Advances in Cancer Research, Volume 29

ADVANCES IN CANCER RESEARCH VOLUME 29

Contributors to This Volume L. F. Chasseaud

Dan H. Moore

Arnold S. Dion

David Naor

A. Clark Griffin

Erkki Ruoslahti

Etienne Y. Lasfargues

Markku Seppala

Carole A. Long

Joel B. Sheffield

J. F. A. P. Miller

Jose Uriel

Akhil B. Vaidya

ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

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

Volume 29-1 979 ACADEMIC PRESS

New York

San Francisco London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT @ 1979, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS,INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMlC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W I 7DX

LIBRARY OF CONGRESS

CATALOG CARD

NUMBER: 52-13360

ISBN 0-12-006629-7 PRINTED I N THE UNITED STATES OF AMERICA

79808182

9 8 7 6 5 4 3 2 1

CONTENTS CONTRlRUTORS TO VOLUhlE 2 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

X i

Influence of the Major Histocompatibility Complex on T-cell Activation J. F. A. P. MILLER I. 11. 111. IV. V. VI. VII.

VIII. IX. X. XI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Major Histocompatibility Complex: A Brief Description T-Lymphocyte Subsets . . . . MHC Gene Products Influence T-cell Activities . . . . H-2 Restriction Phenomena ................................... Models Offered to Explain H-2 Restriction ............................. Level at Which H-2 Restriction Is Imposed . . . . . . . . . . . . H-2 Restriction: Relationsh Possible Models for T-cell Recognition and l r Gene Effects H-2 Restriction and Resista Summary and Conclusions ........... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

11

39

Suppressor Cells: Permitters and Promoters of Malignancy?

DAVIDNAOR I. Background and Introduction . . . . . . . . . . . . ............. 11. Effect of External Intervention on Antitum A Historical Perspective . ........................... nostimulation” and 111. Are Suppressor Cells the I

45 52

“Sneaking through” Phenomena? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IV. Relationships between Malignant Cells and Suppressor Cells V. Suppressor Cells Induced by Nontumorigenic Stimuli or by Tumor Excision.. . . . . . . . . . . . . . . . ................ ..................... .......... VI. Conclusion VII. Appendix .................... ........................... References ....................

104 106 114 119

Retrodifferentiation and the Fetal Patterns of Gene Expression in Cancer Josh URIEL I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Fetal Patterns in Malignant Tumors.. \’

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

127 130

vi

CONTENTS

I11. Fetal Patterns in Noncancerous Growth ................................ IV. The Plasticity of the Differentiated State ............................... V. Retrodifferentiation and Cancer. ....................................... References ...........................................................

147 152 166 169

The Role of Glutathione and Glutathione S-Transferases in the Metabolism of Chemical Carcinogens and Other Electrophilic Agents L. F. CHASSEAUD

I . Introduction I1. Glutathione

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

176

111. Glutathione S-Tr IV. Compounds that V. Discussion . . . . . ............. . . . . . . . . 251 References ........................................................... 255

a-Fetoprotein in Cancer and Fetal Development ERKKIRUOSLAHTI AND MARKKUSEPPALA I . Introduction .......................................................... I1 . Detection and Measureinent of AFP and Its Immunochemical Properties ............................................................ 111. Sites of Physiological AFP Synthesis ................................... .............. IV. Physiological Concentrations of AFP in Body Fluids V. Purification and Chemical Properties of AFP ........................... VI . Biological Properties of AFP .......................................... VII . AFP in Liver Disease ................................................. VIII . Germ Cell Tumors .................................................... IX . Other Clinical Conditions with Informative AFP Levels . . . . . . . . . . . . . . . . X . Tolerance to AFP and Its Abrogation ................................... XI . AFP as a Possible Target Molecule for Tumor Innnunoprevention and Imniunotherapy .................................................. XI1 . Molecular Basis of Regulation of AFP Synthesis ........................ XI11. Areas of Future Research and Concluding Reinarks ..................... References ...........................................................

276 276 282 284 290 305 313 323 327 331 333 334 335 336

Mammary Tumor Viruses DANH . MOORE. CAROLEA . LONG.AKHIL B. VAIDYA.JOEL B. SHEFFIELD. ARNOLD s. DION.AND ETIENNEY. LASFARGUES I . Introduction .......................................................... 347 I1. Morphology .......................................................... 350 111. Structural Proteins and RNA-Directed DNA Polymerase of MuMTV . . . . . 356

vii

CONTENTS Synthesis and Assembly of MuMTV Proteins ........... Genome of MuMTV . . . . . . . . . . . . . . . . . . . . . . . . . Biology of MuMTV ............................... . . . . . . Antigens of MuMTV . . . . . . . . . . . . . . . . . . . . . Interaction of MuMTV with the Host’s Immune System . . . . . . . . . . . . . . . . . IX. Coinments on the Question of a MuhlTV-Related Virus in Humans . . . . . X. Concluding Re1 .............. References . . . . .....................................

IV. V. VI. VII. VIII.

364 367 370 386 395 405 408 410

Role of Selenium in the Chemoprevention of Cancer A. CLARKGRIFFIN

I . Introduction.. . 111. Nutritional Aspects of Seleniu

VII. Suinmary . . . . . References . . . . . . . . . . . . . . . . . . . . .

419 420 421 . . . . . . . . . . . . . . . . . . 423 429

. . . . . . . . . . . 432 ............................... 438 ..................... 440

SUBJECTINDEX . . . , . , . . , . . . . , . . . . . , . , . . , . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . 443 CONTENTSOF PREVIOUS VOLUMES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447

This Page Intentionally Left Blank

CONTRIBUTORS TO VOLUME 29 "umbers in parentheses indicate the pages on which the authors' contributions begin.

L. F. CHASSEAUD, Depurtinent of Aletuholism uiid Phamiacokiiietics, Huiitingdon Research Centre, Huntingdon, United Kingdom (175) ARNOLDS. DION,Institute f o r Medicul Research, Camden, New Jersey 08103 (347) A. CLARKGRIFFIN,Department of Biochemistry, The Univer,sity of Tesus System Cancer Center, J1.D. Anderson Hospitul und Tumor Institute, 6723 Bertner Avenue, Houston, Texas 77030 (419) ETIENNE Y. LASFARGUES, Institute f o r Medical Research, Cumden, New Jersey 08103 (347) CAROLEA. LONG,Depurtment of Jlicrohiology urid Zmmuno1ogy, Huhnemaiiii Aledicul College nnd Hospital, Philadelphiu, Penii.sylvuniu 19102 (347) J. F. A. P. MILLER,The Wulter und Eliza Ha11 Iristitute of Aledicul Re.seurch, P.O. RoUd Jlelhourne Hospitul, Victoria, ~3050,Austruliu (1) DAN H . MOORE, Depurtment of Microbiology unsl Zmmunolog!i, Hahnemunn Medical College and Hospital, Philadelphiu, Pennsylvania 19102 (347) DAVIDNAOR,Lautenberg Center f o r General and Tumor Immunolegg, The Hebrew Uiiiver.sit!l-Hada.s.vtih Aledicul School, Jerusalem, Israel (45) ERKKIRUOSLAHTI,Division of Zmmunology, City of Hope National Medical Center, Duarte, Calijornia 91010 (275) MARKKUSEPPALA,Department of Ohtetrics and Gynecology, University Ceiitrd Hospitul c i n d Depurtinent of Bacteriology c i n d 1111niunology, University of Helsinki, Helsinki 00290, Finland (275) JOEL B. SHEFFIELD,Depurtment of Biology, Temple University, Philadelphia, Penizsylvaiiiu 19122 (347) JOSE URIEL,Znstitut de Recherches Scientifiques sur le Cnncer, B.P. No& 94800 Villejuif, Frunce (127) AKHIL B. VAIDYA,Depurtment of Alicro1,iology und Zmniuiiology, Hahnemann Medical College and Hospitul, Philadelphia, Pennsylvunin 19202 (347) ix

This Page Intentionally Left Blank

ADVANCES IN CANCER RESEARCH VOLUME 29

This Page Intentionally Left Blank

INFLUENCE OF THE MAJOR HISTOCOMPATIBILITY COMPLEX ON T- C E LL ACT I VAT I0N

J. F. A. P. Miller The Walter and Eliza Hall Institute of Medical Research Post Office Royal Melbourne Hospital Victoria, Australia

I . Introduction . . . . . . . . . . . 11. The Major I-iistocoiiipati

111. T-Lymphocyte Stillsets . IV. MHC Gene Products In V. H-2 Restriction Plienoinena . . . . . A. Cytotoxicity . . . . . . . . . . . . . . . . . B. Delayed-Type Hype C. Helper Functions . , D. Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Possible Exceptions VI. Models Offered to Explain H-2 Restriction . . . . . . . . . A. Intinlacy Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Altered Self-Model . . . . . . . . ................................ C. Two Receptor Model . . . . . . VII. Level at Which H-2 Re. A. Priming of T Cells ........ B. Ditferentiation of T ......................... VIII. H-2 Restriction: Relationship to A. Forination of Complex of Antigen and MHC Product Iminunogenic for T Cells . . . . . . . . . . . . . ..................................... B. Generation of the T-cell Repertoire IX. Possible hlotlels for T-cell Recognition L A. Positive Selection for T Cells with Anti-Self-H-2 Receptors . . . . . B. Selection against T Cells with High-Atfinity Anti-Self-H-2 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. MHC Gene Products on Antigen-Presenting Cells Are Obligatory for T-cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. H-2 Restriction and Resistance to X I . Summary and Concliisions . . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations Used . Note Added i n Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11 12

15

21

39 40

I. Introduction

The major stimulus for the activation of most T lymphocytes does not seem to be antigen alone, but antigen in association with one or the other of the gene products of the major histocompatibility complex. 1

2

J. F . A. P. MILLER

These products are present on the surface of the body’s own cells and some of these, for example, cells of the monocyte-macrophage series, are particularly well equipped to present antigen in the appropriate form to T lymphocytes and to deliver the activating signal. Once activated, the T cells have their specificities directed, not to antigen alone, but to both the antigen and some histocompatibility gene product (“H-2 restriction,” Zinkernagel and Doherty, 1974a). This discovery has stimulated a vast number of investigations aimed at defining the precise mechanism b y which T cells are activated, the specificities of the receptors that enable T cells to recognize antigenic detenninants, and the means b y which the repertoire of T-cell reactivities may be generated in the genome. This work has led to a better understanding of the function of histocompatibility gene products and of the advantages of polymorphism of the loci which code for these products. It is the purpose of this review to consider some of these investigations and to offer certain models which may elucidate the role of the histocompatibility gene products in T-cell activation. The effect these genes may have in determining resistance to certain tumors will be briefly considered. 11. The Major Histocompatibility Complex: A Brief Description

The features of the major histocompatibility complex (MHC)’ relevant to this discussion are shown in Fig. 1. The MHC, H-2 in mice or HLA in man, codes for antigens (glycoproteins) many of which are expressed on the surface membrane of most cells. If we disregard S and G, we can divide H-2 antigens into two major classes: those controlled by the peripheral K or D regions (or classical H-2 antigens) and those controlled by the central I region [or I-associated (Ia) antigens]. The genetic control of the classical H-2 antigens is determined by the loci H-2K in the K region and H-2D in the D region. Due to polymorphism, there is a large number of alleles of H-2. Each allele is designated by a small letter as a superscript: e.g., H-2Kb, H-2Kf1, H-2Kk, etc., for the H-2K locus, and H-2Dh, H-2Dd, H-2Dk, etc., for the

-

Chromosome 17

+l5cM+-O.ScM Tlt K

-I

: :

I

S

G

D

TL

L A I-H-H-l

FIG.1. T h e major histocoinpatibility complex or H-2 in the mouse.

HISTOCOhlPATIBILITY COhlPLEX AND T-CELL ACTIVATION

3

H-2D locus. Each haplotype ( a particular combination of H-2 antigens controlled by a single chromosome) is designated also by a small letter as a superscript: e.g., H-2*, H-2d, etc. I-associated antigens or Ia antigens are genetically controlled by the I region and are distributed among five loci (so far detected): I-A, I-B, I-J, I-E, and I-C. Antigens coded by different I regions behave as on separate molecules. In contrast to H-2K and H-2D products which are represented on most cells, I-region products are found predominantly on cells of the lymphoreticular system (subsets of T cells, B cells, macrophages, etc.) and also on the epidermal cells of Langerhans. There are differences in the tissue distribution of individual Ia antigens coded b y different I subregions (e.g., Ia antigens coded by the I-J locus are found predominantly on suppressor T cells). Many of the MHC-linked Zr genes have been mapped in the I-region. On the right of D is the TL locus which codes for antigens expressed on thymocytes and on cells of some thymus-derived lymphomas. On the left of K is the T/t complex. The genes of this complex mediate a series of essential steps in embryogenesis probably b y coding for cell surface components at relevant stages in development. There is a reciprocal relationship between one of these antigens, F9, and the H-2 antigens. F9 but not H-2 is present on sperms and on cells of the very early embryo. Later H-2 is present but not F9. TL has also some reciprocal relationship with H-2D: less H-2D is present when TL antigens are expressed on thymocytes. T L is not expressed on mature T cells. F9, H-2K, H-2D, and T L antigens are glycoproteins of similar molecular weight and three of these have the small molecule p2 m associated with them. It is therefore of great interest that four loci, within a region of some 15 cM on chromosome 17, specify analogous cell niembrane molecules playing an important role in cell to cell interactions. This has led to the hypothesis that this family of four loci are the logical descendants of an ancestral locus which acquired the property of cell surface perception as a condition for metazoan evolution. Further details on the genetic organization of the MHC will be found in Jan Klein’s excellent monograph (1975) and in a number of reviews (e.g., Shreffler, 1977; McKenzie, 1977; Peterson et ul. 1977; Artz and Bennett, 1975). Ill. T-Lymphocyte Subsets

T cells act in different ways in immune responses and may be divided into various subsets (Simpson and Beverley, 1977).Some T cells are directly cytotoxic ( T C ) if they can make intimate contact with ap-

4

J. F . A . P. MILLER

propriate targets. Some are involved in inducing the inflammatory lesions of delayed-type hypersensitivity (DTH, TDcells). Some help B cells produce IgG antibody (TH)and some regulate the immune response by suppressing the activities of other T and B cells (Ts). The various subsets can be distinguished by cell surface markers, such as the differentiation antigens of the Ly series and the differential expression of antigens coded by the MHC (Vadas et al., 1976; Cantor and Boyse, 1977). Thus, in general, T H and TI, cells are Ly-l+ and Ia-; Tc cells are Ly-2, 3+ and usually Ia-, whereas Ts cells are Ly-2, 3+ and express Ia antigens coded by the I-J locus of the MHC. The exact way in which the various T-cell subsets exert their effect is not known but evidence points to the production, after antigenic stimulation, of factors which may be antigen specific or nonantigen specific. For example, TH cells are thought to influence IgG-producing B cells either by a nonantigen-specific factor acting indirectly via macrophages (Miller, 1975), or by releasing antigen-specific factors of various types (Feldniann and Nossal, 1972; Munro and Taussig, 1975). Activated To cells release lymphokines such as the migration inhibitory factor (MIF), which are nonantigen specific and influence the recruitment of cells of the mononuclear-macrophage series (Bloom and Bennett, 1970). Tc cells presumably kill by some nonantigen-specific influence against target cells for which they have antigen-specific receptors (Cerottini and Brunner, 1974). Finally, antigen-specific helper or suppressor factors have been obtained from TH and Ts cells and carry determinants coded by the I-region of the MHC. Unfortunately, none of the factors thought to be elaborated as a result of stimulation of the various T-cell subsets has yet been precisely characterized chemically. IV. MHC Gene Products Influence T-cell Activities

The MHC exerts a profound influence on T-cell activities. This is clear from the following. (1) The frequency of alloreactive T cells (i.e., those directed against H-2 antigens) is generally thought to be 100 to 1000 times as high as the frequency of T cells reactive to non-H-2 antigens (Simonsen, 1967; Lindhal and Wilson, 1977). (2) Different subsets of T cells exhibit the phenomenon of H-2 restriction, i.e., identity at certain MHC gene products is required between cells responsible for immune induction of T cells and targets of immune T cells (Zinkernagel and Doherty, 1974a; Bevan, 1975; Miller et d.,1975; Katz and Benacerraf, 1975; Section V).

~IISTOCOSfPATIBILI1’YCORIPLEX AND T - C E L L ACTIVATION

5

(3) The I r genes, linked fiinctionally to the MHC, influence tlie responsiveness of T cells or T-cell dependent functions (Benacerraf and Katz, 197Fj; Benacerraf and Germain, 1978; Section VII). (4) There are I-region coded determinants on antigen-specific “helper” and “suppressor” factors which can he released or extracted from TH and Ts cells, respectively (Feldniann et uZ., 1977; Tada et NI., 1977).

The first three phenomena can all be accoinmodated in one general model of T-cell recognition and activation. This will be attempted in Section IX. On the other hand, the antigen-specific helper and suppressor fiactors will not be considered in detail in this review. It is felt that much more work is required to characterize these Factors physiologically and 1,iocheniically to appreciate their special role in the network of immune interactions. To account for the high frequency of alloreactive T cells, Jerne ( 1971) proposed that the repertoire of T-cell reactivities is coded b y a set of germline c genes (genes coding for tlie oaricible region of T-cell antigen-binding units) which code for structures essentially complementary to the H-2 alleles of the species. Ater entering the thymus, potentially alloreactive T cells, which form a relatively large proportion ofthe T-cell pool, need not be influenced. By contrast, T cells with anti-self-H-2 reactivities would proliferate i n response to H-2 structures present in the thymus. This must not be allowed to continue, for it was argued that such cells could kill self-H-2-bearing cells. Random somatic mutations in the genes coding for structures complementary to self-H-2 would thus be allowed to accumulate. This would decrease the strength of anti-self-H-2 binding and finally, only those T cells without anti-self-H-2 reactivities would mature. Such cells would have their reactivities directed to non-H-2 antigens and each specific set would thus constitute a much smaller proportion of the total T-cell pool than alloreactive T cells. As will be discussed later (Section IX), the discovery of the phenomenon of H-2 restriction has rekindled interest in this negative selection theory of Jerne. It has in fact been reformulated to account for a variety of the new findings (Langman, 1978; Blanden and Ada, 1978; von Boehmer et ul., 1978). V. H-2 Restriction Phenomena

Most immune phenomena associated with T cells are subject to H-2 restriction. This has been well documented for T cells involved in cytotoxicity, delayed-type hypersensitivity (DTH), and helper func-

6

J. F. A . P. MILLER

tions. By contrast, there is no evidence for H-2 restriction of B cell or natural killer (NK) cell activity. A. CYTOTOXICITY Tc cells may have their reactivities directed to H-2 antigens (other than self) or directed to non-H-2 antigens. For example, C B A (H-2k) mice immunized to DBN2 (H-2d) produce Tc cells which kill any targets bearing either H-2Kd or H-2Dd, no matter what other cell surface antigens these targets may have. Alloreactive T cells are thus clearly not self-H-2 restricted but rather “allo-H-2” restricted in view of their specificities to alloantigens. On the other hand, CBA mice recovering from infection by the virus of lymphocytic choriomeningitis (LCM) have Tc cells which attack LCM-infected H-2k targets, not uninfected H-2k targets or LCM-infected targets of any other H-2 type. There thus appears to lie a requirement for H-2 matching between Tc cells and targets. This constitutes the phenomenon of “self-H-2 restriction” (hereinafter referred to as H-2 restriction) as originally described by Zinkernagel and Doherty (1974a). By using inbred strains of mice, congenic lines and recombinant lines, the genes imposing H-2 restriction of Tc cells could be mapped. The H-2K and the H-2D genes were found to impose H-2 restriction of Tc cells generated against virusspecified antigens (Doherty et al., 1976a), hapten-modified antigens (such as trinitrophenyl, TNP) (Shearer et al., 1976), and minor histocompatibility (H) antigens (non-H-2 H antigens designated H,, HO, H4,etc., perhaps up to HI”,,, and the H-Y antigen which is a sex-linked minor H antigen) (Bevan, 1975; Gordon et ul., 1975). Tr cells have clonally distributed receptors (the word receptor is used loosely to describe a cell surfi‘we structure with a site having some degree of complementarity to a determinant on an antigen molecule). In other words, one particular Tc cell has a receptor endowed with a unique specificity directed against either H-2K or H-2D gene products, but not both. There is therefore genic and allelic exclusion of receptors on the T cell (in contrast to the presence of both H-2K and H-2D gene-coded antigens on the same cell) (Zinkernagel and Doherty, 1975). B. DELAYED-TYPE HYPERSENSITIVITY To achieve successful transfer of DTH, there is a requirement for H-2 matching between donors of sensitized T cells and naive recipients (Miller et al., 1975). In the case of protein and polypeptide anti-

HISTOCOXlP,4TIBILITY COhlPLEX A N D

T-CELL ACTIL’ATION

7

gens, the MHC region involved in restriction was I-A, but in the case of contact chemicals such as dinitrofluorobenzene, the genes were either H-2K, H-2D, or in the I region (Vadas et al., 1977). These restrictions were interpreted as a requirement for H-2 matching between T, cells and cells, such as macrophages or epidermal cells, responsible for presenting antigen to the T cells. More formal evidence for this was obtained in experiments which showed the necessity for Ia matching between antigen-pulsed macrophages used for sensitization and for elicitation of sensitivity in an i l l vivo system (Miller et al., 1978). This is in line with observations of other investigators showing a requirement for Ia matching between antigen-pulsed macrophages and sensitized T cells in vitro to allow these to proliferate (Yano et d . , 1977; Thomas et d., 19771)).The in vitro system enabled the investigators to use specific antibodies to determine the role played by antigen and MHC components in stimulating the proliferation of sensitized T cells. Incubation of T cells, themselves, with anti-Ia antibody had no effect. Antibody directed to Ia antigens, of the appropriate specificities, present on macrophages blocked the ability of the antigen-pulsed macrophages to stimulate proliferation (Shevach et al., 1972; Thomas et d . , 197721). The blocking was haplotype-specific in the F1, indicating that it did not result from some nonspecific effect of the binding of antibody to a cell surface component (Schwartz et ul., 1976a). By contrast, antibody to the native antigen failed to block proliferation (Ellner et d., 1977; Thomas et al., 1978). The antigenic determinants which stimulate T cells therefore appear to do so in association with Ia determinants but are not readily accessible to antibodies directed against imtive antigen. Similar observations have been made in cytotoxic systems: the specific inhibition of lysis of target cells by TC cells was achieved by antibody directed to the H-2K and H-2D gene products ofthe targets (Germain et al., 1975; Schmitt-Verhulst et al., 1976). Antiviral antibodies, on the other hand, consistently failed to protect the target (Blanden et a / . , 1976b; Doherty et d . , 1976a; Braciale, 1977), except in one case (Koszinowski and Ertl, 1976). The implications of these observations are that native antigen is not recognized as such by T lymphocytes. C. HELPERFUNCTIONS It was initially observed that TH cells and B cells must share I-A gene products for successful cooperation in antibody responses (Katz and Benacerraf, 1975). The restriction of THcells occurred both at the level of induction (presumed to reflect activation of TH cells by

8

J . F . A . P. MILLER

macrophage-associated antigen) and during T- and B-cell cooperation (Sprent, 1978a). T-cell recognition of MHC-associated antigen on niacrophages and on specific B cells may thus be either identical or very similar. If, as discussed above, the antigen recognized by T cells on macrophage surfaces is not native antigen, the same must be true of antigen presented by B cells. THcells may recognize the B cells which they help, only if these can associate antigen with their surface Ia determinants in the same way as the antigen-pulsed macrophages. Tand B-cell cooperation thus might not depend on carrier-hapten “focusing” between T- and B-cell receptors specific for determinants of the carrier and the hapten, respectively. The T cells specific for Ia and “processed” antigen would seek out those B cells displaying identical structures. Of course, only B cells with specific receptors for either hapten or carrier determinants should “capture” sufficient hapten-carrier conjugates via their surface immunoglobulin receptors. Only these B cells should be able to “process” carrier determinants and associate them with Ia determinants on their cell surface, thus displaying the correct structure for specific carrier-reactive TH cells. Binding of the specific T-cell receptors to such structures on the surface of the B cells may be associated with the delivery of an activating signal (nonantigen specific) from the T H cell to the B cell, and the induction of synthesis of the corresponding specific antibody by the B cell. Hence, antibodies to both carrier and hapten determinants would be produced by carrier-specific and hapten-specific B cells. There may be a requirement for H-2 matching (also in the I-A region) between TH cells and Tc cells, as suggested by recent work in chimeric mice (Zinkernagel et ul., 1978a) (see Section VII). D. SUPPRESSION The antibody response to protein antigens may be suppressed by an antigen-specific factor, bearing an I-J-coded determinant and obtained from Ts cells. Identity at the I-J subregion between donor and recipient resulted in more efl’ective suppression in some (Tada et ul., 1977) but not all cases (Kapp, 1978). When Ts cells were induced to hapten-modified cell membranes as, for example, when mice were tolerized with dinitrophenyl (DNP)-modified lymph node cells, the Ts cells suppressed recipient mice only if these shared H-2D with the strain providing the lymphoid cells (S. D. Miller et ul., 1978). The reactivity of the Ts cells in this system thus appeared to b e directed to DNP-modified H-2D products. Somewhat similar results were obtained with soluble suppressor factors released from Ts cells

HISTOCOhfPATIBILITY COhfPLEX A N D

T-CELL ACTIVATION

9

(Moorhead, 1977). A requirement for H-2D compatibility was also reported for Ts cells active in virus-infected systems (Pang and Blanden, 1976; Kumar and Bennett, 1977).

E. POSSIBLEEXCEPTIONS TO H-2 RESTRICTION A number of phenomena have not exhibited the classical H-2 restriction described above. They are listed under the following headings and some will be referred to again in Section X. 1. T c Cells f o r 1-Region Deterniinunts I-region determinants can serve both as stimulator and as targets of Tr cells (Wagner et ul., 1975; J. Klein et al., 1977; Billings et al., 1977a). H-2K or H-2D compatibility is not required for the activity of these Tc cells. In addition, I-region determinants are susceptible to the same type of antigen modification (e.g., by TNP) as H-2K or H-2D gene products. In this case, the modified Ia determinants serve as targets for specific Tc cells raised against I-region determinants (Billings et u l . , 1977b). 2. T r Cells f o r F9 Antigen OTT 6050 is a teratocarcinonia line, derived from strain 129 mice. It does not express H-2K or H-2D antigen specificities but produces an antigen, F9, genetically linked to the T/t locus of the MHC (Jacob, 1977). Although virus-infected F9+ cells cannot serve as targets for Tr cells generated in vizjo by infection with the virus (Zinkernagel and Oldstone, 1976; Doherty et ul., 1977), i r t ~jizjopriming of mice with H-2 negative F9+ cells followed by i n citro restimulation with F9+ cells triggers the generation of anti-F9 immune Tc cells able to lyse specifically H-2 negative F9+ cells (Wagner et ul., 1978). Since there may be some evolutionary relationship between the H-2 and T/t complexes, both located on chromosome 17 (Artz and Bennett, 1975), F9+ cells may code for R functional analog of the H-2K and H-2D gene products. Whether it is possible to induce F9-restricted virus-specific or hapten-specific T cells has yet to be demonstrated. 3. Cross-Reuctiue L!/sis by Tc Cells

Tc cells activated to allogeneic cells in zjico or in citro in mixed lymphocyte cultures are very heterogeneous as evidenced by their cross-reactivity patterns. The effector cells kill not only targets syngeneic to stimulator cells but also third-paity allogeneic targets with different H-2 antigens (Lindahl et u l . , 1975). Cross-reactive lysis has

10

J. F . A . P. hlILLEH

also been demonstrated both against minor H antigens (Bevan, 1977) and when normal spleen cells were cultured with irradiated TNPconjugated syngeneic spleen cells. This generated Tc cells which lysed syngeneic TNP-targets efficiently and, to a lesser extent, allogeneic TNP targets (Burakoff et al., 1976). Cold target inhibition experiments indicated the existence of clones of cross-reactive Tc cells and treatment of targets with anti-H-2 antibody blocked lysis thus demonstrating the requirement for recognition of H-2 on targets. If the repertoire of T-cell reactivities evolved to recognize modified self-H-2 antigens (Jerne, 1971), Tc cells with reactivities to alloantigens or xenoantigens might consist of clones of cells with specificities directed to modified autologous MHC products which cross-react with allogeneic and xenogeneic antigens. In an experimental investigation of this possibility, alloreactivity generated by xenogeneic stimulation was shown to result from the activities of separate Tc cell clones, each specific for an allogeneic target (Burakoff et al., 1977). Moreover, alloreactive ‘Tc cells exhibited cross-reactivities with chemically modified target cells syngeneic to the responders (Lemonnier et al., 1977). Such cross-reactive Tc cells may account for the apparent breakdown of H-2 restriction observed in some tumor immunity systems (see Section X).

4. Anomalous Cytotoxicity “

Anomalous’’ cytotoxicity has been detected in a variety of experimental systems. Thus, for example, heterospecific cytotoxic cells were generated in the first 3 to 5 days of acute LCM infection in mice. These cells killed many types of infected or uninfected cultured cells, including syngeneic cells (Pfizenmaier et al., 1975; Blanden and Gardner, 1976; Welsh and Zinkernagel, 1977). Likewise, Epstein-Barr virus (EBV)-genome carrying lymphoblastoid cell lines (LCL) stimulated autologous lymphocytes to generate cytotoxic cells reactive not only against the autologous cell line but also against other unrelated cell lines, whether or not these carried the EBV genome (Viallat et al., 1978a,b). A somewhat analogous situation was described in human mixed lymphocyte cultures (Seeley and Golub, 1978). Here, two distinct types of cytotoxic activities were generated during sensitization to normal allogeneic peripheral blood lymphocytes: (a) allospecific cytotoxicity directed against alloblasts only; (b) “anomalous” cytotoxicity directed against serologically unrelated LCL and against the autochthonous LCL. The peak anomalous cytotoxicity occurred 1-2 days earlier than the peak allocytotoxicity and declined earlier. The anomalous cytotoxicity was not limited to targets sharing HLA

HISTOCOhlPATIBILITY COlIPLEX AND T-CELL ACTIVATION

11

specificities, carrying EBV-associated antigens or fetal calf serum antigens. Substantial evidence was given to relate the anomalous cytotoxicity, in the mixed human lymphocyte culture experiments, to NK cell activities (Seeley and Golub, 1978). Hence, viruses could conceivably activate NK cells and these would account for the nonH-2-restricted cytotoxicity observed in the early stages of LCM virus infections (see above). Whether NK cell activity plays a role in the non-HLA-restricted cytotoxicity generated by stimulation with EBVinfected autologous cells (Viallat et nl., 197th) has to be checked. In these experiments, however, the responder cells were purified from peripheral blood by passage through nylon wool followed by rosetting with sheep erythrocytes and may thus have been T cells. VI. Models Offered to Explain H-2 Restriction

Many questions are raised by the existence of H-2 restrictions. Why should alloreactive T cells not be restricted by self-H-2? What is the mechanism of restriction? Is H-2 restriction limited only to the specificity dictated by self-H-2? Does the requirement for H-2 matching between T cell and stimulator cell (i.e., cell presenting antigen to T cell and able to stimulate it) exist prior to immunization (priming) or is it imposed only as a consequence of priming? Three alternative models have been proposed to explain H-2 restriction. They will be briefly described here. A. INTIMACYMODEL

The Tc cell has a receptor directed against X (e.g., virus-specified antigen). For effective interaction with target, however, the H-2K and H-2D gene products must match, otherwise there is no lysis of the target. This matching is therefore between like molecules. For those who dislike like-like interactions (since like molecules may not have complementary sites) linkage by an intermediate molecule able to bind to both matching H-2 gene products can be envisaged. The intimacy model was discarded by an experiment performed by Zinkernagel and Doherty (1974b) using F, hybrids. F, mice between two H-2 incompatible parents can, when virus infected, generate Tc cells able to kill in vitro virus-infected targets of both parental types. Consider, however, [CBA (H-2’) x C57BL (H-2h)]F1 mice exposed in vivo to virus-infected CBA cells under conditions in which the virus cannot escape the CBA cells to infect the host F, cells. The F, generates TCcells which can be tested on targets in vitro. They killed only

12

J. F . A . P. MILLER

virus-infected CBA targets, not virus-infected C57BL targets, even though the F, T cells possess both H-2k and H-Zd gene products on each cell (there is no allelic exclusion of H-2 antigens in an F,). Hence there was matching of H-2h gene products between F, Tc cells and C57BL targets in the above experiment, and yet no killing. This does not support the intimacy hypothesis. B. ALTEREDSELF-MODEL This model states that non-H-2 closely associates with H-2 to create new antigenic determinants recognized by one T cell receptor. There may thus be something special about H-2ness since T cells will recognize only the new antigenic determinants created by the complex, but will not be concerned with either non-H-2 alone or H-2 alone. If we adopt Jerne’s negative selection theory for the generation of the T-cell repertoire (Section IV), we can readily accommodate this peculiar preoccupation of T cells with H-2ness. Because of the particular way in which T cells were generated, virtually all T cells must retain some memory” of their anti-self-H-2 past and are directed to antigens which contain some measure of H-2ness, The difficulties with the altered self-model relate to the nature of the association between a wide variety of non-H-2 antigens and H-2 gene products. The following possibilities have been suggested to explain this association but three appear unlikely for the reasons given. “

(1) Non-H-2 could modify H-2. For example, a virus or hapten could conceivably alter H-2K or H-2D products in a specific manner. While this may be so for certain cases, it is unlikely that the many unrelated minor H antigens could modify H-2, each in their own unique way. A modification of H-2 from virus infection is also rendered less likely by data showing that viruses inactivated by p propiolactone or ultraviolet light can still provoke H-2-restricted lysis in the absence of viral protein synthesis (Koszinowski et d . ,1977; Schrader and Edelman, 1977; Ennis et al., 1977). (2) H-2 could modify non-H-2. It was proposed, for example, that H-2K and H-2D genes code for a battery of glycosyl transferase enzymes (Blanden et d.,1976a). This concept is no longer tenable since Tc cells could still act in systems where glycosylation had been inhibited by 2-deoxyglucose (Braciale, 1977). (3) New serologically defined H-2 specificities could be derepressed as a result of virus infections or chemical modification (Garrido et id., 1977; Gipson et al., 1978).They may serve as recognition struc-

IIISTOCOSIPATIBILITY COSIPLEX AND T-CELL ACTIVATION

13

tures for Tc- cells. Since many viruses (e.g., influenza) turn off host protein synthesis within a few hours of infection (e.g., Lanzarowitz et al., 1971), it is difficult to imagine how new H-2 specificities could be transcribed and translated in such a short time. Yet virus-infected target cells do become susceptible to lysis by Tc cells as early as 2-3 hours after infection (Jackson et d . , 1976; Ada et m l . , 1976; Kosinowski and Ertl, 1976; Zinkernagel and Althage, 1977). (4)A “hybrid” antigen may form on the cell surface. Strong evidence in favor of this was obtained in one system by the demonstr at’ion of cocapping of virus antigen and H-2 products (Schrader et d . ,1975). The formation of a covalent bond between H-2 and non-H-2 seems unlikely since the two molecules could not be coprecipitated from the same cell surface (Schrader et d., 1977).A hybrid antigen could, however, form on the cell surfice as a result of‘ a noncovalent transient interaction, via diffusion within the membrane, of the H-2 gene products and some components of the non-H-2 antigen. Thus, only a very minor proportion of the H-2 molecules niay become associated with the non-H-2 molecules at any one time. In support of this are the observations that only a very small amount of viral antigen needs to be synthesized for the targets to hecome susceptible to Tc cells (Jackson et ul., 1976; Schrader et nl., 1977), and that inactivated virus can render targets susceptible in the absence of protein synthesis (Schrader and Edelman, 1977; Koszinowski et al., 1977; Ennis et a l . , 1977). C. Two RECEPTOR MODEL The tw7o receptor model was proposed independently by several investigators (e.g.,Janeway et uZ., 1976; Doherty et id., 19761); Wilson et al., 1977a). It states that each T cell has two distinct receptors: one directed to non-H-2 antigen “X” and another to H-2. Both anti-H-2 and anti-X are clonally distributed: anti-X as anti-XI, anti-Xl, anti-X’, . . . , anti-X”, where 1, 2, 3 , . . , , n are different specificities; anti-H-2 as anti-H-BK, anti-H-eD, anti-I-A (and any other subset of MHC gene products which niay have a relevant role, as for example I-J). Of course anti-H-2 (self)specificity is found on a large subset of T cells, whereas anti-X is different in every T cell. A model based on independent recognition of MHC determinant and antigen by two T-cell receptors is of course not beset b y the difficulties encountered by the “altered self” model in relation to the formation of the “hybrid” antigen. Furthermore, it readily accommodates the finding of sharing of idiotypes (immunoglolxdin variable regions) between T cells and humoral antibody (Rajewsky and

14

J. F . A . P. MILLER

Eichmann, 1977; Krawinkel et al., 1977,1978; Julius et al., 1977). The anti-X receptor could utilize the same o region gene pool as the immunoglobulin (Ig) H chains: this would explain the linkage of both T and B cell receptors to the Ig-1 allotype locus. One of the main difficulties with the two receptor theory is to explain the results of cold target competition experiments, Unlabeled cells were found to inhibit the lysis of "Cr-labeled target cells, but only if they had both the appropriate H-2 determinants and hapten or virus antigens (Zinkernagel and Doherty, 1975; Shearer et al., 1975; Forman, 1975). This difficulty disappears, however, if it is assumed that the anti-X and anti-H-2 receptors can work only as a paired unit. Thus, for example, the interaction of one binding site with its specific target antigen may cause a conformational change as a result of which the second binding site can now accommodate its own appropriate antigen. It follows from this that, in addition to linked recognition by the T cell, the H-2 and non-H-2 determinants on the stimulator or target cells must be in close proximity. If this is so, it will be very difficult to devise experiments which can unequivocally support one but not the other of the two models just described. Before discussing these models in greater detail and relating them to the phenomenon of MHC-linked Zr gene control, it may be appropriate to ask whether H-2 restriction exists prior to priming or is imposed only as a result of priming. VII. Level at Which H-2 Restriction Is Imposed

Does H-2 restriction exist prior to immunization, or is it imposed only as a consequence of priming? To answer this question, one could stimulate T cells from CBA (H-2k)mice with antigen X on C57BL ( h - P ) cells and determine whether activated T cells could recognize that antigen only on C57BL cells and not on CBA (self) cells. I f this turned out to be the case, H-2 restriction would clearly have been imposed as a result of priming. The difficulty with such an experiment is that alloreactive T cells would be generated and their activities would interfere with the response to antigen X. To overcome the complication of alloreactivity, tolerance could be induced, either genetically (F, hybrids) or artificially (chimerism). Alternatively, alloreactive T cells could be selectively removed (negative selection experiments). These systems will now be discussed because the results obtained have influenced our thinking on the mechanism of H-2 restriction and of the MHC-linked Zr gene effects.

HISTOCOhlPATIBILITY COhfPLEX AND T-CELL ACTIVATION

15

A. PRIMING OF T CELLS

F, mice, between two MHC-incompatible parental strains (say PI and PJ, can be sensitized to antigen in such a way that lymphoid cells from the sensitized F, can transfer DTH to naive recipients of both parental strains. If, however, the F, is sensitized by antigen-pulsed macrophages derived from one of the parental strains (say P,), lymphoid cells from such F, mice will transfer DTH only to naive P, recipients, not to P, (Miller et ul., 1976). This suggests that, in the F, immunized by antigen in the usual way, some T cells are responding to antigen in association with an MHC component of one parental haplotype, whereas others respond to antigen in association with the MHC product of the other parental type. If the F, is immunized by antigen-pulsed macrophages derived from one parental haplotype, only one of these two populations of T cells will be sensitized, and transfer will therefore be possible only with that subset. Clearly, in this situation, the restriction has been imposed as a result of priming. Similar results have been obtained in other F, experiments in which priming by antigen associated with one haplotype was shown to restrict Tc cells (Zinkernagel and Doherty, 197413; Shearer et d., 1976), TH cells (Swierkosz et nl., 1978; Sprent, 1978a), and T cells proliferating i n vitro in response to antigen-pulsed macrophages (Paul et ul., 1977a,b). Further evidence in favor of H-2 restriction being imposed as a result of priming has come from in vitro antibody responses (Pierce et al., 1976). Syngeneic and allogeneic antigen-pulsed macrophages supported the development of comparable primary antibody responses. Genetic restrictions thus did not limit efficient macrophagelymphocyte interactions in primary responses. By contrast, spleen cells from immunized animals developed secondary antibody responses preferentially when stimulated i n vitro with antigen on macrophages syngeneic to the macrophages used to present antigen during in uivo immunization. The genetic restrictions were shown to be antigen specific, to operate at the level of the immune T cell, and to be controlled by the I-A region (Pierce and Kapp, 1978). Other experiments were designed to test whether T cells, depleted of alloreactive cells, could be immunized when confronted with antigen presented on allogeneic stimulator cells. Alloreactive T cells can be removed by certain “acute depletion” procedures. One method is to employ the technique of antigen-driven 5-bromodeoxyuridine (BUdR) and light suicide. By this method T cells reactive to H-2 incompatible cells can be eliminated and the residual cells tested for

16

J. F. A . P. MILLER

their capacity to respond to antigen on allogeneic stimulator cells. Another method utilizes the “negative selection” system, described by Ford and Atkins (1971), in which parental strain lymphocytes are depleted of alloreactive cells b y “filtration” from blood to lymph in irradiated F, hybrids. The filtered T cells are then tested for their capacity to be activated by antigen on allogeneic cells. Several studies utilizing such acute depleting procedures have shown that T cells can respond to antigens presented on allogeneic cells and are restricted by the particular MHC haplotype of the stimulator cells. In other words, expression of those T cells required MHC identity between the target and the stimulator cell population, even though the T cells themselves were allogeneic to both. These observations were obtained with T cells proliferating i n vitro in response to antigen-pulsed allogeneic macrophages (Thomas and Shevach, 1977) and with Tc cells negatively selected i n v i v o (Wilson et d.,1977b). Unfortunately, however, two independent investigations did not confirm these results but showecl the opposite. Deletion of alloreactive cells by the BUdR technique left a population with little or no reactivity specific for modified allogeneic stimulator cells (Janeway et al., 1978; Schmitt-Verhulst and Shearer, 1977). If these results are true, one might be tempted to conclude that T cells are committed to recognizing only self-H-2 specificities and that this precedes intentional immunization. Further investigations are clearly required to establish whether or not this is indeed the case. B. DIFFERENTIATION OF T CELLSIN THYMUS Recent experiments employing chimeric mice support the idea of some commitment of T lymphocytes to MHC components expressed prior to intentional immunization (Bevan, 1978; Zinkernagel et al., 1978a). F, mice between two H-2 incompatible strains (say P, and P2) were thymectomized, irradiated, and protected with F, bone marrow. An epithelial thymus (the thymus having been depleted of lymphocytes b y irradiation) from one of the parental strains, say PI was grafted and the animals allowed to regenerate their hemopoietic system. They were then infected with virus and the Tc cells generated by such infection i n v i v o , were tested in v i t r o on virus-infected P, targets and virus-infected P, targets. Only the former were killed. This is in contrast to ordinary F, mice infected with virus which can generate Tc cells capable of killing both parental-strain targets. The essential difference between the two experimental systems is that in the former, the F, stem cells differentiated in a thymus expressing only one H-2

HISTOCOSIPATIBILITY COhfPLEX AND T-CELL ACTI\’ATION

17

haplotype, whereas, in the latter, differentiation occurred in a thymus expressing both H - 2 haplotypes. The results were interpreted as follows (Zinkernagel et ( i l . , 1978a). The T cells “learn” in the thymus which anti-self-H-2 receptor to .express. The anti-X receptor is expressed independently. Viewed in this way, H-2 restriction is primarily imposed by the H-2 type expressed in the thymus epithelium and commits the immature T cells differentiating in the thymus. In tlie case of an F,, further restriction can of course be imposed on the mature T cells after priming, if antigen is presented on stimulator cells bearing only one of the parental MHC haplotypes (see Section VI1,A). However, T cells should not apparently be able to recognize antigen i n association with an entirely foreign MHC haplotype. Hence, the importance of reexamining the claims of Pierce et n l . (1976), Thomas and Shevach (1977), and Wilson et (11. (197711)discussed above. The experiments of Zinkernagel et (11. (19784 were repeated b y others using Tc cells (referred to in Section VIII), TI, cells (Sprent, 1978\)),and T, cells (Miller, 1978). The situation inay be summarized a s follows. ( 1 ) Normal l z o n i o q g o z t s T cells of strain 11 consist primarily of a predominant population of anti-self-ci (anti-cl) T cells which recognize antigen presented by cells or macrophages of strain ( I ; the question whether, in the normal N animal, T cells can recognize antigen presented b y cells or macrophages of strain 11 appears unresolved (see above). (2)Normal heterozygous ((1 x 11) F, T cells consist of an approximately 50 : 50 mixture of anti-a and anti-b T cells; the proportion of these can be altered by exposure to antigen on macrophages bearing only one of the haplotypes n or 13 (restriction imposed by priming). ( 3 ) Heterozygous ( ( 1 x b ) F, T cells derived from stem cells differentiating in an (i thymus contain functional anti-ci but not anti-13 T cells (H-2 restriction imposed in thymus). (4)Hornozygous strain (I T cells derived from stem cells differentiating in an ((1 x b ) F, thymus contaiii both anti-ci and anti-13 T cells. These generalizations are illustrated in Table I from data obtained in DTH studies (Miller et (il., 1975, 1976; Miller, 1978). It may be tiseful to ask at this stage whether these experiments support or exclude one of tlie models proposed to explain H-2 restriction (Section VI). Although they do not formally exclude the altered self model, they make it simpler to adopt the two receptor model. The reasons are as follows. The two receptor model simply states that anti-H-2 reactivity is generated in the thymus independently ( i n space and time) of anti-X reactivity. On the other hand, the altered self model (one receptor for altered self) has to assume that tlie spectrum of reactivities generated by an anti-A-like receptor developing in thymus

TABLE I TRANSFEROF DELAYED-TYPE HYPERSENSITIVITY

DTH transfer to Sensitized mice

Antigen for sensitization

Naive

II

hive b

Reference ~

F, b ) Fi Thymectomizrd ( ( I x b ) F, grafted with 0 thynius, irratkitrd, and protwtcd with ((I x h ) F, hone marrow Thyrnectomized (o X b ) F,, grafted with h thymus, irradiated and protected with (a X h ) F, bone marrow lrratiiatcd (a x b ) F, given a bone marrow Irradiated (I] x /I) F, given b bone marrow (a x

17)

(a x

" +, Successful transfer, -, no transfer.

