogeeswaran et aZ., 1972), in 3T3 cells transformed with Friend leukemic virus (Diringer et al.,1972),, and accumulation of lactosylceramide in some clonal isolates of polyomatransformed BHK C13-21 cells (Hakomori and Murakami, 1968; Hakomori, 1970). Also, enhanced synthesis of some sialylfucosylglycopeptides which eluted faster than the corresponding normal fraction has been observed on Sephadex G-50 gel filtration. These changes, either glycolipid synthesis or sialosylglycopeptide synthesis, are closely associated with transformation and can be reversed with reversion of malignancy. How are these membrane changes correlated with altered regulation of cell growth, loss of contact inhibition, reduced intercellular linkages, altered membrane transport, etc? Currently, thc following topics are considered t o be regulatory mechanisms of cell division and cell movement: ( 1 ) enzyme systems that regulate cyclic adenylic acid level of cells, i.e., membrane-bound adenylcyclase and phosphodiesterase ; (2) protein kinase, which is activated by cyclic adenylic acid (DeLange et al., 1968; Kumon et aZ., 1970). The activity of this enzyme can be correlated with regulation of a number of other enzymes including histone kinase; (3) nuclear acidic protein and histone, which supposedly regulates nuclear DNA synthesis (Baserga and Stein, 1971 ; Stein and Baserga, 1972) ; (4) an unknown mechanism which switches on or off polymerization and depolymerization of the protein units of microtubules that regulate motility of cells and membranes, which possibly relates to cellular division and intracellular cytoplasmic movement; (5) plasma membrane loci that can

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regulate transport of sugars and other nutrients. This function is elevated or the number of loci increased in transformed cells. None of these topics can, at this time, be realistically correlated with membrane glycolipid changes or agglutinability change of cells. Some attempts to correlate cyclic AMP level and membrane changes are currently being made. Because enhanced synthesis of some glycolipids (e.g., hematoside and Forssman glycolipid in NIL cells) was observed a t the early stage of cell contact (Kijimoto and Hakomori, 1972), “contact extension” may not result from, but could be a necessary condition for contact inhibition. Further extensive studies are needed to settle this problem. It has been found that morphological and growth characteristics of transformed cells in vitro can be reverted to those of normal or nontransformed cells by addition of cyclic adenylic acid derivatives (Hsie and Puck, 1971; Johnson et al., 1971), and that contact-inhibited 3T3 cells Cell-to-cell contact I

Glycolipid or glycoprotein change (“contact accumulation”)

’

/

/ //

P’

Change of microtubule activity I

Change of agglutinability

J

Contact inhibition of cell movement

/

/

/

1

Intercellular specific linkage

i

Altered Change of membrane conformation - - - - - - +nutrient transport

(Membrane-boundadenylcyclase)

1

(Protein kinase)

I

Contact inhibition of cell growth due to lowered nutrient concentraction

1

Nuclear DNA Bynthesis

Contact inhibition’of DNA replication

FIQ.3. Possible relationship between cell contact, changes in membrane, inhibition of cellular movement, DNA replication, and charge of nutrient transport. These relations are purely hypothetical (see text, Section 111).

GLYCOfdPIDS OF TUMOR CELL MEMBRANE

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contained a higher level of cyclic adenylic acid than did sparse-growing cells (Otten et al., 1971). However, sparse-growing cells whose growth was stopped by lowering serum concentrations in growth medium (sparse, quiescent cells) also showed a high level of cyclic AMP (Seifert and Paul, 1972). The level of G D l a ganglioside in confluent 3T3 cells was five to ten times higher than that of sparse-growing 3T3 cells, with a parallel increase of cyclic AMP level (Hakomori, unpublished observations). The level of G D l a ganglioside in sparse, quiescent cells has not been determined. Some trials have been made to determine whether glycolipid pattern can be changed when cell growth behavior changes with addition of cyclic AMP to the media. So far this attempt has not been successful (Sakiyama et al., 1972; Yogeeswaran et al., 1972). Glycolipid changes in membrane should occur in advance of change of cyclic AMP level, i.e., the change of glycolipid synthesis can affect membrane-bound enzymes that regulate cyclic AMP level. The glycolipid change is also a necessary step to fulfill conditions for complete intercellular linkage (see Roseman hypothesis, Section V) . Glycolipid change could also relate to activity of microtubules in the membrane, as motility of agglutination sites (that is, glycolipid or glycoprotein) on a fluid membrane bilayer is related to microtubule activity. It is speculated, therefore, that contact inhibition of cell movement results from partial inactivation of microtubule activity due to change of membrane glycolipid (see Fig. 3). A number of other factors have been known to alter cell growth behavior, as listed in Table IV. Serum factors believed to be heat stable polypeptides have been isolated which could release contact inhibition of 3T3 cells. Nontransformed cells showed much higher susceptibility to this factor than transformed cells. Unfortunately, the effect of this fact40ron synthesis of glycolipid and glycoprotein is unknown, but is known to correlate with cyclic AMP level (Otten et al., 1972; Paul et al., 1972). Brief treatment of nontransformed 3T3 cells with trypsin or other proteases can induce growth and release contact inhibition. This was interpreted as “cryptic carbohydrate sites” on cell surfaces becoming exposed (Burger, 1970). This growth stimulus from protease treatment is now correlated with a decrease in cellular cyclic AMP levels, and protease-dependent release of contact inhibition can be prevented by dibutyryl cyclic AMP level, not to increased adenylcyclase but rather to increased phosphodiesterase, which brought about DNA synthesis (S phase) and induced mitosis (Burger et al., 1972). Small amounts of sialidase can stimulate cell division and release contact inhibition. It is extremely noteworthy that sialidase also remarkably stimulates transport of 2-dcoxyglucose and glucose (Vaheri et al., 1972). This is the first observation to suggest that function of membrane trans-

TABLE I V

EFFECTS OF VARIOUS REAGENTS WHICHALTERBEHAVIOR OF CELLGROWTH Macromolecules

Cells

Effect

“Serum factor”

3T3 cells, etc.

Release contact inhibition

Trypsin Ficin plus a-galactosidase

3T3 cells BHK cells, NIL cells

Insulin

3T3 cells

Sialidase Trypsinized concanavalin A Dextran sulfate

3T3 cells 3T3 svpy cells 3T6 cells, HA2 cells, NQ-19 cells NIL cells, BHK cells

Release contact inhibition Morphology change to that of NILpy or BHKpy cells Decreased intercellular linkage; release contact inhibitrion Release cont,act inhibition Restore contact inhibition Induce contact inhibition

“Fab” fraction of antiForssman antibody

Low molecular weight components Dibutyrl cyclic AMP Transformed 3T3 cells Prostaglandin (activates adenylcyclase) H+ concentrat.ion Mannose and fucose Galactose

References Todaro and Green (1963); Todaro et al. (1965); Holley and Kiernan (1968); Jainhill and Todaro (1970); Paul et al. (1972) Burger (1970); Vaheri ct al. (1972) Kijimoto, Li, and Hakomori (unpublished observations) Vaheri et al. (1972) Vaheri et al. (1972) Burger (1970) Goto and Sat0 (1972);Goto et al. (1972)

Slowness of cell growth; morphology change

Hakomori and Watanabe (unpublished observations); Laine and Hakomori (unpublished observations)

Restore contact inhibition

Hsie and Puck (1971); .Johnson et al. (1971) Johnson and Pastan (1971); Otten et al. (1972) Ceccarini and Eagle (1971) Cox and Gesner (1965) Kalckar et al. (1973)

Transformed 3T3 cells

Restore contact inhibition

Various cells 3T3 cells BHK cells, NIL cells

Loss of contact inhibition Loss of contact inhibition Increased contact inhibition

GLYCOLIPIDS OF TUMOR CELL MEMBRANE

307

port for sugar is related to membrane-bound glycoprotein or glycolipid. The function is greatly enhanced in tumor cells in general. The effect of serum factors and insulin in releasing contact inhibition has been correlated with cyclic AMP level. Initiation of DNA synthesis in confluent 3T3 cells by the addition of fresh serum is preceded by a decrease in cyclic AMP levels. Cyclic AMP levels also decrease following addition of trypsin or insulin, agents which stimulate DNA synthesis and cell growth. Prostaglandin E, which inhibits cell growth in some cells, raises cyclic AMP levels. Purified a-galactosidase has no effect on cell behavior, but purified a-galactosidase with pepsin or ficin synergistically induces change of cell contact behavior, and NIL or BHK cells grown in regular Eagle’s medium containing a-galactosidase and ficin result in mimic transformed cells (Kijimoto, Li, and Hakomori, unpublished observations). On the other hand, some macromolecular compounds can induce contact inhibition. “Monovalent” concanavalin A, if included in culture medium, can restore contact inhibition in transformed cells. Criticism and support for this finding was already discussed (see Section VI1,C). The “Fab” fraction of anti-Forssman antibody and other anti-glycolipid antibodies slowed cell division of NILpy cells (Hakomori and Watanabe, unpublished observations ; Laine and Hakomori, unpublished observations). An interesting effect was reported using dextran sulfate, which induced remarkable contact inhibition in 3T6 cells, NA-2 cells, and NQ-19 cells (Goto et al., 1972; Goto and Sato, 1972). Thus, the effects of macromolecules which can alter cell growth through cell surfaces have become increasingly apparent, and the idea that carbohydrates of cell membranes can be sites for membrane-mediated control of cell growth has now become plausible. Some simple sugars were reported to change morphology and contact inhibitability of cells. Relatively high concentrations (12 mg/ml) of mannose and fucose were found to release contact inhibition of 3T3 cells, while other sugars did not effect such a change (Cox and Gesner, 1965). If the physiological concentration of galactose replaced glucose, cell growth was significantly changed. BHK or NIL cells appear more contact-inhibited and a remarkable change in morphology of NILpy cells appears a t the later days of Gal-culture. This could result from accumulation of Gal-1-p and galactitol in cell membrane, which is due to the UDP-galactose epimerase choke (Kalckar, 1965; Robinson et al., 1966; Kalckar et al., 1973). It is interesting to note that metabolic changes of simple sugars can effect such a remarkable change in cell contact behavior, which could be a membrane-mediated process. A possible sequence of reactions triggered by the change of cell surface structures which may induce or release con-

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tact inhibition or which may effect transport of sugars through cell surfaces is summarized in Fig. 2. It is hoped that such a figure, although oversimplified, can stimulate further extensive studies on the possible role of membrane glycolipids in controlling various phases of cell physiology. Understanding the membrane-mediated processes via glycolipid is undoubtedly a key to understanding the secrets of malignancy.

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CHEMICAL ONCOGENESIS IN CULTURE Charles Heidelberger McArdle Loboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin

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I. Introduction . . . . . . . . 11. Organ Cultures . 111. Hamster Embryo Cells . IV. Transformation of Fibroblastic Cells Derived from Mouse Ventral Prostate . . . . . . . . . . . V. 3T3-Like Systems . . . . . . . . . . VI. Other Cell Systems . . . . . . . . . . VII. Liver Cell Systems . . . . . . . . . . VIII. Combined Effects of Chemicals and Ontogenic Viruses . IX. Metabolism of Polycyclic Aromatic Hydrocarbons . . X. Binding to Cellular Macromolecules . . . . . . XI. Metabolic Activation of Polycyclic Hydrocarbons . . . XII. Conclusions . . . . . . . . . . . . References . . . . . . . . . . . .

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I. Introduction

In this review of Chemical Oncogenesis in Culture, it is necessary that the subject matter be quite restricted. Although the field depends upon the fundamentals and advances in cell biology, virology, biochemistry, and other related disciplines, it is clearly impossible to cover these other fields. Although most of the work has been done with cell cultures, organ cultures will also be considered. The more important systems will be described, followed by some of the major generalizations that can be drawn from the various researches involving those systems. First let us consider a few definitions. I n almost all cases where chemically transformed cells have given rise to tumors on inoculation into suitable animals, sarcomas have been obtained. Therefore, I prefer to refer to this field as oncogenesis in culture, rather than carcinogenesis in vitro, especially since the term in vitro to the bacteriologist and molecular biologist refers to cell-free systems. Organ cultures consist of small 3-dimensional pieces of differentiated tissues, that are usually maintained on top of the nutrient medium. Cell cultures consist of individual cells submerged in the medium that grow either on a glass or plastic substrate or in suspension. Primary cultures refer to the initial growth of cells taken directly from the animal, and secondary cultures represent the first sub317

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culture of primaries. Many freshly derived cultures have a finite and usually short life-span, in that the growth rate of the cultures decreases and the cells tend to die. Such cultures are generally referred to as strains. Other cultures, usually after passing a crisis when growth stops, grow indefinitely a t a constant rate, and are termed lines. Usually in the formation of lines there is a chromosomal rearrangement. As will become evident, there are advantages and disadvantages to the use of strains and lines for studies of chemical oncogenesis in culture. The first report of chemical oncogenesis in culture was that of Earle and Nettleship (1943), who treated long-term cultures of mouse fibroblasts with methylcholanthrene and eventually obtained cells that gave tumors on inoculation into mice. However, Sanford et al. (1950) observed the same phenomenon without the use of the oncogenic chemical. This was another case of “spontaneous” oncogenic transformation of mouse cells in culture that had first been reported by Gey (1941). This subject has been thoroughly reviewed (Sanford, 1968), but in spite of extensive investigations, I believe that it is safe to say that the mechanism of spontaneous transformation is not presently understood. Nevertheless, this phenomenon is of fairly widespread occurrence, and it is necessary in all studies of chemical oncogenesis in culture to provide evidence that spontaneous transformation is not a factor. I n my laboratory we have long been interested in determining the cellular and molecular mechanisms of chemical oncogenesis (Heidelberger, 1969, 1970a,b). We belatedly became impressed with the limitations of working with the skin of living mice, and in 1962 actively sought to develop a system for chemical oncogenesis in culture. At that time, the most suitable lead I could find in the literature was the work of Lasnitzki (1963), and it was under her wise and patient tutelage that I became inducted into the mysteries of culture techniques. After my return from that sabbatical I set out to develop a quantitative system for chemical oncogenesis in culture. When our system was in its early stages of development, Berwald and Sachs (1963, 1965) described their quantitative system for producing chemical oncogenesis in cultures of hamster embryo cells. 11. Organ Cultures

Lasnitzki (1963) has carried out extensive research on the morphological effects produced by polycyclic hydrocarbons and hormones on organ cultures of mouse and rat prostate glands. Those pieces of prostate cultivated on plasma clots and not treated with the test compounds maintained for periods up to 2 weeks a differentiated appearance consisting of a single layer of epithelial cells surrounding the alveoli. When these

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prostate fragments were treated with microgram quantities of oncogenic hydrocarbons such as 3-methylcholanthrene (MCA) , the epithelial cells underwent massive hyperplasia and squamous metaplasia, which are preneoplastic changes. She also obtained rather similar, but less striking, effects in organ cultures of embryonic mouse, rat, and human lungs (Lasnitzki, 1963). We wished to ascertain whether these morphological effects were associated with malignancy, so we adapted the technique to liquid media and used the ventral prostates from inbred, C3H mice. The appearance of a typical control culture is shown in Fig. 1, and the striking morphological alterations produced by MCA are shown in Fig. 2. In addition to hyperplasia and squamous metaplasia, we also observed pleomorphism of the epithelial cells and invasion through the basement membrane. Some of our slides were read as malignant by pathologists. Disappointingly, however, when 872 of these hydrocarbon-treated pieces from the organ cultures were implanted into as many isologous mice under a variety of conditions, no tumors were produced. Thus, these profound morphological alterations were not associated with detectable malignancy (Roller and

Fro. 1. Photomicrograph of a prostate maintained in organ culture for 7 days (Roller and Heidelberger, 1967).

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FIG.2. Photomicrograph of a prostate maintained in organ culture for 14 days, with 10 pg/ml of DBA present for the first 7 days (Roller and Heidelberger, 1967).

Heidelberger, 1967). Therefore, we believe that morphological transformations cannot be seriously considered as being malignant unless tumor formation can be consistently detected in suitable hosts. However, we established some cell lines from the morphologically altered hydrocarbontreated organ cultures that did give rise to sarcomas on inoculation into C3H mice (Heidelberger and Iype, 1967). Despite the fact that the morphological changes produced by chemical oncogens in organ culture cannot be equated with malignancy, studies of these sorts are considered useful. The changes seen histologically in these cultures often parallel premalignant changes seen in vim. Thus, observation of organ cultures is particularly valuable in the case of epithelial tissues, since when chemical oncogenesis in cell culture has been successful the tumors ultimately produced in animals have almost invariably been sarcomas. Thus, organ cultures havc been used to examinc some aspects of environmental oncogenesis as related to epithelial cells. The work mentioned above has been extended by Lasnitzki (1958) , who showed that cigarette smoke condensate produced hyperplasia and squamous metaplasia in organ cultures of fetal human lung. This illustrates one advantage of such morphological studies in organ culture in that human tissues can be used. Crocker et al. (1965) carried out organ cultures with tracheas from rats 1-5 days old and did a very comprehen-

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sive histological study after treatment with various polycyclic hydrocarbons. They found that 7,12-dimetliylbenz [a] anthracene (DMBA) produced, in addition to toxicity, considerable metaplasia in the epithelial cells, followed by hyperplasia. It also stimulated the incorporation of tritiated thymidine into the epithelial cells as compared to the solventtreated controls. Benzo [alpyrene (BP) and MCA produced similar but less striking effects. Dirksen and Crocker (1968) examined 1l-day organ cultures in the electron microscope and found that when the epithelial cells of the DMBA-treated specimens were compared with the solvent controls there was a decrease in organized endoplasmic reticulum with a concomitant increase in free ribosomes; there was also an increase in autophagic vacuoles and cytoplasmic filaments. These effects were not observed to as marked an extent in cultures treated with nononcogenic hydrocarbons. Crocker and Sanders (1970) produced the above-mentioned morphological changes with B P in organ cultures of hamster trachea. They found that simultaneous addition of citral and vitamin A, which by themselves did not produce such effects, respectively, speeded up and prevented the effects of BP. The preservation of the differentiation of columnar epithelium when vitamin A was added to B P was particularly striking. Palekar et al. (1968) also carried out organ cultures of rat trachea and obtained good survival for periods up to 4 weeks, as determined by thorough histological evaluation. They found that MCA produced hyperplasia, squamous metaplasia, and some pleomorphic cells, which were not seen on treatment with benz [a]anthracene (BA) , a very weak chemical oncogen. These authors discussed their results purely in morphological terms and prudently chose not to relate their findings directly to oncogenesis. Chan et al. (1969) did organ cultures of fetal mouse lung buds and found that polycyclic hydrocarbons produced considerable toxicity; however, they found that 2 pg of B P per milliliter produced a “selective toxicity” with some thickening and disorganization of the tubular bronchial epithelium and increased mitosis. An extensive report by Shabad et al. (1972) summarized several investigations, some of which had been published in the Soviet literature. They carried out organ cultures of fetal mouse kidneys obtained from mothers given various dosage schedules of oncogenic and nononcogenic chemicals; thus, this system can be considered as a possible model for transplancental oncogenesis. Morphological examination revealed that the fetal organ cultures from the mothers treated with oncogenic hydrocarbons exhibited a much greater degree of hyperplasia of the epithelial cells than did those from groups given nononcogenic hydrocarbons ; similar changes were also frequently seen in the group treated with dimethylnitrosamine (DMNA) .

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These authors made no attempts to implant thc cultures into mice and look for tumors. There have been other studies in which various tissues have been placed for brief periods in organ culture with oncogens, implanted directly into isologous mice, and tumors observed a t that site. Although one set of authors states that these experiments are a “straightforward proposition” it is difficult to judge what has actually occurred in these experiments. Jull et al. (1968) removed the ovaries from 3-week-old inbred mice, incubated them from 4 hours to 4 days, and immediately implanted them subcutaneously into isologous mice. They examined the implantation sites periodically by serial sections. After 30 weeks there was a high incidence of luteomas a t the implantation sites in control and DMBA-treated specimens. In addition, granulosa cell tumors were found in the implanted ovaries that had been cultured with 4 p d m l of DMBA. They considered that the treatment with the chemical oncogen somehow altered the susceptibility of the implant to the hormones circulating in the host. Laws and Flaks (1966) carried out organ cultures for 8 days of lungs from embryonic or 1 month BALB/c mice and then implanted the pieces subcutaneously into mice of the same strain. Histological examination revealed no appreciable changes in the control pieces. However, those pieces that had been cultured with 4 pg/ml of MCA gave rise to histologically identified adenomas on implantation, with very little indication of inflammatory changes surrounding the implant. An extension of this work (Flaks and Laws, 1968) led to the observation that during the brief period in organ cultures, no histological changes such as the hyperplasia and squamous metaplasia described above (Lasnitzki, 1958,1963 ; Crocker et al., 1965; Roller and Heidelberger, 1967) were produced by the oncogenic hydrocarbons. The implantation procedure again led to adenomas (Flaks and Laws, 1968) to a greater extent when the lungs of weanling mice were cultured as compared to those of embryo mice. MCA also produced adenomas, and some adenocarcinomas arose later a t the implantation site; some of these were transplantable. Flaks and Flaks (1969) concluded from an electron micrographic study that the ultrastructural changes that occurred in the implants after organ culture with oncogens were similar to those seen during the in vivo induction of lung adenomas. Flaks and Hamilton (1970) without the presentation of any data, asserted that in the same system in which polycyclic hydrocarbons produced the effects already described, urethan and acetylaminofluorene (AAF) did not. Consequently, they suggested that the latter two compounds might require whole-body metabolic activation. The results of the Flaks group were confirmed by Davies et nl. (1970) with MCA.

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The mechanism involved in these studies is difficult to grasp. It seems possible that in such short-term organ cultures with relatively high levels of oncogenic hydrocarbons and immediate implantation into mice, some of the chemical (either free or bound to cellular constituents) was transferred with the piece of tissue. If this were true (none of the authors seriously considered this seemingly obvious possibility or attempted any experiments to clarify this point), then these experiments probably represent another case of in vivo oncogenesis. Koyama et al. (1972) carried out organ cultures of rat mammary glands for 6-12 days and found that insulin was necessary for their maintenance. Addition of estradiol, but not hydrocortisone or prolactin, stimulated some proliferation of ducts and increased the thymidine labeling index. Progesterone strongly stimulated ductal proliferation, low levels of DMBA produced an increase in labeling index, squamous metaplasia, and some anaplastic changes. When mammary gland organ cultures were treated for 9 days in a medium containing insulin, estrogen, prolactin, progesterone, and DMBA and then transplanted into isologous rats, a high incidence of papillary adenocarcinomas was obtained (Dao and Sinha, 1972). The previous suggestion about carry-over of the oncogenic chemical into the implant site may not apply to this case. Ill. Hamster Embryo Cells

The first unequivocal demonstration of transformation of cells in culture with chemical oncogens was that of Berwald and Sachs (1963). They treated Syrian hamster embryo secondary cells with MCA, BP, urethan, and the appropriate solvents as controls. I n mass cultures treated with MCA and BP, but not with urethan or the solvents, and then cloned on feeder layers of irradiated rat cells, some colonies were formed that had small, fusiform cells that piled up in an irregular crisscross pattern. This morphological transformation was comparable to that observed when similar cells were treated with polyoma virus (Vogt and Dulbecco, 1960). When the colonies obtained in the presence of B P and MCA were counted, it was found that the hydrocarbons exerted considerable toxicity (as measured by a decrease in plating efficiency) and that up to 16% of the surviving clones were transformed. It was concluded that the transformation was a consequence of the direct effect of the chemical oncogen. The method of scoring colonies for transformation is illustrated in Fig. 3; these colonies were fixed and stained in a dish that had been treated 8 days previously with MCA. The colony on the left is normal; the cells are flat, the cytoplasm is clearly seen, and there is very little piling-up. The colony on the right is transformed; the cells are smaller, more fusiform, and there is extensive piling-up. However, piling-up some-

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FIG.3. Photomicrograph of two fixed and stained colonies in a mrthylcholnnthrene-treated dish containing hamster embryo cells in the cloning assay described in the text. The colony on the left is normal; the cells are in a strict monolayer and their cytoplasm is evident. The colony on the right is transformed; the cells are smaller, more fibroblastic in appearance, and are heavily piled up.

times occurs in the center of nontransformed colonies. Therefore, the best way to score is to examine the edges of the colonies, which are shown in higher magnification in Fig. 4. I n contrast to the flatness of the normal colony on the left, the random crisscross array of cells is clearly illustrated a t the edge of the transformed colony on the right. Berwald and Sachs (1965) continued and extended their research and published more experimental details of their quantitative system. When they carried out mass cultures of mouse embryo cells, the incidence of transformation in the controls was almost as high as in the BP-treated cells; this finding was also paralleled by the production of sarcomas on inoculation of the cells into mice. Thus, the high incidence of spontaneous oncogenic transformation precluded the use of mouse embryo cells. On the other hand, spontaneous transformation was not observed in hamster embryo cells, yet mass cultures transformed by B P and MCA did produce sarcomas on inoculation into Syrian hamsters. The major emphasis in this paper (Berwald and Sachs, 1965), how-

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FIG.4. Highcr power photomicrograph of the edges of the same two colonies shown in Fig. 3. The crisscross piling up of the transformed colonies on the right is clearly shown.

ever, was on quantitative cloning techniques with the hamster embryo secondaries in which small numbers of cells were plated and individual colonies were scored for transformation. The untreated cells always had a limited life-span, while mass cultures of hydrocarbon-transformed cells had an indefinite life-span and were resistant to the toxicity of the oncogenic hydrocarbons. There was a good correlation between the oncogenic activity of a series of polycyclic hydrocarbons and the number of transformed clones they produced, and the conclusion was again drawn that the action of the chemical upon the cells was direct. With BP, the percentage of transformed to surviving colonies averaged about 10%. Although these workers did not pick individual transformed clones and show that they were tumorigenic, this was done by DiPaolo et al. ( 1969a) ; the transformed clones produced fibrosarcomas on subcutaneous inoculation into weanling hamsters. I n this system, Huberman and Sachs (1966) carried out careful doseresponse studies on the toxicity and transformation produced by BP. They concluded that the two latter processes are unrelated and that transformation is a “one-hit” phenomenon. Thus, transformation could

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not be considered to result from a selection of clones resistant to toxicity. Huberman and Sachs (1968) also reported that polyoma virus-transformed clones of hamster embryo cells have a wide variation in their resistance to B P cytotoxicity; hence, the transformed state does not determine resistance. Moreover, they found that transformed cells that were quite susceptible to BP toxicity in carly passages, became resistant in later passages; thus, this is a secondary change after transformation, probably analogous to tumor progression. Consequently, resistance to cytotoxicity is not a good marker for transformation. Gelboin et al. (1969) showed in a large number of rodent cell clones that there was a direct correlation between the level of aryl hydrocarbon hydroxylase (to be discussed below) and the degree of cytotoxicity produced in the cultures by BP. They also found that 3-hydroxy-BP, a product of the enzyme reaction, was highly cytotoxic to cells that were either susceptible or resistant to the toxic effect of BP. Huberman et al. (1968) treated hamster embryo secondaries with the powerful oncogen dimethylnitrosamine (DMNA) . Although the cells thus obtained did not form the characteristic crisscross piled-up colonies, they did have an indefinite life-span and an increased growth rate. Moreover, these cells did form typically transformed colonies after 5 months in culture; 3 months after that the cells were capable of forming colonies in soft agar and giving rise to fibrosarcomas. These results suggest that under some circumstances oncogenic transformation by chemicals may require several steps. Huberman et al. (1970) found that transformed cells, were in general, resistant to the toxicity of DMNA with some variation among clones; since this was not true for BP, they concluded that the metabolic activation of these compounds was carried out by different enzymes. Nitrosomethylurea (NMU) , a compound that does not require metabolic activation, was always more cytotoxic to transformed than to nontransformed cells, which may be correlated with growth rates. In vitro transformation of hamster embryo cells by X-irradiation was reported by Borek and Sachs (1966a). They treated secondaries plated on a feeder layer of rat cells with 300r of X-irradiation and found in mass cultures the same sort of piling-up and crisscross orientation that had been described for transformation by chemicals and viruses. These cells also could be cultured indefinitely, had an increased growth rate, and were resistant to the cytotoxicity of BP. However, on inoculation into hamsters these morphologically transformed cells produced transient growths that regressed, but no tumors. Similar results were obtained in cloning experiments. Borek and Sachs (1967) demonstrated that cell division was required to “fix” the transformed state induced by X-irradiation, and that the “fixation” of the transformed state was lost if the cells were

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maintained for 2 days in the confluent state in which cell division does not occur. A similar requirement of cell division for transformation with SV40 virus was noted by Todaro and Green (1966). Further experiments led to the conclusion that two cell divisions were required to “fix” the X-ray-induced transformed state (Borek and Sachs, 1968). An interesting study of the specificity by which cells exert contact inhibition of replication of other cells was carried out by Borek and Sachs (1966b). I n order to monitor these interactions, they seeded small numbers of cells marked with India ink on monolayers of unlabeled cells and observed the ability of the marked cells to grow with the others. They observed considerable specificity and their results can be summarized as follows: (1) normal cells were able to inhibit and be inhibited by both transformed and normal cells; (2) cells transformed by X-rays or polycyclic hydrocarbons did not inhibit transformed cells from the same line; (3) two cell lines independently transformed by X-rays inhibited one another; (4) a line transformed by BP inhibited a line transformed by MCA; ( 5 ) cells doubly transformed by X-rays and polyoma virus were inhibited by cells of both parental lines; (6) cells transformed by polyoma virus were able to inhibit and be inhibited by cells transformed by SV40; and (7) cells transformed by polyoma virus were not inhibited by other cells transformed by the same virus. Obviously, much further research is required to elucidate the mechanism of these complicated specificities. The above research with hamster embryo cells was carried out in the laboratories of Leo Sachs in Rehovoth, Israel. There has also been considerable work done along rather similar lines in Japan, where two groups simultaneously announced the transformation of hamster embryo cell cultures with 4-nitroquinoline-N-oxide (NQO) (Kamahora and Kakunaga, 1966; Sat0 and Kuroki, 1966). The latter workers obtained colonies with the typical piled-up crisscrossed morphology within a few days. These cells eventually produced tumors in hamsters. Kuroki et al. (1967) also succeeded in producing malignant transformation with 4-hydroxylaminoquinoline-N-oxide (HAQO) , which is an activated metabolite of NQO. Many further details of the system were reported by Kuroki and Sat0 (1968). Using mass cultures, they found that the control cells eventually stopped proliferating and gave no tumors on inoculation into hamsters. In cultures treated with various oncogenic derivatives of NQO, transformation generally occurred within 20 days after treatment. However, in some cultures morphological transformation preceded the acquisition of malignant properties ; sometimes 50-100 days of cultivation after morphological transformation were required before the cells produced fibrosarcomas on inoculation into hamsters. Since noninbred hamsters

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were used, an immune response was probably involved, and the early transformed cells may have been highly antigenic and have lost some of this antigenicity during further culturing. Yosida et al. (1970) investigated the chromosomal constitution of the various cells. In general, the normal cells had a diploid karyotype, the cells taken immediately after transformation had diploid karyotypes, but on further cultivation they became hypotetraploid. Thus, at least in the early stages of transformation, no marked chromosomal changes were observed. Sat0 et al. (1970) found that cells transformed with NQO showed an increased aerobic glycolysis and Crabtree effect as compared to the normal cells; however, since it was necessary to use large numbers of cells for the biochemical assays, the cells could not be obtained in sufficient quantity immediately after transformation. Thus, it is not known whether these changes are a result of transformation or further progression of the transformed cells. I n our laboratory a t a time when we were fortunate in having both Drs. Huberman and Kuroki in the group, we used the hamster embryo cell system to demonstrate that epoxides, which we postulate (see below) to be a metabolically activated form of polycyclic hydrocarbons, are much more effective than the parent hydrocarbon and other derivatives a t producing oncogenic transformation (Grover et al., 1971c; Huberman et al., 1972a). The other group that has intensively studied the hamster embryo cell system has been that of DiPaolo a t the National Cancer Institute. DiPaolo and Donovan (1967) treated mass cultures of Syrian hamster embryo secondaries with polycyclic hydrocarbons. Following marked toxicity, there was recovery of some cells that eventually grew into multilayers, were resistant to toxicity of the oncogenic hydrocarbons, and produced tumors on inoculation into hamsters. The system was made quantitative by the cloning techniques used by Berwald and Sachs (1965), and there was a good quantitative correlation between the extent of production of piled-up clones and the oncogenic activity of a series of 9 polycyclic hydrocarbons (DiPaolo et al., 1969b). Individual transformed colonies were found to give fibrosarcomas on inoculation into hamsters (DiPaolo et al., 1969a). The morphology of transformation was illustrated more fully (DiPaolo e t al., 1971d), and a thorough study of the chromosomes revealed that while the control cultures were predominantly diploid with a normal karyotype, most of the transformed cell lines fluctuated around the diploid mode with a few changes in chromosomal morphology ; sometimes the tumors induced from hyperdiploid lines were hypotetraploid. They concluded that the chromosomal alterations were random events independent of the transformation (DiPaolo et al., 1971d). However, Benedict (1972) reported that chromosomal abnormali-

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ties could be detected within 72 hours after treatment of similar cells with DMBA, BP, but none after 3-OH-BP and BA. These abnormalities included rearrangements in addition to breaks, and it was concluded that the former may be important in transformation. A number of fibrosarcomas obtained from chemically transformed cell lines were put back into culture; they formed piled-up colonies, and heteroploidy increased with the time in culture (DiPaolo et al., 1971e). Careful dose-response curves were carried out (DiPaolo et al., 1971a) on the transformation of hamster embryo cells by BP, and in agreement with earlier results (Huberman and Sachs, 1966) it was concluded that transformation and toxicity were not directly related, and that the transformation follows single-hit kinetics. It was reported (DiPaolo et al., 1971b) that increased transformation occurred when BP was added 48 hours after irradiation of the cells with 250-500 r, whereas no transformation was observed after irradiation alone. DiPaolo et al. (1972a) demonstrated that transformation could be produced by a number of other oncogenic chemicals including aflatoxin B1, N-acetoxyacetylaminofluorene, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), and methylaaoxymethanol. When urethan and diethylnitrosoamine were given to pregnant mothers, the hamster embryo cells showed a high incidence of transformation that was not observed when the same compounds were added directly to the cultures. This model of transplacental oncogenesis may find future usefulness in host-mediated metabolic activation tests. Finally, Huberman et al. (1972b) demonstrated again that N-acetoxyacetylaminofluorene transformed Syrian hamster cells and was also highly mutagenic to a line of Chinese hamster cells, as measured by the production of clones that were resistant to 8-aaaguanine. The “reversion” of clones of virally and chemically transformed cells has been studied in great detail by Sachs and his colleagues, and only the highlights of their work on chemically transformed clones can be considered here. Rabinowita and Sachs (1970b) carried out extensive studies on clones of hamster embryo cells that had been transformed by DBA, BP, and DMBA. When they were plated on normal cells that had been fixed by glutaraldehyde, variant clones were selected that, as compared with the parent transformed clone, had a lower saturation density, a 500to 1000-fold less malignancy, exhibited specific contact inhibition, and regained the characteristic of a limited life-span. About 85% of the transformed clones gave rise to these less malignant variants. Some of these less malignant variants on further culturing regained malignancy and increased saturation density ; this phenomenon was given the unfortunate term, “re-reversion.” While 85% of the revertant clones obtained from

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clones transformed by the above tlircc chemicals regained a limited lifcspan, reverted polyoma virus-transformed clones, although exhibiting a lower saturation density and less malignancy, did not regain a limited life-span. This suggests that integration of the viral genome prevents the reacquisition of this property (Rabinowitz and Sachs, 1970b). A model was proposed to explain these findings (Rabinowitz and Sachs, 1970a) in which the expression or suppression of the transformed state is determined by the balance of factors E and S, respectively; it was postulated that these factors were chromosomally determined. Some evidence for this chromosomal control was provided by Hitotsumachi et al. (1972), who found that less malignant variants always had different chromosome numbers than the parent malignant clone. They categorized the chromosomes into different groups based on size and morphology, and reported that chromosomes in group 5 carry the factors for expression (E) of malignancy and chromosomes in groups 6, 7, and 9 carry the factor for suppression (S) . Similar results had been found in revertants of polyoma virus-transformed cells (Hitotsumachi et al., 1971). The expression of malignancy was also found to be correlated with susceptibility to treatment with bromodeoxyuridine and visible light and that suppression of malignancy was associated with resistance to such treatment. They suggested (Hitotsumachi et al., 1972) that viral and nonviral oncogens cause malignancy by inducing chromosomal rearrangements that result in a change in balance between E and S. The Sachs group and others have also studied changes in the surface membranes of cells during transformation by the use of plant lectins that bind to specific carbohydrate groups on the cell surface. Again, the literature is extensive, but deals primarily with virally transformed cells. Inbar and Sachs (1969a) demonstrated that concanavalin A, a protein that reacts with a-methylglucopyranosides, agglutinates hamster embryo cells that were transformed by polyoma and SV40 viruses, BP, and X-rays, but a t comparable concentrations did not agglutinate normal cells. The agglutination was specifically reversed by a-methylglucopyranoside, but not other carbohydrates, and also by the removal of the essential divalent cation in the protein. Similarly, Burger (1969) found that a partialIy purified wheat germ lectin agglutinated virally transformed cells, but that normal cells were also agglutinated following mild trypsin treatment. Inbar and Sachs (1969b) then studied the binding of Wi-labeled concanavalin A to various normal and transformed hamster embryo cells. They found a considerably greater binding of the lectin to the membranes of polyoma-, SV40-, and DMNA-transformed cells than to normal cells. However, when the normal cells were treated lightly with trypsin, cryptic sites were exposed and the total binding was about the

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same as was obtained with the transformed cells. Thus, about 85% of the sites on the normal cells were cryptic. Moreover, a number of less malignant “revertant” clones of polyoma virus-transformed cells (see above) were no longer agglutinated by concanavalin A without trypsin treatment; this finding provides further support that the membranes of revertants regain normal properties (Inbar et al., 1969). It was found that concanavalin A was nontoxic to normal hamster cells, but killed polyoma, SV40, and DMNA-transformed cells ; similar results were obtained with mouse 3T3 cells (Shoham et aZ., 1970). Furthermore, injection of large amounts of this protein a t the site of inoculation of polyoma-transformed hamster cells delayed or prevented the appearance of tumors. Burger and Noonan (1970) also found that concanavalin A is toxic to transformed cells. However, when they split the lectin to a monovalent form and treated polyoma-transformed 3T3 cells, without any inhibition of growth rate, the growth properties of the transformed cells were restored to normal with a lowering of the saturation density. This toxic property of concanavalin A has been used to select for revertants of SV40-transformed 3T3 cells, which had a lower saturation density while still expressing the T antigen (Culp and Black, 1972). Inbar et aZ. (1971a) found that concanavalin A agglutinates transformed cells and trypsin-treated normal cells a t 24OC, but not a t 4”C, although the same amount of binding was obtained a t both temperatures. This indicates that there are two metabolic components of concanavalin A action; a temperature-insensitive component of binding, and a temperature-sensitive component that determines agglutination. Other lectins with different carbohydrate specificities did not show this temperature dependence. Thus, transformation changes the organizational structure of the cell membrane. The nature of these changes was studied by BenBassat et al. (1971), who provided evidence too detailed to be described here that during transformation there is a rearragement of the concanavalin-binding sites on the cell membrane, and that it is the density of sites, rather than their total number, which determines the agglutination. A tight correlation between agglutinability of cells by concanavalin A and their malignancy has been demonstrated by Inbar et al. (1972). In addition, it was shown (Inbar et al., 1971b) that the binding of concanavalin A to transformed but not normal cells inhibited the active transport of amino acids into the cells; but the transport of fucose was not affected. I n this way, a kind of biochemical mapping of the cell membrane can be achieved. Nicolson (1971) succeeded in visualizing in the electron microscope that ferritin-conjugated concanavalin A was bound in a dispersed fashion to membranes prepared from normal 3T3 cells. However, in the membranes of polyoma-transformed 3T3 cells, the

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concanavalin A was bound in dense clusters. This provides strong evidence for the rearrangement of binding sites in transformed cells. I n 1971 it was independently discovered in three laboratories that dibutyryl cyclic AMP [ (But),cAMP] produced profound changes in the surface properties of cultured cells. This was not surprising, since cAMP is known to exert its manifold controls a t the membrane level. Hsie and Puck (1971) first reported that when a line of Chinese hamster ovary cells, which grew in a compact well-separated form with no contact inhibition, were treated with (But) zcAMP the cells were converted within 3 hours into an elongated fibroblastic form that produced oriented colonies with strict contact inhibition. This conversion was prevented by Colcemid and vinblastine, was augmented by addition of testosterone propionate; the generation time of the two forms was the same. The effect is completely reversible; within 1 hour after removal of (But),cAMP the fibroblastic cells returned t o the original compact form. Johnson et al. (1971) showed that when tumor-producing mouse L cells that grew in a crowded compact fashion were treated with (But),cAMP, they elongated into a fibroblastic form and regained the growth characteristics of normal mouse fibroblasts. Under these conditions there was no significant effect on DNA, RNA, and protein synthesis, and the morphological effect was reversible. Similar observations were made with rat XC cells, and colchicine prevented the conversion. Sheppard (1971), using polyoma and spontaneously transformed 3T3 cells, found that (But) ,CAMP, without affecting cell growth rate and particularly in the presence of theophylline, brought the saturation density down to normal levels and prevented agglutinability by the wheat-germ lectin. This morphological conversion was also quickly reversible. Sheppard (1972) also showed that in the steady state the cAMP level in contact-inhibited 3T3 cells was twice as high as in virally and spontaneously transformed 3T3 cells. The cAhlP level was not affected by the growth rate of the cells nor by their state of confluency, and prostaglandin E l greatly increased the cAMP levels in all of these cells. Hsie et al. (1971) found that the changes they had previously described in the Chinese hamster ovary cells that were produced by (But) ,CAMP were also accompanied by a decrease in saturation density, the disappearance of knoblike pseudopoda on the cell surface, induction of collagen synthesis, and a decrease in agglutinability by plant lectins and specific antibodies. Mutants were obtained with altered characteristics, demonstrating that the conversion is under genetic control. The system was proposed as a useful one to study the regulation of phenotypic expression in mammalian cells. Further studies of the morphology of the conversion suggested that the organization of the microtubular and

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microfibrillar systems inside the cell was involved in these conversions, and possibly in the regulation of malignancy (Puck et al., 1972). Johnson and Pastan (1972) found that (But),cAMP treatment of normal 3T3 cells further decreased their saturation density, but not that of transformed 3T3 cells. They also reported morphological effects comparable to those already described. It is clear from the foregoing that the cAMP system is of great importance in the regulation of surface properties, growth characteristics, and possibly malignancy of cultured mammalian cells. How relevant these controls are to the properties of tumor cells in animals remains to be seen. Although I am unaware of any reports of the effects of cAMP derivatives on the properties of chemically transformed cells, doubtless such research will soon be forthcoming. IV. Transformation of Fibroblastic Cells Derived from Mouse Ventral Prostate

As mentioned above, attempts in our laboratory to produce oncogenic transformation with polycyclic hydrocarbons in organ cultures of C3H mouse ventral prostates were unsuccessful (Roller and Heidelberger, 1967). However, when treated organ cultures were dispersed into cell suspensions, lines were produced that were malignant (Heidelberger and Iype, 1967). These lines, however, lacked normal controls. Thus, it was necessary to derive lines of cells from untreated mouse prostates, and this was done. Aneuploid lines of cells were maintained indefinitely in culture, reached a saturation density and did not pile up, produced no tumors even when lo7 cells were injected subcutaneously in irradiated C3H mice, and underwent spontaneous malignant transformation rarely and only after very prolonged cultivation (Chen and Heidelberger, 1969a). When these cells were treated in culture with MCA, they piled-up in a crisscross fashion, did not reach a saturation density, and produced tumors in unirradiated C3H mice with as few as 1000 cells inoculated subcutaneously. These tumors were fibrosarcomas that grew progressively and killed the mice, were easily transplantable, and metastasized to the lungs (Chen and Heidelberger, 1969b). Thus, chemical oncogenesis in these cultures was accomplished. The system was then made quantitative. One thousand cells were plated in a dish, and groups of dishes were treated with the solvent as controls and with various concentrations of polycyclic hydrocarbons for 1 or 7 days. After 8 days some of the dishes were fixed and stained; the number of colonies was counted as a measure of toxicity. The remainder of the dishes were cultured from 4 6 weeks and then fixed and

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stained. The appearance of control dishes is shown in the left of Fig. 5. I n MCA-treated dishes, darkly stained piled-up foci of cells appeared that can be counted, as shown on the right of Fig. 5. When such morphologically transformed foci were picked, grown, and injected into C3H mice they all gave rise to fibrosarcomas. Hence, in this system morphological and oncogenic transformation can be equated. The appearance of a control dish a t higher power is shown in Fig. 6, where the lack of piling-up is evident. The edge of a transformed colony is shown in Fig 7; the cells appear out of focus because they are piled up into a multilayer, the transformed cells are stained more deeply, and the extension of the transformed cells into the surrounding monolayer is seen (Chen and Heidelberger, 1969~). A series of dose-response curves for toxicity and transformation were constructed for oncogenic hydrocarbons. I n general, the shapes of the curves were different for those two parameters, leading to the conclusion that toxicity and transformation were different phenomena, in agreement with the earlier conclusion by Huberman and Sachs (1966) with the hamster embryo cell system. I n our study there was an excellent correlation between the number of transformed colonies produced by a given hydrocarbon in this system and its in uivo oncogenic activity (Chen and Heidelberger, 1 9 6 9 ~ ) .

Fro. 5. Photographs of fixed and stained dishes of confluent prostate fibroblasts. The dish on the left is a control, and the monolayer of lightly stained cells is only faintly seen. The dish on the right was treated with methylcholanthrene, and distinct transformed colonies are seen (Chen and Heidelberger, 1969~).

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FIQ.6. Photomicrograph of a monolayer area of the MCA-treated dish in Fig. 5. The appearance of untreated or solvent control dishes is identical (Chen and Heidelberger, 19690).

It now became possible to determine whether polycyclic hydrocarbons directly transform nonmalignant into malignant cells, or whether, as Prehn (1964) has postulated, they selected for preexisting malignant cells. By means of the use of small fragments of cover slips it was possible to plate and detect single cells from prostate lines; these were placed in individual dishes and gave rise to clones with 72% efficiency. When treated a t the single-cell stage with the proper concentration of MCA, 100% of the cultures eventually piled up without appreciable toxicity. This high rate, coupled with the high cloning efficiency, excludes a selective mechanism for the oncogenic transformation in this system. By conducting other experiments in which the descendants of the single cell were recloned twice a t random, it was possible to show that all their progeny were potentially transformed, although for reasons still not understood they expressed the transformed state at different times (Mondal and Heidelberger, 1970). It is well known that most hydrocarbon-induced sarcomas exhibit ccllsurface tumor-specific transplantation antigens (TSTA) that are individual and non cross-reacting (Prehn, 1968). We wished to determine

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FIQ.7. Photomicrograph of the edge of one of the piled-up transformed colonies in the MCA-treated dish in Fig. 5. The deeply stained array of cells and their crisscross orientation in the transformed colony is evident and contrasts with the strict monolayer orientation of the cells outside the transformed colony (Chen and Heidelberger, 1969~).

whether our model system behaved comparably in this respect. Prostate cells were treated with MCA and individual transformed clones were isolated and tested for antigenicity by classical transplantation techniques. The majority of the clones tested were highly antigenic. When pairs of antigenic transformed clones isolated from the same dishes were tested reciprocally, they were found not to cross react (Mondal e t al., 1970). Thus, the model mimics the antigenic behavior of the chemically induced tumors in mice. It was then possible to take the analysis further than had been possible in vivo and ask whether multiple transformants produced with MCA from individual clones had common or individual TSTA’s. Using the techniques of lymphocyte-mediated colony inhibition and indirect immunofluorescence it was found that there was no significant cross-reactivity when 23 transformed colonies, all derived from a single clone, were compared against each other. However, some spontaneously transformed and malignant clones were not significantly antigenic. Hence, this cell-surface antigenicity is not essential for malignancy

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(Embleton and Heidelberger, 1972). It has since become possible to test clones of tumors induced in vivo for cross-reactivity, and the same result was obtained (Basombrio and Prehn, 1972). The mechanism that accounts for this great antigenic diversity remains an enigma. By using some of the techniques described above, it has been possible to select less malignant “revertant” lines from thrice-cloned highly malignant prostate cell clones. These “revertants” had either more or fewer chromosomes than the parent malignant clone, and had also lost their TSTA (Mondal et al., 1971). The use of the prostate cell system to elucidate a mechanism of metabolic activation of polycyclic hydrocarbons will be described below. At this point it is appropriate to compare and contrast the advantages and disadvantages of the two most widely used systems for the quantitative study of chemical oncogenesis in cultures. The hamster embryo cell system has the advantage that the cells are diploid, whereas the prostate cells are aneuploid, and that they can be scored after about 10 days both for toxicity and transformation, whereas the prostate cells can only be scored for transformation after several weeks. On the other hand, the prostate cells are advantageous in that they clone efficiently, and clones of non-transformed cells can be compared with multiple clones of transformed cells derived therefrom with respect to any given biological or biochemical property (as illustrated above for TSTA’s) ; this cannot be done with the hamster embryo cells because the normal cells have a short lifespan. V. 3T3-Like Systems

The 3T3 cell lines (Todaro and Green, 1963; Aaronson and Todaro, 1968) derived from mouse embryo cells are highly contact-sensitive and have been used for a wide variety of studies of oncogenic viral transformation. A thorough quantitative study of the chemical transformation of clones derived from the original BALBJc 3T3 line (Aaronson and Todaro, 1968) has been carried out by DiPaolo et al. (1972b). They obtained morphological and malignant transformation of suitably characterized clones with several oncogenic hydrocarbons, MNNG, N acetoxy-AAF, and aflatoxin B,. They presented dose-response curves that indicate a direct one-hit mechanism of transformation with hydrocarbons, and found that transformed clones had higher growth rates and saturation densities than the untreated controls. Thus, this represents a third quantitative system to study chemical oncogenesis in culture. We have also described in detail the development of new cloned 3T3like cell lines from embryos of the C3H, AKR, and C57JB1 strains (Reznikoff et al., 1973a). One of these from C3H mice is so contact-inhibited

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that it is termed 10T1/2. Properly selected clones of these cells have a remarkably flat background and undergo malignant transformation on treatment with polycyclic hydrocarbons (Reznikoff et al., 1973b). Many parameters affecting transformation have been investigated in detail in this system, and its advantages and disadvantages are being contrasted with the prostate cell lines. A striking cell-cycle phase-dependency for transformation has been discovered with the 10T1/2 cells. When they are synchronized by amino acid deficiency and restoration or by excess thymidine, and are treated a t various times with a short-lived oncogenic chemical, MNNG, there is a strikingly higher incidence of transformation when the chemical is added a t the time of release from the block, prior to the onset of DNA synthesis (Bertram and Heidelberger, 1973). The implications of this finding are under further investigation. VI. Other Cell Systems

A number of other cellular systems have been used to study chemicaI oncogenesis. These, excluding liver cells, will be described here. Borenfreund et al. (1966) treated a long established line of Chinese hamster lung cells with various polycyclic hydrocarbons and observed the production of chromosomal abnormalities. Cultures derived from treated cells produced a slightly higher incidence of tumors in the cheek pouch of Syrian hamsters than the untreated controls. Sanders and Burford (1967) treated the same cells with NMU, which produced considerable toxicity, whereas DMNA and methylurea (MU) did not. NMU also gave rise to piled-up foci that maintained this growth characteristic after further passages; no such colonies were found with DMNA or MU. Cells from the MNU-treated colonies gave rise to tumors in hamster cheek pouches. This work has been extended (Sanders and Burford, 1968) to test the response to MNU of many other cell lines of diverse origins, including those transformed by oncogenic viruses. They found great differences among the cells in their susceptibility to toxicity; in general transformed cells were more susceptible. They also observed a number of morphological effects produced by NMU, including “hyperconversion” of transformed colonies into a more piled-up state, and reversion to a flatter colony. Sanders (1972) has recently proposed a series of quantitative criteria to characterize transformation in culture, by a sophisticated analysis of dose-response curves. Frei and Oliver (1971) showed that NMU accelerated the spontaneous transformation of primary mouse embryo cell structures. Inui et al. (1972) treated primary cultures of lung cells obtained from newborn Syrian hamsters with MNNG and obtained morphological transformation

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and chromosomal aberrations within 2 7 4 8 days after treatment; these cells could not induce tumors in hamster cheek pouches until after 160 days in culture. It is not clear whether the apparent dichotomy of morphological and malignant transformation may be due to the antigenicity of the transformed cells in non-inbred hosts. Gutmann et al. (1972) reported the malignant transformation of rat embryo fibroblasts in mass cultures by N-acetoxy-AAF. However, the system has not yet been quantitated. Chemical transformation of cells infected with oncogenic viruses will be considered below. Sivak and Van Duuren (1968) have carried out studies of cocarcinogenesis in culture. They reported that they got inconsistent results in attempting to transform mass cultures of hamster embryo fibroblasts with B P and DMBA, and obtained no transformation by these compounds of 3T3 cells, although these and croton oil did produce some toxicity. These workers found (Sivak and Van Duuren, 1970a) that cocarcinogenic phorbol esters enhanced the outgrowth in mixed cultures of SV40-transformed 3T3 cells with a large excess of contact-inhibited 3T3 cells. With this as a quantitative assay, they found promoting activity in extracts of tobacco leaf and other cocarcinogenic chemicals. This test is complicated when the compounds are toxic; such toxicity tends to mask the effect on the outgrowth. Sivak and Van Duuren (1970b) also found that cocarcinogenic phorbol esters stimulate nuclear RNA synthesis in 3T3, but not in virally transformed 3T3 cells. There is a t present to my knowledge only one report on attempts to transform human cells in culture. Dietz and Flaxman (1971) exposed outgrowths of human epidermis to MCA and BP. Although there was inhibition of the outgrowth, there were no striking morphological changes in the treated or the cont.ro1 cultures. Clearly, much more work needs to be done with human cells, taking into account knowledge of metabolic activation (see below). VII. liver Cell Systems

There is a voluminous and controversial literature on the subject of growing differentiated liver cells in culture and the question of their reality as parenchymal cells. I n this section attention will be restricted to those studies that are directly concerned with chemical oncogenic transformation of liver cells in culture. The pioneer in this field is Katsuta and his group in Tokyo. In 1963 they described (Katsuta and Takaoka, 1963) the cultivation of rat liver cells; from adult rats the cells were maintained for considerable time without proliferation. When 4dimethylaminoazobenzene (DAB) was added to these stationary cultures, there was an induction of proliferation of parenchyma-like cells, and a

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number of lines were obtained. However, inoculation of these cells into immunosuppressed rats failed to give rise to any tumors (Katsuta and Takaoka, 1965a). It was possible to produce a second stimulation of growth rate with DAB, but again no tumors could be induced from the cells so treated (Katsuta and Takaoka, 1965b). They also obtained spontaneous morphological transformation of these cells (Katsuta et al., 1965). Malignant transformation of these cells was achieved, however, by treatment with NQO (Yamada et al., 1972), although no information was presented as to the histological diagnosis of the tumors induced from those cells. Katsuta and Takaoka (1972) have discussed the fact that many of the criteria normally used for the characterization and detection of malignant transformation in fibroblasts do not apply to epithelial cells; they have suggested that a search for different criteria be instituted. Toyoshima et al. (1970) exposed to aflatoxin B, a proliferating culture obtained from newborn Wistar rat liver. The original culture contained mostly epithelial cells with a few fibroblasts. However, after treatment there was a proliferation and piling-up of fibroblasts in the culture, which on inoculation produced fibrosarcomas. Thus, hepatic parenchymal cells were not transformed in this mixed culture. With the aim of developing a system for oncogenic transformation of rat liver cells in culture, Iype (1971) has carefully described and characterized epithelial cells that grow in monolayer following their preparation from adult livers by a collagenase-hyaluronidase perfusion. These cells retain their epithelial morphology on cultivation, have a normal karyotype, and do not pile up when a monolayer is attained. Further work is continuing to characterize these cells enzymatically. The occurrence of liver-specific cell-surface antigens was demonstrated in freshly isolated cells, and these antigens were maintained for more than 6 months in culture. Evidently the trypsin dispersion used for subculturing did not affect these surface antigens (Iype et al., 1972). Borek (1972) has described in detail the preparation of a cloned line of rat hepatocytes with a generation time of 28 hours, a diploid karyotype, and that grows in contact-inhibited monolayers. I n cultures that had not been refed adequately, foci of less cuboidal cells piled up, leading to multilayered cultures that, unlike the parent lines, grew in soft agar, and were agglutinated by concanavalin A. The karyotype of these spontaneously transformed cells was aneuploid (Miller et al., 1972). There has been no publication on the production of tumors by inoculation of these transformed cells into rats. However, Oshiro et al. (1972) have reported that an aneuploid line spontaneously derived from cultured adult rat liver cells gave rise to carcinomas on injection into isologousi.ats. The cells retained some liver-specific enzymes, and the production of carcinomas confirms that these cell lines are really epithelial.

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Williams et al. (1971) described the preparation of epithelial cells from 10-day-old Fischer rat livers by taking advantage of their stronger adherence to the culture surface than the contaminating fibroblasts. These cells did not produce tumors on inoculation into isologous rats. However, when they were treated repeatedly in mass cultures with aflatoxin B,, NMU, N-OH-AAF, and DMBA the cultures eventually did produce carcinomas on injection into rats, even though no distinct morphological transformation was observed in the cultures (Williams et al., 1972). Although a t the time of this writing these results are only available in abstract form, it appears that this is the only system involving epithelial liver cells in which chemical oncogenesis has been achieved with the production of unequivocal carcinomas. It is evident from these various studies, however, that new criteria of transformation need to be elucidated for epithelial cells. VIII. Combined Effects of Chemicals and Oncogenic Viruses

It has been proposed that the genetic information for cancer is present in “oncogenes” in all mammalian cells and that the information for vertically transmitted C-type RNA oncogenic viruses is carried in the eukaryotic genome as “virogenes.” Oncogenesis, consequently has been visualized as the derepression of the “virogene,” the “oncogene,” or both, and it has been further proposed that chemicals produce oncogenesis by activating or derepressing either or both of the above genes (Huebner and Todaro, 1969; Todaro and Huebner, 1972). Consequently, it has been of interest to investigate interrelationships between chemical and viral oncogens. Freeman et al. (1970) treated mass cultures of a line of rat embryo cells for 21 days with diethylnitrosamine (DENA) and continued to subculture the cells ; howcver, no morphological transformation was observed. When this line was infected productively with the Rauscher leukemia virus no morphological transformation was seen. However, when the infected mass cultures were treatcd with DENA, 23 of 28 showed morphological transformation to aneuploid cells ; however, no tumors were produced on inoculation into isologous rats. Precisely the same experiment was repeated using MCA instead of DENA, and the same result was stated to have occurred without any quantitation, even of the number of cultures examined (Price et al., 1971). The same experiment was repeated using DMBA, and the same result, again expressed without quantitation, was obtained. Moreover, although the transformed cells gave no detectable group-specific viral antigens by complement fixation tests, tumors obtained in rats by inoculation of transformed cells did exhibit these antigens. This host effect is difficult to interpret in mechanistic terms (Rhim et al., 1971b). The same type of nonquantitative experiment

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was reported again, this time using B P and extracts of particulate matter from smog. Morphological transformation was only obtained by these substances in the virally infected cells, and no tumors were induced on inoculation. In this study, hamster embryo cells were transformed morphologically by BP but not by smog. Virally infected cells were transformed by both (Freeman e t al., 1971b). Similar studies have been carried out with early passage N I H Swiss mouse embryo cells. MCA treatment produced toxicity, but no morphological transformation in these mass cultures, a result not in accord with those of other groups that have reported effects in comparable cells. HOWever, when such cells were infected with the AKR mouse leukemia virus, morphologically altered permanent lines were obtained that gave rise to fibrosarcomas on inoculation (Rhim e t al., 1971a). A comparable study using extracts of smog, and also reported only in qualitative terms, gave similar results (Rhim et al., 1972b). Another series of purely quaIitative experiments with a line of rat embryo cells infected with the Rauscher leukemia virus showed that oncogenic transformation was produced only by ( - ) 4rans-A-9-tetrahydrocannabinol among the several cannabis metabolites tested (Price et al., 1972b). When mass cultures of early passage hamster embryo cells were treated with MCA OF cigarette smoke condensate fractions, they became morphologically transformed, and on much later passages produced sarcomas in newborn hamsters. While these cells and the corresponding untreated controls were negative for hamster leukemia virus by complement fixation tests, the tumors they induced and cell lines obtained from these tumors were positive a t low dilutions. Moreover, these cells incorporated tritiated uridine into a fraction with a density of 1.16 gm/cm2, indicating the presence of virus particles. Interpretation of whether or not these chemicals “switched on” the virus is complicated by this host effect (Freeman et al., 1971a). A further complication of this type of work is that spontaneous oncogenic transformation with concomitant “switching on” of C-type RNA viral group-specific antigens has been detected in long passaged uninfected rat embryo cells (Rhim et al., 1 9 7 2 ~ ) . In the above-mentioned studies, polycyclic hydrocarbons, such as BP, did not produce transformation in uninfected early passage rat embryo cells. The same was true for long-passaged cells, both rat and mouse. As previously, rat and mouse cells infected with the Rauscher virus were transformed by BP. However, in later passage uninfected hamster embryo cells oncogenic transformation was achieved (Rhim et al., 1972a). When the sequence of treatment of rat embryo cells with MCA and virus was studied, it was reported in purely qualitative terms that no morphological transformation occurred in cells that were untreated, given MCA