In adjuvant In adjuvant On u macrophages In acljuviiiit

+"

~~~

f

+

+

-

Miller et al. (1975) %filler~f 01. (1975) Miller et al. (1976) kliller (1978)

+

Miller (1978)

+

In adjuvant

-

-

On F, macrophagcs

f

f

Miller {unpublished date)

On F, macrophages

+

+

Miller (unpublished data)

HISTOCOIIPATIBILITY COMPLEX A N D T-CELL ACTIVATION

19

A cannot overlap with that generated by an anti-B like receptor developing in thymus B (otherwise there would be no H-2 restriction). If we adhere to Jerne’s negative selection theory (Section IV) we have to explain how random mutations which preserve “A-ness” could generate specificities which do not overlap with those created by random mutations which preserve “B-ness.” Independent generation of anti-H-2 and anti-X reactivities is clearly logical but may carry the implication discussed later (Section IX) that there is positive selection for anti-self-H-2, rather than negative selection as in the Jerne hypothesis. One important observation resulting from the recent experiments of Zinkernagel et a l . (1978b) was the finding that the phenotypic expression of T cell reactivities depended on the H-2 structures expressed by cells of the lymphoreticular system (LRS). The experiments are summarized in Table 11. In brief, long-term chimeras were used (constitution shown in the left-hand four columns). The lymphoreticular system of these chimeras, including macrophages, had been entirely replaced by the injected stem cells (injected bone marrow cells) and thus had the genotype identical to that of the stem cell donor (fourth column). The spleen cells of these chimeras were, in some experiments, transferred to an acutely irradiated host (fifth column) which was infected with virus. The Tc cells generated (in the first or second host) were tested for cytotoxic activity on virus-infected targets A, B, or C. It is TABLE I1 PHENOTYPIC EXPRESSION OF T-CELLREACTIVITIESIs DETERMINED BY H-2 STRUCTURES EXPRESSED O N CELLS OF T H E LYMPHORETICULAR SYSTEM (LRS)“’b

Stem cell donor

A A A A A x B A x B A x B A x B A A

Irradiated recipient

A A A A

x x x x A A A x A x B B

B B B B C C

Thynins epitlielinm

A A A A

x x x x A A A x A x B B

Second acutely Lysis of virusirradiated infected target term chimera recipient A, B, C

LRS of long-

B B B B C C

‘’ For details see text. Partly after Zinkernagel et a / . (197811).

A A A A

A A A A x x x x A A

B B B B

AXB A B A x B -

A x C A x B

A A, B A B A A A A, C None None

20

1. F. A . P. MILLER

clear that A stein cells developing in an (A x B) thymus could express both anti-A and anti-B receptors. However, infection of the original chimera with virus (line 1)-generated Tc cells which could kill only A targets, presumably because the LRS of the original chimeras was only A-type, being derived from the A stem cells. When, however the spleen cells were transferred froin the original chimera into an acutely irradiated A x B host infected with virus, Tc cells were generated which could kill both A and B targets (line 2). It was only in the infected A x B host that the potential of the T cell with anti-B receptors was revealed. When A x B stem cells developed in an A thymus, the cells expressed only anti-A receptors and killed only A targets, whether infection with virus occurred in the original chimera or after the cells were transferred to an acutely irradiated A x B recipient (lines 5 and 6). Similar principles hold for A x B stem cells developing in an A x C thymus (lines 7 and 8). An interesting situation was seen with the totally allogeneic chimeras composed of A stem cells differentiating in a B thymus. Infecting this chimera with virus could not lead to the generation of Te cells, since the A-derived T cells now expressed an anti-B receptor and since the LRS of the long tern1 chimera was of genotype A. However such cells should be able to generate Tc cells when transferred to acutely irradiated A x B hosts (last line of Table 11).But they did not. The explanation given for this failure is as follows. The T, cells require help from TH cells just as B cells require help from THcells. But in order for THcells to recognize the cells they need to help, they must recognize a specific component on the cell to be helped (cf. Section V,B). Previous experiments had shown that help is I-A restricted and the present experiments of Zinkernagel et al. (1978a,b), with congenic lines of mice differing at I-A, likewise suggested that help between TH and Tc was Z-A restricted. In the particular example given in the last line of Table 11, A-line T cells developing in thymus B express anti-B self receptors: i.e., anti-H-2Kh and anti-H-2Dh in the case of Tc cells and anti-I-A” in the case of TH cells. Hence, although A-line T, cells can recognize virus infected A x B targets in the acutely irradiated hosts since they have an anti-B receptor (an anti-H-2K” or anti-H-2Dh receptor), there is no way in which these Tc cells themselves can be recognized and thus helped by A-line TH cells which developed an anti-I-Ab receptor in the B thymus of the original chimera. Why? Because the A-line Tc cells bear the I-Aa gene product on their surface and this cannot be recognized by A-line T H cells which bear, not an anti-I-Aa receptor, but an anti-I-A” receptor. If this interpretation is correct, I-A gene products must be expressed on the surface of Tc cells. So far, however, Ia antigens have

HISTOCOSlPATlBILITY COhlPLEX AND T-CELL ACTI\.ATION

21

generally been found only on B cells and macrophages and on T, cells, but not on T,. cells (Vadas et NI., 1976; Kimura and Wigzell, 1977). VIII. H-2 Restriction: Relationship to MHC-Linked I r Genes

The immune response to a large number of thymus-dependent antigens is controlled by genes which map in the I region of the MHC (for reviews see Benacerraf and Katz, 1975; Benacerraf and Germain, 1978). These genes influence the activities of T cells (e.g., delayedtype hypersensitivity, i n vitro antigen-induced proliferative responses) or T-cell dependent functions such as antibody responses to T-dependent antigens. No instance of MHC-linked I r gene control of antihody responses to T-independent antigens has ever been reported in spite of the many recorded examples of T-independent antigens which produce clonally restricted antilmdy responses. This section will consider the relationship between the H-2 restriction phenomenon and the MHC-linked Ir gene effects. H-2 restriction was shown to be imposed either during priming, or prior to immunization when T cells differentiate in the thymus (Section VII). Likewise, the MHC-linked I r genes may be considered to exert their effects either at the level of cells presenting antigen to T lymphocytes, or at the level of the generation of the T-cell repertoire during differentiation in the thymus. A. FORMATION OF COMPLEX OF ANTIGEN AND MHC PRODUCT IhfMUNOGENIC FOR

T

CELLS

When responsiveness is under MHC-linked Z r gene control, the trait is dominant and an F, between responder (R) and low responder (LR) strains can respond. In DTH, it will be recalled that lymphoid cells from F, mice sensitized to an antigen not under Ir gene control, could transfer sensitivity equally well to naive recipients of both parental types (Section VII). If, however, the lymphoid cells were obtained from a sensitized ( R x LR) F,, transfer of sensitivity to the terpolymer L-glutamic acidf~"-L-alanine:3"-L-tyrosine"' (GAT) was successful in naive recipients of the F, aiid of the R but not of the LR haplotype (Miller et c i l . , 1977). The naive recipients are essentially passive and only present antigens to the T cells which have been actively sensitized in the F, donors. The sensitized (R x LR) F, behaves essentially as the (PI x P,)F, sensitized by antigen-pulsed P1 macrophages (Section VI1,A). The latter can transfer sensitivity to naive P, not P,, the former to naive R not LR. It seems, therefore, that in LR strains,

22

J. F . A . P. MILLER

stimulator cells (probably macrophages) fail to display antigen under Zr gene control in a form immunogenic for T lymphocytes. Support for this was obtained in experiments using antigen-pulsed macrophages. The same amount of radioiodinated GAT was associated with macrophages of R and LR strains after in vitro incubation. Yet only GATpulsed R macrophages could sensitize and elicit sensitivity in F, mice in viuo; GAT-pulsed LR macrophages were totally ineffective (Miller et (11.) 1978).These results differ from those of Pierce et al. (1977) who were able to stimulate F, cells to produce anti-GAT antibodies with GAT-pulsed LR macrophages. It may be that conditions for induction of antibody and DTH responses are not strictly comparable. The failure to transfer sensitivity by F, lymphoid cells to naive recipients of the LR haplotype is thus considered to reflect a defect at the level of antigen presentation due to a failure to produce a LR I-region determinant-antigen complex immunogenic for T lymphocytes. The alternative possibility of a defect at the level of the coding of the T-cell receptor for antigen is more difficult to reconcile with these data. Thus, if one assumed that the Zr gene codes for the T-cell receptor for antigen, the F, should have T cells with receptors able to recognize antigen associated with the I-region gene product of the LR haplotype, since the Ir gene coding for such a receptor would be inherited from the R parental strain. But if this were so, sensitivity from the F, should be transferrable to naive recipients of the LR haplotype. On the basis of these data, therefore, it was suggested that the Zr genes do not code for the T-cell receptor for antigen but code for some structure which ensures a stable molecular interaction between the I-region gene product and the antigen so that the two can form a complex which is immunogenic for T cells (Miller and Vadas, 1977a). The LR lacks the Zr gene which stabilizes the complex between the antigen and the I-region gene product, no complex is formed and T cells which happen to bear a receptor directed to antigenic determinants of the complex (that should have been formed) cannot be stimulated. Using another antigen, the B isoenzyme of lactic dehydrogenase (LDHB), DTH could be provoked in LR strain mice provided these were pretreated with cyclophosphamide (Miller et d.,1976; Miller and Vadas, 197713).Transfer of sensitivity from cyclophosphamide-pretreated, sensitized LR mice to untreated naive LR strains was achieved. Hence, some mechanism must exist in LR mice, not pretreated with cyclophosphamide, to present LDHB, in a relevant immunogenic form, to sensitized cells. When, however, lymphoid cells were obtained from cyclophosphamide-pretreated, LDH, sensitized (R x LR)F, mice, transfer was observed in naive F, and R-strain recip-

HISTOCOMPATIBILITY COMPLEX AND T-CELL ACTIVATION

23

ients, not in naive LR strains (Miller et d., 1978). The failure of sensitized F, cells to transfer DTH to LDHB to Lfi-strain mice is in agreement with the results obtained with GAT (see above). Thus, it can also be surmised that when LDHB associates with the LR MHC product on the macrophage, it is less stable than when associated with the R MHC product, and therefore less immunogenic to T cells involved in DTH but more stimulatory to T, cells. Therefore there will be, in an F,, more T cells reactive to the “R-form” of LDH,. Hence transfer to R strains will be easy to achieve and transfer to LR strains difficult or impossible with the standard dose of cells given. In the LR, however, there is only one configuration of antigen available, that in association with the LR MHC coded product. This is presumed to be unstable: the antigen will tend to predominate in the nonimniunogenic form and stimulate Ts cells. If these are depleted by cyclophosphamide, the small amount of LDHBin association with the LR MHC product will sensitize T D cells and transfer should eventually be possible to naive LR in which the suppressor mechanism is unlikely to be sufficiently activated in the 24 hours required to observe the transfer reaction. These results thus suggest that low responsiveness can be accounted partly in terms of the operation of a cyclophosphamidesensitive suppressor mechanism, and partly in terms of antigen being less immunogenic when presented by LR-strain than by R-strain macrophages. Further evidence for a defect at the level of antigen presentation was obtained in vitro in the antibody response to the hapten TNP conjugated to the polypeptide poly-L-(Tyr, Glu)-poly-D,L-Ala-poly-L-Lys, (T, G)-A-L (Singer et d.,1978). Spleen cells from (R x LR) F, mice, depleted of adherent cells (presumably macrophages), could not respond to TNP-(T, G)-A-L. The response was restored by the addition of adherent cells from the R, but not the LR, parental strain. The reconstitution was antigen specific, strain specific, and controlled by the same MHC gene known to control responsiveness in this system. The idea of a defect at the level of antigen presentation by macrophages is consistent with the results of in vitro sensitized T-cell proliferation assays. Sensitized cells from (R x LR)F1 guinea pigs could be stimulated to proliferate by antigen-pulsed macrophages but only if these were derived from the F, or R-strain, not from the LR strain (Shevach and Rosenthal, 1973). Similar studies were performed with mice and, here too, the in oitro proliferative response was dependent on antigen-pulsed macrophages derived from mice possessing the I r genes necessary for responsiveness (e.g., Schwartz et al., 1976b). Moreover, the activation of sensitized T cells by antigen-

24

J. F. A . P. MILLER

pulsed macrophages was blocked by anti-Ia antibody directed against the Z-region of the R haplotype, not the LR. In cases where complementing l r genes are involved, situations arise where an F, between two different LR strains can respond in contrast to both its parents which cannot (e.g., Giinther and Rude, 1975; Dorf and Benacerraf, 1975). If the defect occurs at the level of antigen presentation by macrophages, one would expect the two genes to be expressed at the level of one and the same cell, not in different cells. This was shown to be the case in the in vitro T-cell proliferation assay (Schwartz et al., 1978). The implications are that both complementing genes are involved in assembling a structure that can complex with the antigen under their control on the surface of the macrophage. The elegant studies of Rosenthal’s group (Barcinski and Rosenthal, 1977; Rosenthal et d.,1978) add further weight to the concept that the genetic control of immune responsiveness resides at the level of the antigen-presenting cell. Both strain 2 and strain 13 guinea pigs are responders to the polypeptide hormone, insulin, as defined by DTH (Clark and Munoz, 1970), antibody production (Arguilla and Finn, 1963), and i n oitro sensitized T-cell proliferation (Rosenthal et ul., 1977). Strain 13 guinea pigs recognize pork insulin through detenninants on the B chain not recognized by strain 2. Strain 2 animals recognize the hormone through determinants on the A chain not seen by strain 13. By using different species variants of insulin to induce sensitized T-cell proliferation, further definition of the area recognized b y strain 2 animals was possible. Recognition depended on the presence of Thr, Ser, and Ile in position A8, A9, and A10, respectively, in the A chain a loop (Barcinski and Rosenthal, 1977). (2 x 13)F, T cells sensitized to pork insulin could respond to the B chain i t z oitro b y proliferation only when this was presented on strain 13 macrophages. Likewise, they could respond to the A chain only if insulin with a loop identities were given with strain 2 macrophages. The l r genes may thus, at the level of macrophages, play some role in the selection of discrete regions within the antigen molecule for recognition by T cells. In contrast to the specificity of T-cell activation, strain differences were not observed in the specificities of the antibodies to pork insulin. Selection of specificities for the B-cell repertoire is thus different and independent from specific T-cell recognition events essential for T-cell triggering. If MHC-linked l r genes exert their effects at the level of the antigen-presenting cell, one would expect some discrimination in the selection of certain B cell clones. For example, it was shown some time

HISTOCOMPATIBILITY COMPLEX AND T-CELL ACTIVATION

25

ago that immune (R x LR)F, T cells could cooperate with R-type B cells, not with LR-type B cells (Katz et nl., 1973). This was confirmed recently in two different systems. One was the MHC-linked control of cross-reactivity to sheep and burro erythrocytes (Kappler and Marrack, 1977). The other system used chimeras between mouse strains differing for both Ig allotype and for high versus low responsiveness to (T, G)-A-L (Press and McDevitt, 1977).If, as discussed in Section V,C, TH cells recognize those B cells that can associate antigen with their siirface Ia determinants in the same way as the macrophages do, one would expect only R-strain B cells to cooperate with ( R x LR)F, T cells. A selection of this type may explain why MHC-linked Ir genes controlling the response to staphylococcal nucleases appear also to determine the relative responsiveness of B cells to two different determinants on the same antigen (Berzofsky 4 t ul., 1977). If the MHC-linked l r genes are expressed in macrophages, they cannot be expected to display the exquisite specificity of the T or B cell receptors for antigen. The l r gene products are likely to be expressed nonclonally and would interact with antigen with a low degree of specificity. The relative specificity ofthe l r gene effects could be achieved by relatively few loci and multiple allelism. As pointed out by Jan Klein (1975), some l r genes behave like alleles at a single locus: for example, the strain distribution pattern of responsiveness to (T, G)-Pro-L is precisely antithetical to that for (Phe, G)-A-L. The l r gene products and Ia determinants may be identical and may interact with a class of molecules with similar three-dimensional conformations. They may thus have the unique capacity to focus or orient discrete regions, composed of relatively few amino acids, for presentation to T lymphocytes. Alternatively, l r genes may not be Ia determinants but may regulate the activities of enzymes which govern the interactions between separate antigenic determinants and the Ia gene products. In order to be consistent with the above model o f l r gene action, one would have to expect that the Z r genes controlling Tc-cell reactions are localized in the K and D regions of the MHC, since those regions restrict the activities of the vast majority of Tc cells (Section V,A). One l r gene which controls the reactivities of Tc cells for TNP-modified targets has indeed been localized between K and I-A (SchmittVerhulst and Shearer, 1976). Moreover, the fact that it has been impossible to obtain Tc cells recognizing the minor H antigen H-Y in association with either H-2Kh or H-2D' antigens in all strain combinations tested (Simpson and Gordon, 1977) could be interpreted as a failure of the H-Y antigen to associate with these H-2K and H-2D gene products

26

J. F . A . P. SIILLER

in a manner that would render it immunogenic for Tc cells. Even though complete capping of H-2 antigens had no detectable effect on the distribution of H-Y antigens (Geib et d.,1977), the relevant H-Y determinants for Tc cells could still be recognized if a small number of such molecules associated in the fluid membrane of the stimulator cells with H-2K or H-2D gene products. Other genetic restrictions of Tc cells to H-Y antigens do not fit this idea (Hurme et al., 1978). It must be recalled, however, that TF1cells may be essential for the generation of a cytotoxic response and that some Z r genes will be expressed at the level of antigen recognition by these cells. Thus, for example, H-2k mice are low responders to H-Y even though Tc cells can be generated against H-2Kk and H-2Dk.The low responsiveness of the H-2k mice may be due to the inability of H-Y to associate with Iak to form a structure immunogenic for TH cells. Other instances of responsiveness dictated by H-2K and H-2D genes will be given in Section X. B. GENERATION OF THE T-CELL REPERTOIRE The suggestion that MHC-linked Zr genes exert their influence at the level of antigen-presentation to T lymphocytes does not exclude the possibility that there may be situations in which Zr genes are expressed at the level of the generation ofthe T-cell repertoire. One level where H-2 restriction is imposed is the thymus (Section VI1,B). Whether it may also be a site of Z r gene action should therefore be considered. CBA mice are essentially low responders (or nonresponders) to virus-infected C57BL targets (Section V,A). When, however, CBA stem cells are allowed to differentiate in a (CBA x C57BL)F, thymus, the CBA T cells can now lyse virus-infected C57BL targets (Section VI1,B). In fact, the “low responder” CBA becomes a “responder” if its cells can differentiate in an F, thymus. Investigations were thus made of the responsiveness of LR-derived T cells after differentiation in a (R x LR)F, thymus. With both TDand Tc cells, LR-derived cells acquired responsiveness in this way. For example, Tc cells derived from low responder BIO.A (5R) stem cells differentiating in an irradiated (H-2k x H-2”)F1 could now lyse H-Y antigen-bearing H-2b targets. This was stated to be “consistent with the hypothesis that diversity of T cell receptors is generated by somatic mutation of germline genes encoding specificity for self-H-2” (von Boehmer et nl., 1978). The experiment does not, however, exclude a defect at the level of the association of H-Y with H-2Kh to produce an immunogenic complex. BIO.A

27

€IISTOCOXlPATIRILITY COllPLEX AND T - C E L L ACTI\’.ATION

(SR) mice are H-2Kt1,I-A”, and H-2D“. When stein cells from BIO.A (SR) diiferentiate in an (H-2k x H-2”)FIthylnI15, the following T-cell reactivities can potentially be generated (in accordance with the thymus chimera experiments described in Section VI1,B): anti-H-2Kk, anti-H-2Kt’, anti-H-2Dk,and anti-H-2Dh for Tc cells, and anti-I-Ak and anti-I-A” for T H cells. Since the macrophages are derived from the stern cells and bear the Ia” antigens, there will be no difficulty in generating THcells for H-Y in association with I-Ah (anti-I-A”THcells). These will he able to cooperate with Tr cells which express I-Ah, since they are derived from BlO.A(SR). When male H-2’’ targets are used, the target antigens able to stimulate anti-H-Y Tr cells could still be only the H-2D” gene products in association with H-Y, rather than the H-2Kh gene products which may f i i l to associate effectively with H-Y. Lysis of H-2” targets would thus be effected b y those Tc cells that are reactive against H-2Dt’, but may not necessarily be achieved by anti-H-2Kh Tc cells. Hence, although anti-H-2D” reactivity is not expressed in an ordinary BlO.A(SR) but only in a BlO.A(SR)+ irradiated (H-2k x H2h)F,,the results of such an experiment do not exclude a lesion at the level of association of H-Y with H-2Kh, In other words, no manipulation at the thyiiius level might ever create a Tc cell which could lyse a target with such a cornplexed antigen. Experiments with TI, cells have given exactly similar results (Miller, 1978). They are summarized in Table 111. CBA (H-2k)mice are

TABLE 111 DELAYED-TYPE HYPERSENSITIVITY TO GAT Bone illarrow donors CBA A.SW C BA A.SW

Irradiated recipients (A.SW x (A.SW x (A.SW x (A.SW x

CBA) F , CBA) F , CBA) F , CBA) F,

Antigens GAT in adjuvant GAT in adjuvant GAT-F, macrophages GAT-F, macrophages

LIR “SI-labeled UclR nptake“ 3.3 & 1.2 2 1.8 2 1.7 2

0.4 0.1 0.1 0.1

The naive recipients were tested for DTH b y a radioisotopic method, using ‘*JI-labeled 5-ioclo-2’-deo~yuriditie,a s described elsewhere (Miller et nl., 1975). Briefly, the mice were challenged with antigen in the left ear and injected intraperitoneally with the radiolabeled DNA precursor, and the ears were removed 24-48 hours later for radioactivity counting. The results are expressed a s the ratio of ratlioactivity in the left ear (L) to that in the right ear (R). It is unusual to obtain it ratio greater than 1.2 in naive mice not receiving sensitized cells. Results are given a s arithmetic means 2 standard error of six to eight mice per group. From Miller (1978).

28

J . F . A . P. MILLER

responders to GAT and A.SW (H-2’) mice low responders. (A.SW x CBA)F, mice were irradiated and repopulated with bone marrow cells from either A.SW or CBA. DTH response was ohserved in the CBA+ (A.SW x CBA)F1 chimeras, not in the ASW + (A.SW x CBA)F1 chimeras. If, however, these chimeras were primed, not by GAT given only in Freund’s adjuvant, but by GATpulsed F, macrophages, DTH to GAT could be induced. The LRS of the chimeras not primed with macrophages had only A.SW macrophages. The results therefore do not allow us to distinguish between a defect at the level of the generation of anti-GAT receptors or a failure of effective association between GAT and Ias on A.SW macrophages, as the reason why A.SW T, cells cannot respond to GAT. When F, macrophages are provided, TI, cells with reactivities against H-2k are likely to be those cells activated to the complex antigen formed by Iak and GAT on the surface of F, macrophages. A defect at the level of the T-cell repertoire can be envisaged if it is hypothesized that antigen-Ia complexes resemble a self-antigen present on thymus epithelium during the differentiation of T lymphocytes (Schwartz, 1978). If this were so, T cells reactive to such structures would be deleted due to self-tolerance or suppression. However, it is not simple with this hypothesis to explain why LR inice can respond if pretreated with cyclophosphamide (Section VII1,A). Unmasking such a latent responsiveness by removing Ts cells appears to be a strong argument against the idea that the MHC-linked l r genes exert their effect at the level of the generation of the T-cell repertoire. Perhaps the most compelling evidence in favor of some l r genes expressing their influence at the level of the generation of the T-cell repertoire comes from experiments with strains of mice having Tc cells exhibiting cross-reactive lysis of TNP-modified allogeneic targets (Billings et al., 1978a,b). In brief, CBA (H-2k)mice (abbreviated “k”) could lyse only TNP-modified syngeneic targets: i.e., did not lyse, for example, TNP-d targets. C57BL (H-2h) mice (“b”) could lyse TNPmodified syngeneic targets and allogeneic targets, TNP-d, as well. The ability to produce cross-reactive lysis was dominant in (k x b)F, mice and did not reflect an inability of TNP-k cells to stimulate crossreactive lysis by F, cells and by cells of appropriate responder strains. The genes controlling this response were mapped to the K and/or I-A region of the MHC. Chimeras of the constitution k + irradiated (k x b)F, could be stimulated by TNP-k cells to lyse TNP-d targets. The environment in which k T cells differentiated thus influenced the specificity of the primary T-cell response to TNP-modified syngeneic cells.

HISTOCOXIPATIBILITY COAIPLEX AN11 T-CELL ACTIVATION

29

IX. Possible Models for T-cell Recognition and Ir Gene Effects

This section will consider in some detail three possible models to explain H-2 restriction and l r gene effects. The first two models assume T cells have two receptors: one for H-2 structures and the other for non-H-2 antigens. It must, however, be emphasized that the phenonienon of H-2 restriction implies that binding by different sites must produce a qualitatively different effect than binding by similar sites. Therefore, multipoint binding per se cannot be a sufficient requirement for T-cell activation. Some additional interaction, perhaps one between two distinct receptors, is essential for triggering. For example, binding to the appropriate antigen b y one site may produce a conformational change which allows the second receptor to bind to its target antigen.

A.

POSITIVE SELECTION FOR T CELLS WITH ANTI-SELF-H-2

RECEPTORS This model is based on the idea of positive selection for cells expressing anti-H receptors only against those H-2 structures displayed by thymus epithelium. It is constructed in such a way that it accommodates the three phenomena: high frequency of alloreactive T, cells, H-2 restriction, and the H-linked l r gene effect. In order to do this, it assumes the existence of only one 2; region gene pool for both the anti-H-2 and the anti-X receptors. An essentially similar model has been described by Langiian (1978). If T cells with anti-self-H-2 receptors are positively selected rather than purged, the activation mechanism of the cell must not be linked to the anti-self-H-2 receptor, otherwise there would be selfdestruction. It has to be linked to the anti-X receptor. The function of the anti-self-H-2 receptor may be to increase the avidity of binding to the stimulator cell presenting antigen X. If two different biological functions are invoked for the anti-self-H-2 and anti-X receptors (the latter with a site linked to an activation mechanism, the former without such a site), it appears logical to invoke two separate genes coding for these receptors. Since the antiself-H-2 receptor can be selected for any H-2 structure, independently of the genotype of the cell (Section VII,B), it is also logical to suggest that it be coded by two genes: one constant and the other variable. Likewise a constant and a variable gene would code for the anti-X receptor, the constant part assuring that the polypeptide chain be linked to the activation mechanism of the cell, the variable part coding

30

J . F. A. P. MILLER

for structures more or less complementary to any antigenic determinant. It seems also reasonable to surmise that these genes arose by duplication and expansion of an ancestral gene in the primitive precursor of the T cells, whose function must have been to ensure selfintegrity by binding to a self marker (the precursor of H-2 structures). If so, one could assume a common u region gene pool for both antiself-H-2 and anti-X and develop the following model (Fig. 2). (1) The germline genes contain a C gene for the constant part of anti-self-H-2, a C gene for the constant part of anti-X, and a u region gene pool that can code for structures which are essentially complementary to the H-2 alleles of the species. (2) When the pre-T cells enter the thymus, positive selection for proliferation will occur for only those cells which happen b y chance to have translocated to the C anti-H gene the correct o gene, i.e., that which codes for a chain complementary to the H-2 structure expressed on the thymus epithelium. ( 3 ) Random somatic mutation can now affect the rest of the o region gene pool in the proliferating population, and random genes will be translocated to the C anti-X gene to allow production of the anti-X receptor. The model can explain the high frequency of alloreactive T cells and the H-linked Zr gene effects. Provided somatic mutation does not affect each and every u gene in each and every proliferating T cell (which is hardly likely), there will be a high frequency of T cells with an anti-X receptor derived from an unmutated u gene, and therefore alloreactive. 1. Germline genes v4 v5 v6 . . . . . . . . . . v n

C anti+ C anti-X v l v2 v3 u u 2.

H

Translocation of v gene to C a n t i - H C a n t i - H v5 a n t i - H

v l v2 v3 v4 v6

C anti-X

,.

I V l H H - + + + - i L

3.

H

2

Random somatic mutations of v genes C anti-H v5 a n t i - H

C anti-X

v l v2 v3 v4

v6

.. , . . .

H 4.

. . . vn

----- v5 ---__-

vn

H

Translocation of any v gene, mutated or unmutated. to C anti-X C anti-H

-1

v5 anti-H C anti-X v4 anti-X v l v2 v3 v6 . . . .. v n 1

'

4

" " '

L--V4

--A

I '

H

FIG. 2. A genetic iiiodel to explain the development of the genes coding for two antigeii-recogniziiig receptors oil the T cell.

HISTOCOJIPATIBILITY COhIPLEX AND T-CELL ACTIVATION

31

The model can accommodate the H-linked Zr gene effect. A specific example will facilitate the task of explaining this. Consider the antigen GAT. H-25 mice are LR and most other strains are R. Assume that a mutation in a particular 2 j gene, say v 5 , which codes for the structure complementary to H - 2 , would create the polypeptide chain which best fits GAT. In H-2" mice, U S has been translocated to the C anti-H gene to produce anti-H-2". It has therefore been removed from the rest of the gene pool which is now allowed to mutate (if u5 in the C anti-H position mutates, the mutated cell will not function since the cells are selected on the basis of their capacity to express anti-H-2"). Since there is no U S in the mutable v region pool in an H-2s mouse, there is no possibility of creating an anti-GAT receptor in H - 2 mice. Therefore H-2' mice inlist be LR to GAT. It can be argued that mutations in other u genes, say 055, could produce an anti-GAT structure. This, however, may be of such low affinity that the mice would be LR. In this model, therefore, the so called Ir genes could be claimed to code for the T-cell repertoire and would appear to be linked to MHC but only in a functional sense. The structural genes involved need iiot even be on the same chromosome as the MHC since they code for structures complementary to MHC-coded antigens. There are many difficulties with this model and among these may be mentioned the following.

(1) The model allows successful T-cell activation when binding to foreign H-2 occiirs via the anti-X receptor alone. This must be so since alloreactivity is not restricted by self-H-2. On the other hand, the model states that binding by anti-X to non-H-2 antigens does iiot activate unless additional binding occurs via anti-self-H-2. This is necessary to account for H-2 restriction of the responses to non-H-2 antigens. Why anti-X bindiiig alone is sufficieni for T-cell activation in one case (alloreactivity), but not in the other (non-H-2 reactivity) cannot be explained solely on the basis of affinity, since some aiiti-X receptors should have high affinity for X. Hence additional ad hoc assumptions must be introduced. For example, Langnian (1978)has proposed that a channel (for lytic effects, but this presumably can be extended to activation effects) is physically linked on target (or stimulator) cells to MHC gene products and must be brought into close apposition with a similar channel situated between the paired anti-self-H-2 and anti-X receptors on the responding T cell. The binding of anti-self-H-2 to H-2 approximates the two channels but no signal for activation occurs unless anti-X binds to X on the stimulator or target cells. On the other hand, the binding of anti-X to foreign H-2 is associated both with the

32

J. F.

A.

P. MILLER

delivery of an activation signal and the approximation of the relevant channels between stimulator and responder cells. (2) Perhaps a greater difficulty with this model is to explain why products of wild-type H-2Kb and mutant H-2Kt)agenes cross-react at the level of allogeneic Tc cells and B-cell-derived antibodies, but appear distinct when recognized by Tc cells immunized to virus-infected cells (Blanden et al., 197613).If positive selection did occur for T cells with anti-self-H-2 receptors, those cells with high-affinity anti-self-H-2 receptors should exhibit cross-reactivity for other H-2 antigens which would lead to a breakdown of H-2 restriction. It must be recalled that, for a related set of antigens, sites with high-affinity binding have low specificity and thus exhibit cross-reactivity (Eisen, 1964). (3) If we accept the model of gene organization for T-cell receptors given in Fig. 2, we must assume that the spectrum of reactivities for T cells is different from that for B cells. There are many examples in the literature which come to just that conclusion. On the other hand, the idiotype work of Rajewsky and Eichmann (1977) and of others suggests that somewhat similar regions exist on both T- and B-cell receptors for antigen. Whether this proves that these receptors must utilize one and the same u region gene pool remains to be determined. (4)The model allows a novel explanation of H-linked immune responsiveness based on the idea that the Zr gene effect may be linked to the phenomenon of H-2 restriction at the level of the genes coding for the T-cell anti-X receptor. It fails, however, to account for the phenomenon of complementation, particularly since the complementing genes exert their effects at the level of one and the same cell (Section VII1,A). It is also incompatible with the appearance of responsiveness in LR strains following cyclophosphamide treatment. B. SELECTION AGAINST T CELLS WITH HIGH-AFFINITY ANTI-SELF-H-2 RECEPTORS

This model assumes that the anti-H-2 receptors of the T cells are directed not solely to H-2 structures present in the thymus where T cells developed, but also to ciny H-2 alleles of the species. The anti-X receptors are directed to any non-H-2 structures. The model explains the high frequency of alloreactive T cells, but does not demand that the same 2) region gene pool be used by the two receptors. Finally, it explains the phenomenon of H-2 restriction provided some suppressor component is activated. Essentially similar models have been proposed by Janeway et d.(1976) and by Blanden and Ada (1978). The frequency of alloreactive Tc cells is generally stated to be 100 to

HISTOCOhlPATIBILITY COSIPLEX AND T-CELL ACTIVATION

33

1000 times higher than the frequency of T cells reactive to non-H-2 antigens. However, most experiments leading to this generalization were performed with noncongenic H-2 incompatible mouse strains (Skinner and Marbrook, 1976; Linda111 and Wilson, 1977). In congenic strain combinations, incompatible only at H-2 and not at minor H or other cell surface antigens, the freqiiency of alloreactive T cells was less than that found in noncongenic strain combinations. In order to explain this finding, on the basis of a two receptor model, the following suggestions were made (Marbrook et n / . , 1978).The anti-H-2 receptors are directed to any H-2 alleles of the species and display a range of affinities extending from low to high levels. If the activation of precursors is related to the affinity of the receptor for the stimulating antigen, the fixtion of clones against a given H-2 allele which were activated b y the corresponding H-2 antigen could correspond to the high-affinity precursors. These would not require dual recognition to be activated and hinding b y the anti-X receptor would be irrelevant. This implies that the activation mechanism of the cell must be linked to the antiH-2 receptor, not to the anti-X as in the previous model. On the other hand, clones with low affinity for that particular H-2 allele would not be activated by the corresponding H-2 antigen alone. When confronted by stimulator cells which, in addition to H-2, have non-H-2 differences, the avidity of the precursors for the stimulators would be increased by additional binding of non-H-2 antigens via the anti-X receptors. This would activate the low-affinity clones and lead to the increased frequency. This model generally agrees with that proposed b y Janeway et a / . (1976). More recently, Blanden and Ada (1978) have used an essentially similar model to account for the exquisite specificity of virusiminune Tc cells which can distinguish between virus-infected wild type (H-2K”)and mutant (H-2K””)cells. They therefore suggested that only T cells with receptors of low affinity (and hence high specificity) for H-2 structiires displayed on the thymus epithelium are allowed to survive, cells with high-affinity receptors being destroyed (possibly b y contact with the epithelium). I n addition, T cells with anti-H-2 receptors against any allele not expressed on the epithelium will be produced. The anti-X receptor is of course generated independently of the anti-H-2 receptor. To explain the results obtained in (A x B)F, stem cells -+ irradiated A mice (Section VI1,B). Blanden and Ada (1978) proposed the activation of some suppressor component. For example, any cell with an anti-H-2 receptor directed against B (anti-B) which also has an anti-X receptor directed against a component not present on the thymus epithelium but expressed, for example, on the F, cells,

34

J. F . A . P. MILLER

should be stimulated and should proliferate. This would activate an anti-idiotype suppressor system directed against the anti-B receptor on the proliferating cells. Hence, response to virus-infected B cells would not be seen and H-2 restriction, as observed by Zinkernagel et ul. (19784, would appear to have been “learned” in the thymus and not to have been imposed as a consequence of priming. If an anti-idiotype suppression supervenes to prevent cells with anti-B reactivity from operating in vivo, it can be predicted that anti-B reactivity should return when the suppressor component is removed, e.g., if the cells of the chimera are incubated in vitro. This model implies that H-2 restriction is not preexistent but is imposed by priming [as suggested before (Miller and Vadas, 1977a) and in Section VII,A]: hence antigen-pulsed allogeneic macrophages should prime and the reactivities of the primed cells should be restricted by the foreign H-2. This was shown to occur by Pierce et ul. (1976) and by Thomas and Shevach (1977) in vitro, and by Wilson et ul. (1977) in v i v o . Unfortunately, however, two groups could not repeat these observations (Section VI1,A). This model does not require that the anti-H-2 and anti-X receptors share the same u region gene pool. The pool for the anti-X receptor could be the same as that which codes for the u regions of the Ig heavy chains. This would readily accommodate the findings of several groups of a sharing of idiotypes between T cells and humoral antibodies (Rajewsky and Eichmann, 1977). In turn, this would imply that the MHC-linked Zr genes are not expressed at the level of the T-cell anti-X receptor, since LR mice have a selective defect in T-cell functions, not in B-cell reactivities. The model would thus be supported by observations that MHC-linked Ir genes exert their effects at the level of the antigen-presenting cells (Section VII1,A). C. MHC GENEPRODUCTSON ANTIGEN-PRESENTING CELLSARE OBLIGATORY FOR T-CELL ACTIVATION This model is totally heterodox. It was suggested very briefly previously (Miller and Vadas, 1977a). It will be easy to modify or abandon as more data accumulate. It assigns to MHC antigens present on stimulator cells on obligatory role in the activation of T lymphocytes. It does not require any special mechanism for the generation of diversity of the T-cell repertoire. Both T and B cells could use essentially the same u region gene pool. The specificity of selection of the T-cell repertoire, revealed by the phenomena of H-2 restrictions and Ir gene effects, is dictated, not at the level of the u region genes, but at the

HISTOCO3lPATIBILITY COhIPLEX A N D T - C E L L ACTIVATION

35

level of association of antigenic determinants with MHC gene products on antigen-presenting stimulator cells (Section VII1,A). The inodel is based on three essential premises. (1) The frequency of strictly alloreactive T cell clones is tiot as great as the frequency of clones reacting to any non-H-2 antigens (Marbrook et al., 1978). (2) Cells lacking MHC gene products fail to activate T cells (Bevan and Hyman, 1977). ( 3 ) Stimulator cells provide not only MHC gene products but also a nonspecific inductive stimulus acting as a second signal for T-cell activation (Lafferty and Woolnough, 1977). As mentioned in Section IX,B, the frequency of clones of precursors of T, cells in spleen was found to be lower in one congenic strain combination (differing only at H-2) than in noncongenic H-2 incoinpatible mice (differing at H-2 and at minor H loci as well) (Marbrook et crl., 1978).Of course this has to be repeated using many other congenic strain combinations, and trivial explanations, such a s a liniiting number of TH cells in congenics versus noncongenics, must be excluded. But if it is assumed that strictly alloreactive T cells are not as frequent as was previously thought, the necessity of having to generate the T-cell repertoire by special mechanisms must be questioned (e.g., Jerne, 1971; Langnian, 1978; Sections IX,A and B). The H-2 restriction phenomenon need not necessarily be explicable in terms of the T-cell repertoire arising by somatic mutations of germline u region genes coding for structures essentially complementary to the H-2 alleles of the species. It could simply reflect an obligatory role for MHC gene products, present on certain “stimulator” cells of the LRS, in T-cell activation. Is there any evidence for this? Cell lines expressing viral antigens or minor H antigens, but lacking H-2 or HLA gene products are resistant to lysis by Tc cells. Thus, Tc cells generated in uiuo by LCM virus infection failed to lyse LCM-infected F9 targets which do not express H-2 antigens (Zinkernagel and Oldstone, 1976). A C58 ( H-2k)lymphoma variant line lacking H-2 and T L gene products could not b e lysed by Tc cells directed to H-2k or minor H antigens (Bevan and Hyman, 1977). Daudi cells, originating from a human B lymphoma and carrying Epstein-Barr virus (EBV) genome but lacking HLA gene products, were resistant to lysis by Tc cells from patients with infectious mononucleosis. The same Tc cells could lyse two HLA-positive EBV genome carrying cell lines (Tursz et al., 1977). Therefore H-2 and HLA antigens are required on targets for lysis b y Tc cells. Perhaps an even more important question is whether MHC antigens are essential for stimulation of T cells. No schedule of inimunization with the H-2 negative C58 variant lymphoma line could induce activation ofT, cells in vitro orin uivo (Bevan and Hynian, 1977). The only

36

J. F. A . P. MILLER

H-2 negative cell line which could activate Tc cells in uiuo was the teratocarcinoma cell line lacking H-2 but possessing F9 (Section V,E,2). It will be recalled that F9 is coded by genes in the T/t complex which may be related to the H-2 complex (Section 11). F9 may thus conceivably act as a functional analog ofH-2. On the other hand, antiidiotype antibody was shown to induce THcells but whether H-2 is involved in this activation, or is not required, is not known (Eichmann and Rajewsky, 1975). As emphasized by Lafferty and his colleagues (e.g., Lafferty and Woolnough, 1977), MHC antigens, per se, may not be sufficient for T-cell activation. Only MHC gene products on certain cells of the LRS (“stimulator cells”) may be able to activate, perhaps because they are closely associated with some molecular mechanism which can deliver an activating signal to a nearby T cell. Hence, only T cells with receptors having varying degrees of affinity for MHC antigens will bind these and, as a result, will receive an activating signal from the stimulator cell. Langman (1978) also proposed physical linkage, to MHC gene products on target cells, of a channel for the delivery of an activation signal to the T cell (Section IX,A). This third model does not, of course, require two receptors per T cell. One receptor directed toward complexed MHC determinant and antigen is sufficient. The model implies that any H-2 restriction apparent before priming arises only as a result of the necessity to maintain tolerance, as was discussed for the second model above (Section IX,B). Finally, implicit in the present model is the notion that, under normal circumstances, T cells with receptors having varying degrees of affinity to allo-H-2 antigens, which do not cross-react with antigens formed by the association of antigen and self-H-2, will be wasted. This cannot be used as a persuasive argument against the model. Many potential antigens are never encountered in the normal life of an individual and yet the repertoire enabling production of the corresponding antibodies exists. Furthermore, as pointed out by Janeway et ul. (1976), wastage in a biological system may b e a sign of its importance. X. H-2 Restriction and Resistance to Tumors

Tc cells are generated during oncornavirus-induced oncogenesis in the primary hosts (e.g., Leclerc et id., 1973). They probably play some role in tumor cell destruction in uivo. Are they H-2 restricted? In some experimental systems there is clear evidence of H-2 restriction. Thus, for example, Tc cells specific for SV40 tumor-associated antigens generated in C57BL/6 and BALB/c mice killed SV40-transformed cell lines of the same H-2 specificity (Trinchieri et ul., 1976). Likewise,

I-IISTOCOXIP4TIBILITY COlIPLEX A N D T-CELL ACTIVATION

37

H-2 restriction was observed with Tc cells generated by injection of niurine sarcoma virus (MSV) (Gomard et d . , 1976) and Friend virus (Blank et a l . , 1976). I n other experimental investigations, however, H-2 restriction was not always seen. For example, spleen cells from mice immune to MSV, Friend, or Rausclier virus could under certain conditions lyse either syngeneic or allogeneic tumor targets carrying the appropriate tumor-associated antigen. In one such investigation, H-2 restriction was seen i n a 4-hour :'Cr release assay, not in a11 18hour assay (Ting and Law, 1977). I n other studies (Holden and 1978), sonie results showed H-2 Herbemian, 1977; E. Klein et d., preference rather than H-2 restriction and memory responses seenied less restricted than primary ones. Cross-reactive Tc cells could have been generated in some of these situations and account for the apparent lack of H-2 restriction, as was pointed out in Section V,E,3. If memory cells are cells with high-affinity receptors, and if, a s occurs in the ,case of antibodies to a related set of antigens (Eisen, 1964), highaffinity T-cell receptors exhibit cross-reactivity to a greater degree than low-affinity receptors, less H-2 restriction can be expected in a meniory response. In some instances, human T, cells showed no apparent HLA restriction in the lysis of EBV-carrying cell lines. For example, patients with infectious mononucleosis, but not normal individuals, had T cells ahle to kill EBV-genome-carrying LCL specifically and Burkitt's lymphoma cells, but not any EBV-negative cell lines (Svedmyr and Jondal, 1975; Jondal et uZ., 1975). In another study, HLA-, EBV-genome+, Daudi cells could not be killed by T cells from infectious niononucleosis patients. Some HLA restrictions were observed, although T cells from one patient could kill targets with which they shared no HLA antigens (Tursz et d . , 1977). Several reasons may be used to explain the lack of HLA restrictions toward EBV-genome-carrying cell lines. (1) Human individuals may show less MHC restriction than mice. This is unlikely to be the case, because restrictions of human Tc cells have been demonstrated in responses to influenza virus (McMichael et d . , 1977) and in individuals sensitized to diiiitroclilorobenzene (Dickmeiss et a1 ., 1977). In addition, a female patient who had rejected a bone marrow graft from her HLA-identical h o t h e r had Tr cells in her blood with activities restricted to inale targets having the HLA-A2 antigen in common with lierself(Gou1my et d . ,1977).( 2 ) New HLA specificities may be expressed after EBV infection, just as has been found on mouse tumor cell lines (Garrido et d . , 1977; Gipson et al., 1978). This may not, however, be a general phenomenon: for example, no foreign specificities have been found on EL4 mouse lymphoma lines (Flaherty and Rinchik, 1978). ( 3 ) The HLA system may be far more

38

J. F . A. P. MILLER

polymorphic and may exhibit more cross-reactivity than the mouse H-2 system (Dupont et nl., 1976). Thus, there may be cross-reactivity not revealed by known serological cross-reactions between HLA antigens. It was indeed suggested that EBV-transformed LCL expressed target antigens cross-reactive with allogeneic target antigens found on normal cells (Zarling and Bach, 1978). (4) The HLA antigens are not as clearly defined as the mouse H-2 antigens: the determinants of the C locus, for example, are incompletely defined. ( 5 ) Some Tc cells may be directed exclusively to EBV-specified antigens and these alone may provoke Tc-cell activity in the absence of association with HLA. It will be difficult to prove this particularly in view of the third and fourth points listed above. On the other hand, it would be important to establish whether this is the case, since the EBV system can be assumed to reflect a rather ancient encounter of the species. As suggested by George Klein (personal communication) viruses which have evolved to achieve a balanced relationship with their hosts may, when they transform cells, induce membrane changes which per se activate the T-cell system to limit the proliferation of the malignant cells. In mice injected with Friend virus and the radiation-induced leukemia virus (RadLV), association of viral antigens with defined H-2 gene products seem to determine host resistance. Mice of H-2b haplotypes are resistant to Friend virus. A physical association between virus and H-2Dh molecules has been demonstrated on cell surfaces. No affinity was shown to exist between the viral molecules and the H-2Kb, H-2Kd, and H-2Dd molecules (Blank and Lilly, 1977). The H-2 resistance conferred by the H-2h haplotype may thus be mediated b y Tc cells directed against a complex antigen made up of both viral and H-2Db molecules on the cell surface. In the case of the RadLV, resistance was conferred by the H-2W allele, not by H-2q and H-2s. The synthesis of H-2K gene products was found to be increased on the surface of thymus cells after virus infection (the thymus being the target organ for RadLV infection). Significant increases in the expression of H-2D molecules, however, occurred only on thymus cells from resistant strains ( H-2Dd). Resistance, therefore, presumably depends on stimulation of Tc cells by virus in association with the H-2Dd gene products. On the other hand, the diseased state may follow the disappearance of the H-2 antigens since RadLV-transformed cell cultures showed decreased H-2 antigen expression (Meruelo et nl., 1978). That Tc cells play a role in resistance to virus infections has been formally demonstrated in the influenza virus system (Yap et ul., 1978). The same type of resistance mechanism is likely to hold also for oncoviruses. Association of viral molecules with defined H-2K or H-2D

HISTOCOhlPATIBILITY COhlPLEX AND T-CELL ACTIVATION

39

allelic products may provoke a good Tc-cell response and hence a better elimination of the virus-infected cells. Viewed in this way, the H-2K and H-2D genes perform functions analogous to the I-region gene products in determining responsiveness. As suggested above (Section VIII,A), the Z r genes controlling the reactivities of Tc cells may thus be considered to reside in the K and D regions of the MHC. XI. Summary and Conclusions

Genes of the MHC exert a profound influence on T lymphocytes. The MHC-linked Ir genes control T-cell activities and T-cell-dependent functions, and the reactivities of various T-cell subsets are restricted by distinct MHC genes. I-region gene products govern the immune responses of T H and T D cells to antigens presented by macrophages and B cells. H-2K and H-2D gene products influence the response of Tc cells which play an essential role in resistance to virus infections. The exact manner by which the MHC produces such effects is not known, but several possible mechanisms were discussed in this review. One suggestion is that MHC gene products are intimately involved in delivering an activating signal to T cells. Only antigenic determinants able to associate with such products would thus be immunogenic. The existence of several MHC gene loci and of multiple allelism at each locus will ensure effective association of antigenic determinants with MHC gene products in most members of a given species. Polymorphism thus allows most individuals to respond adequately to one or other determinant of a complex antigen. Abbreviations Used: DNP, 2.4-dinitrophenol; DTH, delayed-type hypersensitivity; EBV, Epstein-Barr virus; GAT, terpolymer L-glutamic acid60-L-alanine""-L-tyrosine'"; H, histocompatibility; Ig, immunoglobulin; LCL, lymphoblastoid cell lines; LCM, lymphocytic choriomeningitis; LDHB, isozyme B of lactic dehydrogenase; LR, low responder; LRS, lyniphoreticular system; MHC, major histocompatibility complex; MSV, murine sarcoma virus; NK, natural killer; (Phe, G)-A-L, poly-L-(Phe, G ~ L I ) - ~ o ~ ~ - D , L Alii-poly-~-Ly~; R, responder; RadLV, radiation-induced leukemia virus; T,, cytotoxic T cells; TD, T cells involved in DTH; T,, helper T cells; T,, suppressor T cells; (T, G)-A-L, p ~ l y - ~ - ( T yGr ,l ~ ~ ) - p o l y - ~ , ~ - A l a - p ~ l y(T, - ~G)-Pro-L, -ly~; poly-L-(Tyr, Chi)poly-u,L-Pro-poly-L-Lys; TNP, 2,4,6-trinitrophenol.

Note Atltlerl i r i P J Y J O An ~ . error was made in calculating the frequency of clones of precursors of cytotoxic cells in congenic combinations in the paper b y Marlxook et ( I / . ( 1978). The difference between congenic and noncongenic coml,inations is of the order oftenfold only.

40

J. F . A . P. MILLER

REFERENCES Ada, G. L., Jackson, D. C., Blanden, R. V., Tha Hla, R., and Bowem, N. A. (1976). S C O t t d . J . ~ W ~ t J l l l l ~5,O 23-30. ~ . Arguilla, E. R., and Finn, J. (1963).J . E x p . A4ed. 118, 55-71. Artz, K., and Bennett, D. (1975). Nature (Loitdon) 256, 545-547. Barcinski, M. A., and Rosenthal, A. S. (1977)./. Ex),. A4ed. 145, 726-742. Benacerraf, B., and Germain, R. N . (1978). ~ l J l 7 t i u t l ~ iReo. /. 38, 70-119. Benacerraf, B., and Katz, D. H. (1975).Ado. Coticer Res. 21, 121-173. Berzofsky, J. A., Schecter, A. N., Shearer, G. M., and Sachs, D. H. (1977).]. E s p . Med. 145, 123-135. Bevan, M. J. (1975)./. E x p . Bled. 142, 1349-1364. Bevan, M. J. (1977). Proc. N u t l . Acnd. Sci. USA 74, 2094-2098. Bevan, M. J. (1978). Natirre (Lotidoti) 269, 417-418. Bevan, M. J., and Hyman, R. (1977). Ztnniutiogetietics 4, 7-16. Billings, P., Burakoff, S . J., Dorf, M . E., and Benacerraf, €3. (1977a). J. E x p . Xfed. 145, 1387- 1392. Billings, P., Burakoff, S. J., Dorf, M. E., and Benacerraf, B. (19771)).J. Ex),. Med. 146, 623-628. Billings, P., Burakoff, S . J., Dorf, M. E., and Betiacerraf’, B. (1978a).1.E.v/J..\fed., 148, 341-351. Billings, P., Burakoff, S. J., Dorf, M. E., and Benacerlaf, B. (19781))./. E q i . ,\fed., 148, 352-359. ~ . 181-190. , Blanden, R. V., and Ada, G. L. (1978). Sccltld. /. Z ~ U ~ I I U I I 7, Blanden, R. V., and Gardner, J. (1976). Cell. Zmniutiol. 22, 271-282. 13, 179-191. Blanden, R. V., Hapel, A. J., and Jackson, D. C . (1976~1).Zmmutiochemi.~fr~ Blanden, R. V., Duiilop, M. B. C., Doherty, P. C., Kohn, H. I., and McKenzie, I. F. C. (l976b). I t ~ ~ n , u t i o g e ~ l e3, t i c541-548. .~ Blank, K. J., and Lilly, F. (1977). Nuture (London)269, 808-809. Blank, K. J., Freedman, H. A., and Lilly, F. (1976). Nuture (London) 260, 250-252. Bloom, B. R., and Bennett, B. (1970). S e m i t i . H e m u t o / . 7, 215-224. Braciale, T. J. (1977)./. E x p . A4ed. 146, 673-689. Burakoff, S. J., Gerniain, R. N., and Benacerraf, B. (1976)./. E x p . Med. 144, 1609-1619. Burakoff, S. J.. Ratnofsky, S. E., and Benacerraf, B. (1977).Proc. N n t l . Accid. Sci. USA 74, 45724576. Cantor, H., and Boyse, E. A. (1977). ZntnmrioZ. Rev. 33, 105-124. Cerottini, J. C., and Bninner, K. T. (1974).Adti. Zmtnrtttol. 18, 67-132. Clark, C., and Munoz, J. (1970).J . Zmmtrtiol. 105, 574-583. Dickmeiss, E., Soeberg, B., and Svejgaard, A. (1977). Nnticre (Lotidoti ) 270, 526-528. Doherty, P. C., Blanden, R. V., and Zinkernagel, R. M . (1976~1).Trcltislil. Reo. 29,89-124. Dolierty, P. C., Gotze, D., Trinchieri, C., ancl Zinkernagel, R. M . (19761-3).lnittintiogetiefic.9 3, 517-524. Doherty, P. C., Solter, D., and Knowles, B. (1977). Nutiire (London) 266, 361-362. Dorf, M. E., and Benacerraf, B. (1975). Proc. N u t l . Acnd. Sci. USA 72, 3671-3675. Dupont, B., Hansen, J. A., and Yunis, E. J. (1976).Adu. Zninirrtiol. 23, 107-202. Eichmann, K., and Rajewsky, K. (1975). Eirr. J . Zr~imutio/.5, 661-666. Eisen, H. N. (1964). Ilnrtie!/ Lect. 60, 1-34. Ellner, J. J., Lipsky, P. E., and Rosenthal, A. S. (1977)./. Initnuno/. 118, 2053-2057. Ennis, F. A., Martin, W. J., ancl Verlmnitz, M. W. (1977).Noture (Lotirloii) 269,418-419. Feldniann, M., and Nossal, G. J. V. (1972). TruiispI. Reo. 13, 3-34.

HISTOCOMPATIBILITY COMPLEX AND T-CELL ACTIVATION

41

Feldmann, M., Baltz, M., Erb, P., Howie, S., Kontiainen, S., Woody, J., and Zwaiffler, N . (1977).Progr. Zntincri~01.3, 331337. Flalierty, L., and Rinchik, E . (1978).Ncrture (Lorldort) 273, 52-53. Ford, W. L., and Atkins, R. C. (1971).Ntrture Netc B i d . 234, 178-180. Forinan, J. (1975).J.Exp. Aled. 142, 403-418. Garrido, F., Schirrniacher, V., and Fetenstein, H . (1977).J . Immtinogewt. 4, 15-27. Geil), R., Goldberg, E. H . , and Klein, J. (1977).Nature (Loitdoit) 270, 352354. Germain, R. N., Dorf; M. E., and Benacerraf, B. (1975).J.Ex/,. Med. 142, 1023-1028. Gipson, T. G., Imamiira, M., ConliRe, M.A., and Martin, W. J. (1978).J.E s p . Med. 147, 1363-1373. Gomard, E., Duprez, \’., Henin, Y., and Levy, J . P. (1976).h’rrtiire (London)260, 707709. Gordon, R. D., Simpson, E . , and Samelson, L. E. (1975).J.Ex),. Med. 142, 1108-1120. Goillmy, E., Termijtelen, A,, Bradley, B. A,, and van Rood, J. (1977).Nature (London ) 266, 544-545. Giinther, E., and Riide, E. (1975).J.I i t t m i t i i o l . 115, 1387-1393. Holden, H. T., and Herberman, R. B. (1977).Notirre ( L o i i d o i i )268, 250-252. Hurme, M.,Hetherington, C. hl., Chandler, P. R., and Simpson, E. (1978).J.E s p . Aled. 147, 758-767. Jackson, D. C., Ada, G. L., and Tha Hla, R. (1976).Airstrcrl. J . E s p B i d . Med. Sci. 54, 349-363. Jacob, F. (1977).I Pi n~~ r i t o / .Rec. 33 3-32. Janeway, C. A,, Wigzell, H., and Binz, H. (1976).Scmitd. J . I n i i t i i r i i o / . 5, 993-1001. Janeway, C . A., Murphy, P. D.. Kemp, J., and Wigzell, H. (1978).J . Ex)).M e d . 147, 1065- 1077. Jerne, N . K . (1971).Eirr. J . I i i i i i i i r i i ~ ~ 1, / . 1-9. Jondal, M . , Svedmyr, E., Klein, E., and Singli, S. (1975).Ntrtirre (Loridon)255, 405-407. Julius, hl. H., Cosenza, H., and Augustin, A. A. (1977).Nutitre (Loiidoit ) 267, 437439. Kapp, J. A. (1978)./. E.xp. ,\let/. 147, 997-1006. Kappler, J., and Mtirrack, P. (1977).J.E x / ) . ,\let/. 146, 1748-1764. Katz, D. H., and Benacerriif, B. (1975).T r t r i i . ~ p Ret;. /. 22, 175-195. Katz, D. H., Hamaoka, T., Dorf; hl. E., Maurer, P. H., and Benacerraf, B. (1973).J.Es),. illcd. 138, 734-739. Kimiira, A. K., and Wigzell, H. (1977).Coiiteritp. Toi1ic.v Z t i t i i t i r i i o / ~ i o / .6, 209-244. Klein, E., Becker, S., Galili, N., Devens, B., and Naor, D . (1978). Proc. Erwin Reisch Symp. “Cytotoxic Cell Interiiction and Inimrinostimulation,” in press. Klein, J. (1975). “Biology of the h4oiise HistoconipatiI,iIity-2 Complex.” SpringerVerlag, Berlin, N e w York. Klein, J., Chiang, C . L., and Hauptfeld, V. (1977).J.E.xp. ,\led. 145, 450454. Koszinowski, U., and Ertl, H. (1976).Eirr. J . I i i i t t i i r i i o / . 6, 679-683. Koszinowski, U., Gething, M. J., and Waterfield, M .(1977).Nntirre (Loitdoit ) 267, 160163. Krawinkel, U., Cranier, hl., Imanislii-Kari, T., Jack, R. S . , Rajewsky, K., and Mikela, 0. (1977).EUr. 1. ~ l t 1 l 1 1 ~ ~ l i O8,/ . 566-4573, Krawinkel, U., Crainer, hl., Melcliers, I., Imanishi-Kari, T., and Rajewsky, K. (1978).J . E.xp. ,\fed. 147, 1341-1347. Kuinar, V., and Bennett, M. (1977).Ncrtttrc, ( L o i i d o i i ) 265, 345-347. LaRerty, K. J., and Woolnough, J . (1977).I i ~ t n t i i i i o l .Rec. 35, 231-262. Langnian, R. E. (1978).R e c . P l t ! / s i o / . Riochou. Phuriittrcol. 81, 1-37. Lanzarowitz, S. G., Compaiis, R. W., and Choppiti, P. W. (1971).Viro/og!/46, 830-843.

42

1. F. A . P. MILLEH

Leclerc, J. C., Gomard, E., Plata, F., and Levy, J. P. (1973). I n t . J . Cancer 11, 426-432. Lemonnier, F., Biimkoff, S. J., Germain, R. N. aiid Benacerraf, B. 1977). Proc. Not/. Acc~d.Sci. USA 74, 1229-1233. Lindahl K. F., and Wilson, D. B. (1977).J. E x p . Jled. 145, 508-521. Lindahl, K. F., Peck, A. B., and Bach, F. H. (1975). Sccttid. J . Itimtoiol. 4, 541-553. Marl)rook, J., Nawa, Y., and Miller, J. F. A. P. (1978).J. E x p . Jled. 148, 324-328. McKenzie, I. F. C. (1977). Progr. I n i t n t t t ~ ) /3,, 79-88. MeMichael, A. J., Ting, A., Zweerink, H. J., and Askonas, B. A. (1977).Nature (London) 270, 524-526. Meruelo, D., Nimelstein, S. H., Jones, P. P., Lieberman, M., and McDevitt, H. 0. (1978). J . E.xp. Rletl. 147, 470487. Miller, J. F. A. P. (1975).Anti. N.1’. Accid. Sci. 249, 9-26. Miller, J. F. A. P. (1978). Z t t i t t i i r t i o / . Rec. 42, 76-107. Miller, J . F. A. P., and Vadas, M. A. (1977a). S c u d . J . I n i n i r r t i o / . 6, 771-778. Miller, J. F. A. P., and Vadas, M. A. (19771)).Cold Spritig H u r h r S u m p . Vzicrrit. B i d . 41, 579-588. Miller, J. F. A. P., Vadas, M. A., Whitelaw, A., and Gamble, J. (1975). Proc. Nut/.Acacl. Sci. USA 72, 5095-5098. Miller, J. F. A. P., Vadas, M. A,, Whitelaw, A,, and Gamble, J. (1976). Proc. Nntl. A c c ~ d . Sci. USA 73, 2486-2490. Miller, J. F. A. P., Vadas, M. A , , Whitelaw, A., aiid Gamlile, J. (19771.1. Ex)).Med. 145, 1623- 1628. Miller, J. F. A. P., Mottram, P., Gamble, J., a i d Vadas, M. A. (1978). R/oot/ Cells. 4, in press. Miller, S. D., Sy, M-S., and Claman, H. N. (1978).J. E x p . M e d . 147, 788-799. Moorhead, J. W. (1977).J . Zniniutiol. 119, 1773-1777. Munro, A. J., and Tanssig, M . J. (1975). Noture (Lotidoti) 256, 103-106. Pang, T., and Blanden, R. V. (1976).J. E x p . Med. 143, 469-481. Paul, W. E., Shevach, E. M., Thomas, D. W., Pickeral, S. F. and Rosenthal, A. S. (19774. Cold Spri?ig Hnrbor S ! / t i i p . Quatit. B i d . 41, 571-578. Paul, W. E., Shevach, E. M., Pickeral, S. F., Thomas, D. W. and Rosenthal, A. S. (1977b). J . E x p . Met/. 145, 618-630. Peterson, P. A., Rask, L., and Ostberg, L. (1977). Adc. Cnttcer Res. 24, 115-163. Pfizenmaier, K., Trotsman, H., Riillinghoff, M., mid Wagner, H. (1975).Nature (London ) 258,238-240. Pierce, C. W., and Kapp, J. A. (1978). Fed. Proc. 37, 86-90. Pierce, C. W., Kapp, J. A,, and Benacerraf, B. (1976).J. E x p . &led. 144, 371-381. Pierce, C. W., Germain, R. N., Kapp, J. A,, and Benacerraf, B. (1977).J. E x p . Med. 146, 1827-1832. Press, J. L., and McDevitt, H. 0. (19771.1. E x p . Med. 146, 1815-1820. Rajewsky, K. and Eichinann, K. (1977). Cotitemp. To?>icsImnwnobiol. 7, 69-112. Rosenthal, A. S., Barcinski, M. A,, and Blake, J. T. (1977). Nnture (London)267, 156158. Rosentha1,A. S., Rarcinski, M.A., Rosenwasser, L. J.,andWeiland,O. (1978).Fed.Proc. 37, 79-85. Schmitt-Verhulst, A-M., and Shearer, G. M. (1976).]. Ex)).Rled. 144, 1701-1706. Schinitt-Verhiilst, A-M., and Shearer, G. M . (1977).J. Srtprutnol. Struct. S u p p / . 1, 206. Schmitt-Verhiilst, A-M., Sachs, D. H., and Shearer, G. M. (1976). J . Ex?,. Med. 143, 21 1-217. Schrader, J. W., and Edelman, G. M. (1977).J. Eq1. Med. 145, 523-539.

11ISTOCO~\IPATIBILITYCOhlPLEX AND T-CELL ACTIVATION

43

Schrader, J. W., Cuiiiiiiig1i;iin. B. A . , antl Edelman, G . hl. (1975). Pt-oc. Nutl. Acod. Sci. U S A 72, 5066-5070. Schrader, J. W., Henning, R., Slilner, R. J., and Etlelman, G. h l . (1977). Cold S/jritig fJ(rr/ior S ! / t i i / ) . ( ) i r t r t i t . H i o l . 41, 547-557. Schwartz, R . H. (1978). Sctttid. J. Z t ~ i t ~ t i ~ t t o7,/ . 3-10. Schwartz, R. H., David, C . S.,Sachs, D . € I . , antl Paul, W. E. (1976a).J. Z t n t n u t i o / . 117, ,531-540. Scliwartz, R. H., Dorf, M. E., Benacerraf, B., and Paril, W.E. (1976b).J. E x p . Ale t t t l r i t t o / , Rct;. 40, 181-204. Ting, c - c . , and Law, L. w. (1977)./. ~ l l i t t 2 ~ ~ l i ( J118, /. 1259-1264. Trincliieri, G., Adeii, D. P., and Knowlea, B. B. (1976).Nntrrre (Lotidoti) 261, 312-314. Tursz, T., Fridiiian, W. H., Senik, A . , Tsapis, A , , and Fellous, M. (1977).Notiire (Lotidoti) 269,806-808. Vadas, hl. A., Miller, J. F. A. P., hlcKenzie, I. F. C . , Cliism, S.F., Shen, F-W, Boyse, E. A , , Gamble, J . , and Whitelaw, A . (1976).]. E.s/i. &led, 144, 10-19. Vadas, M. A., Miller, J. F. A . P., Whitelaw, A , , antl Gamble, J. (1977). Zmniictio~ettetic.s4, 137-1 53. Viallat, J., Svedmyr, E., Steinitz, M., and Klein, G. (1978a). Cell Z t n t r w t i d . 38, 6875. Viollat, J., Svedmyr, E., Yefenof, E., Kleiii, G., mid \Veiland, 0. (1978). C c / / ,Z t t i t i t r i t i o / . 41, 1-8. voii Boehmer, H., Haas, W., and Jeriie, N . K. (1978). Proc. N o t / . Accid. Sci. USA, 75, 2439-2442. Wagner, H., Giitze, D., Ptschelinzew, L., and Rollinghoff, M. (1973.J. E s p . Aled. 142, 1477-1487.

44

J. F. A. P. MILLER

Wagner, H., Starzinski-Powitz, A,, Riillingliotf, M., Goldstein, P., and Jakob, H. (1978).J. E x p . Merl. 147, 251-264. Welsh, R. M., and Zinkernagel, R. M. (1977).Nature ( L o n d o n ) 268, 646-648. Wilson, D. B., Heher-Katz, E., Sprent, J., and Howard, J. C. (1977a).Cold Spriiig €fcirbor S y i n p . Qucint. R i d . 41, 559-561. Wilson, D. B., Lindahl, K. F., Wilson, D. H., and Sprent, J. (19771,).J . Ex!).Med. 146, 361-367. Y a m , A,, Schwartz, R. H., and Paul, W. E. (1977).J.E x ] , . M e t f . 146, 828-843. Yap, K. L., Ada, G. L., and McKenzie, I. F. C . (1978). Nntzire (Loiidon) 273, 238-239. Zarling, J . M., and Bacli, F. H. (1978).J. Ex?,. bled. 147, 1334-1340. Zinkernagel, R. M., and Altliage, A. (1977).J.Ex)).hled. 145, 644-651. Zinkernagel, R. M . , and Doherty, P. C. (1974a).Nature (Lotidon) 248, 701-702. Zinkernagel, R. M., and Doherty, P. C. (19741,). Natnre (London)251, 547-548. Zinkernagel, R. M., and Dolierty, P. C. (1975).J.E x / > .Aleti. 141, 1427-1436. Zinkernagel, R. M . , and Oldstone, M. B. A. (1976). Proc. N a t l . Accid. Sci. USA 73, 3666-3670. Zinkernagel, R. M., Callahan, G. N., Althage, A,, Cooper, S., Kleiii, P. A., and Klein, J. (1978a).J. Exp. hfed. 147, 882-896. Zinkernagel, R. M., Callahan, G. N., Altliage, A., Cooper, S. Streilein, J. W., and Klein, J . (1978h).J.E x ) ) . hfed. 147, 897-911.

SUPPRESSOR CELLS: PERMITTERS AND PROMOTERS OF MALIGNANCY? David Naor Lautenberg Center for General and Tumor Immunology The Hebrew University-Hadassah Medical School Jerusalem Israel

I. Background and Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Effect of External Intervention 011 Antitumor Resistance. A Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Are Suppressor Cells the Initiators of “Iininunostiniulation” and “Sneaking through” Phenomena:? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Relationships between Malignant Cells and Suppressor Cells . . . . . . . . . . . . . A. Nonspecific Suppressor Cells B. Specific Suppressor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Undefined Suppressor Cells V. Slippressor Cells Induced b y N Excision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V I I . Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4s 52

60 64 65 92 101

104 106 114 119

I. Background and Introduction

Both activation and suppression of the immune response are essential to the survival of the individual. The immune system, which obeys 110th “on” and “off’ signals, can initiate an effective defense mechaiiisni against hostile stimulators, can maintain this response at an optimal level for a s long as is necessary, ai- ’ w n stou, the response when the stiiiiulators have been destroyer - -.zli an imiiiuiioregulatory mechanism obviates the overproduction of cellular or hurnoral iminuiiological mediators and consequently can prevent both the destruction of innocent self-component bystanders of noiinal tissues and the unnecessary expenditure of‘energy by the organism. Furthermore, the imniunoregulatory mechanism can serve a s a surveillance system against autoimmunity and can also suppress maternal antifetal responses (Oldstone et u l . , 1977). In the higher vertebrates, irnmunoregulation can operate on several levels; this may ensure that the fiilure of one mechanism will not result in the complete collapse of the entire immune system. Products or feedback signals which are delivered by the niature effector cells of the immune system regulate the induction phase of the 45

46

DAVID NAOR

immune response. Thus, hunioral factors such as antibodies can neutralize circulatory antigens and consequently block their ability to stimulate iiniiiunocoiiil?eteiit cells (Uhr and Moller, 1968). Antigen-antibody complexes are probably involved in tlie regulatory niechanisin in one of two ways: either by direct blocking of receptor sites on 1974) or b y delivery of a tolerance lymphoid cells (Gorczynski et d., signal to the receptor sites (Diener and Feldmann, 1972). Alternatively, or in addition, they may stimulate suppressor cells which, in turn, inhibit immune responses. Gershoii et al. (1974) suggested that such antibody-tumor antigen complexes activate suppressor thyniusderived (T)cells which, in turn, abrogate tlie ability of macrophages to exei-t antitumor activity. It is possible that anti-idiotypic antibodies also participate in the regulation of the immune system: they can recognize idiotypic markers on receptor molecules, combine with them, and consequently 1977). deliver activating or inhibitory signals (Krawinkel et d., Other possible components of the iiniiiuiioregulatioii system are nonspecific factors, such as a-fetoprotein (Murgita and Toinasi, 1975) and iionnal iiiiiiiunosuppressive protein (NIP) (Nelken, 1973),which are capable of inhibiting different types of huinoral and cellular immune responses. In addition, some other regulatory mechanisms are involved in the control of tlie iiniiiuiie system. Elimination of cell clones which possess potential iininunological activity against self-components of normal tissues is possibly an important inimunoregulatory process in both the embryo aiid the adult animal (Nossal et al., 1977).Ininiunoregulatoiy mechanisms which depend on the balance between activated helper T lyniphocytes aiid suppressor T lymphocytes may detennine whether the immune response will be augmented or impaired (see Trtrmplont. Reu. 26, 1975).The physiological requirements of the organism may perhaps detennine whether one population or tlie other will be activated and consequently whether tlie immune response will be activated or suppressed. Finally, it should be noted that phagocytic macrophage-like cells also participate in the regulation of the immune system; these cells are capable of delivering either activating or inhillitory signals (Waksman and Naniba, 1976). The relationships between malignant cells and the above-described regulatory cells or their products constitute the subject ofthis review. The relationships between malignant cells and different inhibitory elements, such as ininiunoconiplexes, viruses, iiucleic acids, natural inhibitory substances in the sera, and tumor cell components, have been reviewed in detail by Kaino aiid Friednian (1977). Although

SUPPRESSOR CELLS

47

these authors did include in their article a discussion on the relationships between suppressor cells and tumors, the effect of suppressor cells on antitumor immunity warrants an in-depth review, particularly in the light of the important progress which has recently been made in the understanding of suppressor cell functions (see Trciit.splant. Rev. 26, 1975). The pioneering studies of Gershon (1975) and of Cantor and Boyse ( 1977)showed that in some systems the immune response is regulated by distinct subpopulations of T cells. In these systems the helper T cell, which bears the Lyl marker on its surface, activates the immune response and the suppressor T cell, which bears the Ly23 niarker on its surfke, inhibits the immune respoiise (Cantor and Boyse, 1977). Feedback signals delivered Iiy mature etfector cells of the immune system may stimulate either suppression or activation in accordance with the physiological requirements of the organism (Gershon, 1975; Gershon et ul., 1977). The suppressor cell can inactivate either the bone marrow-derived precursor of the antibody-producer cell (Basten et d., 1975) or the helper T cell (Herzenberg et d . , 1977). It is not yet known whether activation and suppression of the ininiune response are mediated by cell-to-cell contact or liy soluble factors released froin regulatory cells. Tada and his colleagues (1977) extracted froin primed mouse lymphocytes a soluble helper fictor containing an antigenic specificity and an H-2 product coded for by genes present in the I-A subregion of the H-2 complex. This factor enhanced the specific iinniiine response. Tada et (iZ. (1977) also extracted from the primed niouse lymphocytes a soluble suppressor Factor containing an antigenic specificity and an H-2 product coded for by genes present in the I-J subregion of the H-2 complex. This latter soluble factor suppressed the specific immune response. We can speculate that when a ceiTain acceptor site on an iiiimunocyte communicates with the soluble factor carrying the I-A marker an activating signal is transmitted to this cell. In contrast, when a different acceptor site on the same cell or on another cell interacts with the factor carrying the I-J marker, an inhiliitory signal is delivered to this cell. Similarly, it is possilde that intact regulatoiy cells bearing I-A or I-J products communicate with the target cells and deliver to thein enhancing or inhibitory signals, respectively. The noniial physiological response to some antigens at least inay depend on the proper function of both helper and suppressor cells; it is evident that in certain systems these functions are controlled Iiy the imniune response (It-) and immune suppression (Is) genes, respectively. Both genes are linked to the H-2 coniplex (Beiiacerraf and Dorf,

48

DAVID NAOR

1977).Lack of functional Zr genes may provoke selective expression of suppressor cells. Similarly, lack of functional I s genes may cause selective expression of helper cells. Thus, genetic factors can cause the iininunological balance to favor either suppression or activation of the immune response (Benacerraf and Dorf, 1977). It should be noted that the nature of the antigen can also affect the balance between helper and suppressor cells. Turkin and Sercarz (1977) found that cultures of spleen cells from mice injected with P-galactosidase in complete Freund’s adjuvant (CFA) 3 days previously generated an enhanced antifluorescein response after they were incubated with p-galactosidase-fluorescein. In contrast, splenocytes from mice injected with CB2-a fragment cleaved from P-galactosidase by cyanogen b r o m i d e 4 i d not generate an antifluorescein response after similar treatment. The authors suggested that CB2 stiniulates suppressor cells preferentially and consequently the immunological balance favors inhibition of the specific immune response. The modification of an antigen may impair or improve its ability to stimulate a particular T-cell subpopulation. Thus, the modified antigen induces an immunological balance different from that of the native antigen. Chemical modification or enzymatic cleavage of an antigen can therefore provide the laboratory investigator or the clinician with a powerful tool with which to stimulate a desired immune response. [For further reading see review by Naor and Galili (1977).] The activation of suppressor or helper cells can be achieved specifically by antigenic stimuli or nonspecifically by mitogenic stimuli. Antigens stimulate regulatory cells which bear specific receptors while rnitogens stimulate polyclonal regulatory cells. Dutton (1975) found that the mitogen concanavalin A (Con A) stimulated nonspecifically both suppressor and helper cells when cocultivated with spleen cells. It was possible to separate these two subpopulations on a continuous 5 to 20% Ficoll gradient. The lighter cell fraction mediated helper activity; the cells of this fraction enhanced the in vitro antisheep red blood cell (SRBC) response of normal splenocytes. The heavy cell fraction mediated suppressor activity; the cells of this fraction inhibited the i n vitro anti-SRBC response of normal splenocytes. Similarly, Kedar and his co-workers (1978) found that fetal bovine serum induced preferential nonspecific production of suppressor cells which inhibited in vitro allogeneic and syngeneic antitumor responses. Finally, it should be emphasized that cell-mediated immune responses, such as graft-versus-host reaction (GVHR), allograft reaction, mixed lymphocyte culture (MLC), cell-mediated lympholysis (CML), delayed type hypersensitivity, and various antitumor re-

SUPPRESSOR CELLS

49

spoiises are also regulated by helper (amplifier) or 5uppressor cells (Katz, 1977, and the present review). Macrophages are also known to play an important role in the regulation of the immune response. Feldniann (1973) showed that spleen cell cultures which contain excess of macrophages and a specific T-cell soluble factor generated a maximal level of antibodies after antigenic stimulation. In contrast, a deficiency of inacrophages in such cultures resulted in specific suppression of the immune response. Calcleroii and Unanue (1975) found that a supernatant of preincubated peritoneal iiiacrophages contained factors which did not pass through a dialysis membrane and which enhanced the proliferation of phytoheniagglutiiiiii (PHA)-stimulated normal splenocytes. The mechanism of the in uiuo immunoregulation mediated by macrophages aiid its relationship to other regulatory systems are still not understood. Although we do not as yet fully understand the immunoregul,‘1t’I011 systems, the information accumulated to date can serve a s a staitiiig point for further experimental investigations. Thus, the application of the findings discussed above to experimental clinical models is an obvious aiid essential step. Experimental allergic encephalomyelitis (EAE), an autoimmune disease of the central nervous system, can be induced in laboratory animals by the injection of iiiyeliii basic protein (BP) in CFA (Kies, 1965). It was found that iiijection of the animals with BP in solution or in incomplete Freund’s adjuvant (Alvord et d., 1965; Cunningham aiid Field, 1965), or with the 44-89 fragment obtained b y limited pepsin digestion of BP (Swanborg, 1975), protected the animals from subsequent induction ofthe EAE by BP in CFA. It was also shown that the synthetic peptide S42, which is an analog of the encephalitogenic tryptophan region of BP, could both suppress and reverse EAE iiiduced with BP in CFA (Hashini et id., 1976). It is possible that preferential activation of suppressor cells which inhibit the allergic autoimmune response can be achieved by such modified antigens. In agreement with this assumption, Swierkosz and Swanborg (1975) found that lymph node cells from rats injected with BP in incomplete Freund’s adjuvant could be adoptively transferred to normal animals and thus confer on them resistance to EAE. Adda and his co-workers (1977) confirmed this finding by demonstrating that suppressor cells derived froiii the thymus or the spleen confer resistance to EAE. Aged New Zealand mice provided an example of an immunological imbalance which leads to severe clinical manifestations. It is possible that a defective function of suppressor cells in these mice causes the

50

DAVID NAOR

immunological balance to shift toward activation of the autoimmune 1975). In fact, Klassen and his co-workers response (Steinberg et d., (1977) have shown that aged NZBNV mice developed naturally occurring thyinocytotoxic antibody (NTA) which preferentially eliminated suppressor T cells. Con A-stimulated splenocytes of young NZB/W mice could inhibit the ability of normal splenocytes to synthesize IgM after pokeweed mitogen (PWM) stimulation, when the two cell populations were mixed together. However, Con A-stimulated splenocytes of young NZBNV mice which were injected with NTA lost their suppressive effect; this suggests that NTA caused a deficiency of suppressor cells in the young NZBNV mice. Thus, an immunological balance which favors activation over suppression of the immune response may be the cause of several induced and spontaneously occurring autoimmune diseases. The induction of an immunological balance which favors suppressor cells over activating cells may provide a ineans for facilitating allograft survival. Gelfand and Paul (1975) found that in BALB/c mice injected with anti-Thy 1.2 serum allografts survived longer than those in control animals. Furthennore, infusion of splenocytes from BALB/c mice injected with anti-Thy 1.2 serum into syngeneic recipients of the allograft prolonged its survival, whereas splenocytes from normal mice had no effect. The authors suggested that anti-Thy 1.2 serum preferentially activates suppressor cells which in turn abrogate an efficient antigraft response. In many cases the immune system does not ignore antigenic stimuli which are delivered by autochthonous malignant cells. A large body of evidence indicates that in both man and animals immunocompetent cells recognize tumor-specific antigens and react to them (Herbennan, 1974). However, the question of whether the immune system plays a major part in surveillance against malignancy has still not been resolved. The classic immunosurveillance theory claims that most nascent malignant cells are eliminated by the immune system and that the few malignant diseases that are evident reflect a failure of the imniunosurveillance mechanism (Bumet, 1970). Opponents of the immunosurveillance theory reject the idea that during the process of evolution any surveillance mechanism against malignancy was selected for recognizing and eliminating tumor cells, since neoplasia usually appears ufter the reproduction period. The many experimental models which show eradication of tumor cells after immunological treatment or manipulation are considered by them to be laboratory artifacts (see Trunsplunt. Rev. 28, 1976). Furthermore, schools of thought which reject the immunosurveillance theory view the finding

SUPPRESSOR CELLS

51

that the immune response sometimes stimulates a i d enhances tumor growth (Prehn, 1976)a s bringing the theory to a paradoxical dead end. However, it should be remembered that there is reliable evidence that some virus-induced neoplasms are controlled by an iinmunosurveillance mechanism which was probably positively selected during the evolution of the species (Klein and Klein, 1977). The question of whether or not an iinmuiiosurveillance mechanism operates may be irrelevant to the use of inimunotherapy in the treatment of maligriant diseases, since manipulations which permit activation of the autoimmune response may also permit activation of the antitumor response. It has, in fact, been found that elimination of suppressor cells can activate an autoiinniiine response (Cunningham, 1975).Similarly, it is reasonable to assume that the analogous eliniiiiatioti of suppressor cells from a tumor-bearing animal will shift the immunological I)alance toward activation of an efficient antitumor reaction. Such iiiimunological manipulations might even be effective against spontaneous tumors which otherwise completely fiil to denioiistrate any immunogenic capacity (Hewitt et al., 1976). The present review describes briefly the data which demonstrate augmentation of antitumor responses in experimental “imniunocrippled” animals [adult thyniectomized, splenectomized, X-irradiated, or antithymocyte serum (ATS)-injected animals]. Most of these experiments were performed before the recognition of the function of suppressor cells. However, a retrospective picture of these findings may be interesting, since at least a part of the results may be interpreted differently if the function of the suppressor cell is taken into account. As the phenomena of “sneaking through” and “iminunostiniulation” can also be interpreted differently in the light of the function of suppressor cells, they are also discussed in this article. In addition, the review presents the more direct experimental evidence showing the effect of specific and nonspecific suppressor cells on the relationships between the tumor and the host immune system. For the most part, the effect of suppressor cells on syngeneic tumors, and occasionally on the so-called “nonspecific” tumor cells, is discussed. “Nonspecific” neoplastic cells cannot stimulate a detectable allogeneic immune response after inoculation into an allogeneic host and they can grow progressively in such an environment. The suppressor cells are classified arbitrarily into permitter suppressor cells, which populate the host before its confrontation with the tumor, and promoter suppressor cells, which are induced b y the tumor. Finally, the review describes the chemical, physical, and biological properties of some of these suppressor cells and offers various means for their selective elimination.

52

DAVID NAOR

II. Effect of External Intervention on Antitumor Resistance. A Historical Perspective

The balance between helper and suppressor functions can be changed by external intervention. Selective elimination or inactivation of suppressor cells might permit inaximal immune reactivity which can otherwise not be expressed or which can be expressed only partially. Suppressor T cells can be eliminated or inactivated in a number of ways. Since suppressor T cells may have a shorter life span than both helper T cells (Janeway, 1975) and other T cells that participate in cellular immunity (Simpson and Cantor, 1975), it is possible that adult thyrnectomy will lead to the rapid exhaustion of suppressor cells and that the iiiiniuiiological balance will consequently favor activation of the immune response. Similarly, it may be expected that spleiiectomy will change the balance between helper and suppressor functions b y the preferential eliniiiiatioii of suppressor cells (Okuinura and Tada, 1971). In addition, suppressor cells appear to be more sensitive than cytotoxic cells to both X irradiation (Sabbadini, 1974) and cyclophosphamide treatment (Rollinghoff et d.,1977). Consequently, these treatments can be used to eliminate suppressor cells selectively and thus to potentiate cytotoxic immune reactivity. It has also been found that in certain systems suppressor T cells are more sensitive than helper T cells to ATS and that the suppressor cells can therefore be selectively eliminated by this reagent (Okumma et (11 ., 1974). Indeed, an immunological balance shifted toward activation of the autoimmune response was achieved by injecting normal mice with antilymphocyte serum (ALS) (Cunningham, 1975). However, the conclusions that can be drawn from these findings should be restricted to the particular experimental system under investigation. Furthemiore, it should be remembered that even within the restricted system the successful selective elimination of suppressor cells might be entirely dependent on the protocol of the experiment. Selective removal of suppressor cells should be tested separately for each system as it is impossible to extrapolate conclusions froni one systeni to another. Potentiation of the immune response against neoplastic cells might similarly be dependent on an immunological balance which favors inimuiiological activation over suppression. Such an iiiimuiiological status can be achieved by manipulations which eliminate suppressor cells, i.e., adult thymectomy, splenectomy, ATS treatment or X irradiation. Interestingly, these techniques have been used during the last four decades by many research workers, although their experimental

SUPPRESSOR CELLS

53

designs were motivated by entirely different questions. Miller (1961) and Metcalf (1966) have reviewed these experiments in detail and some of the findings relevant to this article are discussed briefly below. It has been found that in AKR (McEndy et ul., 1944; Furth, 1946) and RIL (Law and Miller, 1950a) mice the thymus is nearly always involved in the “natural” leukemic process and that, in niany cases, it is the sole organ involved. Adult thymectomy of these mice at 4 or 6 weeks of age drastically reduced the incidence of leukemia. Transplantation of a thymus from a leukemia-sensitive animal facilitated tumor appearance in a thymectomized mouse (Metcalf, 1966). However, it was found that inany of the nascent neoplastic cells originated from the host and not from the donor. Metcalf (1966) concluded that “the simple mechanistic explanation of the effect of thymectoniy in preventing lymphoid leukemia (by the removal of susceptible cells) is either not coi-rect or not the whole explanation. Clearly, part of the role of the thymus in leukaemogenesis is an indirect one-the thymus of certain strains (but not others) appears to be able to induce the host cells repopulating the thymus graft to become neoplastic.” The present review may give a broader perspective to this statement. Adult thymectoiiiy has also been found to reduce the incidence of “naturally” induced leukemia in C58 mice (Law and Miller, 1950a) and of inethylcholanthrene (MCA)-induced leukemia in DBA mice (Law and Miller, 1950b), although in many of these mice the disease apparently does not originate in the thymus. Law and Miller (1950a) concluded that “the thymus may be the site of origin ofthe transfonnatioii from nomial to leukemic cells and that leukemic cells are transported by way of the circulation within a short time to other sites throughout the body.” The present review inay provide a different perspective to these experiments also. The publication of the iinniuiiosurveillance theory motivated the performing of inaiiy expei-inients aimed at answering the question of whether malignancy is related to the failure of the immune system. In these experiments the immune system was suppressed experimentally by thymectoiiiy, splenectoiny, ATS treatment, or X irradiation, and the development of malignancy in the “crippled” aninials was compared with that in untreated or “shammed” control animals. Stutinan (1975), who reviewed both these experiments and the available related clinical data, reached the conclusion that “the iinniuiiosurveillance theory does not apply to the vast majority of spontaneous or induced tumors ,, in aninials and inan. Furthemiore, in contrast to the iiinnuiiosurveil-

54

DAVID NAOR

lance concept, immunosuppressive manipulations in several experimental models either reduced tumor incidence, or sometimes even eradicated it completely. Thyniectomy (Martinez, 1964; Law, 1966; 1969; Heppner, 1967; Sakakura and Nishizuka, 1967; Heppner et d.,1968a,b; Yunis et aZ., 1969; Squartini and Bolis, 1970; Squartini, 1971), splenectoniy (Batchelor and Silverman, 1962; Squartini and Bolis, 1970; Squartini, 1971), treatment with ALS (Lapp6 and Blair, 1970; Blair, 1972), and X irradiation (Prehn, 1969; Sparck, 1969) have been demonstrated to have antitumor effects on both induced and transmitted mammary tumors of mice. A nonimmunological interpretation of the thymectoniy effect suggests that this treatment induces a hormonal imbalance which causes a deficiency in noduligenesis and mammogenesis. These nodules are the target of the malignant transformation (Sakakura and Nishizuka, 1967). In more general tenns, it was suggested that thymectomy induces a hormonal balance which favors inhibition of tumor growth (Squartini and Bolis, 1970; Squartini, 1971). The immunological interpretation of the resistance of thymectoniized animals to vertically transmitted mammary adenocarcinoina suggests that thyniectoniy prevents the induction of tolerance b y the viral antigens and that an efficient antitumor immune response is consequently developed (Heppner et ul., 1968b). An alternative explanation is that 1969; Heppiier et al., 1968b)or splenectomy thymectomy (Yunis et d., (Batchelor and Silverman, 1962) may prevent the production of enhancing antibodies and consequently tumor growth is inhibited. The effect of ATS on the augmented resistance of ATS-injected mice to Moloney-induced lymphoma was explained in terms of the direct interaction of the antiseruni with the tumor (Bremberg et d.,1967). The increased resistance of thymectomized mice to radiation-induced leukemia was explained in tenns of the removal of the susceptible foci (Kaplan, 1950; Balner, 1971, 1972; van Bekkum, 1972; van Bekkum and Balner, 1973). Table I summarizes experiments showing the increased resistance to neoplasia of immunologically “crippled” experimental animals. Table I also includes the interpretations of these effects given b y the authors. Although many of the interpretations are as valid today as they were when they were originally proposed, reevaluation of the suggested mechanisms of the augmented resistance to neoplasia of “immunocrippled” animals may be beneficial: some of the techniques described in Table I can lead to preferential elimination of suppressor cells and thus pennit potentiation of antitumor responses. Indeed, there is evidence that the immune response of C57BW6 mice to the

TABLE I ASTIMALIGSASTPROTECTIVEEFFECT OF IM~IUSOSUPPRESSIVE PROCEDURES (THYMECTOMY, SPLENECTOMY, ATS TREATMENT, A N D X IRRADIATION)“ Host DBA mice

Al1,ino rats

Ha/ICR Swiss mice. AKR mice pretreated with

s-180 homogenate A l s n , ACA, .%.BY, C57BL, C31-I. A.S\V, DBA mice SpI-ague-Da\\~Ie y rats

Tumor

Treatment

Results

Suggested interpretation

Reference

VCA-induced le 11 k e mia

Thymectomy at 4 weeks of age

Nonspecific graft of SICA-indricecl \ xcoina

Splenectom> before tumor inoculation

Inhibition of tumor Splenectomy reduces the progrowth, as assayed dnction of enhancing b y tumor size antibody-

Moller (1965)

Graft of \Valket carcinoma

Thymectomy at 12 hours of age

Reduction in liver metastases

Fisher et a l .

T h e presence of thymic tissue Law and Xfiller Decrease in incidence of leukemia in the body is a necessary (1950b) antecedent to the development of leukemia 3,1-BenzpyreneThy mectomy at 1 I ticrease in survival Fumarola and induced month of age time after tumor Giordano leukemia appearance (1962) Nonspecific graft of Splenectomy before Decrease in number Splenectomy reduces the pro- Old et al. sarcoina S-180 tumor inoculation of deaths and in duction of enhancing anti(1962a) (raised in tumor size body Ferrer (1968aJ1) HaiICR mice)

AKR mice

“Spontaneous” leukemia

RIL mice

“

1

Spontaneoris” leukemia

The thymic-pituitary interrelationship which Favors tumor growth is affected by th ymectom y Thymectoniy at 4 to Marked decrease in Removal of potentially 6 weeks of age incidence of ma1ignant ce 11s by leukemia thymectorn y Thymectomy at 4 Decrease in inciRemoval of potentially weeks of age dence of leukemia malignant cells by thymectoniy

(1965)

McEndy et (11. (1944); Furth (1946) Law and Miller (1950a, 11)

-

(continued)

TABLE I-(continued) Host

Tumor

Treatment

C58 mice

“Spontaneous” leukemia

Thymectomy at 4 weeks of age

C3H mice

Viral-filtrateinduced leukemia

Thymectomy at 4 to 6 weeks of age

Z mice

Viral-filtrate-induced leukemia

Thymectomy at 4 to 6 weeks of age

AKR, C3H, CBA mice

Viral-filtrate-induced leukemia

Thymectomy at 3 to 4 weeks of age

C57BL mice

Radiation-induced Thymectomy at 1 lymphoma to 2 months of age Radiation- and Thymectomy estrogen-induced lymphoma

cn rn

BALB/C mice

Results

Suggested interpretation

Decrease in inciThe thymus may be the site dence of leukemia of origin of the transformation of cells from normal to leukemic cells Virtual elimination The thymus represents the of leukemia principal target organ in which the leukemic agent is stored and multiplies Virtual elimination The thymus is the focus of the of death from induction of leukemia by the virus leukemia Virtual elimination (1) Removal of cells in which of leukemia the agent can reach a concentration high enough to induce leukemia. ( 2 ) Removal of cells most susceptible to leukemic transformation. (3) Removal of source of a humoral factor involved in leukemogenesis Virtual elimination Removal of the susceptible of lymphoma focus Virtual elimination The radiation effects on the of lymphoma thymus were secondary or humoral and were elicited by estrogenic hormones

Reference

Gross (1959)

Levinthal et al. (1959) Miller (1959a, b, 1960)

Kaplan (1950) Kirschbaum et al. (1953)

(CBA x C57BL)F, Radiation-induced mice lymphoma

Rabbit ALG after X irradiation

Virtual elimination of early thymic lymphoma

RFM mice

Radiation-induced lymphoma

(C57BL x CBA)F, mice

Radiation-induced lymphoma

Rabbit and horse ALG after X irradiation Rabbit ALS after X irradiation

RF mice

Radiation-induced lymphoma

ALG after X irradiation

C3H mice

A strain mammary tumor graft

C3H mice

MTV-transmitted mammary tumor

Splenectomy before tumor inoculation Thymectomy at 6 days or 12 hours of age

Decrease in the incidence of early lymphoma Marked decrease in the incidence of lymphoma. A different batch of rabbit ALS and horse ALS were ineffective Decrease in incidence of lymphoma. Increase in latent period Decrease in tumor size

C3H mice

MTV-transmitted or infected mammary tumor

in

4

Thymectomv at 3 days of age

ALG inactivates precursor Balner (1971); van Bekkun cells of the hemopoietic and Balner system, which are direct target cells for the leukemic (1973) viruses

Rabbit ALS contains antibody Balner ( 1972) to irradiation-induced Lzirus

van Bekkuni (1972)

Splenectomy reduce$ the Batchelor and production of enhancing Silverman antibody (1962) Decrease in tumor Martinez Presence of thymus permits breakdown of tolerance and incidence, in(1964); Yrinis production of enhancing crease in survival et d.(1969) time antibodies Decrease in tumor Tolerance induction cannot Law (1966, frequency and inbe the explanation for tumor 1969) crease in latent growth since Tx tumor-free period adult mice which were infected with MTV also showed a decrease in tumor frequency (continued)

TABLE I-(continued) Host

Tumor

Treatment

Results

(C3H x 129/J)F, (129/J x C3H)F, mice

MTV-transmitted mammary tumor

Thymectomy at 3 days, estrogen stimulation

Decrease in incidence of HAN and of tumors

C3H mice

MTV-transmitted mammary tumor

Decrease in incidence of HAN

BALB/cf C3 1 mice

MTV-transmitted mammary tumor

BALB/c mice

MTV-induced mammary tumor

BALB/cf C3H virgin mice

MTV-transmitted mammary tumox

Thymectomy at 40 days of age, estrogen stimulation Thymectomy at 1 to 3 days of age, stimulation with noduligenic hormones and force breeding Thymectomy at 1 to 3 days of age and infection no later than 1 month of age. Stiniulation with noduligenic hormones Thymectomy at 1 day of age

BALB/cf (R111) force bred mice

MTV-transmitted mammary tumor

Thymectomy at 1 day of age

u1

m

Decrease in incidence of HAN and increase in latent period

Suggested interpretation

Reference

The inhibitory effect of thymectomy on mammary tumorigenesis is possibly of endocrine origin

Sakakura and Nishizuku (1967)

Young thymectomized mice might be incapable of developing specific tolerance or of making adequate quantities of enhancing antibodies

He p p n e r (1967); Heppner

et a l . (1968a, b)

Decrease in incidence of HAN

Decrease in tumor incidence; increase in latent period Increase in latent period

The thymus induces a horSquartini monal balance which favors and Bolis tumor growth (1970); Sqiiartini (1971)

BALB/cf C 3 H virgin mice

MTV-transmitted mammary tumor

Splenectomy at 2 days of age

C3H mice

hlammary tumor graft

X irradiation (550 or 750 R) before or after tnmor inoculation X irradiation (450 R) 24 honrs hefore tumor inoculation ATS

C3H, (C3H x C57BL)F, mice

Mammary tumor graft

BALBicf C3H mice

MTV-transmitted in am mary t tiinor

C3H mice

hl LV-transmitted timior

Thymectoniy at birth or 1 month of age

(A x C57BL/6)Fl (A x ASW)F, C3H specifically immunized mice BALBic mice

MLV-induced lymphoma

ALS after tnnior inoculation

MLV-induced lymphonla

Thymectomy at 3 days of age and infection at 3 weeks of age

Decrease in tumor incidence; increase in latent period Decrease in tumor size

X irradiation affects the in-

Sparck (1969)

flammatory process which is obligatory for tumor development

Inhibition of tumor growth in \IT\' + mice

Prehn (1969)

ATS interferes with the conDecrease in inversion of a hyperplastic cidence of tumor; increase in latent lesion to a neoplastic lesion period Virtiial elimination The immune processes d o not play any positive part in the of leukemia. Resproduction of malignant toration of Tx mice disease with lymphoid cells does not restore malignancy Direct inhibition of tinnor hy Increase in latent period ALS

Lapp6 and Blair (1970); Blair (1972)

\'irtual elimination of lenkemia

Law (1966)

Bremberg ef a / . (1967)

Nonlyniphoid elements ot' the Allison and Law (1968) thymus which facilitate the conversion of normal cells to leukemic cells are eliminated by thyniectomy

" ALG = antilymphocyte globulin; ALS = antilymphocyte serum; ATS = antithymocyte serum; HAN = hyperplastic alveolar nodnles; MCA = methylcholanthrene; MLV = Moloney leukemia virus; hlTV = mammary tumor virns; Ts = thymectomized.

60

DAVID NAOR

3LL lung tumor (Treves et ul., 1974) and of A/J mice to sarcoma (Fujimoto et al., 1976a) is regulated by suppressor T cells. In these mice adult thymectomy (Treves et d . , 1974),X irradiation (Rotter and Trainin, 1975), or splenectomy (Fujimoto et d , 1976a) was found to augment resistance to tumor growth. However, it should be stressed that in some of the experiments described in Table I thyinectomy was carried out during the first week of life or even on the day of birth. This technique causes efficient elimination of helper cells and the precursors of effector cells and, as a consequence, general impairment of the cellular immune response is achieved (Miller, 1962; Good et ul., 1962). In contrast, adult thymectomy may cause preferential elimination of suppressor cells as a result of their shorter life span. Thus, activation of the immune response, which is induced by the elimination of suppressor cells, is better interpreted by adult thymectomy than b y newborn thymectomy. In addition, it should be remembered that immunosuppressive manipulations such as splenectomy (Old et al., 1962a; Moller, 1965; Ferrer, 1968a,b) can also affect the balance between the humoral response, which can promote tumor progression, and the cellular response, which can promote tumor regression. Therefore, if cellular immunity is favored over humoral immunity in immunosuppressed animals, then resistance to malignancy might be increased. Finally, it should not be forgotten that immunosuppression techniques can provoke direct damage to the tumor itself or to the potential site of the malignant transformation. Ill. Are Suppressor Cells the Initiators of "lmmunostimulation" and "Sneaking through" Phenomena?

Normal lymphoid cells can stimulate the growth of tumor cells both i n vivo (Yoshida and Southam, 1963; Deckers et al., 1971; Prehn, 1972) and in vitro (Fidler, 1973a,b; Jeejeebhoy, 1974; Kall and Hellstrom, 1975). Furthennore, in certain systems, specifically immunized lymphoid cells can enhance, rather than suppress, tumor growth. Such enhancement can be achieved in the absence of preexisting blocking humoral factors, although de novo synthesis of such factors b y the sensitized cells cannot be ruled out. Prehn (1972) found that 103-104 splenocytes from DBA mice immunized against MCAinduced sarcoma specifically accelerated tumor growth when injected together with homologous tumor cells into adult thymectomized, X-irradiated syngeneic recipients. Higher doses of immune splenocytes had an inhibitory effect. This finding has been confirmed in several in vitro systems. Low doses of lymphoid cells from tumorsensitized animals were found to enhance the in vitro growth of tumor

SUPPRESSOR CELLS

61

cells after a few days of cocultivation; higher doses of sensitized cells were inhibitory (Medina and Heppner, 1973; Fidler, 1973a,b; Fidler et ul., 1974). Medina and Heppner (1973) found that lymph node cells from mice bearing the fourth passage of tumor cells enhanced in vitro tumor growth, while cells from animals bearing the first passage inhibited in vitro tumor growth. Other workers have shown that lymphoid cells which were removed from tumor-bearing animals enhanced in vitro tumor growth (LeFranCois et uZ., 1971; Bray and Keast, 1975), while cells removed after tumor excision inhibited in vitro ( Barski and Youn, 1969; Le Franqois et ul., 1971) and in vivo (Mikulska et ul., 1966) tumor growth. The source of the sensitized lymphoid cells can detemiine whether they will enhance or inhibit tumor growth. Bartholoinaeus et ul. (1974) showed that lymph node cells from melanomabearing C57BLl6 mice enhanced tumor growth, while spleiiocytes from the same animals had an inhibitory effect. All these experiments provide support for the “immunostiinulation” theory (Prehn and Lapp6, 1971; Prehn, 1976); this theory claims that a weak immunological response to either a small dose of tumor cells or weak tumor antigens stimulates rather than inhibits tumor growth. However, there is no good explanation why a weak iinmunological response should promote tunior growth. Prehn and Lapp6 (1971) suggested that the enhancement of tumor growth might be achieved by direct stimulation of neoplastic cells. Lymphotoxin which is released from the immune lymphocytes might be the cause of such immunostimulation. It has, in fact, been found that very low titers of lymphotoxins stimulate protein synthesis and DNA production in target cells (Kolb and Granger, 1970). “Sneaking through” is another puzzling observation which has caught the interest of investigators of tumor immunology. In several tumor-host systems very low doses of neoplastic cells were found to grow progressively in recipient animals while larger doses were rejected or grew very slowly. This paradoxical phenomenon was explained by Klein (1966): “Small numbers of tumor cells can grow within the shelters of a compact, differentiated tissue relatively undisturbed until they reach irreversible size. Apparently, they present an inadequate antigenic stimulus to the host, unable to induce a sufficiently strong response before tumor growth has passed a threshold, after which it becomes irreversible.” Although this explanation seems to be the most acceptable, several other interpretations have also been postulated. For example, Prehn and Lapp6 (1971) suggested that low doses of neoplastic cells stimulate a weak immunological response which proinotes tumor growth. Table I1 summarizes the different tunior-host systems for which the “sneaking through” phenomenon

TABLE I1 THE“SNEAKING THROUGH” PHENOYENON~

Host Albino mice

BALB/c mice

m N

BALB/c mice

C57UKL mice

Hamsters

Tumor S37 tumor

Inoculuni and results

Suggested mechanism of enhancement

Reference

Higher incidence of tumor growth after Hewitt (1953) injection of 82 tumor cells than after injection of 247 tumor cells MCA-induced Higher number of takes after injection Forty viable tumor cells may es- Old et al. sarcoma tablish themselves as a wellof 40 tumor cells than after injection (196213) vascularized tumor mass. The of 1250 or 12,500 tumor cells amount of antigen from a greater number of cells may be sufficient to initiate an efficient immune response Low doses of leukemic inoculum Humphreys Leukemia L1210 and Leukemic spleen suspension of 0.08does not stimulate homograft 0.016% causes 50% mortality of reet al. (1962) ascitic subline reaction and eventually the M46G carried in cipients. Leukemic spleen suspenDBM2 mice. C82R animals die from their tumors sion of %lo% does not cause mortumor carried in tality (BALB/c x DBA)F, mice Floersheim Small numbers of tumor cells YL1 MoloneyHigher incidence of tumors after inescape host defence reactions (1967 induced lymphoma jection of lo4 or 10; tumor cells than at a critical time after injection of lo6 tumor cells SV40 virus-induced Higher incidence of tumors after inSmall numbers of tumor cells are Potter less active in the stimulation of et al. 1969) tumor jection of 1000 cells (55%) than after the immune mechanism injection of 5000 cells (37%), 6 months after inoculation

C3HIHe mice

CB, MCA-induced rhabdom yosarcoma

TP10-dibenz(a) anthracene-induced rhabdom yosarcoma (C57BL x IF)F, 1 1CA-induced sarcoma mice Swiss-B mice

BlO.A(5R) mice

LAF-17 lymphoma carried in B1O.A mice

B lO.A(2R) mice

S 1033 Iymphoma

BALBic mice

“

SICA

=

carried i n B1O.A mice BM3 niastoc) toma

metliylcliolantlireiie.

Higher frequency of mice with tumors after injection of 10’-10‘ tumor cells than after injection of lo5 tumor cells Higher frequency of mice with tumors after injection of 10’ tumor cells than after injection of lo3tumor cells Higher frequency of mice with tumors after injection of 2 x 10:’ tumor cells than after injection of 2 x lo5 tumor cells

The effect may b e related to immunological enhancement mediated by circulating antibodies since it also occiirrs in preimmunized animals Higher incidence of mortality after inThe production and release of jection of 10’ tumor cells (63%) than depressive factor (from tumor after injection of 10‘ tumor cells ( 3 %) . cells) may be a mechanism favoring the escape of small T h e low dose of tumor cells “conditions” the animals to “take” a dose of populations of antigenic tumor cells 10’ tumor cells which is normally rejected Higher incidence of mortality after injection of 10’ tumor cells than after injection of 10’ tumor cells Higher incidence of tumors after injec- A small inoculrim of tumor cells induces law-zone tolerance tion of 10’-10’ tumor cells than after which is mediated by supinjection of 10‘-10’ tumor cells. A dose of 10’-10’ irradiated hiinor cells pressor cells “conditions” the animal to “take” a dose of loi living cells which is normally rejected

Donner

et al. (1969)

Marchant ( 1969)

Bonmassar et 01. (1971, 1973, 1974)

Kiilsch et (11. (1973, 1975); hlengersen et (11. (1975)

64

DAVID NAOR

has been documented; it also presents some of the authors’ interpretations of this phenomenon. The possible involvement of suppressor cells in the “inimunostimulation” and “sneaking through” phenomena cannot be ignored. It is possible that small tumor inocula or weak tumor antigens preferentially activate suppressor cells which “turn off’ the antitumor immune response and thus enhance tumor progression. Alternatively, the suppressor cells might reduce immunological reactivity from a strong immune response which has an inhibitory effect on tumor growth to a weak immune response which has a stimulatory effect on tumor growth. Indeed, the interpretation of the “sneaking through” phenomenon has recently been challenged by the experiments of Kiilsch and his colleagues (Kolsch et al., 1973; Mengersen et al., 1975).These authors found that low doses ( 10-103) of irradiated mastocytoma cells induced the appearance of suppressor cells in BALB/c mice. Such adoptively transferred suppressor cells prevented the stimulation of a secondary antitumor response in syngeneic immunized recipients. Bonmassar et (11. (1971) found that low doses of LAF-17 lymphoma cells could “sneak through” the immunological control system of BlO.A(5R) mice (which are incompatible with the tumor at the K end of the H-2 region) but not through the control system of BlO.A(BR) mice (which are incompatible with the tumor at the D end region). A simple explanation for this observation might lie in the ability of the small tumor inoculum to induce suppressor cells in BlO.A(5R) mice but not in B10.A(2R) mice. However, much work has still to be done before more definite conclusions can be reached. IV. Relationships between Malignant Cells and Suppressor Cells

Some neoplastic diseases of both man and experiment animals are accompanied by partial or severe immunosuppression (Kamo and Friedinan, 1977). Whether a “crippled” immune system is the cause of tumor induction-as the immunosurveillance theory suggests-or the result of it, is still the object of endless discussions and arguments. Recent reviews by Kamo and Friedman (1977)and by Waldmann and Broder (1977) include discussions on the role of suppressor cells in malignancy. However, the growing body of information on the relationship between the activity of suppressor cells and the appearance of neoplastic states and the better understanding of the general role of suppressor cells in the immunoregulation process justifies a “closeup” look at this problem. The previoiis sections document some of the information which re-

SUPPRESSOR CELLS

65

trospectively may suggest the existence of suppressor cells in tumorbearing animals. This section will present the more recent and direct evidence of the existence of suppressor cells which enhance tumor growth . Suppressive humoral factors which enhance tumor growth, such as NIP (Nelken, 1973), will not be discussed in this review, since their cellular source has not yet been defined. Furthermore, a broad perspective should include in the “suppressor cell” category, bone inarrow-derived cells (B cells) which participate in the production of blocking factors. However, since this topic recently enjoyed intensive attention (Hellstrom and Hellstrom, 1974) it will also not be included in the present review.

A. NONSPECIFIC SUPPRESSOR CELLS Nonspecific suppressor cells of normal or tumor-bearing mice inhibit immune responses which are not specifically directed toward the relevant tumor antigens. It is possible, however, that such nonspecific suppression can also affect the immune responses directed toward the relevant tumor antigens and, as a consequence, tumor growth is permitted or promoted. Table I11 summarizes the most important properties of these cells. 1. Nonspecijic Suppressioii Medicited b y Macrophnges

Nonspecific suppressor cells were found in the spleens, lymph nodes (Kirchner et al., 1974a), and tumor sites (Holden et nl., 1976) of C57BLJ6 mice bearing Moloney-sarcoma-virus (M SV)-induced tumors at about 14 days after virus inoculation. Kirchner and his colleagues reached this conclusion in the light of intensive work on both the nature of the suppression and the character of the suppressor cells. Splenocytes from the tumor-bearing mice generated significantly lower proliferative responses after stimulation with the T mitogens PHA and Con A than spleen cells from normal mice (Kirchner et d . , 1974b). They could also not generate a significant proliferative response to the syngeneic cross-reactive tumor RBL5 (Kirchner et ( ~ l . , 1975a). The proliferative responses of the splenocytes from the tumor-bearing mice to the B mitogen lipopolysaccharide (LPS) were also reduced but less so than the responses to PHA and Con A. The proliferative response of these cells to PWM was the same or slightly lower than that of normal cells (Kirchner et al., 197413). Splenocytes of the MSV-tumor-bearing C57BLJ6 mice did not produce an anti-SRBC plaque-forming cell (PFC) response after in vitro

TABLE I11 CLASSIFICATION AND ACTIVITY OF SPECIFICAND NONSPECIFIC SUPPRESSOR CELLS~

Host

Tumor

Type of suppressor cell

Specificity of suppression

C57BIJ6 mice

MSV-induced tumor

Macrophage

Nonspecific

WIFu rats

Gross-leukemiavirus-induced transplants

Macrophage

Nonspecific

BN rats

In uitro cell line

Macrophage

Nonspecific

a a

transplant of MSV-induced tumor

Effect of suppressor cells

Release of suppressive factor

Inhibition of No PHA,Con A, RILTI, MLC, PFC or secondarq. antitumor cytotoxic responses and tumor pro1iferation Inhibition of PHA, Con A, or MLTI responses and tumor proliferation Inhibition of Con A, LPS, MLC, and CML reponses

Comments

Reference

Suppressor cells inhibit proliferative responses but not direct cytotoxic responses

Kirchner et al. (1974a, b; 1975a, b); Glaser et al. (1976); Fernbach et al. (1976) Glaser et al. (1975); Oehler et al. (1977)

Suppressor cells were found in the spleen but not in the lymph nodes

Veit and Feldman (1976a, b)

DBM2 mice

Macrophage

Nonspecific

Inhibition of Con A and LPS reponses

BALBlc (BALB/c X mice

Macrophage

Nonspecific

Inhibition of IgM anti-SRBC and TNP-MRBC PFC responses. Inhibition of SRBC RFC response. Inhibition ofiti uitro anti-SRBC PFC response Inhibition of PWM-induced synthesis of Ighl

MCA-induced rhabdomyosarcoma and spontaneorl s 1y induced mm1m a n adenocarcinoma transplants MPC-11, TEPCA/J)F, 183, SPQC-11 plasmacytoma

c2 -4

BALBlc mice

TEPC-183, SPQC-11 plasmacytoma

Macrophage

Nonspecific

Man

Multiple myeloma

Monocyte

Nonspecific

Man

Hodgkin’s disease

Monocyte

Nonspecific

Inhibition of PWM- and anti. gen-induced synthesis of immunoglobulins Inhibition of hlLC

Suppressor cells were found in the spleen but not in the lymph nodes

Pope et a1 (1976)

Yes

Cell-mediated im- Zolla et (11. mune function (1974); Tanapatchaiyaof these mice pong and remains intact Zolla (1974); Zolla-Pazner et al. (1976); Kolb et (11. (1977)

Yes

The suppressive extract did not inhibit fibroblast proliferation and cytotoxicity against chicken red cells

Krakauer et (11. (1976)

Broder et (11. (1975); Mackenzie and Paglieroni (1976) Hillinger and Herzig (1977)

TABLE III-(cotttitiued)

Host

Tumor

Type of suppressor cell

Specificity of suppression

Effect of suppressor cells

Nonspecific

Inhibition of proliferative response to PHA and in vitro cytotoxic response to allogeneic cells Inhibition of proliferative response to PHA and LPS Inhibition of proliferative reponse to MTV tumor Inhibition of proliferative response and protein synthesis after PHA stimulation. Blocking of macrophage migration in hi bi tion

C57BL/6 mice

MCA-induced tumor

Nylon-wooladherent cell; 8-

C3H/He mice

MCA-induced tumor

Adherent; Nonspecific non T; Ig+

BALBlcf C3H GR mice

MTV-transmitted tumor

BALB/c C3H/He mice

MSV-induced tumor, polyoma virus-transformed tumor, spontaneously occurring fibrosarcoma, ethylnitrosourea-induced trigeminal nerve tumor

Plasticadherent spleen cell B cell

Nonspecific

Nonspecific

Release of suppressive factor No

Comments

Reference Kirchner et al. (1974b); Eggers and Wunderlich (1975)

Poupon et al. (1976)

Creemers and Bentvelzen (1977) Yes

LPS stimulation is Kilburn et al. not affected by (1974); suppressor Gorczyns ki cells. The sup(1974); pressor cells Gorczynski can inhibit the et al. (1975) effector phase of the immune response

%

C3H/HeN mice

MTV-induced mammary tumor

B cell

Nonspecific

C57BLJ6 mice

3LL Lewis spontaneously induced lung carcinoma

T cell

Nonspecific

C3H/eb C57BLJ6 mice

MCA- or benzpyrene-induced fibrosarcoma

T cell

Nonspecific

Resistant strains of mousebearing FV-2rr gene (i.e., C57BLJ6) Susceptible strains of mouse bearing FV-2” gene (i.e., BALBlc)

Friend virus leukemia

T cell (but Nonspecific adheres to nylonwool column)

Inhibition of proliferative response to Con A and autochthonous mammary tumor Enhancement of tumor growth; increase in number of metastases in lung; decrease in cellular cytotoxicity

Rudczyns ki and Mortensen (1978)

Yes

Enhancement of tumor growth

Inhibition of Con A, LPS, and DS responses

No

Treves et (11. (1974; 1976a. b); Umiel and Trainin (1974); Rotter and Trainin (1975); Small (1977) Cohen et nl. (1971a, b); Small and Trainin (1975); Gabizon et al. (1976) T suppressor cells Kumar et a / . are under the (1974, 1976); control of M Kumar and cells Bennett (1976, 1977)

(continued)

TABLE 111-(continued)

Host

Tumor

Type of suppressor cell

C57BL/6 mice

402 AX testicular T cell teratoma. Spontaneously ininduced in strain 129/J mice but lacking H-2 specificity

Man

Hodgkin’s disease

T cell

C57BL/6 mice

MCA-induced sarcoma

T cell

Man

EBV-infected cell

T cell

Specificity of suppression

Effect of suppressor cells Inhibition of cytotoxic effector cells

Nonspecific

Inhibition of MLC Inhibition of cytotoxic effector cells Inhibition of EBV-infected B-cell proliferation

Release of suppressive factor

Comments

Reference

Isa and Sanders Treatment of (1976) effector cells of the spleen lymph nodes and thymus with anti-8 serum and complement increased their ability to cytoly~e[~H]thymidine-labeled homologous target cells Hillinger and Herzig (1977) SchaafLafontaine (1978) ThorleyLawson et ul. (1977)

BALBic mice

MLV-induced tumor

Nylon-woolnonadherent &-cells

Nonspecific

Inhibition of in oitro antiSRBC PFC reponses

STU mice

MSV-induced tumor

Spleen cells

Nonspecific

Inhibition of Con A and PHA responses

C57BLi6 mice

MCA-induced tumor

Spleen cells

Nonspecific

C57BL/6 mice

B-16 melanoma

Spleen cells

Nonspecific

Owl monkeys

HVS-induced tumor

Nonspecific

Fischer 344 rats

DMBA-induced mammary tumor

Peripheral blood lymphocytes Spleen cells

Inhibition of antiSRBC PFC response of F, recipients after transferring spleen cells from TBH Inhibition of PHA, Con A, and in uitro antiSRBC PFC responses Inhibition of PHA, Con A, and PWM responses Inhibition of in vitro antitumor cytotoxic response

No

Cerny and Suppression of Stiller (1975); the induction Stiller and phase of the Cerny (1976); in oitro immune Cerny et al. response (1977) Weiland and Suppressor cells Mussgay did not inhibit antitumor cyto(1977) toxicity Khaitov et al. (1976)

LPS mitogenic re- Stelzer and sponse was not Wallace inhibited. (1977)

Wallen et rrl. (1975a, b)

Kupermanet al. (1975)

(continued)

TABLE 111-(contiirued)

Host

Tumor

BALB/c mice

MCA-induced sarcoma

A/J mice

S1509a MCAinduced sarcoma

A/J mice

Type of suppressor cell

Specificity of suppression

Effect of suppressor cells

Spleen and lymph node cells T cell

Specific

L1117 spontaneous lymphoma

T cell

Specific

Inhibition of cytotoxic effector cells

N J mice

SaI MCA-induced tumor

T cell

Specific

Inhibition of cytotoxic effector cells

DBN2 mice

P815 mastocytoma T cell

Specific

Inhibition of i n oitro antitumor cytotoxic response

-3

M

Inhibition of cytotoxic effector cells Increase of tumor size

Release of suppressive factor

Comments

Reference Kall et al. (1975)

Yes

Fujimoto et a!. (1975, 1976a, b); Greene et a l . (1977a, b) Fujimoto and Tada (1978); Fujimoto et al. (1978) Fujimoto and Tada (1978); Fujimoto et al. (1978) Takei et al. (1976, 1977)

4

w

T cell

Specific

BALB/c mice

MCA-induced sarcoma

BALBlc mice

SV4O-transformed Nonadfibroblast herent PC

Specific

C57BL/6 mice

EL4 lymphoma

Thymus and spleen cells

Specific

BALB/c mice

MCA-induced sarcoma

PC

Specific

BALB/c mice

BM3 mastocytoma

Spleen cells

Specific

Inhibition of in oitro antitumor cytotoxic response Blocking of the macrophage migration inhibition Inhibition of in oitro antitumor cytotoxic response Blocking of the macrophage migration inhibition Enhancement of tumor growth. Inhibition of secondary antitumor cytotoxic response

Yes

No

Suppressive Nelson et al. factor was iden(1975a, b) tified as immunoglobulin Blasecki and Tevethia (1975) Ferguson and Simmons (1977) Halliday (1972)

Yes

Induction of suppressor cells with low dose (10') X-irradiated cells

Mengersen et al. (1975); Kolsch et ul. (1975)

CML = cell-mediated lympholysis; Con A = concanavalin A; DMBA = 7,12-dimethyIbenz(a)anthracene;DS = dextran sulfate; EBV = Epstein-Barr virus; HVS = herpes virus saimiri; LPS = lipoproteinpolysaccharide'; MCA = methylcholanthrene; MLC = mixed lymphocyte culture; MLTI = mixed lymphocyte tumor interaction; MLV = Moloney leukemia virus; MSV = Moloney sarcoma virus; MTV = mammary tumor virus; PC = peritoneal cells; PFC = plaque-forming cells; PHA = phytohemagglutinin; PWM = pokeweed mitogen; RFC = rosette-forming cells; SRBC = sheep red blood cells; TBH = tumor-bearing host; TNP-MRBC = trinitrophenylated mouse red blood cells.

74

DAVID NAOR

Stimulation with SRBC (Kirchner et d . , 1974a). Spleen cells from these animals did not proliferate significantly in MLC after cocultivation with irradiated BALB/c splenocytes (Fernbach et ul., 1976). In contrast, the 51Cr-release assay showed that the spleen cells of the tumor-bearing mice significantly cytolyzed the syngeneic crossreactive tumor RBL5. Such spleen cells also generated an increased cytotoxic response after PHA stimulation and their antibodydependent cellular cytotoxicity was improved when compared with that of nornial splenocytes (Kirchner et id., 1975a). The removal of cells which adhered to rayon columns or which were attracted to a magnet after phagocytozing iron particles completely restored the ability of the splenocytes from the tumor-bearing mice to proliferate in response to PHA (Kirchner et ul., 1974a,b). Similarly, removal of adherent cells on rayon columns or inactivation of phagocytic cells with carrageenan restored the ability of the splenocytes of the tumor-bearing mice to respond to irradiated BALB/c cells in the MLC reaction (Fernbach et d . , 1976). Spleen cells from the MSV-tumorbearing mice suppressed the PHA (Kirchner et al., 1974a) and MLC (Fembach et d . , 1976) responses of normal spleen cells when mixed together with them. Spleen cells from the MSV-tumor-bearing mice also suppressed the secondary in vitro anti-RBL5 cytotoxic response of splenocytes from mice in which tumors had regressed. Thus, spleen cells obtained from mice 30 days after MSV inoculation and 10 days after tumor regression generated a significant anti-RBL5 cytotoxic response after cocultivation with mitomyein C-treated RBL5 cells. This cytotoxic response was abolished when irradiated splenocytes from the tumor-bearing mice were added to the cultures (Glaser et ul., 1976). Removal of the adherent cells on rayon columns or of the phagocytic cells either by the iron and magnet technique or by pretreatment with carrageenan abolished the suppressive capacity of the splenocytes from the tumor-bearing mice. In contrast, the suppressive activity of these cells was not abrogated by treatment with anti-8 serum and complement (Kirchner et al., 1974a, 1975a; Fernbach et al., 1976). Pretreatment of splenocytes from the tumor-bearing mice with 5-bromo-2’-deoxyuridine (BUdR) and light restored their ability to respond to PHA. Since BUdR inactivates DNA synthesis, Kirchner et (12. (19744 suggested that the suppressor cells are proliferating cells. However, in other experiments they showed that the suppressor cells resisted a radiation dose of 2500 R (Kirchner et al., 1975a; Fernbach et nl., 1976). It was further found that the suppressor cells inhibited the PHA proliferative response only when added during the first 16 hours of culture (Kirchner et uZ., 1975a).

SUPPRESSOR CELLS

75

Tlie followiiig conclusions can be drawn from tlie work of Kircliner aiid his associates: (a) The suppressive effect is nonspecific since the PHA aiid Coil A proliferative responses were inarkedly reduced in tlie spleen cells of tuinor-bearing mice. (11) Tlie suppressive effect is mediated b y inacrophages; it was abolished b y removing adherent or pliagocytic cells, but not by reinoviiig tliymus-derived &bearing cells. (c) Tlie suppressive effect is aimed priinarily at prolifer‘1t’ive responses mediated by T cells rather than at cytotoxic responses. It was found, in fact, that splenocytes from the tuinor-bearing inice (Kirchner et ol., 1975a,l>)or cells from tlie tumor site itself (Holden et (il., 1976) inhibited tlie proliferation of the cross-reactive syiigeiieic RBL5 tumor cells and inarkedly reduced their nuinher following inutual incubation. The effect is not specific since the cells of the tumor-bearing host can also inhibit tlie proliferation of tlie allogeneic tumor P815 (Kirchner et (il., 197511).Tlie characteristics of the inhihitoiy cells were the same as those of the cells which suppressed the specific and nonspecific immune responses. They could be removed on “adherence” coluniiis or b y the iron and magnet technique. Carrageenan treatment also inactivated these cells. In addition, these cells were insensitive to anti-0 seniin and irradiation with 2500 R (Kirchner et (il., 1975a,b). Kirchner et (11. (1975) therefore suggested that the cells which inhibit the immune responses and the cells which inhibit the tumor proliferative responses are macrophages which belong to tlie same cell population. The fact that splenocytes from nude mice bearing MSV tumors also inhibited the proliferation of the RBL5 lymphoma strongly suggests that thymus-derived cells are not involved in this process (Kirchner et (il., 1975a). Thioglycollate-induced peritoneal exudate cells (PEC) from nonnal C57BL/6 inice also inhibited tumor proliferation but less efficiently than splenocytes from tumor-bearing inice (IGrcliner et ( i l , , 19751)). Such PEC also inhibited the niitogenic responses of spleen cells to PHA, Coil A, and LPS (Kirchner et (il., 1976). Although supernatants froin cultures of splenocytes of tumorbearing inice inhibited the [3H]thyniidine uptake of RBL5 lyinphoina cells, the number of tuinor cells was not reduced after such treatment (Kirchner et (il., 1975b). It should therefore be borne in mind that the reduction of [3H]tl~y~nidine uptake might be an experimental artifact caused b y the dilution of the isotope with “cold” thymidine released from tlie cultured cells (Opitz et nl., 1975). Workers who use the thyniidiiie uptake technique in such circuinstances should be aware of misleading interpretat’ions. Seruin froin the tuinor-bearing inice and, to a lesser extent, seniin

76

DAVID NAOR

from normal mice also inhibited the proliferative responses (Fernbach et ul., 1976). However, the relationship between cellular suppression and huinoral suppression has not yet been investigated. This form of immunosuppression cannot be caused by an excess of Moloney leukemia virus (MLV) contaminating the MSV preparation, since MLV-injected C57BW6 mice did not show reduced PHA responses at the first 3 weeks after injection (Kirchner et al., 1974b). The nonspecific suppressive effect is not limited to splenocytes of MSV-tumor-bearing C57BW6 mice. Splenocytes from C57BW6 mice bearing Rauscher-induced RBL5 tumor did not respond efficiently to PHA (Kirchner et ul., 1974a). Similarly, splenocytes of MCA-tumorbearing C57BL/6 mice did not generate an efficient proliferative response after stimulation with PHA (Kirchner et id., 1974b). In addition, they inhibited the allogeneic cytotoxic response of nylon-woolnonadherent spleen cells which had been sensitized in vitro with irradiated C3H splenocytes. The suppressor cells which were described as nylon-wool-adherent cells did not release any effective suppressive factors (Eggers and Wunderlich, 1975). Similar suppressor cells were also found in C3H/He mice bearing MCA-induced fibrosarcomas (Poupon et ul., 1976). Splenocytes of BALB/c mice bearing the MSV-transformed tumor (KA31) inhibited tumor proliferation as did splenocytes of C57BU6 mice bearing the MSV tumor (Kirchner et al., 197513). In parallel studies it was found that splenocytes from W/Fu rats bearing progressive Gross-leukemia-virus-induced lymphoma (C58NT)Ddid not generate a proliferative response after cocultivation with the homologous tumor (mixed lymphocyte tumor interaction, MLTI) or with T-cell mitogens (Con A, PHA). Removal of adherent cells on rayon columns or of phagocytic cells by the iron and magnet technique restored the ability of the splenocytes of the tumor-bearing rats to proliferate in the presence of the (C58NT)D tumor, PHA, or Con A (Glaser et al., 1975). Spleen cells from rats which had been injected with lo9tumor cells and had developed progressive tumors suppressed the Con A proliferative response of normal rat splenocytes and the MLTI proliferative response of splenocytes from rats which had been injected with los tumor cells and in which the tumors had subsequently regressed (Glaser et ul., 1975; Oehler et ul., 1977). These suppressor cells were still active after mitomycin C treatment (Oehler et al., 1977). While splenocytes from MSV-inoculated C57BL/6 mice did not demonstrate suppressive activity after tumor regression (Kirchner et al., 1974a,b), splenocytes of “regressor” rats did exhibit significant

SUPPRESSOR CELLS

77

suppressor cell activity (Glaser et nl., 1975). However, splenocytes from “regressor” rats regained their ability to generate a proliferative response to the (C58NT)D lymphoma after removal of adherent or phagocytic cells (Oehler et al., 1977). Normal splenocytes also produced an enhanced proliferative response to mitogenic stimuli after the elimination of similar cells (Glaser et al., 1975). Thus, macrophages from normal, “progressor,” and “regressor” rats could mediate the suppressive effect. Serum from “progressor” animals, but not from normal animals, inhibited the MLTI and the mitogenic proliferative responses of splenocytes from regressor and normal rats, respectively (Glaser et al., 1975). Splenocytes from “progressor” rats inhibited the proliferation of syngeneic and xenogeneic tumors. Splenocytes from “regressor” rats had a similar inhibitory capacity, while spleen cells from normal rats had a lower but still significant suppressive potential (Oehler et al., 1977). In contrast, splenocytes of normal mice did not inhibit tumor proliferation (Kirchner et a1., 1975b). Splenocytes from both “progressor” and normal rats lost their ability to inhibit the proliferation of tumor cells after removal of adherent or phagocytic cells. Treatment which removed T cells (ATS and complement) did not affect the antitumor inhibitory capacity of spleen cells from normal and “progresSOT’’ rats. In addition, the inhibitory splenocytes of normal rats were resistant to irradiation doses of 1000 to 5000 R and to anti-Ig and complement (Oehler et al., 1977). Thus, in the MSV mouse system and in the (C58NT)D rat system macrophages mediated both the nonspecific suppression of the proliferative immune responses and the inhibition of tumor proliferation. Since suppressor cells were activated only when the tumor reached a certain size, the authors suggested a chain of events to explain their findings. Factors released from the tumor cells are distributed in the circulation and transported into the spleen. When the tumor reaches a certain size, the concentration of this factor(s) reaches a level which permits the activation of the suppressor cells. Teleologically, the normal function of suppressor cells is to inhibit proliferating tumor cells. However, under certain conditions they also inhibit proliferative iminune responses which are directed against the tumor. Therefore, the direction of the balance between the inhibition of tumor proliferation and the inhibition of the antitumor immune response determines whether tumor regression or progression will occur (Kirchner et d., 1974a, 1975a; Fenibach et al., 1976). Much work has still to be done before this theory can be confirmed. However, the observation of the coexistence of suppressor cells and

78

DAVID NAOR

antitumor cytotoxic cells in tumor-bearing hosts (Kirchner et ul., 1974a) casts some doubt on this theory of the dual effect of suppressor cells. This so-called paradoxical finding of Kirchnsr et al. (1974a,b) was confirmed b y Weiland and Mussgay (1977). STU mice injected with MSV-induced tumor cells developed regressing tumors which reached a maximal size 13 days after tumor inoculation. Secondary tumors appeared at the sites of tumor inoculation after regression of the primary tumors. The secondary tumors developed progressively. The PHA and Con A mitogenic responses of normal cells were inhibited when the cells were mixed together with splenocytes from mice bearing maximal primary tumors. The suppressive effect was not observed before the tumors reached their maximal size or after their regression. Antitumor cytotoxic effector cells appeared only when the tumors had attained their maximal size, a time which coincided with the appearance of suppressor cells. Any theory which emphasizes the role of suppressor cells in promoting a neoplastic state will predict just the opposite situation. At the current state of our knowledge it is too early to decide whether this observation is an exception or a more general phenomenon, and further work should be done to throw more light on the problem. Mice bearing secondary progressing tumors did not generate either cytotoxic or suppressor cells. This observation contradicts the finding of Kirchner et al. (1974b) that “progressor” C57BL/6 mice possessed suppressor cells. The disparity between the findings might be related to strain differences (C57BL/6 mice vs STU mice). In agreement with the findings of Kirchner et al. (1974a,b), Veit and Feldman (1976a,b) found that BN rats bearing an in vitra line transplant of MSV-induced tumors possessed suppressor cells which were identified as macrophages. Splenocytes (but not lymph node cells) from these tumor-bearing animals generated reduced proliferative responses after stimulation with Con A, LPS, or allogeneic lymph node cells (MLC). Their ability to cytolyze homologous tumor cells was evident 7 to 10 days after tumor inoculation and was markedly reduced thereafter. The proliferative responses of these splenocytes to Con A or allogeneic cells was restored after filtration through a nylon-wool column. Addition of splenocytes from tumor-bearing rats to normal spleen cells markedly suppressed the ability of the normal cells to be stimulated by Con A. It was suggested that the suppressor cells were macrophages since the inhibitory activity could be removed from the splenic population of tumor-bearing rats by removal of the cells adhering to plastic dishes or nylon-wool columns. In contrast, anti-Ig serum and complement which lyses B cells and anti-rat thymocyte globulin and complement which lyses T cells did not

SUPPRESSOR CELLS

79

eliminate the suppressor cells. Furthermore, macrophages froin the peritoneal exudate of tumor-bearing rats and, to a lesser extent, those from the exudate of normal rats were also able to suppress the Con A proliferative response of iionnal splenocytes. Pope et (11. (1976) have also shown that inacrophages mediate the nonspecific suppressive effect in DBM2 mice bearing MCA-induced rhabdomyosarcomas or spontaneously induced manimary adenocarcinoinas. Splenocytes (but again, not lymph node cells) from these tumor-bearing inice did not generate an efficient proliferative response to Con A or LPS. These results confirmed previous findings that C57BLJ6 iiiice bearing MCA-induced sarcomas generated a low proliferative response to PHA (Kirchner et d.,1974b). Splenocytes from tumor-bearing DBN2 mice, which theniselves show low mitogenic responsiveness, suppressed the mitogen proliferative responses of normal spleen and lymph node cells. The Hypaque-Ficoll light fraction (specific gravity 1.08) of cells from tumor-bearing aninials had an even stronger inhibitory effect. The suppressor cell population which was isolated on the gravity gradient was insensitive both to anti-Ig serum and complement and to anti-8 seniiii and coinplenient, but could be removed by filtration through a nylon-wool column, by adherence to plastic dishes or by means of the iron and niagnet technique. It was therefore concluded that the cell which inhibited the mitogen proliferative response was the macrophage. Zolla et (11. (1974) found that BALB/c and (BALB/c x A/J)F, mice bearing the MPC-11, TEPC-183, or SPQC-11 plasinacytonia produced a lower IgM anti-SRBC response after injection of SRBC than did nonnal control mice. Mice bearing the MPC-11, SPQC-11, or 2P-21~1 plasiiiacytoina produced a lower IgM antitrinitrophenyl response after injection of the thyiiius-independent antigen, trinitropheiiylated mouse red cells, than did normal control animals. In addition, splenocytes from the plasiiiacytoma-beariilg iiiice did not generate a significant i t i .r;itt-o anti-SRBC PFC response after introduction of SRBC into the culture (Kolb et u l . , 1977). In contrast, lymph node cells or splenocytes from tumor-bearing mice exhibited augmented proliferative response after stimulation with PHA ( a T-cell mitogen) or 8Br-guanosine-3',5'-nionophosphate(a B-cell mitogen), respectively. The ability of lymphoid cells from plasmacytoiiia-bearing mice to respond to allogeneic cells also remained intact. l-Fluoro-2,4-clinitrobenzene-sensitized tumor-bearing mice did not show an impaired delayed type hypersensitivity response (Zolla-Pazner et (11., 1976; Kolb et nl., 1977). Addition of small fractions of splenocytes (viable or irradiated) or

80

DAVID NAOR

peritoneal cells (but not lymph node cells or thymocytes) from tumor-bearing mice to a Mishell and Dutton culture of normal spleen cells inhibited their ability to generate an anti-SRBC PFC response. The suppressor cells adhered to nylon-wool or Sephadex G-10 columns or to plastic surfaces, but were not affected by anti-Ig serum and complement or by antiserum to mouse brain-associated thymic antigen and complement. Adherent peritoneal or spleen cells from normal mice had significantly less suppressive potential (Kolb et al., 1977). In the light of the studies described above Zolla and her co-workers proposed that macrophages are the mediators of the suppressive effect (Kolb et ul., 1977). They also found that millipore-enclosed normal splenocytes which had been implanted in plasmacytoma-bearing mice did not generate a significant anti-SRBC response after stimulation with SRBC (Tanapatchaiyapong and Zolla, 1974). They therefore suggested that either the plasmacytoma produced a soluble factor which induced the appearance or the expression of suppressor cells or that the suppressor cells released a soluble factor which mediated the suppressive effect. They further speculated that the suppressive mechanism observed in plasmacytoma-bearing mice is an exaggeration of the “shut off’ regulatory response which is the normal feedback inhibition of plasma cells (Kolb et ul., 1977). Since the number of antigenbinding cells of plasmacytoma-bearing mice was normal (Zolla et ul., 1974) it can be suggested that the suppressor cells do not affect recognition of the antigen but do interfere with the differentiation of precursor B cells into mature antibody producers. It is interesting that lymphoid cells of patients with multiple myeloma exhibited impaired capacity to synthesize immunoglobins while their ability to bind radiolabeled antigens remained intact (MacKenzie and Paglieroni, 1976). In agreement with these findings, Krakauer et ul. (1976) found that a low-molecular-weight fraction of extracts of tumor ascites cells derived from BALB/c mice bearing the TEPC-183 or SPQC-11 plasmacytoina suppressed PWM-derived IgM biosynthesis of mouse splenocytes and human peripheral blood lymphocytes (PBL). Extracts prepared from tumor ascites cells depleted of phagocytes by the iron and magnet technique exhibited a low suppressive activity. It was suggested, therefore, that the hypogammaglobulinemia of plasmacytoma-bearing mice was caused by the formation of immunoregulatory macrophage-like cells which synthesized a suppressive factor. Tumor ascites extracts of a plasmacytoma such as RPC-5 which was not associated with hypogammaglobulinemia did not demonstrate a suppressive effect.

SUPPRESSOR CELLS

81

Like the plasiiiacytoiiia-beariiig mice, many patients with multiple myeloma showed a reduced ability to mount primary immune responses (Cone and Uhr, 1964). Furthermore, the PBL of patients with multiple myeloma synthesized much less IgM, IgA, and IgG after stiinulation with specific antigens or PWM than the PBL from normal individuals. The ability of normal PBL to synthesize immunoglobulin was suppressed after their incubation with PBL of multiple myeloma patients (Broder et nl., 1975; MacKenzie and Paglieroni, 1976). PBL from patients with multiple myeloma lost their suppressive capacity after the reinoval of phagocytic cells by the iron and inagnet technique. In contrast, the removal of T cells did not affect the suppressive activity. Thus, macrophages from MSV tumor-bearing animals suppress proliferative responses, which are mediated principally by T cells, while macrophages from plasmacytoina-bearing mice and patients with multiple myeloina suppress immunoglobulin synthesis which is inediated by B cells. Suppressor cells which were identified as monocytes have also been described in four out of six patients with Hodgkin’s disease (Hillinger and Herzig, 1977). In summary, it is clear froin these studies that macrophages of tumor-bearing hosts can suppress both various immune responses and tumor proliferation. However, it is not clear if the suppressive effect is dependent on a qualitative change in the macrophages or merely on an increase in their number. The latter possibility seeins to be more acceptable, since it has been shown that normal macrophages can also mediate the suppressive effect, albeit to a lesser extent. This idea is further supported by the finding that the spleens of C57BU6 inice bearing MSV-induced tumors contained three times as many macrophages as iiomial spleens (Kirchner et uZ., 1974a).

2. Notzspecijic Suppression Medinted b y B Cells Suppression mediated by soluble factors from tumor-bearing animals such as antibodies or antibody-antigen complexes (Hellstrom and Hellstrom, 1974) will not be discussed in the present review, although B cells which are involved in the production of these factors can be considered to be “suppressor” cells. Only studies in which the B cells of tumor-bearing mice were found to function directly as suppressor cells will be reviewed. Splenocytes from BALB/c mice bearing progressive MSV or polyoma virus-induced tumors did not show enhanced DNA or protein synthesis after stimulation with PHA. In contrast, splenocytes of such tumor-bearing inice exhibited enhanced responses after LPS stimulation. Reinoval of the rapid cellular fraction sedimenting at a velocity of

82

DAVID NAOR

5-6 mmhour by the bovine serum albumin (BSA) velocity gradient procedure restored the ability of splenocytes from tumor-bearing mice to be stimulated by PHA (Kilbum et al., 1974). Splenocytes and lymph node cells from nonnal mice did not respond to PHA after incubation with nonfractionated spleen cells from tumor-bearing hosts (Gorczynski, 1974) or their suppressive cellular fraction (Kilburn et al., 1974). The activity of the suppressor cells from “progressor” hosts could be eliminated with anti-Ig serum and coniplement but not with a n t i 4 serum and coniplement (Kilbum et al., 1974; Gorczynski, 1974). One million splenocytes from “regressor” BALB/c mice (28 days after virus inoculation) inhibited irk vitro macrophage migration in the presence of specific antigen. The same number of splenocytes from “ progressor” mice (10 days after tumor inoculation) did not inhibit i l l vitro macrophage migration in the presence of specific antigen. The inacrophage migration inhibitory effect of splenocytes from regressor mice was suppressed when splenocytes from progressor mice were added to the culture. Splenocytes which sedimented at 5-6 mmhour in the BSA velocity gradient procedure mediated this suppressive effect. The activity of suppressor cells from MSV-induced tumor-bearing mice was nonspecific since they also suppressed the macrophage migration inhibitory function of splenocytes from polyma virus-induced tumor-bearing mice. Splenocytes from “progressor” mice did not suppress the inhibition of macrophage migration after treatment with anti-Ig serum and complement. The suppressor cells of this system were, therefore, defined as “B” cells (Gorczynski, 1974). A culture supematant derived from incubated splenocytes of BALB/c progressor mice suppressed the PHA proliferative response of nonnal spleen cells. Unfortunately, the nature of this suppressive factor was not completely defined (Gorczynski et ul., 1975).These data were interpreted as indicating that suppressor B cells from tumorbearing mice participate in the formation of immune complexes which nonspecifically block the Fc receptor on T cells and subsequently inhibit their immune responses (Gorczynski et al., 1975). In a similar MSV tumor-host system Kirchner et al. (1974a,b) identified a different type of suppressor cell, a macrophage. The fact that one group of workers used C57BU6 tumor-bearing mice and the other group used BALB/c tumor-bearing mice might explain this difference. Suppressor cells which were tentatively defined as B cells were also found in C3H/HeN mice bearing mammary tumor-virus-induced mammary tuinors (Rudczynski and Mortensen, 1978). Splenocytes of these tumor-bearing mice did not generate a significant mitogenic response to Con A; this response was markedly augmented after filtering

SUPPRESSOR CELLS

83

the splenocytes through a glass-wool column. The splenocytes of the tumor-bearing mice suppressed the Con A mitogenic response of normal splenocytes when the two types of cell were incubated together. Treatment of the splenocytes from the tumor-bearing mice with initomycin C did not abrogate their suppressive activity; this indicates that DNA synthesis was not a prerequisite for suppression. The suppressor cells were tentatively defined as €3 cells since the suppressive activity of the splenocytes was removed b y filtration through a nylon-wool coluinn, but it was not abolished by treatment with anti-h, serum and complement. In addition, the suppressor cells were not removed by the iron and magnet technique, b y adherence to a plastic surface, or by treatnient with silica. Rudczynski and Mortensen (1978) did not rule out the possibility that the suppressor cells belong to either the nonphagocytic monocyte or the null cell populat’ion.

3. N ons pec if; c S u p p res.yioi1 Medi rr ted b y T Cel I .s Spleen cells from C57BL/6 mice injected with the 3LL spontaneously induced lung carcinoma had an inhibitoiy activity when tested after the injection of the tumor cells but before the appearance of the visible tumor. These spleen cells delayed tumor growth when injected together with 3LL tumor cells into recipient mice. The inhibitory activity of the donor spleen cells was “eclipsed” after the tumor became visible. After the appearance of the tumors, splenocytes from the tumor-bearing mice enhanced tumor growth (Treves et r i l . , 1974, 1976a,b; Urniel and Trainin, 1974) and increased the number of lung metastases (Umiel and Trainin, 1974) when injected together with 3LL cells subcutaneously or into the footpads of nonnal animals. These sensitized splenocytes enhanced tumor growth even when injected 4 days before tumor inoculation (Treves et ul., 1976a). The enhancement of tumor development in the recipient animals correlated with the size of the local tumor or the number of metastases in the donor mice (Treves et al., 1976a). Splenocytes of the tumor-bearing mice lost their ability to enhance tumor growth after the excision of the primary tumor. However, the splenocytes of the operated mice regained this ability after the appearance of a secondary tumor in the lungs (Treves et al., 1976a). In most cases, splenocytes from nonnal inice inhibited tumor growth when injected together with the tumor cells into syngeneic mice (Treves et nl., 1974; Umiel and Trainin, 1974). When noiinal spleen cells were injected together with sensitized splenocytes and 3LL cells into syngeneic recipients their protective effect was abolished and tumor growth was enhanced. Thus, the enhancing effect of

84

DAVID NAOR

the sensitized splenocytes dominated over the protective effect of normal spleen cells (Treves et al., 1976a). In contrast, thymocytes from normal mice enhanced tumor growth when injected with 3LL cells into normal recipients (Small, 1977). The sensitized splenocytes enhanced tumor growth in recipients which had functional immune systems. Tumor enhancement was not observed in 750-R-irradiated recipients. It can therefore be inferred that the sensitized splenocytes do not stimulate the enhancement of tumor growth directly, but rather affect the immune system of the host in such a way that tumor enhancement results (Treves et ul., 1974). Splenocytes from tumor-bearing mice depleted of T cells by anti-8 serum and complement did not enhance 3LL tumor growth in normal recipients (Treves et d., 1974). A soluble factor which was released into the supernatant of incubated splenocytes from tumor-bearing inice enhanced tumor growth. The supernatant from a sensitized incubated lymphocyte population which had been depleted of T cells b y anti-8 serum and complement was ineffective in promoting tumor growth. In contrast, a sensitized splenocyte population depleted of inacrophages and B cells by incubation on a plastic surface and subsequent passage through nylon-wool column released an effective enhancing factor (Treves et d . , 1976b). The enhancement effect was not specific, since splenocytes from 3LL tumor-bearing mice or soluble factors released from them could enhance the growth of unrelated tumors. Furthermore, both splenocytes from mice bearing different tumors and soluble factors released from these cells could enhance the growth of 3LL cells in normal hosts (Treves et (11., 1976a,b). The possibility that a cross-reactive antigen, which is shared by different tumor cells, is the inducer of the enhancing effect should also be considered. These results suggest that splenocytes of the tumor-bearing mice contain suppressor T cells which antagonize antitumor immune reactivity and consequently promote tumor growth. This suppressive effect is nonspecific and is possibly mediated by soluble factors (Treves et al., 1974, 1976b).The question of whether the soluble factor mediates the suppressive effect directly or merely stimulates the appearance of suppressor cells has not yet been answered. Spleen cells from tumor-bearing mice which were incubated with histamine-coated Sepharose beads were depleted of suppressor cells, since this cell population lost its ability to enhance tumor growth. In contrast, the fraction of splenocytes which was bound to the histamine-coated beads and subsequently eluted from them enhanced

SUPPRESSOR CELLS

85

tuiiior growth more efficiently than the unfractionated cells (Schechter et nl., 1977). Since histamine-coated Sepharose beads preferentially bind suppressor T cells (Shearer et (iZ., 1972; Eichmann, 1975) this finding further supports the supposition that the enhancing effect is iiiediated by suppressor T cells. It was, in fact, possible to eliminate the suppressor cells by culturing splenocytes from the tumor-bearing mice for 24 hours. Spleen cells from the tumor-bearing inice which had been incubated for 24 hours with 3LL tumor cells and then injected together with the tumor cells into normal recipients inhibited, rather than enhanced, tuinor growth 1976b). It can be assumed that the culture conditioiis (Treves et d., selectively damaged the suppressor cells while the effector cells remained intact. An alternative explanation is that suppressor cells may have a very short life span in culture. It is possible that in this system suppressor T cells do not only proinote tumor growth but also induce conditions permissive for tuiiior development. In adult thyniectoniized C57BL/6 mice which had been inoculated with 3LL cells, both the rate of tumor development (Umiel and Trainin, 1974) and the number of lung metastases (Treves et d., 1974) were smaller than in nonthymectoinized tumor-inoculated control animals. The assuinption that thymus cells have a permissive effect on tumor growth was supported by the finding that adult thymectomized irradiated (600 R) mice which had been reconstituted with thyinocytes had more lung metastases after 3LL tumor inoculation than mice which had been treated similarly but were reconstituted with bone iiiarrow cells (Umiel and Trainin, 1974).Moreover, 60% of the lethally irradiated bone marrow-reconstituted mice were able to reject the 3LL tumor while 100% of nornial mice died after tumor inoculation. The immune response to polyvinylpyrrolidoiie (PVP) was also augmented in the irradiated bone marrow-reconstituted mice. It was assumed that a deficiency of certain suppressor T cells in the irradiated bone marrow-reconstituted mice pemiitted a inore efficient iiiimune response against PVP and 3LL tumor antigens (Rotter and Trainin, 1975). Splenocytes from noimal mice delayed tunior growth when injected together with 3LL cells into nornial recipients. Reinoval of T cells b y anti-6 senim and complement improved the protective effect of the nornial spleen cells (Treves et d.,1974) while removal of the inacrophages b y the technique of adherence to a plastic surface iinpaired the protective effect of the nornial splenocytes (Treves et d.,1976a).

86

DAVID NAOR

Thus, the presence of T suppressor cells in a population of normal splenocytes does not permit the expression of maximal antitumor activity. As has been previously mentioned, thymocytes (in contrast to splenocytes) from normal C57BIJ6 mice enhanced 3LL tumor growth, albeit less efficiently than sensitized thymocytes (Small, 1977). Small (1977) suggested that the cells which mediate this tumor enhancement are dividing immature cells. Thus, normal thymocytes lost their ability to enhance tumor growth after treatment with BUdR and light, which eliminates the dividing cells. Small (1977) also observed that thymocytes from mice which had been injected with hydrocortisone acetate (HC) 1 day previously lost their ability to enhance tumor growth. In contrast, thymocytes from mice which had been injected with HC 2 days previously enhanced tumor growth more efficiently than thymocytes from nontreated mice. The enhancing cells which repopulated the thymus 2 days after HC injection were shown to be dividing cells since it was possible to inactivate them by treatment with BUdR and light. Moreover, these enhancing cells also lost their activity after treatment with thymic humoral factor (THF). Since massive dividing activity and sensitivity to THF are both properties of the immature cell, Small (1977) suggested that the enhancing thymocyte belonged to this category of cell. Since THF shifts the balance from immature T cells to mature T cells and therefore leads to exhaustion of the supply of suppressor cells, it has an antitumor therapeutic action in the system described above. Schechter and Feldman (1977) found that splenocytes from C57BL/6 mice which had been injected with HC 4 days after 3LL tumor inoculation enhanced tumor growth if their enhancement capacity was tested 3 days after HC injection. However, if these cells were tested 7 days after HC injection they were found to delay tumor growth iu vivo and kill syngeneic target tumor cells in vitro. The authors suggested that the mature suppressor cell is HC resistant since the spleens of the tumor-bearing mice were enriched with this type of cell 3 days after HC injection. In contrast, precursors of suppressor cells are HC sensitive and therefore the spleens of tumor-bearing mice lacked suppressor cells 7 days after HC injection. At that time, the supply of mature suppressor cells had already been exhausted and new suppressor cells had not appeared since their precursors had been eliminated. Furthermore, the antitumor effector cells were not affected, at least relatively, by HC treatment. It is impossible to compare the experiments of Schechter and Feldman (1977) with those of Small (1977) since the suppressor cell subpopulation of the normal thymus

SUPPRESSOR CELLS

87

might be entirely different from the suppressor cell subpopulation of the spleens of tumor-bearing mice. The difference between these systems is further expressed by the fact that nornial spleen cells (in contrast to normal thyniocytes) from HC-treated mice exert the same effect on tumor growth as normal spleen cells from nontreated inice (Schechter and Feldman, 1977). The experiment of Schechter and Feldman (1977) shows that in tumor-bearing mice HC can produce a balance which favors antitumor effector cell activity over suppressor cell activity. It is possible that the formation of such a balance can cause tumor rejection in this system. Although the results obtained from both systems are unequivocal, the interpretations should be strengthened, if possible, by additional experiments. The establishment of a GVHR in the tunlor-inoculated mice provides yet another way in which to induce an immunological balance which favors retardation over acceleration of tumor growth (Barnes 1972; Carnaud et d . , and Loutit, 1957; Boranii., 1968; Katz et d., 1974). The number of metastases in the lungs of (C3H/eb x C57BL/ 6)F, mice which had been injected with 3LL cells and parental splenocytes was lower than that in control mice which had been injected with 3LL cells and F, cells. Furthermore, splenocytes from 3LL tumor-bearing animals undergoing the GVHR had a reduced capacity for enhancing tumor growth in normal recipients. This can be interpreted as evidence that the GVHR either inactivates cells which participate in the production of huinoral blocking factors or eliminates enhancing cells which promote tumor growth. Since Camaud et (11. (1974) did not find a blocking factor in the 3LL tumor-bearing mice, they favored the latter intei-pret.'3t'1011. In the system described above, the sensitization of the enhancing spleen cells was achieved in the tumor-bearing animals. However, it was possible to imitate this in uiuo sensitization by culturing iionnal spleen cells on monolayers of syngeneic fibroblasts or 3LL tumor cells. These sensitized splenocytes killed syngeneic target tumor cells in uitro,but enhanced tumor growth when injected together with 3LL cells into noniial recipients (Ilfeld et ( i l . , 1973; S l i d and Trainin, 1976).The enhancement was nonspecific since thymocytes which had been semitized on unrelated monolayers also enhanced 3LL tumor growth. This observation is analogous with the consistent finding that sensitized cells from tumor-bearing hosts can efficiently kill tumor target cells irt uitro, although they have no inhibitory effect on the growing tumor iri uiuo (Hellstrom and Hellstrom, 1969). The splenocytes sensitized i n zjitro lost their ability to enhance tumor growth

88

DAVID NAOR

after removal of the fast fraction of the cell population by BSA velocity gradient sedimentation. Furthermore, the isolated slow fraction could protect some of the C57BL/6 mice from 3LL tumor growth, when injected together with the tumor cells into normal recipients (Small and Trainin, 1976). Similarly, fractionation of the 3LL-sensitized lyniphocytes over a histamine-rabbit serum albumin Sepharose colunin impaired or abrogated the enhancing capacity of these cells and augmented their specific in vitro cytotoxicity (Schechter et ul., 1978). These results were interpreted to indicate that the sensitized population of cells contained two different cell lines, the one inhibiting and the other enhancing tumor growth. The cells which enhance the tumor growth dominate the cells which inhibit it. It is possible that the enhancement of tumor growth in the recipients is mediated by a blocking factor fomied by cellular cooperation between the i r k vitro sensitized cells aiid the host cells. Alternatively, the iu vitro sensitized cells might contain populations of suppressor cells which antagonize the antitumor immune response of the host and consequently promote tuiiior growth. Similar results were obtained in another tumor-host system. Sensitized splenocytes from C3H/eb or C57BW6 mice bearing a fibrosarcoma induced with MCA or benzpyrene, respectively, enhanced tumor growth when injected with the tumor cells into irradiated recip1976; Small and Trainin, 1976). In addition, ients (Gabizon et d., spleen cells which were sensitized on fibroblasts or fibrosarcoma monolayers also enhanced tumor growth (Cohen et al., 1971a,b; Small aiid Trainin, 1976). Introduction of THF to the monolayer cultures impaired the enhancing capacity of sensitized cells (Small and Trainin, 1975). Spleen cells from C3H/eb fibrosarcoma-bearing mice inhibited tumor growth when obtained early (2 to 4 weeks) and enhanced tumor growth when obtained late (6 to 8 weeks) after tumor inoculation. The early inhibitory effect was specific for the fibrosarcoma cells. In contrast, the late enhancing effect was not specific, since the growth of an unrelated tumor which was injected into the recipients together with the fibrosarcoma-sensitized cells was also enhanced. The cells which enhanced tumor growth did not adhere to a nylon-wool column, but were eliminated with anti-8 serum and complement. They were therefore classified as T cells (Gabizon et al., 1976). The BSA gradient fast fraction of splenocytes from C3H/eb and C57BW6 mice bearing progressive fibrosarcomas or from C3H/eb splenocytes sensitized on fibrosarcoma monolayers enhanced tumor growth while the slow fraction inhibited tunior growth. Mixtures of slow and fast fractions caused

SUPPRESSOR CELLS

89

tumor enhancement. Thus, cells which enhanced tumor growth doniinated over the cells which inhibited tumor growth (Small and Trainin, 1976). It can be suggested that early after tumor inoculation the iminunological balance completely favors the effector cells which inhibit tumor growth. Later, more suppressor cells appear in the spleens of the tumor-bearing mice; their fiinction dominates over the function of the effector cells and conseqiiently promotion of tumor growth occurs. However, it was found that suppressor cells do not destroy the effector cells, since it was possible to recover the effector cells after cellular fraction,A t'ion. Schaaf-Lafontaine ( 1978) also found suppressor T cells in C57BW6 mice bearing MCA-induced tumors. Lymph node cells of these mice had demonstrated very low cytotoxicity 2 weeks after the mice had been inoculated with the MCA-induced sarcoma, while 1 or 4 weeks after grafting the cells were found to be very cytotoxic. The lymph node cells of tumor-bearing mice which had been grafted 2 weeks previously inhibited these cytotoxic effector cells when the two cell populations were incubated together. The suppressor cells were sensitive to anti-8 seniin and complement, but they were also found to adhere to a glass bead coltinin. The specificity of these suppressor cells was not tested. In a set of elegant experiments, Kuiiiar and his colleagues found that the Friend leukemia virus (FV)complex induced the appearance of nonspecific T suppressor cells which inhibited the ill vitro proliferative responses to T and B niitogens and, probably, the in zjizjo antibody responses to SRBC (Kuinar and Bennett, 1976, 1977; Kuinar et d., 1974, 1976). Strains of mouse which cany the Fzj-2" gene (i.e., C57BL/6) are resistant to the leukemic effect of FV whereas strains of mouse which carry the Fzj-2ss gene (i.e., BALB/c) are susceptible to this effect. Furthermore, spleen cells, thyniocytes, bone marrow cells, and lymph node cells from the susceptible strains did not respond well to the T niitogen Con A or to the B mitogens LPS and dextran sulfate when incubated together with FV.The proliferative responses of the susceptible cells to these mitogens were nomial when FV was not present in the cultures. In contrast, spleen cells from resistant strains generated normal proliferative responses to all niitogens regardless of whether FV was present or not in the cultures. Susceptibility to FV was not linked to the major histocompatibility gene complex (Kumar and Bennett, 1976). The presence of marrow-dependent cells (M cells) which did not

90

DAVID NAOR

share any properties with T or B cells determined the ability of the resistant mouse strains to resist FV infection and the capacity of their spleen cells to be stimulated by mitogens in the presence of the virus. Resistant C57BL/6 mice which were injected with RgSrin order to eliminate the M cells became susceptible to FV and exhibited erythroleukemia when injected with FV (Kumar et ul., 1974). In addition, FV-incubated spleen cells from "Sr-injected C57BU6 mice generated lower mitogenic responses than normal C57BL/6 splenocytes (Kumar and Bennett, 1976). It can be suggested that FV stimulated the appearance of suppressor cells in the spleens of the susceptible and the 89Sr-treatedresistant mice and that consequent impairment of their iinmunoconipetence occurred. In agreement with this assumption, it was found that after removal of adherent cells on a nylon-wool column the ability of cells from susceptible strains to respond to Con A in the presence of FV recovered. In contrast, rayon and glass-wool columns failed to remove suppressor cells. Removal of phagocytic cells by the iron and magnet technique and B cells by anti-Ig serum and complement did not reverse the inability of susceptible cells to respond to mitogens. It was concluded that the suppressor cells were nylon-adherent cells but not phagocytic or B cells. Nylon-wool-filtered spleen cells from susceptible mice generated a normal proliferative response to Con A in the presence of FV. When normal susceptible spleen or thymus cells were mixed with the nylon-wool-filtered cells, this response was abrogated. However, if the nonnal susceptible cells were treated with anti-Thy-1 serum and Complement, they could not impair the mitogenic response of nylonwool-filtered cells. It was therefore concluded that the suppressor cells were a subpopulation of T cells which adhered to a nylon-wool column and carried a Thy-1 marker (Kumar et d., 1976). Thyinocytes (but not spleen cells) from susceptible BALB/c mice, which had been injected with HC 2 days previously, responded norinally to Con A in the presence of FV (Kumar et al., 1976). It was therefore concluded that the thymus suppressor cells were susceptible to HC. In contrast, Sinall (1977) found that the thymus contained suppressor cells which effectively enhanced tumor growth 2 days after HC injection. These findings are not necessarily contradictory since different subpopulations of suppressor T cells might be involved in the two systems. Kumar et u1. ( 1976)found that spleen cells from *gSr-treatedC57BL/6 mice could suppress the mitogenic response of normal C57BL/6 resis-

SUPPRESSOR CELLS

91

tant splenocytes when the two populations of spleiiocytes were iiicubated together in the presence of FV, i.e., spleen cells of 89Sr-iiijected C57BL/6 resistant mice also contained suppressor cells. This suggests that the presence or absence of M cells, which is controlled b y genetic factors, determines whether FV can or cannot induce the appearaiice of suppressor cells. Since activation of suppressor cells provokes impainnelit of the immune response, conditions facilitating malignant infections are established. The suppressor cells of susceptible mice could inactivate the mitogenic response of nonnal cells from resistant mice oiily when they shared the D subregion of the H-2 complex. This genetic restriction could be circumvented b y irradiation of the suppressive cellular population. Thus, irradiated suppressor cells froin DBN2 susceptible mice suppressed the mitogenic response of allogeneic resistant C57BL/6 cells when the two populations of cells were incubated together in the presence of FV. Kumar and Bennett (1977)suggested that X irradiation eliminated the “interfering cell” which coiiferred the genetic restriction. Evidence ofthe existence ofiioiispecific suppressor T cells in iiiaii is also available. It has been found that the blood of patients with Hodgkin’s disease contains iiiitomycin C-resistant suppressor cells which can significantly inhibit the proliferative response of autologous cells to allogeneic stimulator cells in the MLC reaction. Monocytes suppressor cells were identified in four patients (see previous section) while thymus-derived suppressor cells were identified in two other patients (Hillinger and Herzig, 1977). Suppressor T cells that inhibited the transfoniiatioii a i d proliferative response of B cells after i t i oitro Epstein-Barr virus (EBV) iiifection were found b y Thorley-Lawson et (11. (1977). The transformation by EBV of uiifractioiiated lymphoid cells from the peripheral blood of iionnal donors wa? niuch less etfective than that of lymphoid cells from fetal cord blood. In contrast, the peripheral blood cell population which was retained by a Fab’ iminunoabsor1,eiit coluiiiii and was identified as a B-cell population could be efficiently transformed by E BV. T cells which were obtained after filtration of iionnal lymphoid cells through nylon-wool columns and suhsequeiit isolation of E rosettes delayed the transfonnation of B cells b y EBV. Thorley-Lawson et (11. (1977) postulated that a similar in oioo suppression niechaiiism might keep EBV-infected B cells of normal individuals and of patients with infectious mononucleosis under control. It may be further suggested

92

DAVID NAOR

that the “crippled” function of these suppressor cells is associated with Burkitt’s lymphoma and, possibly, other EBV-associated malignant d’iseases. Suppressor macrophages from MSV tumor-bearing mice could also inhibit the proliferation of tumor cells (Kirchner et ul., 1975a; see previous section). It might be speculated, teleologically, that suppressor cells have a dual function: normally they control tumor growth, but, under certain still unknown conditions, they inhibit the proliferative responses of the immune system and thus promote tumor growth (Kirchner et ul., 1975a). An interesting example of iieoplasia of suppressor T cells was found in a child with acute lymphoblastic leukemia and hypogammaglobulinenia (Broder et d.,1978).Neoplastic cells from seven other patients with acute lyiiiphoblastic leukemia failed to show suppressive activity. Cellular cooperation of human irradiated normal T helper cells and nornial B cells following in vitro PWM stimulation resulted in immunoglobulin synthesis. Leukemic cells of the patient of Broder et (11. (1978) could suppress this cooperative response but only when they were added to the culture together with normal iionirradiated cells. The suppressor leukemic cells were sensitive to X irradiation. The authors postulated that interaction o f T cells of the patient with T cells of a nornial donor was essential for activation of the suppressive effect. In suiiimary, nonspecific suppressor T cells have been found in both noiinal and tumor-bearing hosts. These cells could inhibit nonspecifically antitumor responses, various immune responses, and tumor transfonnation. It is not clear, however, whether spleen cells of tumor-bearing hosts, which suppressed both specific and nonspecific antitumor responses (Treves et a / . , 1974; Gabizon et d . , 1976), could also suppress other unrelated iiniiiuiie responses such as niitogenic, PFC, or mixed lymphocyte responses. Such infomiation will shed light on the question of whether the suppressive effect in these systems is limited to tumor antigens or whether it is a more general phenomenon.

B. SPECIFICSUPPRESSOHCELLS Specific suppressor cells inhibit immune responses directed towards the tumor antigens which induced the appearance of these suppressor cells while unrelated immune responses are not affected. Table 111 summarizes the major properties of these cells.

SUPPRESSOR CELLS

93

1. Specific Suppression Medicited by T Cells In a set of very elegant studies, Fujinioto and his co-workers dernonstrated the presence of specific suppressor T cells which accelerated tumor growth in aiiiinals immunized against the homologous tumor (Fujimoto et c d . , 1975, 1976a,b; Greene et ul., 1977a). Fujimoto and his colleagues immunized A/J mice against MCAinduced S 1509a sarcoma cells by subcutaneous injection of lo6tumor cells and subsequent excision of the growing tumor followed by a few more inoculations of the same tumor. Such animals could reject a dose of 10*cells. However, when thymus br spleen cells from the S1509a sarcoma-bearing mice were injected into the immunized animals together with the challenge tumor, the rejection of the tumor by the immunized recipients was delayed and the maximal size of the tumor was inarkedly increased. It was, therefore, concluded that suppressor cells were activated in the tunior-bearing mice. The suppressor cells exerted their effect even when injected into immunized mice 5 days after the tumor iiioculum. Tliyiii~iscells froin the S 1509a sarcomabearing mice suppressed the rejection of the homologous tumor in immunized recipients, while thyniocytes froin unrelated tumorbearing syngeneic mice f d e d to do so (Fujimoto et id., 1975, 1976a). Blocking factors were not involved in the suppression process, since immunized mice which had been injected with sera froin tumorbearing inice usually rejected the tumor challenge. Histological studies revealed that in the iinmunized d c e , which had beeii injected with spleiiocytes of tumor-bearing hosts and S 1509a cells, the infiltration of mononuclear cells into the tumor tissue was inhibited and the s u h e q u e n t destiiiction of tumor cells was impaired (Fujimoto et nl., 1976a). Suppressor cells appeared in the A/J mice 1 day after tumor inoculation and disappeared 5 days after tumor excision. Thus, suppressor cells were activated very rapidly, their regeneration was clependent 011 continuous stimulation b y tumor iuitigens, and their life span was very shoit. The suppressor cells were generated in the spleen, thymus, draining lymph nodes, and bone marrow, h i t not in the peripheral blood. It was therefore suggested that the suppressor cells were not recirculating cells. Spleen cells and thyniocytes froin tumor-bearing mice which had been injected with HC also impaired the antitumor reaction of the recipients. The suppressor cells of this system were therefore described a s mature HC-resistant cells. The suppressor splenocytes of the tumor-bearing mice were found in the

94

DAVID NAOR

top layer of a Ficoll density gradient (Fujimoto et ul., 1976b) and they were therefore described as comprising a low-density cell population. The suppressor cells which were derived from the spleens, thymuses (Fujimoto et a1., 1976a), or even bone marrow (Fujimoto et ul,, 197617) of the tumor-bearing mice were thought to be thymusderived cells since both anti-8 serum and complement and ATS and complement abolished their activity. This conclusion is in line with the finding that A/J mice inoculated with the S1509a tumor and subsequently splenectomized or treated with ATS, could control, to a certain extent, the growth of the tumor. In these mice the tumors developed inore slowly than the tumors in untreated control animals. The therapeutic effect of splenectomy is usually explained in terms of the removal of the main source that generates blocking antibodies (Hellstrom et al., 1970). Since blocking antibodies were not found in the sera of the tumor-bearing hosts, it was suggested that, in this system, splenectomy resulted in the removal of the main pool of immunosuppressor T cells (Fujimoto et al., 1976a). Freezing and thawing and subsequent ultracentrifugation of thymocytes or spleen cells from S1509a tumor-bearing hosts revealed a soluble factor which could mediate the suppressive effect. The suppressive activity of the soluble factor or its Sephadex G-200 smaller fraction (MW less than 7 x lo4)could be absorbed with S1509a cells but not with unrelated tumor cells. This discriminatory effect illustrates the specificity of the factor. Since the fractionated factor was destroyed at low pH or by heating at 56°C and since it was inactivated by pronase, but not by RNase, it was considered to be, at least in part, a protein. Anti-F(ab’)2immunoabsorbent did not absorb the suppressive activity of the soluble factor. In contrast, an immunoabsorbent with a specificity against the K end of the H-2 complex of A/J mice did absorb the suppressive activity of this factor. The factor therefore contained a product encoded in the K end of the H-2 gene complex, but did not contain the immunoglobulin F(ab‘), component. Since antibodies which were generated against the soluble factor and complement could inactivate the suppressive effect of intact thymocytes from tumor-bearing mice, it was further suggested that the factor was an integral part of the suppressor cell (Fujimoto et al., 1975; Greene et ul., 1977a). Both the information accumulated during the study of this model by Fujimoto and his co-workers and new available knowledge about the nature of the suppressor cell have led to the development of another elegant approach for establishing an immunological balance which fivors tumor rejection.

SUPPRESSOR CELLS

95

It was found in some systems that suppressor cells bear a unique product which is encoded in the I-J subregion of the H-2 gene complex (Tada et al., 1977; Pierres et al., 1977). Pierres et (11. (1977) showed that A/J mice which were injected with an antiserum directed against the I-J product produced a PFC response to SRBC higher than that of control mice. It was suggested that anti-I-J serum selectively eliminated suppressor cells and, consequently, a more efficient antiSRBC response was permitted. In parallel studies, Greene et al. 1977b) found that A/J mice injected with an antiserum directed against their I-J product (I-Jk)could control, at least to a certain extent, the growth of MCA-induced S1509a and SaI syngeneic tumors. The tumors in these mice developed much more slowly than tumors in mice treated with an antiserum directed against a different I-J product (I-J‘). Furthermore, splenocytes from S 1509a tumor-bearing A/J mice which had been treated with anti-I-Jk serum lost their ability to promote tumor growth when injected together with tumor cells into immunized mice. The genetic specificity of the anti-1-Jk serum was illustrated by the fact that lymphocytes bearing the H-2k specificity (from B 10.BR mice) could absorb the activity of the antiserum, while lymphocytes bearing the H-2‘’ specificity (from B10.D2 mice) could not absorb the activity of the antisenim. These results were confirmed by Fujinioto and Tada (1978) who also found that spleen cells from tumor-bearing A/J mice which had been treated with anti-1-J k serum and complement lost their suppressive activity. These experiments suggest that anti-1-J semm can selectively eliiiiinate suppressor cells and, consequently, an immunological balance which favors tumor resistance is established. Olwiously, we share the authors’ hopes “that an antisemin specific for the human counterpart of mouse I-J determinants could have useful therapeutic properties” (Greene et ul., 197711). Fujiinoto and his co-workers (Fujimoto and Tada, 1978; Fujimoto et u l . , 1978) extended their studies to a different tumor-host system and again found the antitumor immune response to be regulated by specific suppressor T cells. Mixtures of nomial splenocytes and lymph node cells from A/J mice which were cocultivated with syngeneic spontaneous mitomycin C-treated lymphoma cells (L1117) generated cytotoxic cells. The effector cells which were harvested on the fifth day ofthe culture lysed 51Cr-labeledL1117 target cells after coinculxition for 6 hours. Addition of splenocytes from L1117 tumor-bearing A/J mice to a mixture of cytotoxic effector cells and labeled target cells prevented the lysis of the L1117 target cells. Thus, in this system the

96

DAVID NAOR

suppressor cells antagonized the effector cells; this effect was expressed after a very short time (6 hours). Splenocytes froin L1117 tumor-bearing A/J mice which had been treated with anti-Thyl.2 serum and complement or with anti-I-Jk serum and complement lost their suppressive activity, since they could not antagonize the cytotoxic activity of the effector cells. It was concluded that the suppressor cells were T cells bearing the I-Jkproduct. The Ia product on the cytotoxic T cells was found to be controlled by a locus in the I-A subregion. Effector cells which were generated after sensitization with S 1509a tumor also killed the SaI tumor which cross-reacts with the S1509a tumor. However, splenocytes from S1509a tumor-bearing mice did not suppress this cross-reactive killing. In contrast, suppressor cells froni SaI tumor-bearing mice did suppress the killing of the SaI tumor by effector cells which were generated after sensitization with the S 1509a tumor. It was therefore suggested that the subset of antigens which was recognized b y the suppressor cells was specific to the tumor and different from the subset of antigens which was recognized by the effector cells. It is possible that the H-2 product, which is the structure coininon to both cross-reactive syngeneic tumors, constitutes that part of the antigen complex which was recognized by the effector cells. Finally, it should be emphasized that the suppressor cells which were generated in L1117 lyniphoina-bearing A/J niice inhibited the effector phase of the in uitro cytotoxic response. The question whether the suppressor cells can inhibit the induction phase of this response has not yet been answered (Fujinioto and Tada, 1978; Fujiinoto et a/.,

1978). In yet another tumor-host system Takei et al. (1976, 1977) found specific suppressor T cells that inhibited the induction or the expression of effector cells in culture. The P815 mastocytonia developed progressively after its inoculation into syngeneic DBM2 mice. Specific antitumor cytotoxic cells which appeared 14 days after inoculation of 5 x lo4 tumor cells disappeared very rapidly concurrently with the acceleration of tumor growth. The possibility that suppressor cells were responsible for the “eclipse” of the immune response was tested by the authors. Spleen cells of DBAI2 mice, which had been inoculated with the P815 mastocytoina tumor 10 days previously and which bore small tumors, generated a secondary syngeneic cytotoxic response after cocultivation with mitomyciii C-treated stimulator P815 cells. Spleen cells or thymocytes of DBN2 mice, which had been inoculated with

SUPPRESSOR CELLS

97

the tumor 16 to 19 days previously and which bore large tumors, inhibited this cytotoxic response when mixed together with the responder and the stimulator cells. Since the spleen cells of the mice bearing large tumors did not inhibit the i i i vitro cytotoxic response to unrelated tumors and their proliferative responses to PHA and Con A were normal, they were descrihed as specific suppressor cells (Takei et l/l., 1976, 1977). Treatment of splenocytes from the mice bearing large tumors with anti4 serum and complement abrogated their suppressive activity, while treatment with anti-Ig serum and complement or by the iron and magnet technique did not affect their ability to inhibit the i i i vitro cytotoxic response. The suppressor cells were therefore defined as thymus-derived cells. The suppressor cells were located in the light density fraction of a Ficoll-Hypaque density gradient (cl < 1.08)while the killer cells were located in the heavy density fraction. Thus, the suppressor cells and the killer cells do not belong to the same subpopulation, although it is possible that they express different differentiation steps ofthe same cell line (Takei et al., 1977). Similarly, suppressor cells of S1509a tumor-bearing A/J mice were also found in the light density fraction of a Ficoll density gradient (Fujimoto et ul., 197610). Since the suppressor cells of the P815 tumor-bearing DBN2 mice were added at the beginning of the cultures, it is impossible to decide whether they inhibit the induction phase, the effector phase, or both phases of the immune response. Ferguson and Simmons (1977) found that suppressor and antitumor reactive memory cells develop simultaneously in the same animal, but in different organs. Thymocytes and spleen cells from EL4 lymphoma-beariiig C57BL/6 mice suppressed specifically the in vitro anti-EL4 cytotoxic response of normal cells after their addition to the culture. In contrast, lymph node cells from the same tumor-bearing animals augmented the in vitro anti-EL4 cytotoxic response of normal spleen cells. The net effect which determines whether the tumor will be accepted or rejected depends perhaps on the balance between suppressor cells in the thymus or the spleen and reactive cells in the lymph nodes. Nelson and. his co-workers ( 197Sa,b) described specific thymusderived cells which participate in the generation of a suppressive factor. This suppressive factor was defined as an immunoglobulin. Lymph node cells from BALB/c mice immunized with irradiated MCA-induced sarcoma cells cytolyzed homologous target cells in a microcytotoxicity assay. supernatants which were obtained from cul-

98

DAVID NAOR

tivated splenocytes of mice healing the homologous tumor abrogated this cytotoxic response when added to the cultures. These supernatants did not abrogate the antitumor cytotoxic response of unrelated effector cells. Splenocytes from tumor-bearing mice which were treated with anti-8 serum and complement did not release an inhibitory factor into the culture supernatants. In contrast, splenocytes of tumnr-bearing mice which did not adhere to Sephadex G-10 and anti-mouse immunoglobulin columns yielded an effective suppressive factor. It was suggested, therefore, that the producers of the suppressive factor were thymus-derived cells. The suppressive factor itself was identified as an immunoglobulin since its activity was removed by filtration through an anti-mouse immunoglobulin affinity column. If the possibility that T cells are the producers of the immunoglobulin-suppressive factor is rejected, another (although not the only) alternative is that the T cells help other nonadherent cells to produce the factor. Elimination of thymus-derived cells which results in augmentation of the antitumor response is usually interpreted as evidence for the existence of suppressor cells. However, the possibility of the elimination of helper cells, which participate in the production of blocking factors, should always be taken into account and carefully checked.

2. Specijic Suppressioii Mediated h!y Unclussijied Cells PEC of BALB/c mice in which MCA-induced sarcomas had regressed (regressor mice) inhibited macrophage migration when incubated with a specific antigen. In contrast, PEC of BALB/c mice which bore progressive tumors (progressor mice) did not inhibit macrophage migration in the presence of a specific antigen. Furthermore, both the PEC of the progressor mice and the supernatants of cultures of these PEC specifically suppressed the macrophage migration inhibition assay of PEC from regressor mice (Halliday, 1972). Similarly, the PEC of BALB/c mice bearing SV40-transformed fibroblasts did not inhibit macrophage migration in the presence of a specific antigen, while the PEC of immunized mice (from which tumors had been surgically removed) did exhibit such immune responsiveness. The PEC from the SV40 tumor-bearing mice suppressed the macrophage migration inhibition assay of PEC from immunized mice when the two populations were mixed together. PEC from mice bearing MCA-induced tumors failed to mediate this suppression. The suppressor cells of SV40 tumor-bearing mice lost their blocking activity after being cultured for 4 days (Blasecki and Tevethia, 1975).

SUPPRESSOR CELLS

99

The nature of the suppressor cells from the MCA-induced sarcomabearing mice was not identified (Halliday, 1972), while the suppressor cells of the SV40 tumor-bearing mice were described as glass-surface nonadherent cells (Blasecki and Tevethia, 1975). Both types of suppressor cells exert their activity on the effector phase of the immune response. Since the nature of the suppressive mechanism has not yet been characterized in these studies, the possibility that macrophage migration inhibition is suppressed by an antibody or by immune complexes should not be disregarded. Kolsch and his co-workers (Mengersen et d., 1975; Kolsch et al., 1975) described the induction of specific unresponsiveness to the BM3 mastocytonia in BALB/c mice which was mediated by suppressor cells. Mice which had been injected with low doses of irradiated tumor cells (10' cells), but not with high doses (10" cells) of cells, generated suppressor cells in their spleens which specifically inhibited in uiuo secondary cytotoxic responses against B M 3 cells, following their transfer into immunized syngeneic recipients. Naor and his associates (Galili et id., 1976, 1978; Devens et al., 197th) have found an interesting relationship between the Moloneyinduced lymphoma YAC and the inimunoregulation system of its host, the A/J mouse. N J mice injected with either low doses of YAC, such as 10-103 cells, or with higher doses could not reject the tumor and died 3 to 6 weeks after inoculation. Attempts to immunize the A/J mice with niitomycin C-inactivated tumor cells were, in most cases, unsuccessful, since the injected mice exhibited very low levels of humoral and cellular cytotoxicity; they could also not reject low doses of viable tumor cells. On the other hand, mice which were injected with the chemically modified tumor trinitrophenylated mitomyciii C-treated YAC not only produced significant levels of cytotoxic antibodies against YAC but also rejected a dose of lo3viable tumor cells (Galili et uZ., 1976). The immunogenic properties of the YAC tumor could also be altered by i n vitro cultivation. Cultivated tumor cells, which were termed YAC-1 cells, did not grow or grew very slowly after inoculation into A/J mice. Furthermore, N J mice which had been injected with mitomycin C-treated YAC-1 tumor cells rejected low doses of viable YAC tumor cells (Devens et al., 1978b). Cultures of spleen cells of YAC-1-injected mice generated a syngeneic cytotoxic response after sensitization with YAC-1 tumor cells. The effector cytotoxic cells of these cultures could kill both the in vivo line YAC tumor and the in vitro subline YAC-1 tumor. Splenocytes from YAC-1 injected mice did not cytolyze

100

DAVID NAOR

syngeneic target cells when tested directly in the absence of secondary in vitro stimulation. Nonprimed spleen cells did not generate significant levels of cytotoxic cells after stimulation with YAC-1. The Rauscher-induced tumor RBL5 which is syngeneic in C57BL/6 mice and cross-reacts serologically with the YAC tumor also induced memory cells in A/J mice. Spleen cells from RBL5-primed A/J mice produced anti-YAC and anti-YAC-1 cytotoxic effector cells after stimulation with mitomycin C-treated YAC-1 tumor cells (Galili et al., 1978). In subsequent studies Devens et al. (1978a) found that, while the spleens of A/J mice which had been injected with YAC-1 tumor cells contained reactive memory cells, the spleens of A/J mice which had been injected with YAC tumor cells contained suppressor cells. Splenocytes of mitomycin C-treated YAC-injected mice did not generate anti-YAC and anti-YAC-1 cytotoxic effector cells after sensitization with the YAC-1 tumor. Furthermore, splenocytes from YAC-1-primed mice did not generate a syngeneic cytotoxic response when splenocytes from YAC-injected mice were mixed with them. In contrast, splenocytes of A/J mice which had been injected with the crossreactive tumor RBL5 were not suppressed by the addition of splenocytes of YAC-injected mice and they generated normal cytotoxic responses after sensitization with YAC-1 tumor cells (Devens et d., 19784. We concluded, therefore, that the suppressor cells recognized and inactivated memory cells which were stimulated by the syngeneic tumor, but did not recognize memory cells which were stimulated by the allogeneic tumor. This postulate was confirmed by a parellel experiment in which a different tumor-host system was studied. Spleen cells of RBL5-primed C57BL/6 mice generated an anti-RBL5 cytotoxic response after stimulation by the syngeneic tumor. Similarly, YAC-1, the cross-reactive allogeneic tumor, also induced memory cells in the spleens of C57BL/6 mice, since in vitro stimulation of these splenocytes by RBL5 tumor cells generated cytotoxic effector cells which killed the RBL5 target cells. Nonprimed splenocytes failed to generate such a cytotoxic response. Splenocytes from C57BL/6 mice which had been injected with the YAC tumor suppressed memory cells induced with the syngeneic tumor, RBL5, but not memory cells induced with the allogeneic tumor, YAC-1. A possible difference between memory cells induced by the allogeneic tumor and the memory cells induced by the syngeneic tumor

SUPPRESSOR CELLS

101

lies in the nature of the idiotypic markers on their receptor sites. We therefore postulated that the suppressor cells communicated with their target meniory cells by recognition of idiotypic markers which were expressed on cells induced by syngeneic tumor antigens but not by allogeneic tumor antigens (Devens et d., 1978a). It is possible that the YAC tumor has “learned,” by a positive selection process, to stimulate the suppressor cells which promote its growth. However, preinjection of the AJJ mice with the in vitro modified tumor stimulated the appearance of antitumor reactive cells, which antagonized the activity of the YAC-induced suppressor cells. Therefore, it is perhaps possible to manipulate the balance between suppressor and antitumor activity in such a way as to cause rejection of small doses of tumor cells. It is likely that larger doses of YAC tumor cells will stimulate a more intensive suppressive reaction and thus prevent effective antitumor activity. In summary, it should be noted that in each case for which classification studies were performed, specific suppressor cells were identified as T cells. In contrast, nonspecific suppressor cells were represented by cells of different classes: macrophages, B cells, T cells, and possibly “null” cells. C. UNDEFINEDSUPPRESSOR CELLS Suppressor cells which were not classified, or whose specificity was not defined, will be discussed in the present section (see also Table 111). Cerny and Stiller (1975) found nonspecific suppressor cells in the spleens of BALB/c mice with MLV-induced leukemia. The suppressor cells where found to be nylon-wool-nonadherent cells and to be insensitive to treatment with anti-8 serum and complement. Such cells can be classified as “naught” cells. Cell viability was not required for the suppressive effect: disrupted cells and cell nuclei retained a significant degree of the activity (Stiller and Cerny, 1976). The nonadherent cells of these tumor-bearing mice suppressed the in vitro anti-SRBC response of normal spleen cells when the two types of cell were incubated together even at ratio of 1: 100. The suppressor cells also inhibited, although less efficiently, the mitogenic responses of normal splenocytes to PHA and LPS (Cerny and Stiller, 1975). Addition of splenocytes from the tumor-bearing mice to cultures of normal spleen cells during the first 48 hours of culture suppressed the i n vitro anti-SRBC PFC responses. However, addition of the spleen

102

DAVID NAOR

cells at a later time did not affect the ability of the cultures to respond to SRBC stimulation. Thus, the induction phase of the in vitro immune response was sensitive to the suppressor cells, .while more differentiated phases and the effector phase were not sensitive to the suppressor cells (Stiller and Cerny, 1976). The authors focused special attention on the question of whether the mediators of the suppressive effect were the cells of the tumor-bearing host or extracellular factors. Supernatants of incubated splenocytes from the tumor-bearing mice did not suppress the in vitro anti-SRBC response of normal cells. In contrast, such supernatants suppressed the mitogenic responses of normal cells, although they lost this effect after dialysis (Stiller and Cerny, 1976). It is possible that the inhibitory effect of the nondialyzed supernatant was mediated by an excess of “cold” thymidine released by the cultured cells and blocked the uptake of the radiolabeled thymidine (Optiz et ul., 1975). The absence of suppressive factors in the supernatants of cultures of leukemic cells was further supported by experiments with double Marbrook chambers, in which leukemic spleen cells separated from normal splenocytes by a Millipore filter consistently failed to inhibit the immune response of the responder cells (Stiller and Cerny, 1976). Furthermore, splenocytes from MLV tumor-bearing hosts which had been treated with antiserum to Moloney leukemia antigens and complement did not lose their ability to suppress the anti-SRBC response of normal cells when incubated together with them (Cerny et al., 1977). All these experiments suggest that the suppressive effect was mediated by suppressor cells and not by virus-infected tumor cells or factors released from them. Such careful control experiments are essential in viral-induced tumor-host systems which possess nonspecific suppression of the immune response. In yet another system, the PBL of new world owl monkeys, which had been infected with herpes virus saimiri (HVS) and which subsequently exhibited lymphoma or lymphoma and leukemia, did not generate significant mitogenic responses after stimulation with PHA and Con A. Their response to PWM was also reduced but to a lesser extent (Wallen et al., 197%). The PBL of the diseased monkeys inhibited the PHA mitogenic response of PBL from normal animals when the two types of cell were incubated together. The possibility that the suppressive effect in this system is mediated by viral particles was also considered. However, it was suggested that the effect was mediated by suppressor cells and not by viral infection, since the PBL of nondiseased but HVS-infected monkeys did not exhibit suppressive ability (Wallen et al., 1975b).

SUPPRESSOR CELLS

103

Different types of suppressor cell exert their activity on different phases of the immune response. Suppressor cells whose activity is restricted to the late phase of the i r i vitro culture were demonstrated by Kuperman et u1. (1975).Spleen cells from Fisher rats bearing 7,12-dimethylbenz(u)anthracene-induced mammary tumors which were obtained 2 to 10 days after tumor inoculation generated, after cocultivation with the homologous tumor monolayer, stronger syngeneic cytotoxic responses than did normal spleen cells which had been sensitized under similar conditions. In contrast, spleen cells of tumor-bearing hosts which were obtained 11 days or later after tumor inoculation failed to generate cytotoxic cells after i n vitro cocultivation with the homologous tumor. Addition of such cells to a culture of normal spleen cells 2 days before the end of the culture strongly inhibited the ability of the normal cells to generate cytotoxic cells after stimulation with the homologous tumor. Splenocytes obtained from the tumor-bearing mice 12 days after tumor appearance gained high responsiveness if cultured for 2 days without tumor cells or adoptively transferred for 5 days through irradiated syngeneic recipients. After either of these two treatments the splenocytes generated cytotoxic cells after stimulation with the homologous tumor (Kuperman e t ul., 1975). The interpretation of these results suggests that short-lived or very susceptible suppressor cells dominate the immune system of the tumor progressor animal and block the differentiation of memory cells to cytotoxic cells. Thus, the suppressor cells do not exert their effect by elimination of the reactive cells but by reversible blocking of their activity. Suppressor cells from 3LL tumor-bearing mice exhibited similar properties (Treves et “I., 1976b). Suppressor cells which exert their activity on the effector phase of the immune response were also described by Kall e t (11. (1975). BALB/c mice were immunized against MCA-induced tumors by inoculation and subsequent excision of the tumors. Splenocytes and lymph node cells of the “tumor-excised” mice generated a significant level of cytotoxicity against the homologous tumor cells when cocultivated with them for 6 days. The cytotoxic response was determined by counting the number of surviving target tumor cells after their incubation with the effector cells which were generated in the culture tubes (microcytotoxicity assay). In contrast, lymphoid cells of mice bearing progressive tumors did not generate cytotoxic cells after 6 days of cocultivation with the homologous tumor. Furthermore, these lymphoid cells inhibited effector cells which were differentiated from lymphoid cells of “tumorexcised” mice (Kall et u l . , 1975). Since the suppressor cells of tunior-

104

DAVID NAOR

bearing hosts exhibited their inhibitory effect in the microcytotoxicity assay, it was concluded that in this system, the suppressor cells inhibited fully differentiated cytotoxic cells, perhaps by releasing soluble blocking factors. The specificity and/or nature of these and other suppressor cells (Isa and Sanders, 1976; Khaitov et ul., 1976; Creemers and Bentvelzen, 1977; Stelzer and Wallace, 1977), which are also listed in Table 111, have not yet been determined. V. Suppressor Cells Induced by Nontumorigenic Stimuli or by Tumor Excision

The tumor progression described in the previous sections was either permitted by preexisting suppressor cells or promoted by suppressor cells which were induced by the tumor itself. However, the possibility that suppressor cells are induced by nontumorigenic stimuli and that they, in turn, establish promoting conditions for tumor growth, is exemplified in the experimental systems described below. Kripke and her co-workers (Kripke, 1978) found that chronic UV irradiation of C3H, NJ, BALB/c, and C57BL/6 mice induced the appearance of a fibrosarcoma which grew progressively. Normal mice rejected the UV-induced tumor, whereas UV-irradiated mice did not. Furthermore, lymphoid cells of the UV-irradiated mice which were injected into X-irradiated mice conferred on the recipient mice the inability to reject the UV-induced syngeneic tumor. In contrast, most of the recipients which had been reconstituted with normal cells were able to reject the tumor. A lymphoid cell mixture which contained equal numbers of cells from UV-irradiated mice and normal mice also suppressed the ability of the X-irradiated recipient mice to reject the UV-induced syngeneic tumor but not the allogeneic tumor. It was therefore thought that UV irradiation induced in the mice specific suppressor cells which recognized syngeneic UV-induced tumors. Spellman and Daynes (1977) found that these suppressor cells were thymus-derived cells since they were sensitive to anti-0 serum and complement. It was suggested that UV irradiation damages the skin which consequently delivers antigenic photoproducts and they, in turn, induce the appearance of the suppressor cells (Kripke, 1978). In more general terms, it can be assumed that UV irradiation causes modification of self-antigens in the skin and these modified antigens induce the appearance of the suppressor cells (Spellman and Daynes, 1977). The preliminary finding that a skin graft from UV-irradiated mice could confer on some of the recipient mice an inability to reject the UV-induced tumor (Kripke, 1978) supports these assumptions.

SUPPRESSOR CELLS

105