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alone, given Rauscher virus alone, or treated with MCA first and then with the virus added three subcultures later. However, transformation was obtained in cultures treated first with the virus and then with MCA added from 3 hours to 3 weeks later. No interpretation of this marked timing effect was offered (Price et al., 1972a). A possible explanation might be that treatment of the cells with MCA damages the DNA (which is known) and thus facilitates integration of the horizontally transmitted viral genome. Comparable investigations have been carried out on the interrelationships between transformation produced by chemicals and DNA oncogenic viruses. Casto et al. (1972) have shown that oncogenic transformation of hamster embryo cells by adenovirus SA7 is quantitatively enhanced when they are treated with oncogenic chemicals immediately prior to viral infection. Treatment with the chemicals alone did not produce the transformation, which was scored in such a way as to establish that the virus produced the transformation. The results were interpreted that the chemical facilitated the fixation of the viral genome responsible for transformation. On the other hand, it has been found that DMBA and MCA added to 3T3 cells 24 hours after infection with SV40 significantly inhibited oncogenic transformation by the virus (Docherty et al., 1972). It is evident that one of the major questions about the mechanisms of chemical oncogenesis is concerned with whether chemicals can transform cells directly or act somehow through the intervention of an oncogenic virus or its informational precursors. The above-mentioned as yet nonquantitative research sheds little light on this important question except to show that there are some sorts of additive effects between chemical and viral oncogens in the morphological and (sometimes) malignant transformation of rodent fibroblasts and that under some circumstances viral antigens appear in tumors induced from inoculation of antigen-negative transformed cells. Clearly, more critical and quantitative experimentation is required to elucidate these relationships. IX. Metabolism of Polycyclic Aromatic Hydrocarbons

Before turning to the subject of metabolic activation of chemical oncogens, we shall consider what was known about their metabolism prior to the elucidation of their metabolic activation. Although most of the original work has been done in whole animals and various liver preparations, we shall concentrate on those studies done with cultured cells. The pioneering research in this field was carried out by Boyland and Sims, who found that the metabolism is comparable whether studied in intact rats, rat liver homogenate, or rat liver microsomes. They showed (Boyland and Sims, 1964) that benz [ a ]anthracene (BA) is metabolized

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in rats primarily at the 5,6-position or K-region (Pullman and Pullman, 1955, 1969) to give the trans-dihydrodiol, the phenol, and the glutathione conjugate and mercapturic acid. When BA and DBA were hydroxylated by rat liver homogenates, the main products were also the K-region phenol, trans-dihydrodiol, and glutathione adduct (Boyland and Sims, 1965b). When DMBA metabolism was studied in rat liver homogenates, the main products were the two isomeric hydroxymethyl compounds, showing that metabolism occurred primarily on the methyl groups (Boyland and Sims, 1965a). Sims (1967a) showed that both 7- and 12-methyl-BA’s were converted by rat liver homogenates into the K-region phenol, dihydrodiol, and glutathione conjugates, the corresponding hydroxymethyl compounds, and also into the non-K-region, 8,9-dihydrodiol. Further studies of the metabolism of 7-Me-BA, DMBA and their hydroxymethyl derivatives were carried out in liver and adrenal homogenates (Sims, 1 9 7 0 ~ )The . same products as mentioned above were identified by thin-layer chromatography; the adrenal homogenate gave primarily ring-hydroxylated products. Large increases in the amounts of those metabolites were obtained from the livers, but not adrenals, of rats pretreated with MCA. The metabolism of BP by rat liver homogenates gave rise primarily to various quinones and phenols, and two dihydrodiols tentatively identified as the 1,2 and the 7,8 compounds, neither of which is located on the K-region (Sims, 196713). MCA was metabolized by rat liver homogenates to a variety of compounds identified as K-region dihydrodiol and glutathione adduct, as well as oxidations on the carbocyclic ring (Sims, 1966). The above-mentioned studies were all essentially qualitative, and consequently an attempt was made, using labeled compounds and thin-layer chromatography, to quantitate the metabolism of various polycyclic hydrocarbons in rat liver homogenates (Sims, 1970b). In addition to metabolism a t the K-region, DBA was found to be converted into dihydrodiols a t the 1,2 and 3,4-positions ; dibenz [ a , ~anthracene ] (whict lacks a K-region) gave a dihydrodiol a t the 10,ll-position; B P led to diols a t the 1,2 and 9,lO positions, and RlCA and DMBA were as previously mentioned. However, these compounds that have been identified are minor metabolites; the major amount of radioactivity was in the form of unidentified water-soluble metabolites (Sims, 1970b). Finally, Sims (1970a) studied the metabolism of some of the same hydrocarbons in cultures of mouse embryo cells and found it to bc quite similar to that of rat liver homogenates; again the major radioactivity was found in uncharacterized water-soluble derivatives. Although it had been shown (Morimura et al., 1964) that various tumor cell lines in culture accumulated polycyclic hydrocarbons,

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Andrianov e t al. (1967) carried out the first major study of hydrocarbon metabolism in cultured cells. They studied spectroscopically the disappearance of B P from mouse, hamster, and human embryo secondary cultures and from various transformed cell lines. The highest rate of disappearance was found in the embryonic mouse and hamster fibroblasts, those cells that were most susceptible to the toxicity of BP. The metabolism of tritiated DMBA to water-soluble substances in various cells has been investigated by Diamond et al. (1968). The oncogen exerted much greater toxicity to hamster embryo and mouse embryo cells and was converted by them into water-soluble compounds to a much greater extent than was the case with a spontaneously transformed hamster cell line and a human diploid cell line; HeLa cells were intermediate with respect t o toxicity and metabolism. The water-soluble metabolites were taken up only very slowly by the cells. It was also found that the hydroxymethyl derivatives were much less cytotoxic than DMBA. Diamond (1971) found that B P and DhlBA were metabolized to watersoluble derivatives only by those cells subject to cytotoxicity. The kinetics suggested that the hydrocarbons were metabolized first to alkali-extractable materials (probably phenols) before conjugation to the water-soluble compounds. The extent of metabolism was dependent on the concentration of the hydrocarbon, the time of incubation, and the number of cells present. Duncan e t al. (1969) incubated 8 tritiated hydrocarbons with monolayers of primary mouse embryo cells and studied the time course of their disappearance. At low concentrations all the compounds, whether oncogenic or not, were metabolized a t about the same rates. Duncan and Brookes (1970) also incubated primary mouse embryo cultures with labeled BP, and found that at low concentrations its disappearance was exponential with time, but at higher levels the rate progressively declined. The metabolism of the oncogenic DBA and that of its less oncogenic isomer DB[a,c]A were compared in mouse embryo cell cultures; the former had a lower rate of metabolism (Duncan and Brookes, 1972). Furthermore, it was found that the rate of disappearance of B P in primary cultures obtained from embryonic human lung was comparable to that found in mouse fibroblasts, and that the human fibroblasts maintained the same rate of metabolism after a number of subcultures (Brookes and Duncan, 1971). Huberman e t al. (1971) studied the conversion to water-soluble metabolites of several polycyclic hydrocarbons not only in mouse embryo cells, but also in transformable and chemically transformed prostate fibroblasts. The metabolism was dose- and cell numbcr-dependent, and there was a greater extent of metabolism in mouse and hamster embryo

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cells than in the prostate cells, which in turn carried out more conversion to water-soluble derivatives than did transformed clones or lines derived from tumors obtained from chemically transformed cells. I n all cells investigated, the rate of production of dihydrodiols was higher than for phenols and water-soluble compounds. I n hamster embryo cells the following hydrocarbons were metabolized with decreasing rates: BP, MCA, DMBA, DB[a,c]A, and DB[a,h]A. Thus, there was no correlation between oncogenic activity and metabolism in this series of hydrocarbons. X. Binding to Cellular Macromolecules

It has been axiomatic since the time of Paul Ehrlich that in order for a foreign compound to produce a physiological effect it must interact with the target tissue. It now appears clear in all cases that have been properly studied that there is a covalent binding of the chemical oncogen to the informational macromolecules, DNA, RNA, and proteins of target cells, and it is assumed that one (or more) of those interactions is responsible for the initiation of oncogenesis (Miller, 1970). It is presently unknown which of these macromolecules is the primary target of chemical oncogens. With azo dyes and AAF, the covalent binding to proteins has been known for a long time (Miller, 1970). With polycyclic hydrocarbons it was shown that there was covalent binding specifically related to oncogenic activity to a certain soluble protein fraction of mouse skin (Abell and Heidelberger, 1962) , which has subsequently been partially purified (Tasseron et al., 1970). The binding of polycyclic hydrocarbons to this same h-protein in cultured transformable (but not transformed) cells has also been demonstrated (Kuroki and Heidelberger, 1972). The protein to which azo dyes and polycyclic hydrocarbons are bound in rat liver has been found to be identical with the protein to which a number of anionic compounds and metabolites of steroids are bound, either covalently or ionically; this protein has been called “ligandin,” and its physiological role is under intense investigation (Litwack et al., 1971). It was proposed that this protein may transport bound oncogens from the cytoplasm to the nucleus (Abell and Heidelberger, 1962). The covalent binding of polycyclic hydrocarbons to mouse skin DNA has been studied rather intensively (Brookes and Lawley, 1964; Brookes, 1966 ; Goshman and Heidelberger, 1967; Brookes and Heidelberger, 1969; Lesko et al., 1971; Marquardt et al., 1971). There seems to be a fairly good correlation between the amount bound to DNA and the other macromolecules and the oncogenic activity of the hydrocarbons. Just how good

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is the correlation with binding to DNA has been a matter of some disagreement. The structure of the hydrocarbon derivative (s) bound to DNA has not yet been elucidated, nor has the base(s) to which they are bound yet been identified. Although the fact of covalent binding strongly suggests that some derivative of the hydrocarbon must be bound, some evidence has been provided that DMBA may be bound to mouse skin DNA without change in structure (Marquardt et al., 1971). There have been many studies of the binding of polycyclic hydrocarbons to DNA, RNA, and proteins of cultured cells. Following the observations that B P and MCA exerted toxicity to normal embryonic cells of several rodent species but not to spontaneously or virally transformed cells, and the lack of toxicity they produced to several primate or human cells (Diamond, 1965), a study of the binding was undertaken. Diamond et aZ. (1967) found that the binding of tritiated DMBA to DNA, RNA, and protein occurred to a much greater extent in embryonic rodent cells than in transformed rodent cells and nontransformed and transformed human cell lines. This binding correlated closely with the cytotoxicity that DMBA produced in these cultures, and was not related to the cellular uptake of total hydrocarbon. Rasmussen et al. (1972) found that with repeated passages, hamster embryo cells lost their ability to convert B P into soluble products and to bind it to acid-insoluble components. Treatment of the late passage cells with vitamin A led to a restoration of the binding of BP. As mentioned above, Duncan et al. (1969) studied the rates of disappearance of a series of tritiated polycyclic hydrocarbons in mouse embryo cells. They also studied the binding of these hydrocarbons to DNA, RNA, and total proteins, and expressed the results in terms of a binding index, which was defined as the amount of hydrocarbon bound divided by the amount metabolized. I n general, the binding indices decreased in the following order: DMBA, MCA, 7-Me-BA, BP, DB[a,h]A, DB[a,c]A, benzo [elpyrene, and BA; this correlates quite well with their oncogenic activities. It would have been interesting to correlate these binding results with the ability of the compounds to transform the cells, but the use of primary mouse embryo cells has not been successfully adapted t o oncogenesis in culture because of the rapid onset of spontaneous transformation. In a study of the metabolism and binding of BP, it was found (Duncan and Brookes, 1970) that a t low concentrations the binding was proportional to overall metabolism, but a t higher levels the binding reached a plateau, resulting in a fall in the binding index. There has been some disagreement in the past whether, both on mouse skin or in cultures, the powerfully oncogenic DB[a,h]A is bound to DNA

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to a greater or lesser extent than the very weakly oncogenic isomer, D B [a,c]A (Brookes and Lawley, 1964; Goshman and Heidelberger, 1967; Duncan and Brookes, 1970; Kuroki and Heidelberger, 1971). A careful comparison of the metabolism and binding of these two compounds was carried out in mouse embryo cells. At very low concentrations, the binding index of the DB[a,h]A was considerably higher than that of D B [a,c]A, in accord with their oncogenic activities (Duncan and Brookes, 1972). It was also found that the binding indices of B P to the macromolecules of human and mouse embryonic fibroblasts were similar (Brookes and Duncan, 1971). The covalent binding of labeled DMBA to the preexisting and newly replicated DNA of fetal mouse skin cells was studied, using bromodeoxyuridine-labeled cells and isopycnic centrifugation. It was found that the binding was slightly higher to the nonreplicating DNA (Yuspa et al., 1969-1970). The same was found for BA, although the extent of binding was considerably less than had been found with DMBA (Yuspa and Bates, 1970). The relevance of all the above binding studies t o the process of oncogenic transformation is somewhat questionable, since the cells used have not been successfully applied to oncogenesis in culture. However, binding studies have been carried out in transformable and transformed cells. Kuroki and Heidelberger (1971) studied the binding of several polycyclic hydrocarbons to DNA, RNA, and total proteins of transformable hamster embryo and prostate cells. With both cells the binding of DMBA was highest to DNA, but relatively low to proteins; B P was bound to proteins to the highest extent. There was very little binding to DNA of both DBA’s, but an appreciable binding to protein. There was much less macromolecular binding in chemically and spontaneously transformed clones. It was also discovered that in nondividing cultures the binding to DNA was persistent over a period of weeks in prostate cells, and that the binding of MCA to DNA in dividing and nondividing cells was about the same. Finally, it was found that the binding of MCA to the DNA of hamster embryo and prostate cells was of about the same magnitude as to the DNA of mouse skin following topical application (Kuroki and Heidelberger, 1971). Connell et al. (1971) studied autoradiographically the incorporation of tritiated DMBA into the nuclei of hamster embryo cells in culture. The incorporation was linear with time, and although hydroxyurea inhibited the incorporation of tritiated thymidine, it did not affect the binding of DMBA. This provides further evidence that DNA synthesis is not required for the binding of oncogenic hydrocarbons ; the same thing was observed when DNA synthesis was blocked with excess thymidine.

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The binding of NQO to transformable hamster embryo cell cultures has been investigated by Kuroki et al. (1970), who found that the labeled compound was quickly bound to acid-insoluble material ; this binding persisted for 72 hours after addition and was not inhibited by hydroxyurea. The binding of NQO to DNA, tRNA, and rRNA in two transformed cell lines was determined, and the kinetics reported (Andoh et al., 1971). XI. Metabolic Activation of Polycyclic Hydrocarbons

It is clear from the foregoing that polycyclic hydrocarbons bind covalently to DNA, RNA, and proteins, yet the hydrocarbons themselves do not react in the test tube with these macromolecules. Therefore, it is necessary that they be converted metabolically into chemically reactive products. This concept has been put forth by the Millers (Miller and Miller, 1966; Miller, 1970), who have brilliantly elucidated the mechanism of metabolic activation of aminoazo dye and acetylaminofluorene carcinogens to proximal and ultimately reactive forms. The generalization that is now accepted is that all chemical oncogcns that are not themselves chemically reactive must be converted metabolically into a form that reacts as an electrophile (secks out negatively charged groups to react with, and hence behaves as if it were positively charged). With respect to polycyclic hydrocarbons, the break in the elucidation of metabolic activity came simultaneously and indcpendently whcn it was shown that incubation of labeled BP and several other hydrocarbons with liver microsomes in the presence of oxygen and NADPH followed by the addition of DNA, led to the covalent binding of the hydrocarbons to the DNA; evidently metabolic activation occurred in this system (Grover and Sims, 1968; Gelboin, 1969). This microsomal system is the nonspecific drug metabolizing system that is involved in the detoxication of many foreign compounds and which carries out mainly hydroxylations and oxidative demethylations. The system is variously known as mixedfunction oxidase, aryl hydrocarbon hydroxylase (AHH), henzopyrene hydroxylase, etc. It is a terminal oxidase in which molecular oxygen is utilized by cytochrome P450 and its close relatives. It is also inducible, i t . , pretreatment of the system with polycyclic hydrocarbons, phenobarbital, or related compounds leads to a net increase in activity of the enzymes, which are bound to the membranes of the cndoplasmic reticulum. This system has generated an enormous literature that cannot be dealt with here ; two reviews from the pharmacological viewpoint have been written by Conney (1967, 1972). The activity of this system is highest in liver, but it has been detected in most other tissues. Moreover, the presence and induction of this enzyme system has been dcrnonstrated in various cultured cells (Nebert and Gelboin, 1968), and aryl hydro-

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carbon hydroxylase activity is under strict genetic control as a simple autosomal dominant trait, as demonstrated in several mouse strains (Nebert and Gielen, 1972). The participation of this microsomal system has been demonstrated when MCA was bound to soluble proteins in explants of fetal liver (Burki et al., 1972). It has also been found that p-naphthoflavone (7,8-benzoflavone, 7,S-BF) inhibits the conversion of BP and MCA into water-soluble metabolites, protects against cytotoxicity in hamster embryo cells, and inhibits AHH activity in liver microsomes of preinduced rats (Diamond and Gelboin, 1969). Furthermore, 7,8-BF inhibits AHH activity in the liver microsomes of methylcholanthrene, but not phenobarbital-treated rats; this indicates that there are two forms of AHH activity that can be differentially induced in rat tissues (Wiebel et al., 1971). As mentioned above, the initially formed metabolites of several polycyclic hydrocarbons are K-region trans-dihydrodiols, phenols, and glutathione conjugates. In 1950 Boyland suggested that these products could be accounted for if an epoxide (arene oxide) were an intermediate (Boyland, 1950), as shown schematically for BA in Fig. 8. The conversion by rat liver homogenates of synthetically prepared epoxides of BA and DBA into trans-dihydrodiols phenols, and glutathione conjugates was

HO

Phenol

Dihvdrodiol

0 -Glucuronide Conjugot as

-sulfate

FIO.8. Scheme of the metabolism of benzCalanthracene.

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shown by Boyland and Sims (1965b). Since epoxides are chemically reactive, it seemed possible that they might also be the metabolically active form of polycyclic hydrocarbons. However, it was found that several epoxides were less carcinogenic in mice than the parent hydrocarbons (hliller and Miller, 1967), whereas it had been previously thought that proximal and ultimate carcinogens should be more active than the parent compounds (hliller, 1970) . Nevertheless, because of the high chemical reactivity of the epoxides, it seemed likely that they might react with and be used up by various nucleophiles on or beneath the skin before they could reach the presumed intracellular target. Several recent findings coincided to revive interest in epoxides as a metabolically activated form of polycyclic hydrocarbons. Other possible mechanisms of metabolic activation will be considered later. Jerina et al. (1970) demonstrated that naphthalene oxide is the intermediate in the microsomal hydroxylation of naphthalene, and they suggested that arene oxides are obligatory intermediates in such hydroxylation reactions. Much subsequent work from that group (which cannot be reviewed here) has substantiated that proposal. Grover and Sims (1970) demonstrated that the K-region epoxides of phenanthrene and DBA reacted in neutral solution a t 37°C with DNA, RNA, and histones to give covalently bound complexes, which the parent hydrocarbons and dihydrodiols did not. They postulated that epoxides might be the metabolically active form of hydrocarbons. Grover e t al. (1971a) also found that when labeled K-region epoxides of phenanthrcne, BA, DBA, and 7-Me-BA were added to cultures of BHK2l cells (a malignant line of hamster cells widely used for studies of viral transformation) they were bound to DNA, RNA, and proteins to a much greater extent than were the parent hydrocarbons, dihydrodiols, or phenols. If epoxides were to be established as metabolically activated derivatives of polycyclic hydrocarbons, it was first necessary to show that they are, in fact produced during metabolism. Using microsomal systems in which epoxide hydrase activity was inhibited by various means, it was possible to trap the epoxides of DBA (Selkirk e t aE., 1971), phenanthrene, and BA (Grover et al., 1971b). Furthermore, Grover et al. (1972) have detected epoxides as metabolites of pyrene and B P in a microsomal system. Subsequently, Sims (1971) has synthesized the 8,g-oxide (non-Kregion) of BA, which was converted by microsomes into the corresponding dihydrodiol and glutathione conjugate ; similarly, the 10,ll-oxide of D B [a,c] A was prepared and converted by microsomes into the dihydrodiol and glutathione conjugate (Sims, 1972). Since (see above) the epoxides were less carcinogenic in mice than

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the parent hydrocarbons, it became worthwhik to test their ability to produce malignant transformation in cell cultures. This was done in our laboratory in collaboration with Grover and Sims. It was found (Grover et al., 1971c) that epoxides were more active than the parent hydrocarbons, dihydrodiols, and phenols a t producing transformation of hamster embryo cells and of prostate cells. I n more detailed studies it was found (Huberman et al., 1972a) that in the hamster embryo fibroblast system the K-region epoxides of BA, DBA, 7-Me-BA1 and MCA were more active than the other derivatives and parent hydrocarbons a t producing transformation. The non-K-region (8,9) epoxide of BA was not significantly active a t producing transformation, nor was the chemically reactive model of methyl-group activation, 7-bromomethyl-BA that had been synthesized, and studied by Dipple and Slade (1970). The more oncogenic 7- bromomethyl-12-Me-BA produced some transformation, as did the epoxides of the nononcogenic chrysene and phenanthrene (Huberman et al., 1972a). A detailed study was carried out with a clone of prostate cells that metabolized hydrocarbons to water-soluble derivatives to a very slight extent (Marquardt et al., 1972), and their complete data are given in Table I. MNNG, which does not require metabolic activation, transformed this clone effectively, as did the K-region epoxides of BA, DBA, and MCA. The non-K-region (8,9) oxide of BA was not active, nor were the epoxides of phenanthrene and chrysene; the epoxide of 7-Me-BA was slightly active. Since not all epoxides produce transformation, it is necessary to initiate careful quantitative studies of structureactivity relationships, particularly with respect to rates of chemical reactivity and transformation ; such studies are now under way. Two hydrocarbons, both with methyl groups, were highly active a t transforming these cells, 7-MeBA and DMBA, whereas the model compounds, 7-bromomethyl-BA1 and 7-bromomethyl-12-Me-BA were not active. Thus, these studies are not revealing about the mechanism of metabolic activation of these metliylated hydrocarbons (Marquardt et al., 1972). Sims et al. (1973) incubated hamster embryo cells with labeled BA and DBA and their corresponding K-region epoxides, cis-dihydrodiols, and phenols. The epoxide of BA mainly yielded the corresponding dihydrodiol, whereas DBA epoxide was converted primarily to the phenol. The cis-dihydrodiols were not appreciably metabolized. Kuroki et al. (1971-1972) investigated the binding of epoxides and other derivatives of BA and DBA to the DNA, RNA, and proteins of transformable hamster embryo and prostate cells. In almost all cases the epoxide was bound to these macromolecules to a greater extent than the parent hydrocarbons and dihydrodiols. However, the phenol of DBA was significantly bound

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CHEMICAL ONCOGENESIB I N CULTURE

TABLE I TRANSFORMATION AND TOXICITY PRODUCED R Y POLYCYCLIC HYDROCARRONS AND THEIR DERIVATIVES"

Compound Control MNNG BA BA-5,gepoxide (K-region) BA-8,S-epoxide (non-K-region)

BA-&-5,6dihydrodiol (K-region) BA-truns-5,6-dihydrodiol (K-region)

5-OH-BA (K-region phenol) DBA DBA-5,6-epoxide (K-region) DBA-&-5,6dihydrodiol DBA-trans-5,6dihydrodiol

5-OH-DBA (K-region phenol)

MCA MCA-ll,l2-epoxide (K-region) MCA-cis-ll,12-dihydrodiol

Concentration (Pg/ml)

DMSO 0.5% 0.1 0.2 1.0 5.0 0.5 1.0 0.1 0.5 1.0 5.0 10.0 1.0 10.0 5.0 10.0 1.0 3.0 10.0 1.0 10.0 0.5 1.0 10.0 1.0 10.0 5.0 10.0 1.0 5.0 10.0 1.5 10.0 0.75 1.5 1.5 5.0 10.0 5.0 10.0

Plating efficiency

(%I 25 17 11 18 15 10 3.0 19 17 13 5.0 1.0 22 20 27 26 11

3.0 0 22 20 22 17 6.5 22 20 25 5.5 19 10 0 17 17 17 9.5 23 26 23 27 26

No. of transformed foci/No. of dishes 0/30 16/18 25/17 0113 O/lS 9/19 23/25 0/14 0/18 0/14 2/16 0/18 0/10 0/10 0/10 0/8 0/12

0/18 0/15 0/18 4/20 9/17 12/15 0/12 0/15 0/10 0/12 0/6 0/10 1/13 10/33 38/27 58/26 0/7 0/10 0/18 O/lO 0/12 (Continued)

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CHARLES HEIDELBERGER

TABLE I (Continued)

Compound 11-Hydroxy-MCA (K-region phenol)

Control MNNG Phenanthrene

Phenanthrene-9,lO-epoxide Chrysene

Chrysene-5,Bepoxide

Control MNNG 7-Methyl-BA 7-Methyl-BA-5,Bepoxide (K-region)

7-Bromomethyl-B A DMBA

7-Bromomethyl-12-methyl-BA a

Concentration (rg/ml) 1.0 2.5 5.0 Acetone 0.5% 0.2 1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0 Acetone 0.5% 0.2 0.1 10.0 0.01 0.05 0.1 0.2 0.1 0.2 0.1 1.5 10.0 0.1 0.2

Plating efficiency

(%) 21 13 2.5 24 13 24 20 11 13 1.5 0 22 22 22 17 1.0 0 23 13 21 13 20 17 14 4.0 13 5.5 18 17 10 18 15

No. of transformed foci/No. of dishes 0/8 8/16 0/10 0/20 12/13 0/12 0/12 0/12 0/12 0/15

0/10 0/12 0/12 0/15 0/18 0/18

12/16 5/20 9/17 0/10

4/15 1/10 0/10 0/18 0/20 3/10 6/10 20/19 0/18 1/19

After Marquardt et al. (1972).

to RNA and proteins, by a mechanism not yet understood. Furthermore, the epoxide of DBA was bound specifically to the h-protein of transformable cells to a greater extent than DBA (Kuroki and Heidelberger, 1972). The reason for this specific affinity for this protein is also not clear. The role of the microsomal mixed-function oxidase system in the metabolic activation of polycyclic hydrocarbons must now be examined in more detail, particularly since the system is ordinarily considered to carry out detoxications. Gelboin e t al. (1970) found that mouse skin

CHEMICAL ONCOGENESIS IN CULTURE

355

contains inducible AHH, which is inhibited by 7,8-BF. When the inhibitor was applied to the skins just prior to DMBA, there was a marked inhibition of tumorigenesis. This suggests that the microsomal system is responsible for the activation of DMBA. Kinoshita and Gelboin (1972a) also found that 7,8-BF inhibits the binding of DRlBA to mouse skin macromolecules. By contrast, 7,8-BF did not inhibit skin tumorigenesis by BP, nor its macromolecular binding. Thus, thc relevance of AHH in the metabolic activation of B P is not clear. Wattenberg and Leong (1970) also found that 7,8-BF inhibited the production of pulmonary adenomas and skin tumors in mice treated with BP. However, they interpreted this result as being due to the induction of AHH by the flavone; consequently they consider that the enzyme system functions primarily to detoxify BP. In a further study, Kinoshita and Gelboin (1972b) found that in order for 7,8-BF to inhibit skin tumorigenesis by DMBA, i t was necessary to apply the inhibitor within 12 hours of the oncogen, suggesting that the metabolic activation of DMBA is complete in that time. 7,8-BF also inhibited skin tumorigenesis by 7-hydroxymethyl-12-Me-BA. As mentioned above, AHH induction in mice is under genetic control. Nebert et al. (1972) found that skin tumorigenesis initiated in mouse skin by DMBA and promoted by phorbol esters was unrelated to genetic differences in AHH induction. This suggests that if AHH is responsible for the activation of DMBA, the constitutive level of the enzyme is sufficient for this purpose. The effects of inducers and inhibitors of AHH on oncogenic transformation in culture have been studied by RiIarquardt and Heidelberger (1972). They found that when the prostate cells were plated on an irradiated “feeder layer” of rat or mouse embryo fibroblasts that metabolized MCA, the transformation that it produced was enhanced, whereas that produced by MCA-epoxide was decreased. As shown in Table 11, when the prostate clone was pretreated with BA or diphenyloxazole (PPO) , the metabolism of MCA to water-soluble derivatives was increased, and so was the transformation by MCA; transformation by MCA-epoxide was inhibited. Treatment with 7,8-BF prior to the addition of MCA prevented its metabolism and transformation without affecting transformation produced by the epoxide (Marquardt and Heidelberger, 1972). DiPaolo et al. (1971~)carried out a rather similar study in the hamster embryo fibroblast system. Pretreatment of the cells with BA enhanced the transformation produced by B P and MCA. However, when they added 7,8-BF 24 hours before the chemical oncogen, AHH was induced and transformation was enhanced (DiPaolo et al., 1 9 7 1 ~ ) .However, prctreatment of hamster secondaries with BA was stated in purely qualitative terms to prevent DMBA-produced transformation (Benedict et al., 1972).

TABLE I1 EFFECT OF INDUCERS (BA, PPO) AND INHIBITORS (7,8-BF) OF MICROSOMAL MIXED-FUNCTION OXIDASES ON TOXICITY AND MALIGNANT TRANSFORMATION IN G23 CELLSTREATED WITH MCA AND THE K-REGIONEPOXIDE OF MCA. Plating efficiency (yo)of G23 cells treated with

No. of transformed foci/No. of dishes

MCA

MCA

Pretreatment

Formation of water-soluble productsb

Control, acetone, 0.5%

None BA (1 P M ) PPO (10 &) 7,8-BF (1.5 pg/ml)

36 61 60 5

23.5 26.5 23.5 20.0

b

pg/ml

10.0 pg/ml

K-region epoxide 1.5 pg/ml

Control, acetone, 0.5%

1.5 pg/ml

pg/ml

K-region epoxide, 1.5 pg/ml

20.0 21.5 18.0 22.0

20.0 14.0 15.0 23.0

11.5 11.5 11.0 5.0

0/19 0/17 0/10

2/18 8/7 20/16 0/9

7/18 15/8 37/19 Oil0

44/25 2/8 4/18 25/15

1.5

From Marquardt and Heidelberger (1972). Water-soluble products (in pmoles) formed in 24 hours by 1 W cells from 260 pmoles of tritiated MCA.

10.0

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CHEMICAL ONCOGENESIS IN CULTURE

If we assume that an epoxide represents one mechanism for metabolic activation (of a t least those hydrocarbons lacking methyl groups) in the AHH system, the results described abovc can be explained according to the scheme shown in Fig. 9. Considerable evidence has been presented (primarily with MCA) that induction of AHH incrcascs the hydrocarbonproduced malignant transformation of cell cultures and that inhibition by 7,8-BF prevents it. This shows that activation is carried out by the oxygenase in the microsomal AHH system. The epoxide so produced can react chemically with target macromolecules or be detoxified according to the pathways: rearrangement to the phenol, conversion by microsomal epoxide hydrase to a dihydrodiol, or conjugation by a soluble enzyme with glutathione. Since AHH induction also causes induction of epoxide hydrase activity, the inhibition of the epoxide-produced transformation in the induced cells is explained. Therefore, we can conclude that the AHH system is a two-edged sword, which carries out both metabolic activation and detoxification of polycyclic hydrocarbons. Whether the overall result of enzyme induction is to increase or decrease the noxious effects of the hydrocarbon probably depends on the balance of activities of the various enzymes depicted in this scheme. Much further research needs to be done on the separation, purification, and comprehension of this complicated enzyme system, before it will be possible to prevent cancer in populations exposed to carcinogenic risks by its manipulation (cf. Miller and Miller, 1971). Are there other possible mechanisms of activation of polycyclic hydrocarbons in addition to that involving epoxides? Dipple et al. (1968) have proposed that hydrocarbons such as DMBA are activated by metabolism on the methyl group to form a potential carbonium ion. The aboveBinding to macromolecules

Hydrocarbon

oxygenase E-

Epoxide

hydrase

*Mhydrodiol

soluble enzyme Phenol

Glutathione conjugate

Conjugates

FIG.9. Pathways of hydrocarbon metabolism.