Reinisch et a / . (1977) demonstrated that BALB/c mice which were inoculated with murine sarcoma virus (MuSV) developed a rhabdom yosarcoma. This tumor regressed spontaneously unless the mice were subsequently injected with CFA. The mice injected with MuSV and CFA developed progressive tumors which killed the hosts within 6 weeks. In contrast, adult thymectoinized mice injected with MuSV and CFA rejected the tumor. Thus, adult thymectomy antagonized the enhancing effect of CFA treatment. The authors suggested that CFA induced nonspecific suppressor cells in the BALB/c recipients. These suppressor cells inactivated the antitumor immune response and promoted progression of the tumor. Adult thymectomy removed the source of the suppressor cells or their precursors and consequently antitumor activity could be achieved even in the presence of CFA. This experiment should be called to the attention of the clinicians who use immunological modulators in cancer immunotherapy. They should remember that adjuvant treatment can change the delicate immunological balance but not necessarily in the desirable direction. Corynebacterium pcrrouni ( C . purorim) can also induce the appearance of suppressor cells under certain circumstances (Kirchner et uZ., 1975c; Savary and Lotzovh, 1978). As has been previously mentioned, splenocytes from C57BL/6 mice in which MSV tumors had regressed could generate a strong secondary cytotoxic response after sensitization with the cross-reactive tumor RBL5. This response was abrogated by the introduction into the culture of splenocytes of C. parozrminjected mice. The suppressive effect of the spleen cells was insensitive to X irradiation and anti-8 serum and complement, but it was abolished by filtering the cells through a rayon column or by treating them b y the iron and magnet technique. It was therefore concluded that C. pciroum induced the appearance of macrophage-like suppressor cells. Splenocytes from C. pcrirjuin-injected mice could also suppress the in vitro proliferation of tumor cells (Scott, 1974). The suppressive mechanisms induced by C. purouin which demonstrates the dual effect of suppressor cells might also exist i n oivo. It is possible that under certain conditions C. parounz will induce a stronger tumor inhibiting function than an immunosuppressive function, while under other conditions the reverse situation will occur. Again, the delicate Iialance hetween the two functions will determine the fhte of the tumor (Kirchner et ( r / . , 1975~).In agreement with this assumption, Scott ( 1974) showed that strong antitumor activity occurred under conditions of minimal systemic activation, whereas there was only weak antitumor activity after systemic administration of C . 11 (1 rv zi )) 1 . Suppressor cells induced by C . parouin in C57BL/6 mice also abro-

106

DAVID NAOR

gated the killing of YAC-1 by natural killer cells. Normal spleen cells which were mixed with spleen cells from C. parvzcm-injected mice were unable to mediate natural killing of YAC-1 (Savary and Lotzova, 1978).Interestingly, injection of C. parvum into BALB/c mice did not raise similar suppressor cells. On the contrary, such treatment improved the natural killing capacity of the spleen cells of these mice (Herberman et al., 1977). However, the experimental conditions (dose of C. purvum, route of injection, time of assay after C. parvum injection, and mouse strain) were entirely different in the two sets of experiments. Consequently, it can be assumed that under certain conditions, C. parvum extends the clone of natural killer cells, while under different conditions, it induces suppressor cells. Another interesting phenomenon relevant to the work of both experimentalists and clinicians is that of concomitant immunity. Gershon ( 1974) reinvestigated this phenomenon in hamsters bearing lyniphoblastic lymphomas. Tumor-bearing hamsters could reject inoculated tumor cells, while hamsters from which the tumors had been resected lost their ability to control the tumor growth and distant metastases sometimes appeared rapidly. Furthermore, hamsters which received lymphoid cells from animals which had lost their immunity after their tumor excision failed to generate antitumor immunity. This extraordinary model illustrates another situation which has possible clinical importance: the presence of the tumor induces antitumor immunity which permits rejection of subsequently inoculated tumor cells while excision of the tumor stimulates the appearance of suppressor cells which inhibit antitumor responses and promote the appearance of metastases. VI. Conclusions

Experimental data concerning the effect of suppressor cells on tumor-host relationships are accumulating rapidly and, as a consequence, the role of the suppressor cell may tend to be overemphasized by both experimentalists and clinicians. It is therefore very important to put the role of the suppressor cell into its correct perspective. The suppressive effect which is assumed to be mediated by suppressor cells may, in fact, be mediated by contaminating viruses, such as the lactodehydrogenase virus (Ryan, 1974) or a variant of the minute virus of mice (Bonnard et al., 1976). In the case of virally induced neoplasms, immunosuppression can be associated with the corresponding oncogenic type C viruses (Dent, 1972). In addition, the suppressive factor which is assumed to be produced by a “suppressor cell”

SUPPRESSOR CELLS

107

may be an antibody or an imniunocoinplex. If this is the case, then the suppressive effect is actually mediated by a conventional blocking factor and the “suppressor cell” is either a B cell which participates in the formation of the enhancing factor or a helper T cell which cooperates with a B cell in the production of the enhancing factor. It should also lie remembered that tumor cells can penetrate into the spleens or other lymphoid organs of tumor-bearing mice and the suppressive effects thought to be mediated b y cells of these organs may, in fact, be caused by tumor cells or by components released by them. With these reservations in mind it will lie easier to analyze, in more detail, the mutual relationships between the tumor, the host, and the suppressor cell. An interesting question is whether suppressor cells permit tumor induction or promote the proliferation of tumors which have already been induced. It can be assumed that polyclonal suppressor cells which exist in the animal before the appearance of the tumor can, under certain circumstances, establish conditions permitting tumor growth. In fact, Small (1977) has demonstrated that the normal thymus does contain suppressor cells; she showed that thymus cells enhanced 3LL tumor growth when transferred together with the tumor into normal recipients. This type of suppressor cell is referred to as a permitter suppressor cell. A different type of suppressor cell is that which is induced in the host by the tumor or its inductive factors (i.e., viruses, chemical carcinogens). This type of‘ suppressor cell-a promoter suppressor cell-f’uither stimulates tumor growth. Most of the suppressor cells described in this review are, therefore, promoter cells which are stimulated either in tumor-bearing hosts or on syngeneic monolayers. Their promotion effect has been demonstrated following their transfer together with tumor cells into normal or immunized recipients. It should be emphasized that the distinction between permitter and promoter suppressor cells is, at this time, arbitrary and the definitions are used to distinguish between noninducible and inducible suppressor cells, respectively. Is the ability of tumor cells to induce suppressor cells a general phenomenon? Can all tumors induce suppressor cells which promote their growth? This review focuses on the literature which demonstrates the existence of suppressor cells in different tumor-host systems. The limitation of such a selective review is that it can give the misleading impression that tumor growth promoted by suppressor cells is a general phenomenon. It must be emphasized very clearly that, at present, there is no evidence for such a generalization. It should also be taken into account that experiments for which positive results are obtained are usually published, while reports of negative

108

DAVID NAOR

findings appear in the literature only rarely. Consequently, the small number of negative findings that have been obtained from human studies should be given careful consideration. Thus, Quan and Burtin (1978) found nonspecific monocyte suppressor cells in only 6 of 120 cancer patients who were tested for the presence of these cells. Zembala et nl. (1977) found nonspecific monocytes and T suppressor cells in 15 of 25 patients with different disseminated carcinomas. Furthermore, lymphocytes from patients with chronic lymphocytic leukemia, lymphosarcoma, or multiple myeloma lacked suppressive activity. Twomey et al. (1975) found that the PBL of 16 of 30 patients with Hodgkin’s disease contained nonspecific suppressor cells which inhibited a MLC reaction. These conflicting findings prevent any attempt to generalize the function of suppressor cells at present. The heterogeneity of the suppressor cells in the tumor-host systems studied is another aspect of the problem which is worthy of attention. Almost all types of cell are represented among suppressor cells (macrophages, T cells, B cells, null cells). Table IV summarizes some of the properties of these cells. It is possible to find both similar and different properties among the various suppressor cells. For example, different types of suppressor cell share insensitivity to X irradiation or sensitivity to staying in culture while other types of suppressor cell exhibit either resistance or sensitivity to HC treatment. The inhibitory efficiency also varies among the different suppressor cells. For instance, suppressor cells which were induced in BALB/c mice bearing the MLV-induced tumor significantly inhibited responder cells at 1: 100 ratio (Cerny and Stiller, 1975), while suppressor cells o f N J mice bearing the YAC tumor had a significant inhibitory effect only at 1 : 1 ratio (Devens and Deutsch, unpublished data). Suppressor cells in one tumor-host system are different from the suppressor cells of another tumor-host system even when the suppressor cells of the two systems belong to the same subpopulation, e.g., T cells. It must be stressed that characterization of suppressor cells should b e done separately for each tumor-host system and it is impossible to extrapolate from one system to another. In addition the reader should be aware of the fact that technical variations, such as the dose of the reagents or the time schedule of the treatments, can account for the different conclusions concerning the nature of the suppressor cell. Which cells are the targets of the suppressor cells? Are they precursor nondifferentiated cells, memory cells, or fully differentiated effector cells? This review suggests that cells at different differentiation steps can be recognized and inhibited by suppressor cells, but much

TABLE IV COMPARISON OF PROPERTIES OF SUPPRESSORCELLSO Sensitivity to

Tunior 111

H,,st

C57BLi6 mice WiF rats

DBA mice

wrus

Type of supprrsm r cell

RUdH and light?

S

IF-

radi.1tion'

Mitoinycin C'

HC'

Staving

Adult thymectom! o r

i 11

splenec-

cultiirt.

GVHH

toniy

Anti-

1-1 ATS treatnient

Location

senim

on

treatment

Ficoll gradient

Velocity in BSA gradient

MSV-

induced trinror GrossXiriisinduced 1) mphonra hICAII l d l , ced rh.dxlo-

Induction phase

Kirclrner ef a/. (1974a. 1975a. b)

Pope ef ol (1976)

Light fraction

phage

Reference

Oehler ef a / . (1977)

Ya

hlacro-

Phase of activity of S I I p pressor cell

myosar-

cllllla BALB/c TEPC-183 (BALBic pla\max A/JVI)F, cytom.i

hlacro-

Kolb et al. (1977)

Nu

phage

mlCe

\Ian BALB/c niice C3HiHeN mice

hlyeloma MSV induced tunlor SIT\'Indllcrd

ttmlor

Slonocyte B cell

B cell

S O

5-6 mm/hour No

Bnider ef ul. (1975) Kilbom e t d.(1974)

Rridczynski and Mortensen (1978)

TABLE IV-(cotitiriued) Sensitivity to

Host

s

Tumor or virus

Type of suppressorcell

C57BW6 mice

3LL spontaneous tumor

T cell ofTBH

C57BW6 mice

3LL spontaneous tumor

T cell of normal mice

C3Weh mice

MCAinduced fibrosarcoma Freind leukemia vinis MCAinduced sarcoma ALL Hodgkin's disease

T cell

BALBk mice C57BLi6 mice Man Man

T cell

BUdR and lightb

Yes

X irradie tion'

Mitomycin Cd

Yes'

Yes

Yes

No

GVHR

ATS treatment

Anti14 serum treatment

Location on Ficoll gradient

Velocity in BSA gradient

Yes

No

N on

Reference

mm/hour

5-7 mndhoui

Kumar et a / . (1976); Kumar and Bennett (1977) Schaaf-Lafontaine (1978)

Yes (thymectomy)

Yes: Thymocytes; No: spleen cells

Yes

Phase of activity of suppressor cell

Treves ei al. (1976h); Small and Trainin (1976); Schechter and Feldman (1977) Camaud et al. (1974); Treves et al. (1974); Rotter and Trainin (1975); Small (1977) Small and Trainin (1976)

9-16 mm/hour

9-16

T cell

T cell T cell or monocyte

HC'

Staying in culture

Adult thymectomy or splenectomy

Broder et al. (1978) Hillinger and Herzig (1977)

MI mice

MI mice

DBAIP mice CS7BU6 mice BhLBIc mice Fisher rat

-

S 1509a MCAinduced sarcoma L1117 spontaneous lymphoma P815 martocytoma MCAinduced tumor 51LV leukemia DMBAinduced Irlionln.ll>

T cell

Yes Ye 5 (splenectomy)

N O

T cell

Yes

Fujimoto el ul. (1976a, h)

Light fraction

Fujimoto and Tada ( 1978)

Yes

T cell

Take] et ul. (1977)

Light fraction

Sot known

Induction phase

Eggers and Wunderlrch (1975)

Sot known Not known

Induction phase Effector phase

Stiller and Cerny (1976) Kuperman ef n l . (1975)

Yes

himor

C57BU6 mice BALBic mice

B-16 melaiinma

SV40transformed fihroblasts

Not known sat known

Yes" Yes

Stelzer and Wallace ( 1977) Blasecki and Tevethia (1975)

112

DAVID NAOR

work has still to be done before this question can be answered more completely. How do genetic factors affect the balance between suppressor and antitumor reactive cells? The elegant experiment of Levy and his coworkers (1976) may indicate the importance of this question. The hybrid mouse (AKR/J x DBA/2)F, (H-2k/")did not reject the AKR spontaneously arising carcinoma. Normal spleen cells of this hybrid did not generate an in rjitro cytotoxic response after their cocultivation with the tumor. In addition, splenocytes from these nonresponder tumorbearing mice very efficiently suppressed the ability of normal cells to produce a direct PFC response t6 SRBC when the two populations were incubated together with the red cells. In contrast, the hybrid mouse (AKWJ x C57BL/6)F, (H-2k i b , successfully rejected the AKR tumor, and its spleen cells generated a strong syngeneic cytotoxic response after cocultivation with the tumor. Spleen cells of the responder mice, in contrast to those of the nonresponder mice, did not suppress the in rjitro anti-SRBC PFC response of noimal cells when the two cell populations were incubated together with SRBC. It is possible to speculate that the H-2d allele is associated with the ability to generate nonspecific suppressor cells which antagonize the antitumor activity and promote successful tumor growth, while the H-2" allele is associated with the failure to generate suppressor cells and consequently with the ability to induce an efficient antitumor response. This possibility should be confirmed b y more detailed genetic studies. Thus, it is possible that genetic factors also influence the balance between suppressor and antitumor reactive cells, and, consequently, the ultimate fate of the tumor. How do suppressor cells communicate with their target cells? Do they transmit the suppressive message by cell-to-cell contact or via soluble factors? How do the suppressor cells recognize the target cells? Do they recognize H-2 products, idiotypic markers, or other components on the target cells? Although this review provides some indications as to how these questions may be answered, intensive work has still to be done before the suppressive mechanism can be completely understood. In some of the systems described in this review, the balance between suppressor cells and antitumor reactive cells determines the fate of the tumor (Fig. 1).If the balance favors antitumor reactive cells over suppressor cells, an immune response against the tumor is developed and the neoplastic cells are rejected. Conversely, when the suppressor cells are favored, immune responsiveness is not established and the tumor grows progressively. Both experimentalists and the

SUPPRESSOR CELLS

113

A

Suppressor cells

Suppressor cells Reactive cells

FIG. 1. The immunological balance. Autoimmmnity refers both to immunological activity against autoantigens aiid tumor antigens. For further explanations see text.

clinicians may ask how external intervention can affect the immunological balance between suppressor cells and antitumor reactive cells. A balance which favors antitumor activity can be established either by improving the function of antitumor reactive cells or by impairing the function of the suppressor cells. In fact, many tools are available, at present, for studying both approaches. The function of the antitumor reactive cells can be augmented specifically by injecting the host with inactivated, chemically modified, enzyme-treated (Prager and Baechtel, 1973; Naor and Galili, 1977) or allogeneic neoplastic cells (Bekesi et al., 1976; Galili et d.,1978) and nonspecifically by im-

114

DAVID NAOR

munomodulator agents such as bacillus Calmette-Gubrin, the methanol extraction residue fraction of tubercle bacilli, or C. parvum (Yashphe, 1971). Biological agents such as T H F have also been shown to improve the f h c t i o n of antitumor reactive cells (Small and Trainin, 1975). The present review describes several tools for the selective elimination of suppressor cells from potential or actual tumor-bearing hosts, i.e., adult thymectomy, splenectomy, HC treatment, or injection of ATS or anti-I-J serum. However, it should be emphasized that such nonspecific elimination of suppressor cells may induce, in addition to the antitumor activity, a general state of autoimmune response (Fig. 1) which obviously has a clinical disadvantage. The immunological model shown in Fig. 1 is based largely on experimental data obtained in mouse systems. It is very likely that a similar model exists in man. In fact, suppressor cells have been found in patients with different malignant diseases (Broder et al., 1975, 1978; Twomey et al., 1975; Blomgren et al., 1976; Hillinger and Herzig, 1977; Zembala et al., 1977; Quan and Burtin, 1978). However, we know very little, at present, about the nature of suppressor cells in man and any further applied approach depends on detailed characterization of their physical, chemical and biological properties. VII. Appendix

Since the original review was submitted, both the immunological and the oncogenical literature has been enriched with new information on the mutual relationships between malignant cells and suppressor cells. These new papers have shed more light on some of the aspects reviewed in this article, and they will therefore be described in brief here. The description of macrophages as a unique class of “suppressor” cells has been criticized by Kruisbeek et al. (Eur.].lmmunol. 8,200206, 1978; see also Kruisbeek and van Hees,]. Natl. Cancer lnst. 58, 1653-1660, 1977). These authors indicated that the spleens of rats bearing bladder carcinomas contained more macrophages than the spleens of normal rats. In addition, the splenocytes ofthe tumor-bearing hosts (TBH) did not respond significantly to the mitogens Con A and PHA, and the potential mitogenic responses were elevated by depleting the macrophages. Addition of 10-20% normal PEC to the depleted cellular population reduced the potential mitogenic responses to their original levels. The authors suggested that the suppression effect mediated by macrophages is quantitative rather than qualitative. Elgert and Farrar (I. lmmunol. 120, 1345-1353, 1978; J . Immunol. 120, 1354-1361, 1978) reached a similar conclusion when they found that

SUPPRESSOR CELLS

115

the spleen cells of BALB/c bearing MCA-induced tumors contained a high proportion of macrophages which inhibited the PHA mitogenic responses of normal cells. These macrophages were no different from normal macrophages which also suppressed the mitogenic responses of normal cells when used in similar proportions. The spleens of TBH also contained suppressor T cells. These suppressor T cells released a soluble factor which inhibited the activity of DNA polymerase. This effect might explain the molecular basis of the suppression. Oehler et al. (Cell. Zmmzinol. 29, 238-250, 1977) also claimed that normal rat spleens contained macrophages which could suppress the i n vitro proliferative and cytotoxic responses of lymphocytes to alloantigens. Pope et ul. (I. Immunol. 120, 2033-2040, 1978) found that the spleens of DBNZJ mice bearing MCA-induced sarcomas contained two types of nonspecific suppressor cell which they defined as a mitomycin C-resistant macrophage and a mitomycin C-sensitive suppressor T cell. The mi tom ycin C-re sistan t macrophages inhibited both the primary anti-SRBC response and the DNP-LPS PFC response to normal spleen cells when incubated together with nolmal spleen cells. The mitomycin C-sensitive suppressor T cells released a suppressive factor which inhibited only the in uitro primary anti-SRBC PFC response. It was possible to separate the two types of suppressor cells, since the macrophages were located at the top of a Hypaque-Ficoll column, while the T cells were located at the bottom. The i n viuo significance of macrophage suppressor cells which inhibit mitogenic responses i n vitro was further criticized by Hanna and Kripke (Cell. Zminutd. 43, in press, 1979). These authors found that C3H mice bearing UV-induced tumors exhibited a number of normal in oivo and in aitro immunologic responses, even though their spleens contained suppressor cells which inhibited in uitro mitogenic responses. Varesio et d . [Nature (Loitdon),in press] found that suppressor cell macrophages from the MSV-induced tumors of C57BL/6 mice inhibited the production of MIF, which is independent of proliferation, whereas macrophages from normal mice lacked the suppressive activity. Zolla-Pazner et (11. [“Handbook of Cancer aiid Immunology,” (H. Waters, ed.), vol. 2, pp. 225-239, Garland STMP Press, New York and London, 19781 found that plasmacytoma (PC)-bearing mice, which had been treated with silica prior to immunization with SRBC had an immune response which was 12-fold higher than that of immunized PC mice which had not been treated with silica. Whitney et al. (Eur.J. Cancer 14, 699-705, 1978) indicated that the ability to induce Hypaque-Ficoll light fraction suppressor cells is not limited to hosts bearing transplanted tumors. Within the spleen cell

116

DAVID NAOR

populations of C57BL/6, BALB/c, and CBMJ mice bearing primary MCA-induced sarcomas and C3H/HeJ mice bearing primary “spontaneous” mammary tumors there was a population of Hypaque-Ficoll light fraction suppressor cells which inhibited the Con A and LPS mitogenic responses of normal splenocytes when incubated together with them. Chickens infected with the oncogenic reticuloendotheliosis virus (REV) died shortly after infection with proliferative disease. The splenocytes of these birds suppressed the PHA proliferative response of splenocytes of normal chickens even at a ratio of 1 : 20. The suppressor cells were defined as nonmacrophage non-B cells and were thought to mediate their suppressive effect via direct contact with their target cells. It is evident that the suppression is mediated by the host cells and not by virus or transformed cells. The authors suggested that the disease and the rapid death are related to the suppression induced with REV (Carpenter et al., Cell. Immunol. 33,392401, 1977; Carpenter et al.,J. Zmmunol. 120,1313-1320,1978; Scofield and Bose,J. Zmmunol. 120, 1321-1325, 1978). In another system, Kumar et nl. (1974,1976)and Kumar and Bennet (1976,1977) showed that mice resistant to spleen focus-forming virus (SFFV), which induces erythroleukemia, also have splenocytes which respond to Con A in the presence of FV. In contrast, mice sensitive to SFFV have splenocytes which do not respond to Con A in the presence of Fv.It was therefore thought that both phenomena were mediated by suppressor T cells regulated by the same genetic control. However, genetic analysis revealed that the ability to respond i n uitro to Con A in the presence of FV is regulated by a separate single autosomal gene (Fu-3) which is dominant for susceptibility and which is not linked to the gene (Fu-2) which controls focus formation in uivo (Kumar et al., J. E x p . Med. 147, 422433, 1978). Subramanian et al. (Cancer Res. 38, 1996-2002, 1978) found that cultured spleen cells from C3H/HeJ mice bearing MCAinduced tumors released a soluble factor into the medium which suppressed the mitogenic or antigenic responses of normal lymph node cells or spleen cells. The suppressor factor, produced by nonadherent cells, was heat stable, disappeared on dialysis, and did not appear to be either a tumor antigen or thymidine. Argyris (Cancer Res. 38, 12691273, 1978) suggested that suppressor cells are activated by a factor released from the tumor cells of DBM2 mice bearing P-815 mastocytomas. P-815 tumor cells placed in diffusion chambers impernieable to cells can activate the appearance of suppressor T cells which inhibit the MLC. Primary tumors induced with MCA in C57BL/6 mice (but not in DBM2 mice) can also activate suppressor cells. The sera of melanoma patients contain an immunosuppressive factor which inhib-

SUPPRESSOR CELLS

117

its the PHA response of normal human lymphocytes (Ninnemann,J . Ininiunol. 120, 1573-1579, 1978). It was found that this immunosuppressive effect was mediated by cooperation between B and T cells. The suppressive factor in the sera stimulated B cells, which subsequently regulated the activity of suppressor T cells. Treves et (11. (1976b) claimed that C57BL/6 mice bearing 3LL Lewis lung carcinoma contained suppressor T cells which released an enhancing factor (EF). This finding has been contradicted by the new observations of Isakov et u Z . (Znt.J . Cancer, 22,465-470) and Hollander et nl. ( Z r 1 t . J . Cnncer, 22,471-478). These authors found that the splenocytes of C57BL/6 mice bearing 3LL tumors release an E F which can enhance tumor growth when injected into normal recipients together with the tumor. In addition, the E F can also augment the anti-SRBC response of normal mice injected with SRBC. Splenocytes of TBH, which were treated with anti-8 serum and complement or which adhered to a nylon-wool column, released the E F. Furthermore, splenocytes of nude TBH also released the EF. Supernatants of incubated tumor cells contained E F and splenocytes of normal mice which were injected with these supernatants also released the EF. It might be suggested that the 3LL tumor releases an E F which is absorbed into the spleen cell populations, where it stimulates B cells which subsequently produce enhancing antibodies. These antibodies are responsible for the tumor enhancement. However, it is possible that suppressor T cells function simultaneously with enhancing antibodies in C57BL/6 mice bearing the 3LL tumor. Indeed, Schechter and Feldman (Proceedings of the 12th Leukocyte Culture Conference, 1978) extended their previous studies and showed that the tumors of 3LL-bearing mice injected with hydrocortisone (HC) developed more slowly than those in a TBH control group. The tumor growth could be accelerated in the HC-treated mice by reconstituting them with thymocytes or splenocytes from TBH donors. Spleen cells which were treated with anti-8 serum and complement did not mediate the acceleration of tumor growth in HC-injected mice a s well as untreated cells. It was concluded that HC eliminates the precursors of suppressor T cells and its effect is therefore primarily immunological. Normal thyniocytes also enhanced 3LL tumor growth in C57BL/6 recipients (Small, 1977). This observation was extended by Umiel et (11. (Cell. lnimunol. 37, 134-141, 1978) who found that the peanut-agglutinated fraction of thymus cells which contained immature cells enhanced tumor growth, while the nonagglutinated fraction which contained mature cells delayed tumor growth. Greene and his colleagues further extended their studies on the S 1509a fribrosarconia-induced suppressor cells in N J

118

DAVID NAOR

mice and found that the suppressor cells and their soluble factor exhibited restricted specificity to the tumor(s) which induced their appearance. These results confirm the similar findings of Fujimoto et a1 (1978).It was further found that TBH treated with cpclophosphamide or Bacillus Calmette-Guerin (BCG) exhibited considerable inhibition of tumor growth. It was suggested that these reagents create an immunological balance which favors tumor rejection, since cyclophosphamide eliminates suppressor cells, while BCG augments the antitumor effector mechanism (Greene et al., Am. J . Pathol. 95,255-265, 1979). It was further proved that the suppressive factor of the TBH suppressor cells carries I-Jk and antigen specificities. This suppressive factor stimulated the appearance of suppressor cells which limited effective host immune response against the tumor antigen (Perry et al. J . lmmuiaol. 121, 2144-2147; Greene et al., J . lmmunol. 121, 2363-2366, 1978). After injection of anti-I-Jk serum, A/J mice bearing the MCA-induced fibrosarcoma showed increased leukocyte infiltration into the tumor site and enhanced tumor destruction. It was suggested that anti -,I-Jk serum eliminates suppressor cells and consequently more pronounced cellular immunity against the tumor can be expressed (Perry et al., A1n.J.Puthol. 92,491-506,1978). Takei et (11. ( J . lmmunol. 120,1218-1224, 1978) extracted a suppressive factor from the thymocytes of P-815-mastocytoma-bearing DBN2 mice, which inhibited the in vitro syngeneic primary and secondary cytotoxic responses of nonnal splenocytes. The sonicated suppressive factor (MW 40,000-60,000) which carried a recognition site with a specificity for P-815 did not contain detectable immunoglobulin. Recent results (Levyet al.,J. Immunol., in press) revealed that the suppressor cells and the suppressive factor did contain an Ia product but were not restricted genetically. Hellstrom and Hellstrom (Proc. N a t l . Acad. Sci. USA 75, 436-440, 1978) found that irradiated MCA-induced sarcoma cells enhanced specifically tumor growth in X-irradiated BALB/c mice inoculated with specific antitumor immune cells and normal cells. The enhancement effect was abrogated if the nomial cells were X-irradiated or treated with anti-T serum and complement. The authors suggested that the irradiated tumor activates suppressor T cells which inhibit an effective antitumor response. Spellman and Daynes (Cell. lmmunol. 36, 383-387, 1978; Cell. lmrnunol. 3 8 , 2 5 3 4 , 1978) further characterized the UV-induced suppressor cells which support the growth of UV-induced tumors. The suppressor cells were cells which passed through a nylon-wool column and which had a relatively short life span. The suppressor cells could recognize antigenic structures which were common to different UV-induced tumors, but they were unable to recognize the unique

SUPPRESSOR CELLS

119

antigenic structure of the individual UV-induced tumor. These observations were recently confirmed by Fisher and Kripke ( J . Znimuiiol., 121, 1139-1144, 1978) who showed that UV-induced suppressor cells are T cells which are specific for the UV-induced syngeneic tumor. In contrast to Spellman and Daynes, Fisher and Kripke claimed that their suppressor cells are long-lived. The difference can be attributed to different experimental conditions. Additional evidence for the presence of suppressor cells in human malignancies has recently been reported. Suppressor T cells have been identified in patients with osteogenic sarcoma; these cells inhibited the in uitro activity of cytotoxic lymphocytes (Yu et ul., N . E n g l . ] . Med. 297, 121-127, 1977). Golding et uZ. (Clin. Z.mrnunoZ. Zmmzrnoputhol. 7, 114-122, 1977) found leukocyte inhibitor factor in the supernatants of lymphocytes culture of patients with Hodgkin’s disease and Herberman’s group (Jerrells et nl., J . Nutl. Curicer Inst. 61, 10011009,1978)found that about 40%of38patients with lung or breast cancer showed activity of nonspecific suppressor cells. In some patients adherent suppressor cells (macrophages) were identified while in others, nonadherent suppressor cells were detected. Bean et (11. (J.E s p . Aied. 146, 1455-1460, 1977) identified T suppressor cells in a patient with urinary bladder carcinoma; these T cells suppressed the MLC of 3 of 27 normal donors. The authors suggested a possible genetic restriction associated with this suppressive activity (see also previous report by i ) 145-146, 1976). A prostaglanBerlinger et (rl., Nuture ( Z ~ n i d o ~260, din-producing suppressor cell appears to lie responsible for the hyporesponsiveness to PHA seen in Hodgkin’s disease. This suppression could be reversed iri ~ i t r oby inclomethacin, a prostaglandin synthetase inhibitor (Goodwin et u l . , N.E1igl.J. Mecl. 297,963-968, 1977). Similnr results in tumor-bearing mice (Pelus and Strausser, Z r i t . ]. Crrrrcer- 18, 653-660, 1976) confirm this ohseivation. The effect of‘suppressor cells on the development of neoplastic cells in both man and animals was also recently discussed by Oehler et (11. (Plzurniacol. Tlier. A . 2, 551-593, 1978) and Kirchner ( E i i r . J . Cancer 14, 453-459,

1978).

REFERENCES A d d a , D-H., Beraud, E., and Depieds, R . (1977).E u r . J . lmttLutto/. 7, 620-623. Allison, A . C., and Law, L. W. (1968).Proc. Soc. E.Y/I.B i d . Med. 127, 207-212. Alvord, E. C., Shaw, C. M . , Hruby, S., and Kies, M . W. (1965).Atiti.N.Y. Acud. Sci. 122,

333-345. Balner, H. (1971).Rev. E u r . Etccd. Cliri. B i d . 16, 981-986. Balner, H. (1972).Bellring I t i s t . M i t t . 51, 196-199.

120

DAVID NAOR

Barnes, D. W. H., and Loutit, J. F. (1957). Br. J . Huemuto/. 3, 241-252. Barski, G., and Youn, J. K. (1969).J. N u t / . Cancer Z m t . 43, 111-121. Bartholomaeus, W. N., Bray, A. E., Papadimitriou, J. M., and Keast, D. (1974).J. Natl. Cuticer Znst. 53, 1065-1072. Basten, A., Miller, J. F. A. P., and Johnson, P, (1975). Transplant. Reu. 26, 130-169. Batchelor, J. R . , and Silverman, M. S. (1962). Transplantatioti, Ciba Found. S ! / r n p . , 216-231. Bekesi, J. G., Roboz, J. P., Zimmerman, E., and Holland, J. F. (1976). Cancer Res. 36, 631-639. Benacerraf, B., and Dorf, M . E. (1977). Cold Spring Harbor S!/tn~i. Quunt. Biol. 41, 465475. Blair, P. B. (1972).J. N u t / . Coiicer I m t . 48, 1121-1124. Blasecki, J. W., and Tevethia, S. S. (1975).J. Zmmutiol. 114, 244-249. Blomgren, H., B a d , E., Petrini, B., and Wassennan, J. (1976). C / i i i . O i i c o / . 2, 379392. Bonmassar, E., Goldin, A., and Cudkowicz, G. (1971). Tr~itis)~/antcltior1it~~fi(i~i 12, 314-318. Bonmassar, E., Bonmassar, A., Goldin, A,, and Cudkowicz, G. (1973). Cancer Res. 33, 1054-1061. Bonmassar, E., Menconi, E., Goldin, A,, and Cudkowicz, G. (1974)J. N u t / . Cancer Znst. 50,475-479. Bonnard, G. D., Manders, E. K., Campbell, D. A., Jr., Herberman, R. B., and Collins, M. J. Jr. (1976).J. E s p . M e d . 140, 187-205. Boranii., M. (1968).J. N u t / . Cancer Znst. 41, 421437. Bray, A . E., and Keast, D. (1975). Br. J . Cancer 31, 170-175. Bremberg, S., Klein, E., and Stjernsward, J. (1967). Cancer Res. 27, 2113-2118. Broder, S., Humphrey, R., Durm, M., Blackman, M., Meade, B., Goldman, C., Stroller, W., and Waldmann, T. A. (1975).N . E n g l . J . Med. 293, 887-892. Broder, S., Poplack, D., Whang-Peng, J., Durm, M., Goldman, C., M u d , L., and Waldmann, T. A. (1978). N . Engl. J . Med. 298, 66-72. Burnet, F. M. (1970). Prog. EX?,.Tunlor Res. 13, 1-27. Calderon, J., and Unanue, E. R. (1975). Nature (London) 253, 359-361. Cantor, H., and Boyse, E. (1977). I r i “Contemporary Topics in Immunobiology” (0. Stutman, ed.), Vol. 7, pp. 47-67. Plenum Press, New York. Carnaud, C., Markowicz, O., and Trainin, N. (1974). Cell. Zrnrnunol. 14, 87-97. Cerny, J., and Stiller, R. A. (1975).J. Imrnuno/. 115, 943-949. Cerny, J., Grinwich, K. D., and Stiller, R. A. (1977).J. Zrnmunol. 119, 1097-1101. Cohen, I. R., Globerson, A., and Feldman, M. (1971a). Zsr. J . Med. Sci. 7, 632-633. Cohen, I. R., Globerson, A., and Feldman, M. (1971b). Transplant. Proc. 3, 891-894. Cone, L., and Uhr, J. W. (1964).J. C / i n . Znuest. 43, 2241-2248. Creemers, P., and Bentvelzen, P. (1977). Eur. J . Cancer 13, 261-267. Cunningham, A. J. (1975). Nature (London) 254, 143-144. Cunningham, V. R., and Field, E. J. (1965).Ann. N.Y. Acad. Sc i. 122, 346-355. Deckers, P. J., Pamming, K . P., and Pilch, Y. H. (1971). Cuiicer 27, 897-901. Dent, P. B. (1972). Prog. Med. Virol. 14, 1-35. Devens, B., Galili, N., Deutsch, O., Naor, D., and Klein, E. (1978a). Eur. J . zt~lr1lu?lo/. 8, s73-578. Devens, B., Deutsch, O., and Naor, D. (197%). Submitted for publication. Diener, E., and Feldmann, M. (1972). Transplant. Rev. 8, 76-103. Donner, M., Oth, D., and Burg, C . (1969). C.R. Soc. B i d . (Paris) 163, 1584-1587. Dutton, R. W. (1975). Trunsplant. Reo. 26, 39-55. Eggers, A. E., and Wunderlich, J. R. (1975).]. Zmrnzrnol. 114, 1554-1556.

SUPPRESSOR CELLS

121

Eichmann, K. (1975).Eor. J . Zmtnunol. 5, 511-517. Feldmann, M .(1973).Nature (New Biol.) 242, 82-84. Fergnson, M., and Simmons, R . (1977).Proc. Atti. A.r..r.oc.Cancer Res. 18, 200. Fernbach, B. R., Kirchner, H., Bonnard, G. D., and Herberman, R. B. (1976).Transldantatiori 21, 381-386. Ferrer, J . F. (1968a). T ~ ~ l i , s ) J / a t i f ~ l6, f i o160-166. ll Ferrer, J . F. (196%).Trarisp/antation 6, 167-172. Fidler, I. J. (1973a). Zmmuno/. Commrin. 2, 4 8 3 4 9 3 . Fidler, I. J . (1973b).J.Natl. Cancer Znst. 50, 1307-1312. Fidler, I. J., Brodey, R. S., and Bech-Nielsen, S. (1974).J.Zmtntrnol. 112, 1051-1060. Fisher, B., Fisher, E . R., and Sakai, A. (1965).Cancer Res. 25, 993-996. Floersheim, G. L. (1967).Nature (London) 216, 1235-1236. Fiijinioto, S.,and Taclii, T. (1978).Z I I “Cancer Imiiiuiiotlierapy and Its Immunoloyic~il Basis” (Y, Yam;nnnra, etl.),pp. 11-20, University Park Press, Baltimore. Fujimoto, S., Greene, M., and Sehon, A. H. (1975).Ztnrnunol. Conzmun. 4, 201-217. Fujimoto, S., Greene, M. I., and Sehon, A. H. (1976a1.J.Zmmunol. 116, 791-799. Fujiinoto, S., Greene, M. I., and Sehon, A. H. (197611).J.Ztntt~unol.116, 800-806. Fujinioto, S., Matsnzawa, T., Nakagawa, K., and Tada, T. (1978). Cell. Z t n t n ~ ~ i i o38, l. 378-387. Fnmarola, D., and Giordano, D. (1962).Tuntori 48, 5-12. Fin-th, J. (1946).J . Gerontol. 1, 46-52. Gabizon, A,, Small, M., and Trainin, N. (1976).Znt. J . Cancer. 18, 813-819. Galili, N . , Naor, D., Asjo, B., and Klein G. (1976).Eur. J . Zmnitinol. 6, 473-476. Gahli, N., Devens, B., Naor, D., Becker, S., and Klein, E. (1978).Eur. J . Ztnttizorol., 8, 17-22. Gelkind, M . C., and Paul, W. E. (1975).J.Zmntunol. 115, 1-4. Gershon, R. K. (1974).Zsr. J . Med. Sci. 10, 1012-1023. Gershon, R . K. (1975).Transp/ant.Rev. 26, 170-185. Gershon, R. K., Birnbauni-Mokyr, M., and Mitchell, M. S. (1974).Nature (London)250, 594-596. Gershon, R. K., Eardley, D. D., Naidorf, K. F., and Ptak, W. (1977).Cold Spring Harhor Sytnp. Quatit. B i d . 41, 85-91. Glaser, M., Kirchner, H., and Herberman, R. B. (1975).Znt. J . Cancer 16, 384-393. Glaser, M., Kirchner, H., Holden, H. T., and Herberman, R. B. (1976).J.Natl. Cancer Inst. 56, 865-867. Good, R. A,, Dalmasso, A. P., Martinez, C., Archer, 0.K., Pierce, J. C., and Papermaster, B. W. (1962).J.E x ) ) . h f e d . 116, 773-796. Gorczynski, R. M. (1974).]. Zmmunol. 112, 1826-1838. Gorczynski, R., Kontiainen, S., Mitchison, N. A., and Tigelaar, R. E. (1974).I n “Cellular Selection and Regulation in the Immune Response” (G. M. Edelman, ed.), pp. 143-154. Raven Press, New York. Gorczynski, R . M., Kilbnrn, D. G., Knight, R. A , , Norbury, C., Parker, D. C., and Smith, J. B. (1975).Nature (London) 254, 141-143. Greene, M . I., Fnjimoto, S., and Sehon, A. H. (1977a).J.Zmmcoiol. 119, 757-764. Greene, M . I., Dorf, M . E., Pierres, M., and Benacerraf, B. (1977b).Proc. Natl. Acad. Sci. USA 74,5118-5121. Gross, L. (1959).Proc. Soc. E s p . B i d . Med. 100, 325-328. Halliday, W. J. (1972).Cell. Zmmunol. 3, 113-122. Hashim, G . A., Sharpe, R. D., Carvalho, E. F., and Stevens, L. E. (1976).J.Zmmutiol. 116, 126-130. Hellstrom, K. E., and Hellstrom, I. (1969).Ado. Cancer Res. 12, 167-223.

122

DAVID NAOR

Hellstrom, K. E., and Hellstrom, I. (1974).Ado. Immunol. 18, 209-277. Hellstrom, K. E., Hellstrom, I., and Sjogren, H. 0. (1970).Cell. Immutiol. 1, 18-30. Heppner, G. H. (1967).Proc. Am. Assoc. Cuncer Res. 8, 27. Heppner, G. H., Wood, P. C., and Weiss, D. W. (1968a).Zsr. J. Med. Sci. 4, 1195-1203. Heppner, G. H., Wood, P. C., and Weiss, D. W. (1968b).Isr. J. Med. Sci. 4, 1204-1209. Herberman, R. B. (1974).Ado. Cancer Res. 19,207-263. Herberman, R. B., Nunn, M. E., Holden, H. T., Staal, S., and Djeu, J. Y. (1977). Znt. J. Cancer. 19, 555-564. Herzenberg, L. A,, Herzenberg, L. A., Black, S. J. Loken, M. R., Okumura, K., van der Loo, W., Osborne, B. A,, Hewgill, D., Coding, J. W., Gutman, G., and Warner, N. L. (1977).Cold Spring Harbor S!ymp. Quunt. B i d . 41, 33-45. Hewitt, H. B. (1953).Br. J . Cancer 7, 367-383. Hewitt, H. B., Blake, E. R., and Walder, A. S. (1976).B r . J. Cancer 33, 241-259. Hillinger, S. M., and Herzig, G. P. (1977).Proc. Am. Assoc. Cuncer Res. 18, 152. Holden, H. T., Haskill, J. S., Kirchner, H., and Herberman, R. B. (1976).]. Immunol. 117,440-446. Humphreys, S. R., Glynn, J. P., Chirigos, M. A., and Goldin, A. (1962).J.Natl. Cuncer Irlst. 28, 1053-1063. Ilfeld, D., Carnaud, C., Cohen, I. R., and Trainin, N. (1973).2nt.J. Cancer 12,213-222. Isa, A. M., and Sanders, B. (1976).I n “Neoplasm Immunity: Mechanisms. Proceedings of a Chicago Symposium” (R. G. Crispen, ed.), pp. 39-44. ITR, Chicago. Janeway, C. A., Jr. (1975).J.Zmmutaol. 114, 1408-1414. Jeejeebhoy, H. F. (1974).Znt. J. Cancer 13, 665-678. Kall, M. A., and Hellstriim, I. (1975).J.Immunol. 114, 1083-1088. Kall, M. A., Hellstrom, I., and Hellstrom, K. E. (1975).Proc. Natl. Acud. Sci. USA 72, 5086-5089. Kamo, I., and Friedman, H. (1977).Adoan.Cancer Res. 25, 271321. Kaplan, H. S. (1950).]. N u t l . Cancer Inst. 11,83-90. Katz, D. H. (1977).In “Lymphocyte Differentiation, Recognition, and Regulation,” pp. 470-481. Academic Press, New York. Katz, D. H., Ellman, L., Paul W. E., Green, I., and Benacerraf, B. (1972).Cancer Res. 32, 133-140. Kedar, E., Schwartzbach, M., Unger, E., and Lupu, T. (1978). Traris),/atitcitiori, 26, 63-65. Khaitov, R. M., Petrov, R . V., Gambarov, S. S., Norimov, A. S., and Blinov, V. A. (1976). Cell. Immunol. 22, 1-10, Kies, M . W. (1965).Ann. N.Y. Acad. Sci. 122, 161-170. Kilburn, D. G., Smith, J. B., and Gorczynski, R. M. (1974).Eur. J . Immunol. 4,784-788. Kirchner, H., Chused, T. M., Herberman, R. B., Holden, H. T., and Lavrin, D. H. (1974a).J.E x p . Med. 139, 1473-1487. Kirchner, H., Herberman, R . B., Glaser, M., and Lavrin, D. H. (1974b).Cell. Immunol. 13, 32-40. Kirchner, H., Muchmore, A. V., Chused, T. M., Holden, H. T., and Herberman, R. B. (1975a).J.Immunol. 114, 206-210. Kirchner, H., Holden, H. T., and Herberman, R. B. (1975b).]. N a t l . Cancer Itast. 55, 971-975. Kirchner, H., Glaser, M., and Herberman, R. B. ( 1 9 7 5 ~Nature ). (London)257,396398. Kirchner, H., Fembach, B. R., and Herberman, R. B. (1976). I n “Mitogens in Immunobiology” (J. J. Oppenheim and D. L. Rosenstreich, eds.), pp. 495-507. Academic Press, New York. Kirschbaum, A,, Shapiro, J. R., and Mixer, H. W. (1953). Cancer Res. 13, 262-268.

SUPPRESSOR CELLS

123

Klassen, L. W., Krakauer, R. S., and Steinberg, A. D. (1977).J. Z t t t t r i u t i d . 119, 830-837. Klein, G. (1966). Zsr. ./. ,!led. Sci. 2, 135-142. Klein, G., and Klein, E. (1977). Proc. Ntrtl. Acmd. Sci. USA 74, 2121-2125. Kolb, W. P., and Granger, C . A. (1970). Cell. I m t n i r t i o l . 1, 122-132. Kolb, J-P., Arrian, S., antl Zolla-Pazner, S. (1977).J. Znitnirriol. 118, 702-709. Kolscli, E., Mengersen, R., and Diller, E. (1973).Eitr. J . Concer 9, 879-882. Kiilsch, E., Stumpf, R., and Weber, G. (1975). Trotisplatit. Reo. 26, 56-86. Krakauer, R. S., SiToher, W., and Waldmann, T. A. (1976). Cliti. Res. 24, 377A. Krawinkel, U., Cranier, M., Berek, C., Hamnierling, G., Black, S. J., Rajewsky, K., and Eichniann, K. (1977). Co/d-Spriitg lfurbor S y t i t ) ~ Q. i r c i r i t . B i d . 41, 285-294. Kripke, ill. L. (1979). Z i t “Cmcer Biology Reviews” (J. J. Marchalomis et u l . , ed Rlarcel Dekker, New York, in press. Kumar, V., and Bennett, M. (1976).J . Ewp. Med. 143, 713-727. Kiimar, V., and Bennett, M . (1977). Nofirre (Lotidon)265, 345-347. Kuinar, V., Bennett, M.,and Eckner, R. J. (1974).J. E q ] . .\let/. 139, 1093-1109. Kumar, V., Carriso, T., and Bennett, M . (1976).J. E.v/I.Mecl. 143, 728-740. Knperman, O., Fortner, G. W., and Liicas, Z. J. (1975).J. Z t n m r c i i o l . 115, 1282-1287. Lappk, M. A,, and Blair, P. B. (1970). Proc. An,. As.~oc.Cancer Res. 11, 47. Law, L. W. (1966). Ctrticer Res. 26, 1121-1132. Law, L. W. (1969). Cnticer Res. 29, 1-21. Law, L. W., and Miller, J. H. (1950a).J. N o t / . Coitcer Z m t . 11, 253-262. Law, L. W., antl hliller, J . H. (1950li).J. Ntrtl. Coticer I t i . s t . 11, 4 2 5 3 3 2 . Le Fraiqois, D., Youn, J. K., Belehradek, J., Jr., and Barski, G. (1971).J. N n t l . Cntic,er I t i s t . 46, 981-987. Levinthal, J . D., Butfett, R. F., and Fnrth, J . (1959). Proc. Soc. E x p . B i d . Med. 100, 6 10-6 14. Levy, R. B., Waksal, S. D., and Shearer, G. M.(1976).J. E x ) ) . )\fed. 141, 1363-1368. hlcEndy, D. P., Boon, M. C., and Fnrth, J. (1944). Catrcer Res. 4, 377-383. hlacKenzie, M. R., and Paglieroni, T. (1976). Cliti. Re.u. 24, 148A. hlarchant, J . (1969). B r . ,/. Ctrticer 23, 383-390. Martinez, C. (1964).Noticre (Lotidoti) 203, 1188. Medina, D., and Heppner, G. (1973).Nottrre (Lottdoii ) 242, 329-330. Mengersen, R., Schick, R., antl Kiilscli, E . (1975). E i r r . J . Z t ) i t n t i n o / . 5, 532-537. Metcalt, D. (1966).Recent Resirlts Cuticer Re.v. 5, 92-100. Mikiilska, Z. B., Smith, C., and Alexander, P. (1966).J. Nntl. Caticer Z t i s t . 36, 29-35. Miller, J . F. A. P. (1959a).Nntirre (Lotidoii) 183, 1069. Miller, J. F. A. P. (19591)).Nature (Lottdoii) 184, 1809-1810. Miller, J . F. .4. P. (1960). Hr. J . Cnticer 14, 93-98. . Res. 6, 291-368. Miller, J . F. A. P. (1961).A d t ~ i t i Caiicer Miller, J. F. A. P. (1962). PI-oc.R . Soc. Loridoti B 156, 4 1 5 3 2 8 . Miiller, E. (1965).J. N n t l . Cnticer Z m s t . 35, 1053-1059. Murgita, R. A., and Toniasi, T. B., Jr. (1975).J . E x p . Med. 141, 269-286. Naor, D., and Galili, N. (1977). Prog. Allergy 22, 107-146. Nelken, D. (1973).J. Itntiii~tiol.110, 1161-1162. Nelson, K., Pollack, S. B., and Hellstriim, K. E. (1975a). Z t i f . J . Cniicet- 15, 806-814. Nelson, K., Pollack, S. B., and Hellstriim, K. E. (19751i). Z t i f . J . Cniicer 16, 539-549. Nossal, G. J . V., Pike, B. L., Stocker, J. W., Layton, J. E., and Coding, J. W. (1977).Cold Spritig Horbor S!/titp. Qriutit. Biol. 41, 237-243. Oehler, J . R., Cainpl)ell, 1).A,, Jr., and Herberman, R. B. (1977). Cell. ~ t J l t I l l l t i O / . 28, 355-370. Okiunrira, K., antl Tach, T. (1971).J . Z t t t t t i i c t i o / . 106, 1019-1025.

124

DAVID NAOR

Okumura, K., Tada, T., and Ocliiai, T. (1974). Zrnmtitiology 26, 257-268. Old, L. J., Clarke, D. A., Benacerraf, B., and Stockert, E. (19624. Experientiu 18, 335336. Old, L. J., Boyse, E. A , , Clarke, D. A., and Carswell, E. A. (1962b).Ann. N.1.’.Acud. Sci. 101, 80-106. Oldstone, M. B. A., Tishon, A,, and Moretta, L. (1977). Nature (London)269, 333-335. Opitz, H-G., Niethammer, D., Jackson, R. C., Lemke, H., Huget, R., and Flad, H-D. (1975). Cell. Znininnol. 18, 70-75. Pierres, M., Germain, R. N., Dorf, M. E., and Benacerraf, B. (1977). Proc. “if/. Acnd. Sci. USA 74, 3975-3979. Pope, B. L., Wliitney, R. B., Levy, J. G., and Kilbnrn, D. G. (1976).J. Zrnmunol. 116, 1342-1346. Potter, C. W., Hoskins, J. M., and Oxford, J. S. (1969).Arch. Gescint. Virii.sforschritig 27, 73-86. Portpon, M. F., Kolb, J-P., and Lespinats, G. (1976).J. N o t / . Coticer Ztist. 57, 1241-1247. Prager, M. D., and Baechtel, F. S. (1973). A4ethod.s Concer Res. 9, 339-400. Prehn, R. T. (1969).]. N u t / . Coticer Ztist. 43, 1215-1220. Prelin, €3. T. (1972). Science 176, 170-171. Prehn, R. T. (1976). Trrrrts)ilrint. Rec. 28, 3 4 4 2 . Prehn, R. T., and Lappi., M. A. (1971). Trotisplotit. Reti. 7, 26-54. Quan, P. C., and Burtin, P. (1978). Concer Res. 38, 288-296. Reinisch, C. L., Andrew, S. L., and Schlossman, S. F. (1977). Proc. N a t l . Accid. Sci. USA 74, 2989-2992. Ryan, W. L. (1974).J. N a t l . Coticer Inst. 52, 1673-1677. RGllinghotf, M., Starzinski-Powitz, A,, Pfizenmaier, K., and Wagner, H. (1977).J . E.sp. Jfed. 145, 455459. Rotter, V., and Trainin, N. (1975). Trciris/il~tntcititt~ 20, 68-74. Rudczynski, A. B., and Mortensen, R. F. (1978).J. N a t l . Crtncer Z t i s t . 60, 205-211. Sabbadini, E. (1974).]. E x p . Med. 140, 470480. Sakakura, T., and Nishizuka, Y. (1967). Gatin 58, 4414 5 0 . Savary, C. A., and Lotzovi, E. (1978).J. Z m t n u t i o l . 120, 239-243. Scliaaf-Lafontaine, N. (1978). Znt. J . Cancer 21, 329-333. Scliecliter, B., and Feldman, M. (1977).J . Ztritnuttol. 119, 1563-1568. Schechter, B., Segal, S., and Feldman, M. (1977). Znt. J . Cancer 20, 239-246. Schechter, B., Segal, S., and Feldman, M. (1978).J . Znirriunol., 120, 1268-1273. Scott, M. T. (1974).J. N n t l . Crrttcer Z t t s t . 53, 855-860. Shearer, G. M., Melmon, K. L., Weinstein, Y., and Sela, M. (1972).J. Ex),.Med. 136, 1302-1307. Simpson, E., and Cantor, H. (1975). Ettr. J . ltnniiitiol. 5, 337-343. Small, M. (1977).J. Z t r i m i i t i d . 118, 1517-1523. Small, M., and Trainin, N. (1975). Znt. .I. CNricer 15, 962-972. Small, M., and Trainin, N. (1976).J. Initntitiol. 117, 292-297. SpHrck, J. V. (1969). Actcr Pothol. Microbiol. Sccrttd. 77, 1-23. Spellman, C. W., and Daynes, R. A. (1977). Trcit~s~ilontcitioii 24, 120-126. Squartini, F. (1971). I s r . J . A4ed. Sci. 7, 26-35. Squartini, F., and Bolis, G. B. (1970). Texas R e p . B i d . Med. 28, 115-121. Steinberg, A. D., Gerber, N., Morton, R., Gershwin, M., Goodman, D., Chused, T. M., Hardin, J. A., and Barthold, D. R. (1975). Zti “Suppressor Cells in Immunity” (S. Singlial ;ind N. St. C. Sinclair, eds.), pp. 174-182. University of Western Ontario, London, Canada.

SUPPRESSOR CELLS

125

Stelzer, G. T., and Wallace, J. H. (1977). Proc. Am . Assoc. Cancer Res. 18, 68. Stiller, R. A,, a n d Cerny, J. (1976).J.I i n n i i r r i o l . 117, 889-899. Stutnian, 0. (1975).Adc. Cancer Res. 22, 261-422. Swanborg, R . H. (1975).J. I t n i n t r t ~ o / .114, 191-194. i o l ,631-633. Swierkosz, J . E., and Swanborg, R. H. (1975).J.Z ~ n t n ~ ~ ~115, Tada, T., Taniguchi, M., a n d David, C. S. (1977). Cold S p r i n g Harbor S!/nip, Q t i c i i i t . Biol. 41, 119-127. Takei, F., Levy, J . G., and Kilburn, D. G. (1976).]. I r n n l n i i o / . 116, 288-293. Takei, F., Levy, J. G., and Kilburn, D. G. (1977).J. Irntntrnol. 118, 412417. Tanapatcliaiyapong, P., and Zolla, S. (1974). Scieuce 186, 748-750. Thorley-Lawson, D. A,, Chess, L., and Strominger, J. L. (1977).J. Ex/,.Med. 146, 495508. Treves, A. J., Carnaud, C., Trainin, N., Feldnian, M., and Cohen, I. R. (1974). Eirr. J . I n i m t c t i o l . 4, 722-727. Treves, A. J., Cohen, I. R., and Feldman, M. (1976a). lsr. J . Aled. Sci. 12, 369-383. Treves, A. J., Cohen, I. R., and Feldnian, M. (1976b).J. N a t l . Cnticer I r i s t . 57, 409-414. Turkiii, D., and Sercarz, E. E. (1977).Proc. N o t l . Accid. Sci. USA 74, 3984-3987. Twomey, J. J., Laughter, A. H., Farrow, S., and Douglass, C . C . (1975).J. Cliri. Iituest. 56,467475. Uhr, J . W., and hliiller, G. (1968).Atlc. I i n n ~ t r t i o l .8, 81-127. Uniiel, T., and Trainin, N. (1974).T r c i t t u ) ) l a i i t c i t i o i i 18, 244-250. van Bekkum, D. W. (1972). Beliriiig. Iiist. Jlitt. 51, 199-201. van Bekkrim, D. W., and Balner, H. (1973).I n “Unifying Concepts of Leukemia,” Bihl. Haeiticctol. ( R . M. D u t c h and L. Chieco-Biaiichi, eds.), Vol. 39, pp. 677-688. Karger, Basel. 117, 646-654. Veit, B. C., and Feldman, J. D. (1976a).J. Irninir~~ol. Veit, B. C., and Feldman, J. D. (19761)).J. Immutwl. 117, 655-660. t o l .161-176. Waksman, B. H., antl Namba, Y. (1976). Cell. Z t n ~ ~ i ~ r ~21, . 155-199. Waldmann, T. A , , and Broder, S. (1977).Prog:. Clin, I r n m ~ c r i o l 3, Wallen, W. C., Rabin, H., Neubauer, .R. H., and Cicmanec, J. L. (1975a). J. N a t l . Cuncer I ii . s t . 54, 679-685. Wallen, W. C . , Neiibauer, R. H., a n d Ruhin, H. (19751)).Clin. E x ) , . I m n l c r n o l . 22, 468472. Weilaiid, E., antl Mussgay, M. (1977).E u r . J . Cnt1cc.r 13, 705-711. Yashphe, D. J . (1971).I s r . J . ,\led. Sci. 7, 90-107. Yoshida, T. O., a ~ i dSOt1thaI11, C. M. (1963).J f i ] ) ( i t t J . E.X)J. JIed. 33, 369-383. Yunis, E. J., Martinez, C., Smith, J., Stutinan, O., and Good, R. A. (1969).Cuiicer Res. 29, 174-178. Zembala, &I., Mytar. B., Popiela, T., and Asherson, G. L. (1977). Znt. J . C o m e r 19, 605-6 13. Zolla, S., Naor, D., and Tanapatcliaiyapong, P. (1974).J . l ~ n t ~ t ~ r ~112, i o l .2068-2076. Zolla-Pazner, S., Siillivan, B., and Richardson, D. (1976).J. I ~ J ~ ~ I I I 117, ~ I O 563-568. /.

This Page Intentionally Left Blank

RETRODIFFERENTIATION AND THE FETAL PATTERNS OF GENE EXPRESSION IN CANCER

Jose Uriel lnstitut de Recherches Scientifiques sur le Cancer, B. P. N" 8. 94800 Villejuif, France

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Fetal Patterns in Malignant Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Tumor-Associated Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Enzyme Activities and Metabolic Changes ........................... C . Isozyiiies.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Morphologic and Structure-Depentlent Properties .................... 111. Fetal Patterns in Noiicanceroiis Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Fetal Antigens in Regeneration, Cell Iiijiiry, and Tissue Repair . . . . . . . . B. Fetal Isozymic Transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Morphologic Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Plasticity of the Differentiated State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Differentiation versus Retrodifferentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Diflerentiation of Malignant Cells . . . . . . . . . . ................... C . Retrodifferentiation in Cell Iiijury and Tissue r .................. D. Retrodiffereiitiation in Normal and Transformed Cells i r i Vitro . . . . . . . . . V. Retrodifferentiation and Cancer ... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

127 130 130 138 14 1 146 147 147 150 151 152 152 155 158 162 166 169

I. Introduction

At present no unitary concept can give a satisfactory explanation of the intimate nature of cancer. Moreover, proposals that cancer results from structural changes in genetic material, loss of growth control, abnormalities i n cell differentiation, misprogramming of normal gene products, etc. are all valid and probably not mutually exclusive inferences from data obtained at different levels of observation. Unfortunately, from a utilitarian viewpoint they do not enlighten u s much more about the emergence and development of malignant neoplasms than does the simplest definition of the disease, ix., a tumor that grows, invades, and finally kills the host. The pursuit of a careful exploration of the biological properties of malignant cells and tissues in relation with their normal resting and growing counterparts probably remains the most rational approacli to 127

128

JOSE URIEL

study the intricate problem of cancer. In this regard the resemblance between neoplastic and embryonic growth has attracted the attention of biologists for more than a century; and more particularly during the last 15 years this resemblance has become highly significant since it stems from the convergence of molecular, biochemical, morphologic, and behavioral properties common to many cancerous and embryofetal cells. Embryos and neoplasms first appear as cellular populations with cei-tain autonomy and increased growth rate compared to the cellular dynamics and growth control of the host. Both ontogenic and neoplastic development progress in a stepwise fashion, probably by alternative periods of cell differentiation and division. A characteristic property of malignant cells, but also of numerous embryo-fetal elements, is their ability to migrate which permits them to invade and colonize tissues and organs at considerable distance from their place of origin. One essential feature of tumor development is angiogenesis, the capacity of growing cell populations to elicit neovascularization in host vessels; the same capacity is shared by the trophoblast and by some embryonic tissues. Resistance to aggressive immune responses of the host is a common property of malignant and embryo-fetal tissues. At the structural and ultrastructural level, most neoplastic cells, in uiuo and i n oitro, can be distinguished from their normal counterparts by a variable degree of morphological simplification and a lower internal organization; this is equally characteristic of immature cell populations. Analogies also exist in the metabolic patterns. The gradual predominance of catabolic pathways over anabolic ones in carbohydrate metabolism that parallels the increased growth rate of many tumors correlates well with the high glycolytic activity and lactate production rate associated with embryonic tissues. About 30 years ago, Greenstein (1945) concluded from an analysis of the enzymatic activities of tumoral and nontumoral tissues that “tumors tend to converge to common enzymatic patterns” and that in certain cases these patterns resemble those of fetal tissues. More recently, the convergence at the molecular level of ontogenesis and neoplasia was evidenced by the discovery of embryofetal antigens (Abelev et d., 1963) and fetal-type isoenzymes (Schapira et al., 1963) in adult individuals bearing hepatomas, opening a new and enriching field in cancer research. Work in this area constitutes at present a large body of information that has already been reviewed on several occasions (Criss, 1971; Alexander, 1972; Weinhouse, 1973; Schapira, 1973; Coggin and Anderson 1974; Uriel, 1975; Knox 1976).

HETRODIFFEHE~TIATIORTA N D G E N E EXPHESSION IN CANCER

129

There is an increased awareness that phasing in gene expression and the concomitant changes that it entails in cell coniposition, function, structure, and organization are central not only to the problem of developmental and reparative growth 1)ut to that of neoplasia. Whether cancerous tumors develop preferentially from tlie arrested or the abnormal differentiation of stem cells (Pierce, 1967, 1970; Markert, 1968; Potter 1969) or from mature elements undergoing a process ofretrodifferentiation toward more juvenile phenotypes (Uriel, 1969, 1975) remains to be elucidated. The alternative is not mutually exclusive but tlie elucidation of tlie differentiated stages of target cells from which neoplastic growth emerges has obvious physiopathologicai and therape 11tic i inpl icat ion s . As it appears from a recent analysis (Uriel, 1976) retrodifferentiation is a physiological and a common adaptive process for the maintenance of cell integrity against deleterious agents of varied etiology (physical, chemical, viral). While preserving the entire information encoded in its genome, cells undergoing retrodifferentiation lose morphologic and functional complexity by virtue ofa stepwise process of self-deletion of cytoplasmic structures and the transition to a more juvenile pattern of gene expression. This results in a progressive uniformity of originally distinct well-differentiated cells. Retrodifferentiation is in physiological conditions counterbalanced by a process of redifferentiation, which tends to restore the terminal phenotypes from which the reversion started. This explains why retrodifferentiation remains invariably associated with cell regeneration and tissue repair. There is ever growing evidence that neoplastic transformation in civo and i l l Ljitro is frequently preceded and accompanied by biological transitions characteristic of cells undergoing retrodifferentiation: nevertheless contrary to what occurs in regenerating tissues, the unbalanced character of tumor-associated retrodifferentiation seems to be a property often linked to malignancy. The aim of the present chapter is to review the most representative data accumulated in recent years which strongly suggest that: (a) the resemblance between many cancerous and embryo-natd cells is highly significant since it stems from the convergence of common antigenic, biochemical, inorphologic, and behavioral properties; (11) this resemblance is, nevertheless, not intrinsically specific to neoplasia since it can also be shown by some noncancerous cells and tissues growing under the influence of certain deleterious agents or in response to aggressions of varied etiology; and (c) retrodifferentiation seems to be a unique mechanism of cell rejuvenation underlying both neoplastic development and regenerative processes.

130

JOSE URIEL

It. Fetal Patterns in Malignant Tumors

A. TUMOR-ASSOCIATED ANTIGENS Soluble and structural malignant cell constituents are generally considered as tumor-associated antigens when demonstrated in tumors by virtue of their immunologic properties. They can elicit cell or humoral-mediated immune responses in uivo or in uitro in syngeneic or autologous recipients or they react with antisera evolved in heterologous hosts and made tumor specific by appropriate immunoadsorption procedures using normal tissue counterparts. Thanks to this immunologic approach many cellular constituents of unknown physicochemical, biochemical, and biological properties have been accurately associated with malignant neoplasms. Only a very few of them have been later isolated and fully characterized. For this reason, tumor-associated antigens are often designated and defined in operational terms, i.e., tumor-associated transplantation antigens (TATA) or from topographic data, i.e., tumor-associated surface antigens (TASA). In other cases the designation refers to etiologic factors such as virus-induced or chemically induced tumor antigens, or rises from conceptual considerations, i.e., oncofetal antigens, cell differentiation tumor antigens. This type of terminology is advantageous for descriptive purposes. In certain cases, however, it can be a source of some confusion or at least of ambiguity: the same antigen detected by different unrelated methods (tumor rejection tests, cytotoxicity, immunodiffusion, etc.) can be classified under different columns and, conversely, different antigens may indiscriminately be revealed by a single procedure. There is increasing evidence that the reappearance of embryo-fetal constituents observed in many malignant tumors is closely related not only to cell differentiation, a process which is operative during intrauterine ontogenic development, but also in renewing tissues of the adult (e.g., bone marrow, gonads, endodermal epithelium) and in all kinds of tissues undergoing reparative growth. The following is a tentative classification to fit the most representative types of tumor antigens specified by the cellular genome in line with the phase-specific events accompanying developmental growth. Antigens specified by the cell genome following structural alterations (mutations) or by genetic material extrinsic to the cell (oncogenic virus) form a different class of potential tumor-associated constituents and will not be considered here. Virus-specified antigens in cancer have been, on the other

RETRODIFFERENTIATIONAND GENE EXPRESSION I N CANCER

131

hand, repeatedly reviewed elsewhere (Bailer, 1974; Lanion, 1974; Herberman, 1977). Among the potential antigens shared l ~ tissues y undergoing developmental and neoplastic growth the following can be distinguished.

1. Antigen(.s)Espres.sed iile except in the pancreas and gastrointestinal tract where the values found ranged from 65 to 200 ng/gm of wet tissue in fetuses and from 28 to 86 ng in adults. (I)) Much higher than average amounts were found in a variety of malignant tumors, including carcinomas, sarcomas, and their metastases, and Hodgkin’s disease, with the highest contents in rectocolonic neoplasms (up to

136

JOSE URIEL

almost 3000 ng/gm) and lung carcinomas (up to 500 ng/gni). (c) Moderate or slightly raised levels were observed in several pathological albeit nonmalignant tissues which coiifiiiiis previous work (Bui-tin ct d . , 1972; Mai-tin and Martin, 1972; Kupchik aiid Zainchek, 1972). A current interpretation of these findings is not feasible since the structure and antigenic profile of CEA and related substances, their synthetic and catabolic pathways, their distribution among different cell populations, and also their mechanisms of release from cell membranes are still not completely elucidated. They may be an array of niacromolecules whose polypeptide and/or carbohydrate moieties differ in structure and composition but which share a coninion core of antigenic detenninants (von Kleist et (/I., 1972; Mach aiid Pusztaszeri, 1972; Khoo et d . , 1973; Rogers, 1976; and Chisin et a l . , 1977).During neoplastic development a shift toward the expression of those aiitigenic variants of fetal predominance occurs and consequently a rearrangement in the inorphologic aiid secretory patterns of these substances .

5. A n t i g e n ( s )Espres,sed brl All the Elements of a Cell Lineuge during Ontogenic Dez;elopment b u t Od!y by the Immature Cells of the Liiie in the Adult Orgunism These may reappear in tumors emerging in renewing tissues (bone marrow, endotheiinal epithelium, etc.) of the adult. Teplitz et (11. (1974) denionstrated a cell surface antigen of fetal origin in the peripheral red blood cells (RBCs) of chickens with leukemia induced by avian myeloblastosis. The antigen was absent from mature RBCs of healthy adult animals. It was however, present in erytliroid stem cells and preerythrocytes of adult bone marrow and in all the elements of the cell line at the time of hatching (Sanders and Wiley, 1971). This class of developmental antigens characteristic of renewing tissues of the adult and of their malignant derivatives inay easily escape detection by conventional immunologic methods. There is a great probability that adsorption of antituinor antisera with iiornial tissue, where niature and immature elements coexist, removes such antigens . 6. Antigen (.I ) Expressed as Norinul Product ( s ) of Certain Muture Dijjferentiated Cells i n (in Adult Orgunism und Reappearing i n Tumors Emerging f r o m Eleriieiits which Never Espressed T h e m Either duriiig Ontogenic Developnient or in the Adult State Many malignant as well as benign tumors derived from cell lineages other than those of endocrine tissues may secrete peptidic hormones

RETRODIFFERENTIATION AND GENE EXPRESSION I N CANCER

137

indistinguishable from the homologous products of normal endocrine glands: among the honnones reported to be produced by nonendocrine neoplasms are ACTH, vasopressin, melanocyte-stimiilatiii~hormone, parathormone, erythropoietin, etc. (reviewed by Ode11 and Wolfsen, 1975). The possibility to be discussed later (Section IV,A) that the so-called ectopic production of hormones in cancer might be due to cell transdifferentiation justifies the inclusion of these antigenic substances in the present inventory of developmental antigens.

7. Coiicl usions Since the beginning of this century, when the rejection of tumor transplants by mice immunized with fetal tissues was reported for the first time (Schiine, 1908) inany fetal antigens present in malignant diseases of varied origin have been demonstrated. Their ever-growing number in cancerous tissues has evoked the possibility that all tumorassociated antigens are in fact of embryo-fetal nature. If a positive answer can still not be given to such a question, it is already possible to conclude that embryo-fetal antigens are probably expressed in all malignant neoplasms and that together with the development of antigens outlined above, they account for a great majority of tumor-associated antigens specified by the cell genome. Besides the common property of being transiently expressed soinetime during the intrauterine developmental history of cells and tissues it is worth noting that they span a large spectrum of cell constituents of very diverse properties. Thus certain types of these “oncofetal” antigens appear at the very early cleavage stage of embryogenesis (i.e., F9 mouse antigen). Others such as those expressed in chemically and virus-induced tumors of laboratory rodents are maximally synthesized around the middle of gestation, while antigens such as AFP are produced during relatively long periods covering intrauterine and early postnatal life. Another group is associated with physiological renewal of adult cells and tissues (e.g., endodermal epithelium, red and white blood cells). Many of these transitory cell antigens belong, perhaps due to facilities of detection, to the cell-surface type, although others are localized in the cytoplasm and some are transported out of the cell into the blood stream. They also show a great variability in immunologic properties. The degree of cross-reactivity stretches from a narrow organ or tissue specificity to a broad interspecies antigenic relationship. With rare exceptions (CEA, AFP, isoferritins) most of the oncofetal antigens are immunogenic in syngeneic or autochtonous hosts, although the nature of cell and humoral immune response which they

138

JOSE URIEL

can elicit in zjivo and in oitro may be different. Thus, while virusinduced fetal antigens function effectively as tumor rejection agents, those reappearing in chemically induced or spontaiieously arising tumors generally lack this property. It is unclear whether the competence or the incompetence to display such or such type of immune response is a characteristic inherent to a given antigen or whether it results from its association with cellular structures or its accessibility to effector cells and substances. In fact, many points remain obscure on the immunologic aspect of developmental antigens. Their reappearance in malignant tumors and in cultured neoplastic cells offers an excellent opportunity of using these experimental models to improve our knowledge on the intricate problem of the immunology of cell differentiation.

B. ENZYMEACTIVITIES AND METABOLICCHANGES The classical work of Warburg on the glycolysis and respiration of human and experimental tumors provided deep insight into the field of cancer biochemistry. Warburg observed that most tumors show low respiration rates and, when metabolizing glucose under aerobic or anaerobic conditions, produce lactate at a higher rate than does normal tissue. He thus concluded that damage to the respiratory process of growing cells and the adoption of a fermentative pathway to derive energy necessary for survival were the cause and the mechanism of cancer development. The early experiments of Warburg and his contemporaries also showed that, in addition to a few normal resting and renewing tissues (brain, retina, bone marrow, etc.), whole embryos and fetal lung and liver display high rates of glycolysis. One can easily understand, however, that the enthusiastic search for what was then considered the biochemical clue to neoplastic transformation did not prompt biochemists to carry out a systematic and more careful exploration of glycolysis in tissues during ontogenic development. Only much later did Villee (1954)demonstrate a significantly high aerobic glycolysis in all the human embryonic tissues of the gestational period studied. The relationship between cancer and immature cells was reinvestigated on the basis of individual enzymatic activities by Greenstein. Analyzing data from his own work and that of others, Greenstein (1945) made and formulated a series of observations which can be summarized as follows: enzymatic activities of tumors of different origin tend to converge to a common pattern because the specific functional enzymes of the corresponding normal tissues are deleted during

R E T H O D I F F E R E N T I A T I O ~AND G E N E EXPRESSION I N CANCEI-I

139

neoplastic progre on. In the particular case of hepatomas, Greenstein noted the homologv of their enzymatic pattern with that of fetal liver and conclitded that “neoplasms may revert to a more primitive a n t l less differentiated metabolism.” This was probably the first time that the old concept of embryonic reversion of malignant tumors was postulated from experimental findings. Most ofthe subsequent work on individual enzyme activity has been done with rat hepatoinas and with adult, immature, and regenerating liver a s normal contrds of resting and growing tissues. This choice was i-ctt determined by the arxi1al)il it!. o f : t 1:u.g~s p ~ c t r r i i i ~o it ti-:tiisplantal,lt-it~il~I~~ hepatonxis of Val-icld gro\vtli r a t e , niol-pliologic dit~~~reiitiatioii, cuncl karyotypic composition (Slorl-is, 1965). ‘ I ’ l i i t ~ L\’r~lwr and hi4 colleagues, using a series o t ’ t r ~ ~ i i s ~ ~ l ~ ilwpitonias, t i t ~ t l ~ l c Iiavt, c-splorctl tlw correlation between tumor gi-o\\.tlr r:itc, itirtl the nietalmlic i i n l ~ a 1 : i i i c ~ ~ resulting from changes in the enzymatic pattern. Among the numerous enzymes of carliohydrate, lipid, protc:in, : t i i d niicleic. ;ic.itl Ii-c~tal)olism whose activities were evaluated, thosc slio\\ring ;I coi ix~li~tion with tlic growth rate were selected and integrated in separate but ordered patterns of metabolic behavior. The integrated data led t o the conc111sio~~ (Weber, 1966) that the increasing growth rate of hepatomas parallels a progressive loss of metabolic equilibrium of the cancer cell evidenced by the gradual predominance of catabolic over anabolic pathways in carbohydrate metabolism (glycolytic activity rises whereas gluconeogenesis decreases), and a reverse behavior of protein and nucleic acid metabolism, where synthetic routes become preponderant. Table I is a compilation of some enzyme activities, most, of them involved i n carbohydrate and nucleic acid metabolism, which were selected from studies including comparative data on adult, immature, regenerating, and neoplastic rat liver. The table illustrates that analogous changes in k e y metabolic enzymes may result in developmental and neoplastic rat liver. One demonstrative case is the activity of ribonucleotide reductase. This is an enzyme which plays a critical role in the biosynthetic processes of nucleic acids and which increases greatly in rat hepatomas, fetal and regenerating liver, a s well as in livers from newborn aiiiinals up to 7 days of age (Elford, 1972, 1974; Larsoon, 1969).Another example is provided by liver xanthine oxidase, the rate-limiting enzyme of purine catabolism. The activity of this enzyme was found to have decreased to 50 to 60% of that of adult rat liver in all 17 transplantable hepatomas and in developing liver during the first 3 weeks after birth (Pradja et UZ., 1976). Moreover, in a modern revival of Greenstein’s approach to the relationships between fetal and neoplastic tissues, Knox (1976) has compiled comparative

TABLE I COMPARATIVE DATAON ENZYME ACTIVITIES I N

Enzyme Glucokinase

Hexokinase

Pyruvate kinase

Thymidine kinase

Deoxycytidylate deaminase Thymidylate s ynthetase Ribonucleotide red u cta se Xanthine oxidase a-Glutam yl transpeptidase Arginase

Immature liver Fetal (17 to 21 day) N.M. Fetal (17 to 21 day) 3 Fetal (19 to 21 day)

0.4 Fetal (21 day) 23 Fetal (18 day) 40 Fetal (18 day) N.M. Newborns (7 day) 5.000 New borns (1to 6 day) 0.05-0.1 Fetal (14 to 21 day) 2730 Fetal (14 to 21 day) Traces 0.1

Regenerating live?'

(24 hours) 0.3 (24 hours) 1.5 (24 to 72 hours) 0.5 (23 hours) 25 (48 hours) 17 (48 hours) 1.000 (24 hours) 18.000 (12-96 hours) 1 (14-24 hours) 15-20

Not done

RAT

LIVER"

Hepatomas' (9618A):0.3 (7800):N .M . (5123C):N. M. (9618A):1.8 (7800):2.2 (5123C):1.2 (9611B):0.4 (5123A):0.2 (5123): 24 (5123B) 6 (5123) 960 (5123C) 18.000

References Walker and Potter (1972) Walker and Potter (1972) Farina et al. (1974) Klemperer and Haynes (1968) Bresnick et d.(1964) Maley and Maley (1961) Maley and Maley (1961) Elford et al. (1972) Pradja et al. (1976)

0.05 to 0.6 Primary 25-36 (5123) Traces

Activities in arbitrary units. Normal adult liver taken as 1. Hepatocyte proliferation after partial hepatectomy. ' Number in brackets: Morris hepatomas of medium growth rate. N.M., not measurable.

Cameron et al. (1978 Malkin et al. (1978)

RETRODIFFERENTIATION

A N D GENE EXPRESSION IN CANCER

141

values of 66 individual enzymes in adult and fetal rat liver and in 11 transplantable Morris hepatomas. Fetal liver and growing hepatomas were found not only deficient in the same adult-type enzymes but they also contained the same fetal-type enzymes. In slow growing hepatomas the shift toward fetal-type enzymes was more characteristic than the loss of adult-type enzymes.

C. ISOZYMES There is an ever growing literature, starting from 1963 (Schapira et 1963), pointing to profound alterations of isozymic patterns in neoplastic tissues, namely, the progressive loss of specific tissue forms and their replacement by isozymic counterparts preponderant in other adult or fetal tissues. Biochemical transitions of this type can occur without significant modifications in total enzymatic activities thereby providing a means of revealing fine changes in enzymatic patterns that would otherwise escape notice. The subject has been reviewed on several occasions (Criss, 1971; Knox, 1976, Ono and Weinhouse, 1972; Weinhouse, 1973; Schapira, 1973; Uriel, 1975). We shall limit this discussion to a few representative studies where tumor isozymic patterns were correlated with those characteristic of developmental tissues during either ontogenesis or regeneration and repair. The main data are summarized in Table 11. cil.,

1. Clrtcose-ATP Phosi,liotrurisfernse Isozymic analysis has revealed that in most mammalian tissues there exist four molecular forms of this key enzyme in glucose utilization. Three of them (types I, 11, and 111) with a low K , for glucose are collectively referred to as hesokincises and the fourth (type IV), possessing a high K , for glucose, is designated as glucokinase. Hexokinase type I and, particularly, glucokinase are the predominant forms in adult liver, whereas in fetal liver glucokinase is barely detectable, hexokinase type 11 (Satoet ~ l . 1969a) , or type I (Weinhouse et ( I / . , 1972) being preponderant. The general pictiire of these isozymes in well and poorly differentiated hepatomas and in most primary hepatomas induced by chemical carcinogens is that of a shift from high to low K , fetal-type isozymes with very low levels of glucokinase and rather high values of hexokinase isozymes which, according to Sato et (11. (1969b),shift from type I to type 11. Highly differentiated hepatomas with very slow growth rates showed, in contrast, an almost adult liver pattern ofboth hexokinase and glucokinase (Weinhouse, 1973).

TABLE I1 Liver patterns: : Isozyme variants

Enzymes Glucose-ATP phosphotransferase Phosphofructokinase

Pyruvate kinase

Aldolase

Glycogen phosphorylase

Hexosaminidase Glutaminase Alanine aminotransferase Lactate dehydrogenase a

1-111: Hexokinase IV Glucokinase I: Muscle type IV: Liver type 11-111: Other M: Muscle type L: Liver type K: Kidney type A: Muscle type B: Liver type C: Brain type L: Liver M: Muscle F: Early embryo and many fetal tissues A: Liver and other tissues B: Brain L: Liver type K: Kidney type C: Cytosolic M: Mitochondria1 L, to L,

Adult

I

Fetal or neonatal

.1

lerant isozymes

licspator

'

Hepatectomp

Chemical injuw

I, I1

I, I1

I, I1

I, I1

IV

IV

IV

IV

N.D.

L K (Traces)

L (Traces)

L

L (Traces) K A B C F; L

C F; L

L (Traces) K A B C L; F

L (Traces) K A B C L; F

A ; B (Traces)

B; A

B,A (Traces)

B; A

L

K

K

N.D.

N.D. N.D.

C; M (Traces)

M

M

N.D.

N.D.

_ L5- 40

L, = 2.5 -

-L5 L4

N.D.

N.D.

ZVd

B

L4

L

High or moderate growth rate.

* 16 to 72 hours after Y; liver resection. ' Hepatotoxics or hepatocarcinogenic drugs: early action. N.D., not determined. When several isozymes coexist, the preponderant variant is in italics.

K A B

3

RETRODIFFERENTIATION A N D GENE EXPRESSION IN CANCER

143

Recently, hexokiiiase isozymes of uterine tumors were investigated using analytical electrophoresis (Sato et ul., 1972). Here again, fetal type I1 hexokinase was strongly predominant in cervical and corpus carcinomas, while in control tissues, cervical epithelium, and endometrium, only tlie type I isozyme was revealed.

2 . Pliosplznfructokinuse Chromatographic separation has revealed at least four molecular forms of this glycolytic enzyme (Tanaka et d . ,1971).Their distribution in adult rat tissues has led to three main groups. The first is composed of muscle, heart and brain, where isozyme I (muscle type) is the only form present. A second group, including liver, kidney, and red blood cells, contains isozyme IV (liver type) as the most preponderant constituent. To tlie third group, where isozymes I1 and/or I11 predominate, belong the majority of tissues not included in the two other groups. Isozynie IV was found to be the major constituent in liver from newborns. A series of rat and human tumors, including three of nonhepatic origin (Walker carcinosarcoma and two gastric cancers) were examined and all showed isozyine IV as the preponderent form. Tanaka et (11. (1972) studied isozyme patterns of phosphofructokinase i n neonatal but not in fetal tissues. Nevertheless, if it is assumed that isozynie IV (liver type), which is predominant in newborns, also represents the fetal form of the enzyme, then it would appear that tumors from tissues other than liver shift to a coninion fetal or neonatal molecular fonn of phospliofnictokinase. Isozyme IV was equally preponderant in both normal and neoplastic liver probably because the same isozyniic pattern is expressed in both adult and fetal liver.

3. P!lrriotite Kiiiase By chromatographic and electrophoretic methods. pyruvate kiiiase can be resolved into three major, noninterconvertible molecular forms: isozynies I, I1 and 111, also referred to as liver, muscle, and kidney-type, respectively. Isozynie I predominates in adult liver, where isozyme 111 appears only as a minor constituent. In contrast, a completely inverted pattern is observed in fetal liver. In transplantable hepatomas a transition to the fetal pattern occurs in parallel with tlie increasing growth rate and dedifferentiation of the tumors. The fetal transition is also characteristic of primary hepatomas induced b y chemical carcinogens and of Walker carcinosarcoma, a tuinor of nonhepatic origin (Inamura and Tanaka, 1972; Tanaka et d . , 1972; Weinhouse et al., 1972; Walker and Potter, 1972; Farina et al., 1974). Farron et (11. (1972) have shown that isozyine I1 (muscle type) was

144

JOSE URIEL

almost completely replaced by the fetal form (isozyme 111) in an experimental rliabdomyosarcoma. Only highly differentiated hepatomas showed an isozymic pattern virtually identical to that of adult liver.

4. Aldoluse As indicated previously (Section II,A,2) aldolase exists in multiniolecular forms of tetrametric structure designated as muscle type (isozyme A), liver type (isozyme B), and brain type (isozyme C ) depending on the adult tissue where each form prevails. Isozyme A is the most widely distributed among fetal tissues and the preponderant form in all of them (Farron et al., 1972). There is now evidence that aldolase isozymes revert to the fetal pattern in rat, mouse, and human hepatocellular carcinomas, in rat transplantable hepatomas, and also in tumors of different origin, such as spleen reticulosarcoma (Schapira, 1966), rhabdomyosarcoma, kidney carcinomas, and mammary carcinomas (Farron et ul., 1972). More recently isozyme C (brain type), which is also present in fetal liver although to a lesser extent than isozyme A, was found associated with ascites hepatomas (Schapira and Josipowicz, 1970; Sugimura et ul., 1970).

5. Glycogen Phosphorylnse This enzyme is involved in the important hepatic function of glycogen inobilization and is highly suited to studies on alterations of regulatory enzymes in cancer. Three isozymic forms have been demonstrated in rat tissues, the L and M forms (liver and muscle type, respectively) and a third form called F (for fetal). This latter form is found in early, 10-day-old embryos as the sole isozyme and later on in all fetal tissues together with either the L or the M forms. All the hepatomas examined, including the Yoshida ascites hepatoma (Sat0 et u l . , 1975), contained the fetal-type isozyme, but most of them also retained, as expected, the liver form since this is expressed in both adult and fetal liver. The enzymes described in this section are probably the most representative examples of isozymic reversion to fetal and neonatal patterns in malignant tumors. Others, such as the isozymes of glutaminase (Katununia et d.,1972), branched-chain amino acid transferase (Ichihara and Ogawa, 1972), lactate dehydrogenase (Uriel, 1969; Farron et ul., 1972), carboxylic esterases (Kaneko et al., 1974), and glucosamine-6-phosphate synthetase (Tsuiki and Miyagi, 1975) can also be added. The list is obviously incomplete and ever growing.

HElHODIFFEREI\TTIiZTION .4ND G E N E EXPRESSION IN CASCEH

145

6. Cotidusion We have presented experimental data showing that some metabolic and biochemical patterns specific to developmental tissues are found in malignant tumors. Even though the body of evidence may be convincing, it would be premature to make any generalizations chiefly because we lack systematic studies under standardized conditions. Tumors, like normal, developing, and renewing tissues, are made of heterogeneous cell populations undergoing phase-associated changes a s growth progresses. The extent to which either cancerous cells or nonmalignant elements contribute to the biochemical changes observed is in the majority of cases unknown. Frequently the data 011tained result from the analysis of transplantable tumors maintained in serial passage for several years or even decades (i.e., Ehrlich ascites tumor, Yoshida hepatoma, Walker carcinosarcoma, etc.), thus being exposed to genetic drifts in host histocompatibility as well as being subjected to long-term selection pressures and nietabolic adaptations. A few summary statements can, however, be drawn from the data outlined in this section: (a) Metabolic pathways and isozynie patterns characteristic of the functional activity of adult tissues tend to vanish when they undergo inalignant transformation and to be replaced by the rather common pathways and patterns characteristic of tissues undergoing developmental growth. Thus, when the isozymic pattern of an adult tissue differs from its fetal or neonatal counterpart (liver aldolase and glutaminase, kidney lactate dehydrogeiiase, etc.), the tumor expresses the fetal-type isozyme. However, if the same isozymic form is expressed in both adult and fetal tissue (kidney aldolase and pyruvate kinase, muscle aldolase, glycogen phosphorylase, etc.), the pattern does riot change in the corresponding tumor although quantitative differences may be observed. The isozymic distribution in fetal tissues has not been explored in a systematic way, but one can anticipate that the niolecrilar forms of a given enzyme should converge to a common pattern in most, if not all, the tissues of an individual during the early stages of ontogenic development. (b) The reversion of neoplasms toward iinmature tissues is accentuated to a certain extent a s the tumor’s growth rate increases and as differentiation lessens. It is unclear whether this reflects phase-specific changes in gene expression or the metabolic adaptation of cells to higher growth rate or to special environmental conditions. Probably both processes are implicated. (c) While widely common among tumors, the biochemical transitions discussed here seem to be neither essential nor unique to malignancy: highly differentiated, slowly growing hepatomas show enzyme pat-

146

JOSE URIEL

terns very close to those of normal resting tissues and, in contrast as discussed later, patterns of the fetal or neonatal type can be observed in adult tissues undergoing noncancerous growth. PROPERTIES D. MORPHOLOGICAND STRUCTURE-DEPENDENT The undifferentiated or poorly differentiated morphology of many cancer cells is one of the oldest observations made by pathologists on the phenotypic characteristics of malignant tissues and is the basis of the embryonal concept of cancer advanced late in the nineteen century by Cohnheim (1889). Modern studies at the ultrastructural level on neoplastic cells growing in vivo or in vitro have confirmed their convergence toward standardized patterns of surface structure, junctional complexes, and intracellular organelles [see Easty, 1974; Balls and Monickeiidauii (1976); Franks and Wilson, 19771. This is also true for einbryofetal elements particularly when engaged, like malignant cells, in an active growth cycle. In a recent ultrastructural exploration of rat liver carcinogenesis by diethylnitrosamine (DENA), several distinctive features associated with premalignant and neoplastic hepatocytes (see Table 111) were found to coincide with the morphologic characteristics of fetal or neonatal rat hepatocytes. These fine subcellular similarities were too striking and too prevalent to b e considered a random event. (Bruni,

1973). Another morphologic analogy between fetal and neonatal rat liver tissue and primary hepatomas induced by oral ingestion of dimethylaminoazobenzene (DAB) was observed in a population of transitional parenchymal cells possessing high binding affinity for estrogens (Uriel et d.,1973, 1975).These estrogen binding cells which are present in fetal and neonatal liver, but not in normal adult liver, reappear in hepatomas. Changes in the structure and properties of the cell surface are important because of their consequential effects in the social behavior of cells: mobility, adhesiveness, self-assembly, agglutinability, reactivity TABLE I11 MORPHOLOGICSIMILARITIES BETWEEN FETALAND NEOPLASTIC HEPATOCYTES

I. Microsegregation of nucleolar components 2. Disorganization and scarcity of smooth endoplasmic reticulum 3. Disorganization and decrease in volume of rough endoplasmic reticulum 4. Appearance of free ribosomes 5. Decrease or absence of glycogen content 6. Simplification of the cell surface and of other cytoplasmic structures

RETRODIFPERENTIATION A N D GENE EXPRESSION IN CANCER

147

toward exogenous substances, etc. Concanavalin A (Con A) and other plant lectins that specifically interact with carbohydrates of the cell surface and cause some cells to agglutinate serve as structural probes of cell surface organization and have been used extensively in recent years to explore cell agglutinability and surface properties in relation to ontogenic development and neoplastic transformation (Nicolson, 1974). Early work in this area demonstrated that Con A preferentially agglutinates chemically or virus-transformed cells, but not untransformed cells unless preceded by unmasking of Con A binding sites by trypsin treatment (Burger and Goldberg 1967; Inbar and Sachs, 1969). Moscona (1971) extended these observations to embryonic tissues b y showing that embryonic neural retina cells dissociated with ethylenediamine-tetracetate, a treatment which does not modify the behavior of normal adult cells, are readily agglutinated by Con A. It was suggested that the Con A binding sites are operational during ontogenesis and cell differentiation and probably become clustered in adult resting cells. The unmasking of these sites in neoplastic cells may represent a reversion to a condition resembling that of embryonic cells. Confirmation of the fetal-like behavior of neoplastic cells interacting with lectins was later provided by experiments comparing adult and immature cells from the same origin. Thus Becker (1974) did a comparative study of the response to Con A of rat hepatocytes from adult and fetal livers as well as those derived from five transplantable hepatomas. Fetal hepatocytes and hepatoma cells were highly and equally susceptible to agglutination while hepatocytes from young or adult rats livers, whether quiescent or stimulated to divide after partial hepatectomy, were not. Ill. Fetal Patterns in Noncancerous Growth

Many immunological, biochemical, structural, and behavioral properties related to neoplastic cells and tissues have been discovered in the past. Claims of their specific linkage to malignancy have almost systematically been advanced. The reappearance of phenotypic characteristics of embryofetal origin in cancerous tumors has not failed to uphold this rule. The following is a quick survey of fetal patterns of gene expression associated with nonneoplastic growing cells in adult individuals.

A. FETALANTIGENS I N REGENERATION,CELL INJURY, A N D TISSUE REPAIR Studies on the kinetics of AFP secretion during hepatocarcinogenesis in rats by Watabe (1971), Kroes et al. (1972, 1973),

148

JOSE URIEL

Kitagawa et nl. (1972), de Nbchaud and Uriel (1973), Becker and Sell (1974) and Becker et (11. (1975) among others have established the following points: (a) When rats are fed a basal diet supplemented with an appropriate chemical (azo dye or nitrosainine derivative, N-2-fluorenylacetaniide, etc.) a characteristic kinetic pattern of serum AFP is observed. It begins with the transient appearance of AFP in a high proportion of animals, as early as 1 week after feeding the carcinogenic diet, and this elevation occurs in the absence of any histological sign of neoplastic transformation and even without detectable signs of cell injury or proliferation. (b) As a rule, the precociousness and the frequency of preneoplastic onset of AFP increase with the dose of hepatocarcinogen. Also young animals are more susceptible than older ones. In addition to some individual and strain peculiarities, the response is also influenced by the type of hepatocarcinogen used and its mode of administration. (c) Several waves of AFP secretion can follow the initial one, and finally, even if the carcinogen is discontinued, serum AFP becomes persistently elevated in most animals and increases in concentration or reaches a plateau in many of them. (d) Animals whose serum AFP increases early and persists at high levels later, have a high probability of developing hepatocellular carcinoma. Nevertheless, AFP can be present at physiological levels in the serum of animals with developed hepatomas (up to 30% frequency in certain experimental series). The existence of non-AFP-secreting hepatocarcinomas has been confirmed in rats bearing transplantable hepatomas (Sell and Morris, 1974) with some animals showing as much 18 mg/ml of serum AFP while others had normal values (less than 60 nghnl). (e) Histologic observations during 2-acetylaminofluorene and 3’Me-DAB induction of hepatomas resulted in a good correlation between the early phase of AFP increase and the presence of a special type of liver cell. These cells have been designated as “oval cells” by Farber (1956) and “transitional cells” by Inaoka (1967) and have been shown to contain AFP by immunohistologic techniques (Onoe et al., 1976; Kuhlmaii, 1978) and on the basis of their estrogen binding properties (Uriel e f d . , 1973). Many studies have been conducted to explore the association between elevated levels of AFP and pathological situations other than hepatomas. Evidence has been provided that liver cell damage provoked in mice or rats by either partial hepatectomy (Perova et d . ,

RETRODIFFEHENTIATION AND G E N E EXPRESSION I N CANCER

149

1971; Sell et a / . , 1974) or a great variety of hepatotoxins (Bakirov, 1968; de Nechaud and Uriel 1971, 1972; Pihko and Ruoslahti, 1973; Watanabe et ol., 197611) is followed b y a transient production of AFP starting 2 4 4 8 hours after the initial treatment and returning to normal after about 6-8 days. In human pathology, persistently elevated levels of AFP are associated up to 80% with primary liver cancer and with almost all germ cell carcinomas as well as, at a much lower frequency, with neoplasms of nonhepatic origin, i.e., secondary liver cancer (see for details and complete bibliography reviews on AFP). Raised serum levels of the protein were, however, reported in alcoholic cirrhosis (Abelev, 1971; Hirai et ul., 1973; Ruoslahti et d . , 1974; Lehnian, 1976), acute and chronic hepatitis (Abelev, 1971, Wepsic and Sell, 1974), congenital tyrosinemia (Belanger et (11., 1973; Buffe and Riinbaut, 1973), and ataxia-telengiectasia (Waldman and McIntire, 1972) among others. A recent survey (The People’s Republic of China, 1974) of serum AFP levels covering almost 500,000 individuals revealed that a small proportion (about 10%) of patients with primary liver cancer showed a biphasic elevation of AFP: a transient increase before any symptom of clinical cancer was detectable, followed by a second and persistent rise in AFP concomitant with the onset of disease. The significance of this transient elevation of serum AFP as well a s the early one observed during experimental carcinogenesis or after acute liver injury has not been completely elucidated. As discussed later, it may in part be the consequence of hyperplastic growth following tissue repair and regeneration, but it can also be due to retrodifferentiation, a general process of cell rejuvenation in response to cell and tissue insults of varied etiology (hepatectomy, chemical poisoning, viral infection, etc). Other evidence of the fetal transitions in antigenic patterns accompanying reparative nonneoplastic growth is suggested by cellmediated immune responses to fetal and neoplastic antigens as demonstrated in mice subjected to partial hepatectomy followed by liver regeneration (Hellstrom et d.,1975). Lymph node cells (LNC) from these mice, 10 to 80 days after two-thirds liver resection, were cytoxic to neoplastic cells of varied origin but not to normal ones. Moreover, serum from hepatectomized mice could block the cytotoxic effect of LNC from multiparous and hepatectoinized mice. This seeins to indicate that antigens of the type discussed earlier (Section II,A,l) and shared by embryofetal and neoplastic cells can also be reexpressed in regenerating liver. Other “oncofetal” antigens such as the acidic-type isoferritins of

150

JOSE UHIEL

human origin have been found in increased amounts in both malignant and nonmalignant diseases (Hazard and Drysdale, 1977). Also, elevated concentrations of tissue and serum human CEA (Martin and Martin, 1970; Burtin et al., 1972; Kupchik et ul., 1973) or atypic tissue localization of the antigen (Rogalsky, 1976) have been found.

B. FETALISOZYMICTRANSITIONS

The adult versus fetal or neonatal shift of some enzymatic activities and molecular forms of enzymes which are characteristic of many neoplastic tissues has also been observed in situations of nonneoplastic growth similar to those reported above which lead to the reexpression of embryo-fetal antigens. Early hyperplastic and reparative growth of liver in response to the administration of hepatocarcinogens (3’-MeDAB) to rats was concomitant in rats with an isozymic transition of the fetal type. A progressive lowering of glucokinase, the adult type liver isoenzyme, was observed during the first 6 to 8 weeks of the diet, followed b y a gradual increase in fetal hexokinase activity (Walker and Potter, 1972). A similar shift toward the fetal pattern of pyruvate kinase was observed. The activity of isozyme I gradually decreased while the reverse was true for the type I11 isozyme. By the sixth week of the hepatocarcinogen diet, the activity of isozyme I fell to less than 10% of the initial value and, correspondingly, that of enzyme I11 increased almost 6-fold (Walker and Potter, 1972; Yanagi et ul., 1974; see also h e n , 1977, for a review). The isozyinic changes in aldolase in the precancerous liver of rats fed hepatocarcinogens were also characterized, as in the case of glucokinase and pyruvate kinase, by a progressive loss of the adult pattern and an increase in the muscle-type form characteristic of fetal and neonatal liver (Endo et d . , 1972; Walker and Potter, 1972). The activities of several enzymes (glucose-6-pliosphatase, nonspecific esterase, alkaline phosphatase, and y-glutamyl transpeptidase) in prenoeplastic nodules, considered to be precursors of malignant hepatomas, were reported to deviate from adult patterns and to approach those of fetal or cancerous liver (Onoe et d . , 1975, 1976). These observations are of particular interest because they correspond to a phase characterized morphologically by the appearance of a significant population of oval or transitional cells and of hyperplastic nodules considered as noncancerous precursors of hepatocellular carcinoma (Farber, 1956, 1973). There are several undeniable examples of fetal-type enzymatic transitions associated with liver repair and regeneration after partial hepatectomy .

RETRODIFFERESTIATIOK

A N D G E N E EXPRESSION IN CANCER

151

During tlie 24- to 72-hour period after two-thirds liver resection, isozynie 111 (fetal type) of pyruvate kinase increased to more than twice the normal value with a concomitant drop in the type I/type 111 ratio to below 3 (normal ratio = 11) (Walker and Potter, 1972; Farina et al., 1974). Moreover, an analogous shift from type I to type I1 isozyme was demonstrated in homogenates of isolated parenchymal liver cells from adult rats 2 to 15 days after partial hepatectomy (Boney et ( i l . , 1973; Garnett et al., 1974). Significant deviations from normal adult patterns were reported for adolase in regenerating liver. Bergks et (11. ( 1974) demonstrated b y electrophoretic and immunologic methods the reappearance of fetal liver isozymes A and C and their hybrids, 24 to 72 hours after one-third or two-thirds partial hepatectomy. Analogous transitions were reported in the case of several other enzymes such a s hexokinase and glucokinase (Sato et (11. 1969a), phosphofructokinase (Tanaka et u l . , 1972), and glutaminase (Katunuina et al., 1972). These transitions were, nevertheless, reported absent for other enzymes. Liver regeneration is a very complex phenomenon at the niorphological, biochemical, and molecular levels and is still obscure because of tlie diversity and rapidity of the sequential events that occur in a relatively short period of time. If tlie model is not carefully scrutinized, fine changes can escape experimental observation and perhaps this may partially explain some controversial results. Some quantitative data on enzymatic activities in regenerating rat liver which correlate with those of immature and cancerous liver are given in Table I. The values quoted for regenerating liver appear to appro x i inate in ore close 1y those of he pat oin as with in ode r at e growth rates than those of rapidly growing tumors. Regenerating liver is often compared to rapidly growing hepatomas on tlie basis of DNA incorporation rates of [''C]thymidiiie (Lea et a / . , 1966). In terms ofdifferentiation, however, this correlation seems inadequate. Hyperplastic hepatocytes in regenerating liver show a much less degree of iminaturity or dedifferentiation than malignant cells i n hepatomas of high growth rate. C. MORPHOLOGICCHANCES Owing to olwious experimental difficulties, very few studies have been devoted to the comparative analysis of morphologic modifications associated with ontogenic, neoplastic, and nonneoplastic growth i n a single tissue or cell population. The compensatory hyperplastic capacity of the liver to cell damage and tlie easily inducible stimulation of this capacity by a number of agents of diverse nature explain

152

JOSE UlUEL

the extensive use of it as an excellent physiological model for reparative nonneoplastic growth. As far as niorphologic data are concerned, a series of ultrastructural changes have been reported in hepatocytes from regenerating livers following chemical injury (Meldolesi, 1967) or partial hepatectomy (Bresnick, 1971). These include: (a) dispersion of cytoplasmic basophilic bodies; (b) hypertrophy and disaggregation of smooth endoplasniic reticulum; (c) decrease of rough endoplasmic reticulum; (d) decrease in the number of lipid bodies; (e) loss of stored glycogen; (f) appearance of free ribosomes; (g) increased lysosomal activity; and (h) simplification of the cell membrane. Most of these morphological characteristics are coincidental (see Table 111)with those of juvenile, fetal, or neonatal hepatocytes and were also observed in hyperplastic nodules of rat hepatocytes appearing early in the preneoplastic phase of liver carcinogenesis by diethylnitrosamine (Section 11,D). The information assembled in the present section illustrates what has been repeatedly pointed out in the preceding ones, namely, the molecular and organizational convergence among tissues of the same origin undergoing expanding growth of malignant or nonmalignant nature. Even though the body of evidence may be convincing, it must be taken as a probabilistic approximation, and, consequently, any dogmatic generalization should be avoided chiefly because the data now available are incomplete and result from the analysis of extremely complex material. It seems however possible to state that the progressive acquisition of fetal-type phenotypes, while characteristic of many cancerous cell populations, does not appear to be specifically linked to malignancy since some neoplasms are composed of highly differentiated tissues and because some reparative and renewal processes mimic the fetal transitions observed in malignant tumors.

IV. The Plasticity of the Differentiated State

A. DIFFERENTIATION VERSUS RETRODIFFERENTIATION The hypothesis that malignant transformation of cells may be concomitant with mutational changes induced b y physical, chemical, or biological stimuli has long received wide acceptance as a causal explanation of cancer. However, extensive immunological and biochemical studies reported over the last 15 years have failed to demonstrate

RETRODIFFEREPITTIATIO;U AND G E N E EXPRESSION ISCANCER

153

new antigens other than virus-specified antigens, and macromolecular constituents which would logically be expressed by the cellular genome of the assumed mutated progeny. More recently, an alternative, although not mutually exclusive, hypothesis has been advanced which focuses on cell differentiation as the basic alteration of malignant growth (Pierce, 1967, 1970; Markert, 1968; Potter, 1969; Uriel, 1969; Stonehill and Bendich, 1970). In this regard, while the arrested differentiation of stern cells at some intermediate level before full maturity seems to meet with general agreement, the concept that tumors may derive from adult tissues and that their emergence correlates with a process of retrodifferentiation or stepwise reversion toward juvenile phenotypes has received much less attention (Uriel, 1969, 1976).A variety of connotations, sometimes without clear definition, are associated with the process of differentiation, whereas the term “differentiation” is even more loosely used to describe a loss of morphological properties in formerly recognizable cell phenotypes. We shall consider differenticitioii as a time and space sequence of metabolic and biosynthetic patterns that reflect the diversified activity of the genome during developmental growth, and retrodiferen ticition as a sequence of nucleocytoplasniic events inverse to those of differentiation. This formulation was inferred from an analysis of the dynamics of fetospecific antigens and isozymic patterns in liver during ontogenic, reparative, and neoplastic growth (Uriel, 1969). It postulates that differentiation and retrodifferentiation are alternatives of two convergent directions of a single chain of events and assumes that both directions are potentially reversible. Thus when an adult cell retrodifferentiates or a stem cell differentiates, they probably travel through similar stages of nucleocytoplasmic expression, albeit sequenced in opposite order. Consequently, the initial phase-specific transition in cells entering retrodifferentiation should be similar to that of the terminal step of their previous developmental history. For the same reason, the synthesis of one or another embryo-fetal antigen or enzyme and, inversely, the deletion of certain phenotypic structures and molecular components would depend on how far the reversion has proceeded. Figure 1 illustrates in an oversimplified manner the reversibility and the stepwise character of both differentiation and retrodifferentiation, as well as the rough dynamics of differentiation versus retrodifferentiation changes. As pointed out by Manes (1974) and contrary to early assumptions, development does not involve a progressive restriction of gene expression a s ontogeny proceeds from one phase to the next. Rather, the best evidence yet available, although not abundant, indi-

154

JOSE URIEL

FIG. 1. Differentiation versus retrodifferentiation: alternative of two convergent and reversible directions of similar events. Cell lineages emerging from primordial elements (x) diversify as development proceeds and become morphologically and functionally mature at the terminal stages of differentiation ( A l , A2, B1, . . .). It is postulated that when cells retrodifferentiate they travel through similar patterns of nucleocytoplasmic expression albeit sequenced i n the reverse order to their own developinental history. Under the effect of external or internal deleterious agents cells may revert throiigh the corresponding pathway up to the stationary state (i.e., D1+ d + c -P I)) from where redifferentiation can start in the same (I]+ c+ . , .) or in another direction, the latter leading to a inetaplastic schift ( i e . , b+ B d ) . During preneophstic development cells also retrodifferentiate hut, at a given moment, the process of compensatory redifferentiation remains incomplete or does not occur and leads to the emergence of'a population of malignant, persistently dividing cells. [Reproduced with permission from Uriel (1976).]

cates that the metabolic and structural phenotypes of primordial cells are sustained by relatively small portions of the genome. During embryogenesis phase-specific gene activation occurs and new cell structures, antigens, and enzymes are produced, either transiently or permanently, in the somatic cell lineages until differentiation terminates. Conversely, a process of gradual restriction of gene products specific to adult phenotypes seems to characterize cellular retrodifferentiation. On the other hand it is becoming increasingly clear that differentiation proceeds by following a rigorous program of mutually exclusive changes which through selective DNA-transcription by sets of RNA templates lead to the synthesis of protein patterns characteristic of the different cell phenotypes. Under these conditions it seems unlikely that random deviations from that pattern would be compatible with viability in cells undergoing retrodifferentiation. From the point of view of cell information content, differentiation and retrodifferentiation appear as antagonistic processes. While preserving the entire information content encoded in its genome, a living organism is a thermodynamically open system that uninterruptedly exchanges energy and matter, that is information, with its environment. During ontogenic development, when cells diversify, when new

HETRODIFFEHENTIATION A N D G E N E EXPRESSION I N CANCER

155

phenotypes and functions emerge, when tissues and organs form, and the whole mature organism becomes created, in short, as diflerentiation progresses, more and more information content is bound to the system. Conversely part of this information is lost when cells and tissues retrodifferentiate and shift toward embryofetal patterns of gene expression. In this regard, differentiation and retrodifferentiation may be considered as biological transitions involving respectively gain and loss of information content. The following discussion is to a large extent devoted to reviewing the experimental evidence now available on the nature of retrodifferentiation i n normal and neoplastic cells growing in G ~ O Oand in

vitro. The irreversibility of the differentiated state as cells diversify and mature from primordial elements or committed precursors has in the past been a firmly ingrained and unshakeable opinion for many ernbryologists, associated with the concept of differentiation. Similarly, the association of cancer with mutational events, the existence of experimentally selected, fully autonomous tumor cells, and the common acceptance of the fatal evolution of most spontaneous inalignant neoplasms have contributed to spread the idea of the “frozen” state of cancer phenotypes. Yet there is no formal argument to refute the assumption that differentiation and retrodifferentiation are inherent potentialities in all the somatic cells of an organism as long as their genetic information content is preserved. In fact experimental evidence has accumulated in recent years which emphasizes that phasespecific changes of gene expression may occur in practically all soinatic cells whatever their difierentiated state at the moment when the change started. The competence to modulate ’ differentis‘1 t’ion or retrodifferentiation in one or another direction may, however, vary over a broad range depending on different parameters (i.e., type of the organism, age, cell species, initial stage of diflerentiation, etc.).

B. DIFFERENTIATION OF MALIGNANTCELLS That many kinds of malignant cells from a broad range of plants and animal species may undergo differentiation in cioo and i l l oitro with the loss of neoplastic properties has now been well dociinieiited (see review by Braun, 1975). One example to be considered is that of virus-induced Lucke adenocarcinoma of the leopard frog. Nuclei were isolated from the cancer cells and injected into normal fertilized, but enucleated, frog eggs. Not only did these nuclei participate in normal cleavage but also, in a significant number of cases, their genetic infor-

156

JOSE URIEL

mation was reprogrammed to the development of apparently normal tadpoles without any evidence of malignancy (Mc. Kinell et d.,1969). A different situation is provided by the behavior of some chemically induced tumors in the European newt (Seilern-Aspang and Kratochwil, 1963). All the animals injected with a mixture of hydrocarbon carcinogens developed coalescent, inulticentric tumors from the basal cells of the skin mucosa glands, which infiltrated and metastasized into normal tissues. Somewhat more than half of the injected animals died at this stage of the disease while in the rest the process evolved to the spontaneous maturation and differentiation of the tumor cells that became normal adult elements (pigment cells, mucous glands, fibrous connective tissue, etc.). Pierce and Wallace (1971) studied the squamous cell carcinoma of the rat lip, a tumor derived from the basal cells of the skin or the hair follicles and consisting of pearls of mature, keratinized cells surrounded by large masses of undifferentiated elements. They concluded that only the undifferentiated cells divide actively and then migrate into the pearls with subsequent differentiation to a mature benign form. The latter was confirmed by separately grafting into adequate hosts equal amounts of dissected pearls and undifferentiated cells. About a third of the transplants of undifferentiated cells formed squamous cell carcinomas, whereas none of the pearls developed into a tumor. As pointed out by Braun (1975) the same mechanism seems to operate in cases of partial or complete self-healing of squamous cell carcinomas in h um an s . Similar behavior may result in the spontaneous regression of other malignant tumors in humans. An illustrative example is that of neuroblastomas which in some instances revert spontaneously to ganglion cells probably through a process of differentiation and maturation of neuroblastoma cells (Dyke and Mulkey, 1967). Recent experimental work with cultured lines of neuroblastoma cells support such an assumption. These cell lines are being used as a model system for studying the events involved in neuron differentiation (see Augusti-Tocco, 1976, for a review on the subject). Proliferating cells of neuroblastoma cell cultures express some molecular and biochemical properties of differentiated neurons such as the synthesis of neurotransmitters but lack others such as the formation of axon and specialized junctions. In fact the cells remain in a very immature state of differentiation. Under appropriate culture conditions, it is possible to induce them to differentiate into neurites and to establish contact with other cells (Augusti-Tocco et d.,1973; Ross et uZ., 1975). The formation of functional synapses has also been demonstrated by culturing a hybrid

RETRODIFFERENTIATION

A N D G E N E EXPRESSION IN CANCER

157

neuroblastoma X glioma cell line with muscle fibers (Nelson et d., 1976). Attempts have been made to induce other cultured malignant cells to differentiate either under defined culture conditions or by proper stimulation with naturally or nonnaturally occurring substances. There are several recorded examples of differentiation of certain murine and human myeloid leukemia cells in the presence of appropriate conditioned media or of a “feeder layer” of peripheral blood leukocytes (Fibach and Sachs, 1975; Moore et ul., 1974).Exceptionally, long-term leukocyte cultures derived from myeloid leukemic cells have been shown to differentiate along the myeloid series without exogenous inducers (Collins et al., 1977). On the other hand, cells of a mouse erythroid leukemia were made to differentiate fully after treatment with dimethylsiilfoxide (Friend et ul., 1971) and those of a highly malignant murine melanoma shifted to a normal phenotype when cultured in the presence of 5-broniodeoxyuridine and reverted to the cancerous state when the latter was removed from the medium. (Silagi and Bruce, 1970; Cliristman et d., 1975.) The mouse teratocarcinoina probably offers the best example of malignant cells with multipotential competence for differentiation. This tumor, thought to arise froin primitive germ cells, consists mainly of sheets of undifferentiated cells, called “embiyonal carcinoma cells,” scattered among a great variety of well-differentiated elements derived from all three germ layers but in a completely disorganized array. Teratocarcinonias are frequently transplantable in syngeneic hosts. A variety of permanent cell lines have been isolated from either spontaneous or transplantable tumors. Their investigation has produced great interest in their potential to serve as a model for the analysis of both cell differentiation and neoplastic development. A considerable number of detailed reports and reviews on the subject have already been published (Pierce, 1967; Stevens, 1967; Damjonow and Solter, 1974; Jacob, 1977; Martin, 1975; Mintz, 1978). For the purpose of the present discussion we shall overlook the fact that the so-called embryonal carcinoma cells are tlie malignant elements of the tumor and point out that (a) they resemble early embryonic cells with respect to some morphological, immunological (see Section 11,A,2), and biochemical properties, (b) they can differentiate into derivatives of tlie three germ layers and even appear as developmentally totipotent since they can participate in the production of normal mice when grafted into mouse blastocysts, and (c) once differentiated they lose their malignancy. Other examples could be added to the above very brief outline of

158

JOSE URIEL

cancer reversibility to normality. Nevertheless it seems clear from the data presented that the nucleocytoplasmic functions of malignant phenotypes are neither definitive nor irreversible. This implies that nuclear gene expression is probably regulable in spontaneous tumors and susceptible to manipulation in experimental neoplasms and cancerous cells. Although these conclusions do not exclude mutational events as possible causal agents of malignant transformation, they lend considerable support to the developmental concept of cancer.

c. RETRODIFFERENTIATION I N CELL INJURY AND TISSUEREPAIR An extreme case of reversible changes in retrodifferentiation is illustrated by the experiments of Gnrdon (1966) with the nuclei from gut epithelial cells of Xenoprts tadpoles. When nuclei from these differentiated cells were transplanted into enucleated unfertilized eggs of the same species, normal development into feeding tadpoles was pronioted in some of the transplants. This experiment apparently implies the necessity for the complete phasing of nuclear gene expression to a primordial stage before the reconstituted egg is able to resume the entire program of tadpole ontogenesis. The epiinorphic regeneration of resected parts of certain lower invertebrates has for many years attracted the attention of developmental biologists (see review by Goss, 1969). There is now conclusive evidence that the blastema cells which accuinulate at the site of transection of amphibian tadpole limbs emerge from retrodifferentiated mature elements, e.g., myocytes, chondrocytes (Hay, 1969; Burgess, 1974), and not, as formerly considered, from a reserve of undifferentiated stem cells. Moreover, there is a clear correlation between actual regeneration and the competence to retrodifferentiate. Thus, in nonregenerating limbs such as those of postmetamorphic anurans, amputation stimulates little or no retrodifferentiation, the damaged tissues at the amputated surface undergoing rapid individual healing. Nevertheless, when retrodifferentiation is forced to occur by prolonged injury to the stump, whether mechanical (Polezhayev, 1946) or chemical (Rose, 1946), regeneration follows. At the cellular level stump retrodifferentiation results in the progressive disappearance of cytoplasmic structures characteristic of the mesodermic adult elements of the stump and their replacement by a simplified pattern of organelles common to many “undifferentiated” cells. It is this population of niesenchymatous cells that proliferate and form the regeneration blastoma from which the different tissues of the limb will be reconstituted. Molecular events during the retrodifferen-

HETHODIFFERENTIATION AND G E N E EXPRESSION IN CANCER

159

tiatioii phase of limb regeneration are poorly understood. The increased activity during this period of several hydrolytic enzymes including cathepsins, peptidases, collagenases, and acid phosphatases (Schmidt, 1968) probably reflects tlie marked predominance of autolytic processes. Of particular interest is the decrease in the glycogen content, which seems intimately related to the rise in aerobic glycolysis ancl the respiration rate observed during retrodifferentiation until blastenia formation is completed. The high rate of lactate production attained at this moment gradually drops to normal adult values as regeneration progresses. As discussed in Section 11, the intensification of glycolysis under aerobic and anaerobic conditions is also associated with the embryonic state of most animal species, including man, as well as with many cancerous tissues. Another remarkable example of regenerative capability is provided b y the hydra. Under constant optimal conditions this lower invertebrate bypasses sexual reproduction and regenerates entirely and continuously from a reserve of sinall immature, basophilic cells, the so-called interstitial cells. However, it can also regenerate from practically any amputated part of tlie organism even when depleted of its population of interstitial cells by previous X-ray or nitrogen mustard treatment, to which these cells are particularly vulnerable. In such cases, regeneration occurs b y retrodifferentiation and subsequent moulding of the amputated part. Morpliologic evidence of this mechanism resulted from tlie work of Burnet and his colleagues (1969) who succeeded in promoting inetaplastic regeneration of H!/drci rjiridis after explants consisting only of gland cells and digestive cells were cultivated in appropriate media. The former reverted to the stage of interstitial cells which then dividecl ancl redifferentiated. The digestive cells transformed into epidermal cells without apparent morpliological signs of retroclifferenti,d t 1011. ' Wolfiaii lens regeneration, which provides yet another example, is a property unique to certain kinds of urodel amphibians. After removal of the lens, the organ is conipletely regenerated by a cell population derived from the dorsal part of the iris epithelium. The fully differentiated iris cells convert into lens cells following an ordered, stepwise sequence of morpliological and biosynthetic pattern starting with a retrodifferentiation process of the iris cells: nuclear activation, repliAfterward, lens regeneration begins with cation, and del>i~,rmentatioli. the synthesis of lens-specific antigens (y-crystallins) and progresses by a series of morphological changes until complete lens organogenesis. The subject was reviewed b y Yamada (1967) who also pointed out that "the convergeiice of regenerative and ontogenic leiis forniation

160

JOSE URIEL

implies that Wolffian lens regeneration involves recovery of an embryonic condition by the iris epithelial cell.” These phenotypic changes, which were confirmed in experiments carried out with cultured cells (see below), well illustrate the general assumption (Fig. l) that metaplastic shifts follow strict requirements for limited retrodifferentiation back to a common precursor and then transdifferentiation in another direction. Liver regeneration of laboratory rodents, such as rats and mice, is probably the best investigated i n vivo model of reparative and compensatory growth in mammals and provides an excellent example of restricted retrodifferentiation without apparent metaplastic shifts. Although the cell-reverting phase of liver regeneration has been explored to a lesser extent than the subsequent period of compensatory hypertrophy and hyperplasia, the data already available permit us to draw a general, although incomplete, picture. First, the process of hepatocyte retrodifferentiation in rats starts very soon after partial hepatectomy and seems to be accomplished during the 18- to 24-hour period which precedes the onset of D N A synthesis and the first wave of mitosis. Thus Kostraha and Wang (1973) have shown that RNA species transcribed in vitro from repetitive sequences of DNA in chromatin obtained 6 hours after partial hepatectomy were not present in the transcript from resting liver chromatin. Also, Mayfeld and Bonner (1972) did an interesting study of early molecular events occurring before initiation of DNA synthesis in liver from partially hepatectomized rats. They were able to demonstrate as early as 1 hour after the operation the sequenced production of rapidly labeled, highmolecular-weight RNA and then of chromosomal RNA, both containing sequences not produced by normal adult liver. After 9 hours, a maximal 35% increase in the template activity of chromatin supported the conclusion that during this interval there occurred a significant derepression of previously silent genes. For Mayfeld and Bonner these genes are presumably activated to prepare the cells for D N A synthesis and subsequent cell division. It is probable, however, that such nuclear activation also involves genes directing the synthesis of fetaltype antigens and isozymes whose presence is observed during the 24to 72-hour period of liver regeneration (see Section 111) induced either b y partial hepatectomy or by chemical poisoning. Some evidence in support of this suggestion is given by recent studies on the early production of mouse and rat AFP following acute liver injury. Engelhardt et nl. (1976) studied AFP synthesis in mouse liver after CCI, inhalation. By employing a combination of autoradiographic analysis following repeated pulses of tritiated thymidine and intracellular AFP

RETHODIFFERENTIATIONA N D GENE EXPRESSION I N CANCER

161

localization 11y indirect iniinuiiofluorescence, they were able to demonstrate the production of AFP i n hepatocytes before the onset of D N A synthesis. A similar conclusion was advanced by Watanabe et al. (197Ga) who reported an early increase in AFP serum concentration preceding tritiated thymidine incorporation into liver DNA of rats treated with a single injection of ethionine. As reported in the preceding section, this early period between 18 and 72 hours after partial hepatectomy or liver poisoning is like the period of nonmalignant hyperplasia which follows the administration of chemical poisons characterized by (a) the increased synthesis of fetospecific liver proteins (a-fetoprotein, a,M-fetoprotein, lipoprotein esterase, cell surface antigens, etc.), (11) the transitory shift of some isozymes (glucoseATP-phospliotransferase, phosphofructokinase, pyruvate kinase, aldolase, etc.) to a pattern characteristic of iniinature hepatocytes, and (c) metabolic transitions leading to the predominance, as in fetal liver, of catabolic pathways over anabolic ones, and a reverse behavior of protein and nucleic acid metabolism. I n the absence of neoplastic transformation all these changes are transient and progressively disappear while the phenotypic properties of mature hepatocytes are recovered. The evidence outlined above permits the delineation of certain general traits of retrodifferentiation associated with reparative and regenerating growth in a broad range of organisms throughout living systems. Some kind of disturbance of the normal homeostatic equilibrium of cells and tissues seems to be a necessary requirement for promoting the process. Most frequently, retrodifferentiation is initiated as a response to cell injuries of varied etiology. The question as to whether the cells undergoing retrodifferentiation are those sublethally damaged or those situated in the vicinity of the traumatized area remains to be elucidated. Probably both kinds of elements may participate in the process. The progressive nature of retrodifferentiation that we have assumed seems to be sustained by several well-documented examples. Some common features of cells undergoing retrodifferentiation in v i v o include a variable loss of cytoplasmic organization and complexity, the transition to new embryo-fetal type patterns of gene expression as evidenced by the synthesis of embryo-fetal type antigens and enzymes, and the phase-specific restriction of genes characteristic of the mature state of each differentiated cell. The extent of the process may vary within broad limits ranging from the restricted transition of adult versus fetal or neonatal hepatocytes which takes place in liver regeneration, to the advanced level of retrodifferentiation characteristic of blastema cells from which tadpole limbs regenerate. Between these

162

JOSE URIEL

two extreme cases intermediate levels of retrodifferentiation are provided by the different phenotypes associated with several systems and particularly, as discussed in the next section, with neoplastic cells. Retrodifferentiation is normally counterbalanced by a process of reontogeny which tends either to restore the terminal phenotypes from which the reversion phenomenon started or, in some circumstances, to transdifferentiate in another direction leading to metaplastic shifts, with the appearance of mature elements distinct from those which initially retrodifferentiated.

D. RETRODIFFERENTIATIONI N NORMALA N D TRANSFORMED CELLS i n Vitro The preservation of the integrity and growth potential of cell and tissue explants in tiitro requires rapid adaptive changes to culture conditions which differ greatly from those prevailing in v i u o . The morphologic and behavioral changes in normal and tumor cells that often start immediately after plating are at present well known and have been described in detail in recent reviews on the subject (Macpherson, 1970; Auersperg and Finnegan, 1974; Franks and Wilson, 1977).As a rule, and regardless ofthe cell phenotype at the time of culturing, a variable degree of cell reversion toward a common poorly differentiated state is almost systematically observed (Wilson, 1973). The retrodifferentiated phenotype may continue to revert during subsequent proliferation until it stabilizes or may redifferentiate to some extent depending on the particular coinpetelice ofthe culture cell line or on the adequate manipulation of culture conditions. In fact all cultured cells tend, indiscriminately, to resemble each other due to adaptive loss of cellular and subcellular complexity and organization and the acquisition of behavioral properties comnion to juvenile elements.

1. Norniul Ce1l.s As far as normal cells grown in culture are concerned, evidence of retrodifferentiation at the molecular level has been provided by several recent studies. Parker et al. (1977) have demonstrated that cultured normal muscle and lung fibroblasts from adult mice express fetal-type antigens, absent in the corresponding cells i n viuo, which cross-react with fetal antigens present in murine sarcomas. On the other hand, Thorpe et al. (1977) have shown that normal adult human skin grown in culture shares antigens with first trimester human fetuses. The fetal antigens were found absent in a variety of in uioo human tissues such as skin, muscle, decidua, and lymphocytes. More-

HETHODIFFEHESTIATIOIU ASI) G E N E EXPRESSIOS ISC:ASC:ER

163

over, natural antibodies against these antigens could be demonstrated in many normal adult sera. As pointed out by the same author this reactivity may be a soiirce of error if tissue-cultured tumor cells are used to detect antibodies to tuinor-specific antigens in human sera. Primary cultures of avian tendon have been used for studying environment-dependeiit mocliilations i n collagen synthesis (Schwarz and Bissel, 1977). At high density and i n the presence of ascorbate the cells are well differentiated in that they devote, like tendon cells i t i oco, 25-3010 of their total protein synthesis to collagen. However, either at low density or upon removal of ascorbate the cell retrodifferentiate as evidenced b y a decrease in collagen synthesis to only one-third ofthe initial level. The change is reversible since addition of ascorbate will again restore collagen synthesis to its in o i ~ olevel. Modulations of the differentiated state of normal cells depending on culture conditions have also been demonstrated in the case of calf lens epithelial cells. The direction of the events, differentiation or retrodifferentiation, was identified using specific markers, namely, the activity of metabolizing enzymes and the synthesis of specific structural polypeptides. When lens cells were incubated on plastic surfaces instead of on their natiiral growth substrate, the lens capsule, signs of limited cell reversion were observed a s evidenced by a decrease in the hydroxylating activity of steroids and the absence of synthesis of the A2 chains and B2 chain of a-cristallin, a specific molecular marker of lens differentiation. The findings described were interpreted as originating b y cell surface changes induced h y different culture conditions. Direct evidence for transformation in culture of differentiated iris epithelial cells (Yamada and McDevitt, 1974) and of neural retinal cells (Okada ct a l . , 1977) into lens cells has also been obtained recently . As discussed i n Section 111, these transdigerentiation changes, leading i n ~ i o oor iii oitro to lens formation, imply a previous retrodifferentiation of cells to an embryonal-like state before rediiferentiation starts in another direction. Another interesting example of the plasticity of the differentiated state is offered by studies on the evolution of cultured liver cells taken from 8-day-old quail embryos (Le Douarin, 1975).During the first 6 days of cultivation, two morphological types of cells, fibroblast and epithelial-like cells, could be distinguished. From the sixth day epithelial cells disappeared and the culture showed a homogeneous fibroblastic aspect. Cell sheets at this stage of culture were placed in close contact with hepatic mesenchyme from 7-day-old chick embryos and the organotypic culture was transferred for 4 to 6 days on the chorioallantoic membrane of 6-day-old chick embryos. I n

164

JOSE UFUEL

40 of 60 tests, cords of differentiated hepatocytes were found in the explants. This result indicated that the hepatocytes reverted and became fibroblastic in appearance but the transition was reversible as soon as adequate conditions of redifferentiation were provided.

2. Malignant Cells An ever increasing number of cells derived from normal and neoplastic tissues with different phenotypic properties and at varied states of differentiation can already b e cultured as permanent lines. On some occasions, nomial cell lines may become spontaneously tumorigenic, although most often they must be forced to transform by treatment with physical, chemical, or viral agents. In terms of morphology and cell behavior, both spontaneous or experimentally induced transformation in vitro involves some additional loss of cell organization and differentiation which seems to reflect a new step along the retrodifferentiating pattern (Auersperg and Finnegan, 1974). Thus, chick embryo cells transformed by strain MC29 avian leukemia virus, when compared to their normal cultured counterparts, reveal a series of ultrastructural cytoplasmic alterations characteristic of immature cells and which resemble those accounted for by the adult to fetal-type reversion of hepatocytes following partial hepatectomy (see Section 11,D).Also, studies of a line of epithelial hepatoma-like cells transformed from a cultured line of normal epithelial liver cells (Borek, 1975) have shown a transition to patterns of random multilayered growth, a loss of responsiveness b y growth restriction to high cell densities and to intimate contact, alterations in the cell surface as evidenced by a decrease in the number of tight junctions, and the acquisition of cell agglutinability by plant lectins, among other traits associated with cell rejuvenation. Molecular events of retrodifferentiation in tumor-derived cell lines or in permanent normal cell lines upon their transformation in vitro have not been extensively explored. The available data, however, already represent a significant body of information. Isozynies of the branched-chain amino acid transaminase are useful markers for studies of embryo-fetal transitions in tumors (Section 11,B). In adult kidney tissue of the rat and hamster, isozyme I is the exclusive form, while the newborn kidney of both species also contains isozyme I11 (Roth et al., 1977). These authors examined the isozymes in a cloned, normal rat kidney cell line which was transformed by a mutant of ROW sarcoma virus temperature sensitive for transformation and showed that at 36°C these cells contained predominantly isozyme 111, the newborn kidney form. Upon being grown at 4WC, the isozymic pattern shifted to a predominance of isozyme I as is found in normal adult rat kidney

RETHODIFFERENTIATION A N D G E N E EXPRESSION I N CANCER

165

tissue and the cells reacquired a normal morphology. Both isozyme forms I and 111 were found in a normal baby hamster kidney cell line and its polyoma virus-transformed derivative, although in the latter the relative amount of isozyme I was greater when compared to the nontransfornied cells. All these changes were interpreted as being indicative of cell retrodifferentiation consequent to the viral transformation. Studies on the expression of molecular forms of thymidine kinase in cultured human fibroblasts (Bull et al., 1974) upon transformation b y SV40 virus have shown a shift from adult to fetal thymidine kinase activity. The elaboration of this fetal form with transformation suggests that it is coded by the host genome. Placental alkaline phosphatase (Regan isoenzyme), an oncofetal antigen associated with human cancers of varied origin (Fishinan and Singer, 1976), is expressed in some HeLa sublines (Singer and Fishman, 1974) and in other tumor cell lines, but also in a reduced number of normal, permanent cell lines (Wilson et al., 1977). The isoenzyme has been proven useful in studies of environmentdependent modulations in gene expression (Singer and Fishman, 1976). Thus when HeLa TCRCl, a subline cloned for the exclusive production of Regan isoenzyme, was transferred to grow in neonatal immunosuppressed rats, a phenotypic change in the alkaline phosphatase was observed. After 30 days of growth in vivo, the Regan isoenzyme was absent in extracts of the tumor nodules whereas a different alkaline phosphatase, the oncoaminion isoenzyme, was identified. Upon transferring the tumor back to culture the original Regan phenotype become preponderant. Modern studies at the molecular level on the phenotypic characteristics of normal and neoplastic cells growing in vitro have unquestionably attested to the plasticity of the differentiated state. Under the particular conditions of tissue culture many cells unable to adapt to the stressful effects of plating die quickly, while cells having the capability to retrodifferentiate revert toward more juvenile patterns of gene expression and survive. It is unknown why only some cells in a given population possess such a capability. It seems, however, that the ability to retrodifferentiate, whether in u i v o or i n vitro, decreases with the age of the organism and this probably explains why most permanent cell lines arise from embryo-fetal elements. For the same reason, at least in part, many neoplastic cells which can already be considered as juvenile phenotypes, are more easily cultured than their normal adult counterparts. Whatever the differentiated state of either normal or malignant cells at the time of culturing, some degree of retrodifferentiation seeins to be systematically observed in surviving cells. On the

166

JOSE UFUEL

other hand, normal cells in long-term cultures also show, upon neoplastic transformation, molecular events of retrodifferentiation. Thus, spontaneous or induced malignant transformation i n vitro is accompanied by additional changes leading the cells to a new stationary state characterized by an increased expression of juvenile patterns. Another important point brought to light by these studies is that the differentiated state of cells can be manipulated in vitro and be forced to evolve along a given direction (differentiation or retrodifferentiation) b y adequate adjustment of culture conditions. One can reasonably anticipate that with increasing knowledge of the homeostatic and nutritional factors regulating cell growth and development, a greater number of cell types regardless of the level of differentiation could be successfully maintained in long-term cultures and subjected to neoplastic transformation. They should represent appropriate models for the study of cell retrodifferentiation if, as we have repeatedly pointed out in the preceding pages, the latter is generally associated with neoplastic development. V. Retrodifferentiation and Cancer

In summarizing the observations discussed in the preceding sections it is possible to point out that the immunological, biochemical, morphological, and behavioral embryo-fetal traits common to a variable degree to inany malignant tumors are, nevertheless, neither essential nor unique to cancer. Highly differentiated, slowly growing tumors may show patterns of gene expression very close to those of normal adult resting tissues, while patterns of cell immaturity of fetal or neonatal type can be transitorily observed in tissues undergoing regenerating or reparative growth. With respect to the question of whether adult, juvenile, or stein cells are the target elements in carcinogenesis, no clear-cut answer can be given today. The information already accumulated suggests that the site where the initial events of cancer occur may be provided b y any stationary state attained by a cell along its differentiation pathway. Nevertheless, it seems that for a given cell species, certain levels of differentiation are more susceptible to transformation than others. We have commented above (Section IV,D) that the phase-specific changes accompanying neoplastic transformation i n oitro may be recognized as a manifestation of a process of cell retrodifferentiation. On the contrary, the emergence in vivo of a population of neoplastic cells having a variable degree of resemblance to embryo-fetal elements does not enlighten us about their origin. The direction of the events, differentiation from stein cells or retrodifferentiation from mature ele-

HETRODIFFEHENTIATION

A N D G E N E EXPRESSION IN CANCER

167

inents, is in either spontaneous or experimentally induced neoplasms difficult to establish. The comparative analysis of fetal-type biological transitions accompanying hepatocarcinogeiiesis and liver regeneration led Uriel ( 1969) to envisage them as demonstrative examples of retrodifferentiation. It was speculated that, while stepwise reversion toward inimature cell phenotypes could account for tlie analogies observed between hepatomas and regenerating liver after partial hepatectoniy or cheniical injury, the dynamics of both processes were clearly distinct. In regenerating liver the change is a cyclic one; initial retrodifferentiation of parenchymal cells is “counterbalanced” after a short period of active growth hy a process of redifferentiation which restores the histotypic properties of the adult organ. During the preneoplastic phase of liver carcinogenesis, cells also undergo retrodifferentiation, but at a given moment and by an unknown mechanism the system escapes compensatory redifferentiation and becomes malignant. Thus “unbalanced retrodifferentiation” may chmitcterize neoplastic development arising at least i n adult tissues. The demonstration that liver AFP is synthesized in partially hepatectomized rats (Engelhard et (il., 1976) and i n rats subjected to some hepatotoxic drugs (Watanal)e et u / . , 1976a,b) provides good support for the hypothesis which considers liver regeneration as a process starting with the retrodiffereiitiatiori of adult hepatocytes. More recently Kuhlman (1978) has used double tracer techniques combining imniunocytology and autoradiography to follow tlie synthesis of AFP and D N A replication during hepatocellular carcinogenesis in rats fed N-nitrosomorl)lioliiie ( N N M ) . With high doses of N N M , necrosis of hepatocytes, proliferation of hyperplastic nodules of immature ovalshaped cells, and AFP synthesis occurred, 11s is usually observed with other liel~atocarcinogensduring tlie preneoplastic phase (see Section I I I ,A). When N N hl feed in g was stopped, ova 1-ce 11 pro 1i fe rat ion came rapidly to a standstill and, furthermore, AFP was no longer detected afterwmd until hepatoinas developed. In contrast to what occurred with high N N M closes, upon feeding low doses of the carcinogen neither oval-cell proliferation nor cellular synthesis, nor serum secretion of AFP was o1)served during the sanie period. Since both NNM schedules (low and high doses) led to hepatomas it was concluded that hyperplastic proliferation of oval cells is not a prerequisite for neoplastic development. hlerely oval cells seem to result from restitution of damaged liver due to acute toxic injury. At the stage of inalignant conversion, distinct nodules consisting of neoplastic hepatocytes with a variable degree of retrodifferentiation were observed in rats fed either high or low doses of N N M . AFP-staining and non-AFP-staining

168

JOSE URIEL

nodules, probably of clonal origin, were seen concomitantly in the same liver. These observations provide experimental evidence in support of the hypothesis (Uriel, 1969) that fetal-type transitions characteristic of primary hepatoma may start in adult hepatocytes undergoing a process of retrodifferentiation. If the assumed role of retrodifferentiation in liver carcinogenesis is correct and can be further extended to other neoplasms derived from adult tissues, the question arises as to why a unique mechanism of cell rejuvenation operates in malignant tumors as well as in regeneration and tissue repair. A trait common to these situations is that they emerge in response to cell aggressions of different origin. Under physiological conditions and as a result of internal and external factors, thermally labile cell structures and macromolecular components rich in information content are continuously broken down although they are simultaneously restored by a process of subcellular repair, designated b y Trincher ( 1965) as the antifluctuation internal cell work, which maintains the cell in a stationary state. This mechanism is specific to living systems and vital to the preservation of cell integrity in the stationary state. In multicellular organisms, efficient homeostatic controls which assure the constancy of the internal milieu provide an additional means for damping anomalous fluctuations of external or internal origin. When the homeostatic controls are, however, overcome and the deviation proceeds beyond the antifluctuation capability of the cell system, retrodifl'erentiation seems to be the only alternative to the cell's risk or undergoing irreversibly deleterious changes. It enables the cells to revert temporarily, through adequate nucleocytoplasmic transitions, toward stationary states with higher growth potential as well as lower dependence on normal ecologic controls and, consequently, they become better adapted to stressing environmental conditions. This change is advantageous because retrodifferentiation is a multistep process of cell rejuvenation tending to simplify and to minimize the differences between originally distinct cell phenotypes which progressively leads the system to levels of higher thermodynamic stability. Retrodifferentiation appears then as a unique property of living matter, a general adaptive process for the maintenance of life. This probably explains its contribution to neoplastic development and to reparative growth. Another question brought up by the above discussion is why retrodifferentiation is, under physiological conditions (i.e., regeneration), a transient change followed by a process of redifferentiation while in neoplasia the transition becomes stationary and leads to the emergence of a population of persistently dividing cells. There is ob-

RETRODIFFEHENTIATION AND G E N E EXPRESSION IS CANCER

169

viously no well-sustained answer to this question at present. A trivial explanation could be that some increased susceptibility to neoplastic transformation may lie associated with cells undergoing retrodigerentiatioii. In this regard it is interesting to note, as pointed out b y Ruinyantsev ( 1977),that almost all the very rare rhabdomyosarcomas of cardiac origin were localized in atria. This is consistent with the characteristic ease with which myocytes of this area, unlike ventricular myocytes, may reenter the mitotic cycle following limited but tinequivocal signs of retrodifferentiation. In summarizing this discussion, we wish to emphasize that (a) many properties of cancerous cells may result partly from an adaptive process of live preservation against internal deleterious factors (aging, abnormal metabolism, or biosynthetic errors) or noxious external agents (physical, chemical, viral, mutational), and (b) retrodifferentiation appears to be the most efficient mechanism underlying such an adaptive process. Viewed i n this manner, cancer may represent the cell’s last resource to escape the fatal destiny of multicellular organisms, the ultimate step of an adaptive mechanism which succeeds in selecting a cell population with the highest autonomy and unresponsiveness to regulatory factors operational in normal adult organisms. It is to be hoped that a careful study ofretrodifferentiation in physiological and tumoral models will help to distinguish that which in neoplastic development is relevant to adaptive cell behavior from that which might be the result of intrinsically specific alterations.

REFERENCES Abelev, G . I. (1971). Adc. C o w e r Res. 14, 295-358. Abelev, G . I., Perova, S. D., Khramkova, N. I., Postnikova, 2 . A., and Irlin, I. S. 1963). Trci ti s ~ i l tni tci t io iI 1, 174- 180. Alexander, P. (1972). Notrtre (Lotidoil ) 235, 137-140. Artzt, K.,Dubois, P. H., Bennet, D., Condamine, H., Babinet, C., and Jacob, F. 1973). Proc. Nntl. Accitl. Sci. C‘SA 70, 2988-2992. Auersperg, N., and Finnegan, C. V. (1974). Zti “Neoplasia and Cell Differentiation” ( G . V. Sherbert, ed), pp. 279-318. S. Karger, Basel. Angnsti-Tocco, G. (1976). “Trends in Biological Sciences,” pp. 151-154. Augusti-Tocco, G., Parisi, E., Zucco, F., Casola, L., and Romano, M . (1973). Z r i “Tissue Culture ofthe Nervous System” ( G . Sato, ed.), pp. 87-106. Plenum Press, New York. Bakirov, R. D. (1968). R!yul/. Er.yp. R i d . ‘\fed. (U.S.S.R.) 65 ( 2 ) ,45. Baldwin, R. W. (1973).A d o . Crcticer R e s . 18, 1-75. . J.Cnncer 4, 47-53. Baldwin, R. W., and Embleton, M. J. ( 1 9 6 9 ~ )Znt. Baldwin, R . W., and Embleton, M. J. (1969b). Znt. J . Caticer 4, 430-439. Baldwin, R. W., and Embleton, M. J . (1974). Z t i t . J . Cancer 13, 4 3 3 4 4 3 . Baldwin, R. W., Glaves, D., and Piinin, M. V. (1971). I n “Progress i n Immunology” (B. Amos, ed.), pp. 907-920. Academic Press, New York. Baldwin, R. W., Glaves, D., and Vose, B. M. (1972). I t i t . J . Cancer 10, 233-243.

170

JOSE URIEL

Baldwin, R. W., Glaves, D., and Vose, B. M. (1974a).Int. J. Cancer 13, 135-142. Baldwin, R. W., Embleton, M. J., Price, M. R., and Vose, B. M . (1974b).Trunsjdunt. Rea. 20,77-99. Balls, M., ant1 Monickendaun, M. (etls.) (1976). “Horgan Culture in Bioinedical Research,” Caml~ridgeUniv. Press, London. Baranska, W. P., Kolodovsky, P., and Kaprowski, H. (1970).Proc. N u t / . Accicl. Sci. USA 67, 193-199. Bauer, H . (1974).Adu. Cancer Res. 20, 275-329. Becker, F. F. (1974).Proc. N u t / . Acad. Sci. USA 71, 4307-4311. Becker, F. F., and Sell. (1974).Cuircer Res. 34, 2489-2494. Becker, F. F., Horland, A. A,, Sliurgin, A., and Sell, S. (1975).Cancer R e s . 35, 1510-151.3. Belanger, L., Belanger, M., Prive, L., Larochelle, J., Tremblay, M., and Anbin, G. (1973).Patho/. B i o l . 21, 449-455. Bergts, J., Nechaud, de, B., and Uriel, J. (1974).F E B S Lett. 48, 76-78. Berman, L. D. (1972).I n t . J . Cancer 10, 326-330. Boney, R., Walker, P., and Potter, V. R. (1973).Biochenz. J. 136, 947. Borek, C. (1975).I n “Gene Expression and Carcinogenesis in Cultured Cells” (L. E. Gerschenson and E. B. Thoinson, eds.), pp. 62-93. Academic Press, New York. Brann, A. C. (1975).I n “Cancer. A Comprehensive Treatise,” Vol. 111, pp. 3-20. Plennm Press, New York. Braunstein, G. D., Vaitukaitis, J. L., Carbone, P. P., and Ross, G. T. (1973).Ann.I n t . Med. 78, 39-45. Brawn, R. J. (1970).I i z t . J. Criricer 6, 245-249. Bresnick, E., (1971).I n “Methods in Cancer Research” (H. Busch, ed.), Vol. VI, pp. 347-391. Bresnick, E., Thonipson, U. S., Morris, M. P., and Liehelt, A. G. (1964). Hiocherii. Biojdi!/,~.Hes. Cotnitmtr. 16, 278-284. Brnni, C. (1973).J.N u t / . Cancer Znst. 50, 1513-1528. Buffe, D., and Rimhaut, C . (1973).I I I “Alpha-fetoprotein” (R. Masseyeff, ed.), pp. 191195. INSERM, Paris. Bull, D. L., Taylor, A. T., Austin, D. M . , and Jones, 0. W. (1974).Virology 57, 279-284. Burger, M . M., and Goldberg, A. R. (1967).Proc. N u t / . Accid. Sci. USA 57, 355-366. Burgess, M. C. (1974).In “Neoplasia and Cell Differentiation” (G. V. Sherbet, ed.), pp. 106-152. Karger, Basel. Burnet, A. L. (1969).Z t i “The Stability of the Differentiated State” ( H . Ursprung, ed.), pp. 109-127. Springer-Verlag, Berlin. Burtin, P., Martin, E., Sabine, M. C., and Kleist, von, S. (1972).J.N u t / . Cuncer Znst. 48, 25-32. Cameron, R., Kellen, J., Kolin, A,, Malkin, A., and Farber. (1978). Cancer R E S . 38, 823-829. Chism, S. E., Warner, N . L., Wells, J. V., Crewther, P., Hunt, S., Marcliolamis, J. J., and Fudenberg, H. H. (1977).Cancer Res. 37, 3100-3108. Christman, J. K., Silagi, S., Newcomh, E. W., Silverstein, S. C., and Acs, G. (1975).Proc. N u t l . Acrid. Sci. USA 72, 47-50. Coggin, J. H., and Anderson, N. C. (1974).Ado. Cancer Res. 19, 105-165. Coggin, J. H., Ambrose, K. R., and Anderson, N. G. (1970).J.Irnniunol. 105, 524-526. Coggin, J. H., Anibrose, K. R., Bellamy, B. B., and Anderson, W. G. (1971).J.Zinmunol. 107, 526-533. Collins, S. J., Gallo, R. C., and Gallaghier, R. E. (1977).Nature (London)270, 347-349. Conheiin, J. F. (1889). Lect. Celt. Phys. 2, 1-28. New Sydenliani Soc. London. Criss, W. E. (1971).Cancer Res. 31, 1523-1542. Danijonov, I . , and Solter, D. (1974).Current Topics Puthol. 59, 69-130.

RETRODIFFEHENTIATION AND GENE EXPRESSION IN CANCER

171

Dyke, P. C., and hlulkey, D. A. (1967). Ctrric.c,r 20, 1343-1349. Easty, G. C . (1974). 111 “Neoplasia and Cell Difierentiation” (G. V. Sherbet, ed), pp. 190-233. S. Karger, Basel. Edidin, M., Patthey, H. L., bicGuire, E. J., and Slielfield, W. D. (1971). Z i t “Proc. 1st. Conference and Workshops on Eiiiliryonic and Fetal Antigens in Cancer” ( N . G . Andersoii and J. H. Coggin, etls), pp. 239-248. Oak Ridge, Tennessee. Elford, H. L. (1972). ( A r i i i t J f o i i o g r a i i / i . 13, 205-217. Elford, H . L. (1974).Arch. Bioclieni. B i o ) i / i ! / . ~163, . 537-543. Endo, M., Eguchi, hl., Ymagi, S., Torisu, T., Ikehara, Y., antl Kaiiiiya, T. (1972). G a i i i i ~ / o i t o g r ~ r 13, ~ ~ /235-250. t. Engelhardt, N. V., Lazareva, hl. N., Uryvaeva, I. V., Factor, V. hl., and Brotlsky, V.Ya. (1976).Nrrttrre (Loiidoii ) 263, 146-148. Farlier, E . (1956). Cnttcer Rc.v. 16, 142. Farber, E . (1973). Crrticer H e s . 33, 2537-2550. Farina, F. A,, Shatton, J. B., Morris, H.P., and Weinhorise, S. (1974). Coitcet- Res. 34, 1439- 1446. Farron, F., IIsu, H. H.T., and Knox, W. E. (1972). Coitcrr Res. 32, 302-308. Filmch, E., and Sachs, L. J. (1975). Cell P h ! / s i o / . 86, 221-230. Fishnian, W. H.,and Singer, R. (1976). Ctriicrr Re.r. 36, 42.564261. Fishman, W.H., Inglis, N. I . , Stolhach, L. L., and Krant, M . J. (1968). Cniicer R c a . 28, 150- 154. Franks, L. hl., aiid Wilson, P. D. (1977). Z i t f . Hcc. C ! / t o / . 48, 55-139. Friend, C., Scher, W., Holland, J. G., antl Sato, T. (1971).Proc. N u t / . Accrd. S c i . USA 68, 378-382. Fuks, A,, Banjo, C., Schuster, J., Freedman, S. O., and Gold, P. (1974). f ? i O C / i i l J l . B i o p / i ! / , Actct ~. 417, 123-152. Gaclielin, G., Fellous, M.,Guenet, J . L., and Jacob, F. (1976). Dcc. R i d . 50, 310-320. Garnett, M . E., Dysoii, R. D., and Dost, F. N. (1974).j. Biol. Chein. 249, 5222-5226. Gitlin, 1).J., and Boesrnan, M. (1967). Camp. Bioc/ieni. P/t!/sio/. 21, 327-336. Gitlin, D., Perricelli, A , , : i d Gitlin, G . XI. (1972). Cnttcer Hc.v. 32, 979-982. Gold, P., m t l Freetliiian, S. 0. (1965).J. E . v / i . .\let/. 122, 467-481. Goldstein, S., and Trietlman, G., (1975). E.rperieiititr 31, 177-180. Goss, H. J . (1969). “Principles of Regeneration.” Acadeniic Press, New York. Greenstein, J. P. (1945). Z i t “AAAS Research on Cancer” (F. R. Moulton, ed.), pp. 191-215, AAAS, Washington, D.C. Gurdon,J . B. (1966).[ti “Advances i n h4orphogenesis” ( M . Abercrombie and J. Bracher, eds), Vol. IV, pp. 1-43. Acadeniic Press, New York. Haiina, M . G., Teniiaiit, R. W., and Coggin, J . H . (1971). Proc. N t r t l . Actrd. S c i . USA 68, 1748- 1752. Hay, E. (1969). Z t i “The Stability of the Differentiated State” ( H . Urspriing, ed.), pp. 85-108. Springer-Verlag, Berlin. Hazard, J. T., aiitl Dr).stlale, J. W,(1977). X / r t i i r c ( L o i r r / o t i ) 265, 755-756. Hellstriiin, I., Hellstriiin, K. E., and Nishioka, M.(1975).Nntirre (Lontloti ) 253, 744-746. Herbenilan, R. B. (1977). 211 “Immunology of Human Infections” (A. J. Ndimias anc! R. J. O’Reilly, eds.), Vol. 11. Plenum Press, New York. Hirai, H., Nishi, S., Watabe, hl., and Tsakada, Y. (1973). Cotiii M o i ~ o g r o ~ i /14, t . 19-34. Hogan, B., Fellous, XI., Avner, P., and Jacob, F. (1977).Nntrcre (Lotidoit)270, 515-518. Holden, S., Bernard, O., Artzt, K., Whitmore, W. F., and Bennet, D. (1977). Natrcrc, (Loiidoii ) 270, 518-520. Ibsen, K. H. (1977). Ctrtrcer Re.?. 37, 341-353. Ichihara, A,, and Ogawa, K. (1972). Caiiii Monogrcrpli. 13, 181-190. Inamiira, K., and Tanaka, T. (1972).J. Bioclieiii. (Tok!/o)71, 1043-1051.

172

JOSE UFUEL

Inaoka, Z. Y. (1967). Gann 58, 355. Inbar, M., and Sachs, L. (1969). Nature (Lotidon) 223, 710-712. Jacob, F. (1977). Zmmunol. Reo. 33, 3-32. Kaneko, A., Dempo, K., Yoshida, Y., Chisaka, N., and Onoe, T. (1974). Cancer Res. 34, 1816-1821. Katunuma, N., Kuroda, Y., Yoshida, T., Sanada, Y., and Morris. (1972). Gann Monograph. 13, 143-150. Khoo, S. K., Waener, N. L., Lie, J. T., and Mackay, I. R. (1973). Znt. J . Cancer 11, 681-687. Kitagawa, T., Yokochi, T., and Sugano, H. (1972). I n t . J . Cancer 10, 368-381. Kleist von, S., Chavanel, G., and Biutin, P. (1972). Proc. Nutl. Acad. Sci. USA 69, 24922494. Klemperer, H. G., and Haynes, G. R. (1968). Biochem. J. 108, 541-546. Knox, W. E. (ed.) (1976).“Enzyme Patterns in Fetal, Adult and Neoplastic Rat Tissues.” S. Karger (ed.), Basel. Kostraba, N. C., and Wang, T. Y. (1973). E x p . Cell Res. 80, 291-296. Kroes, R., Williams, G. M., and Weisburger, J. H. (1972). Cancer Res. 32, 1526-1532. Kroes, R., Williams, G. M., and Weisburger, J. H. (1973). Curicer Res. 33, 613-617. Kuhlman, W. D. (1978). Z r t f . J . Cuticer 22, 335-343. Knpchik, H. Z., and Zamcheck, N. (1972). Gastroenterology 63, 95-101. Knpchik, H. Z., Zanicheck, N., and Saravis, C. A. (1973).]. Nut/. Cancer Znst. 50, 17411749. Lamon, E. W. (1974). Biochini. Biophys. Actu 355, 105-118. Larsoon, A. (1969). Eiir. J . Biochem. 11, 113-121. Lea, M . A., Morris, H. P., and Weher, G. (1966). Cancer Res. 26, 465-469. Le Douarin, N. M. (1975). M e d . B i d . 53, 427455. Lehnian, F. G. (1976). 111 “Onco-developmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 407-415. Academic Press, New York. Mach, J. P., and Pusztaszeri, G. (1972). Imrnunochernistr!/ 9, 1031-1033. Macpherson, I. (1970). Arlo. Cancer Res. 13, 169-215. Maley, F., and Maley, G. F. (1961). Curicer Re.u. 21, 1421-1428. Malkin, A., Kellen, J. A,, and Caplan, B. (1978). Personal communication. Manes, C. (1974). Caticer R E S .34, 2044-2052. Markert, C. I. (1968). Cancer Res. 28, 1908-1914. hlartin, G. R. (1975). Cell 5, 229-243. Martin, F., and Martin, M. S. (1970). Znt. J . Cnncer 6, 352-360. Martin, F., and Martin, M . S. (1972). Int. J. C o m e r 9, 641-647. Mayfeld, J. E., and Bonner, J. (1972). Proc. Nut/. Acad. Sci. USA 69, 7-10. Mc. Kinell, R. G., Deggins, B. A,, and Labat, D. D. (1969). Science 165, 394-396. Meldolesi, J. (1967). Bioclzem. Plturmucol. 16, 125-129. blintz, B. (1979). “Advances in Cancer Research.” Academic Press, New York, i n press. Moore, M. A. S., Spitzer, G., Williams, N., Metcalf, D., and Biickley, J. (1974).Blood 4.1, 1-18. Morris, H. P. (1965).Ado. Cnncer Res. 9, 227-302. Moscona, A. A. (1971). Science 171, 905-907. Nathanson, L., and Fishman, W. H. (1971). Cancer 27, 1388-1397. Nechaud de, B., and Uriel, J. (1971). I n t . J. Cancer 8, 71-80. Nechatid de, B., and Uriel, J. (1972). I n t . J . Cancer 10, 58-71. Nechaud de, B., and Uriel, J. (1973).1 t t t . J . Cancer 11, 104-115. Nelson, P., Christian, C., and Niremberg, M. (1976). Proc. Nutl. Acud. Sci. USA 73, 123-127.

RETRODIFFERENTIATION A N D G E N E EXPRESSION IN CANCER

173

Nicolson, G. L. (1974). Znt. Reo. Ciytol. 39, 90-190. Odell, W. D., and Wolfsen, A. (1975). 111 “Cancer, A Comprehensive Treatise” (F. F. Becker, ed.), Vol. 111, pp. 81-97. Plenum Press, New York. Okada, T. S., Yasuda, K., Hayashi, M., Hamada, Y., and Eguchi, G. (1977).Dee. B i d . 60, 305-309. Ono, T., and Weinhonse, S. (1972). I n “Isozyme and Enzyme Regulation in Cancer,” Gann Monograph. 13, Tokyo University Press, Tokyo. Onoe, T., Kaneko, A., Dempo, K., Ogawa, K., and Minase, T. (1975). Aitii. N.1’. Accrd. Sci. 259, 168-180. Onoe, T., Kaneko, A., Yoshida, Y., Dempo, K., Chisaka, N., Yokoyama, S., and Ogawa, K. (1976). I n “Onco-developmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 227-236. Academic Press, New York. Parker, C. A , , Hyatt, C., and Rosenberg, S. A. (1977). TraIt.si)lantcifioti 23, 161. People’s Republic of China (1974). “The Coordinating Group for the Research on Liver Cancer. Studies of Human a-Fetoprotein.” Republica of China. Perova, S. D., Elgort, D. A., and Abelev, G. J. (1971). R y u l l . Ek.s),. Biol. .\fed. (U.S.S.R.) 3, 45. Pierce, G. B. (1967). ZII “Current Topics in Developmental Biology” (A. A. Moscona and A . Monroy, eds.), Vol. 11, pp. 223-246. Academic Press, New York. Pierce, G. B. (1970). Fed. Proc. 29, 1248-1254. Pierce, G. B., and Wallace, C. (1971). Cancer Res. 31, 127-134. Pihko, H., and Ruoslahti, E. (1973). I i i t . J . Cmitcer 12, 3 5 4 3 6 0 . Polezhayev, L. W. (1946). (cited by R. J. Goss). Hiol. Reo. Combridge. Phil. Soc. 21, 141-147. Potter, V. R. (1969). Cuii. Coiicer Coiif. 8, 9-30. Pradja, N., Morris, H . P., and Weber, G. (1976). Criitcer Res. 36, 4639-4646. Price, M . R., and Baldwin, R. W. (1975). Z I I “Cancer, A Comprehensive Treatise” (F. F. Becker, ed.), Vol. I\’, pp. 209-236. Plenum Press, New York. Rogalsky, V. Y. (1976). Z i t “Onco-developmental Gene Expression” (W. H. Fishman and S. Sells, eds.), pp. 593-598‘. Academic Press, New York. Rogers, G. T. (1976). Hiochoii. Hio/)h!/s.,4ctn 458, 355-074. Rose, S. M. (1946).J . E.xii. Zoo/. 108, 337. Ross, J., Olnisted, J. B., and Rosenbaum, J. L. (1975). Tissrre Cells 7, 107-136. Roth, S. L., Delotto, R., and Kaji, A. (1977). Cancer Res. 37, 1147-1153. Runiyantsev, P. P. (1977). Z n f . Rec. C!/tol. 51, 188-273. Ruoslahti, E., Salaspuro, 41..Pihko, H., Anderson, L., and Seppala, M. (1974). Br. Meletf. /. 2, 527-529. Sanders, B. G., and Wiley, K. L. (1971).J. E s p . Zool. 178, 393-398. Sato, S., Matsushima, T., and Sugimrira, T. (1969a). Cancer Res. 29, 1437-1446. Sato, S., Matsushima, T. and Sugimura, T. (19691)). Cniicer Res. 29, 1437-1443. Sato, S., h r u c h i , Y., Takakrira, K., Chien, T. C., and Suginiura, T. (1972). Gniiri Motlogrtiph. 13, 279-288. Sato, S., Sato, T., Morris, H. P., and Weinhouse, S. (1975). Ann. N.1’. .4ccrd. Sci. 259, 273-286. Schapira, F. (1966). Eirr. J . Caiicer 2, 131-134. Schapira, F. (1973). A d c . Cnrtcer Res. 18, 77-153. Schapira, F., and Josipowicz, A. (1970). C . R . Soc. Biol. 164, 310. Schapira, F., Dreyfiis, J. C., and Schapira, G. (1963). Nottcre (Londoii ) 200, 995. Schmidt, A . J. (1968). “Cellular Biology of Vertebrate Regeneration and Repair.” University Chicago Press, Chicago. Schone, G. (1908). .29rrrricheii M e d . Woc/reii.sc/ir.51, 1. Schwarz; R. I., and Bissel, M . J. (1977). Proc. N u t / . Acurl. Sci. U S A 74, 4453-4457.

174

JOSE URIEL

Seilern-Aspang, F., and Kratochwil, K. (1963). Wien. K l i n . Wordienschr. 75, 337. Sell, S., and Morris, P. (1974). Ctrttcer Res. 34, 1413-1417. Sell, S., Nichols, M.:Becker, F. F., and Leffert, L. (1974). Cuticer Res. 34, 865-871. Silagi, S., and Bruce, S. A. (1970). Proc. N a t l . Acud. Sci. USA 66, 72-78. Singer, R. M., and Fishman, W. H. (1974).J. Cell R i d . 60, 777-780. Singer, R., and Fishman, W. H. (1976). In “Onco-developmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 177-184. Academic Press, New York. Stevens, L. C. (1967). A d o . Morphol. 6, 1-31. Stolbach, L., Inglis, N., Lin, C., Tiirksoy, R. N., Fishman, W., Marchant, D., atid Rule, A. Zii “Onco-developmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 433-443. Academic Press, New York. Stonehill, E. H., and Bendich, A. (1970).Notiire (Lotidon) 228, 370-372. Siigimura, T., Sato, S., and Kawabe, S. (1970). Biocheni. B i o p h y s . Res. Coinmiin. 39, 626-630. Tanaka, T., Ann, T., and Sakue, Y. (1971).J. Biocheni. (Tokyo) 69, 609-612. Tanaka, T., Inamiira, K., Ann. T., and Taniuchi, K. (1972). Gaiiii. Xloiiogrctph. 13, 219234. Teplitz, R. L., Sander, B. G., Brodetzky, A. M., Fung, H., and Wiley, K. L. (1974). C o m e r Rex. 34, 1049-1053. Thorpe, W. P., Parker, G . A., and Rosenberg, S. A. (1977).j. Z t i i m i i t i o l . 119, 818-823. Trincher, K. S. (1965). “Biology and Information. Elements of Biological Thermodynamics.” Consultants Bureau, New York. Tsuiki, S., and Miyagi, T. (1975). Ann. N.Y. Acud. Sci. 259, 2 9 8 3 0 6 . Uriel, J. (1969). Potliol. B i o l . 17, 877-884. Uriel, J. (1975).111 “Cancer, A Comprehensive Treatise” (F. F. Becker, ed.), Vol. 111, pp. 21-55. Plenoni Press, New York. Uriel, J. (1976). Concer Res. 36, 4269-4275. Uriel, J., Aussel, C., Bouillon, D., Nechaud de, B., and Loisillier, F. (1973).Nutiire New R i o l . 244, 190-192. Uriel, J , , Aiissel, C., Bouillon, D., Loisillier, F., and de Nechaud, B. (1975). Anit. N.1. Actrtl. Sci. 259, 119-130. Villee, C. A. (1954). Cold Sl)ring Hnrhor S ! / r n p . Qrrcrnt. B i d . 19, 186. Waldman, T. A., and McIntire, K. R. (1972). Ltrricet 2, 1112-1114. Walker, P. R., and Potter, V. R. (1972). Adu. Enzyme Reg. 10, 339-364. Watabe, H. (1971). Cniicer Res. 31, 1192-1194. Watanabe, A., Miyazaki, M., and Taketa, K. (1976a). Esperietitici 32, 470. Watanabe, A,, Taketa, K., Kosaka, K., and Miyazaki, M . (197611). I n “Oncodevelopmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 209-217. Academic Press, New York. Weber, G. (1966). Gaitit kfonogrcr))h. 1, 151-178. Weinhouse, S. (1973). Fed. Proc. 32, 2162-2167. Weinhouse, S., Shatton, J. B., Wayne, E . C., Furina, F. A,, and Morris, H . P. (1972). Gnnn M o r i o g r ~ ~ ~13, i / t . 1-17. Wepsic, H. T., and Sell, S., (1974). Prog. E.rp. Tumor Res. 19, 297. Wilson, P. D. (1973). Caiicer Res. 33, 375-382. Wilson, P. D., Benh;im, F., atid Franks, I,. M .(1977). Cell H i d . Z i t / . Rep. 1, 229-238. Yamatla, T. (1967).Z i i “Current Topics in Developmental Biology” (A. Monroy and A. A. Moscona, eds.), Vol. 11, pp. 267-283. Academic Press, New York. Yamada, T., and McDevitt, D. S. (1974). Deo. R i o l . 38, 104-1 18. Yanagi, I., MakitIra, S., Arai, M., Matsuniura, K., Hirao, K., Ito, N., and Tunaka, T. (1974). Coiicer R e s . 34. 2283-2289.

THE ROLE OF GLUTATHIONE AND GLUTATHIONE S-TRANSFERASES IN THE METABOLISM OF CHEMICAL CARCINOGENS AND OTHER ELECTROPHILIC AGENTS

L. F. Chasseaud

.

.

.

Department of Metabolism and Pharmacokinetics Huntingdon Research Centre Huntingdon United Kingdom

..................................... ..................................... I11. Glutathione S-Transferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Nature ............................................... C . Identity with Ligandin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Mode of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Assay and Stability ................................... F. In trace 1I 11 1ar Locatio ................................... G . Tissue Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . Introduction

H . Species Distribution

176 176 177 178 180 184 186 187 187

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

K . Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . Sex Ditferences .............................. IV. Compounds that Conjugate with Glutathione A . Halogenonitrobenzenes and Congeners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . 2-Chloro-S-Triazines and Congeners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Aryl Nitrocomporinds . . D . Phenol tetrabromphthaleins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................. E . Aralkyl Halides F. Aralkyl Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Alkyl Halides. Sulfates. or Nitrocompounds ................. H . Alkene Halides . . ................. I . Alicyclic Halides .................................. J . Ally1 Compounds K. L . Organophosphorus Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M . Arylhydrocarbon Epoxides (Arene Oxides) N . Arylhalide Epoxides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. Other Epoxide Intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................. P. ap-Unsaturated Compounds es. and Related Compounds Q . Arylaniines. Arylhydroxylam R . Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... .. S . Quinones and Catechols T. Isothiocyanates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . U . Trichloromethylsulfen yls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. v. Thiocarbamates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

194 199 200 201 202 204 205 207 208 213 216 217 219 219 22 1 226 227 230 235 240 241 242 243 244

176

L. F. CHASSEAUD

W. Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Other Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

244 245 251 255

I. Introduction

It is now realized that cancer can result from exposure to certain chemicals that are present in the environment, perhaps as drugs, food additives, industrial intermediates, pesticides, cigarette smoke, or other contaminants (Boyland, 1969; Miller and Miller, 1971; Higginson and Muir, 1973; Preussmann, 1976; Wynder and Gori, 1977). Knowledge of the metabolic fate of these chemicals is necessary because it may provide clues as to the underlying mechanisms of chemical carcinogenesis and an assessment of the carcinogenic risk involved through exposure to such compounds. Chemicals can be converted by a variety of biotransformation reactions into substances that are more, equally, or less toxic than the parent compound, and indeed it is believed that these biotransformation reactions (mainly oxidative) can produce carcinogenic species (ultimate carcinogens) from some compounds that may otherwise be relatively inert chemically (Miller and Miller, 1966, 1971, 1974; Magee, 1974; Sims and Grover, 1974; Heidelberger, 1975). Conjugation with glutathione (GSH), however, is one biotransformation process that generally results in less toxic products (Boyland and Chasseaud, 1969a). In 1969, Boyland and Chasseaud (196913) suggested that a probable function of GSH and its associated enzymes, the GSH S-transferases (EC 2.5.1.18), is the protection of cellular constituents from electrophilic agents. Since many carcinogens or mutagens are strong electrophilic agents (Miller and Miller, 1971, 1974, 1976; Magee, 1974; Heidelberger, 1975) which have the ability to covalently bind with DNA, RNA, and/or proteins of the cell in target tissues, it is appropriate to assess the role that GSH and the GSH S-transferases play in their detoxication. II. Glutathione

Glutathione (GSH) is a tripeptide (I) that is present in nearly all living HSCH,CHCONHCH,COOH

I

NHCOCH,CH,CHCOOH

I

N Hz (1)

GSH CONJUGATION WITH CARCINOGENS

177

TABLE I GLUTATHIONE C O N C E N T R A I I O N S IN SOME TISSUESOF M A L E WHITE MICE"

Tissue

Liver

Spleen Ki dne1.s Pancreas Lungs Heart Adrenals

Blood"

Concentr;ition" (mg/100gn1 tissue)

214 140 125 120 63 57 48 38

From Shaker and Soliman (1966). Determined by the nitroprrisside method: s e e ,Jocelyn (1972) for a survey of methods used to ineiisiire GSH in tissues. Esainples of rat tissue GSH levels were c i t c d 1 ) ~ Boyland ant1 Chasseaiid (1969a). GSH is alinost entirely present in the erythrocytes (Meister, 1975). 'I

cells. It is usually the most abundant sulfhydryl compound present in animal tissues (Meister, 1975), mainly in the cytosol. The concentrations of GSH are relatively high in some tissues (Table I), such as the liver. Concentrations of GSH present in a particular tissue may vary with respect to the nutritional status, hormonal balance, and growth of the organism (Knox, 1960). Histochemical studies have shown that GSH is uniformly distributed in rat liver but not in some other rat tissues such as the kidneys, lungs, testes, or e y e lens (Asghar et al.,

1975). The properties and functions of GSH have been the subjects of several reviews (for recent examples, see Flohe et ul., 1974; Arias and Jakoby, 1976). One recognized function of this substance relates to biological protection, for example, from the toxic effects of hydrogen peroxide through the mediation of glutathione peroxidase (Mills, 1960; Flohe et al., 1976) and from the toxic effects of electrophilic agents and other substances through the action of the glutathione S-transferases as discussed in this review. I l l . Glutathione S-Transferases

Conjugation with the nucleophile GSH is an important process for the elimination of electrophilic foreign compounds from the body (for previous reviews see Boyland and Chasseaud, 1969a; Wood, 1970;

178

L. F . CHASSEAUU

Chasseaud, 1976a; Hutson, 1976; Grover, 1977). The GS H conjugate has the requisite properties to be excreted in the bile (Chasseaud, 1973a,h), and it is also converted by a series of enzymic reactions (Fig. I) to an N-acetylcysteine conjugate (mercapturic acid), which may be excreted in the urine or bile. The first convincing evidence that GSH was the source of the cysteine moiety of mercapturic acids was presented in 1959 (Barnes et nl., 19S9; Bray et d., 1959). Conjugation with GSH is generally catalyzed by the GSH S -transferases (Booth et a / . , 1961; Boyland and Williams, 1965; Johnson, 1966; Boyland and Chasseaud, 1967; Boylaiid and Chasseaud, 1969b),a fairly nonspecific but partly selective group of soluble enzymes (Pabst et ul., 1973; Habig et ul., 1974a) that also function a s binding proteins (Ketley et ul., 1975). Earlier (Boyland and Chasseaud, 1969a; Chasseaud, 1973a) and more recent reviews (Grover, 1977; Jakoby, 1978; Jakoby et d., 1976a,b) of the properties of these enzymes are available. A. SUBSTRATES

The conjugations catalyzed by the GSH S-transferases ( E C 2.5.1.18) occur between the biological nucleophile GSH and compounds possessing a sufficiently electrophilic center. Kosower (1976) has R-X

+

HSCH,CHCONHCH,COOH I NHCOC H,C H,C HCOOH I

NH, glutathione S -transferases

1

R -SC H,C HCONHC H,COOH

I

NHCOCH,CH,C HCOOH I NH,

1

y-glutamylt r anspept idase

R- SCH,CHCONHCH,COOH I

NH, cysteinylglycinase R- SCH,CHCOOH I

N-acetylase

--

R-SCH,CHCOOH

NH,

FIG. 1 . Mercapturic acid formation.

I

NHCOCH,

GSH CONJUGATIOX WITH CARCCVOGENS

179

classified the nucleophilic reactions of GSH a s addition [Eq. ( l ) ] , addition-eliminatioil [Eq. ( 2 ) ] and , displacement [Eqs. ( 3 and 4)]. The resultant product is a thioether in each case. The wide range of substrates for these enzymes undergo one of these reactions. Most of the substrates for GSH S-transferases are synthetic chemicals, such a s halojienonitrobenzenes, or Phase I (Parke, 1968) biotransformation products, such as arene oxides, but some are naturally occurring, such as certain ap-unsaturated compounds, and possibly others are endogenous (Boyland and Chasseaud, 1969a; Chasseaud, 1 9 7 6 ~ Hutson, ; 1976; Grover, 1977). Of the latter type, possibilities include certain ap-unsaturated coinpounds, or alkyl and acyl groups re leased as carbon i iini ion s during 11iot ran s format ion react ion s, or epoxides formed its intermediates during oxidative metabolic reactions (Boyland and Chasseaud, 1969a). It intist be stressed, however, that many of the reactions catalyzed b y the GSH S-transferases also proceed nonenzymically, such a s those shown in Eqs. (1)-(4),and in some cases nonenzyniic reaction at physiological pH (and temperature) is so rapid that enzymic catalysis is difficult to demonstrate.

Qo

+

GSH

-

I

k0 I

0

0

CH,C1

CH,SG

/O\

OCH,HC-CH,

+ GSH

-

O_OCH,CH(OH)CH,SG

(4)

There are other less well-understood reactions of GSH with noncar-

boil electrophilic centers, involving the GSH S -transferases. These include reactions with the vasodilator organic nitrates (Needleman

180

L. F . CHASSEAUD

and Hunter, 1965; Habig et uZ., 1975; Keen et al., 1976) and the organic thiocyanates (Ohkawa and Casida, 1971; Ohkawa et al., 1972; Habig et d., 1975; Keen et al., 1976). Reaction of GSH with the latter leads to a toxic product (HCN) and an intermediate mixed disulfide [Eq. (5)]. The cyanide formed may also partly react with GSH, since one of its metabolites, 2-iminothiazolidine-4-carboxylic acid (Parke, 1968), could be formed by cyclization of the corresponding cysteine conjugate. GSH S -traderases probably also catalyze certain disulfide interchange reactions (Keen and Jakoby, 1978), and they appear to be involved in the conversion of prostaglandin endoperoxides to certain prostaglandins (Christ-Hazelhof et l i l . , 1976). Possibly more unexpected was the recent report that GSH S-transferases may possess isomerase activity toward A5-3-ketosteroids by promoting their conversion to the corresponding up-unsaturated A4-3-ketosteroids, a process that requires GSH which is not consumed in the reaction (Benson et u / . , 1977). GSH S-transferases also appear to possess GSH peroxidase activity (Prohaska and Ganther, 1977). CH3CHZSCN

+ CSH -+ CHSCHZSSC + HCN

(5)

B. NATURE Many of the earlier studies of the GSH S-transferases focused mainly on the types of second substrate involved and were carried out mostly with crude or only partially purified enzyme preparations (see reviews by Boyland and Chasseaud, 1969a; Chasseaud, 1973a). These studies nevertheless revealed that there was an apparent multiplicity of GSH S-transferases and that more than one enzyme may catalyze each reaction (Chasseaud, 1973a). Consequently generic names, such as GSH S-aryltransferase (Grover and Sims, 1964), S-epoxidetransferase (Boyland and Williams, 1965), S-alkyltransferase (Johnson, 1966), S-alkenetransferase (Boyland and Chasseaud, 1968), and Saralkyltransferase (Boyland and Chasseaud, 1969b) were adopted for the enzymes since it was believed that enzyme specificity was directed toward the type of second substrate catalyzed. That this belief was probably an oversimplification was revealed by work on the enzyme-catalyzed conjugation of GSH with various @-unsaturated compounds (Boyland and Chasseaud, 1968; Chasseaud, 1 9 7 3 ~ )Later . purification of the rat liver GSH S-transferases to homogeneity by Jakoby and co-workers (Fjellstedt et ul., 1973; Pabst et uZ., 1973; Habig et al., 1974a; Jakoby et a l . 1976a,b; Jakoby, 1978) enabled them to demonstrate that there was indeed a multiplicity of GSH Stransferases, but that these enzymes did, however, possess broad and overlapping second substrate specificities (Table 11). Any one GSH

181

GSH CONJUGATION WITH CAHCINOGENS

TABLE I1 SPECIFIC ACTIVITIESAND RELATIVE CONCEKTHATIOSS O F T l i E GSH S-TtWNSFERASES O F RAT LIVER" Eiizynie specific acti\it)

A

Substrate

l-Cliloro-2,~-cliiritrol~en~eii~ 62 1,2-DicliIoro-l-iiitrol)eiizeiie 4.3 \i-Ni trol~enzylcliloride 11.4 1,2-E1)o"y-3-(/)-1iitrophenos)) 0.1 propane Benzo [ti ] pyrene 4,ii-oxide 0.087 E t hacry n ic acid ND" Benzylidenr acetone 0.02

I'

B

C

E

14 0.008 0.09

11 0.003 0.1 N D"

10 2.0 10.2 NDh

0.01

0.004 0.3

0.01 1 0.26 0.001

0.22

Relative concentrittion "

AA

1

1 .0

0.14

N D" 4.1 6.7

0.098 0.11 0.40

0.069

0.52

0.10

N D" ND"

From Jakoby et (11. (1976a,b): units of' specific activity are pmoleslniinlmg protein. = not detected.

ND

S -transferase catalyzed the conjugation of GS H with several different types of second substrate (Pabst et al., 1973; Jakoby et ul., 1976a), although there appears to be almost absolute specificity for GSH as the thiol substrate. At the time, the purified enzymes were named GSH S-transferase AA, A, B, C, D, and E on the basis ofthe reverse order in which they were eluted from carboxymethylcellulose columns (Fig. 2)

t

f C

.-e> .c

I

I

,-\

0

I

E D

E

\

E

/

W

//

\

A

\

\

'L/

\

AA

\

\

p\

' \

'\J / I

'./'

/

Fractions

-

I \

\ \

FIG.2. Elutioii patterns of GSH S-transferase activity from carl~oxymethylcellulose coltinins. Enzyme activity toward l-chloro-2,4-diiiitrol~e1izene was assayed ( f r o ~ n Jakohy et ul., 1976;t, i n "Glutathione: Xletalmlism and Function," with pemiissioir t r o ~ n Raven Press, New York).

182

L. F. CHASSEAUD

during purification (Ketley et al., 1975; Jakoby et al., 1976a). GSH S-transferase E was the first of these enzymes to be isolated from rat liver in a pure state (Fjellstedt et al,, 1973), and purification of S-transferase A (Pabstet ul., 1974), B (Habiget al., 1974a), C (Habiget u l . , 1974a),and AA (Habiget al., 1976a) from rat liver followed. A GSH S -transferme M has also been characterized (Gillham, 1973) and small amounts of others may exist. Other purifications of these enzymes in rats and other species have been reported (Askelof et u l . , 1975; Clark et ul., 1977; Simons and Vander Jagt, 1977; Usui et u l . , 1977a; Hayakawa et al., 1977; Asaoka et ul., 1977; Grahnen and Sjoholm, 1977; Motoyama and Dauterman, 1977). These enzymes were calculated to represent about 10% of the soluble protein in rat liver (Jakoby et ul., 1976b) and about 2% in human liver (Nemoto et al., 1975; Habig et id., 1976b). The rat liver GSH S-transferases have a molecular weight of about 46,000 and consist of two subunits which may be identical, of approximately equal molecular weight. They have an isoelectric point above 7 and differ somewhat in amino acid composition and antigenicity, although in these respects GSH S-transferases A and C are closest (Habig et al., 1974a). GSH S-transferases, designated a,p, y , 8, and E on the basis of increasing isoelectric points have been isolated from human liver and were more closely related to each other than were those GSH S-transferases isolated from rat liver (Kamisaka et al., 1975; Habig et ul,, 197613). However, the isolated rat and human hepatic GSH S -transferases have a similar molecular weight which is the sum of two subunits of almost equal molecular weight, a basic isoelectric point, a selectivity for GSH as the thiol nucleophile, and a broad specificity for a variety of electrophilic agents. Further details of the properties of these enzymes may be found in the excellent reviews of Jakoby et al. (1976a,b), Jakoby (1978), and also in the more recent published work cited ab ove. With regard to enzyme nomenclature, GSH S-transferases A, B, etc. were convenient reference terms only for the pure enzyme proteins isolated from rat liver while the earlier names, such as GSH S -aryltransferase, are imprecise since they embrace the activity of several enzymes. However, the older names still may have descriptive usefulness provided that they are defined beforehand; thus GSH S-aryltransferase may be defined as GSH S-transferase activity toward a particular aryl substrate, e.g., 1,2-dichloro-4-nitrobenzene, or a group of aryl substrates. Appreciation of this definition enables the data presented in the literature employing these generic terms to be placed in proper context. Throughout this review, the enzymes in crude or par-

GSH CONJUGATION WITH CARCINOGENS

183

tially purified preparations are described as GS H S -transferases and the substrate(s) concerned is specified. The earlier generic terms are deliberately avoided.

c. IDENTITY WITH LIGANDIN An interesting consequence of the purification of the GSH S-transferases was the demonstration that rat liver GSH S-transferase B was apparently identical to rat liver ligandin (Habig et n l . , 1974b). Identity of ligandin with GSH S-transferases had been suspected from other data (Kaplowitz et u l . , 1973). Ligandin is a cytosol basic protein (pZ 9.0) ofmolecular weight about 45,000 that noncovalently binds a variety of compounds (Table 111), such as bilirubin, porphyrins, sulfobroinoI>hthalein, certain steroids, and benzylpenicillin (Levi et al., 1969; Litwack et al., 1971; Kaplowitz et d . , 1973; Ketterer et ul., 197511; Tipping et ul., 1976, 1978; Fleischner and Arias, 1976; Wolkoffet al., 1978). Ligandin, which is norinally present as 5 % of the total extractable protein of rat liver (Fleischner et ( i l , , 1972) is synonymous with Y-protein (Reyes et ul., 1971), corticosteroid I binding protein (Morey and Litwack, 1969), and azodye carcinogen binding protein (Ketterer et d . , 1967) as shown irnmunologically (Litwack et al., 1971). Thus, in addition to their catalytic role, the GSH S-transferases also serve as binding proteins, and ligandin (rat liver GSH S-transferase B), in particular, is thought to be TABLE 111 SOSIECOMPOUNDS THATARE BOLJVDBY LIGANDIN" C;lutathione Bi 1ir ubi 11 Estradiol Cortisol Testosterone Diethylstil1)estrol Sulfobroinoplithalein Indocyanine green ct~enotleosycl~olate N,N-Diniethyla~ni~ioazol~enzene :3-~letliylcliolanthrene Tetracycline Penicillin Etliacrynic acid

These compounds are among those listed i n the reviews of Arias c't NI. (1976), Fleisa i i d Aris (1976),and Smith c,t (11. (197711).

chner

184

L. F. CHASSEAUD

a major determinant of the net flux of organic anions from plasma to liver. The evidence for this hypothesis has been recently reviewed (Arias et d., 1976; Fleischiier and Arias, 1976). However, ligandin-binding organic anions such as bilirubin and indocyaiiine green, which do not react with GSH, are competitive inhibitors of GSH S-transferase activity (Kaplowitz et al., 1975b; Jakoby, 1978). The half-life of rat hepatic or renal ligandin has been reported to be about 2 days (Arias et d . , 1976). Iniinunodiffusion measurenients have suggested that the levels of ligandin present in the kidney (22 pg/nig supernatant protein) or small intestinal mucosa (17 pg/mg) of rats was about half that in the liver (45 pg/nig) (Fleischner et al., 1972, 1976). Ligandin was reported to be present in rat gonads (Bannikov et ill., 1973), but it could not be measured i n 16 other rat tissues including serum and bile, above a limit of detection of 3 pg/mg supernatant protein (Fleischner et d . ,1972). Rat kidney ligandin has been studied in some detail (Kirsch et ul., 1975). Ligandiii has been detected in a range of transformed, nontraiisformed, and hybrid cell lines (Smith et a l . , 1977a). According to the studies of Sarrifet (11. (1976),mouse liver h-protein, to which polycyclic hydrocarbons covalently bind, is not ligandin, although it possesses similar physical properties and also some apparent GSH S-traiisferase activity. Ligandin and h-protein were distinguished on the basis of irnmunological evidence, amino acid composition, and separation on CM-cellulose. 12-Protein has also been 1978). separated from other GSH S-traiisferases (Sarrif et d.,

D. MODE OF ACTION In contrast to other inajor conjugation processes, such as sulfation or glucuronidation (Parke, 1968), GS H conjugation does not require the initial formation of a high energy intermediate involving ATP, although synthesis of GSH from its component amino acids (Jocelyn, 1972; Meister, 1975) and N-acetylation of the cysteine conjugate (see Fig. 1) do utilize ATP (Green and Elce, 1975). Al-Kassab et (11. (1963), noting the low enzyme affinity for the second substrates that they studied, suggested that the action of the “enzyme” was to provide an activated GSH for conjugation with any suitable electrophile. Boyland and Chasseaud (1968) pointed out that as several GSH S-transferases existed, both GSH and the second substrate are specifically bound to the GSH S-transferases. This may also be concluded from the earlier work of others (Johnson, 1966; Clark et d . , 1967).

GSH CONJUGATION W I T H CARCINOGENS

185

The rate of the nonenzyinic reaction of second substrates with GSH increases in a parallel fishion to the increase in the proportion of GSH ionized, suggesting that GS- is the reacting nucleophile (Fig. 3 ) . Thus it may be argued that a function ofthe GSH S-transferase active site(s) would be (1)to promote ionization of the sulfhydryl group of GSH by lowering its pK (pK 9.2) and thereby increasing its nucleophilicity and (2) to bind the electrophilic substrate which can then react preferentially with GS- rather than with other cellular nucleophiles. Indeed recent studies by Keen et al. (1976) are consistent with the latter view. Other studies of the kinetic mechanisms of the GSH S-transferases have been reported (Gillham, 1973; Pabst et al., 1974; Mannervik and Askelof, 1975; Jakobson et al., 1977; Jakoby, 1978). GSH S-transferases may also covalently bind directly with certain extremely reactive substrates (Jakoby, 1976).This leads to inactivation of the enzymes. It could be argued that it is this particular reaction of the GSH S-transferases that precedes the toxicity to the cell that is caused by some substances, rather than the straightforward depletion of GSH itself, which has been suggested (Mitchell et al., 1975a). HowBO

2.0

70

1.5

60

-2

50 I

U

g

1.0

40

?

!n

0

I

v)

U

30

0.5

20

10 I

F I G . 3. Relationship Iwtween the proportion ot' GSH ionized ( W ) and its coi1jug;ition (0 = nonenzymic. A = etizvmic + tionenzymic) with diethyl m;ileate (from Chasseaud, 19741)).

186

L. F. CHASSEAUD

ever, an adequate cellular concentration of GSH is probably also necessary to maintain the integrity of the GSH S-transferases.

E. ASSAY A N D

STABILITY

Conjugation of GSH with electrophiles may be followed by measurement of the amount of substrate reacted or of product formed. Most of the methods described utilize titrimetric, colorimetric, spectrophotometric, or radioisotopic techniques. Jerina and Bend ( 1977) have surveyed some of the available procedures. Titration with iodate in the presence of iodide in sulfosalicylic acid using sodium starch glycollate as an indicator (Woodward and Fry, 1932) was used extensively in the studies of Boyland and Chasseaud (1967, 1968, 1969b). This method is rapid and accurate provided concentrations of GSH exceed about 1 mM. Colorimetric techniques have been used in the studies of Al-Kassab et (11. (1963) to measure nitrite production during enzyme-catalyzed conjugation of GSH with compounds containing labile nitro groups, such as 4-nitroquinoline N-oxide. Alternatively, the thiol reagent 5,5’-dithiobis-(2-nitrobenzoate) (Ellman, 1959) has been used to measure loss (reaction) of GSH (Johnson, 1966; Chasseaud, 1973c; Fjellstedt et ul., 1973). Spectrophotometric methods were preferred in the studies of Jakoby and co-workers (for details see Habig et ul., 1974a). Changes in absorbance at a particular wavelength that occur when the electrophilic substrate reacts with GSH are measured. Either product formation, for example, S-(2-chloro-4-nitrophenylglutathione) at 344 nm (Booth et al., 1961; Grover and Sinis, 1964) or substrate reaction, for example, ethacrynic acid at 270 nm (Habig et al., 1974a) may be followed. Several radioisotopic assays have been described (Johnson, 1966; Hayakawa et d . , 1974; Nemoto and Gelboin, 1975; Marniemi and Parkki, 1975). I n such assays, the electrophilic substrate may be radiolabeled. After incubation, unreacted radiolabeled substrate is extracted and the radioactivity in the extract and in the aqueous phase containing the GSH conjugate is measured. Alternatively, radiolabeled GSH could be used when a chromatographic separation stage would be required. Although the stability of enzymic activity toward an exhaustive range of substrates has not been studied, the GSH S-transferases appear to be a relatively stable group of enzymes when stored frozen in the original liver (Darby, 1973), crude tissue supernatants (Boyland

GSH CONJUGATION WITH CARCINOGENS

187

and Chasseaud, 1968), or purified preparations (Hayakawa et d . , 1974; Pabst et aZ., 1974). F. INTRACELLULAR LOCATION Like GSH, the GSH S-transferases are located principally in the cytosol. Crude differential centrifugation experiments showed that at least 80% of GSH S-transferase activity was present in the soluble supernatant fraction of rat liver (Booth et al., 1961; Johnson, 1966). The remaining activity was equally distributed among other subcellular fractions, h i t this may have Iieen due to contaminating cytosol. Studies of the enzyme-catalyzed conjugation of benzo[u Ipyrene 4,5oxide with GSH iiidicated that there may be some GSH S-transferase activity in the hepatic eiidoplasmic reticulum (microsomes) of mice constituting, however, only a minor proportion of total hepatic GSH S-transferase activity (Glatt and Oesch, 1977). The more recent purification studies of Jakoby and co-workers (Jakoby et d . , 1976a,b) clearly suggested that the GSH S -transferases are mainly present in the cytosol as had also been shown for ligandin (GSH S-transferase B in rat liver) (Fleischner and Arias, 1976). G. TISSUEDISTRIBUTION Most reports of tissue GSH S-transferase activity describe results obtained using relatively crude tissue preparations. Nevertheless, for ranking purposes, crude tissue preparations are preferable to purified tissue preparations since they probably more closely reflect the situation i n t-iuo. Predictability of the capacity of a given tissue to metabolize foreign compounds is important, particularly if the tissue is the site of entry or exit of foreign compounds and their inetalmlites from the l d y , e.g., lungs, liver, skin, gastrointestinal tract, kidneys. GSH S-transferase activity is generally greater in hepatic than in extrahepatic tissues (Tahles IV and V). Enzyme activity toward 1,2dichloro-4-nitrobenzene was particularly low in extrahepatic tissues (Booth et nl., 1961) but was higher in kiclney toward other substrates, such as methyl iodide (Johnson, 1966), benzyl chloride (Suga et al., 1967), and cyclohex-2-en-l-one (Boylarid and Chasseaud, 1968). GSH S -transferase activity has been examined in some detail in the kidneys (Boyland and Chasseaud, 1968, 1969b; Bend et d . , 1975; Clifton et al., 1975; James ef a / . , 1976), lungs (Gram et d . ,1974; Litterst et ~IZ., 1975; James et al., 1976), intestines (Datta et (/l., 1973a; James et al., 1976,

TABLE IV ACTIVITY TOWARD VARIOUSSUBSTRATES IN DIFFERENTRAT TISSUE PREPARATIONS RELATIVETO LIVER" GSH S-TRAXSFERASE Substrate 1,2-Dichloro-4nitrobenzene Methyl iodideb Benzyl chloride' Diethyl maleate Cyclohex-2-en-1-one Benzylidene acetone Naphthalene 1,2-oxided Methyl parathion" Diazinon Sdfobromophthalein

Kidney

Lung

Heart

Spleen

3

2

3

1

82 73 23 80 21 49 4 4 4

NW 4 29 23 26 10 6 4 1

ND 3 13 16 26 5 4 6 1

ND 13 11

23 25 8 8 1

Blood

Brain

Booth et al. (1961) ND 11 18 24 2 4

9 10 5

6

Calculated from data in the cited references (liver activity is expressed as 100 in each case). Adrenal, 35. Intestine, 4. " Skin, 2. Muscle, 3. ND, not detected. "

(I

Reference

Johnson (1966) Suga et d . (1967) Boyland and Chasseaud (1967) Chasseaud (1967) Chasseaud (1967) Hayakawa et al. (1974) Frikami and Shishido (1966) Shishido et al. (1972) Datta et al. ( 1 9 7 3 ~ )

189

G S H CONJUGATION WITH CARCINOGENS

TABLE V GSH S-TRANSFERASE ACTIVITY TOWARD 1,2-DICHLORO-4-N1TROBENZENEIN THE TISSUES OF ADULT MALE L AB OR AT O RY ANIMALS" Tissueh ~~

Species

Liver

Rat' hl ouse" Guinea pig Ham5ter R d h it hlon k e y

76.4 -c 28.9 74.4i 31.1 57.0 f 13.0 35.0f 11.1 21.9f 4.3 18.0

Kidney

3.8f 36.0t 7.5 It 3.6f 7.4 k 2.6

1.4 14.0 2.2 1.7 2.8

Lung

2.1t 0.3 21.3t 4.2 4.7f 2.9 2.8 t 0.4 5.3f 1.5 1.9

" From Litterst et t r l . (1975) except for monkey (.llocnccifrrsccita) data which were from Asaoka et (11. (1977)and were generated under ditferent experimental conditions. " Results (fSD) expressed iis nmoles of prodrict formed/min/nig protein. " Sprague-Dawley strain. " CDF, strain.

1977), and to a lesser extent in other tissues or fluids, such as the skin (Hayakawa et d.,1974; Mukhtar and Bresnick, 1976a), adrenals (Johnson, 1966), brain (Fukarni and Shishido, 1966; Hayakawa et u l . , 1974; Asaoka e t a l . , 1977), muscle (Fukami and Shishido, 1966), spleen (Boyland and Chasseaud, 1967; Chasseaud, 1967), placenta (James et a l . , 1977; Asaoka et ul., 1977), whole blood (Boyland and Chasseaud, 1967; Chasseaud, 1967; Mukhtar and Bend, 1977), and plasma or serum (Mukhtar and Bend, 1977). GSH S-transferases present in the liver and kidneys of rats were generally similar with respect to Michaelis constant (K,,,),inhibition by organic anions, such as probenecid, and the effects of hypophysectomy which resulted in increased enzyme activities of up to 100% (Kaplowitz et d., 1976). GSH S-transferase activity toward 1-chloro-2,4dinitrobenzene has been detected i n the proximal tubules but not i n certain other rabbit nephron segments (Fine et ul., 1978). The excretion of ligandin (GSH S-transferase B) in rat urine following doses of nephrotoxic agents may provide a useful index of acute injury to the proximal tubule (Feinfeld et al., 1977). The major route of entry of most carcinogens into the body is probably through the gastrointestinal tract, and it is therefore important that this tissue possesses adequate detoxifying enzymes, such as the GSH S-transferases. Clifton and Kaplowitz (1977) detected GSH S transferase activity toward some substrates, such as ethacrynic acid,

190

L. F. CHASSEAUD

in rat sinall intestine, particularly in proximal intestinal segments, but not toward other substrates, such as methyl iodide. Over 90% of GSH S-transferase activity toward l-chloro-2,4-dinitrobenzene was present in the small intestine of rat gastrointestinal mucosa, mainly in the duodenum and jejunum villus and crypt cells (Pinkus et a l . , 1977). As might be expected, GSH S-transferase activity was about 2-fold greater in the former cells than in the latter. Enzyme activity, in both cell types was induced by intraperitoneal treatment of rats with phenobarbital (80 mg/kg for 3 days) (Pinkus et u l . , 1977). Another major route of entry of carcinogens into the body is through the lungs; consequently pulmonary GSH S -transferase activity, particularly toward epoxides, has been examined (Bend et al., 1976; Mukhtar and Bresnick, 1976b,c), styrene oxide being a popular substrate for study. Grover (1974) has reported that rat lung preparations possessed greater GSH S -transferase activity toward the K-region epoxide of benz[n] anthracene than corresponding liver preparations, but this appears to be unusual (see Tables IV and V). GSH S-transferase activity in different regions of monkey brain toward 1,2-dichloro-4-nitrobenzene varied no more than 2-fold (Asaoka et ( i l . , 1977). Bend et (11. (1976) have reported that GSH S-transferase activity toward several epoxides in rat testes was about half that present in the liver, and activity toward 1,2-dichloro-4-nitrobenzene was about 30% ofthat in liver (Hinderer and Menzer, 1976a). The formation of GSH conjugates of beiizo[a Ipyrene 4,5-oxide b y the isolated perfused rat testis has been investigated (Lee et nl., 1978). GSH S-transferase activity toward l-chloro-2,4-dinitrobenzene in human peripheral leukocytes was about 2- to 4-fold lower than that in liver (Kaplowitz et ul., 1978). Juchau and Namkung (1974) have observed that GSH S-transferase activity toward naphthalene 1,e-oxide was higher in the fetal adrenals of pigtail monkeys (day 60) and humans (day 72) than in the liver, a somewhat unexpected finding. Similarly, activity in human fetal kidney exceeded that in liver, by about 3-fold (Juchau and Nainkung, 1974). It would be interesting to determine if this relationship of kidney > adrenal > liver activity also occurred in adults. GSH S-transferase activity toward 1,2-dichloro-4-nitrobenzenzeneand l-chloro-2,4-dinitrobeiizene in a range of nontransformed, transformed, and hybrid cell lines was lower than that measured in tissues (Smith et d . , 1977a). Extrahepatic conjugation with GS H has recently been reviewed (Chasseaud, 1979).

GSH CONJUGATION WITH CARCINOGENS

191

The relatively high concentrations of GSH and the activity of the GSH S-transferases in the liver suggest that generally this organ should be relatively best protected from the toxic effects of those carcinogens that undergo facile enzyme-catalyzed conjugation with GS H. However, this defense mechanism may fail or be overwhelmed in certain situations. The possible relationship between GSH conjugation and the susceptibility of a particular target organ to chemical carcinogens remains to be explored.

H. SPECIES DISTRIBUTION Hepatic GSH S-transferase activity has been detected in the livers of all the species so far examined (Tables V-VII). Enzyme activity in the livers of laboratory animals are generally greater than in those of humans (Table VI). However, further studies of GSH S-transferase activity in human liver are required; the existing data are based on a limited number of samples only. One fairly detailed study of GSH S -trailsferase activity toward l-chloro-2,4-dinitrobenzenzene showed that enzymic activity in rat and human liver ( n = 59) was similar (Darby,

RELATIVE

TABLE \'I DISTRIBUTION OF C L U T A T H I O N E S -TUNSFERASE ACTIVITY IN DIALYZED LIVERSUPERNATANTS FROM VARIOUS SPECIES" Relative activity toward

Species Rat" ( P ) Dog(d + 9 ) Guinea pig ( 6 ) Mouse (6) Rabbit ( d ) Ferret ( 6 ) Pigeon (6) Hamster ( d ) Human, adult ( P ) Human, fetus ( d )

1,2-Dicliloro4-nitrobenzene

Methyl iodide

Benzyl chloride

2,3-Epoxypropyl phenyl ether"

Diethyl maleate

1.00 2.80 2.46 2.40 1.40 1.OO 0.80 0.40 0.08 N D"

1.00 0.16 0.50 1.16 0.36 0.11 3.56 0.56 0.26 0.23

1.00 0.69 3.14 0.82 0.47 0.13 1.43 0.82 0.21 0.30

1.00 0.42 1.06 1.42 0.14 0.38 0.47 0.80 0.19 0.23

1.00 0.39 0.93 1.62 0.22 0.52 0.36 0.90 0.13 0.12

Calculated from the data of Boyland and Chasseaud (1969b). 1,2-Eposy-3-pheiioxy~~r~~~aiie. " Absolute enzyme activities for the rat were 0.5, 3.0, 2.3, 2.1, and 7.5 pinoles ofthiol reactediminigm tissue, respectively. d ND = not detected. "

192

L. F . CHASSEAUD

TABLE VII DISTRIBUTION OF HEPATICGLUTATHIONE S-TRANSFEHASE ACTIVITYTOWARD 1,2-DICHLOHO-4-NITHOBENZENE IN DIFFEHENT SPECIES" Species

Enzyme activity"

Mouse ( d ) ? Mouse ( 0 ) Rat ( ~ 3 ) ~ Rat ( 0 ) Dog Guinea pig ( d ) Guinea pig (P) Cat Hamster Sheep Horse Rabbit Monkey (rhesus) Lizard Pig Man Chicken Go Id fis h Duck Frog

4.6 9.6 6.4 4.1 5.3 3.5 2.9 2.8 1.9 1.1 1.0 1 .0 0.9 0.7 0.4 0.3 0.2 0.2 0.1 0.1

From Grover and Sims (1964). Units, 10' x Fimoles of product tbrmetl/min/mg protein. CBNH strain. " Chester Beatty strain.

"

"

1973). Hepatic GSH S-transferase activity toward sulfobromophthalein was reduced by about 4-fold in human cirrhotic subjects compared to normal controls (Boyland and Grover, 1967; Datta et ul., 197313). Calculation has shown that the GSH S -transferases represent about 10 and 2%, respectively, of the total protein extractable from rat and human liver, respectively (Nemoto et al., 1975), and purification of these enzymes from both these sources has been reported (Fjellstedt et al., 1973; Pabst et ul., 1974; Habig et ul., 1974a; Kamisaka et al., 1975). It is claimed that ligandin-like proteins are present in lungbreathing amphibians, reptiles, birds, and of course mammals (Levine et al., 1971). GSH S-transferase activity is present in vertebrate species such as the lizard and frog (Grover and Sims, 1964) and birds

GSH CONJUGATION WITH CARCINOGENS

193

(Wit and Snel, 1968; Wit, 1968; Hinderer and hlenzer, 1976b), in insects, such a s the grass grub, housefly, and locust (Cohen ef ( I / . , 1964; Clark et cil,, 1967; Goodchild and Smith, 1970; Clark et (/I., 1973, 1976; Motoyama and Dauterman, 1975), in wornis (Douch aiid Buchanan, 1978), and in plants (Frear aiid Swansoii, 1970; Shimaliukuro, 1975). It is interesting that the susceptibility of corn to tlie herbicide atrazine appears to be determined by its GSH S-transferase activity which was much lower in atrazine-siisceptilile corn than in atrazine-resistant corn (Shimabukuro et u l . , 1971). I. MATURATION Existjiig data have shown that hepatic or extrahepatic GSH S -transferase activity is lower in the fetal or neonatal tissues of laboratory animals than in the corresponding iiiaternal tissues (Klaassen, 1975; Bend et a l . , 1975; James et (il., 1977; Bairies et (il., 1977; Pegg and Hook, 1977). By contrast, the rather limited available evidence indicates that human fetal (>14 weeks) and adult liver possess similar GSH S-transferase activity (Grover and Sims, 1964; Boyland and Chasseaud, 1967, 1968, 1969b). If confirmed, this situation parallels the relatively earlier development of drug oxidative capacity i n tlie human fetus compared to those of lalioratoiy animals (Pelkonen, 1977). Glutathione S -transferase activity toward sulfobroniophthalein in rat liver has been shown to increase slowly to adult levels during a 9-week postnatal period (Conibes and Stakelum, 1962; Klaassen, 1975). More extensive studies of the development of GSH S-transferase activity toward 1,2-dichloro-4-iiitrolienzeiieor styrene oxide in the livers, lungs, kidneys, or intestines of guinea pigs showed that enzymic activity developed most rapidly in the lungs which possessed about 80% of adult activity at day 38 of a gestation period of about 68 days. Enzynie activities developed more slowly in the small intestines (Bend et u l . , 1975; James et al., 1977). Parallel studies in rabbits also indicated that enzyme activity developed at different rates in these tissues and that there was an apparent decline in specific activity at around birth (James et a l . 1977). Rat liver and kidney GSH S-transferase activity toward 1,e-epoxy3-(p-nitrophenoxy)propane was < 10% of adult levels at birth, gradually reaching tlie latter at about 40 days of age (James and Pheasant, 1978). However, this maturation of enzyme activity did not appear to influence tlie proportion of the dose (ca. 25%) excreted as the corresponding mercapturic acid in oioo (James and Pheasant, 1978), possi-

194

L. F. CHASSEAUD

bly because hepatic GSH levels were similar in both neonatal and adult animals. GSH S-transferase activity toward styrene oxide or 3-methylcholanthrene 11,12-oxide in the lungs of 18-day fetal rats was about 25% of that in the corresponding liver which was about 20% of that in the adult. Thereafter lung and liver enzyme activities reached maximum levels measured during 20-30 days after birth (Mukhtar and Bresnick, 1 9 7 6 ~ )The . lower GSH S-transferase activity in the tissues of these fetal or neonatal rats may partly explain their apparently greater susceptibility to the carcinogenic effects of polycyclic aromatic hydrocarbons, such as 3-methylcholanthrene. The livers of 5-day-old rats contained about 10% of the adult (35day) levels of ligandin (GSH S-transferase B). Levels increased slowly during the first 15 days of age and more rapidly thereafter (Klaassen, 1975). Neonatal liver ligandin content can be increased at least 2-fold by administration of microsomal enzyme inducers, such as pregnenolone-16a-carbonitrile (75 mg/kg) (Klaassen, 1975). Similar results have been obtained b y Hales and Neims (1976a) who also noted that the percentage increase in GSH S -transferase activity after phenobarbital induction appeared to remain constant from 5 days postnatally to adulthood (Fig. 4). Radial immunodiffusion studies have shown that the concentration of hepatic ligandin in the late human fetus (6.8 pugling ? 2.2 SEM, n = 6) was lower than that in the adult (17.9 pg/mg & 3.6 SEM, n = 13) (Fleischner et al., 1976). GSH S -transferase activity toward l-chloro-2,4-dinitrobenzene in liver, kidney, and small intestine supernatants from newborn homozygous mutant albino mice was more than %fold greater than in those from normal newborn controls and similar to those from control adult mice (Gatmaitan et d,, 1977). Early GSH S-transferase maturation in the mutant homozygous mice may reflect the effects of accumulation of substrates usually metabolized by the cytochrome P-450 system which was virtually absent in these mice.