I

Conjugates

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mentioned bromomethyl compounds react in the test tube with DNA, and 7-bromomethyl-12-methyl-BA is carcinogenic (Dipple and Slade, 1970) . Although DMBA sometimes appears to behave anomalously in activation experiments (see above), it has not yet been possible to establish that activation on a methyl group is responsible for its oncogenic activity. Another mechanism, that of formation of a radical cation by one-electron oxidation has been proposed and documented in purely chemical model systems, but has not so far been implicated in oncogenesis (Fried and Schumm, 1967; Wilk and Cirke, 1969). Finally, evidence has been presented that a free radical may be involved in the metabolic activation of BP. Nagata et al. (1967) demonstrated that when B P was added to a mouse skin homogenate an electron paramagnetic resonance (EPR) spectrum characteristic of a free radical was observed, which was increased in the presence of oxygen. It was then found that when B P was incubated with liver microsomes the free radical was seen, and the characteristics of the EPR spectrum indicated that it was the 6-phenoxy radical (Nagata et al., 1968). Inomata and Nagata (1972) showed that the 6-phenoxy radical was produced from B P by photoirradiation, and it was suggested that it was produced as a result of oxidation with singlet molecular oxygen. Lesko e t al. (1971) prepared 6-hydroxy-BP synthetically and found that it was bound covalently to DNA in the test tube without any additional activation; they postulated that the mechanism involved the phenoxy free radical. Finally, Nagata et al. (1971) stated that, on injection of 6-hydroxy-BP into mice, 2 sarcomas, 1 leukemia, and 1 lung adenoma were found in 50 mice, whereas in 30 controls 2 leukemias and 1 lung adenoma were found. Whether this demonstrates significant oncogenic activity of 6-hydroxy-BP will have to await further experimentation. I n summary, it seems likely to this author at this time that metabolic activation of BA, DBA, and MCA involves epoxide formation. In the case of BP, either an epoxide (Grover et al., 1972) or a free radical (Nagata et al., 1971), or both, may be the metabolically activated forins. At present the mode of metabolic activation of DMBA has not been elucidated. Xll. Conclusions

Among the many advantages of studying biological phenomena in cell cultures, rather than in intact animals, is the ability to control the environment, to do quantitative bookkeeping of the cells, to vary the exposure to chemicals, to synchronize the cells, and t o be free of the immunological milieu of the host. The progeny of individual cells can be studied with respect to the stability of their genetic, metabolic, and immunogenic

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properties. All these advantages have been applied to thc problcms of chemical oncogenesis, and much new information has been gained. It must be recognized, however, that these are model systems that must continuously be scrutinized and validated with respect to their relevance to “real life,” i.e., oncogenesis in animals and man. The quantitative cell culture systems that are now available involve the use of various sorts of fibroblasts; hence the tumors that the transformed cells produce on inoculation into isologous hosts are sarcomas. There is a great need to establish similarly quantitative systems for the malignant transformation of epithelial cells ; much effort toward this end is now being devoted to liver cells. I n the meantime, before true chemical oncogenesis in epithelial cells is accomplished, the study of the morphological effects of oncogenic chemicals on the epithelia of organ cultures is of considerable interest. Over all the cell culture systems, however, hovers the specter of spontaneous transformation, which has precluded the use of most mouse embryo fibroblasts, and makes it essential to use untreated control cultures in all experiments. Much fundamental information on the cellular mechanisms of chemical oncogenesis has already been derived from studies in culture. It is now generally agreed that the chemical oncogen directly transforms nonmalignant t o malignant cells and does not select for preexisting malignant cells. It appears that this direct transformation may be a “one-hit” phenomenon. I n the three most widely used quantitative systems, there has been an excellent correlation between the oncogenic activity of diverse chemicals and their ability to produce transformation in cultures. I n several cases, morphological and malignant transformation occur simultaneously ; in other cases morphological transformation precedes malignant transformation. By various manipulations it has been possible t o obtain from highly malignant transformed clones, “revertant” clones of lesser malignancy and altered cellular behavior. This shows that oncogenic transformation can be reversed, and it appears that cyclic AMP plays some role in the regulation of these properties, which may also be under chromosomal control. The fact that non-cross-reacting TSTA’s are found in culture systems, as in vivo, represents a validation of the model system in a particularly complicated situation, and cloning experiments have advanced our knowledge beyond what can be accomplished in whole animals. By the use of such systems, it has been established that epoxides (arene oxides) are a metabolically activated form of certain polycyclic hydrocarbons, since they are much more active a t producing oncogenic transformation than the parent hydrocarbon or other metabolites. This metabolic activation is carried out by the inducible microsomal aryl

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hydrocarbon hydroxylase system. With future knowledge, its manipulation can provide an exciting opportunity to prevent cancer in exposed animal and human populations, such as has already been accomplished in the cell culture systems. It seems very likely that other mechanisms of metabolic activation will be established by the use of these cell cultures. Moreover, they will also be useful in elucidating the nature of interactions between chemical and viral oncogens, particularly with reference to the question of whether the chemical “switches on” a viral oncogen. And these systems can provide large quantities of clonally related normal and transformed cells as materials for critical comparisons of biochemical or other properties. Such cell culture systems will also become useful in screening large numbers of environmental substances for oncogenic activity ; there is the exciting possibility that human cells may be used for this screening. Before such systems can be adopted with confidence on a large scale, it will be necessary to validate them by a careful determination of the transforming activity of a large number of chemicals of diverse structures and known oncogenic activities and to obtain a good correlation. Furthermore, some means will have to be found to carry out metabolic activation of those compounds that may not be activated by the cells used for the tests. This might be approached by coupling the cultures with some hostmediated metabolic system, or perhaps by the addition to the test cultures of lethally irradiated feeder cells or liver cells. In any event, it seems likely that the cell culture systems will continually be refined and will play an ever more important role in screening for oncogenic chemicals in the environment and for elucidating the fundamental cellular and molecular mechanisms of chemical oncogenesis.

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363

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364

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AUTHOR INDEX Numbers in italics refer t o the pages on which the complete references are listed.

A

Allard, D., 96, 133, 148 Allen, J. M., 81, 124, 156 Allen, R. C.,129, 161 Alleva, F. R., 163, 196 Alleva, J. J., 163, 196 Allison, A. C., 45, 67, 71, 124, 144, 212, 214, 226 Alonzo, N. F., 270, 277,313 Amante, L., 218, 299 Ambilard, P., 220, 226 Ambrose, E. J., 266, 267, 287,308, 309 Ambrose, K. R., 31, 32, 33, 34, 67,69 Amerus, J. L., 182, 908 Amesbury, 0. F., 169, 196 Amiel, J. L., 132, 1 4 , 251, 252, 255, 260,

Aaronsoii, S. A., 223, 228, 337, 360 Abad, A., 170, 204 Abad, R., 245,262 Abarbanel, A. R., 169, 206 Abbondanza, A., 173, 174, 197 Abdel-Rrtouf, M., 162, 196 Abe, N., 113, 114, 148 Abe, S., 283, 312 Abelev, G. I., 30, 31, 36, 67, 131, 132, 133, 136 Abell, C. W., 346, 360 Abercrombie, M., 266, 267, 287, 308 Aberdeen, E. R., 322, 361 Abul-Fadl, M. A. M., 117, 123, 125, 136 262 Acevedo, H. F., 162, 196, 196 Amiet, A., 221, 296 A d a m , A. E., 171, 196 Amirmokri, E., 220, 227 A d a m , D. H., 174,196 Amman, A. J., 219, 220, 296 Adelman, R. C., 94,97, 102, 103, 106, 108, Amos, D. B., 22, 41, 67, 73 136, 137, 139, 1.40, 149 An, T., 110, 160 Adler, C. R., 268, S14 Anan, F., 90, 146 Adler, F. L., 215, 926 Andersen, V., 88, IS6 Adlercreutz, H., 176, 196 Anderson, A. D., 111, 142 Adlersberg, R., 221, 927 Anderson, B., 285, 309 Aebi, H., 84, 86, 89, 104, 106, 147, 161 Anderson, C., 111, 1& Agarwal, M. K., 181, 195, 196 Anderson, D. W., 222,328 Agostoni, A., 88, 138 Anderson, N. G., 31,32,33, 34, 67,69 Aguilar, M. J., 220, 296 Ando, S., 273, SO9 Ahmed, Z., 117, 119, 136 Andoh, T., 349, 360 Amenberg, A. C., 79, 136 Andrada, J. A., 162, 198 Aizawa, M., 5, 41, 74 Andres, G., 45, 74 Aki, K., 112, 113, 136, 141 Andrews, H., 267, 273,275,278,280,283, Alagilie, D., 220, 926 296,310 Albert, A., 162, 196, 234, 261 Andrianov, L. N., 345, 360 Albert, S., 180, 20s Angeletti, P., 124, 125, 126, 136, 144 Alexander, M. K., 171, 196 Angistein, D. M., 212, 226 Alexander, P., 5, 6, 41, 59, 60, 67, 69, 72, Angistein, L., 212, 296 132, 136 Anstiss, C. L., 121, 122, 139 Alfert, M., 182, 183, 196, 200 Anthony, H. N., 42, 70 Alfred, L. J., 43, 67 Anton-Tay, F., 159, 196 Alger, E. A., 168, 197 Aoki, T., 41, 67 Alkins, B. J., 28, 69 Apelo, R., 245, 260 Allan,J. M.,. I&, 196 Appella, E., 80,136 Allan, R. N., 171, 196 367

AUTHOR INDEX

Appelmans, F., 124, 138 h a i l Y., 157, 165, 196 Arber, J., 125, 147 Arce, A., 292, 308, 318 Archibdd, F. M.,268,314 Arm, J., 17, 30,70 Arms, J. C.,35,67,179,196 Arms, M.F., 35, 67, 179,196 Arfors, K.E.,121,156 Arias, I. M.,346,364 Ariga, T.,270, 310 Armstrong, A. R., 117, 14% Armstrong, E.C., 188, 196 Armstrong, M.Y.K., 217, %%6 Arnold, H.,106, 156 Arrillaga, F.,162, 198 b i n , J. M.,89,141,174,goo1 Ashley, D. J. B., 183,187, 188,190,196 Ashmore, J., 85,97, 156,161 Asofsky, R.,178, 199 Aub, J. C.,268,286,295,299,308 Auerbach, V. H.,79, 136,172,%O% Augustinason, K.B., 129,156 Austen, K.F., 220, MO Avis, P.J. G., 25, 76 Avrameas, S.,81, 136, 147 Aw, S. E.,123, 160 Axelrad, J., 175,206 Axelrod, A. E., 179, 203 Axelrod, L.R., 162,196,196 Azar, H.A., 253, $61

B Babson, A. L., 117, 123,126, 136 Baca, Z.V., 167, 168, 196 Bacila, M.,94,1 4 Bacon, R. L., 187, 202 Bagshawe, K. D., 57, 69, 232, 233, 234, 235,237,238,240,243., 245,248,249, 251,252,254,255,256,258,259, 260,

,

861 868, 263

Baguley, B. F., 116, 136 Bahl, 0.P., 234, 260 Bailey, E.,105, 136, 160 Baird, H.W.,220,%%Y Baker, R.,96, 136 Baldwin, R.W., 1, 3, 5, 8, 10, 11, 12, 13, 14, 15, 17, 19, 20, 22, 23, 24, 25, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,

40, 41, 42, 57, 58, 61, 62, 6Y, 68, YO, 64,340,363 Balinsky, D.,136 Ball, J., 159, 204 Ball, J. K., 52, 53, 68 Balner, H.,46, 47, 68 Balogh, A., 164,1%' Banfield, W.,16, Y8 Bank, W.J., 109, 145 Bansal, S. C., 62, 63, 64, 65, 68, Y3, Yq, 218, 8.99 Bao-Linh, D.,178, 196 Bar, U.,89, 136 Baranowski, T.,95,96,143 Baranska, W.,31,32, 68 Barclay, M.,268,314 Bard, D.S., 59, 68 Bardick, R.,243,963 Bardos, T.J., 182, 208 Bargellesi, A., 127,146 Barker, C. R., 10, 11, 12, 13, 14, 17, 20, 30,33,35,37, 38,40,58, 67,68 Barmada, B., 220,1830 Barnes, J. M.,17,Yl, 174, 19Y Baron, D.N.,93,95,97,156, 139, 146 Barraclough, C.A., 161,163,164,196,200 Barrera, B., 184, 189, 196 Barrett, A. M.,167, 196 Barrett, K.,120,148 Barrett, M.K.,255,860 Bar-Sela, M.,167,198 Barski, C.,59,61, 68 Bartholomew, E.M.,129, 136 Bartholomew, W.R., 129, 136 Barto, E.,81,149 Barton, A. G., 182, 183, 808 Bartsch, C. E.,180,201 Barsilai, D.,172, 176, 178,196 Baserga, R.,303, 508,314 Basombrio, M.A., 5, 6,7, 12, 19, 20, 43, 68,337,560 Bastens, B., 131, l4Y Basterk, B., 131,140 Basu, M., 267,290,SO9 Batchelor, J. R.,171, 196 Bates, R.R., 186, 196,348, 566 Bates, R.W., 234,268 Bau, D.,175, 195, Baumann, C.A., 184, 189, 191, 207 Baumler, A., 40, 69

AUTHOR INDEX

Baylis, P., 186, 198 Bearn, A. G., 80, 82, 86, 88, 161 Beautyman, W., 87, 136 Becker, E. M., 318, 366 Becker, F. F., 173, 196 Beckman, G., 123, 124, 136 Beckman, L., 121, 123, 124, 136 Beech, M., 253, 961 Beer, A. E., 246, 960 Beere, A,, 109, 141 Behnke, O., 298, 308 Bekesi, J. G., 57, 68 Beldotti, L., 216, 999 Belehradek, J., 59, 68,68 Belitsky, G. A., 345, 360 Belitsky, P., 90, 146 Bell, E. T., 234, 961 Bell, J. J., 234, 260 Bell, M. E., 178, 196 Bellomy, B. B., 31, 32, 33, 34, 69 Belman, S., 182, 908 Beloshapkina, T., 39, 68 Benacerraf, B., 214, 998 Ben-Bassat, H., 331, 360,363 Bendich, A., 338, 346, 347, 361, 364 Bendixen, G., 258, 963 Benedetti, E. L., 38, 69 Benedict, W. F., 328, 355, 360, 364 Ben-Hur, N., 250, 251, 255, 962, 963 Benirschke, K., 162, 197 Bennett, B., 9, 68 Bennison, B. E., 52, 54, 55, 71, 215, 998 Bensted, J., 60, 67 Beratis, N. G., 119, 136 Berbst, A. L., 167, 909 Berendes, H., 222, 999 Bergel, F., 79, 90, 136, 169 Berger, R., 83, 139 Bergquist, P. L., 116, 136 Berkowitz, E., 182, 908 Berlin, A., 276, 298, 308 Berliner, D. L., 165, 808 Berman, C., 184, 189, 196 Berman, L. D., 45, 67, 212, 213, 296 Bern, H. A., 167, 906 Bernadou, A., 88, 169 Bernecky, J., 35, 70 Bernstein, D. E., 167, 197 Bernstein, J. D., 60, 65, 76 Berthillier, G., 101, 136

369

Bertolotti, R., 92, 134, 136 Bertram, J. S., 338, 360, 366 Berwald, Y., 318, 323, 324, 328,360 Berwick, M., 174, ,909 Beswick, J., 38, 39, 42, 68 Bettane, M., 118, 123, 136 Beutler, E., 100, 101, 102, 149 Bew, M., 110, 160 Bhatty, R. S., 103, 136 Biancifiori, C., 184, 197 Bielka, H., 181, 197 Bielschowsky, F., 184, 185, 192, 193, 194, 197 Bielschowsky, M., 193, 197 Bienenstock, J., 219, 830 Bier, D., 220, 896 Bierman, H. R., 88, 136 Bigelow, N. H., 190, 197 Biggar, W. D., 219, 225, 997, 998 Bigley, R. H., 104, 106, 136, 137 Bigliere, E. G., 167, 206 Bilik, E., 173, 8/37 Billeter, 0. A., 169, 199 Billingham, R. E., 246, 860 Billington, W. D., 256, 961 Bilski-Pasquier, G., 88, 169 Bird, C. W. G., 300, 318 Birge, C. A., 170, 197 Birkett, D. J., 117, 137 Biron, P., 84, 136 Biscardi, A. M., 159, 198 Biserte, G., 81, 136 Bismarck, B. L., 55, 68 Biswas, S., 233, 960 Bittner, J. J., 160, 90.4 Black, J. A., 106, 149 Black, M. N., 286, 299,311 Black, P. H., 217, 997,267, 287, 302,309, 319,313, 316, 331,361 Black, P. L., 217, 296 Blase, R. M., 216,217,220,221,296, 930 Blair, P. B., 31, 45, 68 Blanchaer, M. C., 84, 136 Blanchet, J. P., 118, 123, 136 Blanco, A., 81, 82, 136, 168 Bliss, J. Q., 171, 903 Blk, G., 270,308 Bloch, D. P., 115, 147 Bloch, E., 162, 165, 197 Bloch, H., 283, 284, 295, 310

370

AUTHOR INDEX

Block, M. H., 213, 227 Bloom, B. R., 9, 68 Bloom, E. T., 10, 58, 68 Blostein, R., 94, 95, 97, 136, 147 Bliime, K. G., 106,136 Bluestein, H. G., 51, 68 Blumberg, B. S., 223, 230 Boccabella, A. V., 168, 197 Bock, F. G., 53, 54, 70 Bocquet, J. P., 221, 229 Bodansky, O., 79, 81, 89, 111, 112, 117, 118, 120, 121, 125, 126, 136, 137, 146, 148

Boder, E., 220, 826, 826, 829 Bogovski, S . P., 321, 566 Bohn, L., 90,146 Boiato-Chen, L., 47, 54, 55, 74 Boivin, P., 106, 137 Bollum, F. J., 115, 162 Bombik, B. M., 305, 308 Bond, H. E., 178, 197 Bonetti, E., 173, 174, 197 Bonne, C., 184, 189, 197 Bonnet, H., 220, 286 Bonser, G. M., 188, 196, 198 Boon, M. C., 213, 228 Boone, C., 81, 137 Booth, J., 175, 197 Borek, C., 267, 278, 279, 308, 326, 327, 340,360, 361, 364 Borek, E., 115, 116, 149, 160 Borel, J. A., 87, 140 Borelli, J., 125, 163 Borenfreund, E., 338, 361 Borland, R., 253, 862 Boros, A., 164, 197 Borrell, U., 233, 260 Borsos, A., 164, 197 Borsos, T., 16, 17, 25, 60, 65, 71, 72, 76, 170, 198 Borum, K., 62, 73, 213, 229 Bosman, H. B., 267, 290, 291, 292, 308 Bottomley, R. H., 79, 88, 137 Bouchard, P., 180, 181, 206 Bouen, J., 222, 228 Bousser, J., 88, 162 Boxer, G. E., 79, 85, 149 Boycott, A. E., 180, 197 Boyd, J. D., 237, 261 Boyd, J. W., 86, 111, 137

Boyer, P. D., 93, 94, 138, 143, 160 Boyer, S. H., 81, 118, 119, 137 Boyland, E., 343, 344, 350, 351,361 Boyse, E. A., 2,5, 6,7,8,9, 10, 28,39, 41, 56, 58, 59, 67, 68, 69, 72, 216, 226, 228 Brace, K. C., 244,260 Bradbury, J. T., 166, 197 Bradbury, S., 256, 261 Bradley, R. M., 267, 278, 279, 308, 312 Brady, R. O., 267, 278, 279, 280, 282, 286, 290, 508, 309, 312 Braikevitch, M., 234, 261 Brand, K., 105, 141 Brand, M. M., 220, 826 Brankow, D., 366 Branton, D., 275, 276, 297, 313 Bras, G., 190, 806 Braucher, P. F., 171, 206 Brawn, J., 32, 64, 69, 257, 261 Breckenridge, R. M., 305, 308 Breekveldt-kielich, J. C., 178, 207 Brehm, T. J., 217, 221, 226 Bresnick, E., 110, 111, 137, 144, 350, 361 Bresson, Y., 133, 147 Breuer, H., 176, 205 Brewer, G. J., 100, 159 Brewer, J. I., 232, 245, 860 Brewis, R. A. L., 233, 960 Breyere, E. J., 255, 260, 262, 863 Briber, R., 82, 84, 148 Bridson, W. E., 163, 200 Brierley, G., 180, 199 Brillantes, F. P., 6, 70 Broadfoot, M., 266,312 Brodie, B. B., 175, 806 Brody, J. A., 85, 161 Brookes, P., 43, 69, 345, 346, 347, 348, 357, 361, 562 Broome, J. D., 173, 196 Brough, A. J., 220, 829 Brown, A. L., Jr., 221, 827 Brown, F. C., 99, 137 Brown, J., 92, 101, 103, 137, 164 Brown, M. B., 213, 226, 227 Brown, P. S., 166, 199 Brown, T., 175, 195, 206 Brown, T. J., 89, 141, 174, 201 Brown-Grant, K., 168, 177,197,199 Brox, L. W., 97, 137, 140 Brudenell-Woods, J., 119, 1.63

AUTHOR INDEX

Brunet, P., 220, 226 Brunius, F. E., 270, 308 Brute, G., 252, 255, 262 Bryan, R. J., 342, 362,366 Bryson, M. J., 165, 208 Bubenik, J., 7, 9, 10, 58, 69 Buch, L., 116, 137 Buchheit, R., 285, 311 Buck, C. A., 267, 291, 292, 308, 316 Buck, G. M., 93, 95, 136, 139 Buckley, Z., 81, 149 Bucher, T., 106, 137 Buell, P., 187, 197 Biirki, K., 350, 361 Buffe, D., 90,137 Bullough, W. S., 40, 69 Burbank, R. C., 171, 203 Burdick, J. F., 22, 69 Burford, B. O., 18, 73, 338, 366 Burge, B. W., 292, 313 Burger, A., 84, 89, 147 Burger, M. M., 2, 69, 268, 286, 295, 296, 297, 299, 302,305,306, 308,309, 312, 313, 330, 331, 361 Burgoyne, F. H., 185, 192, 208 Burkitt, D. P., 187, 198 Burmeister, B. R., 214, 215, 226, 229 Burnet, F. M., 19, 39, 66, 69, 211, 226 Burns, R. K., 162, 197 Burr, G. O., 180, $04 Burrill, M. W., 165, 200 Burstein, S., 16, 72, 176, 197 Burtin, P., 90, 132, 137, 161 Busch, D., 106, 136 Butler, H. A., 245, 260 Butler, W. H., 174, 185, 192, 197, 200

c Cagnoni, M., 163, 197 Cahn, R. D., 81, 82, 137, 1.42, 146, 162 Calbert, C. E., 180, 200 Camain, R., 131, 140 Camargo, A. C. M., 181, 197 Cambier, J., 220, 226 Cameron, E. C., 93, 139 Campadelli-Fiume, G., 108, 137 Campbell, J. H., 266, 308, 309 Campos, J. O., 104, 106, 136, 137 Canfield, R. E., 234, 260, 262

371

Cantarow, A., 181, 184, 185, 197, 208 Cantoni, G . L., 173, 197 Caputo, A., 107, 146 Caputto, R., 292, 508, 312 Carayon, F., 110, l& Carbonell, A. W., 126, 144 Cardini, C. E., 113, 144 Cardoso, S. S., 181, 197 Carmichael, G. G., 186, 197 Carminatti, H., 105, 138, 146, 147 carnes, W. J., 213, 226 Carpenter, C. B., 224, 226 Cam, D. H., 162, 207 Carr, J. A., 181, 201 CarriBre, R., 183, 195, 197 Carruthers, C., 40, 69 Carswell, E. A., 3, 5, 6, 7, 8, 9, 16, 27, 59, 72, 214, 228 Carter, H., 270, 308 Carter, J. E., 232, 260 Cartier, P., 106, 157 Case, M. P., 220, 287 Castaigne, P., 220, 226 Casto, B. C., 343, 361 Cattan, A., 87, l4O Ceccarini, G., 306, 308 Celada, F., 170, 190, 197 Cenciotti, L., 86, 137 Cerilli, G. J., 47, 69 Chan, P. C., 321, 361 Chan, W. W. C., 93,94, 108, 156,137,146 Chang, C., 80, 14.6 Chang, G. G., 173,806 Changeux, J. P., 2, 69, 106, 144 Chaplain, M., 134, 16g Chapman, B. A., 171, 196 Chapman, V. M., 81,147 Chapman, W. H., 7,8, 34,61, 74 Chappel, C. I., 166, 206 Chaptal, J., 220, 226 Chase, H. P., 220, 226 Chayen, J., 38, 39, 42, 68 Cheema, R. P., 267, 279, 308 Chen, C., 94, 143 Ch’en, H. C., 184, 189, 198 Chen, R. F., 83, 162 Chen, T. R., 81, 137, 147 Chen, T. T., 333, 334, 335, 336, 361 Cherry, C. P., 187, 197, 200 Chesterman, F. C., 212, 2W

372

AUTHOR INDEX

Chien, T. C., 98, 148 Chiodi, H., 167, 168, 196 Chilson, 0. P., 80, 81, 137, 148 Chrnielewice, Z. F., 182, 208 Cho, H. Y.,341, 342,366 Chong, L., 121, 139 Choppin, P. W., 269, 311 Christen, P., 95, 147 Christensen, E., 89, 240 Christian, W., 96, 161 Chriistiansen, K., 181,198 Christie, D. W., 234, 261 Christie, F. H., 121, 140 Chrktot, J., 158, 203 C h u g , H. L., 184, 189, 198 Churchill, W. H., 16, 76, 170, 198 Cikes, M., 297, 309 Cinader, B., 170, 209 Ciotti, M. M., 82, 83, 84, 148 Ciillo, V. J., 100, 142 Claflin, A., 81, 146 Clark, H. M., 165, 198 Clarke, D. A., 5, 6, 7, 8, 59, 72, 214, 928 Clausen, J., 88, 89, 136, 137, 140 Clawson, C. C., 226 Clayson, D. B., 188, 198 Clayton, C. C., 91, 1@ Cline, M. J., 297, 309 Cochran, A. J., 297, 309 Coffey, C. B., 21, 74 Coggin, J. H., 31, 32, 33, 34, 67, 69,70 Cohen, A. M., 22, 69 Cohen, J., 171, 203 Cohen, M. M., 221, 226, 245, 260 Cohen, P. P., 111, 140 Cohen, R. B., 168,198 Cohen, Y.,187, 198 Coleman, A., 220, 226 Coleman, D., 224, 227 Coleman, W. H., 170, 198 Collins, F. D., 180, 198 Collins, V. P., 238, 960 Colnaghi, M. I., 54, 56, 79 Colobert, L., 101, 136 Colonge, R. A., 162, 209 Colowick, S. P., 102, 142 Columbani, J., 252, 255, 969 Coman, D. R., 266,309 Connell, D. I., 348,361 Connelly, R. R., 186, 198

Conney, A. H., 349,361 Contopoalos, A. N., 169, 196 Convery, H. J. H., 105,162 Conway, M. M., 109, 143 Conyers, R. A., 117,137 Coon, M. J., 106, 138 Cooper, M. D., 214,215,217,220, 996, 999 Cooper, R., 303, 309 Coppola, J. A., 159, 904 Cori, C. F., 79, 85, 99, 137, 149 Cori, G. T., 79,85,95,99,137, 149,160 Cornateer, W.E., 195, 198 Cornelius, E., 214, 226 Cornish, C. J., 119, 146 Corsigilia, V. F., 185, 192, 200 Corson, J. M., 246, 263 Corte, E. D., 173, 174, 197 Cory, J. G., 123, 138 Cosson, G., 88, 162 Costello, L. A., 80, 81, 137, 198 Cote, M. N., 295, 308 Cotlove, E., 118, 160 Cottam, G. L., 106, 138 Cousineau, L., 88, 89, 149 Cowdry, E. V., 184, 209 Cox, R. P., 122, 123, f39,240, 306, 307, 309 Craighead, J. E., 224, 926 Cramer, J. W., 172, 90.4 Crane, R. K., 102, 138 Crawen, E. M., 220, 287 Creasy, B., 342, 366 Cremer, N. E., 215, 926 Cresswell, P., 29, 73 Cribbs, R. N., 83, 138 Criss, W. E., 92, 103, 105, 107, 109, 134, 158,162 Cristofalo, V. J., 79, 85, 102, 106, 107, 139, 14,162 Critchley, D., 267, 282, 287, 288,290, 292, 309,316 Critchlow, V., 167, 198 Crocco, M., 182, 900 Crocker, T. T., 320, 321, 322, 347, 361, 369,364 Croisille, Y., 81, 91, 138 Crowder, S. E., 168, 200 Cruickshank, H., 187, 1.99 Cukier, J. O.,159, 198,208 Culp, L. A., 267, 309, 331, 361

AUTHOR INDEX

Cumar, F. A., 267, 279, 290, 309, S l d Cunanan, R. F., 245, 660 Cunningham, D. D., 290, SO9 Cunningham, G. J., 38, 39, 42, 68 Currie, G. A., 57, 69, 237, 255, 256, 257, 260, 861 Curtis, W. C., 173, 808 Cutler, S. J., 186, 187, 198, 199 Czitober, H., 125, 140

D Dabelsteen, E., 283,309 DaCosta, R. R., 181, 197 Dagg, M. K., 255, 257, 261 Dahl, V., 158, 206 Dahnke, G. S., 220, 627 Dalmacio-Cruz, A. E., 184, 189, 196 Dalton, A. J., 18, 69 Daly, J. W., 351, 355, SBS, S64 Damant, G. C. C., 180, 197 D'Angelo, S. A., 169, 198 Dao, T. L., 323, 361, S6S Daughaday, W. H., 170, 197 Dausset, J., 252, 255, 2668 Davidsohn, I., 283, 285, S09,Sl4 Davidson, 0. W., 162, 198 Davies, D. A. L., 28, 69 Davies, D. S., 175, 198 Davies, J. N. P., 187, 198 Davies, R. F., 322, S61 Davis, B. J., 82, 158 Davis, P. A., 103, l 4 S Davis, R. C., 47, 70 Davison, J. P., 195, 198 Dawood, M. Y., 245, 248, 250, 261, 262 Dawson, D. A., 53, 68 Dawson, D. M., 84, 138 Dawson, G., 270,511 Deak, B. D., 216, 228 Deal, W. C., 93, 106, 1.98, 149 Deane, H. W., 167, 198 de Asda, L. J., 105, lS8, 146, 147 De Baun, J. R., 173, 179, 195,198 De Carvalho, S., 181, 198 Deckers, C., 37, 40, 59, 69 De Duve, C., 124, 138 Defendi, V., 43, 69, 212, 226, 267, 296, 309, 510, 347,361