J. INDUCTION Hepatic GSH S -transferases are inducible by microsomal drugmetabolizing enzyme inducers, such as phenobarbital, polycyclic hydrocarbons, and certain organochlorine compounds. In general, enzyme activity in rats may be induced by up to %fold, but usually less. Earlier studies (Grover and Sims, 1964; Suga and Akagi, 1970; Darby and Grundy, 1972) did not detect significant induction of GSH S-transferases by phenobarbital administration to rats, presumably because

GSH CONJUGATION WITH CARCINOGENS

195

0.06

-

E . E . -z 0

-5a. 0.04 w

L > U

U

E

t W

0 02

I

1

1

1

0

20

40

60

A ao

Age (days)

FIG.4.Phenobarbital induction of rat hepatic GSH S-transferase activity toward 1,2dichloro-4-nitrobenzene during development (from Hales and Neirns, 197621).Phenobarbital-pretreated rats ( 0 )received 75 mg/kg/day intraperitoneally for 3 days and controls (A)received 0.9% (w/v) sodium chloride.

treatment with the inducer was insufficient. That the GSH S-transferases were inducible was first discovered indirectly when it was shown that Y-protein (ligandin, GSH S-transferase B) was induced in rats by phenobarbital treatment (Reyes et al., 1969). This was confirmed by subsequent work (Fleischner et al., 1972; Habig et al., 1974b), and other studies have extended the list of known inducers of ligandin (Arias et al., 1976). However, the extent to which particular inducers enhance ligandin or GSH S-transferase activity has not been rigorously determined. Phenobarbital appears to be a more effective inducer of rat liver ligandin than are several other substances, such as spironolactone or 3-methylcholanthrene (Klaassen, 1975; Hales and Neims, 1977).

196

L. F. CHASSEAUD

Phenobarbital-enhanced biliary excretion of intravenously administered sulfobromophthalein in the rat was partly due to induction of GSH S -transferases responsible for the conjugation of this compound with GSH (Whelan and Cornbes, 1975). Hepatic ligandin content was 2-fold greater in phenobarbital-treated humans (40.6 pglmg 4.7 SEM, n = 5) than in untreated subjects (17.9 pg/mg f 3.6 SEM, n = 13) (Fleischner et uZ., 1976). Kaplowitz and co-workers examined the induction of rat hepatic and renal GSH S -transferase activity by phenobarbital, benzo[a]pyrene and 3-methylcholanthrene (Table VIII). Renal enzyme activities were induced less than those in the liver (Clifton et al., 1975; Kaplowitz et al., 1975a; Kaplowitz and Clifton, 1976). Intraperitoneal administration of phenobarbital (75 mg/kg) or 3-methylcholanthrene (40 mg/kg) to six different mouse strains (C57BU6,C3H, C3Hf-, BALB/c-, A+,and DBA/2+)did not induce GSH S-transferase activities in lung toward styrene oxide or 3-methylcholanthrene 11,12-oxide (Mukhtar and Bresnick, 197613). However, enzyme activity in the liver was induced in all strains of mice by the

*

TABLE VIII INDUCTION OF GSH S-TRANSFERASE ACTIVITY IN MALE RATS AFTER INTRAPERITONEAL INJECTION OF ENZYMEINDUCERS FOR 7-10 DAYS"

Tissue and substrate typeb Liver Alkyl A d Aralkyl Epoxide Kidney Alkyl A d Aralkyl Epoxide

Control

Phenobarbital

Benzo[u I pyrene

3-Methylcholanthrene

16.6 t 0.6 113 2 5 169 t 6 11.6 ? 0.7

23.1 2 0.9* 190 +- 6" 274 t 17* 15.2 i 1.0"

18.9 ? 0.8* 142 t 9* 233 t 12% 14.2 t 1.0*

20.7 t 1.1* 151 i 6 * 240 t 7* 14.7 2 0.7*

15.3 ? 0.8 3.80 ? 0.29 29.5 i 2.7 7.89 2 0.40

16.3 t 1.1 3.80 i 0.21 44.5 t 1.8* 8.30 2 0.47

15.4 t 1.1 6.72 ? 1.32* 42.6 i 0.33* 8.56 t 0.47

16.5 i 1.1 6.58 t 0.66* 54.9 t 0.53* 8.35 ? 0.47

From Kaplowitz et (11. (19754 and Clifton et (11. (1975). used were methyl iodide, 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, and l,e-epoxy-(p-nitrophenoxy)propane, respectively. L. Approximate dose levels were 80, 12, and 12 mg/kg, respectively. Statistically significant increases in enzyme activity (expressed as nmoles of product formed/min/mg protein ? SEM, n = 10) are indicated by an asterisk. 'I

* Substrates

GSH CONJUGATION WITH CARCINOGENS

197

former inducer, but not by the latter, perhaps because the treatment period (2 days) was too short (Mukhtar and Bresnick, 1976b). Baseline GSH S-transferase activity in the liver or lung was similar in all six mouse strains as was the extent of hepatic enzyme induction (about 50%) caused by phenobarbital treatment (Mukhtar and Bresnick, 197613). Intraperitoneal treatment of rats with either phenobarbital (75 mg/kg for 3 days) or 3-niethylcholanthrene (20 mg/kg for 2 days) resulted in about a 50% increase in hepatic GSH S-transferase activity toward styrene oxide and 3-methylcholanthrene 11,le-oxide (Mukhtar and Bresnick, 1976d). Failure of 3-methylcholanthrene administration (20 nig/kg for 2 days) to induce rat liver GSH S-transferase activity toward styrene oxide has also been reported (Mariiiemi and Parkki, 1975). Hepatic GSH S -transferase activity toward styrene oxide was enhanced by about %fold within 1 day of an intraperitoneal dose of a polychlorinated biphenyl mixture (100 mg/kg) or of DDT (160 mg/kg) to rats and, not surprisingly, remained elevated for many days (Parkki et ul., 1977). This activity was also enhanced by about 50% in rats treated with several cutaneous doses (8 mg/kg) of a polychlorinated biphenyl mixture (Marniemi et al., 1977). Significant induction of GSH S-transferase activity toward ethacrynic acid and p-nitrobenzyl chloride occurred in the more distal segments of rat intestine after intraperitoneal treatment with benzo[a]pyrene or 3-niethylcholanthrene at a dose level of about 7 mg/ kg/day for 10 days. Activity toward 1,2-epoxy-(p-nitrophenoxy)propane was not induced by this treatment (Clifton and Kaplowitz, 1977). Mouse liver GSH S-transferases may be more responsive to the inducing etfects of phenobarbital than those in rat liver (Kulkarni ef u l . , 1978), which appear more responsive than those in the livers of nonhuman primates (Table IX). Thus it is possible that the inducibility of the activities of the GSH S-transferases varies in different species and also is a reflection of the substrate used for their measurement and/or the enzyme inducer used for their induction. This aspect merits further study. The individual inducibility of “aryl hydrocarbon hydroxy1ase”-an enzyme system measured by its conversion of benzo[a]pyrene mainly to 3-hydroxybenzo[ci]pyrene at least partly through an intermediate epoxide (Yang et u l . , 1977)-in cultured human lymphocytes was under strict genetic control (Kellermann et t i / . , 1973a; Kellermann, 1976). However, the apparent relationship of this enzyme system’s inducibility with the likelihood of an individual developing bronchogenic carcinoma (Kellermann et d., 1973b) has been questioned

198

L. F. CHASSEAUD

TABLE IX EFFECTOF ORALDOSESOF ENZYME INDUCERS ON HEPATICGSH S-TRANSFERASE ACTIVITY IN RATS AND NONHUMAN PRIMATES" Inducer (dose level)',"

Species and substrate" Rat DCNB DEM BA Rhesus monkey DCNB DE M BA Baboon DCNB DEM

Control

Phenobarbital (15 indkg)

Phenobarbital (50 m d k d

DDT (15 mdka)

492 ? 104 6.4 t 1.4 2.7 k 1.1

695 t 140* 7.3 t 1.0 2.6 k 0.5

911 k 279* 7.9 k 1.0* 2.9 2 0.5

722 t 69* 10.0 t 1.4* 4.5 c 1.0*

129 k 33 2.5 ? 0.4 0.4 2 0.3

189 k 25* 2.6 k 0.4 0.4 k 0.2

133 ? 37 2.6 ? 0.6 0.6 +- 0.3

64 k 33 1.6 k 0.7

7 4 k 14 1.9 ? 1.4

5 7 ? 11 1.9 ? 1.0

Unpublished data of Down and Chasseaud. DCNB, 1,2-dichloro-4-nitrobenzene (nmoles conjugate formed/hr/mg protein SD); DEM, diethyl maleate and BA, benzylidene acetone (pmoles GSH reacted/hr/mg protein t SD). Daily doses during 21 days. 'I Statistically significant increases in enzyme activity are indicated b y an asterisk. "

*

(Paigen et al., 1977). Similar pharmacogenetic studies have yet to b e reported for the GSH S-transferases which also occur in cultured human lymphocytes (Booth et al., 1974; Kaplowitz et al., 1978). Such studies are necessary because of the protective nature of the GSH S-transferases.

K. INHIBITION Although probably of greater consequence toxicologically than induction, inhibition of GSH S -transferases has not been extensively studied. The enzymes are known to be inhibited by phthaleins at least in insects (Clark et ul., 1967), drugs such as the coronary vasodilator benziodarone (Boyland and Grover, 1967), certain radiographic contrast media (Goldstein and Arias, 1976), organotin compounds (Henry and Byington, 1976), nonsubstrate ligands such as bilirubin, indocyanine green (Kaplowitz et al., 1975b, 1976; Ketley et al., 1975),

GSH CONJUGATION WITH CARCINOGENS

199

and steroid sulfates (Oh1 and Litwack, 1977), as well as their reaction products, the GSH conjugates (Johnson, 1966; Boyland and Chasseaud, 1968; Gillham, 1973; Askelof et nl., 1975). Inhibition of GSH S-transferases by their own substrates has been reported (Booth et nl., 1961; Hayakawa et al., 1975), but this is quite likely to be due in many cases to the GSH conjugate formed by the “inhibitor” since it is also a substrate. It is possible that accumulation of GSH conjugates in a particular tissue in oivo such as might occur after excessive exposure to electrophilic agents, could lead to reaction product-inhibition of the GSH S-transferases and their subsequent failure to operate in the detoxication of the electrophilic agents. This phenomenon could be an explanation for the toxicity caused by administration of relatively large doses of certain compounds, rather than straightforward depletion of GSH. Pretreatment of dialyzed rat liver supernatant with “group-specific” agents such as l-fluoro-2,4-dinitrobenzene, acetic anhydride, or formaldehyde destroyed much of the GS H S -transferase activity toward diethyl maleate, whereas treatment with iodoacetamide, bromopyruvic acid, cyanide, ferricyanide, or dithiothreitol were without effect, and phenylmercuric acetate or iodine had only a moderate effect (Chasseaud, 1976b). GSH S-transferases are unlikely to be unduly sensitive to thiol-specific agents or they would be readily inactivated by their own substrates. However, some strong electrophiles, such as l-chloro-2,4-dinitrobenzene (cf. l-fluoro-2,4-dinitrobenzene above), bind to these enzymes and thereby cause their inactivation (Jakoby, 1976). This function of the GSH S-transferases amounts to a “suicide” reaction and may partly explain the toxic consequences observed after administration of certain compounds to animals.

L. SEX DIFFERENCES Differences in hepatic and renal GSH S-transferase activity toward some substrates between male and female rats have been reported (Darby and Grundy, 1972; Clifton et d . , 1975; Kaplowitz et d.,1975a; Kaplowitz and Clifton, 1976). Male rat liver apparently possessed more activity (up to 3-fold) toward certain substrates than female rat liver (Darby and Grundy, 1972; Kaplowitz et nZ., 1975a; Hales and Neims, 1976b), whereas the converse mainly occurred in rat kidney (Clifton et uZ., 1975). The proportion of ligandin (GSH S-transferase B) in a 100,000 g supernatant from female rat liver was 4.5% of the total protein present and was significantly greater than that from male rat liver (3.3%).

200

L. F. CHASSEAUD

Hypophysectoniy of the female rat abolished this sex difference (Hales and Neims, 1976b). At present the implications of these reported sex differences in GSH S-transferase activity are not clear. It would be interesting to determine whether certain substrates for the GSH S-transferases are more toxic to female than to male rats. Sex differences in GSH S-transferase activity have been shown also in other rodent species, such as the mouse (Hayakawa et al., 1974). A nonrodent species for which sex differences in hepatic GSH S-transferase activity have been reported is the tree shrew (Tupaia): the livers of females (25.1 nmoles product/min/mg +- 3.1 SD, n = 6) contained significantly more enzymic activity toward 1,2-dichloro-4nitrobenzene than those of males ( 16.2 nmoles producthinhng t 3.2 SD, n = 6) (Litterst et nl., 1976). No significant sex differences in GSH S-transferase activity toward l-chloro-2,4-dinitrobenzei1e in human biopsy or postmortem liver were detected during a relatively extensive study by Darby (1973). IV. Compounds that Conjugate with Glutathione

A wide range of electrophilic agents are known to undergo enzyme-catalyzed conjugation with GSH. Previous reviews (Boyland and Chasseaud, 1969a; Wood, 1970; Chasseaud, 1973a, 1976a; Hutson, 1976; Grover, 1977) have described and discussed the earlier work. This review is mainly concerned with newer developments. Compounds are grouped using the system adopted previously (Chasseaud,

19764. A. HALOGENONITROBENZENES AND CONGENERS The metabolism of halogenonitrobenzenes has been extensively studied (see Bray et al., 1957). Most, but not all of these compounds, were excreted in the urine of rabbits partly as mercapturic acids, and presumed conjugation with GSH occurred at the position of elimination of a halogen or nitro group. Thus 41% of a dose of 1,2,3trichloro-4-nitrobenzene (11) and 10% of a dose of 1,2,4-trichloro-3nitrobenzene (111) were excreted in rabbit urine as mercapturic acids by elimination of a chloride and nitrite moiety, respectively. The leaving group may be predicted since increased lability of a particular halogen or nitro group during alkaline hydrolysis was related to increased mercapturic acid formation in vivo (Betts et nl., 1955; Bray et nl., 1957). Examination of the tumor-initiatory activity of pen-

GSH CONJUGATION WITH CARCINOGENS

c1

c1

CI

(m

(nI)

(IV)

20 1

C1

(VJ

tachloronitrobenzerie and three tetrachloronitrobenzenes on mouse skin revealed that the compound with a stable nitro group 1,2,3,4tetrachloro-6-nitrobenzene was the most potent (Searle, 1966). Indeed much less mercapturic acid was produced by rabbits treated with this compound than with other tetrahalogenonitrobenzenes (Bray et d., 1957). Important chloroiiitrobenzenes include 1,2-dicliloro-4-nitrobenzene (IV) and l-chloro-2,4-dinitrobenzene (V), both frequently used to assay GSH S-transferases (e.g., Booth et d.,1961; Grover and Sims, 1964; Boyland and Chasseaud, 1969b; Habig et d.,1974a, 197th). The chlorine atom at C-1 is replaced by GS-. Other related substrates for GSH S-transferases include 1,2,3,5- and 1,2,4,5-tetrachloro-6-nitrobenzenes and pentachloronitrobenzene (Al-Kassab et al., 1963). The latter has been reported to cause hepatomas in mice (Innes et ul., 1969) and to be mutagenic to E . coli (Clarke, 1971). GSH conjugates appear to have been formed when yeast cells were exposed to the fungicide chlorothalonil (VI) (Tillman et al., 1973), and GSH seems partly to fulfill a protective role which would also be expected to operate in higher biological systems. CN I

c1

".

CN

B. ~-CHLORO-S-TRIAZINES AND CONGENERS Since the identification of a mercapturic acid and a GSH conjugate as major urinary and biliary metabolites, respectively, of the herbicide cyanazine in rats (Fig. 5) (Hutson et c d . , 1970; Crayford and Hutson, 1972), similar metabolites of other s-triazines have not been reported (Bedford, 1975). However, atrazine (VII) undergoes enzyme-catalyzed

202

L. F. CHASSEAUD NHCOC H, I

A

N/

fGSH

N

CH, CN

R

SCH,CHCOOH

SG

c1

A

N’

R AN’

CH,

N CH,

_____

NHtCN

A

N’

N

*ANANHT;N R

CH,

CH,

FIG.5. Biotransforniation of an s-triazine herbicide (cyanazine) ( R = NHEt) to GSH conjugates excreted in the bile and mercapturic acids excreted in the urine ( R = NHEt or NH,). The major metabolites cont$ined the free amino group ( R = NH,) (Crayford and Hutson, 1972).

conjugation with GSH in plants (Lamoureux et al., 1970; Shimabukuro et al., 1970, 1971) and presumably this also occurs in mammals (see Dauterman and Muecke, 1974). Conjugation of cyanazine with GSH (Fig. 5) was catalyzed by GSH S-transferases in mouse, rat, rabbit liver (Crayford and Hutson, 1972).

c1 N A N C*HSHN

C,H,HN

(VII)

NHC(CH,),CN (VIII)

Formation of a GSH conjugate of cyanatryn (VIII) in rats required initial bioactivation of (VIII), probably to a sulfoxide which could react with GSH by displacement of inethylsulfenic acid (Bedford et ul., 1975). Conjugation of cyanazine with GSH (Fig. 5) exemplifies detoxication. The LD,, of the herbicide was 294 mg/kg in the rat and that of the GSH conjugates exceeded 1000 mg/kg (Crayford and Hutson, 1972). C. ARYLNITROCOMPOUNDS Despite fhcile enzyme-catalyzed conjugation of GSH with aryl nitrocompounds, such as 4-nitroquinoline N-oxide (IX) (Al-Kassab et ul., 1963) and 5-nitrofurfural (X) (Boyland and Speyer, 1970), which contain labile nitro groups, detection of the expected conjugates, such as the mercapturic acid, in the excreta has not been reported. 4-Nitroquinoline N-oxide (IX) and some of its derivatives are potent skin carcinogens in mice (Nakahara et ol., 1957), and (IX) also produces lung tumors in rats (Mori, 1964). It has been demonstrated that

G S H CONJUGATION WITH CARCINOGENS

(rx)

203

(XI)

(X)

carcinogenicity roughly paralleled the extent of reaction of these compounds with GSH (Endo, 1958),although the latter is a probable detoxication route. Indeed rat liver GSH S -transferase activity toward the carcinogenic 4-nitroquinoline N-oxide was about 5-fold greater than toward the noncarcinogenic 4-nitropyridine N-oxide (XI) (Al-Kassab et al., 1963). More recent work has suggested that the ultimate carcinogenic form of (IX) is the corresponding hydroxylamino derivative (Kawazoe et al., 1969; Matsushima and Sugimura, 1971), which can oxidize GSH (Hozumi et d., 1967), and is produced through initial reduction of the nitro group of (IX) (Kato et d . , 1970).

Although several 2-substitutec1-5-nitrofurans [Eq. (6)] are substrates for the GSH S-transferases (Boyland and Speyer, 1970), many others are not, such as the carcinogenic 2-(2-formylhyclrazino)-4-(5-nitro-2fury1)-thiazole (XII) and nitrofurazone (XIII) (Morris et a1 ., 1969). Lability of the nitro group to alkaline hydrolysis approximately correlated with the GSH S-transferase activity toward a particular nitrofuran (Boyland and Speyer, 1970), and this procedure may enable

4 N

CH =NNHCONH,

02N

0

(xm)

prediction of those nitrofurans that would be substrates for the GSH S-transferases. It is not presently known whether there is any correlation between the potential tuinorigenicity of a particular 2-substituted5-nitrofuran a nd the extent to which it conjugates enzymically with GSH. This aspect is surely worthy of investigation in view of the mutagenic (Wang and Lee, 1976; Yahagi et al., 1976; Rosenkranz, 1977) and carcinogenic properties (Morris e t d , , 1969; Cohen et ul.,

204

L. F. CHASSEAUD

1973; IARC, 1974c; Takayama and Kuwabara, 1977) of this class of compounds. Assuming that the ultimate carcinogenic (reactive) species from such compounds is a corresponding hydroxylamino derivative (or a closely related intermediate) arising by initial reduction of the nitro group, then removal of the nitro group by reaction with GSH would be expected to reduce or even abolish the carcinogenic potential of the nitrofuran. However, a relatively stable nitro group may be a prerequisite for the adequate bacteriostatic action of this class of compounds (see Dodd and Stillman, 1944). Compounds analogous to the 5-nitrofurans include the 5-nitrothiophenes and the 5-nitroimidazoles, some of which have been shown to be carcinogenic (Cohen et al., 1973, 1976; Rustia and Shubik, 1972), but no information is readily available regarding their possible conjugation with GSH. The major metabolite (41% dose) of the herbicide fluorodifen in rats was the mercapturic acid (Lamoureux and Davison, 1975) arising by initial enzyme-catalyzed conjugation with GSH [ Eq. (7)] which had earlier been reported for plants (Shimabukuro et al., 1973; Frear and Swanson, 1973).

(7) D. PHENOLTETRABROMPHTHALEINS Among the many substrates known to undergo enzyme-catalyzed conjugation with GSH, sulfobromophthalein (XIV) was one of the first to be described (Combes and Stakelum, 1961) and has since received much attention because of its use to assess hepatic function. Additionally (XIV) has proved of value for studies of ligandin (Y-protein or hepatic GSH S-transferase B in rat). The bromine atom most probably

Br

GSH CONJUGATION WITH CARCINOGENS

205

replaced by GSH is identified in structure (XIV) by an asterisk (Kosower, 1976; Javitt, 1976), and even disubstitution of (XIV) by GSH has been reported (Javitt et al., 1960; Whelan et al., 1969). Not surprisingly, the GSH conjugate of (XIV) is cleared more rapidly from the liver than (XIV) itself (Combes, 1965; Whelan et al., 1970; Yam et al., 1976), but conjugation, although important, is probably not normally rate limiting (Boyland and Grover, 1967; Klaassen and Plaa, 1967). However, this subject remains a controversial issue. The extensive literature on the conjugation of (XIV) with GSH is somewhat divorced from the intended theme of this review and will not be discussed further.

E. ARALKYLHALIDES Aralkyl halides (Table X), such as the carcinogenic benzyl chloride (Preussmann, 1968; Druckrey et d.,1970), are known to be metabolized to mercapturic acids through nucleophilic displacement of halide by GS- [see Eq. (3)]. A competing metabolic pathway is replacement b y OH- resulting, for example, in the formation of a glycine conjugate through the intermediate oxidation of the alcohol to the acid (RCH,Hal + RCH,OH + RC0,H). Mercapturic acid formation from benzyl chloride in rats, dogs, and rabbits (Stekol, 1938; Bray et al., 1958) and from menaphthyl halides in rats and rabbits (Hyde and Young, 1968) has been reported. An aralkyl halide with a sidechain containing two or more carbon atoms, such as phenethylbromide TABLE X ARALKYL HALIDESEXCRETED AS MERCAPTURICACIDS I N THE U R I N E AFTER ORAL ADhlINISTRATION TO

Compoiind Benzyl chloride I orolieii z y1 ch 1ori de 1 1 -N i troben zy I chloride 11 - M e thox yben zy l ch I oride ~ ~ - C l i l o r o i n e t l i y l l ~ ~chloride iiz~l Beiizyl bromide 11 -Ch

Plienethyl bromide 3-Broinapropylbenzene 4-BroiiioI~~ityIl)enzene "

Data of Bray et crl. (1958).

RABBITS" hlercapturic acid excreted (% dose) 39 23 49 1 11 19 31" 11

4

'' Excreted partly a s a h y d r o x y a r a l k y l m e r c ~ l l ~ t ~acid i r i ~ (Jiiiiies and White, 1967)

206

L. F . CHASSEAUD

(XV), may also be metabolized to a hydroxyaralkylmercapturic acid (XVI) (James and White, 1967). Although hydroxylation could have followed conjugation with GSH, it is more likely to have preceded it or occurred simultaneously. C H,C H, Br

(XV)

~H(OH)CH,SCH,~HCOOH

(XVI)

GSH S-transferase activity toward benzyl chloride [Eq. ( 3 ) ]and its p-nitro-analog has been extensively studied (e.g., Suga et al., 1967; Boyland and Chasseaud, 1969b; Habig et al., 1974a; Kaplowitz et al., 1976), but none of the studies has been concerned with the influence that GSH may exert on the potential carcinogenicity of benzyl chloride or related compounds. Rats converted 42% of the anti-inflammatory agent 2-acetamido-4chloroniethylthiazole (XVII) to the mercapturic acid (XVIII) which was apparently further metabolized to the methylthio derivative (XIX) and its corresponding sulfoxide and sulfone (Chatfield and Hunter, 1973).It is possible that some methylthio metabolites may arise through biotransformation of biliary excreted GSH conjugates (or related NFICOCH,

t

L3CH*SCH3

CH,COHN (XIX)

products) in the gut, but evidence for this hypothesis is lacking and other mechanisms are possible. About 20% of an oral dose of the herbicide propachlor (XX) was excreted as a mercapturic acid (XXI) in rat urine (Lamoureux and Davison, 1975) presumably through conjugation with GSH. It is probable that other related a-chloroacetamides are similarly metabolized.

207

CSH CONJUGATION WITH CARCINOGENS (CH,),CH- N - COCH,Cl

I

(cH,),cH-

N - COCH,SCH,CHCOOH

I

NHCOCH,

Enzyme-catalyzed conjugation with GSH has also been implicated in the reductive dechloriiiation of pheiiacyl halides in rat liver [Eq. (8)] (Hutson et ul., 1976; Crawford et ul., 1976).

6"

fGSH

v1 C OCH,S G

COCH,Cl

~

c1

\

c1

fGSH

COCH,

=

$fcl \

+GSSG

(8)

c1

F. ARALKYLESTERS Aralkyl esters, such as l-~iienaphthylsulfate(XXII)were metabolized by rats to niercapturic acids which were excreted in the urine and to GSH and cysteine conjugates which were excreted in the bile (Clapp and Young, 1970). The amounts of mercapturic acid excreted after administration of nienaphthyl esters of carboxylic acids was low, and these esters appear to be poor substrates for the GSH S-transferases ( Boyland and Chasseaud, 1969b). Enzymic conjugation has been investigated with the more reactive 1-nienaphthylsulfate (XXII)(Gillham, 1971).

208

L. F. CHASSEAUD

Metabolism of the antioxidant 3,5-di-tert-butyl-4-hydroxytoluene (BHT) (XXIII)to a mercapturic acid (11% dose) by rats (Daniel et al., 1968)may have proceeded through an intermediate aralkyl carbonium ion (RCH,+) which is probably the reactive species during conjugation of GSH with aralkyl halides and esters [see Eq. (3)]. Only traces ofthis mercapturic acid were detected in the urine of humans receiving much lower doses of (XXIII)(Daniel et al., 1968), and its formation may be dose dependent. G. ALKYLHALIDES,SULFATES,OR NITROCOMPOUNDS Certain alkyl halides, such as methyl iodide (XXIV),sulfates, such as diethyl sulfate (XXV),and nitrocompounds, such as l-nitropropane (XXVI),were metabolized by rabbits and/or rats to mercapturic acids (for references, see Chasseaud, 1973~1,1976a), presumably through initial conjugation with GSH catalyzed by GSH S-transferases (Booth et al., 1961; Boyland and Williams, 1965; Johnson, 1966). Some of these compounds may be carcinogenic, for example, methyl iodide (XXIV)and diethyl sulfate (XXV),to rats b y subcutaneous injection (Preussmann, 1968; Druckrey et al., 1970) and 2-nitropropane to rats by inhalation (Busey et al., 1978). The formation of S-methylglutathione during exposure of erythrocytes (Redford-Ellis and Gowenlock, 1971a) or liver and kidney homogenates (Redford-Ellis and Gowenlock, 1971b) to chloromethane amply demonstrates the role of GSH as an endogenous protecting agent. CHJ (XXIV)

(CH3CHzO)zSOz (XXV)

CH3CHzCHzNOz (XXVI)

These compounds are excreted as apparent mercapturic acid (Table XI) which probably comprised alkylmercapturic acids, hydroxyalkylmercapturic acids, and other related sulfur-containing metabolites. Hydroxyalkylmercapturic acids are formed from alkyl halides containing more than two carbon atoms (Bray et al., 1964; Barnsley, 1966; James et al., 1968a; Grasse and James, 1972) and from halogenocycloalkanes (James et al., 1971), alkenes (James and Jeffery, 1964), 1,2epoxycycloalkanes (James et al., 1971), and cycloalkenes (James et al., 1971; Waring, 1971), and may arise by conjugation of intermediate epoxides with GSH (Fig. 6) (James and Jeffery, 1964). Such epoxides would probably be more reactive and therefore potentially more toxic than their precursors. Barnsley ( 1966) showed that the same hydroxyalkylmercapturic acid (Fig. 6, Alkyl = CH,) was present in the urine of rats dosed with l-chloropropane, l-iodopropane, 1,2-epoxypropane, and l-chloro-

209

GSH CONJUGATION WITH CARCINOGENS TABLE XI

BRO.ZlOALKANES OH hTITROi\LIi.\NES E XCRE T E D A S AI'PAHEN'I' MERCAPTUHIC i\CIDS I S T H E [TRINE

AFTER ORAL ADMI"II'HA-IION

TO

RABBITS OR

hTS"

Apparent mercaptiiric aci tl e xc retetl " ( % dose,)

41 21 23 16 12 23 1

1-Bromol)ittane 1-Bromol)entane 1-Rromollex;lne 1-Bromoheptane 1-Rromooctnne 1-Nitrobutane 1-Nitroliexaiie I'

'I

54 35 52 70 46 Sl 6

Data of Bray ef ( I / . (1964). Also excreted after chlorodkane or iotlnalkane administration (see Bray et (//., 1964).

Alkyl- CHz- CHz- Hal

/

\

- HHal

+"O"

Alkyl- C H Z C H ,

Alkyl- CHOHTCH,-H~I

Alkyl- CHOH- CHz- SG I I

A

I I

I Alkyl- CHOH-CHz-

SCH,CHCOOH I

NHCOCH,

FIG.6. Possible mechanistus for the foniiatioii of hydroxyalkylmercaptut-icacicls frotit alkyl lialicles through intermediate epoxides.

2 10

L. F. CHASSEAUD

propan-2-01. The available evidence (James et al., 1971; James and Needham, 1973) suggests that hydroxylation may also occur after conjugation with GSH. James et a1. (196th) have detected Sbutylglutathione, S -butylcysteinylglycine, and S -butylcysteine in the bile of rats dosed subcutaneously with 1-bromobutane (Table XI), and the (2- and 3-hydroxybutyl) mercapturic acids, as well as butylmercapturic acid, in the urine, thus demonstrating the formation from 1-bromobutane of all the individual conjugates in mercapturic acid biosynthesis. Humans excreted much of an oral dose of the sedative-hypnotic bromvaletone (a-bromoisovalerylurea) in the urine, mostly as a mercapturic acid and some as the cysteine conjugate (Niederwieser et al., 1978), both of which presumably arose by initial replacement of bromine with GSH [Eq. (9)]. tCSH

(CH,),CHCH(Br)CONHCONH2--+

(CH,),CHCH(SG)CONHCONH,

(9)

The further metabolism that mercapturic acids or cysteine conjugates may undergo in vivo such as sulfoxidation is exemplified in the studies of S-methyl-L-cysteine in rats, guinea pigs, and hamsters (Sklan and Barnsley, 1968), of 1-bromobutane in rats and rabbits (James et u Z . , 1968a), and of S-pentyl-L-cysteine in various species (James and Needham, 1973). Conjugation with GSH has been implicated in the detoxication of a variety of other aliphatic halides, some of which have been shown to be mutagenic and even carcinogenic; but the nature of the actual reactive intermediate that supposedly conjugates with GSH has not always been rigorously established. The alkylating antifertility agents, a-chlorohydrin (XXVII)and epichlorohydrin (XXVIII),were metabolized by rats mainly to 2,3dihydroxypropyl-S -cysteine and to the corresponding N-acetyl derivative (mercapturic acid) which were excreted in the urine (Jones et ul., 1969).These studies have been extended to include other a-halogenohydrins, and it was shown that the cysteine conjugate and mercapturic acid probably arose b y initial conjugation of GSH with an epoxide intermediate, glycidol (XXIX) (Jones, 1973, 1975). The mutagenicity of (XXVIII)to E.coZi (Strauss and Okubo, 1960) and of (XXVIII)and (XXIX)to Neurospora (Kolmark and Giles, 1955) have been reported. 0

/ \

CH,(C~)CH(OH)CH,OH

CH,(C~)HC-CH,

(XXVII)

(XXVrn)

0 / \

CH,(OH)HC-CH, (XXIX)

GSH CONJUGATION WITH CARCINOGENS

211

It would be worthwhile assessing whether administration of the mutagenic and carcinogenic chloroalkylethers (Nelson, 1976), such as the human lung carcinogen chloromethyl methyl ether ( CH30CH2CI) (Figueroa et ul., 1973), can affect liver GSH levels in experimental animals and whether such compounds undergo enzyme-catalyzed conjugation with GSH. Mice rapidly excreted up to 50% of intraperitoneal doses of [1,2''C]dichloroethane (Yllner, 1971a), [ 1,1,2-14C]trichloroethane (Yllner, 1971b), or ['4C]chloroacetic acid (Yllner, 1971c) in the urine as S-carboxymethylcysteine [see Eq. (lo)]. Only small amounts of the corresponding mercapturic acid were detected but another major metabolite was thiodiacetic acid possibly formed by oxidative deamination of the cysteine conjugate. In all cases, conjugation with GSH was thought to occur with chloroacetic acid [Eq. (lo)], but as this is a relatively poor substrate for the GSH S-transferases (Johnson, 1966), conjugation mainly with the reactive chloroacetaldehyde would be more likely. The corresponding GSH conjugate, S-carboxymethylglutathione, has been detected in livers of rats dosed orally with 2chloroethanol as would be expected from Eq. (10) (Johnson, 1967). By contrast, formation of a mercapturic acid (and mercapturic acid sulfoxide) in rats dosed orally with 1,2-dibromoethane (ethylene dibromide) (Nachtomi et d., 1966; Jones and Edwards, 1968) was thought to proceed through enzyme-catalyzed displacement of bromide by GSH [Eq. ( l l ) ] because the corresponding GSH conjugate had been detected in the livers and kidneys of' treated rats (Nachtomi, 1970). 1,2-Dibromoethane administered to rats by inhalation was apparently also conjugated with GSH (Watanabe et ul., 197th). CH,(CI)CH,CI

-

CH,(Br)CH,Br

CH,(CI)CH,OH

+GSH

CH,(Br)CH,SG

__t

+OH

CH,(C~)CHO

CH,(OH)CH,SG

(11)

Thus, different mechanisms have been argued for the formation of GSH conjugates of 12-dichloroethane [Eq. (lo)] and 1,2-dibromoethane [Eq. ( l l ) ] ,although there is no obvious reason forthis difference. However, it has been reported recently that the mutagenicity of 1,2-dichloroethane was greatly enhanced in the presence of GSH and GSH S-transferases (Rannug et ul., 1978) implying that conjugation with GSH had resulted in bioactivation rather than detoxication.

2 12

L. F . CHASSEAUD

It appears that reaction of 1,2-dichloroethane with GSH yielded a bioactive sulfur half-mustard (RSCH,CH,Cl) which may be Schloroethylglutathione or the product of further biotransformation, such as the highly mutagenic S -chloroethylcysteine (Rannug et d., 1978). A similar result was obtained with 1,2-dibromoethane. Thus initial conjugation of GSH with these bifunctional alkylating agents (and possibly others) did not result in detoxication but quite the reverse and probably proceeds by the route shown by Eq. (11) rather than that b y Eq. (10). 1,2-Dibromoethane has been reported to be mutagenic to S . typhirnuriurn (Brem et al., 1974) and E . coli (Fahrig, 1974) and to be carcinogenic to rats and mice (Olson et al., 1973; Powers et ul., 1975); its interaction with DNA has also been studied (Nachtomi and Sarma, 1977). The related 1,2-dibromo-3-chloropropane has been shown to produce stomach cancer in rodents (Olson et d., 1973; Powers et al., 1975) and was the probable cause of infertility in exposed fktory workers (Whorton et d.,1977). Major urinary metabolites of mustard gas (ClCH,CH,SCH,CH,Cl) in rodents included GSH conjugates of bis(2-chloroethy1)sulfide and of the corresponding sulfone, presumably formed through replacement of chloride (Cl-) with GS-. Competing hydrolysis reactions led to products such as thiodiglycol (Davison et al., 1961). These studies do not appear to have been extended despite the recognition of mustard gas as being carcinogenic to man (Wada et al., 1968). GSH serves as a cofactor for the metabolism of dihalomethanes, such as bromochloromethane, catalyzed by rat liver cytosol enzymes to formaldehyde and inorganic halide (Ahmed and Anders, 1976). The nature of these enzymes and the exact mechanism of the reaction require further study (e.g., Ahmed and Anders, 1978).A similar comment may be made regarding the GSH-dependant enzyme system in rat liver that defluorinated methoxyflurane (XXX)(Madelian and Warren, 1977). CHC12CF,0CH3

(XXX)

CF,CHClBr

(XXXI)

CH,=CHOCH,CF,

(XXXII)

The observation that mouse liver GSH levels were decreased b y intraperitoneal doses of halothane (XXXI)(Rosenberg, 1971) or inhalation of fluroxene (XXXII)(Fiserova-Bergerova, 1977) suggested that these anesthetics may be partly metabolized by conjugation with GSH. Subsequently Cohen et al. (1975) showed in an unusual study involving (legally “dead”) heart transplant donors that humans partly metabolized (XXXI)to a mercapturic acid, presumably by replacement of fluorine with GSH, but the exact mechanism is uncertain. This result indicated the ability of the human liver to defluorinate the re-

GSH CONJUGATION WITH CARCINOGENS

213

markably stable - CF, group. The biotransformation and hepatotoxicity of this important anesthetic (XXXI)has been summarized recently (Brown and Sipes, 1977). GSH and other thiols appear to serve as cofactors in the metabolism of haloforms, such as chloroform, to carbon monoxide in oitro. This conversion followed the halide order: thus production of carbon monoxide was greatest from iodoform and least from chloroform (Ahmed et d.,1977). GSH is thought to protect rats from the hepatotoxic effects of chloroform (Brown et d.,1974; Docks and Krishna, 1976) and carbon tetrachloride (Rechnagel and Glende, 1973), but whether this protection is mediated through the GSH S-transferases or another GSH-dependent pathway needs to be established, particularly in view of the reported liver carcinogenicity of both these halogenated hydrocarbons in rats and mice (Eschenbrenner and Miller, 1944; Reuber and Glover, 1970; Powers and Voelker, 1976). However, recent evidence indicates that phosgene may be a reactive metabolite of chloroform in rats (Mansuy et u l . , 1977; Pohl et ul., 1978) and would be expected to covalently bind to tissue macromolecules and to react readily with GSH [Eq. (12)], as apparently does thiophosgene formed during captan metabolism (see Section IV,U). The industrial use, environmental distribution, and toxicity of major alkyl halides have been surveyed ( Fishbein, 1976).

- HCI

NADPH. "0"

+ C,SH

H,C-CHCO-GIv

H. ALKENE HALIDES Some of the earlier studies of the conjugation of GSH with aliphatic halides have been mentioned in this review because they provide a useful baseline from which to discuss the newer developments in this area. Furthemiore, they have already eiiabled accurate prediction (see Chasseaud, 1976a) of some of the subsequent findings of more recent studies, for example, that the mutagens and carcinogens vinyl chloride (XXXIII) and vinylidene chloride (XXXIV), and the anesthetic halothane (XXXI) would be metabolized via mercapturic acid biosyntliesis. CH, = CHCl

CH, = CCI,

(XYXIII)

(XXXIV)

Exposure to vinyl chloride monomer (XXXIII)has been implicated as the cause of angiosarcomas in workers (Creech and Johnson, 1974)

214

L. F. CHASSEAUD

and was shown to produce these and other tumors in animals (Viola et al., 1971; Maltoni and Lefemine, 1974). (XXXIII) is mutagenic (Bai-tsch et ul., 1975a; Bartsch and Montesano, 1975) and has been reported to produce chromosomal damage in the peripheral blood lymphocytes of exposed workers (Heath et ul., 1977). Consequently considerable effort has been expended into understanding the metabolic fate (Green and Hathway, 1975; Watanabe et ul., 1976a,b), pharniacokinetics (Withey, 1976), and covalent binding to hepatic macromolecules (Watanabe et ul., 1978b) of (XXXIII) as well as other aspects (see Selikoff and Hammond, 1975). Elimination of (XXXIII) is dose dependent. Increasing oral doses of [14C]vinylchloride monomer to rats resulted in the elimination of a greater proportion of the dose unchanged in the expired air (Green and Hathway, 1975; Watanabe et ul., 1976a) indicating possible saturation of a process for the biotransformation of (XXXIII), presumably that process responsible for its bioactivation. Major urinary metabolites of (XXXIII, dose level 3 x 50 mg/kg p.0.) were reported as thiodiglycollic acid (thiodiacetic acid, cf. Yllner, 1971c) (47% dose), S-(2-~hloroethyl)cysteine(23% dose), and the corresponding mercapturic acid (23% dose) (Green and Hathway, 1975). Other workers (Watanabe et ul., 1976a,b) identified a different mercapturic acid, N-acetyl-S-(2-hydroxyethyl) cysteine, as a major urinary metabolite of (XXXIII) after oral or inhalation administration to rats, and it appears that the conjugates reported by Green and Hathway ( 1975) were probably artifacts of their identification procedure (Watanabe et ul., 1976a; Green and Hathway, 1977). Knowledge of the identity of the sulfur-containing metabolites of (XXXIII) is important in order to ascertain the nature of the bioactive metabolite of (XXXIII), which has been suggested to be chloroethylene oxide rather than its rearrangement product chloroacetaldehyde [see Eq. (13)],on the basis of its mutagenicity to S . typhirnuriurn (Malaveille et ul., 1975a; Rannug et d., 1976) and to yeast (Loprieno et d . , 1977), studies with microsomal enzyme systems (Gothe et ul., 1974; Kappus et ul., 1976), and theoretical considerations (Bonse and Henschler, 1976). However, conjugation of GSH could also occur with the rearrangement product ClCH=CH,-

I

1

ClCH,CHO

GSH CONJUGATION WITH CARCINOGENS

215

chloroacetaldehyde, but, by analogy with [Eq. (101, this should also result in S-carboxymethylcysteiiie via S-carboxymethylglutathione [Eq. (101, and indeed such a conjugate has been detected (Muller and Norpoth, 1975; Green and Hathway, 1977). Maintena'nce of adequate GSH levels appears necessary to protect rats from the toxic effects of (XXXIII) (Conolly and Jaeger, 1978). The related vinylidene chloride (XXXIV) has also been shown to be mutagenic to S. t y p l z i n w - i u n t (Bartsch et al., 1975b; Malaveille et n l . , 1977a), to lie tumorigenic to mice (Maltoni et ul., 1977), and to be metabolized by rats mainly to a mercapturic acid (N-acetyl-S-(2hydroxyethyl) cysteine) and thiodiglycollic acid after oral (McKenna et uZ., 1978a) or inhaled doses (McKenna et nl., 1978b), presumably through initial conjugation of a bioactive nietabolite(s) with GSH. Inhaled (XXXIV) was less hepatotoxic to fed rats than to fasted rats because the greater GSH levels present in the former than in the latter (Jaeger et nl., 1974) would diminish the amount of the bioactive metabolite(s) of (XXXIV) that could bind to biological macromolecules (e.g., McKenna et d.,1977). These results provide further evidence for the protective role of GSH against tissue injury from electrophilic agents. Comparison of the metabolism of (XXXIV) in inice and rats appeared to explain the greater toxicity of this monomer to the former species: mice and rats excreted 50 and 28%, respectively, of an oral dose (50 mg/kg) of (XXXIV) as a mercapturic acid which was identified as an N-acetyl-S-cysteinylacetyl derivative (Jones and Hathway, 1978). Greater production of the mercapturic acid implies greater formation of the bioactive metabolite(s) for conjugation with GSH and for covalent binding with tissue macromolecules (McKenna et ul., 1977). The detoxication of a number of other mutagenic halogenated alkeiies, such as the pesticide 1,3-dichloropropene (XXXV) (De Loreiizo et d . ,1977),the synthetic rubber intemiediate 2-chloro-l,3-butadiene (chloroprene, XXXVI) (Bartsch et al., 1975b), and the industrial solvent trichloroethylene (XXXVII) (Greini et (11., 1975), could involve conjugation with GSH and the concomitant protection that this affords. (XXXVII) has been shown to produce hepatocellular carcinoma in mice (DHEW, 1976). Fishbein (1976) has reviewed the industrial production, environmental distribution, and toxicity of major alkene halides including (XXXIII), (XXXIV), (XXXVI), and (XXXVII). The maintenance of adequate protective levels of GSH can be appreciated from the observation that the hepatotoxic etfects of certain alkeiie halides, such as (XXXIV), (XXXVI), arid (XXXVII), were

2 16

L. F . CHASSEAUD

greater in fasted than in fed rats (Jaeger et al., 1975; Moslen et ul.,

1977). CH(CI)= CHCH,CI

CH, = CHC(C1) = CH,

(XXXV)

ClCH = CCI,

(XXXVI)

(XXXVII)

Henschler and Bonse (1977) have argued that epoxides of’chlorinated ethylenes bearing unsymmetrical chlorine substitution are more electrophilic than those having symmetrical substitution. Thus epoxides of the former group would react with GSH and with tissue macromolecules more readily than those of the latter. Covalent interaction with biological macromolecules of “metabolites” of several of these halogenated hydrocarbons, such as (XXXI) (Subba Rao, 1977), (XXXIII)(Kappus et al., 1976), (XXXVII)(Banerjee and Van Duuren, 1978), and carbon tetrachloride (Uehleke et al., 1977), have been reported. However, covalent interaction cannot be expected to predispose to tissue damage unless it occurs with a “ target” site. Most measurements of covalent binding or interaction are nonspecific by nature and their interpretation can therefore be somewhat speculative. I. ALICYCLICHALIDES The formation of isomeric GSH conjugates and/or related metabolites from hexachlorocyclohexane by insects (Sims and Grover, 1965; Ishida, 1968; Clark et ul., 1969; Tanaka et al., 1976) and by rats (Grover and Sims, 1965; Koransky et al., 1975; Kurihara et al., 1977) has been extensively studied, but the exact nature of the chemical species that conjugates with GSH has not been established yet (Freal and Chadwick, 1973; Tanaka et al., 1976; Brooks, 1979). An intermediate pentachlorocyclohexene is possible [Eq. (14)] (Kurihara et al., 1977). The rate of metabolism in vitro of hexachlorocyclohexane isomers was a > y > 6 9 p (Ishida and Dahm, 1965). The a-isomer, which produced liver tumors in rats (It0 et ul., 1975) and mice (Ito et al., 1976), undergoes enzyme-catalyzed conjugation with GSH (Portig

c1

c1

c1 *cl c1

c1

c1

GSH CONJUGATION WITH CARCINOGENS

217

et d . , 1973; Kraus et ul., 1973; Kraus, 1976). Other isomers are also reported to be Carcinogenic to mice (IARC, 1974a).

J. ALLYL COMPOUNDS Ally1 halides, such as allyl chloride (XXXVIII), and allyl esters, such as allyl formate (XYXIX), are partly metabolized to allylmercapturic acid and/or 3-hydroxypropylmercapturic acid (Kaye et nZ., 1972; Kaye, 1973). The mechanism for the fonnation of these mercapturic acids and the allyl ester compounds from which they result has been discussed previously (Kaye, 1973; Chasseaud, 1976a).Their formation appears to involve either alkyl-oxygen or acyl-oxygen fission (Kaye, 1973). The fonner results in a carbonium ion (CH,=CH-+CH,) as the reactive intermediate whereas the latter involves acrolein (CH,=CHCHO), an irritant and probably mutagenic ap-unsaturated aldehyde (Epstein and Shafner 1968;Izard and Libennann, 1978).Thus acrolein is the possible bioactive metabolite responsible for the hepatic necrosis observed in rats after administration of allyl fonnate (XXXIX) (Rees and Tarlow, 1967) or of allyl alcohol (Reid, 1972). Hepatotoxicity (e.g., by allyl alcohol, Hanson and Anders, 1978) would be enhanced if protective GSH levels were depressed by other compounds. The derivative acrolein diethylacetal reacted rapidly with GSH and this reaction was also enzyme catalyzed (Boyland and Chasseaud, 1967). Rapid reaction of acrolein with GSH i n vitro has also been reported (Alarcon, 1976). CHI = CHCHZCI

(XXXVIII)

CH, = CHCH,OCOH

(XXxIX)

A proximate carcinogenic metabolite of the hepatocarcinogenic (Long et nl., 1963) and naturally occurring flavoring agent safrole in rodents was 1’-hydroxysafrole (Borchert et nl., 1973a), and the ultimate carcinogen could be a corresponding ester (Borchert et ul., 1973b), or 1’-ketosafrole formed from 1’-hydroxysafrole (Fig. 7) just as acrolein from allyl alcohol. 1’-Ketosafrole reacted more readily with various nucleosides, particularly guanosine, than did other possible safrole metabolites (Wislocki et al., 1976) but was apparently not carcinogenic to rats after dietary administration for 22 months or mutagenic to S. typhirnuriurn (Wislocki et d.,1977). Its 2’,3’-epoxide derivative (Fig. 7) was mutagenic, however, as were the 2’,3’-epoxides of other safrole metabolites (Wislocki et nl., 1977). 1’-Ketosafrole would be expected to react readily with GSH and this may be a means for its detoxication. Recent metabolism studies of safrole in rats, guinea pigs, and humans (Stillwell et d.,1974; Strolin-Benedetti et

218

L. F. CHASSEAUD

C H,CH =CH,

CH,CH - CH, ‘0’

Saf r o l e

Safrole-a’, 3’-oxide

PCOCH=CH,

1

C H(0H)C H =C H,

1’-Hydroxysafrole

1’-Ketosafrole

\

CH(OS0,H)CH Z C H , Safrole- 1‘ -sulfate

CH(0H)CH- CH, ‘0’

1’-Hydraxysafrole2’, 3’-oxide

P \

bOCglCH, 0

1’-Ketosafrole-2

‘, 3’-oxide

FIG. 7 . Formation of‘ possible carcinogenic metabolites of‘ safrole in rat and nioiise (Wislocki et nl., 1977).

ul., 1977) have not reported metabolites that could arise through conjugat ion with GSH . Allylic double bonds can be converted to the corresponding epoxides (cf. Fig. 7 )and such intermediates have been detected in the urine of rats dosed with allyl-substituted barbiturates (Harvey et ul., 1972, 1977). The metabolism of allylbenzene is believed to involve an intennediate phenyl vinyl ketone as well as a corresponding epoxide (Peele and Oswald, 1977) (cf. Fig. 7). Both types of intermediate would be expected to conjugate with GSH (Boyland and Williams, 1965; Boyland and Chasseaud, 1967) and thereby undergo mercapturic acid biosynthesis.

GSH CONJUGATION WITH CARCINOGENS

2 19

K. ALKYL METHANESULFONATES Roberts and Wanvick ( 1958) first reported that ethyl methanesulfonate [ Eq. ( 15)] was partly inetabolized to ethyliiiercapturic acid and related conjugates, presumably by initial conjugation of an intermediate ethyl carbonium ion with GSH. A competing pathway appeared to be reaction of the carbonium ion with OH- to produce ethanol which was subsequently converted to CO, (Robei-ts and Wanvick, 1958). Conjugation of ethyl inethanesulfonate with GSH appeared to be enzyme catalyzed (Booth et nl., 1961).Other lower alkyl inethanesulfoliates (Pillinger et c i l . , 1965; Jones and Edwards, 1973) and alkylene me thanes u Ifonat e s ( Ed wards e t ( I I., 1970a,l)) were sim i larl y me tabolized to mercapturic acids aiid related compoimds b y rodents. Jones ( 1973) h a s reviewed the nietal)olisni ofthis group of compotmds. Their mutagenicity (Loveless and Howarth, 1959) and carcinogenicity (Walters et a/., 1967; Clapp et ( i l . , 1968; Dnickrey et d . , 1970; IARC, 1974c) have also been reported. CH,CH,OSO,CH,,

-

[CH,CH,+]

tGSI1

CH,CH,SG

(15)

Biotransfomiation of the antileukemia drug ['4C]niyleran in rats, rabhits, aiid mice is novel. The major urinary metal)olite, 3-hydroxytetrahydrothiophene-1,l-dioxide[Eq. (16)], was probably fornied b y initial coiijugatioii with GSH (Roberts and Wanvick, 1961), but the nature oftlie urinary metabolite does not suggest that this had occurred. Similar reactions of GSH with other I)ifiinctional alkylating agents could also occur. Carcinogenesis b y alkylating agents was the subject of a recent comprehensive review (Lawley, 1976).

3

I CH,CHCO. Gly I NH- Glu

L. ORGANOPHOSPHORUS COhlPOUNDS The report that methylparathion was biotransformed to desniethylparathion b y a GSH-dependent enzyme system (Fig. 8) present in rat tissues (mainly liver) aiid insect larvae (Fukami and Shishido, 1966) showed that GSH a i d GSH S-transferases may be involved in the detoxicatioii of organophosphorus compounds by vertebrates and by insects (see reviews of Fukunaga et al., 1969; Dauterman, 1971; Hollingworth, 1971; Chasseaud, 1973a; Hutson, 1976 for other examples).

220

L. F. CHASSEAUD

H /o \ ! - O ~ N 0 2 CH,O

\

CH,O

+

CH,SG

s

\II

P-OH

+

GS

CH,O'

FIG. 8. Enzyme-catalyzed GSH-dependent dealkylation and dearylation of methyl parathion (Hollingworthet o / . , 1973; Usui et d., 1977b).Dearylation of methyl paraoxon has also been demonstrated (Benke and Murphy, 1975).

Removal of a single alkyl group from organophosphorus triesters reduces their anticholinesterase activity and hence their acute toxicity (Hollingworth, 1970). This GSH-dependent pathway (Fig. 8) apparently dealkylates the 0-methyl group more readily then 0-ethyl or 0-isopropyl groups (Hollingworth, 1969; Hutson et al., 1972; Hutson, 1977; Motoyama and Dautennan, 1977) to presumably initially form S -alkylglutathione conjugates (Fig. 8) and is probably more active in organophosphate-resistant insect strains than in susceptible strains (Motoyama and Dauterman, 1972; Oppenoorth et al., 1977), but possibly not toward all substrates (Motoyama et d . , 1977). The mutagenicity of some organophosphorus compounds, particularly dichlorvos(CH,O),PO(OCH=CCl,)has been reviewed (Wild, 1975). Small amounts of niethyhnercapturic acid and S -methylcysteine were detected in the urine of rats dosed with dichlorvos (Hutson and Hoadley, 1972),presumably the result of S-methylation of GSH (Dicowsky and Morello, 1971). Similarly the alkylating agent trimethylphosphate which was mutagenic to mice (Epstein et ol., 1970) and other simple trialkyl phosphates were metabolized by rodents to the corresponding S-alkylcysteines (Jones, 1970) probably through initial S-alkylation of GSH, a reaction which may have been enzyme catalyzed. GSH-dependent dearylation of organophosphorus compounds also occurs (Fig. 8 ) (Shishido et ul., 1972; Hollingworth et al., 1973; Motoyama and Dauterman, 1977; Usui et d.,197%). A study of the acute toxicity of parathion and methylparathion and their rates of detoxication by GSH S-transferases in rats of different

GSH CONJUGATION W I T H CARCINOGENS

22 1

ages showed that toxicity decreased and rates of dealkylation and dearylation (Fig. 8) increased with increase in age (Benke and Murphy, 1975).

M. ARYLHYDROCARBONEPOXIDES (ARENEOXIDES) The polycyclic aromatic hydrocarbons constitute an important group of environmental carcinogens (IARC, 1973b) whose presence in cigarette smoke (Wynder and HofFmann, 1959; Severson et al., 1976) implicates them as a possible contributory factor in the production of cancer of the human respiratory tract (Doll, 1955; WHO, 1975). The mechanisms by which these relatively inert chemicals can cause cancer has intrigued researchers for many years. Boyland ( 1950) recognized that metabolic activation of these hydrocarbons occurred, probably to an epoxide (arene oxide), and subsequent research has confirmed this view (for reviews see Daly et d., 1972; Sims and Grover, 1974; Jerina and Daly, 1974; Garner, 1976). Currently vicinal diol-epoxides (Sims et al., 1974; Jerina and Daly, 1977) are thought most likely to be the ultimate carcinogenic forms of these hydrocarbons, and their reactivity toward nucleophilic centers has been predicted as a result of stereocheniical (Hulbert, 1975) and quantum mechanical (Lehr and Jerina, 1977) considerations. Hydrocarbon epoxides or diol-epoxicles have been shown to react covalently with nucleic acids and proteins in biological systems (Grover and Sims, 1968; Gelboin, 1969; Grover et al., 1971c; Kuroki et d . , 1972; Heidelberger, 1975; Tierney et d . , 1977; Meehan et al., 1977), to be mutagenic to S. typhinturiun1 (Ames et u / . , 1972; Sinis and Grover, 1974; Wood et ( I / . , 1975, 1977a,b; Malaveille et d . , 1975b), to produce malignant transforniations of rodent cells in culture (Grover et n / . , 197121; Huberman et u / . , 1972; Heidelberger, 1975), and to cause tumors in mice (Sims and Grover, 1974). Stereochemical aspects of reactions of epoxides with nucleophiles have been investigated (Jeffrey et a / . , 1974), a s have their enzyme-catalyzed conjugation with GSH (Boyland and Williams, 1965; Hayakawa et a / . , 1975). More details of the biological properties and chemistry of hydrocarbon epoxides are given in the excellent reviews of Sims and Grover (1974) and Jerina and Daly (1974, 1977) and elsewhere (Freudenthal and Jones, 1976; Jollow et u l . , 1977). Brookes (1977) has reviewed the mutagenicity of polycyclic aromatic hydrocarbons and their epoxides. Pol ycyclic aromatic hydrocarbons, such a s naphthalene (see Fig. 9) (Boyland et al., 1961) and benz[a]anthracene (XL) (Boyland and Sims, 1964), are partly metabolized to acid-labile hydroxyarylmercapturic

222

L. F. CHASSEAUD

a

NADPH

+ "0"

microsomes

[mH] +

\

GSH

\

rases

\ A'-acetylase

'\

NHCOCH, I

H

NHCOCH,

SCH,CHCOOH

-

SC H,C HCOOH

+HCl

FIG. 9. Mercapturic acid formation from naphthalene through initial conjugation of naphthalene 1,2-oxide with GSH. The various conjugates have been detected in the bile of rats dosed with naphthalene (Boyland et al., 1961). GSH is probably attached at position C-2 of the dihydronaphthalene ring as shown (Jeffrey and Jerina, 1975)and not at position C-1 as was first thought. During dehydration in the presence of acid, the N-acetylcysteine moiety migrates to position C-1.

acids (Fig. 9) through initial conjugation of reactive epoxide intermediates with GSH (Table XII). The evidence for this route has been presented in previous reviews (Boyland and Chasseaud, 1969a; Chasseaud, 1973a; Sims and Grover, 1974).The earlier evidence, for example, included the discovery that similar metabolites were produced by animals dosed with 1,2-dihydronaphthalene (XLI) or its corresponding epoxide 1,2-epoxy-1,2,3,4-tetrahydronaphthalene (XLII) (Boyland and Sims, 1960; Boyland et ul., 1961).Furthermore, Booth et ul. (1961)

(XL)

(XLII)

showed that reaction of naphthalene with GSH in the presence of rat liver cytosol (containing GSH S-transferases) occurred only when mi-

223

GSH CONJUGATION WITH CARCINOGENS

CARCINOGENIC AROMATIC

TABLE XI1 HYDROCARBONS THATCONJUGATE

Hydrocarbon Benzene" Ben z[(/]anthracene 7-Meth y lben z[c/ ]an thracene 12-Methylbenz[a] antlrracene 7,12-Ditnethylbenz[o ]an thracene Dibenz[a,h ]anthracene Benzo[cr lpyrene 3-Me t h y Icholm th rene

WITH

GSH

Reference to conjugation with GSH"." Jerina et nl. (1968) Siins (1971) Siins (1972) Sims (1967) Booth et crl. (1973) Boyland and Sinis (1965) Waterfdl and Sims (1972) Sinis (1966)

" Only recent references are given. See reviews of S i n i s and Grover (1974) and Garner (1976) for citation of earlier work. " Conjngation occ~irswith an intermediate epoxide [see Table IV of S i m s and Grover (1974) and Jerina and Bend (1977)l. ' Suspected leitkemogenic agent (Aksoy et ( I / . , 1972; Vigliani and Forni, 1976).

crosonies (containing mixed-fiinction osiclases), NADPH, and oxygen were availalile (Fig. 9) presumably to generate first the intei-mediate naphthalene epoxide for conjugation with GSH (Jerina et ( / l , , 1970). Also the bile of rats treated with various hydrocarbons, such as naphthalene and benz[a]anthracene (XL) (Boyland and Sims, 1964) contained the hydroxyiiiercapturic acid together with the corresponding cysteine, cysteiiiylglycine, and GSH conjugates of the appropriate hydrocarbon epoxides (see Fig. 9), thereby indicating the nature of the intermediate conjugates involved in mercapturic acid biosynthesis from these hydrocarbons. S-substituted arylcysteines, such a s the cysteine conjugate of (XLII), are apparently incorporated into protein via the usual pathways of protein synthesis (Morrison et ( i l . , 1977).While this suggests that an alternative mechanism may exist for the binding of aromatic hydrocarbons to proteins, it is probable that Iiinding through reactive epoxide interiiiediates is of much greater importance. Epoxide formation typifies bioactivation since most epoxides are reactive and therefore potentially toxic to biological systems, whereas their conjugation with GSH results in detoxication. Conipeting with GSH conjugation catalyzed b y GSH S-transferases is hydration of the epoxide catalyzed by the epoxide hydratase system (Oesch, 1973, 1979; Bentley et ul., 1976; Kapitulnik et ul., 197713) to produce a di-

224

L. F. CHASSEAUD

hydrodiol, or even enzyme-catalyzed reduction to the parent hydrocarbon (Booth et nl., 1975). The intrinsic reactivity of an epoxide with respect to spontaneous isomerization to a phenol and the relative affinities of these and other enzyme systems for that epoxide in different species would decide which routes of metabolism are dominant and may influence the carcinogenicity or toxicity of that epoxide in a particular biological location. The biotransformation of polycyclic aromatic hydrocarbons involves a complex interplay of metabolic processes resulting in the production of various phenols, dihydrodiols, quinones, and conjugates, which have posed many technical problems in their isolation and characterization. The recent advent of high performance liquid chromatography should greatly facilitate the study of such metabolites (e.g., Selkirk et ul., 1975; Thakker et al., 1977). Earlier studies of polycyclic aromatic hydrocarbon epoxides involved those formed at the K-region, a bond in the molecule of relatively high electron density (Boyland and Chasseaud, 1969a; Sims, 1970), but more recent attention has focused on non-K-region epoxides, particularly those vicinal diol-epoxides (e.g., XLIII) of benzo[a]pyrene (XLIV), whose synthesis (Yagi et d.,1975, 1977), formation (Yanget d . , 1976; Thakker et ul., 1977), binding to DNA (King et al., 1976; Meehan et nl., 1977; Straub et al., 1977), mutagenicity (Wood et nl., 1976; Huberman et ul., 1976; Marquardt et al., 1977; Malaveille et ul., 1977b), and carcinogenicity (Kapitulnik et nl., 1977a, 1978; Slaga et ul., 1977a,b) have been studied. Relatively high biological activity is expected to be possessed by an epoxide on a saturated, Bay-region \

K-region (XLIII)

angular benzo-ring that forms part of a “bay-region” of the hydrocarbon [eg., (XLIII)] (Jerina and Daly, 1977).(XLIII) is thought to be an ultimate carcinogenic metabolite (Kapitulnik et al., 1978). The mutagenicity of other “bay-region” epoxides has also been reported (Malaveille et ul., 1977b; Wood et ul., 1977a,b). The reactivity of such epoxides with GSH needs investigation. GSH would be expected to

GSH CONJUGATION WITH CARCINOGENS

225

conjugate at position C-10 of (XLIII) (see Yagi et al., 1977) and this is the position at which binding to nucleic acids occurs (Koreeda et al., 1978). The formation of K-region epoxides that can react with GSH was demonstrated for several hydrocarbons (Grover et d . ,1971b; Sims and Grover, 1974) including benzo[a]pyrene (XLIV) (Grover et ul., 1972) and 7,12-diniethylbenz[a]anthracene (XLV) (Keysell et al., 1973). K-region epoxides may be more stable than epoxides fonned at K-region

\\

1

\

K-region (XLIV)

K-region (XLV)

other regions of the molecule (Booth et al., 1973; Bruice et al., 1976) which would partly explain why they were the first hydrocarbon epoxides to be detected in biological systems. Furthermore, K-region epoxides such as benz[n]anthracene 5,6-oxide may conjugate with GSH more readily than non-K-region epoxides, such as benz[u]anthracene 8,g-oxide (Booth and Sims, 1974). K-region epoxides appear to be less tumorigenic than the parent hydrocarbons (Grover et d . , 1975) which may partly reflect the protective influence of GSH. Grover (1974) has found that GSH S-transferase activity toward the K-region epoxide of benz[a]anthracene was slightly greater in rat lung than in liver. Purified human and rat liver GSH S-transferases catalyzed the conjugation of the K-region epoxide, benzo[a]pyrene 4,5-oxide with GSH (Nemoto et al., 1975; Nemoto and Gelboin, 1975).Addition of GSH and mouse liver cytosol (containing GSH S-transferases) to an S. t!/ph i ) i t u ri II IN iissay sy s teni containing micro somes reduced the mu tagenicity of benzo[tr Ipyrene which was mediated, in this case, mainly through its K-region epoxide (Glatt and Oesch, 1977). Conjugation of 3-niethylcholanthrene 11,12-oxide with GSH has been studied in rodent liver, lung, and skin (Mukhtar and Bresnick, 1976a,b,c,d). Treatment of rats with polycyclic hydrocarbons, such as 3-methylcholanthrene, induced hepatic GSH S-transferase activity by about 2-fold (Mukhtar and Bresnick, 1976d; Hales and Neims, 1977). In a

226

L. F. CHASSEAUD

limited study, rates of conjugation of GSH with various arene oxides, catalyzed by partially purified sheep liver GSH S-transferases, steadily decreased a s the size of the hydrocarbon ring system increased (Hayakiiwa et (11,, 1975), thereby arguing against an o1)vious protective role of GSH S-transferases against the larger hydrocarbons. However, whether the ease with which a particular epoxide or diolepoxide conjugates with GSH is related in any way to its carcinogenic or mutagenic potency remains to be established.

N. ARYLHALIDEEPOXIDES Historically, the first niercapturic acids reported were those fonned from chlorobenzene (Jaffe, 1879) and bromobenzene (Baumann and Preusse, 1879) and excreted as acid-labile hydroxyarylmercapturic acids (Knight and Young, 1958) that have since been isolated (Gillham and Young, 1968). In general, the extent of metabolism of mono-, di-, and trilialogenobeiizeiies to mercapturic acids (Smith et d . , 1950; Azouz et ul., 1955; Jondorf et d . , 1955; Parke and Williams, 1955) decreased with increased halogen substitution in the ring ranging, for example, from 20% of the dose in the urine of rabbits after oral administration of chlorobenzene, to 11% after nz-dichlorobenzene, and to 0.3% after 1,2,3-trichlorobenzene. The N-acetylcysteine moiety is usually located at a free para position; this may explain why little (Koch-Weser et ul., 1953) or no mercapturic acid (Parke and Williams, 1955) was detected in the urine of animals dosed with p-di-substituted arylhalides. A study of chloro, showed benzene metabolism in rabbits (Lindsay-Smith et ~ l . 1972) that the relative distribution of isomeric mercapturic acids was ortho-2.5%, meta-2.5%, and para-95%, which may suggest stereoselectivity in the formation of the reactive intermediate that conjugated with GSH. Metabolism of arylhalides, like that of arylhydrocarbons, proceeds through intermediate epoxides [eg., XLVI (see Selander et nl., 1975; Hayakawa et al., 19751 that can conjugate with GSH (Brodie et ul., 1971).Rats excreted a greater proportion (70%) ofa nontoxic parenteral dose of bromobenzene as bromophenylmercapturic acid than of a toxic dose (48%) (Zampaglione et ul., 1973), which presumably had caused depletion of hepatic GSH through conjugation with bromobenzene 3,4-oxide (XLVI) (Jollow et d.,1974a). Thus when hepatic levels of GSH became rate limiting for the protective conjugation reaction, increased covalent binding of the reactive epoxide (XLVI) to vital tissue macromolecules occurred resulting in hepatic necrosis (Mitchell et

GSH CONJUGATION WITH CAHCLVOGENS

227

Br

QHH

d.,1975a; Mitchell aiid Jollow, 1975). Pretreatment of rats with phenobarbital enhanced tlie hepatic necrosis caused b y bromobenzene (Reid et ul., 1973a; Jollow et ul., 197421) and the conjugation with GSH (Suga mid Akagi, 1970; Brodie et al., 1971; Jollow et al., 1974a), both effects being due to induction of the niicrosonial oxidases producing more of the intermediate epoxide (XLVI) for covalent binding with tissue macromolecules and conjugation with GSH. Pretreatment of rats with tlie microsomal oxidase inhibitor SKF 525A expectedly exerted the opposite effects (Jollow et ul., 1974a).The ainouiit of covalently bound “hroiiiobenzene” was considerably greater in the necrotic than in the iioniiecrotic areas of liver (Reid aiid Krishna, 1973).Studies of the tissue distribution of cytotoxicity and the covalent binding observed after administration of broniobenzene to mice suggested that binding and necrosis. which were observed in the liver, lungs, and kidneys, were related events (Reid et d . , 1973b). Hepatic necrosis caused by other arylhalides (Brodie et al., 1971; Toranzo et d . , 1977) presumably arose in a similar way, namely, metabolic activation and covalent binding to vital macromolecules. It is probable that metabolism of the ubiquitous enviroiiineiital pollutants, the polyhalogenated biphenyls (Fishbein, 1974),also proceeds tlirougli intermediate epoxides (e.g., XLVIIa, Chen et ( i l . , 1976). These epoxides would be expected to conjugate with GSH, to bind covalently to tissue macromolecules (Seymour et ( I / . , 1976; Wyiidham et a / . , 1976; Shimada and Sato, 1978), and may he implicated in the cause of the mutagenicity (Wyndham et al., 1976) aiid tumorigeiiicity of some polyhalogenated biphenyls in mice (Kimbrough aiid Linder, 1974).

0. OTHEREPOXIDEINTERMEDIATES The major aflatoxin metabolite of certain strains of Aspergillus flnvus is the very potent hepatocarcinogen aflatoxin B, (Wogan, 1973). Its ingestion by man is associated with the high primary liver cancer incidence in certain regions of Africa (Peers et al., 1976). That this

228

L. F. CHASSEAUD

mycotoxin exerts its biological effects through an active metabolite (Garner, 1976; Campbell and Hayes, 1976), probably a 2,3-oxide [Eq. (17)], has been suggested by results from studies of its binding to nucleic acids (Swenson et al., 1977; hlartin and Garner, 1977; Essigmann et ul., 1977), mainly to N-7 of guanine (Essigmann et al., 1977). 0

0

0

0

The presence of GSH was reported to reduce the extent offonnation of aflatoxin B1 adducts with DNA (Allen-Hoffmann and Campbell, 1977), and conjugation of GSH with this mycotoxin in the presence of rat liver preparations has been claimed (Raj et al., 1975). Styrene was partly metabolized to a mercapturic acid (up to 10% dose) by rats and rabbits (James and White, 1967)probably through an intermediate styrene oxide [Eq. (IS)], whose enzyme-catalyzed conjugation with GSH (Boyland and Williams, 1965; Hayakawa et al., 1975) has been subjected to extensive study. It appears that GSH can conjugate to either carbon atom of the oxirane ring, and not only to the tenninal carbon atom (Seutter-Berlage et ul., 1978) as suggested by the i n vivo studies of James and White (1967) [see Eq. (lS)]. Styrene oxide has become a popular substrate for assay of GSH S-transferase activity toward epoxides (see Section 111,E). Styrene oxide was mutagenic when tested in various assay systems (Loprieno et d.,1976; Milvy and Garro, 1976). Similarly, about 40% of an oral dose of thiophene was excreted in the urine of rats and rabbits as 2-thienylinercapturic acid and its likely immediate precursor 3-hydroxy-2,3dihydro-2-thienylmercapturic acid (Bray et (11 ., 1971)which was probably fornied through an intermediate epoxide [Eq. (19)]. 0

8 / I

CH=CH,

HC- CH,

NADPH "0," microsomes

~

0

CHOH- CH,SG

+GSH

~

(18)

GSH CONJUGATION WITH CARCINOGENS

229

Only about 4% of a dose of the carcinochemotherapeutic agent triethyleiieglycol-bis-2,3-epoxypropyl ether was eliminated by rats as niercapturic acids in the urine. However, as administration of this epoxide caused liver GSH levels to decline to 25% of nonnal (Janies and Solheini, 1971), GSH conjugation with the epoxide (Boyland and Williams, 1965) was probably extensive, and much of the conjugate may have been excreted in the bile. A similar comment probably applies to the metabolic fate of 1,2-epoxy-3-phenoxypropaneand its p-nitro analog which have been studied in rats and rabbits (James et n l . , 1978). Boyland and Williams (1965) have reported that 1,2,3,4diepoxybutane which was mutagenic to S. typhimum'unz (McCann et al., 1975) and carcinogenic to rodents (Van Duuren, 1969) underwent enzyme-catalyzed conjugation with GSH and would therefore be expected to be partly metabolized by mercapturic acid biosynthesis (James et d.,196813). Mercapturic acids and related conjugates should be expected as metabolites of compounds containing aromatic rings or isolated double bonds (alkenes) that can be biotransformed to epoxides (Leibman and Ortiz, 1970; Maynert et d . , 1970; Watabe and Yaniada, 1975). The properties of a particular epoxide that would favor its conjugation with GSH have not yet been elucidated. It is likely that its ability to reach the cytosol (where GSH is located) from where it is generated (endoplasmic reticulum or nuclear membrane?) is important together with its intrinsic chemical reactivity and the relative affinities of the GSH S-transferase and epoxide hydratase systems for it. Thus mercapturic acids detected or isolated after administration to animals of the herbicides 2,6-dichlorobenzonitrile (Griffiths et (11., 1966) and carbaryl (Bend et al., 1971), the carcinogens trurzs-4-dimethylaminostilbene (Metzler and Neumann, 1977) and P-naphthylamine (Boyland and Manson, 1966), the drugs 6,6,9-trimethyl-9-azabicyclo[3,3,l]non-3~-yl a,a-di(2-thienyl)glycolate (XLVIIb) (Meshi et n l . , 1972)aid acetanilide 1974), and various alkyl halides (James et d . ,1968a; (Granthani et d., Grasse and James, 1972), alkenes (James and JeEery, 1964), halogenocycloalkanes (Jameset ol., 1971), and cycloalkenes (James et uZ., 1971; Waring 1971) were probably fomied through inteniiediate epoxides (see Figb. 6 and 9) that can conjugate with GSH (Boyland and Williams, 1965; Fjellstedt et ul., 1973; Hayakawa et u l . , 1975; Jerina,

230

L. F. CHASSEAUD

1976). GSH conjugation coiistitutes an available protective niechanism for the detoxication in tissues of those potentially toxic furan and thiophene derivatives (Mitchell et ul., 1974a, 1975a, 1976; Wirth et ul., 1975; Boyd, 1976; McMuitry aiid Mitchell, 1977; Gueiigerich, 1977), such a s the naturally occurring pulmonary toxin 4-ipomeanol (XLVIII) (Boyd, 1977) or the smog contaminant 3-methylfuran (Statham et d . ,1978),which are presumed to act through intermediate epoxides. The extent to which conjugation with GSH niay occur could influence the toxicity of the epoxide. Indeed the presence of GSH reduces the covalent 1)indiiig of at least sonie of these compounds to tissue macromolecules. The carcinogenicity of various epoxides has been reviewed (Van Duuren, 1969; Lawley, 1976).

P. ~P-UNSATURATED COMPOUNDS ap-Unsaturated compounds react with GSH through addition of the iiucleophile (GS-) to the p-carbon atom of the cup-double bond strongly polarized b y electron-withdrawing substituents, such as carbonyl, nitrile, sulfone, or nitro groups [Eq. (ZO)]. Although sometimes exceedingly rapid nonenzymically (Chasseaud, 1967), this reaction is nevertheless usually catalyzed by the GSH S-transferases (Boyland aiid Chasseaud, 1967, 1968; Chasseaud, 1967, 1973c; Habig et al., 1974a; Clifton and Kaplowitz, 1977). GS-%&=6&RCgO

-

+HI

[GS- CR,- C R = R C -0-1 -----+ GS-CR,-

CHR-RC=O

(20)

The nature of the electron-withdrawing group and the position and nature of substitutents 011 the a- or p-carbon atoms of the carboncarbon double bond greatly influence the nonenzymic and enzymic reactions of such compounds with GSH. The nonenzymic reaction of GSH with certain ap-unsaturated carbonyl compounds, including the carcinostatic 4-hydroxy-2-alkenals (Conroy et ul., 1975), has been reported (Hashimoto and Aldridge, 1970; Chasseaud, 1973c, 1974a; Esterbauer et al., 1975). Enzyme-catalyzed conjugation of GSH with diethyl maleate has been examined in some detail (Boyland and Chasseaud, 1967, 1968; Chasseaud, 1976b).This compound has since become a useful agent to deplete liver GSH levels (Boyland and Chasseaud, 1970; Reid et al., 1973a; Jollow et ul., 1974a), even in isolated rat hepatocytes (Hogberg and Kristoferson, 1977) where the protective role of GSH has been studied (Reed and Orrenius, 1978). The cis-ester diethyl maleate ap-

23 1

GSH CONJUGATION WITH CARCINOGENS

peared to cause a greater fall in liver (Boyland and Chasseaud, 1970) or lung (Bruce and Murphy, 1977) GSH levels in rats and mice, respectively, than did the trans-isomer diethyl fumarate. Rat liver GSH S-transferase activity was greater toward the former than toward the latter ester (Boyland and Chasseaud, 1967). As might be expected, inhalation of aciylate esters, such as ethyl aciylate (Table XIII), b y rats resulted in a greater depletion of the nonprotein thiol levels (mainly GSH) in their lungs than in their livers (Silver and Mui-phy, 1978). This depletion was enhanced when hydrolysis of the acrylate esters was inhibited. The studies with subcellular preparations (Boyland and Chasseaud, 1967, 1968; Chasseaud, 1973c) and the report that administration of such compounds to rats (Table XV) could severely deplete liver GSH levels (Boyland a i d Chasseaud, 1970) implied that ap-unsaturated compounds (e.g., Table XIII) would be at least partly metabolized by

TABLE XI11

QP-LJNSATUHATED COMPOUNDS THAT CONJUGATE ENZYMICALLY WITH

GSH AND ARE PROBABLY PARTLY METABOLIZED BY MERCAFTUIUC ACID BIOSYNTHESIS"

Compound type

Ex amp I e

Structure

Ester

Ethyl acrylate

Aldehyde

Cinnamaldeli y de

Ketone

Ethyl vinyl ketone

Lactone

Parasorbic acid

Nitrile

Acrylonitrile

Nitro-compound

P-Nitrostyrene

Sulfone

Methylvinyl sulfone

CH,=CHSO,CH,

Aniide

Acrylamide

CH,= CHCONH,

CH,

C HCOOC H,CH, C H =C HCHO I

CH,= CHCOCH,CH,

CH=CHNO, I

" From Boyland iintl Chasseaud (1967)where otherex~lmples are also cited. Acrylainitle was stndiecl b y Edwards (1975).

232

L. F. CHASSEAUD

TABLE XIV EXCRETION OF 3-HYDROXYPROPYLMERCAPTURIC ACID IN THE URINES OF RATS DOSED WITH CYCLOPHOSPHAMIDE OR RELATED COMPOUNDS~

Compoundb

Mercapturic acid excretion (% dose)

Ally1 alcohol Acrolein Cyclophosphamide Triphosphamide Isophosphamide ASTA-5607 ASTA-5122

14.7 11.0 9.3 8.9 5.1 4.0 2.3

Data of Alarcon (1976). Parenteral doses (i.p. or s.c.) at 179.1 gmoles/kg.

mercapturic acid biosynthesis. Indeed some recent work has begun to confirm this suggestion. At least 11% of a subcutaneous dose of acrolein (XLIX) (Table XIV), an important vapor phase component of cigarette smoke, was nietabolized by rats to N-acetyl-S-(3-hydroxypropyl)~-cysteine (Kaye, 1973; Alarcon, 1976).Reduction of the aldehyde moiety presumably occurred after conjugation of (XLIX) with GSH. The next higher alkenal homolog, crotonaldehyde (L), was similarly excreted by dosed rats as the inercapturic acids of the corresponding alcohol and acid (Gray and Barnsley, 1971); in this case reduction or oxidation of the aldehyde moiety occurred after conjugation of (L) with GSH, which was catalyzed by GSH S-transferases (Boyland and Chasseaud, 1967). A study of the enzyme-catalyzed conjugation of GSH with members of a homologous series of alk-Ben-l-als showed that nonenzymic reaction rates decreased with increasing alkyl chain length, as expected, but that enzymic activity increased to a maximum with the C , member, non-2-en-l-a1 (LI) (Chasseaud, 1967). CH,--CHCHO

CH,CH=CHCHO

CH,( C H,),CH=CHCHO

(XLIX)

(L)

(LI)

The bile of both rats or dogs dosed intravenously with the diuretic ethacrynic acid contained, as major metabolites, the corresponding GSH conjugate [Eq. (21)] and inercapturic acid (Klaassen and Fitzgerald, 1974) which were predictable from the results of an earlier in vitro study by Boyland and Chasseaud (1967) with this compound.

GSH CONJUGATION WITH CARCINOGENS

233

Formation of the GSH conjugate in the proximal tubules of the rabbit nephron has been reported (Fine et al., 1978). C- CH,CH, I

CH,SG I CH- CH,CH, I

OCH, C OOH

OCH,COOH

fiHZ

co

co

Although a small proportion of an intraperitoneal dose of the betel nut alkaloid arecoline (LII) was metabolized by rats to a mercapturic acid, conjugation of this ap-unsaturated ester (LII) with GSH did not appear to be enzyme catalyzed (Boyland and Nery, 1969). The possibility that the chewing of betel nut preparations may be linked to oral cancer has been discussed (Muir and Kirk, 1960).Parasorbic acid (Tac1

I

CH, (LD)

(LW

ble XIII) is one of several lactones found to be carcinogenic on subcutaneous injection into rats (Dickens, 1964), but no carcinogenic effect was observed when this compound was administered orally 1966) although the total dose was nearly 10 times (Dickens et d., greater. Possibly when administered orally, the lactone was more readily detoxified by the greater concentrations of GSH in the liver (see Table I). That conjugation of the cyclopentenone moiety of prostaglandin A 1 with GSH occurred in the human red cell (Cagen et nl., 1976) and was also catalyzed by homogeneous hepatic GSH S-transferases [Eq. (22)] (Cagen et al., 1975) is not unexpected. It has been known for some time that cyclopent-2-en-l-one itself undergoes enzyme-catalyzed

234

L. F. CHASSEAUD

conjugation with GSH (Boyland and Chasseaud, 1967). Other apunsaturated prostaglandins would also be expected to conjugate with GSH, e.g., 15-keto-prostaglandin FZoi(Chaudhari et al., 1977). Within 1 hour of intraperitoneal administration of acrylonitrile (60 mg/kg) (Table XIII) to various rodent species, hepatic nonprotein thiol levels (mainly GSH) had declined to less than 20% of normal (Vainio and Makinen, 1977) thereby indicating a hepatoprotective function of GSH against this nitrile which has been reported to be mutagenic to E . coli (Venitt et al., 1977) and even carcinogenic (Finklea, 1977). Acute administration of almost lethal intravenous doses (150 mg/kg) of acrylonitrile to rats rapidly depleted GSH concentrations in the liver, kidneys, adrenals, and lungs (Szabo et ul., 1977). This depletion was also dose dependent. The suggestion of Szabo et (11. (1977) that a reactive (“probably epoxy derivative”) may also be involved in GSH depletion is unlikely. Acrylonitrile is sufficiently electrophilic to conjugate with GSH (Friedman et nl., 1965) without prior metabolic activation and this conjugation is known to be catalyzed by GSH S-transferases (Boyland and Chasseaud, 1967). The riot control agent o-chlorobenzylidenemalononitrile (LIII) undergoes enzyme-catalyzed conjugation with GSH (Chasseaud, 1973c), but a mercapturic acid or related conjugates were not among reported metabolites of (LIII) (Upshall, 1977). (LIII) has been shown to yield a positive result when tested in a mouse skin bioassay system for screening for potential tumorigenicity (Barry et al., 1972). The corresponding GSH conjugates were excreted in the bile of rats dosed intravenously with the neurotoxic agents acrylamide (Table XIII) or N-hydroxymethylacrylainide. Administration of either compound depleted rat hepatic GSH levels to about 40% of normal (Edwards, 1975). Conjugation of GSH with the carcinogenic nitro-olefins which occur as air pollutants (Deichmann et al., 1965) has not been reported. Such compounds would be expected to react rapidly with GSH by analogy with the related 6-nitrostyrene (Boyland and Chasseaud, 1967) (Table XIII). Like (LIII), the latter compound also yielded a positive result when tested in a mouse skin bioassay system for screening for potential tumorigenicity (Chasseaud et al., 1975). The antitumor activity of P-nitrostyrene derivatives has also been reported (Do& et ul., 1976). The origin of various S-carboxyalkylcysteines that are present in normal human urine or in the urine of certain patients (Ohmori et ul., 1965; Kuwaki, 1965; Kuwaki and Mizuhara, 1966; Kodama, 1968) is intriguing. They may have arisen through conjugation of GSH with a 2,3-unsaturated acylCoA thiol ester, which was shown to be enzyme

GSH CONJUGATION WITH CARCINOGENS

235

catalyzed (Speir and Barnsley, 1971). These cysteine conjugates may also be N-acetylated (Rogers and Barnsley, 1977). The exact origin of one of these conjugates, isovalthine (S-(1-carboxyisobutylcysteine), has recently heen questioned (Niedeiwieser et a/., 1978).

Q. ARYLAMINES,ARYLHYDROXYLAMINES, CARBAMATES, AND RELATED COMPOUNDS Since Jagenburg and Toczko ( 1964) reported that humans excreted

2% of a dose of phenacetin (acetophenetidin) (LIV) in the urine as a cysteine conjugate of paracetamol (acetaminophen) (Fig. lo), consid-

NHCOCH,

(HO-

I

NHCOCH, -t GSH

y- COCH, 1

“5’:

QS7 OH

I I

+tissue

I

macromolecules (TM)

I

I

T NHCOCH,

NHCOCH, I

SC H,C HCOOH

+TM OH

I

OH

(Detoxication)

NHCOCH, (Covalent binding and toxicity)

FIG. 10. Covalent binding and fonnation of a GSH conjugate of paracetamol (adapted from Mitchell et a / . , 1975a). The GSH conjugate 1x1s been detected in rat bile (Malnoe et u / . , 1977).

236

L. F. CHASSEAUD

erable interest has centered around the possible biotransformation mechanism involved and its relevance to the hepatotoxicity of these analgesics (Potter et al., 1973; Mitchell et al., 1974b). Boyland and Chasseaud ( 1969a) speculated that the cysteine conjugate was formed through the reaction of a hypothetical N-hydroxy intermediate with GSH (Fig. 10).This intermediate was also later postulated b y Jollow et al. (1974b) and supported by data from Hinson and Mitchell (1976), as a precursor of the likely reactive species which was covalently bound to important proteins in the liver (Fig. lo), thereby eliciting hepatotoxicity. It was also shown that there was a dose threshold for hepatotoxicity which did not occur until hepatic GSH had been sufficiently depleted (Mitchell et al., 1973; Davis et al., 1974)to about 30% of normal levels (Mitchell et al., 1974b), indicating the effective protective function of GSH when paracetamol is ingested in normal therapeutic doses. For example, intraperitoneal doses of paracetamol exceeding about 200 mg/kg were sufficient to deplete hepatic GSH levels in hamsters to about 20%of normal when the extent of covalent binding to tissue macromolecules increased markedly (Fig. 11) (Jol-

I00

80

-8 c

5

c

60

c) 0

I v)

(3

40

20

0

25

50

I00

200

400

Dose of' H- paracetamol (mg/kg)

FIG. 11. Depletion of hepatic GSH ( 0 )and covalent binding (A)of radioactivity to liver proteins at 3 hours after intraperitoneal administration of several doses of [3H]paracetamol to hamsters (from Jollow et u / . , 1974b).

GSH CONJUGATION WITH CARCINOGENS

237

low et d . , 1974b). A species relationship has been observed between the severity of paracetamol-induced hepatic necrosis and the extent of depletion of GSH resulting from intraperitoneal administration of the compound (300 mg/kg). After 90 minutes, this depletion was about 80% in hamsters and mice, 30% in guinea pigs, and 10% in rats, the most resistant of these species to the hepatotoxic effects of paracetamol (Davis et al., 1974). There have been numerous reports of the metabolism of this drug to the cysteine conjugate and/or mercapturic acid in normal and in paracetamol-intoxicated humans (Mrochek et al., 1974; Andrews et al., 1976; Davis et ul., 1976; Knox and Jurand, 1977). Humans excreted as much as 6% of an oral dose of paracetamol (1.5g m ) in the urine as the cysteine conjugate and a similar amount as the mercapturic acid (Fig. 10) (Jagenburg et al., 1968). The proportions of these conjugates excreted in the urine appeared to increase when “large” amounts of paracetamol were ingested by overdose patients (Davis et al., 1976). In another study, it was found that the proportion of the former conjugate, but not the latter, was significantly increased in the urine of patients with severe liver damage (7.6% dose) compared to those without (3.4% dose) (Howieetal., 1977).Indeed Jollowet al. (197413)suggested that the 4-fold greater excretion of the mercapturic acid by hamsters and mice than b y rats after a dose ofparacetaniol reflected the increased susceptibility of the former two species to the hepatotoxic effects of this drug. Pretreatment of hamsters with the microsomal enzymeinducer 3-methylcholanthrene to enhance formation of the hypothetical reactive intermediate (Fig. 10) raised mercapturic acid output 2-fold. More recent studies of paracetamol metabolism to thiol conjugates have been facilitated by the advent of high pressure liquid chromatography (Buckpitt et al., 1977; b o x and Jurand, 1977). Clinical interest has been considerable (see Goulding, 1976; Williams and Davis, 1977) because of the relatively large number of cases of paracetamol poisoning, and indeed thiols, such as cysteamine (Prescott et al., 1976) or N-acetylcysteine (Prescott et al., 1977), have been employed with varying degrees of success to treat such cases. In theory the most effective thiol for treatment should be cysteine or even GSH itself (Strolin-Benedetti et al., 1975) provided the latter adequately penetrates the hepatocyte and particularly if its conjugation with the reactive intermediate is catalyzed by the GSH S-transferases. However, this has yet to be determined as has the exact nature of the reactive intermediate although sonie progress has been made (Hinson et nl., 1977). The urinary excretion of the corresponding cysteine conjugate (see Focella et al., 1972) was greater from rabbits (8% dose) then from

238

L. F. CHASSEAUD NHCOCH, I

(LW

(LV)

ferrets (5% dose), humans (4% dose), rats (3%dose), or guinea pigs ( ~ 1 % dose) given [14C]phenacetin(LIV) orally (Smith and Timbrell, 1974). On the basis of studies of '*O incorporation, it has been argued that the reactive metabolite arising from phenacetin (LIV) was different to that from paracetamol (see Fig. lo), and may have been an epoxide (Hinson et d . , 1977), although this would be contrary to chemical arguments (Calder et d . , 1973, 1974). This possible difference in reactive metabolites is of interest in view of the reported renal pelvic carcinoma claimed to be associated with ingestion of large amounts of analgesic preparations containing (LIV) (Angervall et (11., 1969) and the renal necrosis that also occurs (Abel, 1971). Mitchell et (11. (19754 have discussed the toxicity, covalent binding, and detoxication by GSH of (LIV). Mercaptiiric acids detected in the urines of animals treated parenterally with aniline (Boyland et al., 1963) and the bladder carcinogen (Case et al., 1954) P-naphthylamine (LV) (Boyland et nl., 1963; Boyland and Manson, 1966) may have been formed partly through initial conjugation of GSH with an intermediate N-hydroxy derivative. Depletion of mouse liver GSH levels after administration of (LV) was considered to result either from conjugation with GSH or from the toxic effects of (LV) (Ungeret d . , 1977). In either event, the protective function of GSH could be impaired. The mechanism of metabolic activation of carcinogenic arylamines and arylamides continues to attract great interest (for reviews, see Weisburger and Weisburger, 1973; Miller and Miller, 1974; Clayson and Garner, 1976), and N-oxidation appears to be an obligatory step in their metabolism to ultimate carcinogens. As yet, none of the reported studies appears to assess whether conjugation of GSH with active metabolites of such compounds occurs or is of any biological consequence. Metabolites present in the urines of animals and humans dosed with the carcinogen urethane (LVI) (Mirvish, 1968; IARC, 1974c) included ethylmercapturic acid and N-acetyl-S-carbethoxycysteine (LVII) (Boyland and Nery, 1965). Ethylglutathione was also detected in the bile of rats dosed with (LVI) (Boyland and Nery, 1965). These metabolites were probably formed through ethylation or carbethoxylation, respec-

GSH CONJUGATION WITH CARCINOGENS

239

tively, of GSH by an intermediate N-hydroxyurethane which has been shown to react in vitro with cysteine to produce S-ethyl and S-carbethoxy derivatives (Boylaiid and Nery, 1965), and also with nucleic acids or proteins (IARC, 1 9 7 4 ~ ) .Siniilar compounds were fornied during reaction of GSH or cysteine iri vitro with N niethyl-N-nitrosourethane (LVII) (Schoental and Rive, 1965),which is representative of a class of carcinogenic compounds (Schoental and Magee, 1962). The possibility that such reactions occur in vioo requires inve stigatiori. CH,C H,OCON H,

C H(COO H)CH, SCOOC H,C H,

I

NHCOCH, (LVI)

(LVII)

(LYIII)

Metabolites of azodye carcinogens such as N,N-dimethyl-4-aniino3-niethylazobenzene (LIX) bind covalently to liver proteins, for example, to the cysteinyl residues of ligandin (GSH S-transferase B in rat liver) (Ketterer and Christodoulides, 1969; Ketterer et d.,1975a, 1976a,b). The biological consequences of this binding are not known. It could be regarded as an inactivation mechaiiisin for azodye carcinogens but the possibility that these liver proteins transport such carcinogens to target receptors cannot be excluded. The presence of ligandin (azodye-binding protein) in various rat tumors seemed to de1973). pend on the degree of cell differentiation (Bannikov et d.,

A small proportion of an oral dose of the herbicide amitrole, 3-aiiiiiio-1,2,4-triazole (LX, R = H) was eucreted in rat urine a s ;i mercapturic acid (LX, R = SCH,CH(COOH)NHCOCH,,)(Grunow et ul., 1975). In view of the goitrogeriic and tumorigenic properties of (LX, R = H) (IARC, 1974c; Tsudaet (/I., 1976),the mechanism of'foiniat'1011 of' t 11 is me tabo 1i tc de serve s cletai 1ed i ii vest i gation .

240

L. F. CHASSEAUD

High concentrations of GSH, which is present in the erythrocyte, substantially diminished the binding of 4-dimethylaminophenol to hemoglobin, by the formation of an unusual tris-glutathione conjugate (Eyer and Kiese, 1976) which was converted to the tris-cysteine conjugate in dogs (Eyer and Gaber, 1978).A "bioactive" bis-glutathione conjugate was also formed when lower concentrations of GSH were present (Eyer and Kiese, 1976), but it was not reported whether GSH Stransferases were involved in the formation of either ofthese conjugates.

R. STEROIDS Incubation of estradiol-17p with GSH in the presence of rat liver preparations resulted in GSH conjugates that were C-1 and C-4 monoand dithioethers of 2-hydroxyestradiol-17P and 2-hydroxyestrone [Eq. (23)] (Ktlss, 1970, 1971). 2-Hydroxy-3-deoxyestradiol underwent siniilar conjugation (Numazawa et a1 ., 1977) as did 2-hydroxyestradiol-17P 3-methyl ether (Hoppen et al., 1973). It is believed that 2-hydroxylation of estradiol-17P precedes conjugation with GSH, but this view has recently been questioned (Numazawa and Nambara, 1977). The exact mechanism has not yet been established, but the available evidence suggests that formation of the reactive intermediate, which is possibly an o-quinone, semiquinone, or arene oxide, takes place enzyinically whereas its conjugation with GSH occurs nonenzyniically. SG

OH

-[ : a i l

GSH CONJUGATION WITH CARCINOGENS

241

Up to 8% of an intraperitoneal dose of [14C]2-hydroxyestradiol-17p was excreted in rat bile as the GSH conjugates [Eq. (23)] (Elce and Harris, 1971; Elce and Chandra, 1973). When the C-1 GSH-estrogen conjugate, S-(2-hydro~yestradiol-l-yl)[~~S]glutathione was administered intraperitoneally to rats, this conjugate together with the corresponding cysteinylglycine and cysteine conjugates and mercapturic acids (Elce, 1970) were excreted in the bile (Elce, 1972). The mercapturic acids were also present in the urine of bile duct-ligated rats (Elce, 1972). However, GSH conjugates were not detected in the bile of humans dosed intravenously with [14C]2-hydroxyestradiol-17p (Elce et ul., 1973). A GSH conjugate of estrone or estradiol has been reported to be a hydrophilic metabolite of 3-deoxyestrone in the urine of rats dosed intraperitoneally (Nambara and Numazawa, 1971). Certain sex hormones are tumorigenic in experimental animals (IARC, 1974b), and the protective action of GSH in the disposition of steroids, such as estradiol-17p (Bolt et ul., 1974; Nelson et nl., 1976a), ethinylestradiol (Bolt et ul., 1974; Bolt and Kappus, 1974), or norethisterone (Kappus and Remmer, 1975; Bolt, 1977), would be to restrict the covalent binding to proteins, of their reactive intermediate metabolites.

s. QUINONESA N D CATECHOLS Conjugation of menadione (vitamin K3)with GSH was catalyzed by rat liver preparations [Eq. (24)] although oxidation of GSH b y the quinone or a reaction product

WcH3 . mcH3 0

0

+GSH

0

SG

(24)

0

(possibly H202)was a competing reaction (Chasseaud, 1974b). However, m ercapturic acid fonm at ion has not been detected after adm i nistration of menadione or other quinones to animals (Bray and Garrett, 1961).The latter finding does not preclude the possibility that conjugation with GSH occurs i i i oioo to provide a means of eliminating reactive and potentially toxic quinones in the bile a s GSH conjugates which , however, may not undergo coin ple te in e rcaptu ric acid biosynthesis.

242

L. F. CHASSEAUD

Quinone intermediates may b e involved in the conjugation of the catechol 2-hydroxyestradiol with GSH (see Section IV,R). Analogously, 5-S-cysteinyldopa, the characteristic amino acid of the inelanocyte, whose urinary excretion is elevated in patients with melanoma metastases (Agrup et nl., 1978), probably arose through initial conjugation of GSH with dopaquinone in melanocytes [Eq. (25)l CH,CH(NH,)COOH

+GSH

C H,C H (NH,)COOH

(25)

HO

SG

yL c H,CCOOH &H3 -0

HO

(LXII)

(Agrup et d., 1975, 1977). The implications of this reaction have yet to be evaluated and other examples no doubt exist as indicated by the studies of Dybing et nl. ( 1976).An expected example includes adrenochrome (LXI) whose reaction with GSH has been studied (Mattok and Heacock, 1965).It is tempting to speculate that conjugation with GSH is a means of inactivating reactive quinones generated from endogenous precursors or foreign compounds, which may otherwise react with biologically important macromolecules. A reactive quillone or semiquillone intermediate is possibly involved in the hepatotoxicity of the antihypertensive agent a-methyldopa (LXII), since the presence of GSH almost abolished covalent binding of the drug with rodent microsomal protein in uitro),when the conjugate of GSH and (LXII) was also detected (Dybing et ul., 1976). Although its structure was not established, this conjugate was probably similar to 5-Sglutathionyldopa [see Eq. (25)l.

T. ISOTHIOCYANATES Conjugation of the naturally occurring benzylisothiocyanate with GSH was enzyme catalyzed, and this compound was metabolized by rats to the mercapturic acid [Eq. (26)l. As expected, the mercapturic acid was the major urinary metabolite of the cysteine conjugate of benzylisothiocyanate in rats (62% dose), but not in dogs when it was mainly hippuric acid (40% dose) (Brusewitz et d.,1977). Humans also

243

GSH CONJUGATION WITH CARCINOGENS

produced this mercapturic acid (Brusewitz et ( i l . , 1977). Studies in the rat after radiolabeling of its body sulfur pool with 3.iS suggested that the cysteine conjugate was partly cleaved to the parent isothiocyanate which was reconjugated with [S5S]GSHand excreted as the [:j5S]mercapturic acid (Brusewitz et al., 1977). Benzylisothiocyanate has been reported to inhiliit the carcinogenic eftect of’ 7,l~-tlimetliylhen z [(I ]anthrxene in rats and i n ice (Wattenberg, 1977), lmt the mechanism involved lius not heen elucitlated. H,NC S

C H,NH$SC HzCHCOOH

CH,NH$SG

Some (e.g., allylisothiocyanate) but not all other isothiocyanates (e.g., a-naphthylisothiocyanate) may be metabolized to the mercapturic acid by rats (Brusewitz et ul., 1977). A major urinary metabolite of the fungicide S-n-butyl S’-p-tertliutylbeiizyl N-3-pyridylditliiocarbonimidate (LXIII) in rats was 2(3’-pyridylimino)-4-carboxylthiazolidine(LXIV) (Ohkawa et nl., 1975) which could have been formed through conjugation of a pyridylisothiocyanate inteiiiiediate with GSH and cyclization of the corresponding cysteine conjugate. Other precursors of isothiocyanates may give rise to metabolites similar to (LXIV).

f)/N=Qco

SCH,CH,CH,CH,

/

a N = c \ s H 2 c o -

c(cH,),

(LXIII)

HN

H

(LXW

U. TFUCHLOROMETHYLSULFENYLS The fungicides captan [Eq. (27)] and folpet (LXV) may owe their biological activity to reaction with cellular thiols, such as GSH, which results in the liberation of thiophosgene (Richmond and Somers, 1966, 1968; Siegel, 1970). Studies of the metabolism of captan in rats suggested that further reaction of liberated thiophosgene with GS H prob-

244

L. F. CHASSEAUD

ably occurred since a major urinary metabolite was thiazolidine-2thione-4-carboxylic acid (LXVI) (DeBaun et al., 1974) which could arise through removal of glutamyl and glycyl moieties from the GSH conjugate [possibly SC(SG)C1] and cyclization of the resultant cysteine conjugate. Similar involvement of GSH in folpet (LXV) metabolism would be expected.

Q \ -f;Nsccl,

co S (LJW

Both captan [Eq. (27)] and folpet (LXV) were mutagenic to E . coli (Legatoret al., 1969; Fahrig, 1974) and S. typhimurium (Shirasuet al., 1976), and their reaction with GSH may represent a detoxication mechanism in mammals. V. THIOCARBAMATES The major pathway for the detoxication of herbicidal thiocarbamates in mice and rats appeared to be initiated by sulfoxidation followed by GSH S-transferase-catalyzed metabolism of the sulfoxide [Eq. (28)l (Casida et al., 1975; Hubbell and Casida, 1977), which also occurred in corn (Lay and Casida, 1976). The exact mechanisms involved merit further study. S-Ethyl dipropylthiocarbamate sulfoxide [Eq. (28)l depressed hepatic GSH levels in treated mice to a greater extent than did the parent compound (Lay and Casida, 1976). Rats metabolized both compounds to the corresponding cysteine conjugate and mercapturic acid [Eq. (28)l. The latter was the major urinary metabolite (Hubbell and Casida, 1977).

Certain thiocarbamate herbicides are reported to be mutagenic to S. typhimurium only after metabolic activation (Sikka and Florczyk, 1978) possibly to the sulfoxides which are carbamoylating agents. W. METALS

Since it was suggested that a major urinary excretion product of the mercurial diuretic mersalyl in dogs was possibly a mersalylcysteine

GSH CONJUGATION W I T H CAHCINOGENS

245

complex (Weiner and Muller, 1955), there has been little reported concerning the involvement of GS H in the detoxication and elimination of mercury compounds. Norseth and Clarkson (1971) showed that an intravenous dose of methyl [““Hg]mercuric chloride was excreted in the bile of rats as a cysteine complex which also underwent enterohepatic circulation. Parallel ytudies in mice indicated that the major biliary metabolite was probably methylmercury glutathione (Norseth, 1971), which was also present in the bile of treated rats (Refsvik and Norseth, 1975).More recently it was demonstrated in rats that an adequate concentration of hepatic GS H was probably necessary for the normal translocation of methylmercury from liver to bile (Refsvik, 1978). Renal mercury accumulation and renal GSH levels have been correlated in rats dosed with methylmercury chloride, and it appears that GSH may be a determinant of the deposition of mercury in the kidneys, since depletion of renal GSH resulted in decreased concentrations of administered mercury in the kidneys (Richardson and Murphy, 1975). A cadmium-GSH complex was apparently excreted in the bile of rats treated with cadmium chloride (Cherian and Vostal, 1977), and GSH complexes of cobalt (Co”), nickel (Ni’+) and platinum (Pt4+)may also be produced since these metals depleted hepatic GSH b y 30-50% within 6 hours of administration to rats (Maines and Kappas, 1977). Between 12 and 24 hours, however, hepatic GSH concentrations reached 200% of’ control values due to rebound phenomena (Maines and Kappas, 1977)which may also occur in other tissues (Sasanie et NZ., 1977), at least in those of animals treated with cobaltous chloride. These results suggest that GSH may function as a means of complexiiig and thereby eliminating some persistent metals from the body. Certain metal compounds are suspected to be carcinogenic to humans (IARC, 1973a; Lemen et al., 1976; Sunderman, 1978).

X. OTHERCOMPOUNDS

1. A x t Ir i o p r - i t i c . Rats metabolized an oral dose of the immunosuppressant [‘4C]azathioprine (LXVII, R = 6-mercaptopurine) to a mercapturic acid [LXVII, R = SCH,CH(NHCOCH,)COOH] through initial conjugation with GSH (de Miranda et ul., 1973) which was probably catalyzed by GS H S-transferases (Kaplowitz, 1977). About 35% of the radioactivity in 6-hour urine was associated with the mercapturic acid. The GSH conjugate (LXVII, R = SG) was detected in the blood and in the bile obtained from a liver perfusion experiment. Apparent uptake

246

L. F. CHASSEAUU

isNo2 NH

I

CH,

I CH,

OHN Z N X J

I :HCOOH

I HOOCHC

I CH,

C H~ s -s -C H ~

(LXVII)

(LXVIII)

of the GSH conjugate into blood cells may have arisen by conjugation of azathioprine with GSH in erythrocytes (de Miranda et d . , 1973)

since azathioprine treatment of rats depleted red cell GSH levels by almost 80'70 (Kaplowitz, 1977). Dogs and humans dosed with azathioprine produced less mercapturic acid than did rats, and a major urinary metabolite in these species was the novel disulfide (LXVIII) which may have been derived from the GSH conjugate (LXVII, R = SG) (de Miranda et u l . , 1975). The latter was probably also the precursor of mother major iirinary metal)olite i n dogs and man, 5-mercapto-liiietliyl-4-iiitroi1iiitlazole (LXVI1, R = SH) (Ch~lmers, 1974). The mutagenicity of azathioprine (Clark, 1975) m a y be caused b y the 6mercaptopurine moiety which has heen reported to be mutagenic to S. t ! / ? ~ / ~ ~ (Reneclict ~ ~ ~ z ~ /et- u~/ .~, 1977). ~ ? ~ t 2. R e i t ~ ~ ) t l i i c i z o l e - 2 - S z i ~ ~ ) / i ( i ? ~ i i c l Urinary metabolites of benzothiazole-2-sulfonamide (LXIX, R = SO,NH,) in the rat, ralhit, and dog were the corresponding 2mercapturic acid [LXIX, R = SCH&H(NHCOCH,)COOH], 2mercaptan (LXIX, R = SH), and its S-glucuronide (Colucci and Buyske, 1965), which prolxilily all arose b y initial replacement of the sulfonamide group 1)y GSH since the same "S-labeled metabolites were produced by a rat dosed with benz0thiazole-[2-~~S]glutathione (LXIX, R = SG) (Colucci and Buyske, 1965). However, initial conjugation of (LXIX, R = SO,NH,) with GSH did not appear to be enzyme catalyzed, at least in the rat (Colucci and Buyske, 1965; Boyland and Chasseaud, unpublished results, 1965). Replacement of the sul-

NHCOCH, SCH,C HCOOH

GSH CONJUGATION WITH CAHCINOGENS

247

fonaniide group of (LXIX, R = SO,NH,) by GSH appears somewhat novel and is unlikely to represent a common nietaliolic pathway for s ti 1fon ani i de s .

3. n ~ ~ ~ l ~ l ~ r ~ c ~ ~ ~ l l ~ The methylthio metabolite of bromazepam in rats (Tateishi and Shimizu, 1976) probably arose through initial conjugation with GSH: formation of the corresponding niercapturic acid (LXX) in rats has recently been reported (Tateishi et ~ l . 1978). , These workers suggested that (LXX) was enzyniically converted to the niethylthio derivative in the presence of S-adenosylmethionine.

4. Crr 11 1 be1 1 tl(1zo 1c Enzyme-catalyzed 4-substitution of cainbendazole (LXXI) b y GS H d,, 1977). The mechanism of this reaction and its possible occurrence with other benzimidazoles is of obvious interest in view of the mutagenicity of some members of this class of compounds (Seiler, 1975, 1976).

i n z;itro has been reported (Baer et

(LXW

s.

Chlor-otollt t”Ot1

The herbicide chlorotoluron (LXXII, R = CHJ was partly metabolized by rats to thioniethyl derivatives (LXXII, R = CH,SCH,) (Muecke et al., 1976), whose mechanism of formation is not known. They could conceivably have arisen through initial conjugation of an intermediate carbonium ion (LXXII, R = CH,+) with GSH.

GC1 NHCON(CH,),

?, p -C\H2

M-P

\

FH“

HN- CH,

R

(LxW

(LXXIII)

6. C!lcloi,lios),llciiIlidc. A probable metabolite of the antitumor agent cyclophosphamide [LXXIII, M = N(CH,CH,Cl),] is the cytotoxic acrolein (LXXIV) (Alar-

l

248

L. F. CHASSEAUD