373

De Grouchy, J., 125, 1% De Gutierrez Moyano, M. B., 159, 198 De Harven, E., 41, 6'7 Deichman, G. I., 32, 69 De La Balze, F. A., 162, 198 de la Haba, G., 84, 139 Delain, D., 82, 138 De Lange, R., 303, 309 Delhanty, J. D. A., 161, do4 Della-Cork, E., 108,137 Della Porta, G., 54, 56, 79 Delorme, E. J., 6, 60, 67, 69 Delory, G., 117, l&? Demartial, M. C., 106, 137 De Moor, P., 177, 191, 198 DBmos, J., 84, 138 Den, H., 267, 290, 309 de NBchaud, B., 91, 131, 148, 161 Denef, C., 177, 191, 198 Dent, P., 213, 214, 215, 666 Denys, J., 212, B O Denys, P., 45, 74 De Recondo, A. M., 115, 138 De Reviers, M. M., 162, 604 Dersjant, H., 46, 47, 68 Desmet, G., 87, 148 de Somer, P., 45, 74, 212, 213, $30 de Souza, E., 80, 146 Deuels, H. J., Jr., 180, dGU Dev, V. G., 340, S64 Devalle, J. J., 105, 138 De Wulf, H., 113, 138, 141 Diamond, L., 43, 69, 345, 350, 354, 361, S62, 566 Dickey, A,, 85, 1S9 Dickie, M. M., 184, 201 Diczfalusy, E., 165, 208, 234, 961 Diermeier, H. F., 183, 198 Dietrich, S., 188, 6068 Dietz, A. A., 88, 92, 147 Dietz, M. H., 339, S61 Dikow, A. L., 95, 97, 98, 138, 146 Dioguardi, N., 88, 138 Di Paolo, J. A., 43,67,325,328, 329,337, 340, 343, 348, 355, 361, S62, 563,S64 Di Pietro, D. L., 99, 100, 102, 124, 158 Dipple, A., 345, 347, 352, 357, 358, 362 Diringer, H., 267, 280, 281, 282, 303, 809, 346, 366

374

AUTHOR INDEX

Dirksen, E. R., 321,362 Dishon, T., 286, 314 Di Stefano, H. S., 198 Docherty, J. J., 343, 362 Docke, F., 157, 198 nod, B. J., 269, 309 Dodds, M. L., 171, 198 Dodge, B. G., 185,206 Dodge, 0. G., 187,198 Doida, Y . , 340, 363 Doljanski, F., 182, 198 Doljanski, Z., 299, 311 Dolkart, R. E., 245, 260 Doll, R., 45, 69, 184, 198 Domaniewska-Sobczak, K., 248,262 Donnelly, A. J., 102, 144, 149 Donoso, A. O., 159, 198,208 Donovan, P. J., 43, 67, 325, 328, 329, 355, 36f,368,365

Dore, J. F., 132, 144 Dorfman, It. I., 177, 199 Dorner, G., 157, 161, 199 Dortebach, C., 162, 207 Douglas, S. D., 221, 226, 275, 276,513 Douthwaite, R. M., 256, 261 Drechsler, E. R., 93, 138 Dreyfus, J. C., 82,84,94,96, 97, 133, 134, 138, 146, 148 Dubbs, C.A., 125, 138 Dubbs, D. R., 32, 71 Dubert, J. M., 116, 146 Du Bois, K. P., 175, 209 Duchesne, E. M., 318, 366 Dudman, N. P. B., 81, 138 Duff, R., 31, 32, 69 Dufour, D., 178, 196 Dugois, P., 220, 226 Dulbecco, R., 323, 366 Dulin, W. E., 166, 204 Duncan, M. E., 345, 348, 561,362 Dunn, H. G., 220, 2.96 Dunn, T. B., 190,2001 Dunne, J., 121, 139 Durbin, P. W., 188, 209 Dux, A., 190, 199, 216, 288

E Eagle, H., 306, 308 Eagles, P. A. M., 94, 139 Earle, W. R., 318, 362, 366

Earthy, H., 193, 199 Eaton, G. M., 100, 138 Eaton, R. H., 117, 118, 146 Eaton, S., 348, 366 Eckroade, R. J., 224, 289 Ederer, F., 187, 199 Edgcombe, J. H., 232, 253, 262 Edgerton, B. W., 59, 69 Edidin, M., 275, 297, 309 Edstrom, J. E., 156, 199 Edwards, J. G., 266, 308,509, 314 Eguchi, M., 98, 139 Eguchi, Y., 167, 199 Eichwald, E. J., 170, 199 Eidson, L. L., 170, 204 Eisenberg, E., 158, 199 Eisenberg, H., 187, 199 Eiteman, D. V., 220, 28.9 Ekert, H., 245, 262 Elger, W., 164, 165, 206 El Hilali, M. M., 90, 146 Elliott, B. A., 86, 88, 159 Elliott, H. W., 158, 199, 200 Elliott, J., 341, 366 Elson, N. A., 122, 139 Elston, C. W., 253, 254, 261 Elwood, J. C., 79, 85, 99, 102, 139, 1.44 Embleton, M. J., 5,6,10,11,13, 14, 15, 17, 18, 19, 20, 25, 33, 35, 40, 41, 57, 61, 62, 64, 67, 68, 69, 78, 337, 362, 364 Emery, A. E. H., 83, 139 Emmelot, P., 38, 69 Emory, E., 88, 156 Endo, H., 95, 97, 98, 108, 139, 141, 142, 162

Engelhardt, N. V., 35, 69, 131, 140 Engelstein, J. M., 170, 190, 199 Engle, E. T., 160, 20'7 Engstrom, L., 117, 139 Enomoto, M., 172, 204 Enser, M., 127, 139 Epstein, C. J., 182, 199 Epstein, S. S., 184, 199 Epstein, W. L., 220, 289 Epstein, Y., 125, 147 Erbland, J., 179, 204 Errera, M., 128, 139 Espadaler, J. M., 220, 229

AUTHOR INDEX

Estabrook, R. W., 175, 207 Estborn, B., 118, 124, 139 Eto, T., 273, 309 Etzler, M. E., 119, 1& Evans, C. A., 63, 70 Evans, E. S., 193, 207 Evans, H. M., 195, 200 Evans, R. W., 187, 199 Everett, J. W., 159, 199, 204 Evers, C. G., 219, 220, 827 Everse, J., 83, 139 Eyler, E., 267, 291, 292, 308

F Fagundes, L. A., 168, 198 Fainer, D. C.,81, 157 Falk, H. L., 344, 364 Fantini, F., 163, 197 Farina, F. A., 102, 103, 106, 107, 159, 144, 152 Farron, F., 108, 139 Favarger, P., 179, 199 Fawke, L., 16% 199 Feder, H. H., 162, 206 Feigen, R. D., 220, 226 Feinstein, R. N., 93, 139 Feixi, T., 285, 309 Fell V. J., 40, 69 Feldman, J. D., 168, 199 Feldman, M., 6, 7'0, 170, 199 Fennelly, J. J., 121, 139 Fenyo, E. M., 41, 69 Fernando, J., 127,139 Fernstrom, I., 233, 260 Ferreira, A. C., 157, 800 Fetterman, P. L., 21, 74 Ficher, M., 162, 208 Field, S. M., 224, 227 Fieldhouse, B., 81, 84, 91, 129 Fildes, R. A., 108, 130 Fine, A. W., 268,314 Fine, I. H., 84, 139 Fink, L. M., 21, 75, 181, 195, 199 Finlayson, J. S., 178, 179, 1S9 Finster, J. L., 170, 20.9 Fiorelli, G., 88, 158 Firminger, H. I., 184, 185, 191, 192, 1.9.9, 205, 206

375

Fischer, C., 35, 70 Fisher, J. C., 20, 47, 70 Fisher, J. N., 170, 201 Fishman, L., 118, 138 Fishman, P. H., 290, 309 Fishman, W. H., 117, 118, 119, 121, 122, 130, 140, 146, 150 Fitxgerald, M., 121, 139 Flaks, A., 322, 362, 364 Flaks, B., 322, 362 Flaxman, B. A., 339, 361 Flehmig, It. W., 168, 203 Fleischer, S., 180, 19.9 Fleisher, G. A., 111, 139 Fleisher, M., 121, 126, 148 Flerk6, B., 156, 808 Fletcher, E. K., 193, 197 Fletcher, F., 298, 318 Flexner, J. B., 84, 139 Flexner, L. B., 84, 139 Florsheim, W. H., 168, 199 Fogel, M., 285, 302, 309 Foglia, V., 158, ,805 Fois, A., 220, 286 Foley, E. J., 1, 3, 70, 266, 309 Fondy, J. P., 80, 81, 139 Foneo, D., 167, 199 Forchielli, E., 177, 199 Ford, D. H., 168, 199 Forer, A., 298, 308 Forni, L., 218, 229 Forrester, J. A., 267, 309, 351, 368 Forsberg, J. G., 163, 199 Forthomme, J., 221, 929 Fortner, J. G., 187, 199 Foster, D. O., 266, 309 Fothergill, J. E., 35, 40, 72 Fottrell, P. F., 84, 139 Foulds, L., 188, 199 Fountain, J. A., 85, 139 Fox, H. W., 53, 54, 70 Fox, T. O., 297, 309 Foxwell, C. J., 93, 95, 136, 139 Fralick, R. L., 162, 209 Franchimont, P., 235, 861 Franks, L. M., 126, 144 Fraser, D., 121, 140 Fraser, K. J., 219, 226 Fraumeni, J. F., Jr., 188, 199 Frayssinet, C., 30, 74, 115, 138

376

AUTHOR I N D m

Fredrickson, D. N., 214, 999 Freedberg, J., 80, 81, 139 Freedman, S. O., 132, 1.60 Freeman, A. E., 341, 342, 343,369, ,964 Freeman, A. I., 221,996 Frei, J. V., 338,369 Frendenberger, C. B., 169,199 Frey, I., 175, 907 Friberg, S., 297, 309 Fried, J., 358, S69 Friedell, G. H., 129,143 Friedman, E., 129,161 Friedman, R., 304,306,311,332,363 Friedman, S., 284, 309 Friedman, S. M., 166,907 Friend, C., 85,140 Frilley, M., 161, 906 Fritz, P. J., 81, 83, 140 Frohwirth, N., 346, 366 F r o m , H. J., 100,14O Fudenberg, H. H., 220,221, 996,99'7 Fuhrer, J. P., 267,.291,292,916 Fujii, T., 181, 182, 199, 909 Fujimoto, Y., 87,140 Fujimura, S., 21, 76 Fulling, H. J., 283,309 Furth, J., 188, 909,213, 215, $27, 898 Furukawa, K., 283,310

G Gacon, G., 110, 144 Gabrielsen, A. E., 217, 997 Gaebler, 0. H., 174, 199 Gaensbauer, F., 166,197 GafN, J., 221,9%' Gahmberg, C. G., 268, 269,275, 276,294, 301, S09,S13 Gajl-Peczalska, K. J., 219, 997 Gala, R. R., 167, 168,199, 900 Galand, C., 106, IS7 Galbraith, H., 84, 140 Gallagher, R. E., 116, 140 Gallo, D. G., 195,198 Gallo, R. C., 115, 116, 140, 147 Galton, M., 171, ZW Gan, M. V., 181,198 Ganong, W.F., 166,903 Garbe, E., 87, 140 Garcia, J. A., 157, 900 Gardner, M. B., 342,366

Gardner, S. D., 224, 997 Gardner, W.U., 170, 190, 900, 908 Garisoain, M., 17, 30, 70 Garren,L. D., 182,900 Gasic, G., 267,309 Gastineau, C. F., 234,961 Gatens, E. A., 182,199 Gatti, R. A., 216,217,218,221,223, f297, 9B Gaudry, R., 166, ,?W Gaugss, N. J. M., 212, 997 Gayle, R., 125, 1S6 Geel, S., 158, ,904 Gelboin, H. V., 326, 349, 350, 354, 355, 361, 36.9, 963,364, 366 Genowa, V. G., 95, 1S8 Georgiev, P., 84,140 Gerbie, A. B., 232,245, 960 Gerhardt, W.,88, 89, 136, 137, 140 Gerschenson, L. E., 340,364 Gerstley, B. J. S., 223, &O Geschwind, I. I., 182, 183, 195,196, 900 Gesner, B. M., 306,307,309 Gey, G. O., 234, 237, 961, 969, 318, 369 Ghose, T., 42, 70 Ghosh, N. K., 119,140 Ghostine, S., 220, lpB0 Gianetto, R., 124, 158 Giden, J. E., 350,355,360,364 Gigon, P. L., 175,198, W Gilchrist, C., 212, 997 Gilden, R. V., 266,310, 342,S66%,866 Giles, G., 45,74 Gillette, J. R., 175, 180,197, 198, S'00, 901, 209 G h n , J., 162, ,%!@I Ginsburg, A., 93,140 Ginsburg, V., 267,268,287,309,313 Giraldo, G., 47, 7s Girke, W.,358, 366 Girkin, G., 174, Glasky, A. J., 116, 149 Glass, R. M., 185, 191,209 Glaves, D., 10, 11, 13, 14, 17, 20, 24, 30, 31, 32, 33, 34, 39, 40, 41, 58, M,07, 68,340, S63 Gleich, G. J., 221, 930 Gleichmann, E., 216, 997 Gleichmann, H., 216,997 Glenister, D. W.,167,900

AUTHOR INDEX

Glick, M. C., 267, 269, 292, 293, 508, 516 Globerson, A., 6, 70 Glowinski, J., 159, 901, 20.3 Glucksmann, A., 187,197, 900 Gockerman, J., 124,156 Goddard, J. W., 181, 197 Gold, J. M., 284, 309 Gold, P., 132, 140, 284, 300 Goldberg, A. F., 126, 140 Goldberg, A. R., 268,295,308 Goldberg, E., 81,140 Goldberg, L. S., 221, 926 Golding, P. R., 245,255,258, 260,261 Goldman, A. S., 221, 227 Goldman, L., 3, 6, 7, 16, 72 Goldman, R. D., 89, 90, 140 Goldman, S. S., 119, 140 Goldsmith, M., 214, 228 Goldstein, D. J., 256, 261 Goldstein, D. P., 233, 245, 961, 26s Golub, N. I., 321, 366 Golub, S. H., 297, 509 Gomez, C. J., 158,906 Gomori, G., 127,140 Gonzalea, C., 100, 140 Good, R. A., 211,213, 214, 215,216,217, 218, 219, 220, 221, 222, 223, 224, 926, 226,227, 928,929,%?0 Goodall, C. M., 132, 140, 185, 192, 194, 200,20s Goodfriend, L., 84, 158 Goodfriend, T. C., 82, 84, 140 Goodman, H. C., 178, 179,207 Goodman, S. B., 213,997 Goodmann, N., 224,229 Goodwin, M., 183,906 Gordan, G. S., 158,199,900 Gordon, J., 12, 70 Gordon, J. A., 297, SO9 Gordon, R. J., 342, 562, S66 Gorski, R. A., 160,161, 163,164,165,166, 196, 200, 202 Gosh, N. K., 121, 122, lS9 Got, R., 101, 1S6 Goshman, L. M., 346, 348, 562 Goto, K., 306, 307, SO9 Goto, M., 327,363 Gotoff, s. P., 220, 227 Goussev, A. I., 131, 140 Govan, D., 96,136

377

Goy, It. W., 162, 163, 200, 206,209 Grabar, P., 30, 74, 81, 131, 147, 161, 178, 1.96 Gracy, It. W., 94, 97, 137, 140 Grady, R. O., 290, 309 Graf, C., 270, 315 Graf, L., 270, 277, 278, 302,315 Graff, R. J., 171, 200 Graffi, A., 181, 107, 268, 300,513 Gram, T. E., 175, 180, 200, 901 Grand, L. C., 6, 70 Grant, G. A., 46, 70, 72 Grant, W. C., 171, 200 Grantham, H. H., Jr., 174, 901 Grantham, P. H., 172, 179, 185, 191, 909 Gray, G. M., 181, 201, 269, 270, SO9 Green, A. A., 95, 160 Green, B. A., 86, 149 Green, H., 222, 230, 266, 279, 287, 306, 313, Sl4, 327, 337,366 Green, H. N., 42, 70 Green, I., 51, 68, 221, 227 Green, M., 299, 315 Green, P. A., 221, 927 Green, S., 117, 121, 122, 1S9 Greenberg, D. M., 99, 146, 148 Greenberg, E., 126, 148 Greenberg, S. M., 180,200 Greenblatt, M., 174, 197 Greene, R. R., 165,900 Greenstein, J. P., 79, 140 Greenwood, F., 270, SO8 Greep, R. O., 160, Gregerman, R. I., 168, 200 Gregori, C., 105, 107, 148 Gregory, K. F., 82, 83, 86, 146, 162 Grey, R. D., 119, 164 Griesbach, L. M., 5, 6, 20, 79, 336, S64 Griesbach, W. E., 188, 200 Griffin, A. C., 185,186, 192, 193, 200,206, 206 Griffin, M. J., 123, 140 Grimes, W. J., 267,291, 309 Gronroos, J., 267, S10 Gross, J., 286, Sl4 Gross, L., 213,2H Gross, S. J., 247, 261 Gross, S. K., 267, 281, 288,290,SlS Grossbard, L., 100, 101, 140, 148 Grossman, L. J., 92, 160

378

AUTHOR INDEX

Grover, P. L., 328,349,351, 352, 353, 358, 362, S63,364, 366 Gruenstein, M., 186, 200 Grumberger, D., 21, 76 Grundig, E., 125, 140 Grunt, J. A., 169, 200 Guelstein, V. I., 36, 70 Gurtler, B.,. 94, 140 Guttler, F., 86, 140 Guin, G. H., 220, 226 Gupta, S. L., 182, 195, 200 Gurtman, A. I., 162, 198 Gutfreund, H., 94, 139 Gutman, A. B., 125, 140 Gutman, E. B., 125, 140 Gutmann, H. R., 339, 362

H Haas, T. A,, 106, l4l Habel, K., 213, 227 Haberman, R. B., 70 Hacker, B., 116, 140 Hackett, E., 253, 261 Haeckel, R., 105, l 4 l Hakkinen, I., 267, 284,310 Hammerling, U., 41, 67 Hayry, P., 296,310 Haenseel, W., 186, 200 Haerer, F., 219, 220, 227 Hagen, A. A., 167, 176, 177, 200 Hagens, S. J., 215, 226 Hager, E. B., 246, 863 Hager, S. E., 111,l4O Haggard, M. E., 221,227 Hagopian, A., 267,291, 292,308 Hague, P., 168, 169, 202 Hakomori, S., 266,267,268,269,270,271, 272, 273, 274, 275, 276, 278,279, 280, 281,282, 283,284,285,287,288,290, 291,294,295,296,299, 300, 301, 302, 303, 304, 309, 310,311, 312,314, 316 Halasz, B., 156, 208 Halber, W., 277, 310 Halberg, F., 167, 200 Halbrecht, I., 254, 261 Hale, H. B., 166, 200 Halgrimson, C. G., 45, 74 Halina, L., 2, 73 Hall, J. G., 60, 67, 69

Hall, J. W., 184, 200 Hall, K., 171, 203 Hall, L. C., 111, 140 Hall, T. C., 90, 140 Hall, W. H., 185, 192, 193, 194, 137 Halliday, W. J., 9, 26, 70 Halperin, M., 286, 314 Halpern, R. M., 116, 137 Hamada, H., 164, 207 Hamada, M., 90,146 Hamilton, J. B., 165, 209 Hamilton, J. M., 322, 362 Hamilton, W. J., 237, 961 Hammond, C. B., 245, MI Hammond, W. G., 20, 59,68, 70 Hamolsky, M., 82, 84, 142 Hamon, M., 159, 801 Hampers, c. L., 246, 863 Hanafusa, H., 266, 281,810 Hancock, R. L., 173, 184, 901 Handa, N., 271,310 Handa, S., 270, 271, 277, 310, 316 Handler, P., 115, 149 Hanna, M. G., 31, 70 Hansen, A. E., 218, 220 Hansen, R. J., 100, 101, 146 Hanshaw, J. B., 224, 926 Hanson, T. L., 100, 140 Haran-Ghera, N., 49, 51, 52, 54, 7'0, 123, 146 Harano, Y., 104, 106, 108, 160 Harder, F. H., 10, 70 Hargreaves, R., 65,68 Harkness, D. R., 117, 119, 141 Harley, R. D., 220, 227 Harris, G. W., 158, 160, 163, 201 Harris, H., 108, 119, 121, 124, 125, 139, 141, 148,147 Harris, J., 121, 149 Harris, J. R., 24, 30, 58, 68 Harris, P. M., 180, 201 Hart, Z., 220, 229 Hartley, J. W., 215, 223 Hartmann, H. A., 172, 2O4 Hartree, A. S., 234, 261 Hartstein, M., 168, 199 Harvey, J. J., 212,227 Hashinotsume, M., 123, 141 Haskell, J., 165, 908 Haskova, V., 256, 261

AUTHOR INDEX

379

Hervet, E., 252, 255, 262 Hass, L. F., 93, 149 Hess, B., 82, 86, 105, l 4 f Hatanaka, M., 266,310 Heston, W. E., 184, 190, 201 Hatzfeld, A., 95, 96, 98, 141, 148 Heuer, K., 271, 311 Hauck-Granoth, R., 123, 146 Hewer, A., 351, 358, 362 Hauge, M., 252, 262 Hiasa, Y., 340,366 Haughton, G., 50, Y6 Hickie, R. A., 103, 136 Haung, A. T., 41, 73 Higashino, K., 123, 141 Haus, E., 167, 200 Higginson, J., 184, 189, 201 Hawkins, R. L., 101,140 Hilburn, J. M., 125, 138 Hawryluk, A., 322, 363 Hildebrand, J., 286, 310 Hawtrey, A. D., 92, 141 Hildemann, W. H., 10, 58, 68, Y4, 171, Hay, J. B., 181, 201 200,216, 230 Hayakawa, T., 90,145 Hill, B. R., 85, 86, 88, 136, 141 Hayashi, S., 134, 150 Hill, R. B., 224, ,929 Hays, E. F., 181, 201 Hindocha, P., 233, 260 Haywood, G. R., 40,57, YO, Y3, 294, 314 Hinks, M., 84, 141 Heald, P. J., 84, 140 Hins, G., 161, 199 Heath, E. C., 268, S10 Hiramoto, R., 35, YO Hecht, F., 220, 22Y Heidelberger, C., 5, 6, 18, 19, 20, 35, 69, Hirata, Y., 97, 103, 160 70, 72, 318, 319, 320, 322, 328, 333, Hirsch, B. B., 170, ,901, 213, 226, ZZY 334, 335, 336, 337, 338, 345, 346, 348, Hirsch, M. S., 212, 217, 257 351, 352, 353, 354, 355, 356,360, 361, Hirschhorn, K., 119, IS6 Hirszfeld, L., 277, S10 562,563, 364,366 Hitotsumachi, S., 330, 362,363 Heinrikson, R. L., 124, 141 Hixukuri, S., 113, 161 Heldt, H. W., 108, 141 Hoch-Ligeti, C., 89, 141, 174, 201 Hellbaum, A. A., 162, 201 Heller, H., 221, 2@ Hodes, S., 111, 142 Hodgett, J., 60, 67, 69 Hellerqvist, C. G., 270, 310 Hodson, A. W., 118, 120, 141 Hellman, L. M., 234,2661 Hellstrom, I., 5, 6, 7, 8, 10, 21, 34, 41, 58, Hoe, S., 187, 198 61, 62, 63, 64, 70, Y3, Y4, 218, 229, Hoeg, K., 233, 261 Holzer, B., 18, Y6 257, 261 Hellstrom, K. E., 2, 3, 5, 6, 7, 8, 10, 18, Hoffbauer, R. W., 106,136 Hoffman, F., 17, Y 8 21, 34, 41, 58, 61, 62, 63, 64, YO, Y l , Y3, 74, 218, 229, 257, ,961 Hoffman, G. C., 221, 230 Hoffman, H. D., 346, 358,364 Helmstein, K., 7, 69 Hoffman, H. E., 185,806 Helweg-Larsen, H. F., 182, 183, 801, 203 Hoffman, H. S., IT, 221, 2 f l Henderson, C. S., 222,2f?0 Hoffmann, D., 346, 347, 364 Hendrickson, R., 213, 214, 229 Herberman, R. B., 33, 41, 58, Y4 Hoffmann, J. H., 183, 195, ,901 Herbst, A. L., 177, 209 Holcomb, R. M., 220, 828 Herman, G., 178, 196 Holeysovska, H., 88, 1.64 Hermans, P. E., 221, ,987 Holland, J. F., 57,68 Herndon, R. M., 224, 230 Hollenberg, P. F., 106, 138 Hers, H. G., 93, 94, 96, 113, 114, 1S8, 141 Holley, R., 287, 305, 306, 310, S f 2 Hershey, F. B., 90, 92, 141 Hollinshead, W. H., 159, 204 Herskovits, J. J., 95, 141 Halloway, M. T., 101, 137 Hertig, A. T., 253, 261 Holm, G., 21, Y,9 Hertz, R., 233, 234, 247, 262, 263 Holman, 11. T., 180, 181, 198, ,906

380

AUTHOR INDEX

Holm=, E. C., 7, 9, 16, 19, 27, 70, 71 ~olmes,E. w., 100, 141 Holmea, R. S., 84,140 Holmstrom, E. G., 165, ,908 Holtzman, J. L., 180, m1 Hornan, J. D. H., 234, 86.3 Homburger, F., 51, 70 HOIWCS, F. A., 109,141 Hong, R., 219, 886 Hooker, C. W., 162, 801 Hopkinson, D. A., 124,125, 141 Horecker, B. C., 93, 1S7 Horecker, B. L., 93, 94, 95, 97, 108, 127, 136, isg, 140, 141, I@, 146, Horn, I(.H., 18, 70, 76 Home, M., 119,148 Homer, M. W., 124, 146 Horowita, M. L., 128,141 Houssais, J. F., 81, 141 Houstek, J., 220, 889 Howard, J. G., 216, 827 Hsie, A. W., 304, 306, 310, 332, 333, S6SJ 364 Hsu, H. H. T., 108,1S9 Hu, C. K., 1 7 1 , W Huber, J., 220, 8.W Huberman, E., 43, 70,325, 326, 328, 329, 334, 345, 351, 352, 353, 369, SBS,S64, 366 Hudson, R. P., 221, 837 Huebner, R. J., 341,342, 362, S6S, S64,366 Huggins, C., 6,70 Hughes, J. S., 169,198 Huhtaniemi, I., 162, 901 Huijing, F., 113,161 Huizenga, I(.A., 221, 827 Hulbert, B., 43, 7S Hulb, V., 83,88, I@ Hulme, B., 224, 887 Humiston, C., 270, SO8 Hunter, R. L., 80, 141 Hurwitz, R., 45, 74 Huseby, R. A., 188,901 Huzino, A., 128, I@? Hydbn, H., 156, 801 Hyyppa, M., 157, 159, 901

I Ibsen, K. H., 106,141 Ichihsrs, A., 112, 113, 136, 141,146

Ichikowa, Y., 273,309 Ideo, G., 88,138 Ievleva, E. S., 10, 71 Ifft, J. D., 156, 901 Igel, H. J., 341, 369 Ikehara, Y., 95,97,98,108, 141, 142, 162 Ikonen, M., 162, 901, 206 Ikura, Y., 109,160 Iliya, F. A., 253, 861 Imamura, K., 134, 149, 160 Imbert, R., 220, 986 Imna, J. K., 298,318 Imssnde, J., 115,148 Inbar, M., 268, 296, 297, 298, SlO, SiS, 330, 331, 360, 383,366 Inglis, N. R., 117, 118, 121, 122, 139 Ingram, H. C., 88,137 Inokuchi, K., 98,141 Inomata, M., 358, 363,364 Inone, H., 99,149 Inone, S.,298,310 Insull, W.,Jr., 180,801 Inui, N., 338,363 Iouchim, H., 215, 8,97 Irk, R., 270, 271,316 Irlin, I. S., 30, 67, 131, 156 Irving, C. C., 181, 195, 201 Is&, S., 283,910 Ishida, Y., 98,148 Ishidate, M., 12, 13, 70 Ishihara, M., 90, 148, 174, 809 ~ShikMa,K.,283, $10 Ishikawa, E., 162,196, 196 Ishikawa, H., 298,310 Ishizuka, I., 272, 310 Isojhe, S., 15, 16, 70, 71 Isselbacher, K. J., 266, S10 Ito, K., 111,160 Ito, N., 340,566 Itoh, H., 111,144 Ivanova, D. J., 345,360 Ivanyi, J., 9, 10, 69 Ivanyi, P., 251, M1 Iviiskovti, E., 251, 861 Iwamura, Y., 115, 148 Iwanaga, M., 270, 271, 316 Iype, P. T., 5,6,20, 72, 320, 333, 336, 340, 368,363,364

AUTHOR INDEX

381

J

Jones, 0. W., 111,149 Jones, P. C. T., 181, %I,?? Jackson, C. D., 195,201 Jones, R. T., 104, 106,136 Jackson, J. F., 219,220,997 Jonsson, N., 214, 989 Jacob, F., 134, 14.8 Jordan, R. T.,'85,141 Jacobs, B. B., 188, 901 Josimovich, J. B., 170, 209 Jacobsohn, D., 160, 166,901 Josipowicz, A., 91, 98, 148 Jacobson, K. B., 81,140 Josse, J., 115, 1.6s Jacobson, K. W.,106,14.a Jost, A., 161, 162, 163, 208 Jacobson, M., 176, 901 Joynson, M. A., 94,139 Jacoby, B., 233,961 Joysey, V. C., 253, 969 Jacques, P.,93, 124, 141, 161 Jull, J. W., 322,363 Jacqueaaon, M., 131,147 Junien, C., 91,98, 133, 1.6s Jarvi, D., 267,310 Junkmann, K., 164, 165,801,908 Jagannathan, S. N., 180, 201 Jurandowski, J., 35, 70 Jainhill, J. L., 287, 306,310 Jakoubkova, J., 251,961 K Jamdar, S. C., 103, 143 Kaariain, L., 269, 313 Javoy, F., 159,801,20S Kabat, E. A., 285, 309 Jean, R., 220, 996 Jeanloz, R., 272, 278, 283, 284, 295, 296, Kadotani~T-J254; Kadowaki, J. I.. 221. 227 300,310.311 Kagiyama; H., ill, @ ; Jeejeebhoy, H. F., 47, 73 Kahan,B.D.,9,22,25,27,28,29, 70, 71, Jegatheesltn, K. A., 125,149 79 Jenkins, M. D., 115,146, 182, 906 Kaku, M., 245,961 Jenkins, V. K., 188, 901 Kakunaga, T., 327,363 Jenkins, W. T., 112,160 Kalant, 0. J., 168,902 Jensen, D. S., 293, 303,313 Kalckar, H. M., 266, 311 Jeremy, D., 224, B O Kalckar, M. M., 307,313 Jerina, D. M., 351,363 Kalii, N., 58, 71, 171, 209,255, 257, 961 Jezyk, P. F., 181, 195,203 Kalnins, V. I., 36. 71 Joassin, G., 94,96,141 Kamahora, J.; 327, 363 Joglekar, M. H., 342, 366 Kamei, T., 169, 20.2 Johnson, B. W., 305,306, ,919 Kamenoff, R. J., 171,909 Johnson, C. E., 234,961 Kamimura, M., 270,316 Johnson, D. C., 160, 9W Kamiya, T.,88, 108,149, 168 Johnson, G., 304,306,310,311 Kamoshita, S., 220, 996 Johnson, G. A., 271,310 Kampschmidt, R. F., 91,142,174, 9W,909 Johnson, G. S., 305,319, 332,333, 363 Kanamaru, R., 349,363 Johnson, H., 91, 1&? Kanda, N., 174, $09 Johnson, L. N., 94, 139 Kang, K. Y., 123, 141 Johnson, R. C., 111, 140 Kano-Seuoka, T., 195, 908 Johnson, 'R. M., 180, %" M., 299,311 Kapeller, Johnson, R. T., 224, 230 Kapitola, J., 168, 907 Johnson, S., 5, 46, 47, 71 Kaplan, H. S., 213, 216, 996, 297 Johnston, G., 90,92, 141 Kaplan, J. C., 100, 101, 102, 1@ Jones, A. E., 170,901 Kaplan, N. O., 80, 81, 82, 83, 84, 89, 90, Jones, C., 332,363 92, 95, 137, 138, 139, 140, 141, 14.2, Jones, E. E., 188, 908 146,148, 160,168 Jones, M. E., 111, 140, 148 Karcher, D., 82, 91, 149, 169

"'

382

AUTHOR INDEX

Katayama, C., 126,142 Katona, F., 89,146 Kato, J., 159,202 Kato, K., 349,360 Kato, R.,175,176,202 Katsunuma, T., 128,142 Katsuta, H., 339,340,349,360,363,366 Katunuma, N., 128,129,132,148 Katzen, H.M., 100,101,102,142 Kaufman, D.G., 220,226 Kawabe, S.,97,98,103,133,142,144,160 Kawachi, T., 97,103,150 Kawahara, K., 93,1.42 Kawai, S.,98,148 Kawanami, J., 270,285,311,Sl4 Kawasaki, H., 283,311,312 Kawashima, S.,165,202 Kawauchi, H., 268,300,511 Kay, H.D., 117,14.9 Kay, H.E.H., 283,511 Kellen, J. A., 120,144 Keller, N.,168,20.9 Kelloff, G. J., 342,562 Kelly, W. D., 217,220,228, 229 Kemp, R.G., 110,160,303,309 Kenkare, V. W., 102,1.48 Kennedy, G. C., 168, 169,202 Kennedy, J. R., 298,511 Keogh, J. R.,187,199 Kerr, S.J., 116,1& Kersey, J. H., 216,217,223,227,228 Ketcham, A. S.,22,69 Ketterer, B., 38,71,564 Khesina, A. Y., 345,360 Khoo, J. C., 109,142 Khramkova, N.I.,30,35, 36,39,67,68, 69,70 Khritovo, S. S., 345,360 Kido, I., 234,261 Kidson, C., 181,202 Kierman, J. A., 287,306,310 Kijimoto, S.,266,267,273,274,290,291, 301,302,304,310,511 Kikuchi, Y., 5,41,74,103,14.2 Kikuyama, S.,165,202 Kildeberg, P., 220,228 Kim, U., 6,71 Kimura, T., 306,307,309 Kincl, F.A., 164,165,202

King, E. J., 117,119,123,124,125,136, 142,146,160 King, J.,79,113,123,125,148,149 Kinlen, L., 45,65' Kinoshita, N., 355,363 Kirby, A. H.M., 184,202 Kirby, D. R. S., 256,261 Kirby, K. S.,181,202 Kirkman, H., 187,188,190,191,202 Kirkman, S.K., 92,149 Kirschbaum, A., 184,20.9 Kisic, A., 270,278,311 Kiso, N.,270,277,316 Kissmeyer-Nielsen, F., 22,71,251,252, 26.9

Kit, S.,32,71 Kitagawa, M., 37,38,39,42,71,74 Kitay, J. I.,167,177,20.9 Kitchener, P. N., 119,l4S Klebe, R.J., 81,147 Klein, E., 2,3,5,6,8, 10,18,22,32,41, 58,69,71,74 Klein, F., 87,88,165 Klein, G., 2,3, 5,6,8,18,41,69,71, 266,311 Klenk, E., 270,271,277,311,314 Klenk, H. D., 269,311 Klethi, J., 108,143 Kletzien, R.,267,31.9 Kline, E.S.,83,91,158,143 Klionsky, B.,270,51.4 Kloppenburg, M.,272,S10 Klovda, P. T., 251,252,261,262 Klouwen, H., 180,199 Knox, W. E., 79,103,108,111,128,129, 132,139,141,145,144,162,172,202 Knudsen, F. U., 89,14s Kobata, A., 268,309 Kobayashi, F., 165,202 Kobayashi, I.,168,209 Kobayashi, K., 132,149,277,311 Kobayashi, T., 159,163,202,209 Koch, M.A., 267,280,281,282,303,309 Kochen, J., 179,204 Kochman, M., 94,95,105,146,160 Kochman, U.,95,96,1-43 Kodama, M., 181,203,358,364 Kodama, T., 181,203 Koen, A., 81,143 Koff, E. B., 176,206

383

AUTHOR I N D W

Koga, K., 245, 261 Koida, U., 94, l @ Koike, M., 90, 146 Koldovsky, P., 7, 9, 10, 31, 32, 58, 68, 69, 71, 251, 261 Koler, R. D., 104, 106, 136, 137, 220, 227 Kolodny, E. H., 267, 279, 290, 30.0 Kolstad, P., 233, 261 Komlos, L., 254, 261 Koneff, A. A., 169, 196 Kongshavn, P. A. L., 171, 203 KopeE, A. C., 248, 261, 862 Koprowski, H., 31,32, 68, 212, 226 Kordon, C., 159, 161, 201, 903,206 Korenchevsky, V., 171, 203 Kornberg, A., 115, 143 Kornberg, S. R., 115, 14.3 Korner, A., 179, 195, 203 Korosteleva, T. A., 42, 71 Koscielak, J., 278, 283, 284, 295, 296, 300,310, 311 Kostic, D., 285,311 Kotin, P., 344, 364 Kovarik, S., 283, 309 Kowalsky, A. G., 93, lJ8, 14s Koyama, H., 323,363 Kraemer, P. M., 268,293, 294,311 Kragt, C. L., 166, 203 Krahl, M. E., 101, 146 Kramer, M., 164, 206 Kramkova, N. I., 131,136 Krant, M. J., 121, 122, 139, 150 Kraus, A. P., 81,143 Kraus, S., 286, 299, 311 Krebs, E., 303, 309 Kremer, W. B., 41, 73 Krim, M., 338,361 Kripke, M. L., 56, 71 Krishnamurthi, S., 187, 207 Krivit, W., 220, 221, 223, 826, 228, 230 Krodek, O., 220, 22.9 Krokowski, E., 168, 203 Kronman, B. S., 16, 17, 71, 75 Kruger, W., 300, 313 Krystal, G., 110, 143 Krzywda, U., 95, 96, 14s Kuff, E. L., 18, 71 Kuftinec, D., 84, 139 Kuhn, R., 27ll272,s11 Kumar, M., 179, 803

Kumaresan, P., 169, 203 Kumon, A., 303,311 Kun, E., 91, 92,1@, 14.9 Kunii, A., 188, 209,215, 2q7 Kuntzman, R., 176, Nl Kunze, H., 168, 203 Kunsel, B., 174, 203 Kupchyk, L., 81,162 Kurimura, T., 32, 71 Kurland, A. A., 169, 183, 206 Kuroda, Y., 129, 132, 148 Kurokawa, T., 268, 296, 298,314 Kuroki, T., 327, 328, 340, 346, 348,349, 352, 353, 354, 562,363, 364,366, 366 Kusama, T., 157, 196 Kuschner, M., 321,364 Kutzbach, C., 105, l 4 l Kvamme, E., 128, 143, 162 Kwatkowska, D., 96, 96, 143

1 Labhsetwar, A. P., 166, 903 Lacko, A. G., 94,97,137,140 Ladosky, W., 166, 903 La Due, J. S., 85, 88, 14,162 Laga, E., 117, 160 Lagarde, E., 220, 226 Lai, C. Y., 94, 143 Laine, R. A., 270, 278, 302,311 LalBgerie, P., 87, 91, 160 Lam, K. W., 124, 126, l&,168 Lambrecht, A. F., 220,928 Lampert, F., 220, $98 Lamvik, J., 221, ,888 Landing, B. H., 220, 996 Lane, W. T., 341, 342,369, 364, $66 Langman, M. J. S., 121, 143 Langvad, E., 90, 143 Lankester, A., 268, 295, 308 Lanson, B. L., 82, 136 Lantz, R. S., 57, 76 LappB, M. A., 6, 43, 44, 45, 47, 51, 52, 54, 55, 71 Lardy, H. A., 94, 146 Lamer, J., 113, 161 Laskin, S., 321, 364 Laskowski, J., 277,310 Lasnitzki, I., 318, 319, 320, 322, 561, 364 Laszlo, J., 115, 146, 182, 206 Latarjet, M. R., 181, 203

384

AUTHOR INDEX

Latner, A. L., 83, 86, 87, 90, 91, 118, 120, 121, 141, 143 Lauenstein, K., 270, 811 Laurence, E. B., 40, 69 Lausch, R. N., 41, 59, 71 Lavrin, D. H., 33, 74 Law, L. W., 45, 67, 71, 212, 213, 214, 226, 228 Lawler, S. D., 248, 249, 251, 252, 260, 261, 262 Lawley, P. D., 346, 348, 357, 861, 562 Laws, J. O., 322, 862, 364 Layzer, R. B., 109, 14s Lazar, G., 287, 306, 3rd Lea, M. A., 103, 105, 114, 130, 149, 161, 162

Leathern, J. H., 163, 184, 196, 208 Leavy, M., 243, 268 Lebherz, H. G., 93, 95, 96, 143 Leblanc, L., 131, 161 Leblond, C. P., 182, 193, 199, 207 Lebovici, S., 251, 260 Le Breton, E., 115, 188 Leclerc, M., 88, 162 Ledeen, R., 271, 811 Lee, C. L., 283, SO9 Lee, K. Y., 185, 203 Lee, N. M., 92, 149 Lee, S. H., 186, 20s Lee, S. S., 173, 206 Lee, T. C., 180, 20s Leese, C. L., 90, 144 Leffert, H., 305, 306, 312 Lehninger, A. L., 95, 96, 14.9 Lehmann, V. W. D., 176,20S Leibowitz, S., 217, 227 Leikin, S., 220, 226 Lejneva, 0. M., 10, 71 Leloir, L. F., 113, 14.4 Leonard, E. J., 25, 72 Leonardi, R., 159, 204 Leong, J. L., 355, S66 Lerner, L. J., 191, 20s Leroux, J. P., 106, 137 Lesko, S. A., 346, 358, 364 Lespinats, G., 18, 71 Leuchtenberger, C., 183, 186, 20s Leuchtenberger, R., 186, 20.3 Leuthardt, F., 94, 95, 140, 147, 162 Leuthold, E., 121, 148

Leutz, J. C., 350, 866 Leve, G. O., 101, 137 Leveille, G. A., 106, 144 Leveque, T. T., 156, 203 Levey, R. H., 45, 67 Levi, C., 85, 141 Levin, E., 180, 208 Levin, S., 220, 228 Levine, A. J., 297, 318 Levine, L., 82, 187 Levine, P., 248, 262 Levy, A. I., 220, 226 Levy, R. H., 212, 213, 226 Lewis, D. A., 176, 208 Lewis, J. L., 233, 251, 254, 262 Lewis, L. A,, 171, 203 Lewis, U. J., 170, 201 Leyten, R., 45, 74, 212, 230 Li, C. H., 195, 200 Li, C. Y., 124, 126, 142,144, 162, 182, 20s Li, F. P., 222, 230 Li, M. C., 255, 262 Li, S.-C., 270, 310, 311 Li, Y.-T., 270, 278, 310, 311, 814 Liberti, J. P., 181, 195, 208 Libertun, C., 158, 203, 206 Liebelt, A. G., 110, 137, 188, 203 Liebelt, R. A., 167, 169, 188, 196, 20ty, 207 Liebner, E. J., 220, 227 Lijinsky, W., 174, 197 Likely, G. D., 318, 366 Lilly, F., 215, 216, 228 Lilly, J., 45, 74 Lin, E. C . C., 172, 202 Lin, Y. C., 79, 85, 99, 102, 189, 144 Linder, M., 129, 148 Linder, 0. E. A., 52, 71 Linder-Horowitz, U., 128, 144 Linker-Israeli, M., 47, 54, 55, 74 Linscott, W. D., 9, 71 Linsell, C. A., 187, 204 Lipkin, G., 188, 204 Lippmann, W., 159, 204 Lipschultz, M. L., 256, 268 Lipscomb, H. S., 167, 198 Lipsett, M. B., 162, 204, 233, 234, 262, 268

Lis, H., 268, 295, 296, 297, 809, 811, S13 Lisk, R. D., 157, 166,204 Litwack, G., 109, 188, 864 Litwin, S., 221, 227

AUTHOR INDEX

Livingston, D. C., 297, 309 Livingstone, c. S.,220, 226 Llewellyn-Jones, D., 248, 262 Llorente, P., 105, 144 Lloyd, C . W., 162, 166, 204,206 Lloyd, W . D., 162, '164, 166, 204 Lo, C. H., 102, 103, 106, 107, 108, 136, 139,144 Lo, J . S., 120, 144 Locke, S. J., 88, 137 Loeb, H . G., 180, 204 Loeffler, R. K., 238, 260 Lohr, G. W., 106, 136 Loewenstein, W . R., 266, 311, 312 Logan, G. B., 221, 230 Loisillier, F., 90, 131, 149, 161 Loke, Y . W., 187,204, 253, 262 Loken, M. K., 180, 203 London, W . T., 223,230 Longman, E . S., 181, 195,203 Lonsdale, D., 221, 230 Lopez, A. M., 245,262 Lopez, C., 224, 225, 228 Loraine, J . A., 234, 262 Lorentz, M., 159, 201 Lotlikar, P. D., 172, 204 Love, W . C., 85, 161 Lowenstein, J. M., 92, 1& Lowenthal, A., 82, 91, 149, 162 Lowman, J. T., 220,226 Lucke, B., 174, 209 Ludovici, P. P., 343,362 Lueders, K. K., 18, 71 Luffman, J., 121, 125, 1.41, 143 Lund, G. H., 166, 20.4 Lunden, L. G., 121, 136 Lundgren, E., 123, 136 Lundgren, G., 57, 73, 294, 314 Lundh, B., 87, 144 Lundin, L. G., 124, 144 Luongo, L., 21, 74 Luppis, B., 127, 146 Lurie, M., 49, 51, 52, 70 Lyman, K., 111, 144 Lyman, R. L., 180, 181, 206 Lyster, S. C., 166, ,804

M McArthur, W . P., 214, 228 McBride, R. Z., 21, 74

385

McCann, S. M., 158, 167, 204, 206 McCarter, J . A., 53, 68 Maccioni, H . J., 292, 308, 312 McCluer, R. H., 271,310, 314 McCollester, D. L., 26, 71 McCue, M. M., 21, 74 McDevitt, H . O., 216, 228 McEndy, D. P., 213, 228 McEntegart, M. G., 35, 40, 72 McFadden, J., 221, 227 MacFarland, V . W., 267, 278, 279, 290, 309,312 McGeeney, K., 121, 139 McGuire, E. J., 289, 293, 313 Machado, E., 55, 68 McKay, R. H., 81,146 McKee, R. W., 85, 139 McKenzie, B. F., 221,297 McKhann, C. F., 10, 40, 57, 70, 73, 224, 228, 294, 314 McKinley, T. W., 52, 54, 55, 71, 215, 228 MacKinnon, K . J., 90, 145 McLean, E. P., 3, 5, 9, 16, 27, 68,72 McLean, P., 92, 101, 103, 144, 161 MacLeod, R. M., 170, $04 McMurray, C. H., 94, 139 McPherson, A., 106, 144 Macpherson, I., 267, 282,287, 288, 290, 292, 309,313,316 Maeda, K., 245, 261 Maekawa, K., 165,204 Makela, O., 215, 298 MLrtensson, E., 270, 312, 314 Magee, P. N., 17, 21, 71, 116, 144 Maggi, V., 126, 144 Magill, G. B., 88, 144 Maher, V . M., 20, 71, 346, 358, 364 Mahler, R., 114, 148 Mahnert, A., 162, 207 Mahy, B. W . J., 85, 1& Main, J . M., 1, 3, 6, 20, 73, 266, 313 Major, I. R., 322, 361 Makita, A., 267, 270, 271, 277, 302, 312, 316 Malaskova, V., 88, 144 MalejkaGiganti, D., 339, 362 Malinow, M. R., 158,204 Mallady, J., 99, 137 Malmgren, R. A., 52, 54, 55, 71, 215, 228 Malone, J . J., 100, 141 Manahan, C. P., 245, 262

386

AUTHOR INDEX

Mancini, R. A., 162,198 Mandel, L. R.,116,140 Mandel, P., 108, 143 Manjeshwar, R.,99, 100, 102,144,149 Mannick, J. A., 47, 70 Mannucci, P. M., 88, 138 Mantel, N., 184,199 Mantaavinos, R.,115, 144 Maqueo, M., 164, 165,208 Marchant, D., 245, 260 Marchesi, V. T.,275,276,318 Marco, R.,105,1.44 Marinello, E., 94,143 Marinetti, G.V.,179,20.4 Marinkovich, V. A., 220, 286 Mariotti, A., 86, 137 Mariz, I. K., 170, 197 Markee, J. E., 159, 204 Markert, C.L., 2,41, 71,80,82,84,133, 1361 1411 14.6 Markowitz, A. S., 220, 289 Markowitz, H.,221, 227 Marnorston, J., 178,179, 207 Marquardt, H.,5,6,20,72,328, 346,347, 352, 353,356,362,363,364,366 Marsch, J. B.,269, 293,316 Marsden, A. T. H., 184,904 Marsh, W.L., 285,309 Marshall, C. M., 105, 1.44 Martin, G.,223, 230 Martin, G.S., 266, $12 Martin, R. G., 195,904 Martin, W.T., 298,318 Martinez, C., 160,204 Martinez-Carrion, M., 111, 144 Martinezde-Dretz, G.,94,l4S Maryak, J. M., 341,368 Mesamune, H., 283,318 Masopust, J., 220,229 Masseyeff, R.,131, 132, 133,140, 144, 147,161 Masters, C.J., 81,84,91,94,95,96, 13% 140,141, 1.64,146,149 Mastuda, J., 283,312 Masuji, H.,328,366 Mesukawa, A., 283,318 Math&,G.,43, 71,132, 1&,252,255, 862 Mathies, J. C., 174, 199 Matson-Williams, E. J., 81,137 Matauda, A., 163,209

Makuda, Y., 128, 132,1.42 Matsumoto, T.,26, 71 Matsushima, T.,97,98, 103, 133, 142, 1441 148, 160 Matsuyama, E., 166,204 Matthew, 0. D., 234, 263 Matthews, R. E. P., 116, 136 Matthijsen, R.,234, 863 Mauleon, P., 162,804 Mawer, H.M., 9 4 , I S r Mayfield, E. D., 111, 144 Medenis, R.,220,829 Meezan, E., 267,287,318, 316 Mehler, A. H., 93, 140 Meienhofer, M. C.,110,144 Meighen, E. A., 94, 14.4 Meister, A., 80,144 Meites, J., 194,$09 Melnick, I., 181, 197 Meltzer, M.S.,25, 72 Meranze, D. R., 186,200 Merrill, J. P.,246, 263 Merwin, R. M., 18, 69 Mess, B., 156,208 Metzgar, R. S., 41,73 Meuwisen, H. J., 216, 220,826, 888 Meyer, P. C., 188,204 Michaeli, D.,221, 8.%+ Micheau, C.,91,98, 133,148 Michuda, C.M., 111, 144 Midgley, A. R., 235, 262 Mikulska, Z. B., 5,41,59, 78 Miljanich, P.,180, 181,206 Millar, I. B., 187,204 Miller, D. A., 340,364 Miller, D. G.,220,888 Miller, D. M., 101,137 Miller, E. C., 20, 21,35,42,71,7.2,73, 79,1.44, 172, 173,174, 179, 195,198, 804,349,351, 357,364 Miller, J. A., 20, 21, 35,42, 71,7%,73, 79,1.44, 172, 173, 174, 179,195, 198, 804, 346, 349,351,357,864 Miller, J. F. A. P., 46,70, 72 Miller, K., 267,51% Miller, 0.J., 340,364 Miller, R.W., 220,887 Miller, W. L., 184,189, 191,807 Mims, R. B., 167,199 Minaguchi, H.,159,909

387

AUTHOR INDEX

Minielly, J. A., 221, 230 Mints, D. H., 170, 202 Mirvish, S. S., 346, 366 Mitani, M., 218, 223, 22.9 Mittwoch, U., 161, 204 Miwa, S., 106,160, 161 Miyatake, T., 270, 310 Moberger, G., 7, 69, 233, 260 Moe, P. J., 221, 928 Msller, F., 80, 82, 84, 144 Moller, G., 9, 10, 58, '72 Mogensen, B., 251, 252, 962 Moguilevsky, J. A., 158, 903,204, 906 Mondal, S., 5, 6, 20, '72, 335, 336, 337, 364 Monod, J., 106, 134, I@, 144 Montemurro, D. G., 194, 906 Moog, F., 119, 144 Moon, R. C., 169, 906 Moore, B. W., 124, 125, 126, 136, 144 Moore, C.R., 162, 168, 902,206 Moore, D. J., 129, 161 Moore, E. G.,299, 319 Moore, M., 8, 10, 14, 20, 22, 23, 24, 25, 27, 31, 33, 34, 35, 38, 40, 61, 67, 68 Moore, W. W., 166, 206 Mora, P.,267, 278, 279, 280, 282, 290, 308, 309,312 Morace, G.,163, 197 Morawiecki, A., 275, 319 Moretti, R. L., 45, 68 Morgan, D. L., 348, 366 Morgan, F. J., 234,262 Morgan, J. L., 220, 298 Mori, R., 104, 106, 160 Mori-Chavez, P., 186, 206 Morimura, H., 104, 105, 106, 108, 160 Morimura, Y., 344, 364 Morin, R. J., 180, 206 Morino, Y., 111, 144,146 Morissey, R. W.,220, 228 Morn, P., 290, 309 Morris, H. P., 37, '72, 79, 85, 91, 97, 99, 100, 101, 102, 103, 106, 107, 109, 110, 111, 112, 114, 115, 128, 129, 134, 135, 156, 156, 137,138, 139, 142, 143, 144, 1461 1471 148, 149, 160,161, 162, 182, 184, 186, 191, 206, 9U7,267, 279, 308 Morse, 1). E., 93, 94, 95, 108, 136, 137, 146

Morton, D. L., 3, 6, 7, 8, 9, 16, 19, 27, ro, 71,w, rs Morton, J. I., 215, 229 Morton, M. E., 217, 221, 226 Moscona, A., 299, 312 Moser, H. W., 220, 930 MOSS,D. W., 117, 118, 119, 124, 146, 160 Mosser, D. G., 180, ,903 Mossman, H. W., 237, 961 Mosekowska, A., 161,906 Mountcastle, W., 167, 198 Mourant, A. E., 248,969 Moustacchi, M. E., 181, 903 Moy, P., 215,987 Muheback, O., 216, 928 Muller, M., 12, 42, '79 Muhlbock, O., 190, 199 Muir, C. S., 184, 187, 906 Mukerjee, D., 222, 998 Mulay, A. S., 185, 192, 908 Muller, H. K., 40, '72 Muller-Oerlinghausen, B., 174, 903 Munson, B., 115, 1.44 Murakami, W. T., 267, 278, 285, 295, 303, 310 Muramatsu, T., 29, 72 Murasawa, Y., 163, 909 Murmanis, L., 183, ,903 Murphy, M. L., 220, 998 Murphy, P., 114, 143 Murphy, S. M., 90, 92, 141 Murray, J. E., 246, 963 Murray, R. K., 267, 269,279, 280, 281, 282, 293, 303,305, 308,316 Musella, A., 103, 143

N Nabo, R. C., Jr., 165, 908 Nachlas, M. M., 80, 146 Nadal, C.,182, ,906 Nadel, E., 16, 79 Nadler, H. L., 245,960 Nagablushanam, A., 99, 146 Nagai, Y., 283,319 Nagasawa, H., 170, 194,909 Nagata, C., 358,363,364 Nagel, B., 254, 26'9 Nagle, R. G., 245,960 Nagy, A., 89, 146 Nahas, S., 220,930

388

AUTHOR INDEX

Nair, V., 175, 195, 206 Nairn, R. C., 35,40, 72 Najarian, J., 224, 228 Najman, A., 106, 1.97 Nakagawa, Y ., 134, 148 Nakamura, K., 5, 41, 74, 268, 300, 311 Namba, M., 340, S66 Nan, K. H., 182,203 Nance, W., 81, 146 Nandi, J., 167, $06 Narayan, O., 224, 230 Nathanson, L., 122,146 Nathenson, S. G., 28, 29, 72, 7S, 76 Natori, Y., 173, 806 Nau, F., 116,146 Navon, R. S., 181, 195,20S Nazarian, I., 87, 140 Neale, F. C., 117, 119, 1.97, 143 Nebert, D. W., 349, 350, 355, S60, 364 Neely, C. L., 81, 1.6s Negelein, E., 85, 161 Neil, M. W., 124, 146 Neilands, J. B., 80,146 Nelson, B. D., 124,146 Nelson, D. H., 167, 199 Nelson, R. L.,325, 328, 329, 355,361,562 Neri, G., 107, 146 Nettleship, A., 318, S62 Neuhaus, 0. W., 178, 179, 206 Neuman, Z., 250, 251,255, 262, 263 Neumann, F., 164, 165, 201,202, 205 Neumann, H., 123,146 Newlon, M., 157, 204 Newsholme, E. A., 127, 146, 161 Ni, L. Y., 283, 309 Nicolson, G., 268, 275, 297, 298, 312, 314, 331, S64 Nielsen, B. I., 320, 322, S61 Niemeyer, H., 100, 140 Niemi, M., 162,206 Nigam, V. N., 114, 146 Nilsson, L., 234, 261 Nishikawa, M., 109,160 Nishimura, K., 273,283, 309,312 Nishimura, S., 181, 195, 199, 270, 516 Nishino, H., 128, 142 Nishiyama, H., 188, 206 Nishiauka, Y., 303,311 Nisselbaum, J. S., 81, 89, 111, 112, 117, 118, 137, 146

Nissen, N. I., 90, 146 Nitowsky, H. M., 100, 101, 142 Noach, E. L., 168, 169, 206, 209 Noda, L., 146 Nomoto, K., 46, 72 Noonan, K. D., 2,69, 286, 296, 299, 308, S12, 331,561 Noordhoek, J., 175, 206 Nordling, S., 305, 306, si4 Nordmann, Y., 94, 96, 97, 146, 148 Norman, N., 233,261 Noumura, T., 162,206

0 Ober, W. B., 232,253, 262 Odaka, T., 283,318 Odell, T. T., Jr., 188, 201 Odell, W. D., 233,234, 262, 26s O’Dwyer, E. O., 84,139 Oesch, F., 355, S64 Oettgen, H. F., 3, 5, 9, 16, 27, 68, 72 Oettle, A. G., 184, 201 Ogawa, K., 113, 136, 146 Ohama, K., 254, 262 Ohlson, L., 233, 260 ohmori, Y., 117, 146 Ohuti, K., 283, S12 Okabe, K., 90,146 Okada, Y., 97, 142 Okey, R., 180, 206 Okuno, G., 109, 160 Old, L. J., 3, 5, 6, 7, 8, 9, 10, 16, 27, 28, 39, 41, 56, 58, 59, 67, 68, 69, 72, 214,216,226, 228 Oleinick, A., 188, 207 Oliver, J. A., 90, 146, 338, 362 Omi, J., 90, 146 Omori, Y., 176, 202 @Neal, M. A., 185, 186, 192, 193, 200, 206,206 ONeill, C. H., 302, 312 Ono, T., 115, 142, 146 Onoda, K., 175, 202 Opie, L. H., 127, 146 Opler, S. R., 16, 72 Oppenheim, J. J., 17, 72, 254, 262 Oren, M. E., 58, 70 Orr, A. H., 235, 243, 245, 260, 263 Orr, G. R., 173, 206 Osawa, T., 268, 295,296,319, Sl4

AUTHOR INDEX

Oshiro, Y., 340, S64 Oski, F. A., 100, 141, 220,229 Ostroff, G., 217, 229 Ostwald, R., 180, 181, 206 Otani, T. T., 111, 146 Otten, H., 296,314 Otten, J., 284, 305, 306, 312 Otto, P., 112, 148 Ove, P., 115, 146, 182, 206 Oyama, K., 283, 312 Ozanne, B., 297, S12 Ozer, J. H., 22, 72

P Padberg, G., 271,311 Padgett, B. L., 224, 229 Page, A. R., 218, 222, 229 Page, J. G., 175, 206, 206 Palekar, L., 321, 364 Pan, F., 173, 206 Pandov, H., 97, 98, 146 Pardee, A. M., 266, 309 Pardoe, G. I., 300, 312, 314 Park, B. H., 219, 224, 225, 227, 228 Park, W. W., 232, 253, 262 Parker, J., 188, 202 Parker, R. T., 245, 261 Parks, M. E., 85,139 Parmiani, G., 54, 56, 72 Paschkis, K. E., 181, 184, 185, 197, 208 Passeron, S., 105, 146 Pastan, I., 304, 305, 306, 310,311, 312, 332, 333, 363 Pasternak, G., 9, 17, 18, 32, 70, 72, 76 Patterson, c., 84, 146 Patterson, M. K., Jr., 173, 206 Pattillo, R. A., 237, 262 Paul, D., 305, 306, 312, 313 Paul, J., 266, 312 Payet, M., 96, 148 Payne, L. N., 214, 226 Payne, P., 184, 198 Peake, G. T., 170, 197 Peanasky, R. T., 94,146 Pearce, L., 171, 206 Pearlmann, P., 284, 285, 314 Pearson, H. A., 220, 229 Peeler, T. C., 181, 195, 201 Peled, A., 54, 70 Pelham, R. W., 159,196

389

Pelkonen, R., 221, 229 Pellegrino, M. A., 25, 73 Peng, W. T., 182, 203 Penhoet, E. E., 93, 95, 146 Penn, I., 45, 73, 74, 217, 224, 229 Perdue, J. L., 267, 312 PerezSoler, A., 220, 229 Perlmann, P., 7, 21, 69, 72 Perlov, S., 220, 228 Pernis, B., 218, 229 Perova, S. D., 30, 67, 131, 136 Perry, D. J., 185, 192, $06' Perry, S., 254, 262 Pesce, A., 80, 81, 139, 146 Peters, E. L., 173, 208 Peterson, J. A,, 134, 146 Peterson, R. D. A., 213, 214, 215, 217, 220, 226, 229 Petrissant, G., 116, 146 Pfaff, D. W., 157, 206 Pfeiffer, C. A., 160, 161, 170, 200, 206 Pfleiderer, G., 80, 81, 82, 84, 89, 106, 137, 146, 147, 161, 162 Philip, J., 82, 146, 161 Philips, F. S., 346, 347, 364 Phillips, G. E., 123, 126, 136 Phillips, M., 217, 227 Phoenix, C. H., 163, 209 Pi, A. F., 165, 202 Picon, L., 161, 162, 202 Pieczynski, W. J., 343, 361 Pieprzyk, J. K., 167, 206 Pierce, G. B., 131, 134, 146 Pierce, G. E., 8, 10, 61, 65, 70, 72 Pierce, J. G., 234, 261 Pierce, J. E., 118, 120, 147 Pietra, G., 186, 207 Pietruszko, K., 97, 146 Pike, M. C., 248, 249, 260 Pilch, Y. H., 26, 59, 68, 69, 72 Pilkis, S. J., 100, 101, 102, 146 Pimm, M. V., 10, 11, 13, 14, 17, 20, 31, 32, 33, 34, 40, 67 Pincus, G., 178, 196 Pincus, T., 215, 229 Pinto da Silva, P., 275, 276,297,513 Pitot, H. C., 37, 73, 79, 99, 142, 146 Plagemann, P. G. W., 82, 85, 146 Planinsek, J., 15, 37, 42, 70, 71 Plummer, D. T., 83, 146