con and Meienhofer, 1971; Connors, 1976), and the protection afforded by N-acetylcysteine against cyclophosphainide-induced bladder toxicity in rats (Botta et a l . , 1973; Levy and Harris, 1977) may partly reflect its coiijugation with xrolein.

c I I,=C

HC € I 0

CH,(OI-I)CH,CH,SCH,CHCOOH

I

NHCOCH,

(LXXIV)

(LXXV)

Subcutaneous doses of acrolein (LXXIV) were inetabolized by rats to 3-hydroxypropylmercapturic acid (LXXV) (Kaye, 1973; Alarcon, 1976). The latter (LXXV) was also excreted in the urines of rats (Table XIV) or patients treated with cyclophosphamide, thereby providing further evidence of the production of acrolein during nietulmlisni of cyclophospliainide and its prohalde detoxication b y conjugation with GSH. The proportion of the mercapturic acid (LXXV) excreted by rats dosed with cyclophosphamide was constant over the dosage range 12.5-75 mg/kg. The formation of acrolein as a reactive metabolite of other related antitumor agents was implied by the excretion of the mercapturic acid (LXXV) in the urines of rats treated with these cornpounds (Table XIV) (Alarcon, 1976). The crp-unsaturated compounds, methyl vinyl ketone and crotonaldehyde, were apparently produced as metabolites of 4-methyl- and 6-niethylcyclophosphamide,respectively, by rat liver microsomes (Cox et al., 1975). Both these compounds would conjugate with GSH (Boyland and Chasseaud, 1967). In view of the nornitrogen mustard moiety present in cyclophosphainide and related compounds, they would be expected to cause mutagenic events, as was reported recently (Benedict et u Z . , 1977). It is possible that reactive metabolites such as (LXXIV) also contribute to mutagenicity. 7.

C!/teiiibeiitr

Metabolism of the antitumor agent cytembena (LXXVI) by rat or rabbit liver preparations apparently involved enzyme-catalyzed conjugation with GSH, but the GSH moiety was not retained after further metabolism (Mitoma et al., 1977). The structure of the conjugate was not established. However, the thiol could react with (LXXVI) by replacement of the bromine atom or more probably by addition to the ap-unsaturated double bond. The GSH conjugate of cytembena has been isolated in other studies and was less cytotoxic than the parent compound (Jackson et d.,1976). Cytembena (LXXVI) has been shown

GSH CONJUGATION WITIi CAHCINOGENS

249

to depress intracellular GSH levels (Jackson et nl., 1976), and may be active.by virtue of its reaction with essential thiol groups, whereas its conjugation with GS H reflects its detoxication.

8 . DOT

A GSH-dependent enzyme system D D T dehydrochlorinase (EC 4.5.1.1) converts DDT (LXXVII) to DDE by removal of hydrogen chloride (Brooks, 1979). Although this enzyme system has been compared to the GS H S-transferases in insects (Ishida, 1968; Balabaskaran and Smith, 1970; Goodchild and Smith, 1970), it has not yet been definitely established whether it is synonymous with the GS H S-transferases. The controversy surrounding the interpretation of the tumorigenicity of DDT in mice and its usefulness a s an insecticide (Ginin, 1975; Anon., 1976) should prompt further studies ofthe role of GSH in its metabolism.

9. Is o t i itr z id Metabolic activation has been implicated as a cause of the hepatotoxicity produced by therapeutic doses of isoniazid in some patients (Mitchell et al., 197511; Mitchell and Jollow, 1975), who were mostly fast acetylators and probably produced inore acetylisoniazid and its further metabolites, isonicotinic acid a n d acetylhydrazine [Eq. (29)],than did slow acetylators (Mitchell ct ([I., 1975~). The latter compound is an acylatiiig agent and potent hepatotoxin (Nelson et al., 1976b), and could possibly also be responsible for the observed tumorigenicity of isoniazid in experimental animals (Peacock and Peacock, 1966; Severi and Biancifiori, 1968).

The formation ofS -acetylglutathione from acetylhydrazine and GS H in microsomal reactions has been reported (Nelson et ul., 1976b), but whether GS H has any protective action against isoniazid-inducible hepatitis in uivo remains to be established.

250

L. F. CHASSEAUD

10. 3-Phe~iyl-5-~leth~~1-1,~,4-O.~~~cli~~zole A niajor urinary metabolite of the potential tobacco additive 3-phenyl-5-methyl-l,2,4-oxadiazole in rats, mice, and dogs was a mercapturic acid, presumably formed by initial conjugation with GS H, sinee the corresponding GSH conjugate [Eq. (30)] was detected in rat bile (Schickedantz et al., 1976). The reactive intermediate conjugating with GSH was not identified but it may have been a carbonium ion [Eq. (30)l. N-0 H,C, P N A C H ~

-[

1=

H 5 C . ~ N ~ C H ; N-o

N-0

H,C, ANACH,SC

(30) 11. P-Propioluctone Johnson (1966) has reported that conjugation of GSH with the mutagenic (McCann et ul., 1975) and carcinogenic P-propiolactone (Dickens, 1964; Boutwell et al., 1969; Van Duuren, 1969) was enzyme catalyzed. As the product of the reaction between /I-propiolactone and cysteine [Eq. (31)] was much less carcinogenic (Dickens, 1964), conjugation with GSH can be regarded as a means of detoxication of this lact one. p- Prop iol act one probal~l y reacts with GSH to f o m similar proportions of the tliioether [e.g., Eq. (31)] and the thioester (Dijkstra, 1975). HSCH,CHCOOH I NH,

H,C -CH, I 1 - 1 0-CO

CH,CH,COOH

i-

SCH,$HCOOH NH,

12. M iscell a neo us C on1p ou izds There is also evidence that GSH may afford some protection against the effects of toxic (reactive) metabolites arising from a variety of other compounds, such as the antibiotic chloramphenicol (Pohl and Krishna, 1978), the carcinogenic pyrrolizidine alkaloid retrorsine (White, 1976), the hepatotoxin a-naphthylisothiocyanate (El-Hawari and Plaa, 1977), the anticancer agent adriamycin (Olson et al., 1977), and other such drugs (Fujita, 1977), and even ethanol (MacDonald et al., 1977). Whether this protection is mediated through the GSH S-transferases or

GSH CONJUGATION WITH CARCINOGENS

25 1

some other GS H-dependent or associated pathway remains to be investigated. Another carcinogenic pyrrolizidine alkaloid, the metabolite, dehydroretronecine has been shown to react readily with GSH i n vitro [Eq. (32)] (Robertson et a / . , 1977) and it is possible that this also occurs in L i i w . V. Discussion

The previous pages illustrate the wide range of electrophilic agents, including several known mutagens and carcinogens, that conjugate with GSH, a process usually catalyzed by the GSH S-transferases. This conjugation is probably a protective mechanism, and it is the initial stage in inercapturic acid bios ynthesis for the elimination of foreign compounds from the body. The protection of tissues by thiols from the toxic effects of alkylating agents or radiation has long been recognized (Connors, 1966). GSH S-transferases provide protection not only by catalyzing the conjugation of a potential toxicant with GSH, but also by preferentially binding, even covalently, that toxicant. It seems likely that the former process would be more etfective. GSH conjugates have the requisite propertie5 to be excreted in the bile, but the subsequent fate of the biliary-excreted products has not been fully investigated. It is likely that diiring their passage through the gut, GSH conjugates are converted to cysteine conjugates and/or other related products, or possibly even methylthio derivatives (see Section IV,E and IV,X,3) which can be extensively realmxbed to he excreted eventually in the urine, or partly in the feces. In theory, all electrophilic agents can conjugate with GSH. Undoubtedly the extent to which this occurs, and its protective usefulness, will depend on the affinity of the GSH S-transferases for the electrophile and on the latter’s chemical reactivity (electrophilicity), which may have been created by initial metabolic activation, such as epoxidation. Some clue as to whether GSH is likely to be involved in conjugation with particular electrophiles can be provided by studies of the depletion of hepatic (Table XV), and sometimes extrahepatic, GSH that may occur when such electrophiles or their precursors are administered to laboratory animals. Depletion of GSH by the administration of some cornpounds may also cause impairment of GSHdependent defensive systems other than the GS H S -transferases, for example, GSH peroxidase. In this connection, the studies of Bus et ul. (1975) are of interest. They showed that paraquat toxicity was enhanced in mice pretreated with diethyl maleate to lower GSH levels

TABLE XV AFTER THE ADMINISTRATIONOF COMPOUNDS WHICH ARE PROBABLY PARTLY DEPLETIONOF HEPATICGSH CONCENTRATIONS METABOLIZEDBY CONJUGATION WITH GSH AND ,vERCAPTURIC ACID BIOSYNTHESIS"

Compound

Dose level (nig/kg) and route

Species

Time after dosing GSH concentration (% of control) (hour)

Reference

Acrylamide Acrylonitrile Azathioprine Benzyl chloride Bromobenzene Bromocycloheptane Chloroethanol Chloroform Cinnarnaldehyde

100 i.v. 60 i.p. 277 i.p. 500 p.0. 500 p.0. 389 p.0. 10 i.p. 1000 p.0. 525 i.p.

Rat Rat Rat Rat Rat Rat Rat Rat Rat

Cyclo heptene Cycloheptene epoxide Cyclohex-2-en-1-one

250 p.0.

Rat

2

53

168 p.0. 121 i.p.

Rat Rat

2 0.5

52 17

396 p.0. 1000 ppni

Rat Rat

2

4"

52 51

James et al. (1971) Boyland and Chasseaud (1970) Johnson (1965) Jaeger et (11. (1974)

250 p.0.

Rat

2

58

Barnes et al. (1959)

1,2-Dichloroethane l,l-Dichloroethylene" 1,2-Dichloro-4nitrobenzene

40 16 16 18 28 78 54

53 35

Edwards (1975) Vainio and Makinen (1977) Kaplowitz (1977) Suga et a / . (1966) Suga et al. (1966) James et d . (1971) White (1976) Johnson (1965) Boyland and Chasseaud (1970) James et a / . (1971)

D ich 1orvo s Diethy lmaleate S-Ethyl clipropylthiocarlianiate Ethyl iodide a-Hexachlorocyclohexane Methyl iodide Naphthalene 1-Ni trobu tane Paracetaniol Parasorbic acid

2

Phenanthrene Styrene Snniithion Trichloroeth ylene 1,l,l-Trichloropropene oxide Vinyl chloride

30 i.p. 644 i.p.

Mouse

Rat

0.5 2

81 10

Ehrich and Cohen (1977) Boyland and Chasseaud (1970)

308 i.p. 126 p.0.

Mon st'

Rat

1 2

34 54

Lay and Casida (1976) Johnson (1965)

200 p.0. 75 11.0. 500 P.0. 170 p.0. -200 i.p. 142 i.p.

Rat Rat Rat Rat Mouse Rat

12 2 6 4 2 2

66 17 10

Rat Rat &louse Ratd

6 3 2 2'

42 42 65 49

Noack and Portig (1973) Johnson (1965) Snga et ul. (1966) Barnes et (11. (1959) Mitchell et al. (1973) Boyland and Chasseaud (1970) Suga et (11. (1966) Vainio and Makinen (1977) Hollingworth (1969) Moslen et ul. (1977)

Rat Rat

2 7'

43 39

Oesch et u2. (1973) Hefner et ul. (1975)

500 p.o. 600 i.p. 335 p.0. 10,000 ppm

45

44 26

In some cases metabolic activation is necessary before coiljugation with GSH can occur. chloride. Animals exposed for 4,2, and 7 Iiours, respectively, by inhalation. " Animals pretreated for 7 d a y s with phenobarbital.

"

* Vinylidene

254

L. F. CHASSEAUD

(Cagen and Gibson, 1977). Repeated exposure to electrophilic agents may cause increased synthesis of GSH to afford further protection but the extent to which this can occur does not appear to be known. The statement that conjugation with GSH represents a protective mechanism can be challenged because some compounds that conjugate readily with GSH are mutagens or carcinogens, whereas others are apparently not. In this situation the mutagenic or carcinogenic activity of the compound could be permitted as a result of several factors quite apart from those related to conjugation with GSH. Included among the latter are: (i) failure of a “microsomally” generated electrophile to reach the cytosol where GSH and its associated S-transferases are located, (ii) low affinity of the GSH S-transferases for the electrophile allowing the latter to reach vital macromolecules in the cell, (iii) inadequate activity of the GSH S-transferases present in the target organ, (iv) inactivation or inhibition of the GSH S-transferases I,y the electrophile, another electrophile, or their GSH conjugates, and (v) depletion of the GSH levels necessary to ensure adequate protection thereby reaching a “GSH-threshold” below which toxic effects then occur. The latter has been suggested as an explanation of the hepatoxicity of higher doses of compounds such as bromobenzene or paracetamol (Mitchell et ul., 1975a), but has not yet been demonstrated for mutagens or carcinogens. In fact, it appears that there are no studies which clearly show that GSH and its associated S -transferases have prevented a potential carcinogen from exerting its effects, and the evidence that this does occur is, at present, purely circumstantial. It has been noted, however, that there is an apparent inverse correlation between the level of ligandin-binding activity in the liver cell, which is affected by certain drug or hormonal manipulations, and the susceptibility of the cell to chemically induced carcinogenesis (Smith et d.,1977b). Furthermore the recent development of convenient microbiological screening tests for mutagenicity (e.g., Ames et al., 1975; McCannet al., 1975) provides a good means of assessing whether the presence or addition of GSH can reduce, abolish, or possibly even enhance the mutagenic effects of particular electrophilic agents (e.g., Glatt and Oesch, 1977; Rannug et al., 1978). An analogous approach has been successfully employed during studies of the covalent binding of certain electrophilic agents (e.g., Mitchell et nl., 1975a). The reactive electrophiles which conjugate with GSH may also be those that bind to DNA, RNA, and/or protein, and identification of GSH conjugates can provide information on the nature of these biologically active intermediates or even their immediate precursors. Thus

GSH C0NJUC;ATION WITH CAHCIiiOGENS

255

knowledge of the way in which mutagens arid carcinogens are metabolized is essential to a better understanding of their mode of action and of the processes for their detoxication. Structural modification of vital cellular components by electrophilic chemical carcinogens could represent initial and necessary events in neoplastic transformation, and because conjugation with GSH is the process generally available for the detoxication of potentially harmful electrophilic agents, it must rank as a major protective metabolic pathway of the body.

ACKNOWLEDGMENTS The author is most grateful to Drs. P. L. Grover and P. Siins of the Chester Beatty Research Institute, London, for niany usefiil clisciissions and to Mrs. M . Murphy for her help in the preparation of this review.

REFERENCES Abel, J. A. (1971). Cliii. Phccrniuco/. 7’her. 12, 583-598. Agrup, G., Fnlck, B., Kennedy, B. RL., Rorsrnan, H., Hosengren, A. M., and Rosengren, E. (1975). Actcc Derniutocetier. 55, 1-3. Agrnp, G., Falck, B., Rorsman, H., Rosengren, A. M., and Rosengren, E . (1977). Actci Dermatoortier. 57, 221-222. Agmp, G., Hansson, C., Rorsman, H., Rosengren, A. M., Rosengren, E., and Tegner, E. (1978)./. Iiiue.vt. Uertticitol. 70, 25-26. Ahined, A. E., and Anders, M ,W. (1976). Di-iig .\letti/). Dispos. 4, 357-361. Ahmed, A. E., and Anders, M .W. (1978). Riochcitt. Z’hclrttiucd. 27, 2021-2025. Ahmed, A. E., Knbic, V. L., and Anders, M. W. (1977). Drug Metcib. D i s p m . 5, 198-204. Aksoy, M., 13inco1, K., Erdem, S., and Dincol, G. (1972).A m . J . Med. 52, 160-166. Alarcon, R. A. (1976). Cancer Trecitment Rep. 60, 327-335. Alarcon, R. A,, and Meienhofer, J. (1971). Nature ( N e w Biol.) 233, 250-252. AI-Kassab, S., Boyland, E., and Williams, K. (1963). Biocheni. J. 87, 4-9. Allen-Hoffiixinn, R. L., antl Cainpliell, T. C. (1977). Fed. Proc. 36, 1116. Ames, B. N., Siins, P., and Grover, P. L. (1972). Science 176, 47-49. Anies, B. N., McCann, J., and Yamasaki, E. (1975). M u t n t . Re.7. 31, 3 4 7 3 6 3 . Andrews, R. S., Bond, C. C., Burnett, J., Saunders, A,, and Watson, K. (1976).]. Znt. Mecl. Res. 4 (Suppl. 4), 34-39. Angervall, L., Bengtsson, U., Zetterluntl, C. G., and Zsigmond, M . (1969).B r . J . Uro!. 41, 401405. Anon. (1976). Cliii. Toxicol. 9, 827-833. Arias, I. M., and Jakoby, W. B. (eds.).’(1876). “Glutathione: Metabolism and Function.” Raven, New York. Arias, I. M., Fleischner, G., Kirsch, R., Mishkin, S., and Gatmaitan, Z. (1976). Z n “Glutathione: Metabolism and Function” (I. M. Arias and W. B. Jakoby, eds.), pp. 175-188. Raven, New York. Asaoka, K.,Ito, H., antl Takahashi, K. (1977).J. Riochetti. 82, 973-981. Asghar, K., Reddy, B. G., and Krishna, G. (1975).J. Histochem. Cytochem. 23, 774-779. Askelof, P., Cuthenberg, C., Jakobson, I., and Mannervik, B. (1975).Riocheni. J . 147, 513-522.

256

L. F. CHASSEAUD

Azouz, W. M., Parke, D . V., and Williams, R. T. (1955).Bioclieni. J. 59, 410415. Baer, J. E., Jacob, T. A , , and Wolf, F. J. (1977).J. Tosicol. Enairon. Hecrlfh 2, 895-903. Baines, P. J., Bray, H. G., and James, S. P. (1977).Xetiohioticcr 11, 653-663. Balabaskaran, S., and Smith, J. N . (1970).Biochem. J . 117, 989-996. Banerjee, S., and Van Duiiren, B. L. (1978).Cancer Res. 38, 776-780. Bannikov, G. A,, Guelstein, V. I., and Tchipysheva, T. A. (1973). I t i t . J. Concer 11, 398411. Barnes, M. M., James, S. P., and Wood, P. B. (1959).Biocheni. J . 71, 680-690. Bamsley, E . A. (1966).Biocheni. J. 100, 362-372. Barry, D. H., Chasseaud, L. F., Hunter, B., and Robinson, W. E . (1972).Notitre (Lotidon) 240, 560-561. B;irtacli, H., and Montrsano, R. (1975).. \ J i t ! t r f . Re.u. 32, 93-113. Bartsch, H., Malaveille, C., and Montesano, R. (1975a). Znt. J . Caticer 15, 429437. Bartsch, H., Malaveille, C., Montesano, R., and Toinatis, L. (19751-3).Nature (Lotidoti) 255,641-643. Baumann, E., and Preusse, C. (1879).Ber. Dtsch. C h m . Ges. 12, 806-810. Bedford, C. T. (1975).Z r i “Foreign Compound Metabolism in Mainmals” ( D . E. Hathway, ed.), Vol. 3, pp. 365448. Chemical Society, London. Bedford, C. T., Crawford, M. J., and Hutson, D. H. (1975).Chetrzosphere 4, 311-316. Bend, J. R., Holder, G. M., Protos, E., and Ryan, A. J. (1971).Aztst. J . B i d . Sci. 24, 535-546. Bend, J. R., James, M. O., Devereux, T. R., and Fouts, J. R. (1975). In “Basic and Therapeutic Aspects of Peririatal Pharmacology” (P. L. Morselli, S. Garattini, and F. Sereni, eds.), pp. 229-243. Raven, New York. Bend, J . R., Ben-Zvi, Z., Van Anda, J., Dansette, P. M., and Jerina, D. M. (1976). I r i “Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogenesis” (R. I. Freudenthal and P. W. Jones, ecls.), pp. 63-75. Raven, New York. Benedict, W. F., Baker, M . S., Haroun, L., C h i , E., and Ames, B. N. (1977).Cancer Res. 37,2209-2213. Belike, G. M., and Murphy, S. D. (1975).Tosicol. Appl. Pharmucol. 31, 254-269. Benson, A. M., Talalay, P., Keen, J. H., and Jakoby, W. B. (1977).Proc. Notl. Accrd. S c i . U.S.A. 74, 158-162. Bentley, P., Schniassmann, H., Sims, P., and Oesch, F. (1976). Etrr. J . Bioclaeni. 69, 97-103. Betts, J. J., James, S. P., and Thorpe, W. V. (1955).Biocheni. J . 61, 611-617. Bolt, H. M. (1977).Arch. Tnxicol. 39, 13-19. Bolt, H . M . , and Kappus, H., (1974).J.Steroid Biochem. 5, 179-184. Bolt, H. M., Kappus, H., and Kasbohrer, R. (1974).J . Cliti. Etidocrinol. Metulh. 39, 1072-1080. Bonse, G., and Henschler, D. (1976).CRC Crit. Rev. Toxicol. 4, 395408. Booth, J., and Sims, P. (1974).Biochetn. Phurnicicol. 23, 2547-2555. Booth, J., Boyland, E., and Sims, P. (1961).Biochem. J. 79, 516-524. Booth, J., Keysell, G. R., and Sinis, P. (1973).Biocheni. Phurnicicol. 22, 1781-1791. Booth, J., Keysell, G. R., Pal, K., and Sinis, P. (1974).F E B S Lett. 43, 341-344. Booth, J., Hewer, A., Keysell, G. R., and Sims, P. (1975).Xeriobioticcz 5, 197-203. Borchert, P., Miller, J. A., Miller, E. C., and Shires, T. K. (1973a). Cancer Res. 33, 590-600. Borchert, P., Wislocki, P. G., Miller, J. A., and Miller, E. C. (1973b). Cuticer Res. 33, 575-589. Boutwell, R. K., Colburn, N. H., and Muckerman, C. C. (1969).Ann.N.Y. Acad. Sci. 163, 751-763.

GSH CONJUGATION WITH CARCINOGENS

257

Botta, J. A., Jr., Nelson, L. W., and Weikel, J. H., Jr. (1973).J. N a t l . Caiicer lti5t. 51, 1051-1057. Boyd, M. R. (1976). Enoiron. Health Perspect. 16, 127-138. Boyd, M. R. (1977). Nuttrre (London) 269, 713-715. Boyland, E. (1950). BiiJcheln. Soc. Symp. 5, 40-54. Boyland, E. (1969). Prog. Exp. Tumor Res. 1 1 , 222-234. Boyland, E., and Chasseaud, L. F. (1967). Biochem. J. 104, 95-102. Boyland, E., and Chasseaud, L. F. (1968). Biockenr. J. 109, 651-661. Boyland, E., and Chasseaud, L. F. (1969a).Ado. Etiz!/moZ.32, 173-219. Boyland, E., and Chasseaud, L. F. (196913).Biochem.J. 115, 985-991. Boyland, E., and Chasseaud, L. F. (1970). Biochem. Pharmacol. 19, 1526-1528. Boyland, E., and Grover, P. L. (1967). Clin. Chitn. Act0 16, 205-213. Boyland, E., and Manson, D. (1966). Biochem. J. 101, 84-102. Boyland, E., and Nery, R. (1965). Biochem. J. 94, 198-208. Boyland, E., and Nery, R. (1969). Biochem. J. 113, 123-130. Boyland, E., and Sims, P. (1960). Biochem. J . 77, 175-181. Boyland, E., and Sims, P. (1964). Biochem. J. 91, 493-506. Boyland, E., and Sims, P. (1965). Biochem. J. 97, 7-16. Boyland, E., and Speyer, B. E. (1970). Biochem. J. 119, 463-472. Boyland, E., and Williams, K. (1965). Biochem. J. 94, 190-197. Boyland, E., Ramsay, C . S., and Sims, P. (1961). Biochem. J . 78, 376-384. Boyland, E., Manson, D., and Nery, R. (1963). Biochem. J. 86, 263-271. Bray, H. G., and Garrett, A. J. (1961). Biochem. J. 80, 6P. Bray, H. G., James, S. P., and Thorpe, W. V. (1957). Biochem. J. 67, 607-616. Bray, H. G., James, S. P., and Thorpe, W. V. (1958). Biochem. J. 70, 570-579 Bray, H. G., Franklin, T.J., and James, S. P. (1959). Biochem. J. 71, 690-696. Bray, H. G., Caygill, J. C., James, S. P., and Wood, P. B. (1964).Biochem. J. 90, 127-132. Bray, H. G., Carpanini, F. M. B., and Waters, B. D. (1971).Xenohiotica 1, 157-168. Brem, H., Stein, A. B., and Rosenkranz, H. S. (1974). Cancer Res. 34,2576-2579. Brodie, B. B., Reid, W. B., Cho, A. K., Sipes, G., Krishna, G., and Gillette, J. R. (1971). Proc. N a t l . Acad. Sci. U.S.A. 68, 160-164. Brookes, P. (1977). Mutat. Res. 39,257-284. Brooks, G. T. (1979).In “Progress in Drug Metabolism” (J. W. Bridges and L. F. Chasseaud, eds.), Vol. 3, pp. 151-214. Wiley, London. Brown, B. R., Jr., and Sipes, I. G. (1977). Biochem. Pharmucol. 26, 2091-2094. Brown, B. R., Jr., Sipes, I. G., and Sagalyn, A. M. (1974)Aiiesthesioloa!! 41, 554-561. Bruce, M. C., and Murphy, S. D. (1977). Toxicol. Appl. Pharmacol. 41, 184. Bruice, P. Y., Bruice, T. C., Yagi, H., and Jerina, D. M. (1976).J. A m . Chem. Soc. 98, 2973-298 1. Brusewitz, G., Cameron, B. D., Chasseaud, L. F., Gorler, K., Hawkins, D. R., Koch, H., and Mennicke, W. (1977). Biochem. J. 162, 99-107. Buckpitt, A. R., Rollins, D. E., Nelson, S. D., Franklin, R. B., and Mitchell, J. R. (1977). Anal. Biochem. 83, 168-177. Bus, J. S., Aust, S. D., and Gibson, J. E. (1975).Res. Commun. Chem. Pathol. Pharmacol. 11, 31-38. Bnsey, W. M., Ulrich, C. E., and Lewis, T. R. (1978). Toxicol. Appl. Pliarmacol., 45, 286. Cagen, S. Z., and Gibson, J. E. (1977). Toxicol. Appl. Pharmacol. 40, 193-200. Cagen, L. M., Pisano, J. J., Ketley, J. N., Habig, W. H., and Jakoby, W. B. (1975). Biochint. Biophys. Acta 398,205-208. Cagen, L. M., Fales, H. M., and Pisano, J. J. (1976).J. Biol. Chem. 251, 6550-6554.

258

L. F. CHASSEAUD

Calder, I. C., Creek, M. J., Williams, P. J,, Funcler, C . C., Green, C . R., Ham, K. N., and Tange, J . D. (1973).J . Med. Clieni. 16, 499-502. Calder, I. C., Creek, M. J., and Williams, P. J. (1974). Clzem. B i o l . Interoct. 8, 87-90. Campbell, T. C., and Hayes, J . 13. (1976). To.xico/.Apfr/. Pkunriuco/. 35, 199-222. Case, R. A. M., Hosker, M . E., McDonald, D. B., and Pearson, J. T. (1954). B r . ] . Zndtrstr. Rled. 11, 75-104. Casida, J. E., Kimmel, E. C., Ohkawa, H., and Ohkawa, R. (1975). Pest. Biocliern. P / l ! / S i O I . 5, 1-11. Chalmers, A. H. (1974). Biocheni. Plicirniocol. 23, 1891-1901. Cliasseaiid, L. F. (1967). P1i.D. Thesis, London University. Chasseaiid, L. F. (19734. Drug Metuh. Rec. 2, 185-220. Chasseaud, L. F. (197311).Z. Physiol. Cheni. 354, 829-830. Cliasseautl, L. F. (19734. Biocheni. J. 131, 765-769. Chasseaud, L. F. (1974a). Biochern. Phurntacol. 23, 1133-1134. Chasseaud, L. F. (19741,). In “Glutathione” (L.Flohb, H. Ch. Benohr, H. Sies, H. D. Waller, and A. Wendel, eds.), pp. 90-108. Thieme, Stuttgart. Chasseaud, L. F. (1976a). Z i t “Glutathione: Metabolism and Fnnction” (I. M . Arias and W. B. Jakoby, eds.), pp. 77-114. Haven, New York. Chasseaud, L. F. (197611).Z n “Glutathione: Metabolism and Function” (I. M . Arias and W. B. Jakoby, eds.), pp. 281-282. Raven, New York. Chasseaud, L. F. (1979). Z r i “Extrahepatic Metal,olisin of Drugs and Other Foreign Compounds” (T. E. Gram, ed.), in press. Spectriim, New York. Chasseaiid, L. F., Hunter, B., Robinson, W. E., and Barry, D. H. (1975). Erlrerientio 31, 1196-1197. Chatfield, D. H., and Hunter, W. H. (1973). Biochem. J. 134, 879-884. Chaudhari, A., Eling, T. E., Anderson, M . W., and Hart, L. G. (1977).Fed. Proc. 36, 308. Chen, P. R., McKinney, J. D., and Matthews, H. B. (1976). D r u g Metdi. Dispos. 4, 362-367. Cherian, M . G., kind Vostd, J. J. (1977).J. 7’o.xicol.Etit;iroit. H e t i l t h 2, 945-954. Christ-Hazelliof, E., Nrigteren, D. H., and Van Dorp, D. A. (1976). Biocliint. B i o p h y s . Actci 450, 450-461. Clapp, J. J., and Young, L. (1970). Biocheni. J. 118, 765-771. Clapp, N. K., Craig, A. W., and Toya, R. E., Sr. (1968). Science 161, 913-914. Clark, J . M. (1975). M r r t c r t . Hes. 28, 87-89. Clark, A. G., Darby, F. J., and Smith, J. N. (1967). Biocheni. J. 103, 49-54. Clark, A. G., Mtirphy, S., and Smith, J . N. (1969). Biocheni. J. 113, 89-96. Clark, A. G . , Smith, J. N., and Speir, T. W. (1973). Biochem. J. 135, 385-392. Clark, A. G., Cropp, P. L., Smith, J. N., Speir, T. W., and Tan, B. J.Xl976).Pest. Biocliem. P h y s i o l . 6, 126-131. Clark, A. G., Letoa, M., and Ting, W. S. (1977). Life Sci. 20, 141-147. Clarke, C. H. (1971). M i r t c i t . Res. 11, 247-248. Clayson, D. B., and Garner, R. C. (1976).Z r i “Chernical Carcinogens” (C. E . Searle, ed.), pp. 3 6 6 4 6 1 . American Chemical Society, Washington. Clifton, G., and Kaplowitz, N. (1977). Cancer Res. 37, 788-791. Clifton, G., Kaplowitz, N., Wallin, J. D., and Kuhlenkamp, J. (1975). Biocheni. J. 150, 259-262. Cohen, A. J., Smith, J. N . , and Turbert, H. (1964). Bioclicni. J. 90, 4 5 7 4 6 4 . Cohen, S. M., Erturk, E., Von Esch, A. M., Crovetti, A. J., and Bryan, G. T. (1973)J. N u t / . Cmncer Znsf. 51, 403-417. Colien, E. N., T r d e l l , J. H., Ediniinds, H. N., and Watson, E. (197S).Aiiest/iesio/o~!/ 43, 392401.

GSH CONJUGATION WITH CARCINOGENS

259

Cohen, S . M., Ertiirk, E., antl Bryan, G . T. (1976).J. N a t l . Cartcer Z t t s t . 57, 277-282. Colucci, D. F., and Buyske, D. A. (1965).Riocltetit. Plicirntcicol. 14, 457-466. Combes, B. (1965).J . Clitt. Zticec.t. 44, 1214-1224. Combes, B., m d Stakeluni, G. S . (1961)./. Clitt. Zttcest. 40, 981-988. Combes, B., and Stakeliim, G. S . (1962j.J. Clitt. Ztrce.st. 41, 750-757. Connors, T. A. (1966).Eirt-. J . Cnticer 2, 293-305. Connors, ’L A. (1976). 111 “Progress in Drug X1etnl)olisni” (J. W. Bridges antl L. F. Chasseaud, eds.), Vol. 1, pp. 41-75. Wile?, London. Coiiolly, R. B., and Jaeger, R. J. (1978). Torico/. Apjd. P l t o r t t w c d . , 45, 3.38. Conroy, P. J., Nodes, J. T., Slater, T. F., and White, G. W. (1975).E i t r . J . Cnttcer 11, 231-240. Cox, P. J., Farmer, P. B., and Jarman, XI. (1975).Hiocltcnt. Phurmclcd. 24, 599-606. Crawford, hl. J., IIutson, D. H., and King, P. A. (1976). Xettohioticrr 6, 745-762. Crayford, J . V., and Hutson. D. H. (1972). Pest. R i o c l w t ~ P~ h. y . r i o / . 2, 295-307. Creech, J. L., Jr. and Johnson, M . N. (1974).J . Occir),. Jled. 16, 150-151. Daly, J . W., Jerina, D. hl., and Witkop, B. (1972).E.xperieiificr 28, 1129-1149. Daniel, J . W., Gage, J. C., a n d Jones, D. I . (1968). Riocltem. J . 106, 783-790. Darhy, F. J. (1973).Foreti.vic Sci. 2, 451-457. Darby, F. J., and Grundy, R. K. (1972).Biodtetri J. 128, 175-177. Datta, D. V., Singh, S., ;md Chhuttani, P. N . (1973a).C/iri. Chitit. Acto 49, 247-249. Datta, D. l’.. Singli, S., Samanta, A. K. S., Saha, S., hliikherjee, hl., Nirankari, 0. P., Chhrittani, P. N., and Nair, C. R. (197311).Clirt. Cltini. Acto 47, 133-137. Datta, D. V ~ Singh, , S.. and Chhuttani, I? N. ( 1 9 7 3 ~ )ZtidicrttJ. . Jled. Re.r.. 61, 1351-1359. Dauterman, W. C. (1971). B i t / / . \V/ci. Hecilth O r g . 44, 133-150. Ihitteriiimi, W.C., and Xluecke, W.( 1974).Pest. Riocltetti. Ph!/sio/. 4, 212-219. Davis, D. C.. Potter. Lf’. Z., Jollow, D. J., and Xlitchell, J . H. (1974).L i f e Sci. 14, 20992109. Da\is, M,,Simmons, C . J., Harrison, N. G., and Williams, R. (1976). ()rrcirf. J . .\fed. 45, 181-191. Davisou, C., Rozman, R. S., and Smith, P. K., (1961).Biocltertt. Phnrntnco/. 7, 65-74. De Bnrin, J. R., hliaiillis, J . B., Knarr. J., Mihailovski, A,, and Menn, J. J . (1974). Xeitobiotictr 4, 101-119. De Lorenzo, F., DegI’Innocenti, S., Ruocco, A , , Silengo, L., and Coi-tese, R . (1977). crrtlcer nc>s.37, 1 ~ 1 5 - 1 ~ 1 7 . d e Mirantla. P., Beacham, L. &I., 111, Creagh, T. H., and Elion, G. B. (1973)./. Pltcrrtitcrco/. E.x)J.Tltrr. 187, 588-601. de hliranda, P., Beacham, L. bl., 111, Creagh, T. H., a n d Elion, G . B. (1975)./. k‘harn l c i C o / . E.r/t. T/ter. 195, 50-57. Deichnia~rn,W. B.. hlacDondtl, W. E., Laiiipe, K. F., Dressler, I., iind Anderson, W.A. 1). (1965)./ t t d i r . y t . .\fed. Sitrg. 34, 800-807. DHEW ( 1976). U . S . Departnwnt of Heulth, Education and Welfare. National Cancer Institute, Carcinogeiiesis Technical Heport Series No. 2 . Carcinogeiiesis Bioassay of Tricliloroetliyleiie, CAS N o . 79-01-6,NCI-CC-TH-2. U.S. Goveriiiiient Printing Office, Washiiigton. Dickeiis, F. (1964).f i r . Jfed. B u l l . 20, 96-101. Dickens, F., Jones, H. E. H., and Waynforth, H. B. (1966). B r . J . Crritcet-, 20, 134-144. Dicowsky, L., antl Morello, A . (1971). Life Sci. 10, 1031-1037. Dijkstra, J . (1975).Clietit. B i d . Ztrtertrcf. 10, 115-121. Docks, E. L., and Krishna, G . (1976).E s p . Mo/. P o t l i d . 24, 13-22. Dodd, hl. C., and Stilliiian, W. B. (1944).J. Pltctrntrrcol. Eslr. Tlter. 82, 11-18. Doll, R. (1955).A h . Cnttcer RPS.3, 1-50.

260

L. F. CHASSEAUD

. 451-460. Dore, J . C., Chalvet, O., and Viel, C . (1976). E t i r . J . Med. C h e n ~ 11, Douch, P. G . C . , ant1 Buchanan, L. L. (1978). Xenohioticcr 8, 171-176. Druckrey, H., Kruse, H., Preussmann, R., Ivmovie, S., and Landschutz, Ch. (1970). Z. Krebsforsch. 74, 241-273. Dybing, E., Nelson, S. D., Mitchell, J . R., Sasame, H. A,, and GLllette, J. R. (1976). Mo/. Plicrrntocol. 12, 911-920. Edwards, P. M. (1975). Riockem. Phnrnttrcol. 24, 1277-1282. Edwards, K., Jackson, I I . , and Jones, A. R. (1970a).Biochetti. Phcrrtnncol. 19, 1783-1789. Edwards, K., Jackson, H., and Jones, A. R. (1970b).Biocliefn.P/iarnincol. 19, 1791-1795. Ehricli, M., and Cohen, S. D. (1977). Biochetfi. Plinrntcrcol. 26, 997-1000. El-Hawari, A. hl., and Plaa, G . L. (1977). Riochctri. Phtirintrco/. 26, 1857-1866. E k e , J. S. (1970). Bioclieit-r.J . 116, 913-917. Elce, J. S. (1972). Biocliettl. J . 126, 1067-1071. Elce, J. S.,and Chandra, J. (1973). Steroid.P 22, 699-705. E k e , J. S., and Harris, J. (1971). Steroitls 18, 583-591. Elce, J. S., Bird, C. E., a n d Chandra, J. (1973).J. Clitt. Eiitlocritiol. Afctmb. 36, 10271030. Ellman, G. L. (1959).Arch. Rioclieni. Biop/i!/.s. 82, 70-77. Endo, H. (1958). Gotiti 49, 151-155. Epstein, S. S., and Shafner, H. (1968). Ncrtirre (Lotttlori) 219, 385-387. Epstein, S. S., Bass, W., Arnold, E . , a n d Bishop, Y. (1970). Scietice 168, 584-586. Eschenbrenner, A. B., and Miller, E . (1944).J. N u t / . Cuncer Ztnt. 4, 385-388. Essigniann, J. M., Croy, R. G., Nadzan, A. M., Btisby, W. F., Jr., Reinhold, V. N., Buchi, G . , and Wogan, G. N . (1977).Proc. N o t / . Acad. Sci. U.S.A.74, 1870-1874. Esterbarter, H., Zollner, H.,and Scholz, N. (1975). Z. Notrirforsclt. 30, 466-473. E y c ~ P., , and Galier, H. (1978). Bioclietti. Plitrrrtiacol. 27, 2215-2221. Eyer, P., and Kiese, M. (1976). Clitwi. B i d . Ititeruct. 14, 165-178. Fahrig, R. (1974).Z t t “Cheniical Carcinogenesis Essays” ( R . Montesano and L. Tomatis, eds.) pp. 161-178. International Agency for Research on Cancer, Lyon, Ffiince. Feinfeld, I). A., Bourgoignie, J. J.. Fleischner, G., Goldstein, E. J.. Riempica, L., arid Arias, I. M. (1977). K i d t i e ! / Z t t t . 12, 387-392. Figueroa, W. G., Raszkowski, R., and Weiss, W. (1973). New Etigl. J . Med. 288, 10961097. Fine, L. G., Goldstein, E . J., Trizna, W., Rozmaryn, L., and Arias, I. M. (1978). Proc. Soc. E s p . B i o l . Alad. 157, 189-193. Finklea, J. F. (1977). Vet. Htrttiott Tosicol. 19, 219-220. Fiserova-Bergerova, V. (1977). Xenobiotictr 7, 113-1 14. Fishbeill, L. (1974). Anti. Rev. Phartntrcol. 14, 139-156. Fishbein, L. (1976). M u i t r t . Res. 32, 267-307. Fjellstedt, T. A., Allen, R. H . , Duncan, B. K., and Jakoby, W. B. (1973).J . A i d . Cliem. 248,3702-3707. Fleischner, G . M., and Arias, I. M . (1976). Prog. Lioer Uis. 5, 172-182. Fleischner, G . , Rohl>ins, J., and Arias, I. M . (1972).J. Cliti. Zttocst. 51, 677-684. Fleischner, G. M., Kamisaka, K., Gatmaitan, Z., and Arias, I. M . (1976).111 “Glutathione: Metabolism and Functiou” (I. M. Arias and W. B. Jakoby, eds.), pp. 259-264. Raven, New York. FlohL., L., Benohr, H. Ch., Sies, H., Waller, H. D., and Wendel, A. (ed “ Glutathione.” Thierne, Stuttgart. FlohC, L., Gunzler, W. A , , and Ladenstein, H. (1976). I n “Glut:ithione: Metabolism and Function” (I. M . Arias and W. B. Jakoby, eds.), pp. 115-135. Raven, New York. Focella, A,, Heslin, P., and Teitel, S. (1972). Catt. J . Chem. 50, 2025-2030.

GSH CONJUGATION W I T H CARCINOGENS

26 1

F r e d , J . J., and Cliadwick, R. W. (19731.1. A g r . Food Cltem. 21, 4 2 4 4 2 7 . Frear, D. S., and Swanson, H. R. (1970). Ph!/tochentistr!/9, 2123-2132. Frear, D. S., and Swanson, H. R. (1973). P e s t . Hiochent. P / t ! / , y i o l . 3, 473-482. Freudenthal, R. I., and Jones, P. W. (eds.)(1976) “Pol!~tii~c/ecir Aromatic Hydrocarbons: Chemistiy, Metabolism and Carcinogenesis.” Raven, New York. Friedman, M., Cavins, J . F., and Wall, J. S. (1965).J. Atti. Clterrt. SOC. 87, 3672-3682. Fiijita, 14. (1977). T\urtit~iiU . Dent. J . 3, 1-7. Fiikami, J., a n d Sliishido, T. (1966).J. Ecott. E t t t o t i t o l . 59, 1338-1346. Fiikunaga, K., Fukami, J.. and Shisliido, T. (1969).Re.sit/tre Rec. 25, 223-249. Garner, R. C. (1976). 111 “Progress in Drug hletabolism” (J. W.Bridges and L. F. Chasseaiid, e d ~ . )Vol. , 1, 1111. 77-128. Wiley, London. Gatmaitan, Z., Lewis, S., Tiirchin, H., and Arias, I. M. (1977). Bioc/tet?t.Bioi)/t!/.y.Res. cor?ttrtlltt. 75, 337-341. Celboin, H. V. (1969).Cottcer Res. 29. 1272-1276. Gillham, B. (1971). H i o c l i r t t i . J . 121, 667-672. Gilllinm, B. (1973). Hioclict~t.J . 135, 797-804. Gillham, B., and Young, L. (1968).BiocJteitt. J . 109, 143-147. Glatt, H., and Oesch, F. (1977).Arclt. Toxicol. 39, 87-96. Goldstein, E. J., and Arias, I. h l . (1976). 1itae.r.t. Rtrdiol. 11, 594-597. Gocitlchiltl, B . , and Sinith, J. N . (1970). Biochetit. 1. 117, 1005-1009. Gotlie, R., Callem;in, C. J., Ehrcwlwrg, L., and Wachtnieister, C. A. (1974). At?t/Jio 3, 234-236. Goulding, R. (ed.). (1976).J. Z t t t . ,\lc,tl. R P S . 4,(Suppl. 4). Grahnen, A,, and Sjoholm, I . (1977). E i t r . J . Hioc/to)i. 80, 57.3-580. Gram, T. E., Litterst, C. L., antl hlimnaiigli, E. G . (1974). I>rtrg J f e f t r b . 1lis))o.r.. 2, 254-257. Gr;intliain, P. H., \lohan, I,. C., Weisl)iirger, E. K,,Fales, H. %I., Sokoloski, E. A., antl Weisburger, J. H . (1974).X o t o h i o t i c c r 4, 69-76. Grasse, F. R., antl James, S. P. (1972). Xertohiotictr 2, 117-127. Gray, J . hl., and Barnsley, E. A. (1971). Xettohiotictr 1, 55-67. Green, R. M.,antl Elce, J. S. (1975). Bioclietii. J . 147, 283-289. Green, T., and Hathway, D. E. (1975). Chetit. R i o l . Ztitct-met. 11, 545-562. Green, T., antl Hathway, D. E. (1977). Cltetit. B i o l . Ititcrcrct. 17, 137-150. Greim, H.,Boiise, G., Hadwan, Z., Heichert, D., and I-lenschler, I). (1975). Riocltetit. P h (1 rnt ocol. 24, 20 13-20 17. Griffiths, hi. H., Moss, J. A,, Rose, J. A., and Hathway, D. E. (1966). Hioc/ict?t. .I. 98, 770-78 1. Grover, 1’. L. (1974). Hiochettt. P/ictrtittrc.o/. 23, 3.33-343. Gro\,cr, 1’. I,. (1977). 111 “Dritg XIet;il)itlisiii-trc,m k1icroI)e to .\litin” (11.L’. 1’;irke ;ind 11. L.Sniitli, etls.). 1111, 105-122. Taylor a t i t l Fr;uncis, 1,onclon. Grover, P. L., antl Sims, P. (1964).Biocltetit. ,[. 90, 603-606. Grover, P. L., ;tnd Sims, P. (1965). Bioc/wtii. J . 96, 521-525. C;ro\.er, 1’. I.., i i n d Siiiis, P. (1968). Hiochc,itt. J . 110, 159-160. Grover, P. L. S i m s , P., €Iitl)erman, E., hlarqiiartlt, H., Kuroki, T., and Heitlell)erger, C. (1971a). Proc. S n t l . Accrtl. Sci. I J . S . A . 68, 1098-1101. Grovci-. k’, L., Hewer, A,, i i i i t l Siiiis, P. (19711)).I;EHS Lett. 18, 76-80. Grover, P. L., Forrester, J . A , , and Sims, P. ( 1 9 7 1 ~ Hioc/tet?t. ). P/tcrrttitrcd. 20, 1297-1302. 21, 2713-2726. Grover, P. L., Hcwc>r,A., ; ~ i i t lSinis, P. (1972). Riochettt. P/tortrt~rco/. Grover, P. L., Siins, I’., hlitclile)., R. C. l’.,m t l Roe, F. J. C . (1975). Br. /. Cottcer 31, 182-188. Griiiiow, W., Altmann. H. J., and Bolimc~,Chr. (1975).At-ch. Toaico/. 34, 315-324.

262

L. F. CHASSEAUD

Guengericli, F. P. (1977). Biocliern. PIr~ritincol.26, 1909-1915. Gnnn, 13.I,. (1975).Z i t “Foreign Compound Metaliolisni in Mainmals” (D. E. Hatlrway, e d . ) , Vol, 3, pp. 1-82. Chemical Society, London. Hahig, W. H., Pahst, M . J . . and Jakoby, W. B. (1974a).J. B i d . Clreiii. 249, 7130-7139. Habig, W. H., Palxt, M. J., Fleiscliner, G., Gatmaitan, Z., Arias, I. M., and Jakoby, W. B. (19741)). Proc. Ntrtl. Acotl. Sci. C1.S.A. 71, 3879-3882. Habig, W. H., Keen, J. €I., and Jakoby, W. B. (1975). Biochetit. Bioph!/.s. Res. Coniini~it. 64, 501-Fj06. Habig, W. IH., Palxt, M. J., and Jakoby, W. B. (1976a). Arch. Bioclietn. R i o p h ! / s . 175, 7 10-7 16. Habig, W. H., Kamisaka, K., Ketley, J. N., Palist, M. J., Arias, I. M., and Jakoby, W. B. (1976b).Z r i “Glntatliione: Metabolism and Function” ( I . M .Arias and W. B. Jakoby, etls.), pp. 225-231. Raven, New York. Hales, B. F., and Neims, A. H. (1976a). Bioclient. J . 160, 231-236. Ikiles, B. F., and Neims, A. H. (19761,). Hiocherrt. J . 160, 223-229. Hales, B. F., and Neims, A. IH. (1977). Hiocliern. Pharniocol. 26, 555-556. Hanson, S. K., and Anders, M. W. (1978). ?b.xico/. Lett. 1, 301-305. Harvey, D. J., Glazener, L., Stratton, C., Johnson, D. B., Hill, R. M., Homing, E. C., and Homing, M. G. (1972). Res. Conrtnuri. Chertt. P o t l i o l . P/icirntcicol. 4, 247-260. Harvey, D. J.. Glazener, L., Johnson, D. B., Butler, C. M., and Horning, M . G. (1977). Zlriig M e t t r b . V i s p o s . 5 , 527-546. Hashimoto, K., and Aldriclge, W. N. (1970). Hioc/ietii. Phnrtnacol. 19, 2591-2604. IHayakawa, T., Leniahieu, R. A., and Udenfriencl, S. (1974). Arch. Riocliern. Biop/i!/s. 162, 223-230. Hayakawa, T., Udenfriend, S., Yagi, H., and Jerina, D . M . (1975). Arch. Biochetn. B ~ O ) J / L !170, / S . 438-451. Hayakawa, T., Myokei, Y., Yagi, H., and Jerina, D. M. (1977).J. Bioclieirt. 82, 407-415. Heath, C. W., Jr., Dnniont, C . R., Gamble, J., and Waxweiler, R. J. (1977). Ettciroii. Rcs. 14, 68-72. Hefiier, R. E., Jr., Watanabe, P. G., and Geliring, P. J. (1975).Aiin. N . Y . Accrd. Sci. 246, 135-148. Heidelberger, C. (1975). Anit. Rec. Biochern. 44, 79-121. Heniy, R. A., and Byington, K. H. (1976). Hiocltem. Phcirnrtrco/. 25, 2291-2295. Henschler, D., and Bonse, G. (1977).Arch. Toxicol. 39, 7-12. Higginson, J., and Mnir, C. S. (1973). Z t r “Cancer Medicine” (J. F. Holland and E. Frei 111, eds.), pp. 241-306. Lea Llr Feliiger, Philadelphia. Hinderer, R. K., and Menzer, R. E . (1976a). Pest. RiocIteiir. P h y s i o l . 6, 148-160. Hinderer, R. K., and Menzer, R. E. (19761)). Pest. Biochem. P h y s i o l . 6, 161-169. Hinson, J. A., and Mitchell, J. R. (1976). Drug Aletuh. V i s p o s . 4, 430-435. Hinson, J. A , , Nelson, S. D., and Mitchell, J. R. (1977). Mol. Phnrrncicol. 13, 625-633. Hogberg, J., and Kristoferson, A. (1977). E u r . J . Biocheni. 71, 77-82. Hollingwortli, R. M . (1969).J. Agr. Food Clreni. 17, 987-996. Hollingworth, R. M. (1970). In “Biochemical Toxicology of Insecticides” (R. D. O’Brien and I . Yamamoto, eds.), pp. 75-92. Academic, New York. Hollingworth, R. M. (1971). B i t / / . Wid. Heolth Org. 44, 155-170. Hollingworth, R. M., Alstott, R. L., and Litzenberg, R. D. (1973). Life Sci. 13, 191-199. Hoppen, H. 0.. Ball, D., Hoogen, H., and Knnppen, R. (1973). Z. Ph!/.siol. Clzern. 354, 771-780. Howie, D., Adriaensseris, P. I., anti Prescott, L. F. (1977). J . Pharm. Phnrnzncol. 29, 235-237. Hozunii, M., lnuznka, S., and Sngimura, T., (1967). Cancer Res. 27, 1378-1383.

GSH CONJUGATION WITH CAHCINIOGENS

263

Hiil)l)eIl, J . P., and Casich, J . E. (1977).J.Agr. F ~ o dChc~rii.25, 4 0 4 4 1 3 . Hiiherriian, E., Kiiroki, T., hlarqwudt, H., Selkirk, J. K., Heidellierger, C . , Grover, P. L., and Sims, P. (1972). Cnticer Res. 32, 1391-1396. Hiihetmati, E., Sachs, L., Yiuig, S. K., m d Gell)oin, H. V. (1976). Proc. ,Vat/. Acud. Sci. ci.s.fi.73, 607-611. Hull)ert, P. B. (1975).Nntrrrc (Lotidoti) 256, 146-148. Hiitson, D. H. (1976).I r i “Bound and Conjugated Pesticide Residiies” ( D . D. Kaufinan, G. C . Still, G. U. Paulson, mid S. K. Baiidal, eds.), pp. 103-131. Aiiierican Chemical Society , LVash ing ton. Hiitson, D. H. (1977).Chcnt. H i o l . Zriterrrct. 16, 315-323. Hutson, D. H., and Iloadley, E . C. (1972). Xeiroliioticcr 2, 107-116. Hutson, D. H., H o d l e y , E. C., GriEitlia, M. H.,ancl Donninger, C . (1970).J.A g r . Food Clieni. 18, 507-512. Hutson, 11. H., Pickering, B. A,, and Donninger, C. (1972).Biocltem. J . 127, 285-293. Hutson, D. H., Holtnes, D. J., iiiid Crawford, M.J . (1976).Chemo.spIiare 5, 79-84. Hycle, C . W., aiitl Yoring, L. (1968). Bioclicrn. J . 107, Sly-522. IARC ( 1973a). “ k f o I l O g R q J h S on the Evaluation of Carcinogenic Risk of Chemicals to Man,” Vol. 2 . International Agency for Research on Cancer, Lyon, France. IARC (197311).“Monographs on the Evaluation of Carcinogenic Risk of Chemicals to hlan,” Vol. 3. International Agency for Research on Cancer, Lyon, France. IAHC (1974a). “Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man,” VoI. 5. Interiiational Agency for Research on Cancer, Lyon, France. IARC (19741)). “Moiiographs on the Evaluation of Carcinogenic Risk of Chemicals to Man,” Vol. 6. International Agency for Research on Cancer, Lyon, France. . on the Evaluation of Carcinogenic Risk of Chemicals to IARC ( 1 9 7 4 ~ )“hlonographs Man,” Vol. 7. International Agency for Research on Cancer, Lyon, France. Innes, J. R. M . , Ulland, B. hl., Valerio, M. G., Petrucelli, L., Fishbein, L., Hart, E. R., Pallotta, A . J., Bates, R. R., Falk, H. L., Gart, J. J., Klein, M., Mitchell, I., and Peters, 1. (1969).J.N o t / . Cariccr I n s t . 42, 1101-1114. Ishida, M. (1968).A g r . B i d . Client. 32, 947-955. Ishida, M., and Dalim, P. A. (1965).J.Econ. Eritomol. 58, 383-392. Ito, N., Nagasaki, H., Aoe, H., Sugihara, S.. hliplta, Y., Arai, bl., and Shirai, T. (197s). J . .Vtrt/. Ctriicc,r I t r u t . 54, 801-805. Ito, N., Hananouchi, M.,Sugihara, S., Shirai, T., Tsutla, H., Fukushinia, S., and Nagasaki, H. (1976).Cnricer Res. 36, 2227-2234. Izartl, C., and Liberinann, C . (1978). d4trtnt. Res. 47, 115-138. Jackson, R. C., Taylor, G. A , , and Harrap, K. R. (1976).Neo/i/n.sniu 23, 3 5 5 3 6 2 . Jaeger, R. J., Conolly, R. B., and Murphy, S. D. (1974). E s p . Mol. P a t h o / . 20, 187-198. Jaeger, R. J., Conolly, R. B., and Murphy, S. D. (1975).Arch.Eiwiroii. H e d t h 30,26-31. Jaffe, M. (1879). Rer. Dfscli. Chrni. G e s . 12, 1092-1098. Jagenburg, 0. R., and Toczko, K. (1964).Riochent. J . 92, 639-643. Jagenl,iirg, R., Nagy, A,, and Rodjer, S. (1968). Sccrrid. J . Clin. Lo/>.Invest. 22, 11-16. Jakobson, I., Askelof, P., Warholm, M., and Mannervik, B., (1977). E u r . J . Biocheni. 77, 253-262. Jakolly, W. B. (1976).111 “In Vitro Metabolic Activation in Mutagenesis Testing” (F.J. de Serres, J . R. Foiits, J. R. Bend, and R. M. Philpot, eds.), pp. 207-212. Elsevier, Amsterdam. Jakoby, W. B . (1978).A d o . E n a / m o / . 46, 3 8 3 4 1 4 . Jakoby, W. B., Habig, W. H., Keen, J. H., Ketley, J. N., and Pabst, M. J. ( 1 9 7 6 ~ )I .i r “Glntathione: Metabolism and Function” ( I . M. Arias and W. B. Jakoby, eds.), pp. 189-202. Raven, New York.

264

L. F. CHASSEAUD

Jtikoby, W. B., Ketley, J. N., and Habig, W. H. (19761)).I r i “Glutathione: Metalmlism and Function” (I. bl. Arias atid 1%’. B. J a k o b y , eds.), pp. 213-220. Raven, New York. James, M. O., Fouts, J. R., and Bend, J . R. (1976). Biochcni. P h ~ r i ~ t ~25, c d 187-193. . James, M. O., Forireman, G. L., Law, F. C., atid Bend, J. R . (1977). D r u g Alettib. L)is))os. 5, 19-28. James, S. P., and Jetfery, D. A. (1964). Biocliein. J . 93, 16P. Jaiiies, S. P., id Needham, D. (1973).Xeriobioticci 3, 207-218. James, S. P., and Pheasant, A. E. (1978). Xoiobioticcc 8, 207-217. James, S. P., and Solheim, E. (1971). Xeimbioticci 1, 43-53. James, S. P., and White, D. A. (1967). Biochem. J . 104, 914-921. James, S. P., Jeffery, D. A , , Waring, R. H., atid Wood, P. B. ( 1 9 6 8 ~ )Biocheiii. . J . 109,

727-736. Jaiiies, S. P., Waring, R. H., White, D. A., Harris, G. L. A,, and Needham, D. (19681)).

A I ~ I IR. e ) ) . R r . E l i t / > ,Caiicer ~ f ~ f t l ~ J46, ~ ~ 251. i ~ l i Jaiiies, S. P., Jeffery, D . A,, Wiring, R. H., and White, D. A . (1971).Bioclteni. Pltcirnicico/. 20, 897-907. Jiiiiies, S. P., Pheasant, A . E., and Solheiln, E . (1978). Xettobioticci 8, 219-228. Javitt, N. B. (1976). Zii “Clittathione: Metalx)lisin and Function” (I. M. Arias and W. B. Jakoby, eds.), pp. 309-314. Raven, New York. Javitt, N. B., Wheeler, H. O., Baker, K. 1.. Rainos, 0. L., and Bradley, S. E. (1960). J . Cliii. Zttcc.yt. 39, 1570-1577. Jetfrey, A. M., and Jerina, D. M. (1975).J. A m . Cheiti. Soc. 97, 442711428. Jeffrey, A. M., Yeh, H. J. C., Jerina, D. M., de Miirinis, R. M., Foster, C. H . , Piccolo, D. E., antl Berchtold, G . A. (1974).J. A i i i . Client. Soc. 96, 69294937. Jerina, I). M. (1976). 111 “Glutathione: Metabolism and Function” (I. M . Arias and W. B. Jakoby, eds.), pp. 267-279. Raven, New York. Jerina, D. M., and Bend, J. R. (1977). Z i t “Biological Reactive Intermediates” (D. J. Jollow, J. J. Kocsis, R. Snyder, and H. Vainio, eds.), pp. 207-236. Plenum, New York. Jerina, D. M., and Daly, J. W. (1974). Science 185, 573-582. Jeriiia, D . M., and Daly, J. W.(1977). I r i “Drug hletal)olism-From h4icrol)e to Man” (D. V. Parke antl R. L. Smith, ecls.), pi). 13-32. T;1y10r and Francis, Lontlon. Jerina, D., Daly, J., Witkop, B., Zaltztnan-Nirenberg, P., and Udenfriend, S. (1968). Arch. Biocliem. B i o p h ! / s . 128, 176-183. Jerina, D. M., Daly, J. W., Witkop, B., Zaltznian-Nirenberg, P., and Udenfriend, S. (1970). Biocheinistry 9, 147-155. Jocelyn, P. C. ( 1972). “Biochemistry of the SH Group.” Academic Press, London. Johnson, M. K. (1965). Riocliem. Plicirmcicol. 14, 1383-1385. Johnson, M. K. (1966). Biocltem. J . 98, 44-56. Johnson, M. K. (1967). Biocltewt. Pliarmacol. 16, 185-199. Jollow, D. J., Mitchell, J. R., Zampaglioiie, N., and Gillette, J. R. (19744. P/icrrtncico/ogy 11, 151-169. Jollow, D. J., Thorgeirsson, S. S., Potter, W. Z., Hashimoto, M., iiiid Mitchell, J. R. (1974b). Plinrnincology 12, 251-271. Jollow, D. J., Kocsis, J. J., Snyder, R., and Vainio, H. (eds.). (1977). “Biological Reactive Intertnediates.” Plenum, New York. Jondorf, W. R., Parke, D. V., antl Williams, R. T. (1955).Hiocliena. J. 61, 512-521. Jones, A. R. (1970). E r p r i e i i t i a 26, 4 9 2 4 9 3 . Jones, A. R. (1973). Drug A4etcil7. Rec;. 2, 71-100. Jones, A. R. (1975). Xenobioticci 5, 155-165. Jones, A . R., and Edwards, K. (1968).Esperientio 24, 1100-1101. Jones, A. R., and Edwards, K. (1973). Eaperieiitici 29, 538-539.

G S H CONJUGATION WITH CARCINOGENS

26!5

Joties, A. H., Davies, P., Edwartls, K., a i i d Jackson, 11. (1969).N f t t r t t . r ( I , ~ J I 224, I ~ ~83. J~I) Jones, B. K., and Hiithwii)’, D. E. (1978). Br-.J. C c t t i c c v 37, 411-417. Juchaii, hl. R., ~ i r i dNamkung, XI. J. (1974).D r u g . Jfettr6. Uisp0.c.. 2, 380-385. Kainisaka, K., H h i g , W.I-I., Ketley. J. N . , Arias, I . h l . , ;ind Jakoby, W. B. (197.5).Errr-. / . !3iOC/lCllt.

60, 153-161.

KiipitiiIiiik, J.. Levin, W.. Conney, -4.I I . , Yagi, I l . , and Jeriiin, D. hI. (1977n).S t t t r t t - c , (Lotidoti) 266, 378-3380, Kapitulnik, J., Levin, W., Lii, A. 11.. hlorecki, H., Ihiisette, P. hl.. Jeriiia, I). hl., ;itid Coniiey, A. 11. (19771,).Cliti. Plrttt-ttrtrc~ol. T/ic>r.21, 158-165. Kapitiiliiik, J., Wislocki, P. G.. Leviii, m’.,Yagi. H.,Jerina, D. hl., and Conney, A. 11. (1978).CotlCc,r l3e.c.. 38, 354-358. Kaplowitz, N. (19771.J. Pltttrtttcic~ol.F:.v/>. Tlter-. 200, 479-486. Eaplowitz, N.. and Clifton. G. (1976).Z t i “Glutathioiie: X1etal)olism and Fiinction” ( I . hl. .4ri. .IS. .m c l W.B. Jakol)y, eds.), pp. 301-306. H a \ w i . N e w York.

T.

Kaplowitz, N . , Percy-Rol)b, I. W., and Javitt, N . R . (1973).J.E X I J 3lcvl. . 138, 4 8 3 4 8 7 . Kaplowitz, N.. Kiililenkamp, J., untl Clifton, G. (1875~1).Riocltettr. J . 146, 351-356. Kaplowitz, N., Kiihlenkamp, J., and Clilton, G. (19751)).Pro(,.S O C . E.xp. Hiol. .\lc~l.149,

234-237. Kaplowitz, N., Clifton, G., Kuhlenkniirp, J., and W d l i n , J. D. (1976). Hiocltcttr. J . 158,

243-248. Kaplowitz, N., Spina, C . , Grdiam, M., and Kiihlcnk;iiiip, J. ( 1978). H i o c l w t t t J . 169,

465470. Kappiis, H., and Hemmer, 1. (1975).llr-rtg .\frtcih. 1 l i s ) ) m . 3. 3338-334. Kappiis, H., Bolt, H. X I . , Bricliter, A,, and Bolt, W. (1976).Tosicol. A ) I ~ /P/ttrrtticico/. . 37,

46 1 4 71. Kato, R., T;ikaliaslii, A,. and Osliima, T. ( 1970).Bioclrc,ttt. Plittr-tttcicol. 19, 45-55. Kawazoe, Y., I T e l ~ r a N., , Araki, bl., and Tamiira, 14. (1969).G t t i t r GO, 617-626. Kaye, C. hl. (1973).Bioclicni. J . 134, 1093-1101. Kaye, C. Xl., Clapp, J . J., and Young, L. (1972).X e t i i h i o t i c r t 2, 129-139. Keen, J. H., antl Jakoby, W. B. (1978).,1.H i o l . C/tc~itr.250, 5654-5657. Keen, J. H., Habig, W.H., and Jiikoby, W. H. (1O76).J. H i d . Clic.tt1. 251, 6183-6188. Kellermann, G. H . (1976).It, “Screening Tests i n Cheiiiical Carcinogei~esis”(R. \lolltesaiio, H . Biirtsch, ;tiid L. Tomatis, etls.), pp, 297-317. IARC, L p r , Frmrce. Kellermiinn, G., Luyten-Kellermann. X I . , iuid Shaw, C . R. (1973a).i Z t t t . 1.I f I l J t l . C o i e t . 25,327-33 1. Kellerinanii, G., S h a w , C. R., and Liiyten-Kelleriiiann, h l . (19731)).N e w E t i g l . J . Med.

289,934-937. Ketley, J . N., Habig, W.H., and Jakoby, W. B. (1973.5. R i d . Client. 250, 8670-8673. Ketterer, B., and Cliristodoulicles, L. (1969).Cheni. B i o / . Ztitercict. 1, 17.3-183. Ketterer, B., Ross-Mansell, P., and Whitehead, J . K. (1967).Rioclicni. J . 103, 316-324. Ketterer, B., Tipping, E., Beale, D., Meuwissen, J., antl Kay, C. M .(1975a).Pr-oc. I t i t . Cctttcc~rCotigr-. 11t h 2, 25-29. Ketterer, B., Tipping, E., Meiiwissen, J., and Beale, D. (19751)).Biochetn. SOC.Z’r-uti,v. 3,

626-630. Ketterer, B., Tipping, E., Beale, D., and Meiiwissen, J. A. T. P. (1976:~).I n “Glutathione: bletabolisiii and Function” ( I . M. Arias and W. B. Jakoby, eds.), pp. 243-253. Raven, New York. Ketterer, B., Tipping. E . , Hackney, J. F., and Beale, D . (197611).Biochettl. J. 155, 511-

521. Keysell, G. R., Booth, J., Grover, P. L., Hewer, A,, and Siiiis, P. (1973). Bioclieni. PhclrttlOC(J/.

22, 2853-2867.

266

L. F. CHASSEAUD

Kimhrough, R. D., and Linder, R. E. (1974).J. N u / / . Ctrticer I t i s t . 53, 547-552. King, H. W. S., Osborne, M. R., Beland, F. A,, Harvey, R. G., and Brookes, P. (1976). Proc. Nntl. Acrid. Sci. U.S.A. 73, 2679-2681. Kirsch, R., Fleiscliner, G., Kamisaka, K., and Arias, I. M. (1975).J . C l i n . Znoe.~t.55, 1009-1019. Klaassen, C. D. (1975).J.Plzcirtticictd. E . x ~ JTlier. . 195, 311-319. Klaassen, C . D., and Fitzgerald, T. J. (1974).J . Phcimicrcol. EX^. Ther. 191, 548-556. o / . 1322-1326. Klaassen, C. D., iind Plaa, G. L. (1967).A m . J . P / ~ ! / . F ~213, Knight, R. H., and Young, L. (1958). Biocltem. J . 70, 111-119. Knox, W. E. (1960).1ti “The Enzymes” (P. D. Boyer, H. Lardy, and K. Myrback, eds.), Vol. 2, 2nd ed., l i p . 253-294. Academic Press, New York. Knox, J. H., and Jurand, J. (1977).J. C/tronrtrtogr. 142, 651-670. Koch-Weser, D., de la Huerga, J., Yesinick, c.,and Popper, H.(1953). Metcibo/ism 2, 248-260. Koclama, H,(1968).Biochitii. Bioph!/.c..Actti 165, 432-437. Kolmark, G., and Giles, N . H. (1955). Cetietics 40, 890-902. Koransky, W., Munch, G . , Noack, G., Portig, J., Sotlomann, S., m t l Wirsching, M. (1975). Arch. Phcirincicol. 288, 65-78. Koreetla, M., Moore, P. D., Wislocki, P. G., Levin, W., Conney, A. H., Yagi, H., and Jerina, D. M . (1978). S c i m c e 199, 778-781. Kosower, E. M. (1976). Z I I “Gliitathione: hletul)olism and Fnnction” (I. M. Arias m d W. B. Jokol)y, etls.), pp. 1-15 Haven, New York. KMIIS, 1’. (1976)..Ar~/t.Z ’ / i e ~ ~ t t i c r ~ 296, ~ ~ / . 67-72. K r ~ i i s ,P., Noack, G., inid Portig, J. (1973). Arch. Pltorrtttrcol. 279, 199-202. Kulkarni, A. P., Fabacher, D. L:, and Hodgson, E. (1978). ? b . ~ i c / A ~ / .J ? I /Ph~irtttcrco/. . 45, 32 1. Knrihara, N., Tanaka, K., and Nakajima, M. (1977). Agr. B i d . Chetn. 41, 1317-1319. Knroki, T., Hnliernian, E., Marqtiardt, M., Selkirk, J. K., Heidelberger, C., Grover, P. L., and Sims, P. (1972). Choir. R i d . Ztitercict. 4, 389-397. Kuss, E. (1970). R e s . Stooitls 4, 49-59. Kriss, E. (1971). %. Z’h!/siol. C/reiir. 352, 817-836. Knwaki, T. (1965).,\.Rioclzet)r. 57, 125-130. Kiiwaki, T., ;ind Miznhara, s. (1966). b ’ i t J C / t i j i t . Bi(J/J/l!/,F. i l c l t f 115, 491-49.3. Lamoureux, G. L., iind Davison, K. L. (1975). Pe.r.t. Biochetn. P / i ! / s i o l . 5, 497-506. Lamoiireux, G. L., Shimal)nkuro, R. H., Swanson, H . R., and Frear, D. S. (1970).J.Agr. Food Chertt. 18, 81-86. Lawley, P. D. (1976). 1tr “Chemical Carcinogens” (C. E. Searle, ed.), pp. 83-244. Anierican Chemical Society, Washington. Lay, M . M.,and Casida, J. E . (1976). Pest. Biocltem. P l i ! / s i o l . 6, 4424.55. Lee, I. P., Suzuki, K., Mnkhtar, H., Bend, J. R., and Dixon, R. L. (1978). Toricol. A / J / J / . Phnrtitcrcol. 45, 361. Legator, M. S., Kelly, F. J., Green, S . , and Oswald, E. J. (1969).Aiiii. N.1’. Acarl. Sci. 160, 344-35 1. Lehr, R. E., iind Jerina, D. M . (1977). Arch. Toricol. 39, 1-6. Lemen, R. A., Lee, J . S., Wagoner, J. K., a n d Blejer, H. P. (1976). Atilt. N.Y. Accid. Sci. 271,273-279. Leilmian, K. C., and Oitiz, E. (1970).J. Phunizcictd. E s p . Tlier. 173, 242-246. Levi, A. J., Gatmaitan, 2.. and Arias, I. M. (1969).J.Clin. Ziisest. 48, 2156-2167. Levine, R. I., Reyes, H., Levi, A. J., Gatmaitan, Z., and Arias, I. M. (1971).Nnttrre ( N e w R i o l . ) 231, 277-279. Levy, L., and Harris, R. (1977). B i o c h i r . Pltcirtncicol. 26, 1015-1020.

GSH CONJUGATION W I T H CARCINOGENS

267

Lindsay-Smith, J. R., Shaw, B. A. J.. and Foulkes, D. M . (1972).Xeriobioticu 2, 215-226. Litterst, C. L., Mimnaugh, E. G., Reagan, R. L., and Gram, T. E. (197Fj).D r u g . kletrrh. Z1i.c.po.v.3, 259-265. Litterst, C. L., Gram, T. E., Mimnaugh, E. G., Leber, P., Einmerling, D., and Freudenthat, R . I. (1976).D r u g . ,\fetcrh. Uispo.s. 4. 203-207. Litwack, G., Ketterer, B., and Arias, I. M .(1971). Nntrrre (Loridori) 234, 466-4637. Long, E., Nelson, A. A., Fitzhiigh, 0. G., and Hanseii, W. H. (1963).Arch. Patltol. 75, 595-604. Loprieno. N., A\d~ondandolo,A , , Barale, R., Baroncelli, S., Bonatti, S., Sronzetti, G., Cammellini, A , , Corsi, C., Coiti, G., Frezza, D,, Leporini, C., hlazzaccaro, A , , Nieri, H., Roselliiii. D., and Rossi, A. hl. (1976). .\futot. Re.v. 40, 317-324. Loprielin. S . , Barale, R., Baroncelli, S., Bartsch, H., Bronzetti, G., Caniniellini, A,, Corsi, C., Frezza, D., Nieri, R., Leporini, C., Rosellini, D., antl Rossi, A. hi. (1977). coiicl~r-:36, 253-257. Loveless, A , , and Howarth, S. (1959). Ntrtrtt-e ( L o r i d o r i ) 184, 1780-1782. MacDonald, C. XI., Dow, J., and Moore, % R.I(1977). . Rioclrerti. Plwrriicrcol. 26, 15291531. tr, Putliol. Plicrrrtitrcol. 16, hladelian, V., a n d Warren W. A. (1977). R m . C o ~ ~ i r ~ i t rC/ietn. 385388. Xlagee, P. N. (1974). In “Essays i n Biochemistry” (P. N. Campbell and F. Dickens, eds.), Vol. 10, pp. 105-136. Bioclieiniciil Society, London. hdaines, %I. D., and Kappas, A. (1977). Proc. N o t / . Accrrl. Sci. L!.S.A. 74, 1875-1878. hlalaveille, C., Bartsch, I{., Barbin, A , , Camus, A. M., Montesano, R. (1975a). Riochern. B i o p / i ! / s ,R e s . C r i i t i n i r r i i . 63, 363-370. h4alaveille, C., Bartsch, H., Grover, P. L., and Sims, P. (197Fjb).Rioclieni. R i o / h / , y . Re.c.. C o t i t n i I I n . 66, 693-700. Mala\~eille,C., Planche, G., and Bartsch, H. (19774. C/rertl. Biol. Zritercrct. 17, 129-136. Mahveille, C., Kiiroki, T., Sims, P., Grover, P. L., and Bartsch, H. (19771)). M t r t c t f . Re.9. 44, 313-326. Malnoe. A , , Strolin-Benedetti, M., Smith, R. L., and Frigerio, A. (1977). Z r r “Biological Reactive Intermediates” ( D . J. Jollow, J. J. Kocsis, R. Snyder, and H. Vaiiiio, eds.), pp, 387-393. Plenum, New York. hlaltoiii, C., nnd Lefemine, G. (1974).Eritjirori. Res. 7, 387-405. hlaltoiii, C , , Cotti. G,, Morisi, L., antl Chieco, P. (1977). .\led. Ltrz;. 68, 241-262. htannervik, B., and .4skelof, P. (1975). FEBS Lett. 56, 218-221. h l m s i i y , D., Beaiine, P., Cresteil, T., Lange, M., and Leroox, J. P. (1977). Riochem. R i o ~ ) / z ! /R, ~u. . Cottiriirrrt. 79, 513-517. Marniemi, J , , and Parkki, M . G. (1975). Rioclterir. P/icirnicico/. 24, 1569-1572. Marniemi, J., Nokkala, M.,L’ainio, H., Hartiala, K. J. W. (1977).Chein. B i d . Itiferuct. 18, 247-251. Miarqiiardt, H., Baker, S., Grover, P. L., and Sims, P. (1977). CNricer Lett. 3, 31-36, ) 863-865. Martin, C. N., and Garner, R. C . (1977). Nntrrrc ( I m i r ~ o r i267, M;itsushinia, T., and Sugimur;i, T. (1971). 111 “Chemistry and Biological Actions o f 4-Nitroquiiioline N-Oxide” (H. Endo, T. Ono, and T. Sugimiira, eds.), pp. 53-60. Springer, Berlin. klattok, G. L., and Heacock, R. A. (1965). Crrtr. /. Cherfi. 43, 119-125. hlaynert, E. W., Foreman, R. L., and Watalx, T. (1970).J. B i d . Client. 245, 5234-5238. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. (1975).Proc. N n t l . Accirl. Sci. [I.S.A. 72, 5135-5139. hlcKeiina, hl. J., Watanabe, P. G., and Cellring, P. J. (1977). Etioirolt. Heulth Perspect. 21, 99-105.

268

L. F. CHASSEAUD

McKenna, M. J., Zempel, J. A,, Madrid, E. O., Braun, W. H., and Gehring, P. J. (19784. Toxicol. A p p / . Phnrttinco/. 45, 821-835. McKenna, M. J., Zempel, J. A., Madrid, E. O., and Gehring, P. J. (1978h). Toxicol. A p p / , P/inrinuco/. 45, 599-610. McMurtry, R. J., and Mitchell, J. R. (1977). Toxicol. A p j i / . Phcirnincn/. 42, 285-300. Meehan, T., Straub, K., and Calvin, M . (1977). Nature (London)269, 725-727. Meister, A. (1975). I n “Metabolism of Sulfur Compounds,” Vol. VII, 3rd ed., pp. 101188. Academic Press, New York. Meshi, T., Nakamnra, S., and Sato, Y. (1972). Cheni. Phurni. B u l l . 20, 1687-1698. Metzler, M., and Neriniann, H . G. (1977). Xeiiokioticn 7, 117-132. Miller, E. C., and Miller, J. A. (1966). Phormuco/. Reo. 18, 805-838. Miller, E. C., and Miller, J. A. (1974). Iri “The Molecular Biology of Cancer” (H. B ~ i ~ c h , ed.), pp. 377-402. Academic Press, New York. Miller, J. A,, and Miller, E. C. (1971).J. N u t / . Cancer Inst. 47, v-xiv. Miller, J. A., and Miller, E. C. (1976).I I I “Screening Tests in Chemical Carcinogenesis” (R. Montesano, H. Bartsch and L. Toinatis, eds.), pp. 153-176. IARC, Lyon, France. Mills, G. C. (1960). Arch. Biochem. B i o p l i y s . 86, 1-5. Milvy, P., and Garro, A. J. (1976). M u t u t . Res. 40, 15-18. Mirvish, S . (1968). A d o . Cuncer Res. 11, 1-42. Mitchell, J. R., and Jollow, D. J. (1975). G~.r.troeiitero/ofi!/ 68, 392-410. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R. and Brodie, B. B. (1973).J . Pharnitrco/. E x p . Tlier. 187, 211-217. Mitchell, J. R., Potter, W. Z., Hinson, J. A., and Jollow, D . J. (197421).Nature (Loiit/oti) 251, 508-511. Mitchell, J. R., Thorgeirsson, S. S., Potter, W. Z., Jollow, D. J., and Keiser, H. (19741)). Clin. Pliurinucol. Tker. 16, 676-684. Mitchell, J. R., Potter, W. Z., Hinson, J. A., Snodgrass, W. R., Timlxell, J. A., and Gillette, J. R. (1975a). 111“Handbook of Experimental Pharmacology” (J. R. Gillette and J. R. Mitchell, eds.), Vol. 28, pp. 383-419. Springer, Berlin. Mitchell, J. R., Long, M. W., Thorgeirsson, U. P., and Jollow, D. J. (197511).Chest 68, 181-190. Mitchell, J. R., Thorgeirsson, U. P., Black, M., Tinihrell, J. A., Snodgrass, W. R., Potter, W. Z., Jollow, D. J., and Keiser, H. R. ( 1975~)C. / i i i . Pliurniucol. Tlier. 18, 70-79. Mitchell, J. R., Nelson, W. L., Potter, W. Z., Sasame, H. A . , and Jollow, D. J. (1976).J. Phcrrinncol. E x j i . Ther. 199, 41-52. Mitoma, C., Saito, T., and Howd, R. A. (1977). Xetio/>ioticu 7, 165-179. Morey, K. S., and Litwack, G. (1969). Biochertiistr!/ 8, 48134821. Mori, K. (1964). Gatin 55, 277-286. Morris, J. E., Price, J. M., Lalich, J. J., and Stein, R. J. (1969).C m c w Res. 29,2145-2156. Morrison, W. C., Whyhrew, W. D., Sobhy, C. M., Morrison, J. C., Trass, T. C., and Bucovaz, E. T. (1977). Br. J. Caticer 35, 218-225. Moslen, M. T., Reynolds, E. S., Boor, P. J., Bailey, K., and Szabo, S., (1977). Res. C o m tnu t ~ Chein. . Pothol. Pliunticicol. 16, 109-120. Motoyama, N., and Dantemian, W. C. (1972). Pe.st. Biocheni. Ph!/siol. 2, 113-122. hlotoyaina, N., and Dauternian, W. C. (1975). Pest. Biochem. PIi!/sio/.5 , 489-495. Motoyama, N., and Dauterinan, W. C. (1977). Insect. Biochetn. 7, 361-369. Motoyama, N., Dauterinan, W. C., and Plapp, F. W. Jr. (1977).Pest. Biochem. P h ! / d o / .7, 443-450. Mrocliek, J. E., Katz, S., Christie, W. H., and Dinsmore, S. R. (1974). C/iti. Chem. 20, 1086- 1096. Muecke, W., Menzer, R. E., Alt, K. O., Richter, W., and Esser, H. 0. (1976). Pest. Biochein. Physiol. 6 , 430441.

GSH CONJUGATION WITH CARCINOGENS

269

Muir, C. S., a n d Kirk, R. (1960). Br. J . Cnitcer 14, 597-608. Mukhtar, H . , and Bend, J. R. (1977). Life Sci. 21, 1277-1285. Mukhtar, H., a n d Bresnick, E. (1976a).J. Irtccst. Derrtitrtol. 66, 161-164. Mukhtar, H., and Bresnick, E. (197613).Cltettt. H i d . Itttercict. 15, 99-67. Mitkhtar, H., and Bresnick, E. ( 1 9 7 6 ~ )Cancer. Res. 36, 937-940. Muklitar, [I., and Bresnick, E. (1976~1).Bioclic.ttt. Phcir-tit~i~~ol. 25, 1081-1084. hfiiller, G., and Norpoth, K. (1975). Nntrrric;isset~sc/ici~fte~t 62, 541. Nachtomi, E. (1970).Riocltent. Pltctrnt(ico1. 19, 2853-2860. Nachtomi, E., antl Sariiin, D. S. H. (1977). Riocltcttt. Plturtttocol. 26, 1941-1945. Nachtonii, E., Aliiniot. E., and Bondi, A. (1966). Zsr. J . Cltetn. 4, 239-246. Nakahara, W., Fiikuoka, F., and Suginiura, T. (1957). Cntiti 48, 129-137. Naml~ara,T., and Numaznwa, M . (1971). C/iettt. Plt~tr-ni.R n l l . 19, 855-856. Needleman, P., and Hiinter, F. E., Jr. (19%). M o / . P/ittrmoco/. 1, 77-86. Nelson, N. (1976).Aitjt. N.1’. Acnd. Sci. 271, 81-90. Nelson, S. D., ?\litchell, J. R., Ilybing, E., a i i d Sasame, H . A . (1976a).Biocltetti. BiopltIp. H a . Cotiitttirtt. 70, 1157-1165. Nelson, S. D., Mitchell, J . R., Tiiiil>rell,J. A , , Snotlgrass, W. H., a n d Corcoran 111, G. €3. (197611).Sciotcc, 193, 901-903. Nemoto, N., and Cklboin, H. (1975). Arch. Biochettt. B i o p / t ! / s . 170, 739-742. Nemoto, N., Gelboin, H. \I., Hal)ig, W. H., Ketlev. J. N., m d Jakol)y, W. B. (1975). Nntrrre (Loitdoti) 255, 512. Niederwieser, A., Steiniiiann, B., and Xlatasovic, A. (1978).J . Chror~itrtogr~147, 163176. I c o / . 183-189. Noack, C . , and Portig, J. (1973). Arch. P / ~ N I - ~ ~ I I 280, Norsetli, T. (1971). .Act(/ P / l c / ~ t l i t / c ’ c J /7iJSiCO/. . 29, 375-384. Norseth, T., a n d Clarkson, T. W.(1971).At-di. Eticir-ott. IIco/tli 22, 568-577. Num;tz;i\x-ii, h l . , ;uid Nnml);ira, T. ( 1977).J . S t e r o i d . HioclicJttr.8, 835-840. Numizitwii, X I . , Soedn, N., hlorn, S.,mid Namlxirn, T. (1977).Bioc/te~it. Phc~rtitrrtd.26, 769-773. OeSch, F. (1973).S C l l O / J i f ) f i C t t 3, 305-340. Oesch, F. (1979).111 “Progress in Drug Metabolism” ( J . W.Bridges and L. F. Cliaasealitl, etls.), k701. 3 , pp. 253-301. Wiley, I ~ o ~ ~ d o n . Oesch, F., Jerina, D. A t . , Daly, J. W., ;ind Rice. J. M .(1973).Chet~t.B i d . Iiifcrttcl. 6, 189-202. Ohkawa, H., antl Casidii, J. E . (1971). Hiocltettt. Plttrrtnc/c.o/. 20, 1708-1711. Ohka\va, H., Ohkawa, R., Y;un;inioto, I., antl Casitlii. J. E. (1978). Pcvt. Riocltoiti. P/i!/.vio/. 2, 95-112. Olikmva, I-I., Sliil)aike, R., Hatannku, -I., mid hliyiinioto, J. (1975).Agr. H i d . Chc,ttt. 39, 1605-1615. 0111, V. S.,and I,it\vack, G . (1977).A r ( ~ / iHioclic~~tt. . H i o p h ! / s . 180, 186-190. Ohmori, S., Shimomiira, ‘I.,Azumi, T., and hlizuhara, S. (1965). Rioc/tc~tn.%. 343. 9-15. Olson, R. I),, I\lacDonald, J. S., ~ i i r b i s o t i .H. D., \WI Roxtel, C. J., Boerth, R. C., Sloniiii, A. E., iuitl Oiites, J. A. (1977). Fed. P ~ - o c .36, 303. Olson, W. A , , Halxrniann, R. T., Weisl)urger, E. K., Wurtl, J . M,,a n d Weisburget-, J . 13. (l973).]. N t r t l . C ~ t t ~ eItt.Et. t - 51, 1993-1995. Oppenoorth,F. J., Sinissaert, I f . R., Welling, W., \hn der Pas, L. J. T., and Ilitnian, K. T. (1977). P N f . BiftCh(,tft.P/t!/SiOl.7, 34-47. Pabst, M ,J., Hal)ig, W.H., a n d J;ikol,y, W. B.. (1973). Hiocliettt. Rioplt!/.c..Hes. C o t l i t n i t t i . 52. 1123-1 128. Palxt. X I . J., Habig, W.H . , mid Jnkoby, W. B. (1974)../. B i d . Cliettt. 249, 7140-7148. Piiigen. B., Guttoo, H. L., hlino\v&, J., Hoiitrir, I.., \’iiic.eiit, R., Paigcn, K., PnrkeI, N. B., W:trd, E.. >uld Ihyller, K . 1’.(1977)..Yr,lc E t t g l . J . .\Ic,tl. 297, 346-350.

270

L. F. CHASSEAUD

Parke, D. V. ( 1968). “The Biochemistry of Foreign Conipoiiiids.” Pergamon, Oxford. Parke, 1). V., and Williains, R. T. (1 ). B k i c h e ~ iJ~. .59, 415-422. Parkki, M. G., Marnienii, J., and Vaiiiio, H. (1977).J. Tosicol. E t i o i r o i i . Heultli 3, 903911. Peacock, A., and Peacock, P. R. (1966). H r . J . Ctrncer 20, 307-325. Peele, J . D., Jr.. and Oswald, E. 0. (1977). 13iochirn. Biop/i!/,T.Actcr 497, 598-607. Peers, F. G., Gilman, G. A , , and Linsell, C. A. (1976). Znt. J . Cancer 17, 167-176. Pegg, D. G., and Hook, J . B. (1977).J. Pliurtiicrcol. E s p . Tlier. 200, 65-74. Pelkonen, 0. (1977). I I I “Progress in Drug Meta1)olism” (J. W. Bridges and L. F. Chasse;uid, etls.), Vol. 2, 111). 119-161. Wiley, London. Pillinger, I). J., Fox, B. W., and Craig, A. W. (1965). 111 “Isotopes in Experimental Pharniacology” (L. J. Roth, ed.),pp. 415-432. University of Chicago Press, Chicago, Ill. Pinkus, L. M.,Ketley, J. N., ant1 Jakohy, W. B. (1977). Hiockem. Phnrniucol. 26, 23592363. Pohl, L. R., and Krishna, C . (1978). Bioc/teiii. Phrrr~ncrcol.27, 335-341. I’ohl, L. R., Bhooshan, B., and Krishna, G. (1978). Tosicol. A p p / . Pliorniircol. 45, 238. Portia, J., Kraus, P., Sodomann, S.,and Noack,G. (1973).Arch. Phnrniucol. 279, 185198. Potter, W. Z., Davis, D. C., Mitchell, J. R., Jollow, D. J., Gillette, J. R., and Brodie, B. B. (1973).J. Pliurnirrcol. Esji. Tlier. 187, 203-210. Powers, M. B., and Voelker, R. W. (1976). Tosicol.A\)/)/. Plturtnucol. 07, 179. Powers, M. B., Voelker, R. W., Page, N. P., Weisburger, E. K., and Kraybill, H. F. (1975). Toxicol. Appl. Plirrntiucol. 33, 171-172. Prescott, L. F., Park, J., Sritlierl;mtl, G. R., Smith, I. J., mtl Proudfoot, A. T. (1976). Lrrncet 2, 109-111. Prescott, L. F., Park, J., Ballantyne, A , , Adriaenssens, P., and Proudfoot, A. T. (1977). Lrr?lcrt 2, 432-434. Preiissmanti, R. (1968). Food Co.r.twt. Toxicol. 6, 576-577. Preussniann, R. (1976). Oticolofi!/33, 51-57. Proliaska, J. R., and Gnnther, H. E. (1977). Biocheni. Hio p h ! /s . Res. Cornniun. 76, 437445. Raj, H. G., Santhanam, K., Gupta, R . P., and Venkitasuhramanian, T. A. (1975). C h i n . Riol. Znteruct. 11, 301-305. Rannng, U., Gothe, R., Wachtmeister, C. A. (1976). Chern. R i o l . Znteruct. 12, 251-263. Rannug, U., Sundvall, A., and Rarnel, C . (1978). Client. B i d . Znteruct. 20, 1-16. Recluiagal, R. O., and Glentle, E. A., J r . (1973). CHC C r i t . Rec;. Tosicol. 2, 263-297. Redfnrd-Ellis, M., and Cowenlock, A. H. (19714. Actcr Phurntucol. Toxicol. 30, 49-58. Redford-Ellis, M., nnd Gowenlock, A. H. (19711)). Actu Phurnicicol. Tosicol. 00, 36-48. Reed, 1).J., and Orrenitis, S. (1978). Toxicol. Appl. P/a(irntocol. 45, 241. Rees, K. R., and Tarlow, M. J. (1967). Biochem. J. 104, 757-761. Refsvik, T. (1978). Actci Pltarmctcol. Toxicol. 42, 135-141. Refsvik, T., and Norseth, T. (1975).Actrr Pharrncicol. Toxicol. 36, 67-78. Reid, W. D. (1972). Experientin 28, 1058-1061. Reid, W. D., and Krishna, C . (1973). E x p . Mol. Pethol. 18, 80-99. Reid, W. D., Krishna, G., Cillette, J. R., and Brodie, B. B. (1973a). Pharmacolog!/ 10, 193-2 14. Reid, W. D., Ilett, K. F., Click, J. M., and Krishna, G. (197313).A m . Rev. Res. Dis. 107, 539-551. Reuber, M. O . , and Glover, E. L. (1970).]. N n t l . Cancer Znst. 44, 4 1 9 4 2 7 .

GSH CONJUGATION WITH CARCLIOGENS

27 1

Reyes, H., Levi, A. J.. Gatniaitan, Z., and Arias, I. M . (1969).P r o c , . N n t l . Awd. S c i . [J.S.A. 64, 168-170. Reyes, H., Levi. A. J., ant1 Arias, I . !vf. (1971)./. C/itt. Z t i m s t . 50, 2242-2252. Richardson, R. J., and hlrirphy, S. D. (1975). Tosicol. A/)])/. Pltnt-titcicol. 31, 505-519. Richinontl, D. V’., and Soniers, E . (1966). Atit?. A/)/)/. Hiol. 57, 231-230. Richmond, D. V., antl Soniers, E. (1968).Attti. A/)/)/. Hiol. 62, 3 5 4 3 . Roberts, J . J., and Warwick, G. P. (1958). Hiocltettt. Phiirtilcicol. 1, 60-75. Roberts, J. J., antl Warwick, G. P. (1961). Biochetii. Pliciriticicol. 6, 217-227. Rol)eitson, K. A , , Seymour, J. L., IIsia, M. T., antl Allen, J. R . (1977). Cnticer Re.7. 37, 3 141-3 144. Rogers, K. M., and Raliisley, E. A. (1977). Xetlobioticu 7, 4 0 9 4 1 4 . Rose~iberg,P. H. (1971).A t t t i . Med. E x / ) . B i d . Fvtttt. 49, 84-88. Rosenkranz, H.S. (1977). Riochetii. Phcirmucol. 26, 896-898. Riistia, M., and Shitl)ik, P. (1972)./. Natl. Cnticer Itisf. 48, 721-729. Sasame, H. A., Boyd, hl. R., Mitchell, J. R., iund Gillette. J. R. (1977).Fed. Proc. 36,405. Sarrif, .4. hl., D;inenl,erg, P. V., Heitlelberger, C., and Ketterer, B. (1976). Biocliettt. R i o p h ! / . s . Rev. Cotti i t t i t 11. 70, 869-877. Sarrif; .4.M.,XlcCarthy, K. Id., Nesnow, S., and Heidell)erger, C. (1978).Ciiticer Rcu. 38, 1438-1443. Schickedantz, P. D., Skladanowski, M . A., Zaletel, J., Maiiiior, R. S., and Minnenieyer, H. J. (19761.5. 4gr. F o o d Chetii. 24, 876-881. Schoental, R., and Xlagee, P. N . (1962). Hr. J . Cnticer 16, 92-100. Schoental, R., and Rive, D. J. (1965). Bioclzent. 1. 97, 466-474. Searle, C. E. (1966). Caticcr Rec. 26, 12-17. Sciler, J. P. (1975). :\futcif. Res. 32, 151-167. Seiler, J. P. (1976). A l t r f c i t . Res. 40, :339-347. S c ~ I u i t l c ~ H. r , G., Jerina, D. hl., Piccolo, D. E., arid Berchtoltl, G. .4. (1975)./.A t t i . Chettt. S o c . 97, 44284430. Selikotf, I. J., and Hammond, E. C. (eds.).(1975). Attti. N . 1 ~Accid. . Sci. 246. Selkirk, J. K., Croy, R. G., and Gelboin, H. V. (1975). Arch. Hiocltetii. H i o p h ! / s . 168, 322-326. Seutter-Berlage, F., Dellxessine, L. P. C., Snieets, F. L. 11,. and Ketelaars, H. C. J. (1978).Xetiol)iotictr 8, 41.3-418. Severi, L., and Biancifiori, C. (1968).1. N u t / . Cottcc>r.I t i s t . 41, 331-349. Severson, R. F., Snook, M. E., Hignian, H. C., Cliortyk, 0.T., and Akin, F. J. (1976). I t t ‘ ‘ Po I y n II c 1e a r Aromatic Hydro carbon s : C h e ti1 is t ry , Met abo 1 is m a 11d Carcinogenesis” (R. I. Freudentlial and P. W. Jones, eds.), pp. 253-270. Raven, N e w York. Seymour, J . L., Schmiclt, S. P., and Allen, J. R. (1976). Proc. Soc. E x ) ) . R i d . Med. 152, 62 1-625. Shaker, XI., and Soliin;w, 11. K. (1966). Itid. ./. E . Y / ) .B i o / . 4, 176-178. Shinialmkuro, R. H. (1975). Ettcirott. Q i i c i l . Strfef!/ 4, 140-148. Sliimal~ukuro,R. H., Swanson, 11. R., atid Vlialsh, W. C . (1970).PIottt Plt!/.vio/.46, 103107. Shimaliukuro, R. H., Frear, D. S., Swanson H. R., and Walsh, W. C. (1971).P/utit Pliysiol. 47, 10-14. Shimabukuro, R. H., Lamoureux, G. L., Swanson, H. R., Walsh, W. C., Stafford, L. E., and Frear, D. S. (1973). Pest. Biocliem. Plz!/s.iol.3, 483-494. Shiniada, T., and Sato, R. (1978). Biocheni. Phartnucol. 27, 585-593. Shirasu, T., Moriya, h l . , Kato, K., Furuhashi, A , , and Kada, T. (1976). M u t i i t . Re.9. 40, 19-30.

272

L. F. C H A S S E A U D

Shishido, T., Usui, K., Sato, M., a n d Fukanii, J. (1972). Pest. Biochei~i.P!/sio/.2, 51-63. Siegel, M. R. (1970).J.Agr. Food Chetti. 18, 819-822. Sikka, H . C., a n d Florczvk, P. (1978).J. A g r . F o o d Chein. 26, 146-148. Silver, E. H., and Murphy, S. D. (1978). To.xicol. A p p / . Phorniacol. 45, 312. Simons, P. C., a n d Vander Jagt, D. L. (1977). A n n / . Biocheiii. 82, 334-341. Sims, 1’. (1966). Hiocliein. J . 98, 215-228. Sinis, P. (1967).Bioclieni. J . 105, 591-598. Sims, P. (1970). Bioclwni. P/iorni(rco/. 19, 795-818. Sims, P. (1971). Bioclteiri. J. 125, 159-168. Sinis, P. (1972).XetioIJioticcr 2, 469-477. Sims, P., and Grover, P. L. (1965).Riocltein. J . 95, 156-160. S i m s , P., itnd Grover, P. I,. (1974). Atlc. Ctriicer Res. 20, 165-274. Sklan, N . M., and Bamsley, E. A. (1968).Bioclieni. J . 107, 217-223. Slaga, T. J., Viaje, A,, Bracken, W. N., Berry, D. L., Fisclier, S. N., Miller, D. R., a i i d Leclerc, S. M . (1977a). Cniicer Lett. 3, 23-30. Slags, T. J., Bracken, W. N., Viaje, A , , Levin, W., Yagi, H., Jerina, D. X I . , and Conney, A. I t . (197711).Crriicrr Rev. :?7, 4130-4133. Smith, R. L., a n d Tiinbrell, J. A. (1974).Xeiiobioticci 4, 489-501. Smith, J. N., Spencer, B., a n d Williams, R. T. (1950). BiCJc/ie1?7.J. 47, 284-293. Smith, G. J., H u e b n e r , K., and Litwack, G. (1977a). Bioclieni. B i o p / i ! ~ ~Rse. s . Conitriiiit. 76, 1174-1 180. Sliiith, G. J.. Ohl, V. S., atid Litwack, G . (197711).Coiicer R ~ . Y37, . 8-14. Speir, T. W., aiid Barnsley, E. A. (1971).Biocliem. J. 125, 267-273. Stathmi, C. N., Franklin, R. B., and Boyd, M. R. (1978).I’osicol. Ap p / . Phnr?iitrcn/. 45, 267. Stekol, J. A. (19381.1. B i d . Cltern. 124, 129-132. Stillwell, W. G . , Carman, M . J., Bell, L., a n d Horning, M . G. (1974). Drug Xlef~rrh. Dispos. 2, 489-4138, Straub, K. M., Meehan, T., Burlingame, A. L., a n d Calvin, M. (1977). Proc. N u t l . Actrd. Sci. U.S.A. 74, 5285-5289. Strauss, B., a n d Okubo, S. (1960).J. Rncteriol. 79, 464473. Stroliii-Benedetti, M., Louis, A., Malnoe, A., Schneider, M., Lam, R., Kreber, L., and Smith, R. L. (1975).J . Pharnt. Phnrmcico/. 27, 629-632. Strolin-Benedetti, M., Malnoe, A,, and Broillet, L. (1977).Toxico/og!/ 7, 69-83. Sriliba Rao, G . (1977).J. Med. Chem. 20, 262-265. Suga, T., a n d Akagi, M. (1970).Cltetn. Phariri. E d / . 18, 115-119. Suga, T., Oliata, I., a n d Akagi, M. (1966).J. Biocliem. 59, 209-215. Suga, T., Oliata, I., Kumaoka, H., and Akagi, M . (1967). Clietti. P h r n t . Bull. 15, 10591064. Sunderinan, F. W., Jr. (1978). Fed. Proc. 37, 40-46. Swenson, D. H., Lin, J. K., Miller, E. C., a n d Miller, J. A. (1977). Caitcer Res. 37, 172-181. Szabo, S., Bailey, K. A,, Boor, P. J., a n d Jaeger, R. J. (1977). Bioclrem. R i o p h ! / s . Res. Coiiiirizrii. 79, 32-37. Takayama, S., a n d Kuwabara, N. (1977). Cnncer Lett. 3, 115-120. Taniika, K., Kurihara, N., a n d Nakajinia, M. (1976). Pest. Biocltem. Pla!/sio/. 6, 392-399. Tateishi, M., a n d Shimizu, H. (1976).Xetiobioticn 6, 431-439. Tateishi, M., Suzuki, S., a n d Shimizu, H. (1978). B i O C h E ? ? l . Phurmncol. 27, 809-810. Thakker, D. R., Yagi, H., Akagi, H., Koreeda, M., Lu, A. Y. H., Levin, W., Wood, A. W., Conney, A. H., a n d Jerina, D. M. (1977).Chem. B i d . Iiitercict. 16, 281-300.

G S H CONJUGATION WITH CARCINOGENS

273

Ticriw>,, B., Ilewt.r, ,4., \Valsli, C., Grovel-, P. L., ;uid Siiiis, 1'. (1977). C l i e t r t . Biol. Zti/crnc,t.18, 179-193. Tillinan, R . W., Siegel, hl. R., and Long, J. W. (1973).P c ~ s /H i o c ~ l t c ~ tPli!/.c.iol. i~. 3, 160-167. Tipping, E., Ketterer, R., Cliristotloiilides, L., and Enderby, C;. (1976).E t ~ rJ.. Biocltc,ttt. 67, ,583-590. Tipping, E., Ketterer, B., and Koskelo, 1'. (1978). Riocltptit. /. 169, ,509-516. Toraiizo, E . G., ,P. G., XlcC~ow;in,C;. 11.. \lxlrid, E. O., and Gelrring, 1'. J. (19761)).To.xico/. A / , / I / . " / i t r f ~ t l l o c ~ o l36, . 49-59. Wataiial)e, P. G., Yoring, J. D., Schlachtrr, M. XI.. Zeiiipel, J. A,, and Karlxiwski, R. J. (1978;i).To.xk'O/. / \ / > ] I / . P / t ~ r l i i < / ~45, d . 224. Wataiial)e, P. G., Zeiiipel, J. A,, Pegg, D. G., and Gehriiig, P. J. (19781)).To.xico/.: \ / I / J / . Plltrrfllftcol.44, 571-579. Waterfhll, J . F., iund Sims, P. (1972). Biodietii. /. 128, 265-277. Watteiil)erg, L. M'. (1977)./. N u t / . Crrticer Z t r s ( . 58, 395-398. Weiner. I. M . , and Miiller, 0. H. (1955).J. Plicrrtticrcol. E s / I . Tlter. 113, 241-249. Weishiirger, J . H., and Weisburger, E . K. (1973).P/~crrr~rrrco/. Ret:. 25, 1-66. 24, 1283-1286. Whelaii, G., and Coinlies, €3. (1975). Bioclietti. P/tortit~~co/, Whelm, G,, Hocli, J., and Coiiil)es, €3. (1969).Z'roc. Sot. E.x/).Biol. .\fed. 132, 704-707. Wlielan, G., Hoch, J., a i i d Coinhes, B. (1970).J . Lnh. C/iti. .\fed. 75. 542-547. White, I. N . H. (1976). CItetti. B i d . Ztttercict. 13, 333-342. WHO (197.5). Smoking and its Effects on Health. Technical Report Series 568. WHO, Geneva. Whorton, D., Kraiiss, R . hl., Marshall, S . , and Milby, T. H., (1977).Lnticef 2, 1259-1261. Wild, D. (1975). .\lrcttr/. Res. 32, 133-149. Williains, R., antl Davis, hl. (1977). Actcr Phctrttitrcol. ?b.xico/. 41, (Sappl. IV), 282298.

274

L. F. CHASSEAUD

Wirth, P. J., Bettis, C. J., and Nelson, W.L. (1975).Mo2. Phunttcicol. 12, 759-768. Wislocki, P. G., Borchert, P., Miller, J . A , , and Miller, E. C. (1976). Caitcer Re.%.36, 1686- 1695. Wislocki, P. G., Miller, E. C., Miller, J . A , , McCoy, E. C., and Rosenkranz, H. S. (1977). Cancel- Res. 37, 1883-1891. Wit, J. G. (1968).E u ~J.. Pliurrricrcol. 5, 100-102. Wit, J. G., and Snel, J . (1968).E u r . J . Plicirntucol. 3, 370-373. Withey, J. R. (1976).J . Tosicol. E ~ i v i r o nH. e ~ l t l i1, 381-394. Wogan, G. N. (1973).Metltods Cnricer Re.%.7, 309-344. Wolkotl’, A. W., Ketley, J . N., Waggoner, J. G., Berk, P. D., and Jakoliy, W. B. (1978).J . Cliri. Ztiaest. 61, 142-149. Wood, J . L. (1970). 1 1 1 “Metabolic Conjugation and Metabolic Hydrolysis” (W. H . Fishman, ed.), Vol. 2, pp. 261-299. Academic Press, New York. Gootle, R. L., Chang, R. L., Levin, W., Conney, A. H., Yagi, H., Dansette, Wood, A. W., P. M.,;ind Jerinki, D. hl. (1975).Piwc. Ntrfl. Acnd. Sci. U.S.A. 72, 3176-3180. Wood, A . W., Wislocki, P. G., Chang, R. L., Levin, W., Lit, A . Y. H., Yagi, H., Hernandez, 0.. Jerina, D. M., and Conney, A . H. (1976). Ctrticer Re.%.36, 33583366. Wood, A . W., Cliung, R. L., Levin, W., Lehr, R. E., Schaefer-Ridder, M.,Karle, J. M., Jerina, D. M., and Conney, A. H. (1977a). Proc. N(rtl. Actid. Sci. U.S.A. 74, 27462750. Wood, A . W., Levin, W., Ryan, D., Thomas, P. E., Yagi, H ., Mah, 13. D., Thakker, D. R., Jerina, D. M., and Conney, A . H. ( 19771)).Biocheni. Bioph!/.?. Re.?. C o n i i n r i r t . 78, 847-854. Woodward, G. E., ;ind Fry, E. G. (1932).J.R i d . Cheiri. 97, 465-482. Wynder, E. L., and Hoffinann, D . (1959).Caricel- 12, 1079-1086. Wynder, E. L., and Gori, G. B. (1977).J.Nrrtl. Coiicer f i i s t . 58, 825-832. Wyndham, C., Devenish, J., and Safe, S. (1976). Res. Cotnrnurr. Cheni. Pothol. Phcrrtncicol. 15, 563-570. Yagi, H., Hernandez, O., and Jerina, D. M. (1975j.J.A m . Cliem. Soc. 97, 6881-6883. Yagi, H., Thakker, D. R., Hernandez, O . , Koreeda, M., and Jerina, D. M. (1977).J. A t i t , Clteni. Soc. 99, 1604-1611. Yahagi, T., Matsushima, T., Nagao, M.,Seino, Y., Srigimimi, T., and Bryan, G. T. (1976). Jlrrtrrt. Res. 40, 9-14. Yam, J., Reeves, M., and Roberts, H. J. (1976).J.L t r h . Clin. M e d . 87, 373-383. Yang, S. K., McConrt, D. W., Roller, P. P., and Gelboin, H. V. (1976).Proc. N t r f l . Accrrl. Sci. U.S.A.73, 2594-2598. Yang, S. K., Roller, P. P., FII, P. P., Harvey, R. C . , and Gelboin, H.V. (1977).Biochern. BiO)Ih!/.S.Re.?. ~ O ? J l ? t l t l t t 77, . 1176-1182. Y h e r , S. (1971a).Actrr P/turviaco/. Toricol. 30, 257-265. Yllner, S. (19711)).Actcr Plicirnitrcol. Toricol. 30, 248-256. Yllner, S. (1971~). Act0 Plitrrrttcrcol. T O S ~ C 30, O ~ .69-80. Zampaglione, N., Jollow, D. J., Mitchell, J . R., Stripp, B., Hanirick, M., iind Gillette, J . R. (1973).J.P/t/rrm(rco/. E x ) ) . Tlter. 187, 218-227.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT'

Erkki Ruoslahti Division of Immunology City of Hope National Medical Center Duarte California

and Markku Seppala Department of Obstetrics and Gynecology University Central Hospital and Department of Bacteriology and Immunology University of Helsinki Helsinki Finland

I. Introduction . , , , , . , . . . . . . , . . , . , , . . . , , . . . . . . . . . . . . , . . . . . . . . . .

276

11. Detection and Measurement of AFP and Its Imniunochemical Properties 276 276 A. Antibodies to AFP . . . . , . . , . . , . . . . . . . . . . . . . . . . . . . . . . . . €3. Interspecies Cross-Reactions . . . . , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277

C. Inimunological Cross-Reactions between AFP and Albumin . . . D. Imniunoassay of AFP in Biological Fluids . . , . . . . . . . . . . . . . . . .

........... 111. Sites of Physiological AFP Synthesis .

,..........

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

C. hlaternal Senini

............... F. Normal Semm . . . . . . . . . . . . . .

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

.......

B. Physical and Chemical Properties of' AFP. . .

278 279 280 28 1 28 1 282 284 284 285 286 287 287 288 290 290 292 305

306 308 313 31:3 315 323 VIII. Geriii Cell Tuniors ............................ IX. Other Clinical Conclitio~rswith Informative AFP Levels . . . . . . , . . . . . . . . 327 :327 A. Liver Diseases . . , , , . . , . , . . , , . . . . . . . . 327 327 C. AFP in the Diagnosis of Fetal Pathology . . . B. Possible Immunoregulatory Role of AFP . . . . . . . . . . . . . . . . . VII. .4FP i n Liver Disease ............................. ............................... A. Liver Iiijurq . . , . . . . . . , . . . . . . .

I

' The original work reported i n this manuscript was supported b y Grants CA 19894 and CA 16434 from the National Cancer Institute, DHEW, and from the Finnish Cancer Society and the Research Council for Medical Sciences, the Academy of Finland. 275

276

EHKKI RUOSLAHTI AND MARKKU SEPPALA

X . Tolerance to AFP and Its Abrogation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. AFP as a Possible Target Molecnle for Tumor Inimunoprevention and Iminunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XII. Molecular Basis of Regulation of AFP Synthesis ........................ XIII. Areas of Frihire Research mid Concluding Remarks Heferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

332 333 334 335 33G

I. introduction

Gene products with a nornial expression restricted to the embryonal and fetal periods are frequently reexpressed in tumors. To recognize this dual expression, such components are grouped under the heading of carcinoembryonal, carcinofetal, or oncodevelopmental gene products. a-Fetoprotein (AFP) is one of the most thoroughly characterized oncodevelopmental proteins. A vast amount of work has been done since this protein was first observed by Bergstrand and Czar (1956) 20 or so years ago and brought into the limelight by the observation of Abelev and co-workers (Abelev et al., 1963) that AFP was associated with liver cancer. In the following, we will summarize the salient features of the characteristics and expression of AFP as well as give sonie details from our own work. II. Detection and Measurement of AFP and its lmmunochemical Properties

Despite the fact that AFP was first detected by paper electrophoresis of fetal serum (Bergstrand and Czar, 1956), the definition of AFP is still mainly immunochemical. It is detected, quantitated, and even purified using antibodies. It, therefore, seems appropriate to begin this review with a discussion on the immunochemical properties and assay of AFP.

To AFP A. ANTIBODIES AFP is strongly immunogenic when injected into a foreign species but does not elicit an immune response in the species of origin (Seppalit and Seppalii, 1969; Nishi et al., 1972).The latter restriction can be circunivented (see Section X). As is the case with most globular proteins, the antigenicity of AFP seenis to depend on the three-dimensional conformation of the polypeptide chain. Human AFP unfolded b y reduction in urea loses its inhibitory activity in the usual iniiiiunoassays (Ruoslahti and Engvall, 1976). In the early work, antibodies to AFP were obtained by

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

277

immunization with fetal serum followed by absorption ofthe resultant antiserum with normal adult serum. The concentration of AFP in fetal serum is in the range of milligrams per milliliter, and it usually gives rise to production of antibodies, even in an admixture with other fetal serum proteins. In most species, AFP is the only fetal serum protein present in appreciable quantities which is not shared by the adult serum. Consequently, elimination of antibodies to adult serum proteins by absorption leaves behind an antiserum which is operationally specific for AFP. Such antisera react with a single immunodiffusion line against fetal and liver cancer serum and give no reaction against normal adult serum. A similar approach is possible with liver cancer sera containing high concentrations of AFP (Abelev, 1971).With the availability of purified AFP from most species, the use of purified protein for immunization has become the method of choice. The methodological aspects of preparation of antisera to AFP have been recently discussed (Ruoslahti, 1976).

B. INTERSPECIE s CROSS-REACTIONS AFPs from different mammalian species are cross-reactive. Chicken antibodies against human AFP react in immunodiffusion with AFPs from all mammalian species tested (Nishi et al., 1975). Antisera to human AFP produced in rabbit, horse, and rat also react with AFPs from different mammalian species, but less extensively. Sheep and goat antisera against human AFP are highly cross-reactive with rabbit and bovine AFP (Pihko et ul., 1973, Fig. l),and sheep antibodies against rabbit AFP react strongly with human AFP. Antisera against rat AFP produced in horses, rabbits, and goats react strongly with mouse AFP but not with other mammalian AFPs. Chicken AFP does not cross-react with mammalian AFPs in immunodiffusion (Lindgren, 1976), but antisera to mammalian AFPs show binding of radiolabeled chicken AFP (Lindgren and Ruoslahti, unpublished). Such binding assays seem to be by far the most sensitive way of detecting crossreactivity. It is obvious that the cross-reactivities observed depend on the origin of AFP, i.e., rabbit and dog AFPs are highly cross-reactive with human AFP, and mouse and rat AFP, with each other. The crossreactivities also depend on the origin of antibodies. It seems that the strongest cross-reactions are obtained with antisera from a species remote to the ones tested. The interspecies cross-reactions have proved useful in the purification of AFP from different species (Section V).

278

ERKKI RUOSLAHTI AND MARKKU SEPPALA

FIG. 1. Immiiiiotliffusioii in agtirose. An example o f cross-reactivity of AFPs from different species. Hmman (A) and I>ovine (B) react with a goat antiserum (AS) to hunian AFP.

C. IMMUNOLOGICAL CROSS-REACTIONS BETWEEN AFP AND ALBUMIN AFP and albumin show sequence homology (Ruoslahti and Terry, 1976, Section V). In spite of this, antisera produced against purified human AFP do not react with albumin in immunodiffusion, nor do human AFP and albumin reveal any cross-reactions in radioimmunoassay (Ruoslahti and Engvall, 1976). Unfolding of the polypeptide chain of AFP and albumin by reduction followed by carboxamidomethylation produces derivatives with immunochemical properties different from those of the native proteins. Precipitating reactions and radioimmunoassays (Fig. 2) performed using antibodies to such unfolded derivatives show strong cross-reactions between albumin and AFP (Ruoslahti and Engvall, 1976). The assays lack species specificity; AFPs and albumins from different species are equally active. Several other proteins unrelated to albumin or AFP do not react or react much less in these assays. The implications of these findings will be discussed below (Section V). It is important to note in this context that, since there is no cross-reactivity between native AFP

tr-FETOPROTEIN IN CANCER AND FETAL DEVELOPSIENT Q

2 3 0

loo

279

1

m

10-2

10-3

10-4

10-5

10-6

DILUTION OF ANTI - RC-HSA SERUM

2. Cross-reactivity of AFP and albiuiiin. Binding of '""I-ld~eledhiurnan AFI' and all~~iiiiiii (HSA) I~efbreand after recluctioii and carl~oxamidomcthyIation (HC derivatives) to iiii mtiserrim prepared against RC-humanalbrimin (Ruoslahti and Engvall, 1976). AFP acquires reactivity with the RC-AFP, and RC-albrimin inhibit antiRC-IISA seriiiii after retlrictioii and carl~ox~tmidomethylation. Several unrelated proteins hkive not shown such binding to this antiserum after a siniilar treatment. FIG.

hrimiiii

and albumin, the immunological assays used for detection and quantitation of AFP are not affected.

D. IMMUNOASSAY OF AFP

IN

BIOLOGICAL FLUIDS

All currently used methods for AFP measurement rely on its antigenicity. A great variety of methods have been applied (Table I). Immunodiffusion was the first method introduced for AFP detection 1963). This method is qualitative and suitable for the (Abelev et d., demonstration of AFP when the concentration exceeds 2.5 pglml. Since the development of radioimmunoassay for AFP (Ruoslahti and Seppali, 197111; Nishi and Hirai, 1973), this method has allowed the demonstration and quantitative estimation of AFP in normal human serum. The sensitivity of radioimmunoassay for AFP is about 0.25 nglml, and the method allows detection of small elevations from the normal level. Practically all radioimmunoassays applied to measurement of hunian AFP (Jonsson and Kronwall, 1974; Leek and Chard, 1974; Nishi and Hirai, 1973; Purves et ul., 1973; Ruoslahti and Seppila, 1971b; Silver et al., 1974; Waldmann and McIntire, 1972) and AFPs of various species (Lindgren, 1976; Masseyeff et al., 1974; Pihko

280

ERKKI RUOSLAHTI AND MARKKU SEPPALA

TABLE I METHODSOF AFP MEASUREMENT Method

Sensitivity

Reference for AFP estimation

Double digusion Coimter electrophoresis Rocket iinmunoelectroplioresis

2.5-5 pg/ml 0.25-0.5 pg/inl 0.5-1 &nil

Latex agglutination inhibition Imiiiunoautoradiograph y ELISA

250 ngiml 50 ndinl 20 ngiml 3 ndm l

RIA

0.5 ng/nil

Abelev et u l . , 1963 Smith, 1971 Nprgaard-Pedersen and Gaede, 1975 Cahill et d . ,1974 Abelev, 1971 Nprgaard-Pedersen, 1976 Bdlanger et ( I ! . , 1973b; Masseyeff et d., 1976 Ruoslahti and Seppiilii, 19711)

Radioiinmunoelectrophoresis

and Ruoslahti, 1973; Sell and Gord, 1973)are based on the principle of competition between labeled and unlabeled antigen for a limited amount of antibody. A noncompetitive radioimmunoassay lmed on the sandwich principle has been described (Johansson et d.,1974). A noncompetitive sandwich method of enzyme-linked immunosorbent assay (ELISA) (Engvall and Perlmann, 1971) has also been applied to AFP (Bklanger et nl., 197311; Masseyeff et al., 1976; Hevey et ul., 1976). Results of ELISA are compatible with those obtained by radioimmunoassay, and the specificity and reproducibility are of the same order. The advantages of ELISA over radioimmunoassay include the greater stability of reagents and avoidance of radioactive materials. When AFP is measured at high concentrations, the rocket electrophoresis technique of Laurel1 has some advantages over radioimmunoassay. The technique is rapid, and high dilutions of samples are avoided. This technique can be made more sensitive by applying a radioactive label and autoradiography (Ngrgaard-Pedersen, 1976). The aggregation of AFP in native samples and purified preparations results in the formation of higher molecular weight polymers, which may interfere with quantitative tests, particularly with those based on diffusion or mobility in gel. The aggregation phenomenon has also been reported to affect the sensitivity of radioimmunoassay by increasing the proportion of nonbound radioactivity (Young et al., 1976).

E. INTERNATIONAL STANDARD FOR AFP Collaborative studies have shown that there is a large variation between the AFP values obtained in different laboratories for the same

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPhlENT

281

sample (Sizaret et ul., 1975a).This is not a feature peculiar of AFP but rather seems to be an inherent characteristic of many protein radioimmunoassays. An international standard preparation consisting of pooled human cord serum has been prepared and designated a s Standard 72/225 (Sizaret et d . , 1975a). The average value of 64.35 pg/ml was obtained for this standard in different laboratories. Because of the high variation, it was Gecided to express the International Reference Preparation in terms of arbitrary units. More unifomi results would probably be obtained if the standardization were to be repeated now that a great deal more experience on the AFP assay has accumulated. It is recommended that all published work should include a reference to how the local standard compares with the International Standard. However, the use of International Standard does not allow individual laboratories to escape from estimating their own normal ranges, since diEerences in methodology and antisera may affect the level even when the same standard is used. Racial differences in AFP levels may exist a s well. OF AFP SYNTHESISBY INTERNAL LABELING F. DETECTION

Addition of radiolabeled amino acids in organ or cell cultures producing AFP results in the incorporation of radioactivity to AFP. This has been utilized to detect AFP synthesis (Gitlin and Perricelli, 1970), to study the rate of synthesis of AFP (Kekomiiki et d . , 1971), to study the relation of cell proliferation to AFP production by fetal hepatocytes i i i vitro (Leffert and Sell, 1974), and for comparison ofthe properties of AFP synthesized by the yolk sac and the liver (Zimmernian and Wilson, 1976). Immunoprecipitation of internally labeled AFP seems to be a more sensitive way of detecting AFP production than radioininiunoassay (Leffert et d . , 1978). O F AFP IN TISSUES G. DETECTION

The cellular basis of AFP production has been studied by inimunofluorescence (Abelev, 1971; Engelhardt et uZ., 1969, 1971; Linder and Seppiili, 1968; Onoe et ul., 1975; Tchipyshevaet nl., 1977) and more recently by using the imniunoperoxidase technique (Kuhlmann, 1978). The latter technique seems to allow more accurate observation of the histology. Both have the drawback that distinction between proteins synthesized within the cell and those taken up by the cell is difficult. Utilizing the technique in which parallel staining for IgG was used to reveal nonspecific attachment of proteins, Engelhardt et

282

ERKKI RUOSLAHTI AND MARKKU SEPPALA

al. (1971) obtained a better distinction between specific and nonspecific fluorescence in tissue sections stained for AFP. A technique which allows detection of AFP production by a single cell has been devised by Abelev and co-workers (Abelev, 1979) and de Nkchaud et (11. (1977). The cells are embedded in an agar layer containing red blood cells with anti-AFP on their surface. AFP will react with the antibody and, in the presence of complement, the red blood cell around a source of AFP will be lysed. A halo is thus produced around a cell producing AFP. The full potential of this hemolysis-ingel technique in answering questions on the cellular basis of AFP production is yet to be realized. Uriel et aZ. (1973) exploited the estrophilic property of rat AFP for the localization of AFP-producing cells in the fetal liver and hepatoma tissues by incubating fixed histological sections with radiolabeled estrogens. Autoradiography revealed radioactivity in small groups of single hepatocyte-like cells. No radioactivity was localized in the hematopoietic and reticuloendothelial cells. However, this technique is unlikely to be specific for AFP since other estrogen-binding proteins are present in tissues. Ill. Sites of Physiological AFP Synthesis

The synthesis of AFP by various fetal organs has been studied by incorporation of radioactivity from labeled amino acids to proteins in tissue culture with subsequent detection of radioactive AFP by antibodies. The synthesis of AFP has been found to take place in the fetal liver, the gastrointestinal tract, and the yolk sac (Gitlin and Boesman, 1967; Gitlin and Perricelli, 1970; Gitlin et d . , 1972; reviewed in Gitlin and Gitlin, 1975). In humans, AFP is first synthesized by the yolk sac and the liver and then predominantly by the liver. Synthesis in the yolk sac has been demonstrated between the fifth and fourteenth weeks of gestation. It seems to cease as the yolk sac becomes involuted during the first part of the second trimester. The liver contributes to the AFP synthesis from the fourth week on. In the second trimester of human pregnancy, the fetal liver is capable of producing AFP at a rate which reaches 30 mg/day (Kekomiki et d., 1971). Minor amounts of AFP are being synthesized by other tissues in the gastrointestinal tract (Gitlin et al., 1972). It is generally agreed that the placenta does not produce AFP to any significant degree, although, in some experiments, small amounts of labeled amino acid precursor were incorporated into AFP by this tissue (van Furth and Adinolfi, 1969). Molar trophoblast appears sometimes to be capable of produc-

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

283

ing AFP (Grudzinskas et ul., 1977). Evidence for this comes from a high AFP concentration observed in the molar vesicle fluid obtained by needle aspiration. However, nonnal AFP levels in molar tissue have also been reported (Seppiilii et ul., 1972). The same pattern of AFP synthesis is found in other species with some variations. In the mouse, rat, chicken (Gitlin and Gitlin 1975; Sell and Skelly, 1976), and in rabbits (Branch and Wild, 1972) synthesis hy the yolk sac continues until birth. Sharks have also been ported to possess a serum a-globulin that could be a homolog of mammalian AFP (Gitlin, 1975). Like human AFP, this protein is synthesized primarily in endodermal embryonic and fetal tissues-the liver, yolk sac, and gastrointestinal tract. Thus, an AFP homolog may exist as far back in evolution as 400 million years. In chicken, the AFP synthesis takes place almost exclusively in the yolk sac (Gitlin et al., 1973; Lindgren, 1976). Small but significant amounts are produced b y the liver (Lindgren, 1976). Immunofluorescence shows that it is the hepatocytes, not the hematopoietic cells, that synthesize AFP in the fetal liver. Up to 80% of the hepatocytes in fetal liver have been found to synthesize AFP (see Abelev, 1979). The question of whether the embryonal liver represents a cell mosaic consisting of functionally heterogeneous cells, each synthesizing only one protein, or whether the same cell is capable of synthesizing several proteins has been studied with immunofluorescence and the hemolysis-in-gel technique (Eraizer et (11 ., 1977; Abelev, 1978; Nayak and Mital, 1977). In these studies, the same hepatocytes from human and rat fetuses appeared to synthesize both albumin and AFP. Results from other experiments using double staining with fluorescent-conjugated antibodies to AFP and albumin have suggested that AFP and albumin are synthesized by different cells in cultures of malignant hepatocytes (Tsukada and Hirai, 1975).This and other studies (Isaka et nl., 1976), however, have demonstrated that clonal cell lines derived from rat hepatomas produce both AFP and albumin. Taken together, this evidence suggests that a single cell is capable of producing both AFP and albumin or can differentiate into both AFP- and albumin-producing cells. It seems that hepatocytes form a relatively homogeneous population of cells where the proportion of AFP to albumin produced becomes smaller as they differentiate toward adult type of hepatocytes. The initial studies showing the presence of small amounts of' AFP in normal human sera (Ruoslahti and Seppilii, 1971b) and adult sera of other species suggested that there could be a stem cell-like population of undifferentiated hepatocytes in the liver that would produce AFP. The studies cited

284

ERKKI RUOSLAHTI AND MARKKU SEPPALA

above seem to eliminate this possibility. No AFP-producing cells have been found in the normal adult liver, suggesting that the normal serum AFP is produced in small quantities by many cells and not by a specialized population of cells.

IV. Physiological Concentrations of AFP in Body Fluids

A. FETALSERUM

The main compartments of interest in terms of AFP concentrations are fetal serum, amniotic fluid, maternal serum, cerebrospinal fluid, and urine. The AFP concentrations in fetal serum, amniotic fluid, and maternal serum in human, bovine, and rat pregnancy are depicted in Fig. 3. During human gestation, the highest AFP concentrations in fetal serum occur at 12-14 weeks, where the peak levels reach 1-3 nig/ml. The decrease in fetal serum AFP concentration between 14 and 22 weeks gestation is attributable to an increase in the rate of growth of the conceptus which exceeds the increase in the amount of AFP synthesized during this period. The total amount of AFP in the human fetus is highest between 22 and 32 weeks (Gitlin and Boesman, 1966). At terni, the AFP concentration in cord blood shows wide variation from 4 to 188 pglrnl (Seppala, 1975). The AFP concentration in the fetal serum of other species follows the same general pattern but differs in details. Concentrations in exlo,ooo

1

HUMAN

r

BOVINE

tWEEKS OF GESTATION

RAT

Amnlollc Fluld Slrum

MntemOI Swum

4

r

B 12 16 20 24 26 32 16 4 0 4 4

WEEKS OF GESTATION

1

WEEKS

2

3

OF GESTATION

FIG.3. AFP concentrations in the fetal serum, amniotic fluid, a n d maternal serum in human and Imvine pregnancy and in rat pregnancy serum. Data for bovine pregnancy were adapted from Jalanko ct ( I / . (197%) and for rat pregnancy from Sell (197.3).

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

285

cess of 5 mg/ml of AFP have been observed in fetal rat (Sell and Skelly, 1976),rabbit (Branch, 1972), and bovine (Jalanko et nl., 197813) serum. The AFP levels in rat and rabbit fetal senim peak later in relation to birth than in humans. This presumably reflects the shorter gestation period and relative immaturity of the newborn in the former species. It should be pointed out in this context that in spite of some confusion even in the recent literature (Yachnin, 1976), bovine AFP and fetuin are different proteins (Kithier and Poulik, 1972) with quite different biochemical properties (Ruoslahti and Seppala, 197la).

B. AMNIOTICFLUID In human amniotic fluid, the AFP concentration is 100-300 times smaller than in fetal serum, but the decreasing slope with advancing gestation parallels that of fetal serum AFP concentration. It is assumed that AFP enters amniotic fluid in fetal urine, where the concentration is higher than in amniotic fluid (Seppala and Ruoslahti, 1972b), and the nomial amniotic fluid AFP concentration is presumably maintained through fetal urine. AFP has also been found in the urine of adults with elevated serum AFP levels (Okon et d., 1973).The AFP concentration in amniotic fluid correlates significantly with gestational age, but not with fetal weight at birth. While fetal urine seeins to be a source of AFP in amniotic fluid, AFP is found in the ainiiiotic fluid long before the end of the first trimester when urine is first voided (Sutcliffe, 1975) indicating that another source of amniotic fluid AFP must exist. It has been suggested that the soluble proteins of amniotic fluid which are of fetal origin may also be derived from skin, amnion, chorion, umbilical cord, gastrointestinal tract, and buccal and bronchial secretions (Sutcliffe, 1975). It would seem that some of the amniotic fluid AFP could be derived directly from the yolk sac tissue by diffusion. This assumption is supported by our recent finding that teratocarcinoma AFP, which is presumably of yolk sac origin, has a large fraction not reactive with concanavalin A (Con A) and this property is shared by second trimester amniotic fluids (Ruoslahti et al., 1978a, see Section V). That all AFP in amniotic fluid is not derived from the fetal urine is also supported by the finding of Seller and Berry (1978), who described a normal amniotic fluid AFP level in a case of fetal renal agenesis, and AFP has been demonstrated in the amniotic fluid in the case of blighted ovum (Leek et al., 1974), where the fetus is coinpletely lacking. However, this is not a usual situation, since in most cases of empty sac, the amniotic AFP level is very low (Aflan et nl., 1973). It is likely that very early i n gestation,

286

ERKKI RUOSLAHTI AND MARKKU SEPPALA

part of amniotic fluid AFP is formed by yolk sac elements, and perhaps by the placenta (van Fui-th and Adinolfi, 1969; Leek et al., 1974; Linder and Seppda, 1968).Transfer of AFP from the mother into amniotic fluid is probably insignificant in the first half of pregnancy and rather the opposite is likely to happen, i.e., AFP is transferred from the amniotic fluid into maternal circulation. While AFP in the second'trimester amniotic fluid in human pregnancy represents only about 0.1% of the total protein present, it represents more than one-third of the protein in mouse amniotic fluid (e.g., Murgita and Tomasi, 1975a). This may be a result of the greater activity of the yolk sac in rodents. The pattern of changes in the amniotic fluid AFP concentrations resembles that of the fetal serum AFP levels. The highest levels are seen at 12-14 weeks of gestation. Elevated AFP levels in amniotic fluid may theoretically result from increased fetal AFP synthesis, increased excretion of AFP, decreased elimination, or leakage from fetal or placental lesions. Such disturbances do occur, and measurement of amniotic fluid AFP is now an important adjunct to the monitoring of the fetal development (see Section IX,C).