390

AUTHOR I N D m

Pogell, B. M., 127, 160 Pogson, C. L.,106, 146 Pokier, L. A., 20, 37, 73 Polackova, M., 170, 190, 206, 209 Pollack, R. E., 279, 313 Pontremoli, S., 127, 139, 146, 149 Pool, P. E., 83, 161 Popescu, N. C., 337, 369 Porta, G. D., 184, 186, 206, 807 Porter, J. B., 255, 262 Porter, J. C., 158, 204 Porter, K. A., 45, 74, 253, 262 Posen, S., 117, 119, 137, 1.43, 146 Posener, K., 85, 161 Post, O., 83, 149 Postnikova, Z. A., 30, 35, 67, 69, 131, 136 Potter, C. S., 111, 139 Potter, M., 18, 69, 178, 179, 199, 213,228 Potter, V. R., 79, 133, 146 Pouillart, P., 88, 162 Poulik, M. D., 220,229 Powell, H. R., 245, 962 Poznanska-Lumde, H., 90, 147 Prehn, R. T., 1, 3, 5, 6, 18, 19, 20, 36, 43, 44, 45, 47, 52, 55, 66, 68, 71,73, 215, 229, 255, 2668,266, 313, 335, 337,360,364 Pressman, B. C., 124, 138 Pressman, D., 15, 16, 35, 37, 38, 39, 42, 70, 71, 74 Price, E. B., 232, 253, 262 Price, M. R., 23, 24, 30, 58,64,68 Price, P. J., 341, 342, 343, 368, 364 Prokop, O., 268,300,515 Puck, T. T., 304, 306,310, 332, 333,363, 364 Pudelkewicz, C., 180, 206 Pulkkinen, M. O., 176, 206 Pullman, A., 344, 364 Pullman, B., 344, 364 Pump, K. K., 220, 226 Purchase, H. B., 214, 215, 229 Purchase, I. F. H., 174, 189, 206 Puro, K., 271, 313 Putnam, C. W., 45, 74

Q Quabbe, H. J., 170, 206 Quelin, S., 131, 133, 147 Quenum, C., 131, 161

Quimby, E. H., 168, 206 Quinn, P. G., 175,206

R Rabbat, A. G., 47, 73 Rabinowitz, Y., 88, 92, 147 Rabinowitz, Z., 329, 330, 331, 362,365, 364 Rabstein, L.,342,366 Rach, K., 168,203 Radl, J., 220, 229 Radzichovskaja, R., 214, 229 Raine, L., 118, 120, 141 Rajewsky, K., 81, 136, 147 Rajkumar, T. V., 93, 94, 95, 146, 147 Ramirez, V. D., 167, 906 Ran, M., 62, 73 Rand, H. J., 181,198 Randall, L. M., 234, 261 Randoux, A,, 87,140 Rankin, J. B., 219,226 Rao, S., 99, 161 Rapp, F., 31, 32,41, 59, 69, 71 Rapp, H. J., 16, 17, 25, 60, 65, 71,72, 76, 170, 198 Rapport, M. M., 270, 277, 278, 302, 311, 313 Rasmussen, R. E., 347,364 Ratnam, 5. S., 245,248, 250, 261, 262 Rawlins, G., 248, 249, 260 Rawson, R. W., 243, f?63 Ray, F. E., 186, 906 Ray, P., 294, 314 Ray, P. X., 57, 75, 256, 265 Raynaud, A., 161, 206 Read, P. A., 117, 123, 126, 136 Rebeyrotte, M. N., 181, 203 Reckel, R. P., 170,207 Reece, R. P., 162, 207 Reed, M., 158, 201 Reed, W. B., 220, 229 Rees, R. J. A., 212, 227 Reichlin, S., 170, 207 Reif, A. E., 121, 122, 139 Reifenberg, U., 284, 296, 314 Reiner, J., 6, 7, 11, 73 Iteinhardt, L. R., 88, 136 Iteisfeld, R. A,, 22, 23, 27, 28, 29, 71, 72 Reisman, R., 123, 161 Rejal, T. H., 116, 149

AUTHOR INDEX

Remmer, H., 175, 207 Renkonen, O., 269,313 Rennels, E. G., 212, 926 Rennkampf, F., 270, 277,311 Rensing, U., 95, 147 Resko, J. A., 162, 206 Reuber, M. D., 36, 73, 96, 98, 148, 184, 185, 191, 192, 193, 194, 199, 206 Revess, C., 166,206 Reynolds, R. D., 112, 147 Resnikoff, C. A., 366 Rhim, J. S., 341, 342, 366 Rhines, R., 168,199 Ribacchi, R., 47, 73 Rich, ., 106, 1& Rich, M. A., 213, 229 Richard, M., 101, 136 Richards, F. F., 217,226 Richards, F. M., 94, 149 Richards, 0. C., 93, 147 Richardson, A. P., Jr., 182, 200 Richardson, H. L., 185, 186, 192, 193, 200,206

Richardson, U. I., 168, 902 Richmond, H. G., 35,40, 72 Richterich, R., 84, 86, 89, 104, 106, 147, 161 Richters, A. R., 109, 141 Riddick, D. H., 115, 116, 147 Riddle, O., 171, 206 Riechens, L. A., 348,361 Riedkin, S. T., 193, 907 Riesman, L. E., 218, 223, 229 Rieu, D., 220, 226 Rife, U., 82, 136 Rifkind, D., 224, 229 Rigas, D. A., 220, 2 f l Riley, V., 85, 147 Riley, W., 303, 309 Rinfret, A. P., 185, 192, 200 Rioche, M., 131, 147 Rios, A., 57, 73, 256, 263,294, 314 Rivlin, R. S., 234, 262 Robb, D. A., 84, 140 Robbins, P. W., 267, 281, 287, 288,'289, 290, 316,313,316 Roberts, J. I)., 165, 2ff6 Roberts, It. B., 84, 159 Robertson, C. H., 185, 186, 192, 193, 200,206

391

Robertson, H. T., 302, 313 Robertson, M. R., 224, 230 Robinet, M., 220, 226 Robins, R. A., 41, 61, 62,. 64, 68 Robinson, D. S., 180, 201 Robinson, E., 250, 251, 255, 962, 263 Robinson, E. A., 307,313 Robinson, J. C., 118, 120, 147 Robison, R., 116, 147 Robson, E. B., 119, 143, 147 Roche, A. F., 170,206 Roche, J., 84, 116, 147, 160 Roe, F. J. C., 46, 72, 187, 206 Itoller, M.-It., 319, 320, 322, 333, 366 Rolley, R. T., 20, 70 Rdne, E., 89, 146 Roosa, R. A., 212, 226 Root, W. S., 171, 200 Rosa, J., 89, 147 Rosado, A., 91,99, 14,147 Itosahn, P. D., 171, 206 Rosalki, S. B., 83, 86, 147 Rose, A., 101, 147 Rose, N. R., 129, 136 Rosell-Perez, M., 113, 161 Roseman, S., 267, 289, 290, 293, 309,318 Itosenau, W., 8, 73 Rosenberg, A., 293, 303,313 Itosenthal, I. M., 220, 229 Rosenthd, K. L., 126, 140 Ross, G. T., 233, 234, 238, 263 Ross, M. A., 171, 203 ROSS,M. R., 190, 2M Itossi, E., 86, 147, 161 Ross-Mansell, P., 38, 71 Rossell, J. A., 99, 137 Roth, A., 217, 999 Roth, J. S., 112, 149 Roth, S., 289, 293, 313 Rothblatt, G. M., 345,361 Rothenberg, M. S., 184, 185, 208 Rowe, W. P., 215, 929 Rowland, L. P., 109, 143 Rowlatt, U., 187, 206 Rowley, J. Y., 173, 198 ltowson, K. E. K., 85, 14.4 Roy, A. B., 176, 206 ltoy, A. K., 178, 179, 2ff3 Itoychondhury, R., 115, 147 Itoyer, P., 220, 226

AUTHOR INDEX

Rozengurt, E., 105, 138, 147 Rozenszajn, L., 125, 147 Rubin, B. A., 53, 54, 73 Rubin, B. L., 176, 206 Rubin, H., 266, 312, 313 Rubin, I., 221, 227 Rubinstein, H. S., 169, 183, 206 Rubinstein, L., 158, 205 Ruch, F., 186, 203 Ruddle, F. H., 81, 137, 147 Rudolph, R. H., 254, 263 Rumke, C. L., 175,205 Riimke, Ph., 178, 207 Rumsfeld, H. W., Jr., 184, 189, 191, 207 Runner, C. R., 179,199 Ruoslahti, E., 305, 306, 314 Rushede, J., 89, 140 Rushworth, A. G. J., 243, 260, 263 Russell, A., 322, 363 Russell, P. J., 109, 142 Russell, P. S., 256, 263 Rustigian, R., 121, 122, 139 Rutter, W. J.;93, 94, 95, 96, 97, 105, 106, 136, 138, 143, 146, 147, 160

S Sachs, L., 2, 38, 41, 43, 70, 73, 268, 285, 296, 297, 302, 309, 310, 311, 313, 318, 323, 324, 325, 326, 327, 328, 329, 330, 331, 334, 360, 361, 362, 363, S64, 365 Saheki, R., 114, 150 Saini, P. K., 119, 146 Saito, H., 267, 273, S l S , S14 Saito, M., 80, 103, 147, 267, 291, 313 Saito, T., 271, 281, 282, 288, ,910 Sakakibara, F., 268, 300, 311 Sakane, Y., 110, 150 Sakiyama, H., 267, 281, 288, 289, 290, 292,813 Sakurai, Y., 268,296, 314 Salaman, D. F., 168, 169, 202 Salas, J., 127, 147 Salas, M., 99, 127, 147, 149, 151 Salmon, R. J., 180, 203 Salsman, K., 271, S l l Salthe, S. N., 81, 148 Salzberg, S., 299, 31S, 326, 363 Sambrook, J., 297, S l l Sams, B. F., 182, 183, 208

Sanada, Y., 129, 142 Sanchez, R., 100, 140 Sanders, F. K., 18, 73, 321, 338, 361, 365 Sanders, L. L., 321, 361 Sanderson, A. R., 28,29, 73 Sandler, R., 159, 207 Sanel, F. T., 215,227 Sanford, B. H., 57, 73, 256, 263, 286, 293,294, 295,299,308,313 Sanford, K., 307, 313, 318, 365 Santamarina, E., 162, 207 Santolaya, R. C., 159, 198 Sapico, V., 109, 138 Sardet, C., 345, 361 Sarles, H., 120, 148 Sasovetz, D., 103, l 4 S Sato, G., 305, 306, 312 Sato, H., 267, 291, 298, 306, 307, 309, S10, 313, 327, 328, 349, 363, 565, 366 Sato, J., 340, 366 Sato, K., 20, 73, 113, 114, 115, 127, 135, 148,150, 328,366 Sato, S., 98, 103, 134,l48, 147, 148, 150 Savage, E. E., 180,200 Sawyer, C. H., 159, 204 Sazama, K. P., 255, 263 Scacchi, P., 158, 206 Schabel, F. M., Jr., 239, 263 Schachman, H., 93,94, 144, 149 Schafroth, P., 84, 86, 89, 147 Schalch, D. S., 170, 207 Schapira, F., 82, 84, 89, 91, 93, 94, 95, 96, 97, 98, 105, 107, 133, 138, 141, 145, 147,148 Schapira, G., 84, 96, 97, 133, 138, 148 Scheinberg, S. L., 170, 207 Schelsinger, D., 300, S13 Schengrund, C.-L., 293, 303,313 Schenkman, J. B., 175, 2007 Schiaffini, O., 158, 203,205 Schidlovsky, G., 7, 16, 19, 70 Schier, W. W., 217, 229 Schiller, K. W., 106, 141 Schilling, E. L., 318, 365 Schimke, R. T., 100, 101, 102, 140, 142, 148 Schindler, W. J., 169, 207 Schlamowitz, hl., 117, 118, 120, 121, 145, 148 39, , 73, 120, 148 Schlesinger, ill.

AUTHOR INDEX

Schloss, G. T., 343, 362 Schmid, A., 95, 147 Schmid, R., 114,148 Schmidt, C., 168, 206 Schmidt, E., 89, 112,136, 148 Schmidt, F. W., 89,136 Schmidt, P. J., 247,263 Schmidt, W., 112,148 Schmitt, hf., 90, 92, 141 Schneck, S. A., 45, 73, 224, 229 Schneid, V., 251, 261 Schneider, H., 270, 278, 313 Schnitzler, S., 268, 300, 313 Schobel, B., 125, 140 Schoch, H. K., 86, 88, 89, 149 Schooley, C., 183, 200 Schooley, R. A., 193, 207 Schreiberova, O., 168, 207 Schriefers, H., 177, 207 Schrift, M. H., 217, 229 Schroter, G. T., 45, 74 Schrub, J., 221, 229 Schultz, A. M.,267, 290, 309 Schumm, D. E., 358,362 Schwalbach, K., 108, 141 Schwartz, M. K., 79, 111, 112, 121, 126, 137, 148 Schwartz, R. S., 216, 217, 219, 227, 229 Schwarz, E. L., 149 Sciarra, J. J., 234, 260 Scott, J. S., 247, 263 Scott, R. W., 275, 276, 312 Scott, T. W., 180, 195, 207 Scow, R. O., 193, 207 Seagers, W., 119, 136 Seck, I., 87, 148 Seck, J., 131, 147 Sedgwick, R., 220, 225, 226, 229 Seegar, G. E., 234,261 Sefton, It. M., 266, 313 Segal, S. J., 160, 162, 207 Segrest, J. O., 275, 276, 312 Sehrbundt, H., 268, 300, 314 Seibert, R. A., 350, 561 Seifert, H., 285, 313 Seifert, W., 305, 313 Seigler, H. F., 41, 73 Sekely, L. I., 339, 362 Sela, B. A., 21, 73, 268, 296, 311 Seligman, A. M., 80, 145

393

Selim, A. S. M., 99, 148 Selkirk, J. K., 328, 345, 351, 352, 353, 363, 364, 365 Sellers, A. L., 178, 179, 207 Sellers, E. A., 168, 202 Sells, B. H., 195, 201 Selye, H., 166, 207 Sendelbeck, L. It., 168, 202 Senfert, J., 180, 206' Seyama, Y., 302, 312 Shabad, L. M., 321, 345,360, 365 Shanmugaratnam, K., 187, 207, 208 Shannon, A,, 180,205 Shanta, V., 187, 207 Shapiro, S., 127, 159 Sharma, C., 99, 100, 102, 138, 144, 149, 152 Sharma, R. M., 102, 148 Sharon, N., 2, 73, 268, 295, 296, 297, 309, 511, 313 Shatton, J. B., 100,101,103,107, 134,149, 152 Shaw, C. R., 81, 143, 149 Shea, J. R., Jr., 182, 207 Sheedy, R. J., 95, 149 Sheid, B., 112, 149, 173, 207 Sheinin, R., 267, 269, 280, 281, 282, 293, 303, 305, 315 Shelton, E., 318, 365 Shen, L., 267, 287, 313 Sheppard, J. R., 297, 305, 308, 309, 313, 332, 365 Shevket, F., 171, 196 Shibasaki, K., 193, 208 Shibuya, M., 97, 133, 144 Shichijo, K., 168, 209 Shigeno, N., 28, 69 Shih, H. E., 178, 207 Shimada, A., 28, 29, 73 Shimazu, T., 195, 207 Shimkin, M. B., 183, 186, 200, 207 Shimojo, H., 267, 312 Shinton, N. K., 186, 207 Shiozima, S., 283, 312 Shirai, A., 113, 141 Shiraishi, T., 97, 150 Shoham, J., 296, 297, 313, 331,365 Shoham, O., 125,147 Shonk, C. E., 79,85, 149 Short, B. F., 170, 207

394

AUTHOR INDEX

Shows, T. B., 81, 149 Shreffler, D. C., 216, 228 Shubik, P., 186, 2U7 Shulman, J., 250, 262 Shulman, N. R., 220, 829 Shuster, J., 220, 229 Sia, c. L., 127, 149 Sibley, J. A,, 95, 96, 149 Siddiqui, B., 267, 270, 271, 272, 273, 274, 279, 280, 285,310, 314 Siddiqui, S. A., 83, 143 Sidransky, H., 184, 207 Siegel, B. V., 215, 229 Siegel, S., 175, 195, 206 Siegler, R., 213, $29 Siegmund, H., 162, 207 Sigman, B., 101, 149 Sigurjonsson, J., 187, 207 Silberberg, M., 43, 73 Silmser, C. R., 170, 199 Silverstone, H., 186, 190, 197, 208 Simmons, D. A. R., 284, 285, 314 Simmons, R. L., 57, 73, 224, 288, 256, 263, 294,314 Simon, L. N., 116,137,149 S i o n s , K., 269,313 Sims, P., 328, 343, 344,349, 351,352,353, 358, 361, 362,363, 364, 366 Sinclair, N. R., 53, 68 Sine, H. E., 93, 149 Singer, S. J., 275, 297, 314 Singer, S. T., 298, 312 Sing-Fie, C., 173, 806 Singh, R. P., 162, 2U7 Singhal, R. L., 127, 162 Sinha, D., 323, 361, 363 Sinks, L. F., 221, 226 Siurala, M., 221,229 Sivak, A., 339, 366 Sjogren, H. O., 2, 3, 5, 6, 7, 8, 18, 41, 61, 62, 63, 64,65, 68, 70, 71, 73, 74, 213, 214, 218, 229, 266, 314 Skillen, A. W., 83, 86, 143 Skilleter, D. N., 92, 149 Skipper, H. E., 239, 263 Skipski, V. P., 268, 270, 277,313, 314 Skom, J. H., 245,360 Skoryna, S. C., 185, 192, 193,207 Slade, T. A., 352, 358, 362 Sladen, W. J. L., 84, 14.4

Slein, M. W., 99, 149 Small, M., 47, 54, 55, 74 Small, P. A., 118, 160 Smelby, B., 220, 229 Smid, I., 164,197 Smith, C., 5, 41, 59, 72 Smith, C. H., Jr., 220, 896 Smith, D. F., 57,74 Smith, J. D., 94,143 Smith, J. K., 117, 118, 146 Smith, J. Y., 172, 174, 204 Smith, M., 178, 179, 2U7 Smith, P. E., 160, 162,807 Smith, R. A., 116, 137 Smith, S. R., 92, 144 Smithies, O., 80, 81, 146 Smollich, A., 157,198 Snell, G. D., 171, 800, 216,230 Snell, I(.C., 186, 206 Sniffen, R. C., 162, 807 Snoijink, J. J., 220, 8.98 Snyder, A. L., 222, $30 Sobey, W. R., 170, 807 Soderman, D. D., 100, 101,142 hborg, M., 258, 863 Siiren, L., 254,263 Soetens, A., 91, I.@ Sokol, D. M., 82, 140 Solnick, C., 217, 287 Solomon, A., 221, ,%87 Solomon, M. L., 183, 806 Sols, A., 99, 102, 105, 127, 138, 144, 147, 149, 161 Soria, J., 88, 162 Sorof, S., 21, 74 Sorokina, J. D., 321,366 South, M. A., 217, 2,86 Southam, C. M., 6, 7, 11, 73, 217, 2.90 Spahn, G. J., 342,364 Spain, J. D., 185, 186, 192, 193, 200 Spaull, G.. V., 234, 261 Spector, B. D., 217, 828 Spellman, C. M., 84, 139 Spencer, H. H., 86, 149 Spencer, N., 124, 125, 141 Spolter, P. D., 94, 149 Srinivassn, P. R., 115, 116,1.@, 160 Srivastava, S. K., 127, 162 Stadlan, E. M., 220,221,230 Staehelin, M., 116, 136, 182, 207

395

AUTHOR INDEX

Stafford, M. A., 111, 149 Stafford, R. O., 166, 804 Stagg, B. H., 90, 149 Stalmans, W., 113, 138, 141 Stambaugh, R., 81, 83, 149 Stamm, N. B., 105, 16.9 Stanislawski-Birencwajg,M., 30, 74, 90, 118, 123, 131, 133, 136,149, 161

Stark, C. R., 188, 907 Starkie, S. J., 45, 74 Starkweather, W. H., 86, 88, 89, 149 St. Ameadt, C., 57, 68 Starzl, T. E., 45, 74, 217, 224, 8.99 Stasney, J., 184, 185, 908 Staudt, J., 157,199 Steck, T. L., 294,314 Stefano, F. J. E., 159, 198, 908 Stegink, L. D., 81, 149 Stein, A. M., 92, 149 Stein, G., 303, 308, 314 Stein, J. H., 92, 149 Steinbeck, H., 164, 806 Steinberger, E., 162, 908 Steiner, D. F., 113, 149 Steinman, H. M., 94,149 Steinmetz, M. H., 106, 149 Steinmdler, D. S., 54,71 Stellner, K., 269,272, 273, 310, 314 Stellwagen, E., 93, 149 Stenzel, P., 104, 106, 136 Stern, K., 285,314 Sternberg, S. S., 338,361 Stewart, H. L., 186, 806 Stewart, L. C.,17, 76 Steyn, M., 174, 189, 806 Stick, H. F., 36, 71 Stimson, C. W., 220,899 Stirpe, F., 108, 137, 173, 174, 197 Stjernswiird, J., 5, 47, 52, 53, 59, 74 Stock, C., 268,314 Stockert, E., 28, 41, 69, 216, 826 Stocks, P., 188, 808 Stoffyn, A., 270, 314 Stoffyn, P. J., 270,286,310, 314 Stolbach, L. L., 121, 122,139, 160 Stolaenbach, F. S., 80, 81, 139, 146 Storpe, F., 105,136 Stott, D. F., 112, 147 Stotz, E., 179, M4 Stout, M. G., 116,137

Strecker, H. J., 176, %" Streeter, D., 116, 137 Strober, W., 217, 220, 221, 296, 830 Strobel, G., 267, 280, 281, 282, 303, 309 Stryckmanns, P. A., 286,310 Stutman, 0.S., 52, 61, 74,215,830 Stutzman, E., 174,801 Suda, M., 99, 107, 108, 109, 134, 160 Sue, F., 105, 108, 160 Sueoka, A., 195, M8 Sugimura, T., 97, 98, 103, 133, 134, 148, 1.48, 160, 338, 363

Sugino, Y., 268, 298, 314 Suk, W. A., 342, 343, 364 Sumi, T., 110,160 Sun, S. C., 184,900 Sur, B. K., 124,160 Suretzeff, V., 125, 136 Susor, W. A., 94, 105, 106, 160 Sussman, H. H., 117, 118, 121, 160 Sutherland, It. C., 40, 78 Sutnick, H. I., 223, 930 Suzuki, C., 270, 318 Suxuki, M., 169, 193, 808, ,908 Suauki, N., 97, 160 Suzuki, R., 114, 160 Suxiiki, S., 269, 270, 271, 316 Svenneby, G., 128, 143 Svennerholm, L., 270, 271, 274,324 Svoboda, J., 7, 71 Swain, A. P., 342, 368 Swanson, H. E., 163, 908 Swanson, H. H., 169,808 Swarte, F. J., 182, 183, 195, 901, 808 Sweat, M. L., 165, 808 Sweeley, C. C., 270, 278, 311, 314 Sweeney, M. J., 97, 136 Swenson, A. D., 94, 160 Swiech, K., 41, 69 Swift, M. R., 222, 830 Swin, H. E., 85, 146 Symeonidis, A., 185, 192, 808 Szent&gothai, J., 156, 808 Saulman, A. E., 247, 963 Szwarcfarb, B., 158, 806 Szybalski, W., 20, 71

T Tackwia, A., 300,313 Taeuber, K. E., 186, 800

396

AUTHOR INDEX

Tagashira, Y., 358,364 Takahashi, A., 175, 176, 202 Takahashi, H., 113, 141 Takahashi, Y., 123, l 4 l Takaki, F., 162, 196, 196 Takakura, K., 98, 126, 140, 148 Takanaka, A., 175, 202 Takano, K., 337,562 Takaoka, T., 339, 340, 349, 360, 363, 366 Takasugi, M., 10, 22, 32, 58, 74 Takasugi, N., 166, 208 Takayama, S., 338, 363 Takayanagi, M., 175, 202 Takeda, H., 114,160 Takeda, K., 5, 7, 41, 74 Takemura, Y., 168, 209 Taketa, K., 127, 160 Taketomi, T., 286, 31.4 Takeuchi, M., 268, 298,314 Takeya, K., 46, 72 Tal, C., 286, 314 Taleb, N., 220, 230 Talwar, G. P., 182, 195, 200 Tan, K. K., 123, 160, 187, 208 Tanaka, K., 186, 203 Tanaka, K. R., 106, 160, 161 Tanaka, T., 104, 105, 106, 108, 109, 110, 134, 142, 160, 186, 203 Tanford, C., 93, 142 Tanigaki, N., 37, 38, 39, 42, 71, 74 Taniuchi, K., 134, 160 Tannenbaum, A., 208 Taranger, L. A., 7, 8, 34, 61, 74 Tarui, S., 109, 160 Tashian, R. E., 100, 139 Tashima, C. K., 243, 263 Tasseron, J. G., 346, 366 Tata, J. R., 179, 180, 181, 182, 195, 208, 209 Tatarinov, Y. S., 131, 160 Tatibana, M., 111, 160 Taylor, A., 111, 142 Taylor, C. B., 105, 107, 110, 136, 160 Taylor, D. 0. N., 215, 226 Taylor, J. F., 95, 160 Taylor, R., 234, 261 Taylor, R. B., 45, 67, 212, 226 Taylor, R. T., 112, 160 Teather, C., 267, 275, 278, 280, 296, 510 Temin, H. M., 299,312

Temkine, H., 106, 137 ten Bensel, R. W., 220, 221, 230 Tennant, J. R., 216, 230 Tennant, R. W., 31, 70 Teoh, E. S., 245, 248, 250, 261, 262 Tepperman, J., 183, 198 Terracini, B., 184, 206 Terranova, T., 107, 146 Terasaki, P., 45, 74, 251, 262 ThBret, C., 87, 91, 160 Thoai, N. V., 84, 150 Thomas, B. S., 59, 69 Thomas, E. D., 254, 263 Thomas, L., 211, 230 Thomas, M., 234, 261 Thompson, R., 221, 227 Thompson, U. B., 110, 137 Thonier, M., 59, 61, 68 Thorne, C. J. R., 92, 160 Thorsbecke, G. J., 214, 228 Thorsby, E., 22, 71 Thudicum, J. L. W., 270, Sld Thung, P. J., 178, 207, 208 Tierfelder, H., 270, 314 Tieslau, C., 268, 295, 308 Tildon, J. T., 101, 149 Tillack, T. W., 275, 276, 312 Tillinger, K. G., 165, 208 Tilney, C. G., 298, 314 Timberger, R., 243, 263 Timiras, P. S., 158, 204 Ting, C. C., 33,41, 74 Ting, R. C., 32, 45, 71 Ting, R. C. Y., 116, 140 Tinoco, J., 180, 206 Tisdale, V., 220, 227 Tissenbaum, B., 162,197 Tivey, H., 188, 208, 238, 260 Tock, E. P. C., 187,208 Todaro, G. J., 222,223, $28, 830,266, 279, 287, 306, 310, 313, 314, 327, 337, 341,360,363,366 Toh, Y. C., 180, 182, 183, 185, 192, 194, 206,208 Tohme, S., 220, 230 Tomasi, T. B., 219,230 Tomino, I., 128, 129, 142 Tomita, M., 268,296,314 Tonoue, T., 169,202 Torok, E. E., 245, 260

397

AUTHOR INDEX

Toth, B. A., 184, 208 Tousimis, A. J., 16, 72 Towatari, T., 129, 142 Tower, D. B., 173, 208 Toyoshima, K., 340,366 Tracy, G. S., 217, 2H Trahan, E., 7, 16, 19, 70 Trainin, N., 47, 54, 55, 74 Tran Ba LOC,P., 98,148 Traniello, S., 127, 146, 149 Traut, M., 326,363 Treat, R. C., 47, 69 Treger, A., 51, 70 Tremblay, A., 178, 196 Trentin, J. J., 190, 208 Troedsson, H., 307, S1S Troll, W., 182, 208 Troop, R. C., 167, 176, 177, 200, 208, 209 Tsai, M. Y., 110,160 Tsikarishirli, T. N., 40, 69 Ts'o, P. 0. P., 186, 203, 346, 358, 364 Tsou, K., 80, 146 Tsubura, Y., 340,566 Tsuda, M., 268,298,914 Tsuiki, S., 113, 114, 127, 148, 160 Tsunematsu, K., 97,160 Tsutui, E., 116,160 Tullner, W. W., 162,204 Tung, K. A. K., 221, 2S0 Turner, C. D., 165, 209 Turner, C. W., 169, 203, 206 Turner, D. M., 90, l4S Turner, H. C., 342, 566 Tus, L., 164, 197 Tveit, B., 128, 14s Tyndall, R. L., 129, 161

U Udenfriend, S., 159, 2009,351, 365 Uhlenbruck, G., 268, 284, 285, 293, 296, 300, Sl2, S13,314,316 Ui, M., 110, 160 Ukita, T., 267, 268, 291, 296, SlS, 314 Ulbrey, D., 266, 311 Umberger, E. G., 163, 196 Umehara, Y., 115, 146 Upton, A. C., 188, 201 Urbach, G., 170, 209,256,261 Ureta, T., 100, 140 Uriel, J., 30, 31, 74, 131, 133, 161

Urquhart, J., 167, 177, 209 Ursprung, H., 82,144 Utsugi, K., 174, 209 Uaman, B. G., 267, S1S

V Vaage, J., 59, 60, 65, 74 Vachter, J., 164, 197 Vaes, G., 124, 161 Vagner, D., 88, 162 Vaheri, A., 305, 306, Sl4 Vaitukaitis, J. L., 234, 26s Valavaara, M., 159, 201 Valentine, R., 95, 146 Valentine, W. N., 106, 160, 161 Vance, D. E., 270, $14 Van den Berghe, G., 113, 1.41 Van Den Broecke-Siddre, A., 178, 207 Vandeputte, M., 45,74,212,213, 230 Van der Helm, H. J., 82,162 Vanderlaan, W. P., 170, 201 Van Der Straeten, M. E., 86, 162 van der Werff ten Bosch, J. J., 163, 208 Van Dieten, J. A. M. J., 169, 209 van Doorninck, W., 256, 261 Van Duuren, B. L., 339,366 van Hall, E. V., 234,26S Vanha-Pertulla, T., 124, 161 van Hell, H., 234, 26s Van Holde, K. E., 93, 138 Vanhoude, J., 286, S10 Van Hove, W. Z., 86, 162 Van Maercke, Y ., 86, 162 van Potter, R., 112, 147 Van Rees, G. P., 169, 209 Van Sande, M., 82,91, 149, 162 Van Wijhe, M., 84, 136 Varco, It. L., 217, 228 Vasiliev, J. M., 345, 360 Vass, W., 341, 366 Vassent, G., 159, 203 Veazey, It. A., 181, 195, 201 Vedder, I., 161, 199 Venuta, S., 266, 312 Verdin, A., 245, 260 Vernon, M. L., 342, 366 Vesell, E. S., 80,81, 82, 83,85,86,88, 146, 161, 162, 175, 206,206 Vesselinovitch, S. D., 184, 209 Vestling, C. S., 81, 149

398

AUTHOR INDEX

Vioari, G., 285, 309 Vicker, M. J., 314 Vieillard, A., 87, 140 Vietti, T. J., 220, 2.96 Vihko, R., 162, 901 Vilar, O., 162, 198 Villm-Palasi, C.,113, 161 VillBe, C. A., 84, 161, 181, 182, 199, 909 Vifluela, E., 99, 127, 147, 149, 161 Virgilio, L., 55, 68 Vivoina, C., 125, 138 Vlahakis, G., 184, 901 Vogt, M., 323,366 Vogt, P., 281, 282, 288,310 Vojtiskova, M., 170, 906,908 von Bermuth, G. V., 221, 930 von Fellenberg, R., 104, 106, 161 von Gr&, A., 17, 18, 70, 72, 76 Von Kleist, S., 132, 161 Von Zuppinger, K., 86, 147, 161 Vose, B. M., 11, 32, 33, 68 Vuopio, P., 221, 999

Wang, L., 286, 295, 299, 308 Wang, M., 9, 76 Warburg, O., 79, 85, 96, 161 Ward, F. E., 41, 73 Warms, J. V. B., 101,147 Warner, G., 63, 70, 74 Warner, J., 222, 999 Warnock, M. L., 85, 121, 123, 161 Warren, J. C., 165, 906 Warren, L., 267, 269, 282, 291, 292, 293, 308,316 Wassarman, P. M., 95, 141 Watanabe, H., 268, 300,311 Watanabe, K., 269, 272,310, 314 Watanabe, M., 85, 146 Waterhouse, J., 184, 198 Wattenberg, L. W., 355, 366 Wattiaux, R., 124, 138 Way, S. A., 90, 143 Webb, M., 9, 26, 70 Webb, T. E., 110,143 Weber, A., 133,161 Weber, C. S., 97, 147 W Weber, G., 85,105,107,127,130,136,160, Wachsmuth, E. D., 80, 81, 84, 89, 1&, 161, 169 Weber, H., 89, 147, 266, 319 147, 161 Webster, A., 245,260 Wads, H., 111, 1.6.6, lg Weiler, E., 35, 40, 70, 76 Wagner, B. P., 184, 186, 906,907 Weiner, L., 224, 930 Wagner, J. L., 50, 76 Weinhouse, S., 79, 85, 91, 94, 97, 99, 100, Wagner, J. W., 160, 161, 164, 165, 900 Wagoner, J. K., 188,199 101, 102, 103, 106, 107,108, 109, 115, Wahren, B., 58, 76 131, 134, 135,136, 138, 139,144, 147, Wakman, H. A., 79,136 '@I l-69, 16' Weinstein, B., 21, 76 Walborg, E. F., 57, 74, 76 Waldenstriim, J., 132, 161 Weinstein, D. B., 269, 293, 316 Walder, B. K., 224, 930 Weinstein, H. G., 85, 169 Waldmann, T. A., 217, 220, 221, 930 Weinstein, I. B., 181, 195, 196, 199 Waldren, C. A., 333, 364 Weintraub, R. M., 170, 198 Weisburger, E. K., 172,179,185,191, N9, Walford, R. L., 216, 930 Walker, D. G., 99, 101, 105, 1.6.6,161 341,366 Walker, D. L., 224, 999 Weisburger, J. H., 172, 179, 185, 186, 191, Walker, P., 266, 319 192, 909, 341,366 Walkim, K. G., 111, 139 W e b , D. W., 56,71 Weiss, J., 162, 906 Wallace, B. H., 283, 311 Wallace, J. C., 127, 161 Weiss, L., 57, 76 Wallach, D. F. H., 22, 38, 79, 76 Weiss, M. C., 92, 134, 136, 146, 169 Walter, S. I., 82, 86, 141 Weissman, I., 170, 199 Walters, E., 101, 161 Weisz, J., 162, 164, 166, 904 Weksler, M., 101, 140 Wane, A. B., 87, 148 Wang, I. Y., 347,364 Wells, L. J., 162, 909

AUTHOR INDEX

Welsch, C. W., 194,209 Welsh, K.I., 29, 73 Welshons, W. J., 170, 190, 197 Wenner, C.E.,79,169 Wepsic, H.T.,16,60,65,76 Werner, S. C.,168, 9&3 Westman, A., 165,908,234,961 Westort, C., 176, 197 Westphal, U.,167, 168, 199, 200 Westrop, J. W., 42, 70 Whang, J., 254,969 Wheeler, N.,170,199 Wherrett, J., 267,269,273,280,281,282, 293, 303,305,316 Whitby, L. G., 117,118, 146 White, D.,289,293, 313 White, L. P., 188, 909 Whitehead, J. K.,38, 71 Whitehead, R., 168,909 Whitford, T.W., 123, 138 Whitten, W. K.,234,&33 Whyley, G.A., 90, 149 Wicks, L.F., 178, 909 Widnell, C.C., 182;908, 909 Wiebel, F. J., 350,354,362,366 Wiepndt, H.,271,272,273,310,311,916 Wieland, T., 80, 81,82,16% Wieme, R. J., 82,86, 121, 163 Wigzell, H.,28, 76 Wilcox, W. S., 239, 963 Wade, C. E., 235,960,M3 Wiley, C. E., 101, 14% Wilk, M., 358,366 Wilkins, L.,166, 909 Wilkinson, J. H.,79,82, 83,86, 87,88,90, 120, 121, 139, 140, 146, 147,162 Wdliams, D.,266,309 Williams, 0. M.,341,366 Williams, T. L., 181, 197 Williams-Ashman, H.G.,179, 180, 182, 195, 208 Williamson, S., 253, 281 Wilson, A. C., 82, 169 Wilson, K.J., 123,146 Wilson, R.B.,234, 961 Wilson, R. E., 246,965 Wilson, S. J., 221, 237 Windhorst, D.,222, 230 Winegrad, A. J., 100, 141 Winstead, J. A., 93,169

399

Wintzer, G., 293,516 Wimler, R. J., 57, 76,268, 275, 293, 316 Witabsky, E. Z., 277,316 Withycombe, W.A., 83, 90, 146,147 Witkop, B.,351, 363 Witz, I. P.,62, 73, 76 Wivel, N. A., 18, 71 Wold, M.F., 93,162 Wolf, A., 25, 76 Wolf, H.,95,162 WoM, E.,90,137 Wollaeger, E.E.,221,897 Wollemann, M.,89, Wollman, A. L.,165, ED9 Wonderlich, J. R.,298,919 Wood, D.,3, 6, 7, 16, 79 Wood, W.A., 127,146' Woodfm, B. M., 93,94,97,147 Woodfin, R.,94,169 Woodruff, M.F.A., 216,997 Wray, V. L., 267, 512 Wray, V. P., 57, 76 Wright, A. W.,190,197 Wroblewski, F., 82, 83, 85, 86, 88, 140, 1441 146, 1471 166 Wu, H. C., 267, 287,313,516 Wuntch, T.,83, 168 Wurster, D. H., 187, ,909 Wurtman, R.J., 159, 196 Wurzor, D.J., 94,1& Wyatt, R.G., 220, 296 Wyman, J., 106,1& Wynder, E.L.,321,561

Y Yagi, Y., 15, 16, 37,38, 39,42, 70, 71, 74 Yaginuma, T.,163,909 Yam, K. M.,175,909 Yam, L.T., 124, 126, I.@, 1 4 , 168 Yamada, T., 168,909,340,366 Yamaguchi, Y., 283,312 Yamakawa, T., 269, 270, 271, 273, 277, 309, 310, 312,316 Yamamoto, K., 169,909 Yamamoto, M.,306,307,309 Yamamoto, R.S.,185, 186,191, 909 Yamamoto, S., 174, ,909 Yamamura, H.,303,311 Yamamura, Y., 123, 141 Yamane, K.,28,29,75, 76

400

AUTHOR INDEX

Yamawaki, S., 5, 41, 74 Yanagi, S., 05, 98, 108, 134, 139, 141, 160, 162 Yanai, R., 170, 209 Yang, H.J., 273,283,316 Yasin, R., 90, 162 Yasuhira, K., 46, 76 Yaks, F. E., 167, 168, 177, 200, 202,209 Yazaki, I., 160, 161, 209 Yen, S., 184, 209 Yendt, E. R., 121, 140 Yip, M. C. M., 111, 162 Yogeeswaran, G., 267, 269, 279, 280, 281, 282, 293, 303, 305,308, 316 Yokojima, A., 112, 113, 136, 146 Yokoro, K., 188, 209 Yokoyama, S., 270,277,316 Yoneda, M., 115, 162 Yong, J. M., 121,162 Yoshmura, Y., 129, 162 Yosida, T. H., 328, 366 Yosieawa, Z., 270, 283, 312 Young, E. M., 2I, 74 Young, J. c., 341, 362 Young, R. R., 220,230 Young, W. C., 163,200, 209

Younger, L., 113, 140 Yunis, E. J., 216, 227 Yuspa, S. H., 348, 366

Z Zaltzman-Nirenberg,P., 159,209,351,263 Zarafonetis, C. J., 88, 89, 14.9 Zavadil, M., 251, 261 Zbar, B., 16, 17, 60, 65, 72, 76 Zeckwer, I. T., 174, 209 Zelickson, A. S., 222, 230 Zemplenyi, T., 171, 209 Zengerle, F. S., 124, 138 Zilber, L. A., 10, 71 Zmbelman, R. G., 163,209 Zimmerman, H. J., 85, 162 Zimmerman, S. B., 115, 143 Zinkham, W. H., 81, 136,152 Zittoun, R., 88, 162 Zondag, H. A., 87, 88, 163 Zucker, L. M., 176, 197 Zucker, M. B., 125, 163 Zuckerman, H., 250, 251, 255, 262, 26C Zuelzer, W. W., 218, 221, 223, 227, 229 Zurhein, G. M., 224, 229 Zwilling, E., 82, 137

SUBJECT INDEX A ABO blood groups, in trophoblastic neoplasia, 247-250 2-Acetylaminofluorene, immunogenicity of tumors induced by, 15-16 Adenohypophysis, sex differences in physiOlog~of, 159-167 S-Adenosylmethionine synthetase, sex differences in, 173 Adenylate kinase, isozymes of, in cancer, 108-109 Adrenal gland, sex differences in physiology of, 167-168 Adrenocortical steroids, metabolism of, sex differences in, 176-177 Acid phosphatases isozymes of, 123-125 in cancer, 125-127, 131 Aflatoxin B,, metabolism of, sex differences in, 174 Agglutinin, tumor-cell glycolipid reactmion with, 300 Alcohol dehydrogenase isozymes, in cancer, 92-93 Aldehyde dehydrogenase isosymes, in cancer, 93 Aldolase isozymes of, 93-96 in cancer, 96-98, 130 Alkaline phosphatases isozymes of, 116-121 in cancer, 121-123, 130 properties of, 120 Alkylnitrosamine, immunogenicity of tumors induced by, 16-18 Amino acid transferase isozymes, in cancer, 112-113 Aminoazo dyes, rejection antigens in tumors induced by, 12-14 isolation of, 22-25 Androgen metabolism, sex differences in, 176 Antigens, tumor-associated, see Tumorassociated antigens Antiglycolipid antisera, tumor-cell glycolipid reaction with, 300-302

Antilymphocyte serum, effect on chemical carcinogenesis, 47-52 hbparaginase, sex differences in, 173-174 Aspartate aminotransferase isozymes, in cancer, 111-112

B Biochemical funct,ions, sex differences in, 171-183 Blood, isozymes in, in cancer, 85-89 Blood groUP, glycolipids and glycoproteins of, in human tumors, 283-285 B. K. in immunodeficiency, 224-225

C Cancer (See also Tumors) host resistance to, genetic factors in, 215-216 immunodeficiency and, 211-230 isosymes and, 77-153 fetal forms in, 131-132 modifications in serum, 130 Carbamyl phosphate synthetase isozymes, in cancer, 111 Carcinogenesis (chemical) cell antigen changes during, 42-45 in culture, 317-366 embryonic antigen expression in, 30-35 in hamster embryo cells, 323-333 immunological aspects of, 1-75 immunosurveiliance and, 41-65 in liver cell cultures, 339-341 normal tissue antigen deletion in, 35-41 oncogenic virus combined with, 341-343 in organ cultures, 318-323 sex differences in, 155-209 in 3T3-like systems, 337-338 tumor antigen expression in, 3-21 Carcinogens enzymes sex differences in, 172-174 protein binding by, 179 Catalase, sex differences in, 174

401

402

SUBJECT INDEX

Cell membrane molecular changes in, in malignant transformation, 267-268 of tumors, glycolipids of, 265-315 Cellular immunity, of hydrocarbon-ind u o 4 tumors, 8-9,56-65 Cerebrosides, in animal cells, 270 Choriocarcinoma brain metastases of, immunochemical detection of, 243-244 cellular reaction to, 253-254 cytotoxic therapy of, immune responses to, 258-260 diagnosis and monitoring of, 235-242 fetomaternal immunity and, 256-258 immunological aspectg of, 246-260

D N-Demethylation, sex differences in, 175 Deoxyribonucleic acid (DNA), sex differences in, 182-183 Diethylnitrosamine, rejection antigens in tumors induced by, 25-27 isolation of, 25 DNA polymerase, isozymes of, in cancer, 115 DNA viruses, cancer induced by, immunodeficiency and, 212-213 Drugs, enzymes metabolizing, sex differences in, 174-175 E Enzymes carcinogen-metabolizing type, sex differences in, 172-177 sex differences in, 172-177 Esterases, isozymes of, in cancer, 129 Ehterifying enzymes, sex differences in, 172-173

F Fibroblastic cells, from mouse prostate, carcinogenesis in cultures of, 333-337 Forasman glycolipid, in human tumors, 285-286 Fructose 1,Wiphosphatase isozymes of, 127 in cancer, 127-128

G Galactose oxidase, reactivity with normal and tumor cell glycolipids, 294-295 Ganglioside, in human tumors, 285 Genetics, human disorders involving cancer risk in, 222-224 8-Glucuronidase, sex differences in, 174 Glutaminase isozymes,in cancer, 128-129 Glycogen synthetase, isozymes of, in cancer, 113-114 Glycolipids in experimental tumors, 277-283 of human leukemic cells, 283-286 of human tumors, 283-286 of surface cells reactivity of, in normal and tumor tkue, 293302 .significance of changes in, 302-308 synthesis of, cell contact in tumors and, 286-290 of tumor cell membrane, 265-315 Glycoproteins, of surface cells, reactivity of, in normal and tumor tissue, 293302 Glycosphingolipids, in cell membrane, structure and organization of, 269277 Glycosylhydrolases, reactivity with normal and tumor cell glycolipids, 293294 Glycosyltransferases, changes in tumor cells, 290-293 Growth hormone, sex differences in physiology of, 169-170

H Hamster embryo cell cultures, chemical carcinogenesis in, 323-333 Hexokinase isozymes of, 99-102 in cancer, 102-104 HL-A antibodies, in troblastic tumors, 252-253 HL-A antigens, in trophoblastic tumors, 250-252 Human chorionic gonadotropin (HCG) after hydatidiform mole, 244-246 detection methods for, 235 as tumor index substance, 234-260

403

SUBJECT INDEX

Humoral antibody, hydrocarbon-induced tumors and, 9-12,5645 Humoral immunity, disorders involving, cancer development in, 217-219 Hydatidiform mole, HCG assays in, 244246 Hydrocarbons (polycyclic, aromatic) binding to cell macromolecules of, 346349 metabolic activation of, 349-358 metabolism of, 343-346 rejection antigens in tumors induced by, 3-7 isolation of, 25-27 Hydrolases changes in tumor cells, 290-293 isozymes of, in cancer, 116-129 N-Hydroxylation, sex differences in enzymes for, 172, 175 Hypothalamus, sex differences in physiology of, 155159

I Isocitrate dehydrogenase, isozymes of, in cancer, 92 Immunochemistry, of choriocarcinoma, 246-260 Immunodeficiency cancer and, 211-230 graft vs..host models in, 216-217 human diseases, 217-225 virus- and chemical-iiduced, 214-215 drug-induced, 224 virus isolation in, 224-225 Immunology, of chemical carcinogenesis, 1-75 Isozymes cancer and, 77-153 fetal forms and, 131-132 definition of, 77-78

1 Lactic dehydrogenase ~ S O Z of, ~ ~80-85 W in cancer, 85-91, 130 Lactosylceramide in animal cells, 270 serum factor relative to, 286 Lectin, w e Phytoagglutinin

Leukemia glycolipids in cells of, 283-286 high risk of, in genetrichuman disorders, 222-224 Lipids, sex differences in, 179-181 Liver carcinogenesis in, sex differences and, 155-209 cell cultures of, chemical carcingenesis in, 339-341 tumors of, sex differences in, 184-186 Lyases, isozymes of, cancer and, 93-99

M Malate dehydrogenase, isozymes of, in cancer, 91-92

N Nucleic acids, sex differences in, 181-183

0 Oncogenesis, w e Carcinogenesis Oncogenic viruses, effects on chemical circinogenesis,. 341-343 Organ cultures, chemical carcinogenesis in, 318-323 Organs, isozymes in, in cancer, 89-91 Oxidoreductases, isozymes of, cancer and, 80-93

P Phosphofructokinase, isozymea of, in cancer, 109-110 Phosphorylases, isozymes of, in cancer, 114-115. Phosphotransferases, isozymes of, cancer and, 99-111 Physical agents, immunogenicity of tumors induced by, 18-19 Physiology, sex differences in, 156-171 Phytoagglutinin, tumor-cell glycolipid reaction with, 295-299 Proteins, sex differences in, 177-179 Pyruvate kmase isozymes of, 104-106 in cancer, 106-108

R Ribonucleic acid (RNA), sex differences in, 181-182

404

SUBJECT INDEX

tRNA methylases, isozymes of, in cancer, 115-116 R.NA viruses, cancer induced by, immunodeficiency and, 213-214

s Serine dehydratase, isozymes of, 99 Sex differences in biochemical funct,ions, 171-183 in carcinogen-metabolizing enzymes, 172-174 in carcinogenesis, 155-209 in drug-metabolizing enzymes, 174-175 in lipids, 179-181 in liver tumors, 184-188 in nucleic acids, 181-183 in physiological functions, 156-171 in proteins, 177-179 in steroid-metabolizing enzymes, 175177 Skin grafh, from husbands of choriocarcinoma patients, immunoreactivity of, 255-256 Steroids, enzymes metabolizing, sex differences in, 175-177

T 3T3-like systems, carcinogenesis in cultures of, 337-338 T-cell-dependent immunity, disorders involving, cancer development in, 219222 Thymectomy, effect on chemical carcinogenesis, 46-47 Thymidine kinase, isozymes of, in cancer, 110-1 11 Thyroid gland, sex differences in physiology of, 168-169 Tissues, isozymes in, in cancer, 89-91

Transferases, isozymes of, in cancer, 99116 Trophoblastic tumors ABO blood groups in, 247-250 brain metastases of, immunochemical detection of, 243-244 cytotoxic therapy of, immune responses in, 258-260 diagnosis and monitoring of, 235-242 general properties of, 232-234 HL-A antibodies in, 252-253 HL-A antigens in, 250-252 husband skin graft rejection in, 255-256 immunochemistry of, 246-260 Tumor-associated antigens cellular and humoral immune responses to, 56-65 from chemically induced tumors, 3-21 embryonic antigen expression of, 30-35 isolat,ion and characterization of, 21-30 membrane-associated, 28-30 Tumors (See also Cancer) cell surface membranes of, biological properties of, 266 chemically induced tumor-associated antigens on, 3-21 glycolipids in cell membrane of, 265-315 rejection of, in hydrocarbon-induced tumors, 3-7

U Uridine kinase, isozymes of, in cancer, 110 Urinary proteins, sex-associat,ed, 178-179

V Viruses isolation in immunodeficiency, 224-225 oncogenic, sce Oncogenic viruses

CONTENTS OF PREVIOUS VOLUMES Carcinogenesis and Tumor Pathogenesis I . Berenblum Electronic Configuration and Carcin- Ionizing Radiations and Cancer ogen esis Austin M . Brues C. A . Coulson Survival and Preservation of Tumors in Epidermal Carcinogenesis the Frozen State E. V . Cowdry Jtrmes Craig2 The Milk Agent in the Origin of Mam- Energy and Nitrogen Metabolism in mary Tumors in Mice Cancer L. Dmochowski Leonard D. Fenninger and G. BurHormonal Aspects of Experimental roughs Mider Tumorigcnesis Some Aspects of the Clinical Use of T . U. Gardner Nitrogen Mustards Propcrties of thc Agcnt of Rous No. 1 Calvin T . Klopp rind Jeanne C. Sarcoma Buteman R . J . C. Harris Gcnctic Studics in Experimental Canccr Applications of Radioisotopes to Studics L. 11.’ Law of Carcinogenesis and Tumor Tlic Rolc of Viruses in the Production Metabolism of Cancer Charles Heidelberger C . Oberling and M . Guerin The Carcinogenic Aminoazo Dyes Experimental Cancer Chemotherapy James A. Miller and Elizabeth C. C. Chester Stock Miller AUTHOR INDEX-SUBJECT INDEX The Chemistry of Cytotoxic Alkylating Agents M . C. J . Ross Volume 3 Nutrition in Relation to Canccr Etiology of Lung Cancer Albert Tannenbaum und Herbert Richard Doll Silverstone The Experimental Development and Plasma Proteins in Cancer Metabolism of Thyroid Gland Richnrd J. IVinzbr Tumors AUTHOR INDEX-SUBJECT INDEX Harold P. Morris Electronic Structure and Carcinogenic Activity and Aromatic Moleculcs: Volume 2 New DeveloDments A . Pullman and B. Pullman The Reactions of Carcinogens with Macromolecules Some Aspects of Carcinogenesis Peter Alexander P . Rorrdoni Chemical Constitution and Carcinogenic Pulmonary Tumors in Experimental Activity Animals Michael B. Shimkin G. 11.1. Badger Volume 1

405

406

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues Sidney Weinhouse A U T H O R INDEX-SUBJECT

Volume 4

INDEX

The Newer Concept of Cancer Toxin Waro Nakahara and Fumiko Fukuoka Chemically Induced Tumors of Fowls P. R. Peacock Anemia in Cancer Vincent E. Price and Robert E. Greenfield Specific Tumor Antigens L. A. Zilber Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizabeth K . Weisburger and John H . Weisburger

Advances in Chemotherapy of Cancer in Man Sidney Farber, Rudolf Toch, Edward Manning Sears, and Donald Pinkel The Use of Myleran and Similar Agents in Chronic Leukemias D. A. G. Galton AUTHOR INDEX-SUBJECT INDEX The Employment of Methods of Inhibition Analysis in the Normal and Tumor-Bearing Mammalian OrganVolume 6 ism Abraham Goldin Blood Enzymes in Cancer and Other Diseases Some Recent Work on Tumor Immunity P. A. Gorer Oscar Bodansky Inductive Tissue Interaction in Develop- The Plant Tumor Problem ment Armin C. Braun and Henry N . Wood Clifford Grobstein Cancer Chemotherapy by Perfusion Lipids in Cancer Oscar Creech, Jr., and Edward T. Frances L. Haven and W . R. Bloor Krementz The Relation between Carcinogenic Viral Etiology of Mouse Leukemia Activity and the Physical and Ludwik Gross Chemical Properties of Angular Radiation Chimeras Benzacridines P. C. Koller, A. J. S. Davies, and A. Lacassagne, N. P. Buu-Hoi; R. Sheila M . A. Doak Daudel, and F. Zajdela Etiology and Pathogenesis of Mouse The Hormonal Genesis of Mammary Leukemia Cancer J. F . A. P. Miller 0. Muhlbock Antagonists of Purine and Pyrimidine AUTHOR INDEX-SUBJECT INDEX Metabolites and of Folic Acid G. M. Timmis Behavior of 'Liver Enzymes in HepatoVolume 5 carcinogenesis George Weber Tumor-Host Relations AUTHOR INDEX-SUBJECT INDEX R. W . Begg Primary Carcinoma of the Liver Charles Berman Volume 7 Protein Synthesis with Special Reference to Growth Processes both Normal Avian Virus Growths and Their Etiologic Agents and Abnormal J. W . Beard P. N. Campbell

CONTENTS OF PREVIOUS VOLUMES

407

Mechanisms of Resistance to Anticancer The Relation of the Immune Reaction to Cancer Agents Louis v. Caso R. W. Brockman Cross Resistance and Collateral Sensi- Amino Acid Transport in Tumor Cells R. M . Johnstone and P. G. Scholefield tivity Studies in Cancer Chemotherapy Studies on the Development, BiochemDo& J . Hutchison istry, and Biology of Experimental Hepatomas Cytogenic Studies in Chronic Myeloid Ilnrold P. Morris Leukemia W. M . Court Brown and Ishbel M . Biochemistry of Normal and Leukemic Tough Imwocytcs, Thrombocytes, and Bone Marrow Cclls Ethionine Carcinogenesis I. F. Seitz Emmanuel Farber Atmospheric Factors in Pathogenesis of AUTHOR INDEX-SUBJECT INDEX Lung Cancer Paul Kotin and Hans L. Falk Progrcss with Some Tumor Viruses of Volume 10 Chickens and Mammals: The ProbCarcinogens, Enzyme Induction, and lem of Passenger Viruses Gene Action G. Negroni II. V . Gelboin AUTHOR INDEX-SUBJECT INDEX I n Vitro Studies on Protein Synthesis by Malignant Cells A. Clark Griffin Volume 8 The Enzymatic Pattern of Neoplastic The Structure of Tumor Viruses and Its Tissue Bearing on Their Relation to Viruses W . Eugene Knox in General Carcinogenic Nitroso Compounds A. F. Howatson P. N . Magee and J . M . B a r n s Nuclear Proteins of Neoplastic Cells The Sulfhydryl Group and CarcinoHa& Busch and W i l l k m J . Steele genesis Nucleolar Chromosomes : Structures, J. S. Hanngton Interactions, and Perspectives The Treatment of Plasma Cell Myeloma M . J. Kopac and Gladys M . Mateyko Daniel E. Bergsagel, K . M . Grifith, Careinogenesis Related to Foods ConA , Haul, and W . J. Stuckey, Jr. taminated by Processing and Fungal AUTHOR INDEX-SUBJECT INDEX Metabolites H. F. Kraybill and M . B. Shimkin Experimental Tobacco Carcinogenesis Volume 11 Ernest L. Wynder and Dietrich Hof m a n n The Carcinogenic Action and Metabolism of Urethan and N-Hydroxyurethan AUTHOR INDEX-SUBJECT INDEX Sidney S. Mirvish Runting Syndromes, Autoimmunity, and Volume 9 Neoplasia D. Keast Urinary Enzymes and Their Diagnostic Viral-Induced Enzymes and the Problem Value in Human Cancer of Viral Oncogenesis Richard Stambaugh and Sidney TVeinSaul Kit house

408

CONTENTS OF PREVIOUS VOLUMES

The

Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology William Regelson Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Joseph C . Arcos and Mary F. Argus AUTHOR INDEX-SUBJECT

INDEX

CUMULATIVE INDEX

Volume 12

Antigens Induced by the Mouse Leukemia Viruses G . Pasternak Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses G. I . Deichman Replication of Oncogenic Viruses in Virus-Induced Tumor Cells-Their Persistence and Interaction with Other Viruses H . Hanafusa Cellular Immunity against Tumor Antigens Karl Erik Hellstrom and Ingegerd Hellstrom Perspectives in the Epidemiology of Leukemia Irving I . Kessler and Abraham M . Lilienfeld AUTHOR INDEX-SUBJECT

INDEX

Volume 13

The Role of Immunoblasts in Host Resistance and Immunotherapy of Primary Sarcomata P . Alexander and J . G.Hall Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Oswald Jarrett

The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Haim Ginsburg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gajanan V . Sherbet The Characteristics of Animal Cells Transformed in Vitro Ian Macpherson Role of Cell Association in Virus Infection and Virus Rescue J . Svoboda and I. Hloidnelc Cancer of the Urinary Tract D . B. Clayson and E . H . Cooper Aspects of the EB Virus M . A . Epstein AUTHOR INDEX-SUBJECT

INDEX

Volume 14

Active Immunotherapy Georges Math6 The Investigation of Oncogenic Viral Genomes in Transformed Cells by Nucleic Acid Hybridization Ernest Winocour Viral Genome and Oncogenic Transformation: Nuclear and Plasma Membrane Events Georges Meyer Passive Immunotherapy of Leukemia and Other Cancer Roland Motta Humoral Regulators in the Development and Progression of Leukemia Donald Metcalf Complement and Tumor Immunology Kusuya Nishioka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G. I. Abeler Low Dose Radiation Cancers in Man Alice Stewart AUTHOR INDEX-SUBJECP

INDEX

409

CONTENTS OF PREVIOUS VOLUMES

Volume 15

Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J. S. Butel, S. S. Tevethia, and J. L. Melnick Nasopharyngeal Carcinoma (NPC) J. H . C. H o Transcriptional Regulation in Eukaryotic Cells A. J . MacGillivray, J . Paul, and G . Threljall Atypical Transfer RNA’s and Their Origin in Neoplastic Cells Ernest Borek and Sylvia J. Kerr Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females Philip J. Fidlkow Electron Spin Resonance Studies of Carcinogenesis Harold M . Swartz Some Biochemical Aspects of the Relationship between the Tumor and the Host V . S. Shapot Nuclear Proteins and the Cell Cycle Gary Stein and Renato Baserga AUTHOR INDEX-SUBJECF

INDEX

Volume 16

Polysaccharides in Cancer Vijai N . Nigam and Antonio Cantero Antitumor Effects of Interferon Ion Gresser Transformation by Polyoma Virus and Simian Virus 40 Joe Sambrook

Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing? Sir Alemnder Haddow The Expression of Normal Histocompatibility Antigens in Tumor Cells Alena Lengerovci 1,3-Bis(2-chloroethyl) -1-nitrosourea (BCNU) and Other Nitrosoureas in Cancer Treatment: A Review Stephen K. Carter, Frank M . Schabel, Jr., Lawrence E. Broder, and Thomas P . Johnston AUTHOR INDEX-SUBJECT

INDEX

Volume 17

Polysaccharides in Cancer : Glycoproteins and Glycolipids Vijai N . Nigam and Antonio Cantero

Some Aspects of the Epidemiology and Etiology of Esophageal Cancer with Particular Emphasis on the Transkei, South Africa Gerald P. Warwick and John S. Harington Genetic Control of Murine Viral Leukemogenesis Ffank Lilly and Theodore Pincus Marek’s Disease: A Neoplastic Disease of Chickens Caused by a Herpesvirus I<.Nazerinn Mutation and Human Cancer Aljred G. Knudson, Jr. Mammary Neoplasia in Mice S. Nandi and Charles M . McGrath AUTHOR INDEX-SUBJECT

INDEX

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