C . MATERNALSERUM Evidence from the rat shows that AFP in maternal serum is of fetal origin. In the pregnant rat, radiolabeled AFP injected into the amniotic sac or fetal peritoneal cavity was subsequently demonstrated in the maternal circulation (Sell, 1974).In rat tissue cultures in witro, specific incorporation of radiolabeled amino acid precursor into AFP has been observed to take place in the yolk sac, the fetal liver, and in small amounts by the fetal gastrointestinal tract, but no incorporation by maternal tissues was demonstrated (Sell and Skelly, 1976). In the human products of conception, AFP has been demonstrated as early as at the fourth postconceptional week (Seppala and Ruoslahti, 1973), and, in normal pregnancy, the maternal AFP concentration may increase above the normal nonpregnant level as early as at the seventh week (Seppaliet nl., 1972).The increase in maternal serum AFP level is probably a consequence of transplacental and transamniotic passage of AFP from the fetus to the mother. The highest maternal AFP concentrations occur in the midthird trimester, where the mean level is 150-250 ng/ml. At birth, the maternal AFP level is 200-600 times lower than the corresponding fetal level (Fig. 3). In the postpartum period, maternal AFP concentration decreases at a rate corresponding to a half-life of 4-5 days (Seppala and Ruoslahti, 1972a, 1973).At term, the concentration of AFP in maternal serum seems to be related to the

WFETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

287

AFP level in the fetal circulation as well as to the placental size (Seppala, 1975). AFP levels in maternal serum in Rh-immunized mothers generally have been observed to exceed the normal median value, and the corresponding maternal serum levels in toxeniic mothers are distributed more frequently below the iioniial median (Seppala, 1975). The same trend has been observed in the case of fetal levels. Like AFP in amniotic fluid, the AFP level in maternal serum reflects the fetal condition in many disturbances of the fetal development and provides an important diagnostic tool (see Section IX,C). While maternal seruni in human pregnancy contains too little AFP to be detectable with methods less sensitive than radioimmunoassay (Seppiilii and Ruoslahti, 1972a),concentrations of AFP in the serum of pregnant rats, mice, and rabbits reach much higher levels. Serum AFP levels in pregnant rats (Sell and Skelly, 1976) are shown in Fig. 3. Similar results have been obtained in rabbits (Seppala and Seppalii, 1969).The high maternal serum levels of AFP may be due to high fetal and amniotic fluid levels in these species. In pregnant cows, the AFP levels in maternal serum are barely above the nonpregnant levels (Jalanko et [ i l . , 197811).This presuniably reflects the limited placental excllange of macromolecules in cows.

D. NEWBORNSERUM The average AFP level in the human cord blood at term is 50 pglnil. After birth, the level decreases at a rate corresponding to a half-life of 3-5 days during the first postnatal week, and a normal level of 4-25 ng/ml is attained at the age of 1-2 years of life (Masseyeff et d., 1975). Elevated values of AFP in newborn serum may have diagnostic significance (see Section IX). In rats there is a fd! of the serum AFP level immediately after the birth. This is thought to be d u e to the loss of the contribution of the yolk sac to the total AFP synthesis (Sellet al., 1974). This is followed b y an increase associated with proliferation of the liver cells. After that, a sustained decrease takes place until the nomial adult level is reached at the age of about 10 weeks (Sell et d., 1974; Masseyeffet LIZ., 1974). A similar situation is seen in mice, where the normal level is reached approximately at day 25 postpartum (Olsson et al., 1977). E. BIOLOGICAL HALF-LIFEOF AFP Disappearance of AFP after birth and tumor removal has been utilized to measure the half-life of AFP in humans. All observations

288

ERKKI RUOSLAHTI AND MARKKU SEPPALA

have given values of 4-6 days (SeppBlB and Ruoslahti, 1972a; Mukojimaet al., 1973).The half-life of AFP in mice and rats is shorter, about 24 hours (Olsson et ul., 1977; Sell, 1974). Determination of the rate of disappearance of AFP from serum after removal of an AFPproducing tumor may have prognostic significance (Mukojima et a1 ., 1973). F. NORMAL SERUM Trace amounts of AFP can be detected by radioimmunoassay in normal human serum, where the concentration ranges from 2 to 25 ng/ml (Masseyeff et ul., 1974; Ruoslahti and Seppala, 1971b; Purves et d . , 1973). This shows that the suppression of the AFP gene is not complete in human adults. Adult human AFP has been isolated and found to show characteristics similar to those of human fetal AFP (Ruoslahti et d., 1974b). However, studies by crossed immunoelectrophoresis (Ngrgaard-Pedersen and Axelsen, 1976) have revealed AFP inimunoreactivity in normal human serum showing gamma mobility instead of alpha mobility. A more recent analysis of this “gamma AFP” activity has suggested that it is an artifact (Nishi et al., 1978). The chemical nature of the normal human serum AFP is discussed in more detail below (Section V). In the adult mouse (Pihko and Ruoslahti, 1973),rat (Masseyeffet al., 1974; Sell and Gord, 1973), and rabbit (Ruoslahti et aZ., 1975), the serum AFP concentration is about 5-50 times higher than in adult human sera and can be readily detected in radioimmunoassay. Olsson et al. (1977) found a mouse strain (BALB/c/J)which had an AFP level in adult serum about 10 times higher than any other of the 26 strains tested. These included two other substrains of BALB/c. Kinetic studies showed that the rate at which AFP disappears from serum after birth is reduced in BALB/c/J mice as compared to other strains. The increased AFP level of BALB/c/J mice appeared to be due to an increased rate of synthesis of AFP, since the rate of catabolism of AFP was found to be normal in these mice. Genetic analysis performed by crossing BALB/c/J mice with mice having an ordinary AFP level (DBA/2),followed by determination of AFP levels in mice of the F, and F2generations, as well as in back-cross mice, indicated that the increased AFP level in BALB/c/J mice is controlled by a single recessive Mendelian gene. This conclusion was confirmed by inore recent experiments (Lindahl et d . , 1979) where the BALB/c/J mice were crossed with another low AFP strain (TF/Gn). The gene was named Raf (for regulation of a-fetoprotein). The Raf gene could be directly in-

GENETIC h A L Y S I S OF T H E

T.4BLE 11 HIGHSERU\I .4FP LEVELC.4USEO BY

THE

RAF

GESE I S

BALBIcIJ MICEa

Number of mice with

Lmv i\FP level

BALBlcIJ DBNB TFlGn F,(BALBlclJ x DBNB) BALBlcIJ x (BALB/c/J x DBA/2)F, (BALBIclJ x DBA/B)F, BALB/c/J x (BALBlclJ x TF/Gn)F, (BALBlclJ x TF/Gn)F,

High .4FP level

0

36

31 15 12

0

37 121 34 51

38 58 28

Ol,ser\wl ratio low : high

‘Espectrtl” ratio lo\v : high

0

0

15

1:1 2.1 : 1 1.2: 1 3.4 : 1

1:1 3:1 1:1 3 :1

” .4dapted from data b y

Olsson et ol. (1977) and Lindahl et ((1. (1979). Expected ratio of mice with low and high AFP levels assuming that the AFP level is controlled gene. b

I)?.

a single, recewive

\lendelian

290

ERKKI RUOSLAHTI AND MARKKU SEPPALA

volved in the regulation of AFP synthesis, but it may also control AFP levels only indirectly, e.g., by regulating the synthesis of a hormone that controls AFP synthesis. The BALB/c/J mutant may prove to be helpful in studies on the molecular basis for the regulation of AFP synthesis. The back-cross population, which is divided into two groups with low and high AFP levels, should be particularly useful in the elucidation of the in vivo effects, such as possible immunosuppression, of high AFP levels. Table I1 summarizes the results of Olsson et ul. (1977) and Lindahl et (11. (1978) on the genetic regulation of AFP. The rate of decline of' AFP in the serum of neonatal animals can be manipulated by hormonal treatments. Glucocorticoids and some other hormones given to newborn rats markedly reduce the serum AFP level but increase the serum albumin level (Belanger et d.,1975). Iron deficiency has the opposite effect. It seems that the expression of AFP is closely linked to the stage of differentiation of the liver. Expression of AFP has been found to correlate with the presence of other fetal markers in the liver (Taketa et al., 1976). Conversely, liver cells that express AFP lack markers of differentiation such as ligandin (Leffert et d.,1978) and the major urinary protein (Ruoslahti et ul., 1977; Sippel et d.,1976).

V. Purification and Chemical Properties of AFP

A. PURIFICATION The availability of purified AFP from several species has been the basis of the studies that have led to the present understanding of the characteristics and expression of this protein. The relative abundance of AFP in the fetus and in occasional tumor sera has made its purification a relatively simple task. Human AFP was first isolated from AFP-anti-AFP immunoprecipitates by gel filtration at low pH (Nishi, 1970; Ruoslahti and Seppiilii, 1971a). More recently, immunoadsorbents have been utilized to purify AFP in large quantities (Nishi and Hirai, 1972; Ruoslahti et d.,1974a). The use of low affinity antibody is essential for this procedure (Ruoslahti, 1976). A recent development in this area is the use of iminunochromatography on antibodies with very low affinity (Ruoslahti, 1978). This method makes use of the fact that AFP is present in all mamm a1'ian species (and in chicken) as iininunologically and chemically related, but not identical, molecules (Section 11,B).Antibodies against AFP of

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

291

one species have a lower affinity to AFP from another species than they have to the AFP used for immunization. Such cross-reactive antibodies can be utilized to prepare immunoadsorbents possessing a very low affinity. A major part of the AFP elutes from such columns with phosphate-buffered saline, and the rest can be eluted as a sharp peak with a small quantity of 4 or 6 M urea (Fig. 4). The elution of AFP with the initial column buffer has made it possible to prepare pure AFP which has not been subjected to the chaotropic elution buffers usually employed in affinity chromatography. The ease of elution of the bound AFP from the absorbent column has allowed purification of AFP in gram quantities. A different approach with similar results was utilized by Nishi et nZ. (1976) who first saturated an anti-human AFP immunoadsorbent column with human AFP from an AFP-containing sample. AFP in a similar sample, chromatographed on this presaturated colunii~,was retarded in its elution position relative to other proteins. Although the authors did not discuss the basis of this, it is likely that the presaturation occupied the high affinity antibodies leaving only the antibodies with the lowest affinity available for the subsequent fractionation. These would behave the same way as the cross-reactive antibodies used in the experiment shown in Fig. 4. Among the various chemical procedures that have been employed, isoelectric focusing (Ruoslahti and Seppala, 1971a; Sell et d.,1972),

ml of eluate FIG.4. Affinity chl.oniatography of AFP from fetal calf serum on ii colunin of v c ~ ylow ;iffinit), antil)odies (Ruoslahti, 1978). A goat anti-human AFP serum wiis absorl)ed wit11 I)ovint% ,4FP a s described in the test. The remaining aiitil)odies were coupled to Sepharose and used for the isolation of 1,oviiie AFP. A siibstaiitial fraction of Imvine AFP ( x-x) calutes with phosphate-bu~eredsaline well separated froln the iiiain protein peak (*---j. The AFP remaining 1)oiiiid to the c o l i i ~ ~ u eluted i iis a shaq) peak with 4 .\I urea.

292

ERKKI RUOSLAHTI AND MARKKU SEPPALA

affinity chromatography on Sepharose-linked concanavalin A (Con A) (Pagb, 1973),DEAE cellulose chromatography ( Parmelee et al., 1978), and removal of albumin by affinity chromatography on SepharoseBlue Dextran (Gold et al., 1978) appear to result in an appreciable enrichment of AFP and should be useful in conjunction with immunochemical or other procedures.

B. PHYSICAL AND CHEMICAL PROPERTIES OF AFP There are reasons to believe that AFPs obtained from different sources within the same species show reproducible differences in terms of the quantitative proportions of the microheterogeneous subfractions (see Section IV,D). However, the general characteristics of AFPs from various tissues are the same. Comparison between AFPs from the human fetus and liver cancer has revealed no major physicochemical differences. AFPs from these two sources are immunologically indistinguishable. Their tryptic digests are identical in peptide mapping (Fig. 5 ) (Ruoslahti et al., 1971),and their amino acid and carbohydrate compositions are similar (Hirai et al., 1973; Goussev et ul., 1971b; Ruoslahti et al., 1971). Analysis of the N-terminal (Aoyagi et d . , 1977; Ruoslahti et d . , 197411; Ruoslahti and Engvall, 1978) and C-terminal (Aoyagi et al., 1977) amino acid sequences has also shown that AFPs from these two sources are indistinguishable. AFP from yolk sac tumors has not been characterized beyond the demonstration that it is immunologically indistinguishable from fetal or hepatoma AFP (Fig. 6).

1. Alolecrrlar Weight utid Plz!l.siculProperties of AFP The main physical and chemical characteristics of AFP of various species are listed in Table 111. Early studies using gel filtration of AFP-containing serum showed that AFP has a molecular weight close to 70,000. Molecular weight analysis of purified AFP by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) shows that AFP is composed of a single polypeptide chain with a molecular weight slightly larger than that of albumin (Alpert et d., 1972; Pihko et al., 1973; Pihko and Ruoslahti, 1973; Ruoslahti and SeppLlB, 1971a, Watabe, 1974). Human AFP gives a slightly lower apparent molecular weight than rat or mouse AFP (Table 111) by SDS gel electrophoresis. Bovine AFP also has a lower mobility (=higher apparent molecular weight) than human AFP (Ruoslahti, unpublished). The various AFPs have not been sufficiently well characterized to allow any conclusions about the

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPXlENT

293

A

h

FIG.5. Comparison of' fetal ( A ) aiid liver c ~ i i c e r( B ) AFP 114'peptide mapping. The protein.; were d i g e ~ t e dwith trypsiti and the resulting peptides separated l)y chromatogr;qili!. (vertical) ;uid electrophoresis (horizontal), No significant differences itre f'i)untl. Reprodiiced from Hiroslahti t't t i / . (1971) with pcwnission from I t i f . .I.Ctrtrccr.

reason for this difference. Recent studies with improved separation of the SDS gel electrophoresis have revealed that rat AFP (Kerckaert et fil., 1977; Ruoslahti and Engvall, 1978) can be separated into two closely spaced lxinds with this method. This and other foiins of' microheterogeneity will be discussed in detail below. Using ultracentrifugation to determine the molecular weight of AFP, Nishi (1970) arrived at a molecular weight of 64,500 for hunian AFP. The S value obtained was 4.50. Similar molecular weight and a slightly higher S value have been found for rat AFP in one of the first

294

ERKKI RUOSLAHTI AND MARKKU SEPPALA

FIG.6. Comparison of iinmunological reactivities of human fetal (A), 1 iver cancer (B), and teratocarcinoma serum (C) AFP tested against a goat antiserum to fetal AFP.

studies on the chemical properties of AFP by Kirsch et nl. (1967).The significance of the slight discrepancy between the molecular weights obtained by SDS gel electrophoresis and ultracentrifugation is not clear, but probably reflects the difficulties generally encountered in the determination of molecular weights of glycoproteins. Purified AFP shows a tendency to aggregate similar to that of albumin. An additional band is seen in gel electrophoresis when purified preparations of human AFP are separated by electrophoresis under nondenaturing conditions. This band has a slower mobility than the main AFP band and has been shown to have a molecular weight of approximately 140,000. This evidence and the fact that this material is immunologically indistinguishable from the main AFP fraction indicates the material to be a dimer of AFP (Ruoslahti and Seppala, 1971a)

TABLE 111 PHYSICAL CHARACTERISTICS OF HUMAS,hfOUSE, h T

Characteristic Molecular weight -gel filtration, SDS gel electrophoresis -ultra centrifiigation Sedimentation constant (S20.w) Partial specific volume ( V 2 J DiEusion constant (D,o,w

)

Isoelectric point

E

1'% 1 CIII

(278 11111)

Human AFP

Ref."

hlonse AFP

69,00070,000

1

74,000 70,000

64,600

2

4.5

2

4.6

, AUD

Ref."

5 6

6.7

CHlCKES ~-FETOPROTEINS (AFPS)

Hat AFP

Ref."

70,000 77,000

68 9

64,800

10

4.4

10 9

4.5

0.726

2

6.18 4.7-4.8 (major) 5.2-5.3 (minor) 5.30

2 1,3 3,4 2

4.7

5,6

4.15

6

4.76 and 5.05 4.27

Chicken .4FP

Ref."

70.000

11

6 6

(1) Ruoslahti and Seppala, 1971a; (2) Nishi, 1970; (3)Alpert et ( I / . , 1972; (4) Parnielee ct ( I [ . , 1978; ( 5 ) Pihko and Rnoslahti. 197.3; (6) Watabe, 1974; (7) Attardi and Rrioslahti, 1976; ( 8 ) Sell et ( I / . , 1972; (9) Anssel et ol., 1973; (10) Kirsch et ( I [ . , 1967; (11) Lindgren c>t ( j / . , 1974. I'

296

ERKKI RUOSLAHTI A N D MARKKU SEPPALA

(Fig. 7). Even bands attributable to higher aggregates of AFP may appear. The aggregates are stable enough to allow fractionation by gel filtration and gel electrophoresis but disappear in the presence of SDS and 2-mercaptoethanol or reducing agent alone (Ruoslahti and Seppili, 1971a; Yachnin et al., 1977) and are, therefore, likely to result from disulfide exchange. The extent of AFP aggregation seems to depend on the number of manipulations such as freezing and thawing and lyophilization of the sample. It may affect the results obtained with some types of immunoassays of AFP. A similar aggregation phenomenon has been observed in the case of mouse and rabbit AFPs (Hassoux et nl., 1977; Pihko et al., 1973). Not much is known about the tertiary structure of AFP. One study is available on the circular dichroic spectra of rat AFP. AFP was found to have a high content (60%)of a-helix (Aubert et al., 1977). The corresponding value for albumin is 50-55% (see Peters, 1975).

2. Chemical Coniposition and Primary Structure The amino acid compositions of human (Nishi, 1970; Ruoslahti and Seppila, 1971a; Yachnin et d . , 1977), inurine (Watabe, 1974; Zimmerman et aZ., 1977), rat (Hirai et al., 1973; Watabe, 1974), dog (Hirai et al., 1973), rabbit (Pihko et id., 1973), porcine (Carlsson et al., 1976), bovine (Marti et al., 1976), ovine (Lai et al., 1977; Marti et al., 1976),

15-

E u) 0

10b-

-7

8 2

5

2

0

05-

q

+

-

Migration

-

FIG. 7. Polyacrylamide gel electrophoresis of himian AFP in Tris buffer showing the dinier Ixmd. The gel was scanned to quantitate the diiner (large arrow). About 1% ofthe total AFP was pwsent in this I~aiid.A higher aggregate, prolxibly a trinier, is barely disceniihle (small arrow).

a-FETOPROTEIN IN CANCER AND F E r A L DEVELOPMENT

297

and chicken (Lindgren, 1976) AFPs have been reported. They show significant resemblance to one another. The available data are compiled in Table IV. The amino acid composition of AFP also resembles the composition of albumin. Evaluation of this resemblance using the method of Marchalonis and Welbiiiin ( 1971) suggests that it is significant (Ruoslahti and Engvall, 1978). The number of accessible siilfliydry1 groups h a s been detemiined for rat AFP and was found to be 0.35 in native AFP and 0.8 in the presence of 8 hl urea (Grigorova et (/I., 1.977). H~i~iiaii AFP contains about 4% carbohydrate (Tiihle IV). The presence of N-~~cetylglucosamiiie and the absence of N-acetylgalactosaniine indicates the linkage of the carhohytlrate chains to asparagine. The variation observed in the carbohydrate portion of AFPs from different species is discussed below. The limited amino acid sequence data that are available have indicated that AFP is structurally related to serum albumin. Clearly significant homology (about 50%) between AFP and albumin was found in fragments obtained from human AFP by cleavage with cyanogen bromide (Ruoslahti and Terry, 1976). The N-terminal sequence analysis of human AFP indicates that it exists in at least two forms, one of which gives the sequence shown in Table V. The other seems to lack the first three amino acids (Ruoslahti et d.,19741,). Aoyagi et (11. (1977) confiiined this and found evidence for another type lacking only two of the first three amino acids. Similar heterogeneity of the N-terminus has also been found in mouse but not in rat AFP. The first four amino acids in mouse AFP are the same as in human AFP, but the rat sequence differs from human and mouse in two out of these four positions (Peters et nl., 1978, 1979). It seems possilde that the N-terminus of human and mouse AFP is being degraded by an aminopeptidase. Whether this has any physiological significance is not clear. It should be pointed out that Yachnin et al. (1977) did not observe heterogeneity in the N-terminal sequence of human AFP. Their results differed from the results of two other groups in the amino acids assignment at 2 out of the 17 positions (5 and 13). While the N-terminal sequence of AFP does not show much homology with albumin, there are some similarities. Albumin has a histidine residue which complexes with metals such as copper at position 3 (Peters, 1975). Human AFP has a histidine residue at position 4,and it also hinds copper (Aoyagi et nl., 1978). Furthermore, a peptide synthesized in our laboratory according to the 11 first amino acids in the Nteiininal sequence of human AFP has been shown to bind copper (Aoyagi, personal communication), It seems that apart from the presence

AMINO

Human AFP

Rabbit AFP

Rat AFP

Mouse AFP

Dog AFP

Porcine AFP

Bovine AFP

Ovine AFP

Chicken AFP

Bovine albumin

Human albumin

ASP Thr Ser Glu Pro Gly Ala cys Val Met Ile Leu Tyr Phe LYS His '4% Trp

42 38 33 92 25 35 50 28 31 6 33 58 18 28 49 17 20 2b

42 27 30 83 28 24 44 30 27 9 35 63 18 28 51 23 31

54 29 34 92 29 28 52 31 24 13 28 59 13 23 48 19 21 1

41 49 33 83 24 43 42 23 26 9 30

45 31 45 97 38 32 47 22 30 5 28 53 17 26 41 13 26 2

40 30 44 101 34 32 49 28 34 10 21 59 17 26 36 12 28 ND

49 31 32 91 32 33 40 24 35 5 34 53 16 23 40 15 26 ND

57 37 49 87 36 43 43 12 47 5 26 55 16 26 36 14 22 ND

59 30 40 95 29 37 40 33 32 6 48 14 23 43 17 24 24 ND

54 29 23 83 25 12 63 35 39 6 60 18 30 58 16 22 22 1

53 34 28 78 28 15 46 35 36 4 61 19 26 59 17 23 23 2

Sugar Mannose Galactose N-acetylglucosaniine Sialic acid

3 3 5 2 -3

Amino acid

g Q'

TABLE IV ACID AND CARBOHYDRATE COhlPOSITIONS OF AFPS AND ALBUMINSFROM DIFFERENTSPECIES"

ND

62 10 26 48 17 23

3

6 6 8 4 -6

'I Nnmber of residues per molecule (calculated assuming a MW of 67,000 for the peptide part of AFP). For amino acid compositions of human AFP, see Ruoslahti and Seppala, 1971a; rabbit AFP, Pihko et al., 1973; rat AFP, Watabe, 1974; mouse AFP, Zimmerman et al., 1976; dog AFP, Nishi et al., 1975; porcine AFP, Carlsson et a l . , 1976; bovine AFP, Marti et al., 1976; ovine AFP, Lai et al., 1977; chicken i\FP, Lindgren, 1976; for human and bovine albumin, Brown, 1976; for carbohydrate composition of human AFP (Ruoslahti and Seppala, 1971a; Ruoslahti and Engvall, 1978), and of rat AFP, (Kerckaert et ul., 1977). " Our iinpublislied results and Hirai e t a l . (1973). ND = not determined.

N-TERMINAL AMINO ACID SEQUENCES FROM

TABLE V HUMANAFP" AND

TWO OF

ITS

C N B r PEPTIDESb AND

COMPARISON W T H CORRESPONDING SEQUENCES FROM ALBUMIN' AFP S-tcnnin.rl

I

,5

S

T

D

A

I, I3

n

w s

Ii

S

E

V

Y

c,

10 I

.I

s

.A

I3

R

F

Ii

(' Ruoslahti rt u / . , 197411;Aoyagi

* Rrioslahti and Ten?, "

ef

15 . A " L D D Id G

s

Y

s

c

E

E

S

F

al.; 1977.

1976.

Brown, 1976. Yachnin ef (11. (1977) determined position 5 as serine and position 13 as isoleucine.

THEIR

300

ERKKI RUOSLAHTI AND MARKKU SEPPALA

of a histidine residue near the N-terminus, the N-terminal sequence of AFPs from those species analyzed, humail, mouse, rat, and bovine (E. Ruoslahti and J. Shively, unpublished), although clearly homologous, is also quite variable. This suggests rapid%evolutionin the Nterminus of AFP and may be the reason for the absence of the homology with albumin. Further sequence work is needed to clarify the relationship between AFP and albumin. The C-terminal amino acid of human (Aoyagi et al., 1977; Ruoslahti et ul., 1974a) and mouse AFP (Peters et ul., 1978) is valine. A sequence of the S C-terminal amino acids of human AFP has been pro1977), and it shows similarities with that of posed (Aoyagi et d., albumin. Further support for the structural similarity of AFP and albumin comes from the demonstration of immunological cross-reactivity between these two proteins (Ruoslahti and Engvall, 1976). AFP and albumin do not cross-react immunologically in their native form. However, unfolding of their polypeptide chains by reduction of the disulfide bonds followed by carboxamidomethylation produces derivatives which are cross-reactive. The sequence homology and the immunological cross-reactivity strongly indicate that AFP and albumin genes had a common ancestor. It is possible that their relationship is similar to that of fetal and adult hemoglobin. There are also some similarities between AFP and albumin as regards binding of various small molecular weight ligands (see Section VI).

3. Microheterogeneity of AFP Like most glycoproteins, AFP has variants with slightly different properties. Such microheterogeneity usually results from heterogeneity in the structure of the carbohydrate moiety (Clamp, 1975), e.g., incompleteness of carbohydrate side chains. In human, murine, and rat AFPs there is heterogeneity based on variation in the electrophoretic mobility, size, and the structure of the carbohydrate moiety (Alpeit et u l . , 1972; Smith and Kelleher, 1973; Zimmerman et ul., 1976; Kerckaert et ul., 1977; Ruoslahti and Engvall, 1978; Ruoslahti et nl., 197th). The microheterogeneity has been most extensively studied in the case of rat AFP. At least nine different forms of rat AFP exist. Fractionation of rat AFP on insolubilized concanavalin A results in a bound and unbound fraction (Smith and Kelleher, 1973).The bound fraction can be divided into a loosely bound and tightly bound fraction (Bayard and Kerckaert, 1977), and chromatography on Ricinu.9 cornniuiiis lectin allows even further subdivision based on variation in the carbohydrate moiety. Another type of microheterogeneity in rat AFP is

CI-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

301

demonstrable by polyacrylamide gel electrophoresis under nondenaturing conditions (Goussev and Yasova, 1970), where it appears as two bands. The same heterogeneity is also demonstrable in SDS gel electrophoresis (Kerckaert et ul., 1977; Ruoslahti and Engvall, 1978) indicating that it does not depend on charge differences since the separation in this system is almost exclusively due to size differences. The two electrophoretic variants have been isolated and found to be indistinguishable in amino acid and carbohydrate compositions (Kerckaert et ul., 1977). While Kerckaert et ul. (1977) found valine to be the C-terminal amino acid of both variants, Peters et al. (1979) found a C-terminal valine in the slow variant, and glycine in the fast one. Rat hepatomas seem to produce all the variants, but widely differing proportions of the Con A variants are found in AFPs from different tumors (Smith et ul., 1977). Individual clonal cell lines derived from rat hepatomas are able to synthesize at least four AFP variants, the electrophoretic and Con A ones (McMahon et ul., 1977) suggesting that a single cell may be capable of producing several AFP variants simultaneously. The electrophoretic and carbohydrate-derived heterogeneities of rat AFP are partly, but not completely, independent of one another. The Con A binding fraction is enriched in the slow electrophoretic variant (Belanger and Dufour, 1974).The molecular basis of the heterogeneity of the carbohydrate moiety in rat AFP with respect to binding to various lectins is not completely known. Recent results from our laboratory show that the Con A reactive and nonreactive variants of rat AFP differ in the composition of their sugar moiety. They have approximately the same total amount of carbohydrate, but the reactive variant has more mannose, while the nonreactive variant seems to contain more galactose (Table VI). The higher quantity of mannose in the reactive variant is in agreement with the known specificity of Con A for niannosyl (and glucosyl) residues. The reactivity of AFP with the Riciiius coiiimuiii,~lectin presumably depends on the amount of sialic acid present. This lectin reacts with galactosyl residues, and the reactive variant has been found to have less sialic acid. Sialic acid, when present, is linked to galactose and makes the galactose nonreactive (Kerckaert et ul., 1977). Several electrophoretic variants can be discerned in human AFP (Purves et al., 1970; Lester et al., 1976) and isoelectric focusing (Alpert et ( i l . , 1972; Alpert and Perencevich, 1975). Part of this heterogeneity persists after treatment of AFP with neuraminidase, indicating that it is not solely dependent on variation in the number of sialic acid

302

ERKKI RUOSLAHTI AND MARKKU SEPPALA

TABLE VI A N D REACTIVE CARBOHYDRATE COMPOSITION OF THE CON A NONREACTIVE FRACTIONS OF RAT AFP"

Sugar Galactose Mannose N -Acetylglucosam in e Sialic acid Total carbohydrate "

Con A nonreactive fraction (moles of sugar/ mole of protein)

Con A reactive fraction (moles of sugar/ mole of protein)

3.4 3.7 2.8 1.6 -

1.2 5.3 3.0 1.7 -

3.4%

3.4%

E. Engvall, M . Wrann, and E . Ruoslaliti, unpublished data.

residues. Parmelee et (11. (1978) have produced evidence that at least some of the observed microheterogeneity in human AFP is due to the presence of fintty acids tightly bound to the AFP molecule. They found that the major electrofocusing peak obtained with human AFP at pH 4.7 contains AFP with 2.4 moles of fatty acids bound to one mole of protein. The minor fraction at pH 5.3 was fatty acid free. Removal of fatty acids from the AFP focusing at pH 4.7 converted its isoelectric point to 5.3. The original isoelectric point could be restored by adding back fatty acids to the defatted AFP. The amount of certain electrophoretic variants in a given AFP preparation has been reported to correlate with the activity of the AFP as an immunosuppressive agent (Lester et d . , 1976). Developmental changes in the sialic acid content of AFP have been described in mice. AFP from early mouse embryos contains less sialic acid than in later stages of development (Zimmerman et al., 1976). Five different variants can be detected depending on the number of sialic acid residues present. Polyacrylamide gel electrophoresis of AFP secreted by mouse fetal liver and yolk sac indicated that the yolk sac was the principal source of the sialic acid microheterogeneity of AFP. This correlated with a lower sialyltransferase activity in the yolk sac tissue as compared to the fetal liver at the same stage of fetal development. Heterogeneity with respect to Con A binding has been present in all species we have examined (human, mouse, and bovine). While there is a large Con A nonbinding fraction in fetal bovine and mouse AFP (our unpublished results), human fetal (Page, 1973) or hepatoma (Gold et al., 1978) AFP is mainly of the Con A binding type. However,

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

303

a minor Con A nonreactive fraction is present in human AFP, and our recent studies (Ruoslahti et al., 19784 suggest that this fraction is greatly enriched in AFP of yolk sac derivation. We have recently noticed that AFP from human cord serum is divided by concanavalin A (Con A) binding into three fractions. These fractions are (1) nonreactive, (2) loosely bound, and ( 3 ) tightly bound on insolubilized Con A. About 2-3% of' AFP in cord seruiii does not bind to Con A, and this is a stable characteristic of the nonbound fraction. Upon refractionation, it is all nonbound, whereas the bound fractions show complete binding when eluted and refractionated. Amniotic fluid AFP shows a different distribution. Up to one-half of it is of Con A nonbinding type. Figure 8 shows a comparison of fetal seruni and

A

Volume of eluate

50

B

100

150

200 ml

Volume of eluate

FIG.8. AEnity chromatography on Con A-Sepharose. Second trimester human amniotic fluid ( A ) has inore Con A nonreactive AFP (har) than fetal serum (B). The arrows indicate the beginning of elution with 1 JI tu-~~iethyln~an~~osicle.

304

ERKKI RUOSLAHTI AND MARKKU SEPPALA

amniotic fluid AFPs when fractionated on Con A-Sepharose under comparable conditions. In early pregnancy, the Con A nonbinding fraction of human amniotic fluid AFP constitutes 20-40% of all AFP, whereas, in late pregnancy, almost all amniotic fluid AFP is of the Con A binding type. This difference is independent of sialic acid, since neuraminidase treatment of the samples does not affect the results. It seemed possible that the basis of the difference between AFP from amniotic fluid and fetal serum during the second trimester was due in part to a different origin of these AFP pools. We decided to test the hypothesis that AFP produced b y the yolk sac would be different from AFP produced by the liver. Several lines of evidence support this notion. We have tested three sera with elevated AFP from patients with teratocarcinoma. In this tumor, production of AFP occurs in yolk sac tissue which often forms part of such a tumor (see Section VIII). About 50% of the AFP in these sera was of the Con A nonreactive type, while the proportion of Con A nonreactive AFP in two liver cancer sera was much less. Similar results have been obtained in mice A

Froclion number

Froclion number

FIG. 9. Comparison of AFP isolated from 500 ml of normal human serum with imniunoadsorption (A) (Ruoslahti et al., 1974b) and purified fetal AFP ( B ) in electrofocusing. The norinal human serum AFP was determined by radioimmunoassay.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPkIENT

305

(E. Ruoslahti, in preparation). Mouse AFP during fetal development is about 50% Con A nonreactive. After birth, the proportion of the nonreactive variant drops precipitously until, at about 1week of age, most of the AFP is Con A reactive. Since the contribution ofthe yolk sac to the AFP production is lost at birth in mice (Section HI), this result is in agreement with the idea that the Con A nonreactive AFP would mainly originate in the yolk sac. One mouse teratocarcinoma that has been available to us was associated with serum AFP which was mainly Con A nonreactive. This is also in agreement with the hypothesis stated above. The significance of these findings is in two areas. It is possible that the diagnostic accuracy of the AFP test can be increased by assaying separately for the variants. In obstetrics, it may be possible to differentiate between AFP normally present in the amniotic fluid (large Con A nonreactive fraction) and AFP that has accumulated because of seepage from a fetal lesion such as spina bifida (see Section IX). It will I)e impoi-tant to compare AFP in a benign liver disease, such as cirrhosis, with AFP from liver cancer in terms of the microheterogeneity to establish whether the appearance of malignancy is associated with changes in the distribution of AFP in the subfractions. The significance of the microheterogeneous differences may extend beyond AFP. If this molecule can be glycosylated differently in different tissues, it is possible that other proteins behave similarly. There is evidence from studies on the carcinoembryonic antigen (CEA) that CEA and the various cross-reactive antigens present in normal tissues represent the same or very similar polypeptide chains glycosylated to a different degree (Kessler et d , , 1978; Engvall et al., 1978). 4. Chemical Nature of Normil Hunicm Serum AFP AFP has been isolated from large amounts of normal human serum in quantities sufficient to allow its partial characterization (Ruoslahri et d . , 1974b). A 2000-fold concentration of normal human serum AFP was achieved in a single step of immunoadsorption. About 1pg of AFP was obtained, and this was enough for partial characterization of the isolated AFP b y immunodiffusion, radioimmunoassay inhibition curves, and isoelectric focusing (Fig. 9). No differences could be detected between this AFP and AFP in fetal serum. VI. Biological Properties of AFP

The biological role of AFP is far from clear. The proposed functions of AFP include the binding of steroids and other substances and immunoregulatory effects.

306

ERKKI RUOSLAHTI AND MARKKU SEPPALA

A. BINDINGOF ESTROGENS AND OTHER SUBSTANCES

The estrogen binding property of rat and mouse AFP was first suggested by experiments which revealed the presence of an estrogen binding component in fetal and pregnancy sera of these species (Nunez et aZ., 1971; Soloff et al., 1971). Subsequent studies showed that this component was AFP (Uriel et al., 1972). Similarly, the socalled fetoneonatal estradiol binding protein in mouse brain cytosol has been identified as AFP (Attardi and Ruoslahti, 1976). The binding of estrogens to rat and mouse AFP is strong and specific. The association constants for rat AFP are on the order of 1 x lo8M-' for estradiol with a pH optimum of 5.5-6.0 (Aussel and Masseyeff, 1977; Benassayag et ul., 1975; Raynaud et al., 1971). In the rat, AFP from hepatoma and from the fetus have been reported to show two types of binding sites with different affinities for estradiol (Benassayag et ul., 1975; Vallette et d.,1977). Others have not found more than one type of binding site (Aussel and Masseyeff, 1977). Some laboratories report the presence of a single binding site for estradiol per molecule of rat AFP (Aussel and Masseyeff, 1977; Soloff et al., 1976) and find no difference between the microheterogeneous subfractions, while another laboratory finds the slower electrophoretic variant to be more active than the fast one (Benassayag et al., 1975). Further work will be needed to resolve these discrepancies. While the estrogen binding properties of rat and mouse AFP are well established, reports on estrogen binding by human AFP are conflicting. Most workers would seem to agree that when equilibrium dialysis and density gradient centrifugation are utilized, only rat and mouse AFPs show high affinity binding sites for estrogens. According to most studies, human, guinea pig, hamster, bovine, rabbit, and chicken AFPs are devoid of any demonstrable estrogen binding activity (B. Attardi, E. Ruoslahti, unpublished observations; Nunez et al., 1976; Savu et al., 1974; Schwartz and Soloff, 1875). However, a small fraction of AFP from human hepatoina and cord serum has been reported to bind to estradiol-Sepharose columns (Anion et d.,1973; Uriel et d.,1975). Mouse AFP is completely bound to estradiolSepharose columns (Hassoux et nl., 1977). While the binding of mouse AFP to estradiol-Sepharose was confirmed in the author's laboratory, no evidence of binding of a small fraction of human AFP to such columns was observed (E. Ruoslahti, unpublished). It thus seems that any quantitatively significant binding of estrogens to AFP is limited to some of the rodent species. The biological significance of this phenomenon is not known. Rat AFP has been shown to inhibit estrogen metabolism (Aussel and Masseyeff,

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

307

1976) and it has been suggested that in rodents the fetoneonatal estradiol binding protein, now known to be AFP, may protect the fetal brain against estrogen-induced sexual maturation (McEwen et ul., 1975). Two types of estrogen binding have been demonstrated in the cytosols from the brain tissue of newborn mice and in the uteri of immature rats. One binder is in an 8S, and the other, in 4-5s fraction. The 4-5s binder has been identified as AFP (Attardi and Ruoslahti, 1976; Uriel et al., 1976). The 4-5s binder diminishes with increasing age, whereas the 8 s protein increases. It has been reported that AFP is also a component of the 8 s receptor in the uterus of immature rats (Uriel et al., 1976), but this has been contested by others (Attardi and Ruoslahti, 1977; Radanyi et al., 1977). Human AFP binds substances other than estrogens, such as various dyes (Endo et al., 1974) and copper (Aoyagi et al., 1978). Parmelee et al. (1978) have found that fatty acids are bound to human AFP, and recent studies by our group show that bovine AFP binds bilirubin (Ruoslahti et al., 1978b). The spectral properties of bilirubin are altered in the presence of albumin and bovine AFP (Fig. 10). While albumin causes a shift of the absorption maximum toward a higher wavelength, AFP gives a change to the opposite direction. Both increase the absorption by bilirubin. These changes have made it possible to study the characteristics of the interaction between AFP and bilirubin. The results indicate that AFP has one binding site with K , about one order of magnitude lower than that of albumin. Whether the binding of bilirubin by AFP has biological significance remains to be

350

400

450

500

550

X(nm) FIG. 10. Shift of the absorption maximum of bilirubin ( 0 4 )by human albumin and bovine AFP (-0). Control proteins such as ovalbumin do not alter the absorption spectrum of bilirubin. An intermediate bilirubin spectrum is obtained in the presence of a mixture of all~uininand AFP ( 1 : 10) ( L m ) .

(A-A)

308

ERKKI RUOSLAHTI AND MARKKU SEPPALA

seen. Parmelee et u2. (1978) have shown that purified human AFT contains fatty acids. They found 2.4 moles of predominantly polyunsaturated fatty acids associated with 1mole of AFP. Similar fatty acids were found to be associated with albumin isolated from fetal sources, but in lesser amounts. The fatty acids found in fetal albumin and AFP differ from those associated with albumin isolated from adult serum. The latter are mainly saturated.

B. POSSIBLEIMMUNOREGULATORY ROLE OF AFP 1. Evidence that AFP I s Immuizosuppressive

The idea that AFP would be immunosuppressive originates with the report of Parmely and Hsu (1973) on the immunosuppressive properties of serum fractions rich in AFP. Murgita and Tomasi (1975a,b) subsequently demonstrated in convincing studies that purified mouse AFP is immunosuppressive. They assayed the effect of AFP purified from amniotic fluid, as well as of other proteins, on antibody response of spleen cells in vitro. AFP and amniotic fluid strongly suppressed the plaque-forming cell response to sheep red blood cells in such cultures. Primary IgM and secondary IgM, IgA, and IgG responses were all inhibited. The IgG and IgA responses were suppressed to a higher degree than the IgM responses. The suppression of the IgG and IgA responses was complete at AFP concentration of 200 pg/ml. It was found that AFP must be added at the initiation of antigenstimulated spleen cell cultures for maximal inhibition of primary antibody synthesis to occur. These workers (Murgita and Tomasi, 1975b) further reported that mouse AFP suppresses the mitogenic effects of phytohemagglutinin, concanavalin A, and lipopolysaccharide on mouse spleen cells and inhibits allogeneic lymphocyte stimulation in the one-way mixed lymphocyte reaction. While mouse AFP suppresses the specific antibody response to sheep red blood cells, which is a T-cell-dependent antigen, and inhibits DNA synthesis initiated with phytohemagglutinin and concanavalin A in purified T lymphocytes, it does not inhibit the T-cell-independent antibody response to dinitrophenyl-substituted Ficoll or the lipopolysaccharide-stimulated polyclonal B-cell antibody synthesis (Murgita and Wigzell, 1976). These results suggest that mouse AFP suppresses certain T-lymphocyte functions in vitro but does not directly inhibit B-cell functions. This suppression may be through induction of suppressor T cells with capacity to inhibit helper T cells (Murgita et d.,1977). Alpert et nl. (1978) obtained evidence for a similar stimulation of suppressor cells b y human AFP.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

309

Dattwyler and Tomasi (1975)reported suppression of generation of killer cells to a tumor cell line by as little as 1 ngml of mouse AFP. Using immunofluorescence, Dattwyler et al. (1975) found binding of AFP onto the surface of T cells, and Keller and Tomasi (1976) demonstrated, also with immunofluorescence, cell surface AFP on lymphocytes undergoing graft-vs-host reactions. They also found that such lymphocytes synthesized AFP. The lymphocytes having surface AFP responded poorly to mitogenic stimulation. Immunosuppressive activities have also been reported to be associated with human AFP (Yachnin, 1976; Lester et uZ., 1976). These workers found AFP purified from fetuses more active than AFP derived from adult liver cancer. A 1000-fold variation was found in the potency of different AFP preparations and this seemed to correlate with the ratios of electrophoretic variants in these preparations. The more active fetal preparations had a higher proportion of an acidic variant (Lester et al., 1976). Alpert et d. (1978) also found that AFP from fetal and liver cancer sera suppressed mitogenic and antigen-induced proliferative responses of lymphocytes but did not find them different. They considered the effects pharmacological rather than physiological. The immunoregulatory function of AFP would have potentially important implications in the maternal-fetal relationship, in the immune capabilities of the fetus and newborn, and in malignant and nonnialignant diseases where AFP is elevated. However, other workers have not found AFP consistently immunosuppressive.

2 . Evideiice that AFP I s Not lninzunosuppressive Sheppard et nl. (1977) and Sell et (11. (1977) in extensive studies on the effect of mouse and rat AFP on immune responses could not confirm the findings reported by Murgita, Tomasi, and co-workers. While they did find inhibitory effects, some in vitro systems were unaffected, and others stimulated by purified mouse AFP. Secondary in vitro plaque-forming cell responses which were completely inhibited in the experiments of Murgita and Tomasi (1975a) were inhibited 2- to 4-fold by AFP in the study of Sheppard et (11. The induction of cytotoxic effector cells was stimulated by AFP. Using sera from tumor-bearing mice and rats which had different amounts of AFP, some as much as 5 mghnl, Sheppard et ( 1 1 . and Sell et nl. found no correlation between suppressive potential of a serum and its AFP content. Normal sera (AFP concentration 10-100 ng/ml) were as suppressive as the tumor sera with the highest AFP levels. This group found no evidence for uptake of AFP by spleen cells, nor did they find active synthesis of AFP in mixed lymphocyte culture. They also showed that

310

ERKKI HUOSLAHTI AND MARKKU SEPPALA

the conditions used by Murgita and Tomasi for mitogenic stimulation with Con A were not optimal for the mitogenic response but did give a higher suppression by AFP than was obtained under conditions optimal for mitogen stimulation. Others, working with human AFP, while able to show some suppressive effects in tests such as the phytohemagglutinin stimulation, have concluded that these effects are likely to be of no (Alpert et ul., 1978; Littman et ul., 1977; Charpentier et al., 1977), or doubtful (Gupta and Good, 1977) biological significance. The latter authors found an increase in the percentage of lymphocytes forming T rosettes in AFP-treated cultures, while Alpert et al. did not find any effect by AFP on rosetting. Baumgarten (1976) found no effect by AFPcontaining fluids on the formation of T rosettes. Several laboratories have observed a mitogenic effect on lymphocytes by AFP (Charpentier et ul., 1977; Littman et al., 1977; Sheppard et al., 1977). AFP has also been found to be mitogenic to fibroblasts (Vaheri et ul., 1973). 3. Are the Differences Reconcilable? There seem to be several possible ways of reconciling these discrepant results from different laboratories. Some of the claims about the immunoregulatory properties of AFP seem rather extreme. It is difficult to see how 1 ng/ml of mouse AFP could inhibit the induction of cytotoxic effector cells as has been reported (Dattwyler and Tomasi, 1975). This is 100 times less than the serum concentration of AFP in normal adult mice (Pihko and Ruoslahti, 1973). Also, the tests were apparently carried out in the presence of 5% fetal calf serum. Fetal calf serum contains about 1 mg/ml of bovine AFP (Jalanko et al., 1978b). The immunosuppressive properties of AFP would have to have a remarkable species specificity to allow detection of the effect on mouse cells by 1 ng/ml of mouse AFP in the presence of a 50,000-fold excess of bovine AFP. The presence of bovine AFP in fetal calf serum also complicates the interpretation of the original work on the immunosuppressive effects of mouse AFP (Murgita and Tomasi, 1975a,b; Murgita, 1976). It should be pointed out that more recent work has been carried out using lymphocytes cultured in homologous adult serum (Yachnin, 1976; Peck et al., 1978), and this has not affected the detection of immunosuppressive effects by added AFP. An obvious question is the purity of the AFP preparations used. Could the observed effects be due to the presence of macromolecules other than AFP? Immunosuppressive materials active in the microgram range have been reported to be present in serum (e.g., Chisari, 1977), and Parmely and Thompson (1976) have emphasized the fact

a-FETOPROTEIN

IN CANCER AND FETAL DEVELOPhfENT

31 1

that, while partially purified AFP preparations may have immunosuppressive effects (Parmely and Hsu, 1973), they disappear in further purification of AFP. It should also be pointed out that part of the reported work on the properties of AFP has been done using amniotic fluid, not AFP (e.g., Murgita et ul., 1977). However, some of the studies demonstrating immunosuppressive activity in human AFP (Yachnin, 1976; Lester et d.,1976) have been carried out using AFP which has been extensively characterized (Yachnin et al., 1977). This tends to make it unlikely that the effects seen would be caused by macromolecular contaminants. The effects of AFP on immune functions have recently been found to be more selective than was initially thought. It was already pointed out that T cells seem to be mainly affected, and that the IgG and IgA responses seem to be more sensitive than the IgM responses to suppression by AFP. Peck et ul. (1978) have, quite recently, found a remarkable restriction of the suppressive effect of AFP to the mixed lymphocyte culture responses caused by the I region of the main histocompatibility locus, while reactions caused by the K and D regions were unaffected. These results make it possible that some of the discrepancies between results from different laboratories could be due to differences in the test systems employed. However, the amount of suppression observed in these experiments was only to about 50% of the control level, and no other protein was tested to exclude nonspecific effects of added protein. Also, these results do not explain the divergent observations with respect to inhibition of lymphocyte responses to mitogens. Two groups have reported restriction of the immunosuppressive effects to certain microheterogeneous subfractions of AFP (Lester et ul., 1976; Zimmerman et al., 1977).The latter group found that the activity of mouse AFP was associated with sialylated AFP. Since it is known that the mouse yolk sac produces AFP which is incompletely sialylated compared to AFP produced by the liver (Zimmerman and Wilson, 1976), it is obvious that the source of AFP could greatly influence the results obtained. This has been found to be the case. AFP from fetal sources is more active than liver cancer AFP (Lester et u l . , 1976; Yachnin and Lester, 1976, 1977). However, the suppressive effects observed by Zimmerman et al. were only marginally significant, and Yachnin and co-workers, using human AFP, found that desialylation did not affect the immunosuppressive activity and attributed the differences in activity to the presence of electrophoretic subfractions independent of sialic acid. While this is an attractive explanation for the observed differences in the potency of different AFP preparations, it is

3 12

ERKKI RUOSLAHTI AND MARKKU SEPPALA

not entirely satisfactory. The immunosuppressive activities varied within three orders of magnitude, while the differences between the amount of the presumably active subcomponent were 5-fold or less. It is well established that mouse and rat AFP bind estrogens with a high affinity (Nunez et al., 1976), and recent results suggest that bovine AFP binds bilirubin (Ruoslahti et ul., 1978b). It has been reported that in order to be immunosuppressive, mouse AFP has to be associated with estrogen (Keller et ul., 1976). They found that AFP dialyzed in 0.5 M KC1 lost its immunosuppressive activity, and that this was restored if the dialysate or estradiol was added to the dialyzed AFP. Since AFP from species other than rats and mice does not seem to bind estrogen, it is unlikely that these AFPs would require estrogen. However, some other ligands, tenaciously bound to AFP, could be involved. This could explain the finding that different AFP preparations differ greatly in their immunosuppressive properties within a given laboratory and between laboratories. Those not using strong solvents in their purification procedure (Murgita and Toinasi, 1975a) would be more likely to retain the putative ligand than those who elute their AFP from immunoadsorbents with denaturing solvent. Some support for this possibility comes from the experiments of Auer and Kress (1977) who found that AFP eluted from an immunoadsorbent column by treatment at pH 10 was more active than AFP subsequently eluted by further treatment of the column with sodium thiocyanate. Anything bound to AFP would be more likely to remain associated with it in the milder eluting buffer. Goeken and Thompson (1977) reported immunosuppressive activity in AFP as well as in albumin that was isolated from human cord serum, while albumin isolated from adult serum was inactive. This, again, raises the possibility of a ligand bound to AFP and albumin. This may be the reason why others find fetal AFP more active in immunosuppression than liver cancer AFP (Lester et al., 1976). The presence of different bound ligands in fetal and adult AFP could also explain the differences in the electrophoretic microheterogeneity observed between fetal and liver cancer AFP (Lester et al., 1976). Evidence to support this possibility has recently appeared. Parmelee et al. (1978) have shown that purified human AFP contains fatty acids. These fatty acids are mainly unsaturated and are also found associated with albumin isolated from fetal, but not adult sources. It would seem necessary to study such fatty acids and other substances that show affinity to AFP and albumin for their effects on in vitro immune functions.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

313

4. S ig t i ijic(i t ice of the P o s rilile I r i i m ii iio.rii / ) / I m s s i tie Eakct of AFP in Vivo

Although immunosuppressioii of' the mother by fetal AFP would offer a convenient explanation for the survival of the fetus as ail allograft, there is no direct evidence that AFP woukl be immunosuppressive in vivo. Evidence to the contrary has been presented. Mice bearing AFP-producing tumors and having AFP levels in the range of 10-100 p g h l mount a normal direct plaque-fomiing cell response to sheep red blood cells and produce 2-mercaptoethanol sensitive and resistant hemagglutinins in quantities similar to controls (Sheppard et d . , 1977). Rats injected with serum containing AFP mounted equally good responses to sheep red blood cells as rats that received AFP poor serum (Sell et d . , 1977; Bklanger et nl., 1976). No deficiencies were detected in the immune functions of five children with tyrosineinia-associated elevation of' serum AFP (Bklanger et ( i l . , 1976). It seems that we will be left with confusion and controversy as regards the immunosuppressive properties of AFP for some time to come. The authors' conclusion from the evidence summarized in the foregoing is that AFP does have demonstrable effects in various in vitro tests used to measure immune functions. These effects could be due to a ligand bound to AFP and found in different amounts in AFP preparations from different sources and purified by different procedures. There is at present no evidence that AFP is immunosuppressive in vivo. VII. AFP in Liver Disease

A. LIVERINJURY

The proliferation of hepatocytes in the adult liver is generally associated with AFP production. This has been demonstrated in animal experiments following hepatectomy and chemically induced liver necrosis (Abelev et al., 1963; Bakirov, 1968; de Nkchaud and Uriel, 1971; Engelhardt et d.,1976a,b; Perova et ul., 1971; Pihko and Ruoslahti, 1974; Sell et al., 1974). In humans, elevated serum AFP values are found in hepatitis and liver necrosis (Abelev, 1971; Ruoslahti et al., 1974a,c; Silver et al., 1974).The elevation associated with restitutive liver cell proliferation is transient and tends to occur during the regenerative phase after the elevation of the liver enzymes is subsiding (Ruoslahti et al., 1973, 1974c; Silver et al., 1974).

314

ERKKI RUOSLAHTI AND MARKKU SEPPALA

A correlation between the extent of liver regeneration and the serum AFP level has been observed in mice in toxic liver injury caused by CCl, and the high AFP levels following such injury. When the CCl, dose is increased, the resulting AFP response is smaller, perhaps because necrosis becomes too extensive for a maximal recovery to occur, and this leads to a decreased survival rate (Pihko and Ruoslahti, 1974). A similar situation has been found in galactosamine-induced liver damage of rats (Sell et al., 1976b), and AFP levels seem to give an indication of prognosis also in fsllminant human hepatitis (Karvountzis and Redecker, 1974; Ruoslahti et al., 1974~). However, Bloomer et al. (1975) think that the AFP level correlates with the length of survival rather than the final outcome in such cases. Although the correlation between liver cell proliferation and AFP production seems fairly straightforward in the light of the foregoing, there are some unexplained observations relating to this subject. The increase of serum AFP level is much more pronounced after toxic liver injury than after partial hepatectomy, and partial hepatectomy in humans leads only rarely to an elevation of serum AFP (Alpert, 1972; Sell and Becker, 1978),while such elevations are more frequent in hepatitis (Abelev, 1971; Ruoslahti et al., 1973). Watanabe et ul. (1976) found that while the rates of DNA synthesis after partial hepatectomy and CC1,-induced injury in rats were similar, the AFP levels differed about 10-fold. Thus, other factors than the extent and rate of regeneration may be important in determining the resultant AFP production. The rate of elimination could also be altered in different situations. When rats undergo hepatectomy, as many as 90% of the hepatocytes may go into mitosis within 48 hours. Still, chemically induced liver necrosis in adult rats or mice brings about a greater AFP elevation than partial hepatectomy (Smuckler et al., 1976). In the rat, the magnitude of AFP response to partial hepatectomy is related to age; younger rats have a higher elevation than older rats (Sell et al., 1974). Engelhardt et al. (19764 studied the relationship between hepatocyte cell cycle and AFP synthesis by combined autoradiography to detect [3H]thymidine incorporation and immunofluorescence for cellular localization of AFP. They used regeneration of mouse liver following CC1,-induced necrosis for these studies. They concluded that AFP synthesis is associated with hepatocyte proliferation, but it can be induced in adult differentiated hepatocytes and is detectable prior to the onset of DNA synthesis. Phenobarbital injection is followed by a rise of serum AFP concentration 6 hours before liver cell proliferation (Smuckler et a1., 1976; Taketa et (11 ., 1973). The results cited above show that during liver regeneration, the

~-FETOPROTEININ CANCER AND FETAL DEVELOPMENT

315

AFP synthesis takes place in mature differentiated hepatocytes. This suggests that reelevation of AFP level in adults is caused by derepression of the AFP gene in adult cells. The possibility that an activation of a dormant embryonic cell population existing in adults would cause the resurgence of AFP production associated with liver injury has been entertained by many investigators, including the authors. The above results of Engelhardt et (11. seem to make the existence of such a population less likely.

B. HEPATOCARCINOGENESIS In analogy with embryonal sites of AFP synthesis, tumors arising in adult hepatocytes and those consisting of yolk sac elements elaborate AFP under experimental and natural conditions. The reappearance of AFP in mice with hepatomas was described as early as 1963 (Abelev et ul., 1963). Studies on the association of AFP production with experimental liver carcinogenesis have revealed a complicated relationship between AFP and the various phases of carcinogenesis. The increase of serum AFP during chemical carcinogenesis is biphasic. An early rise occurs within a few weeks of the coiiiinenceineilt of the exposure of the animals to a carcinogenic regimen. Small elevations are detectable within days or even hours after the treatment is initiated (Watabe, 1971; Kitagawaet d,, 1972; Kroes et d . ,1972, 1975; Fig. 11). While most hepatocarcinogens are also toxic to the liver, the early elevation of' AFP associated with exposure to carcinogens seems to have characteristics that set it apart from the elevations of AFP caused by pure liver cell proliferation. Such elevations have been observed even with subcarcinogenic, nontoxic doses of acetylaminofluorene (Becker and Sell, 1974) and in the absence of apparent liver injury in ethionine-treated rats (Watanabe et al., 1976b). The early elevation frequently occurs prior to any significant alteration in morphology and then subsides or achieves a plateau despite continued administration of a hepatocarcinogen. It exceeds in magnitude the elevation that is seen during the intense liver cell proliferation caused by partial hepatectomy (Becker et nl., 1975). Subsequent elevations of AFP may occur in the absence of any detectable malignancy if the administration of the carcinogen is continued. The final elevation of AFP is usually associated with the appearance of malignant cells and tends to be progressive with increasing size of the tumor and with metastatic spread. The same basic mechanism that causes the disproportionate elevation of AFP production during carcinogenesis may be behind the unexplained difference between the intensity of AFP production in

3 16

EHKKI RUOSLAHTI AND MARKKU SEPPALA

0.06% o-AT

\

3”

,

10

MONTHS

FIG. 11. Early rise at 1 to 3 months of semm AFP during chemical carcinogenesis with ortho-aminoazotoluene in three female C3H mice. The carcinogen treatment was started on day 0 and continued for 8.5 nionths. All three mice had a hepatocellular carcinoma at the end of the experiment ( € 3 . Jalanko, unpublished results).

CC1,-induced liver injury and hepatectomy in animals (Abelev, 1971; Watanabe et d., 1976a)and in hepatitis vs hepatectomy in humans (Sell and Becker, 1978). That such an elevation does not necessarily have to be associated with a subsequent development of liver cancer is shown by the fact that the initial rise occurs even with subcarcinogenic doses of 2-acetylaminofluorene (Becker and Sell, 1974). The significance of the early elevation of AFP to the subsequent development of malignancy is not clear. The correlation of the two phenomena is not absolute. No elevation of AFP occurs during the initial weeks of exposure to the potent hepatocarcinogen diethylnitrosamine (Sell and Becker, 1978). The tumors induced by this carcinogen usually are AFP producers. There seems to be a general agreement that so-called oval cells (Inaoka, 1967; Onok et aZ., 1975; Shinozuka et d., 1978; Kuhlmann, 1978) and possibly also transitional basophilic hepatocyte-like cells (Tchipyshevaet aZ., 1977)are responsible for the early rise. The nature of these cells is not completely known, but it seems that the oval cells originate from ductal epithelial cells and convert through transitional cells to mature hepatocytes and possibly to cancer cells. This is especially so in carcinogenesis induced by azo dyes. Hepatocarcinogenesis induced by other substances is not associated with so prominent ductal cell proliferation, and it has been suggested that altered hepato-

317

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPhfENT

cytes rather than epithelial cells produce AFP in this case (Farber, 1976).This is supported by the fact that during carcinogenesis induced by some carcinogens, increase in serum AFP occurs before any histological lesions (Becker and Sell, 1974). At later stages of carcinogenesis, the hyperplastic nodules produced b y 2-acetyl-aminofluorene have been found to be AFP-positive (Okita et d., 1974), but hyperplastic areas were found to be AFP-negative in a later study (Kuhlmann, 1978). An important question is whether elevation of AFP precedes the appearance of a tumor in spontaneous carcinogenesis. Becker et nl. (1977) studied this in a mouse strain with a high incidence of spontaneous hepatocellular carcinomas and found no general elevations of serum concentration of AFP with aging in these mice. Only mice with liver tumors had elevated AFP levels. Recent results by Jalanko et (11. (19784, while confirming the finding that no general elevation of AFP occurs with age in the liver of the cancer-prone mice, indicate that small elevations of AFP do occur during a short period preceding the appearance of a tumor in these mice. Premalignant lesions were present in the livers of 11 out of 16 mice that had elevated AFP but no detectable cancer, while, among the same mice, only 1 out of 14 mice with normal AFP had such alterations (Table VII). Interestingly, some of the mice that developed a liver cell cancer seemed to have a biphasic elevation of AFP reminiscent of the pattern seen during administration of carcinogen. TABLE VII

LIVERPATHOLOGY DURING

THE EAnLY ELEVATION OF AFP IN MICE WITH I500 26 23

3 4 1 1 1 -

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

\

HEPATITIS

321

11500

-c

1000

0,

C

500

1

A

2

3

4

week5

Cirrhosis

0

B 700

2

1 months

1

Liver Cancer

0

C

1

2

months

FIG. 12. Serum AFP (M and )aspartate transferase (0-0)in a patient with hepatitis ( A ) , alcoholic cirrhosis (B), and liver cancer (C) illustrating the transient elevation of AFP typical of hepatitis and cirrhosis and the sustained elevation associated with nialignancy. I n the cirrhosis case, the patient's alcohol intake ceased at the time point prior to the beginninx of the olxervation. The elevation of serum AFP coincided with marked clinical improvenient and presumalily reflected regeneration of the liver.

may prove to be an effective measure to prevent liver cancer (Larouzi. et al., 1977; Ziegler et n l . , 1978). Prolonged elevation of serum AFP concentrations in humans has been associated only diagnostically with tumor burden. The possible significance of elevated serum AFP concentrations in populations with presumptive exposure to hepatocarcinogens has not been fully explored. It has been reported that the serum AFP concentrations among individuals from areas of Africa with high incidence of priniary hepatocellular carcinomas were higher than the concentrations in

322

ERKKI RUOSLAHTI AND MARKKU SEPPALA

Caucasians (Elgoit et d . , 1972; Purves, 1976; Purves et al., 1973). However, the significance of these findings is not clear since the magnitude of hepatocarcinogenic exposure and the incidence of endemic hepatitis are likely to affect the AFP levels, and there may also be ethnic differences in the serum AFP concentrations. In a systematic study of men from several populations, Sizaret et al. (1975b)found no association between the apparent risk for hepatoma and elevated AFP levels, although a slight increase of the serum AFP concentration with age was noted. In another study (Masseyeff et id., 1974), however, the AFP levels were found to decrease slightly after 60 years of age. The low frequency of elevated AFP levels in individuals without a tumor (excepting pregnant women) brings up the possibility of mass screening using the AFP test. This is unlikely to be rewarding in the United States and in Europe where AFP-producing tumors are relatively uncommon. Williams et al. (1977) studied serum samples collected over a period of 30 years by repeated sampling of blood from a cohort of about 5000 individuals. A single elevated AFP was found in a sample collected 10 days before clinical diagnosis of gastric cancer. A survey of 500,000 individuals by the AFP assay in China where primary liver cancer is common, uncovered several new cases of primary liver cancer (Coordinating Group for the Research on Liver Cancer, 1974). It is interesting that in about 10% of these cases, the elevation of AFP was biphasic, a transient increase followed by a sustained increase concomitant with the appearance of clinical symptoms of liver cancer. As described above, this is similar to the dynamics of serum AFP observed in chemical carcinogenesis and in spontaneous liver carcinogenesis of mice. The screening program continues with apparent success in China. Tumors measuring less than 0.5 cm have been detected and removed (Nature, editorial, 1977). Cirrhosis, which is commonly regarded as a premalignant condition, is often associated with slightly elevated AFP values (Abelev, 1974; At least in some cases, this Lehmann, 1976; Ruoslahti et al., 1974~). seems to reflect ongoing liver regeneration rather than premalignant changes (Abelev, 1974; Ruoslahti et ul., 1974~). Tyrosinemia, which is also associated with elevated AFP levels, often leads to development of primary liver cancer in the affected individuals (Bhlanger et al., 1976). These and other (Okuda et al., 1975) results suggest that AFP can be a forerunner of overt malignancy also in spontaneous carcinogenesis. However, Lehmann (1976),in an extensive study on AFP in cirrhosis patients, found, during an observation period of up to 4 years, no liver cancers in 24 cirrhosis patients who had had transiently elevated AFP serum levels. In this study and in others (Okuda et al.,

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

323

1975), sustained abnormal AFP levels were associated with malignancy. Repeated testing of individuals at risk of developing liver cancer for AFP is, therefore, necessary and may reveal tumors still at a curable stage. VIII. Germ Cell Tumors

The association of elevated serum AFP levels with teratocarcinomas of the ovary and the testis was first observed by Abelev et al. (1967) and Masopust et al. (1968). Since then, this finding has been amply confirmed and extended. Embryologically differentiated forms of the multipotent embryonal carcinomas have been subdivided into three histological groups: (1) endodermal sinus tumor, (2) choriocarcinoma, and (3) teratoma. In later studies, the AFP synthesis by these tumors has turned out to be compatible with the histogenetic interpretation. AFP synthesis has been found to correlate with the presence of endodermal sinus tumor (yolk sac tumor), which is analogous to the AFP-producing fetal yolk sac in early embryogenesis (Gitlin and Perricelli, 1970). Some types of embryonal carcinoma and teratocarcinoma of the adult testis contain extraembryonic vitelline structures (Teilum, 1965) accounting for the AFP synthesis frequently seen in these tumors. The pure form of endodermal sinus tumor occurs most often in the ovary of young women and children and in the testis of children, but it is rare in the adult testis. Immunohistochemical studies have shown that AFP is localized in the yolk sac tumors (Kurman et al., 1977; Mawas et ul., 1969; Teilum et ul., 1974). The positive AFP reaction is found in the cells lining the endodennal sinuses and in the PAS-positive hyaline globules (Fig. 13). Similar localization is observed in mouse teratocarcinoma (Engelhardt et al., 1973). AFPproducing testicular tumors develop spontaneously in certain mouse strains and yolk sac tumors can be readily induced by implantation of embryonal tissue outside the uterus (Damjanov et al., 1977; Sakashita et al., 1977; Vandeputte et ul., 1973). Such tumors are becoming an important experimental model for studies on the control of AFP synthesis. In accordance with the immunohistochemical findings, it has been shown that undifferentiated embryonal carcinoma cells cloned from a mouse teratocarcinoma do not produce AFP, but their differentiation i n vitro to endodermal cells (Adamson et al., 1977) leads to AFP synthesis. In published reports, 36436% of patients with germ cell tumors show elevated serum AFP values (Bagshawe, 1978; Grigor et d . , 1977; Kohn et al., 1976; Kurman et al., 1977; Ngrgaard-Pedersenet al.,

324

ERKKI RUOSLAHTI AND MARKKU SEPPALA

c

.

n

FIG.13. Sections of an ovarian endodemial sinus tumor from a 17-year-old woman. (A) I~Ieiiiatoxylin-eosin stain. (B) Immunoperoxidase stain for AFP in an adjacent section. The epithelial cells forming the einlxyoicl bodies stain for AFP. (C) An imm~inoperoxidasestain for AFP of the next serial section stained with anti-AFP serum completely absorbed with purified AFP.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

325

C

".id

'E

.-

...

4

FIG.13C

1975; Talerman, 1977; Talerman and Haije, 1974; Tsuchida et al., 1975; Wahren and Edsmyr, 1974). In a recent study on 44 patients with malignant teratoma, Bagshawe ( 1978) reported elevated AFP levels in 86% of cases. Human chorionic gonadotropin (hCG) was found in 89% of these patients, and 84% had both elevated AFP and hCG. AFP alone was raised in 4% only, and neither of these markers was elevated in 4%. Elevated serum AFP values have been reported in occasional germ

cell tumor patients with no detectable yolk sac elements. However, considering the variety of histologic patterns seen in germ cell tumors, it is possible that the presence of yolk sac elements has been missed. Pure seminomas and teratomas are likely to be AFP-negative. In patients with yolk sac tumors, the serum AFP concentration reflects the activity of disease (Fig. 14). In several studies (Bagshawe, 1978; Lange et d., 1977; Ngrgaard-Pedersen et al., 1978; Scardino et d.,1977; A. Sell et nl., 1976), AFP has been found to be useful in the monitoring of treatment. After therapeutic intervention, the decline in the serum AFP level gives an indication of the conipleteness of the removal of the tumor. The rate of decline of the AFP level may also be important. The biological half-life of AFP is 4-6 days after surgical removal of tumor or after delivery. In cases in which tumor removal is not complete, a longer half-life can be expected. In the case shown in Fig. 14, the AFP

326

ERKKI RUOSLAHTI AND MARKKU SEPPALA

ENDODERMAL SINUS T U W R (EST) OF THE OVARY

&I1

Chemotherapy

1 1 1 1 \

FIG. 14. Kinetics of serum AFP in a patient with ovarian teratocarcinoina during therapy.

level decreased by 50% every 7-8 days, suggesting that the tumor was not completely removed by surgery. The value of half-life determinations is not established and warrants further study. Renewed elevation of AFP is an indication of tumor recurrence, and it often occurs before clinical evidence for a tumor. In the case shown in Fig. 14, chemotherapy was instituted merely on the basis of reelevation of AFP in a clinically symptomless patient. The AFP level normalized subsequent to chemotherapy, and the patient is still in remission. Sell et al. (1976) described a case in which both AFP and hCG were initially elevated and both markers decreased to normal after chemotherapy. When clinical recurrence appeared, the AFP level only was increased, whereas hCG remained negative until death. Discordance of AFP and hCG production was also described in testicular teratocarcinomas by Braunstein et al. (1973), and a similar phenomenon has been reported to occur in choriocarcinoma (Seppala et al., 1978; Searle et al., 1978), where the secretion of hCG and pregnancy-specific P-1-glycoprotein showed a discordant reappearance after chemotherapy. These cases exemplify the survival of a tumor cell population capable of synthesizing one marker only, possibly as a result of cloning of tumor cell populations during chemotherapy. They also illustrate the fact that tumors may produce multiple markers, all of which should be followed during and after treatment.

a-FETOPROTEIN IN CANCER

AND FETAL DEVELOPMENT

327

IX. Other Clinical Conditions with Informative AFP Levels

A. LJVER DISEASES Hereditury tyrosirzemiu is associated with raised serum AFP levels in the newborn (Bklanger et d . , 1973a; Dommergues et nl., 1972). This condition frequently leads to cirrhosis of the liver, and eventually liver cancer may develop. It has been proposed that the AFP test should be used as a discriminating test for hereditary tyrosinemia in iieoiiatal mass screening in high incidence areas (Grenier et al., 1976). The AFP test has also been proposed for use to discriminate between neonatnl hepatitis and hiliary utresiu (Zeltzer et d., 1974). Neonatal hepatitis is associated with higher values, but considerable overlap between the two groups has been observed (Andres et ul., 1977; Johnston et ul., 1976). Atuxia telungiectusia is another neonatal condition in which elevated serum AFP levels have been reported (Waldmann and McIntire, 1972). This may reflect retarded or arrested differentiation of the fetal hepatocytes. It has been argued that patients with cystic fibrosis and even their heterozygous parents could be revealed by the elevated AFP levels in serum. More recent studies (Ngrgaard-Pedersen, 1976) have failed to confirm this.

B. TROPHOBLASTIC DISEASE Trophoblastic diseuse, in which the fetus is absent, usually shows nonpregnant serum AFP levels, and it has been suggested that the test could be useful in differentiating between a molar and normal pregnancy (Seppala et ul., 1972). However, we have seen one case in which serum AFP concentration was markedly raised in a 16-week hydatidiform mole with no fetus. Grudzinskas et ul. (1977) have observed high AFP levels in molar vesicle fluid, and Ishiguro (1975) reported an elevated AFP level in a mole with coexistent macerated fetus. Nonetheless, elevated AFP levels do not usually occur in molar tissue, and patients with choriocarcinoma have consistently shown AFP levels within the nonpregnant range.

c. AFP IN THE DIAGNOSISOF FETALPATHOLOGY The AFP test has recently become important in the prenatal diagnosis of certain fetal defects and a number of other situations in which it

328

ERKKI RUOSLAHTI AND MARKKU S E P P A L A

is necessary to monitor the fetal condition. This aspect is briefly summarized here. The reader is also referred to recent reviews on this topic (Brock, 1977; Seppala, 1977; Kjessler and Johansson, 1977). Early work on immunodiffusion techniques predicted that AFP may become useful in the diagnosis of aberrant pregnancies. Tatarinov (1964) reported a positive AFP reaction in the serum of a patient with spontaneous abortion. Seppala et (11. (1967) described a positive AFP reaction in the amniotic fluid of a 36th-week pregnancy ending in birth of a child with congenital nephrosis. Amniotic fluid samples from normal pregnancies at this stage were AFP-negative when tested b y immunodiffusion. The first quantitative data on maternal serum AFP concentrations were published in 1972 (Seppala and Ruoslahti, 1972a), and it soon became evident that maternal AFP concentration increases in the case of intrauterine fetal death (Seppala and Ruoslahti, 1972b). The application of RIA technique enabled quantitative measurement of AFP in the near term amniotic fluid, and the elevation of amniotic fluid AFP level in congenital nephrosis was confirmed (Seppala and Ruoslahti, 1972~). In threatened abortion, where the outcome of pregnancy is uncertain, abnormally high or low maternal AFP levels were found to be an unfavorable prognostic sign (Seppalii and Ruoslahti, 1972d). In 1972, Brock and Sutcliffe published the classic paper describing elevated AFP levels in the amniotic fluid of pregnancies where the fetus had anencephaly or spina bifida. All these findings have now been confirmed and extended. Neural tube defects occur with varying incidence in various parts of the world. In the U.K., the average incidence is about 4.5 per thousand (Report of the U.K. Collaborative Study, 1977). In the United States, the highest reported incidence is 1.6 per 1000 for anencephaly (MacMahon and Yen, 1971). In Scandinavia, the estimated incidence is around 1 per 1000 or less. The recurrency rate after one affected child is about 5% in Britain, and 2-370 elsewhere in Europe if one affected child has been born. Open neural tube defects almost consistently show elevated amniotic fluid AFP concentrations in the second trimester, but, in closed defects, the levels may be normal or only slightly elevated (Brock, 1977).Estimation of amniotic fluid AFP level offers, therefore, a means of reliably identifying anencephaly and open spina bifida for selective termination of pregnancy in the second trimester. It is assumed that the abnormal AFP level in these cases is caused by leakage of AFP to amniotic fluid through the lesion. A similar situation seems to exist in all fetal conditions where elevated AFP levels are found. The abnormal values are caused by altered distribution of AFP in the different

(U-FE?'OPHOTEIN IN CANCER AND FETAL DEVELOPMENT

329

compartments rather than by changes in the production of AFP b y the fetus. Neural tube defects can also be initially recognized by assay of AFP in the maternal serum. The results of the U.K. Collaborative Study (1977) showed that 88% of cases of anencephaly and 79% of cases of open spina bifida had niaternal AFP concentrations at the level of 2.5 times tlie nonnal median or higher. Of unaffected pregnancies with one fetus, 3.3% had a value above this limit. The diagnosis in these groups is filmed up with AFP deterniinations performed on amniotic fluid and other diagnostic measures. The mutant curly-tail mice have a high incidence of spina bifida, and this is accoinpanied by ahno~inallyhigh AFP levels in the miniotic fluid (Adinolfi et (//,, 1976).This mutant may be a relevant model for tlie human disease. Coiigenitd iiephrosis is inherited as an autosomal recessive trait. The disease is resistant to any treatment and leads to death during early childhood. The incidence of congenital nephrosis shows wide variation. So far, about 200 cases have been reported. Half of these have come from Finland (Hallman et d.,1973), and the rest mainly from the United States, Italy, Switzerland, and Sweden. The early finding of raised aniniotic fluid AFP level in congenital nephrosis (Seppalii et ul., 1967) led to the anticipation that this elevation could be used for the prenatal diagnosis of congenital nephrosis in the second trimester.The amniotic fluid AFP level was assumed to become elevated through fetal proteinuria. This has now been shown in several studies (Kjessler et d . , 1975; Kjessler and Johansson, 1977; Milunsky et d . , 1977; Seppalii et al., 1976; Wiggelinkhuizen et ul., 1976). Figures 15 and 16 illustrate the results obtained with the AFP assay in diagnosis of congenital nephrosis. The authors have seen 24 women who had had a previous child with congenital nephrosis, and who sought genetic counseling in their subsequent pregnancy. Amniotic fluid AFP concentration was strikingly elevated in all eight cases where the fetus was subsequently found to be affected (Fig. 15).Normal amniotic fluid AFP concentration was associated with birth of a healthy child in 16 cases. Six of eight affected pregnancies also showed an elevated maternal AFP concentration (Fig. 16). The maternal serum AFP level probably becomes raised in congenital nephrosis as a consequence of elevated amniotic fluid AFP levels, and perhaps also because the placenta is abnormally large. Screening of all pregnancies by maternal serum AFP is now contemplated in several countries. Such screening would yield about 3000 cases with elevated values per 100,000 pregnancies. In a repeat serum

330

ERKKI RUOSLAHTI AND MARKKU SEPPALA

500 3

100

0

0

0 .

.

50

P915 0.1

P50 P2.5 15 20 25 30 WEEKS OF GESTATION

35

40

FIG.15. Amniotic fluid AFP levels and the outcome of pregnancy in women at risk of having another child with congenital nephrosis. The solid lines are 2.5, 50, and 97.5 percentiles of values in normal pregnancy. Data from previous publications have been included. ( 0 )congenital nephrosis; (0)healthy child. Broken lines connect points from multiple determinations i n the same case.

5000 8 0

1000.

A

12

16

,

0

,

20

,

,

24

,

,

20

,

,

32

,

,

36

,',

40

Weeks of pregnancy

FIG.16. Maternal serum AFP levels and the outcome ofpregnancy in wonien at risk of having another child with congenital nephrosis. The solid lines are 2.5, 50, and 97.5 percentiles of values in noimal pregnancy. Data from previous publications have been included. ( 0 )congenital nephrosis; (0)healthy child.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

33 1

sample, many of these cases would have a normal level, and careful elimination of erroneous dates and multiple pregnancies would further reduce the number of cases requiring amniocentesis (Kjessler and Johansson, 1977) to an estimated 500. Assuming that 80-90% of anencephaly and open spina bifida would have elevated maternal serum AFP levels (The Report of U.K. Collaborative Study, 1977), such screening would reveal at least 50 cases of neural tube defects per 100,000 pregnancies, even in a low incidence population. If congenital nephrosis occurs in the screened population, most of such cases would also be revealed. Aside from these benefits, a carefully conducted screening program would reveal a considerable proportion of twin pregnancies and erroneous dates (Kjessler et nl., 1977). Furthermore, recent reports (Brock et d., 1977; Wald et d.,1977) describe an increased frequency of low birth weight infants among the cases where the maternal AFP level was elevated in the second trimester, but the fetus had no neural tube defect. The knowledge gained by alerting the obstetrician to this problem could prove to be beneficial in terms of prevention of prematurity. An elevated value may also be a sign of imminent fetal death. Intrauterine fetal death in the second trimester is frequently associated with raised amniotic fluid and maternal serum AFP levels, and sometimes the level in maternal serum becomes elevated before fetal death (Seppala and Ruoslahti, 1973). Some other fetal conditions which may be detected because they can be associated with elevated AFP concentrations have been listed in Table IX.

TABLE IX MISCELLANEOUSMALFORMATIONSAND OTHER CONDITIONS WHICH CAN BE ASSOCIATED WITH HIGHAFP LEVELSIN MATERNAL SERUM AND/OR AMNIOTIC FLUID Condition

Reference

Exomphalos Meckel syndrome Sacrococcygeal teratoma Turner’s syndrome Esophageal atresia Hydrocephaly (occasionally) RH immunization Diabetes Multiple pregnancy

DeBruijn and Huisjes, 1975; Seppala, 1977 Seller, 1975; Aula et ul., 1977 Schrnid and Miihlethaler, 1975 Milunsky and Alpert, 1974 Seppila, 1975 Seppali and Uni.rus, 1974 SeppLla, 1975 SeppLlH, 1975 Garoff and Seppala, 1973; Kjessler and Johansson, 1977

332

ERKKI RUOSLAHTI AND MARKKU SEPPALA

These benefits will have to be weighed against the cost of such a program, and the small, but definite risk carried by amniocentesis. False positive results in amniocentesis would also result in loss of healthy fetuses. However, it is apparent that inadvertent fetal loss in such a program would be considerably smaller than the number of affected cases detected. What impact, if any, the utilization of the microheterogeneous variants of AFP (Section V,B) will have in the diagnostic accuracy of the AFP test will have to be determined. X. Tolerance to AFP and Its Abrogation

Transiently raised serum AFP levels in pregnancy and hepatitis have raised the question whether AFP is immunogenic. Autologous anti-AFP antibodies have not been found after the transient elevations of AFP occurring in pregnancies or in hepatitis (Colquhoun et al., 1974; Seppala and Ruoslahti, 1972a). There is a report describing the occurrence of autologous anti-AFP antibodies after hepatitis (FlorinChristensen and Arana, 1973), but no further cases have been reported. Immunization of animals with homologous fetal serum or purified AFP has not resulted in demonstrable antibody formation against AFP (Nishi et ul., 1972; Ruoslahti et a l . , 197421; Seppalii and Seppali, 1969).These results suggest tolerance to homologous AFP. This tolerance may be explained on the basis of high embryonal AFP levels, and it is apparently maintained by the small AFP levels present throughout life. Tolerance to autologous AFP can be terminated by immunization with heterologous AFP or chemically modified homologous AFP. Antibodies to human AFP produced in rabbits cross-react with honiologous (rabbit) AFP in immunodiflitsion (Nishi et al., 1972), and antibodies to mouse AFP induced in rats precipitate rat AFP (Goussev and Yasova, 1974). Sera of mice immunized with rat, human, or rabbit AFP show binding of radiolabeled mouse AFP (Ruoslahti et al., 1974a). Haptenation of rabbit AFP with 3-nitro-5-iodo4-hydroxy-phenyl-acetylazide (NIP) or BOC-(p-azohenezene arsenate)-L-tyrosine converts the homologous AFP to an innnunogen in rabbits (Ruoslahti et ul., 1975). That these antiliodies, reactive with homologous AFP, also react against autologous AFP, is shown by the fact that the immunized animals do not have measurable serum AFP (Ruoslahti et al., 1974a, 1975). Highest titers of' auto-anti-AFP have been obtained by immunization with the most cross-reactive AFPs. Absorption studies have demonstrated that all antibody elicited by these

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

333

immunizations is directed against the immunizing antigen, and the reaction with the animal’s own AFP is based on cross-reactivity (Pihko, 1976). These antibodies, therefore, are not autoantibodies in a strict sense of the word. XI AFP as a Possible Target Molecule for Tumor lmmunoprevention and Im m u not herapy

Attempts have been made to use both heterologous and homologous antibodies in inimunoprevention and immunotherapy of AFPproducing tumors. The report of Tsukadaet nl. (1974), confinned by others (Mizejewski et al., 1975), showed that heterologous antibodies to AFP inhibit the growth of hepatoma cells in uitro. This has encouraged attempts to find out whether nutologous antibodies against AFP would protect against tumor growth in vivo. A few reports on the effect of anti-AFP on transplanted hepatomas in vivo have been somewhat encouraging, but not conclusive (Goussev and Yazova, 1974; Mizejewski and Allen, 1974; Sell et ul., 1976a). More recently, promising preliminary results have been obtained in studies on inimunoprevention of carcinogeninduced liver tumors. Heterologous anti-AFP given to rats during the stage of early AFP elevation caused by the carcinogen treatment reduced the number of liver tumors that developed subsequently (H. Hirai, personal communication). In an extensive experiment, Engvall et crl. ( 1977) induced autologous anti-AFP in mice b y immunization with rat AFP and then inoculated the mice subcutaneously or intraperitoneally with a controlled number of cells of a syiigeneic AFP-producing hepatoma. A linear relationship between the level of serum AFP and tumor mass was found. The AFP levels of immunized mice were lower than those of control mice, but no protection against the transplanted liver carcinomas was obtained. Jalanko et (11. ( 197%) induced autologous antibodies to AFP in a group of C3H mice in a similar fashion. These antibodies reduced the level of normal serum AFP but had no effect on the incidence of spontaneous hepatomas. The sanie was found to be true of mice with chemically induced liver tumors (H. Jalanko, personal communication). The apparent lack of resistance of AFPimmunized mice to these tumors may be due to the fact that the autologous anti-AFP antibodies have a low affinity (Pihko, 1976). It is also possible that AFP is secreted too rapidly from malignant hepatocytes to allow killing by the anti-AFP antibody. Effects of anti-AFP on pregnancy have been equally elusive. De-

334

ERKKI RUOSLAHTI A N D MARKKU SEPPALA

generative changes have been reported to occur in the liver of fetuses of rabbits with autologous anti-AFP antibodies (Nishi et al., 1973). Goussev and Yazova (1974) demonstrated antibodies to rat AFP in rats immunized with mouse AFP. Natural tolerance could be broken even in experiments in which immunization was initiated 10 days after termination of a normal pregnancy where the animals had had high AFP levels. There was no difference in the immune response between females that had had litters and those with no previous pregnancies. Five of eighteen immunized rats became pregnant. All these pregnancies went normally to term, and apparently normal litters were born. Injections of heterologous antisera into pregnant animals have been argued to have an abortogenic effect (Slade, 1973),but this has not been confirmed (Leung et ul., 1977; S. Sell, personal communication). AFP provides one of the few instances where a well-characterized antigen and antibodies are available for studies of experimental tumor therapy. For this reason, new approaches, such as selective stimulation of cellular immunity to AFP or the use of anti-AFP as a carrier of antitumor agents should, perhaps, be explored. XII. Molecular Basis of Regulation of AFP Synthesis

The striking variations of AFP production that accompany differentiation and carcinogenesis indicate that effective mechanisms of turning the AFP synthesis on and off must exist. It also makes AFP an attractive model for studies on gene regulation. Next to nothing is known at present about the exact molecular nature of the events that are behind the expression of AFP production. Tumyan et al. (1975) reported the presence of a factor in extracts of adult mouse organs that, when injected into newborn mice, caused an accelerated rate of disappearance of AFP from their serum. They hypothesized that this could be a regulatory factor, but these studies have not been followed up by further characterization of the factors involved. Administration of various hormones, most notably glucocorticoids, to newborn animals have been shown to cause faster decline of their AFP levels (Bdangeret ul., 1975). The tissue extracts of Tumyan et ul. (1975) could have contained more of such hormones than the fetal extracts used as controls. The expression of AFP may be under the control of a single gene. Olsson et ul. (1977) identified a mouse strain which has a hereditary persistence of high AFP levels during adulthood. Genetic experiments have indicated that this characteristic is determined by a single 1978). How direct Mendelian gene (Olsson et al., 1977; Lindahl et d.,

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

335

the regulation of the AFP level by this gene is, remains to be seen. If it is direct (as opposed to being effected, e.g., through a homione), this mutant may prove helpful in the elucidation of the molecular basis for the regulation of the AFP gene. It is of interest that patients with hereditary lack of albumin do not seem to have a compensatory elevation of AFP (Irunberry et al., 1971). Several laboratories have, during the past couple of years, been tooling up to study the regulation of the AFP gene using methods of molecular biology. The messenger RNA for AFP has been isolated and a complementary DNA probe prepared (Iiinis and Miller, 1977). Studies utilizing this probe and earlier investigations on AFP synthesis directed by isolated messenger RNA from fetal and adult liver (Koga and Tamaoki, 1974; Iio and Tamaoki, 1976) have demonstrated that the control of AFP synthesis takes place at the transcriptional level. Messenger RNA from fetal liver, but not from adult liver, directs the synthesis of AFP, and this correlates with the absence of messenger RNA detectable with the complementary DNA probe in adult liver. XIII. Areas of Future Research and Concluding Remarks

AFP has a well-established position as a diagnostic tool in the diagnosis and follow-up of liver cancer and germ cell tumors. It is also extremely useful in the early prenatal diagnosis of fetal anomalies such as neural tube malformations and congenital nephrosis. While abnomial AFP values are not entirely specific for the situations mentioned above, the expression of AFP in other conditions causing ahnormal values is well understood and can be taken into account. AFP is a well-characterized protein, and its quantitation is on a firm basis. The main question on AFP that remains open concerns its function. A strong and specific binding of estrogens to mouse and rat AFP is firmly established, but its physiological significance is not clear. Since AFPs from species other than rat and mouse do not show similar estrogen binding, this is unlikely to be the main function of human AFP. The albumin-like chemical properties of AFP suggest that AFP may bind and transport other substances in the fetus. Some ligands that bind to AFP have been identified, but whether this has any physiological significance is not known. It may be possible to reconcile this with the reported immunosuppressive effects of AFP by studying the possible immunosuppressive activity of the ligands that bind to AFP. Another area where our knowledge is limited is the significance of AFP production during the early stages of chemical carcinogenesis. Understanding of this phenomenon could mean better understanding

336

ERKKI RUOSLAHTI AND MARKKU SEPPALA

of the changes that occur in gene regulation in general during carcinogenesis. Such studies would be greatly facilitated if permanent cell lines from normal liver cells were available. It will also be necessary to establish probes for the analysis of the expression of the AFP gene at the level of the genome. The elevation of AFP that is observed within days or weeks ofcommencement of carcinogen treatment in animals suggests that exposure of humans to environmental carcinogens could lead to a similar change. This might be useful in testing of certain occupational groups where such exposure is known to occur or is suspected. The time of AFP elevation in relation to the appearance of a malignant tumor in the liver is not clear. Studies cited above suggest that elevated AFP is a forerunner of an overt malignancy, and it often allows the detection of small liver cancers still at a curable stage. The main limitation in the use of AFP for the screening of at risk populations (and as panels of tumor markers become available, even larger populations) is the AFP production associated with liver regeneration. AFP is microheterogeneous, and there is some evidence that AFP from different sources may be different with regard to the relative amounts of the subfractions. It will be important to explore the possibility that AFP produced by malignant hepatocytes is different from that associated with the benign conditions causing elevated AFP levels. This emphasizes the need for more structural work on AFP. As a well-characterized protein, AFP provides an important model system in the studies of the phenomena associated with gene expression. Such studies are likely to further the general understanding of the expression of fetal proteins in cancer.

ACKNOWLEDGMENT We thank Miss Merijane Funiya for secretarial assistance.

REFERENCES Abelev, G. I. (1971).Ado. Cancer Res. 14, 295-358. Abelev, G. I. (1974). Transplant. Reu. 20,3-37. Abelev, G.I. (1979).Zti “Immunodiagnosis of Cancer” (R. Herberman and R. McIntire, eds.), pp. 76-101. Marcel Dekker, New York. Abelev, G. I., Perova, S. D., Khramkova, N . I., Postnikova, Z. A,, and Irlin, I. S. (1963). Trcrnsplantrrtion 1, 174-180. Abelev, G. I., Assecritova, I . V., Kraevsky, N . A,, Perova, S. D., and Perevodchikova, N. I. (1967). Znt. J. Cancer 2, 551-558. Adamson, E. D., Evans, M. J., and Magrane, G. G. (1977).E u r . J .Biochem. 79,607-615. Adinolfi, M., Beck, S., Embury, S., Polani, P. E., and Seller, M. J. (1976).J. Med. Genet. 13,511-513. Akai, S., and Kato, K. (1973).Gariii Monogr. Cancer Res. 14, 149-154.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPJlENT

337

Allan, L. D., Ferguson-Smith, hl. A., Donald, I.. Sweet, E . hl., and Gibson, A. A. 51. (1973). Loricet 2, 522-525. Alpert, E. (1972). Ntrtl. Coricer Z r i s t . A\foriogr.35, 415-420. Alpeit, E., and Perencevich, R. C. (1975).A t i t i . N.1.. Accrtl. S c i . 259. 131-135. Alpert, E., Hershberg, R., Schur, P. H., and Isselbacher, K. J. (1971). Gostroeriterolog!/ 61, 137-143. Alpert, E., Drysdale, J. W., Isselbacher, K. J., and Schiir, P. H. (1972).]. B i d . C/ietti. 247, 3792-3798. Alpert, E., Dienstag, J. L., SFpersky, S., Littman, B., and Rocklin, R. (1978). I t n n i u u o l . C O T T I 7, ~ I163-185. I. Andres, J. M., Lilly, J. R., Altman, R. P., Walker, W. A., and Alpert, E. (1977).J. Pedicitr-. 91,217-221. Aoyagi, Y., Ikenaka, T., and Ichida, F. (1977). Cniicer Res. 37, 3663-3667. Aoyagi, Y., Ikenaka, T., and Ichida, F. (1978). Cciticcr Rc.v. 38, 3483-3486. Arnon, R., Teicher, E., Bustin, M., and Sela, M. (1973). FEBS Lett. 32, 335-338. Attardi, B., and Ruoslahti, E. (1976). Nntttre (London) 263, 685-687. Attardi, B., and Riioslahti, E . (1977). Steroitfs 30, 711-716. Aubert, J. P., Kerckaert, J. P., Bayard, B., Loucheux-Lefebvre, M. H. (1977). FEBS Lett. 78, 194-198. Auer, I. O., and Kress, H. G. (1977). Cell. Itnrtiutiol. 30, 173-179. A da, P., Kajalainen, O., Rapola, J., Lindgren, J., and Sepp , M. (1977).Arn.J. O h t e t . Gytiecol. 129, 700-702. Aula, P., Rapola, J., Kajalainen, O., Lindgren, J., Hartikainen, A.-L., and Seppnli, hl. (1978).A m . J . Di.7. Child.,138, 984-987. Anssel, C., and hlasseyefl’, R. (1976). Biocliitnie 58, 737-741. Aussel, L., and Masseyeff, R. (1977). FEBS Lett. 81, 363-365. Anssel, C., Uriel, J., and Mercier-Bodard, C. (1973). Biochirnie 55, 1431-1437. Bagshawe, K. D. (1978). I n “Internationale Arbeitstagung iiber die Klinische Bedeutung der AFP-Diagnostik” (J. Schneider and H. Weitzel, eds.). Georg Thieme Verlag, Stuttgart, in press. Bakirov, R. D. (1968). Birr/!. Ek.vp. R i o l . ,\led. (USSR) 65, 45-47. Baumgarten, A. (1976). Clin. Initnunol. I,tiniu,ioptithol. 6, 42-46. Bayard, B., and Kerckaert, J. P. (1977). Biochem. B i o p h g s . Res. Comniuri. 77, 489-495. Becker, F. F., and Sell, S. (1974). Crrticei- Res. 34, 2489-2494. Becker, F. F., Klein, K. M., Wohnan, S. R., Asofsky, R., and Sell, S. (1973). Cuticer Res. 33, 3330-3338. Becker, F. F., Horland, A. A,, Shurgin, A., and Sell, S. (1975). Cancer Re.7. 35, 15101513. Becker, F. F., Stillman, D., and Sell, S. (1977). Cirricer Res. 37, 870-872. Bklanger, L., and Dufour, D. (1974). 111 “C. R. ConE Int. L’Alpha-foetoproteine” (R. Masseyeff, ed.), p. 25. INSERM, Paris. Belanger, L., Bklanger, M., Prive, L., Larochelle, J., Tremblay, M., and Aubin, G. (1973a). Pothml. R i d . 21, 449-455. Bklanger, L., Sylvestre, C., and Dufour, D. (1973b). Cliri. Chirn. Actn 48, 15-18. Bllanger, L., Hainel, D., Lachance, L., Dufour, D., Tremblay, M., and Gagnon, P. M. (1975). Noture (Loridon) 256, 657-659. Bklanger, L., Daguillard, F., Waithe, W. I., and Dufour, D. (1976). 191 “OncoDevelopmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 329-335. Academic Press, New York. Benassayag, C., Vallette, G., Cittanova, N., Nnnez, E., and Jayle, M. F. (1975). Biochirn. Biophys. Acta 412, 295-305.

338

ERKKI RUOSLAHTI AND MARKKU SEPPALA

Bergstrand, C. G., and Czar, B. (1956). Scand. J . Clin. Lab. Med. 8, 174. Bloomer, J. R., Waldmann, T. A., McIntire, K. R., and Klatskin, G. (1975). Castroenterology 68, 342-350. Branch, W. R. (1972).Int. J . Cancer 10, 451-457. Branch, W. R., and Wild, A. E. (1972).Z . Zellforsch. 135, 501-516. Braunstein, G. D., McIntire, K. R., and Waldmann, T. A. (1973).Cancer 31, 1065-1068. Brock, D. J. H. (1977).Eur. J. Clin.Ino. 7, 465-472. Brock, D. J. H., and Sutcliffe, R. G. (1972).Lancet 2, 197-199. Brock, D. J. H., Barron, L., Jelen, P., Watt, M., and Scrimgeour, J. B. (1977).Lancet 2, 267-268. Brown, J. R. (1976).Fed. Proc. 35, 2141-2144. Cahill, J., Cohen, H., and Starkovsky, N. (1974). Am. J . Obstet. Cynecol. 119, 10951100. Carlsson, R. N. K., Ingvarsson, B. I., and Karkson, B. W. (1976).Int.J. Biochem. 7, 13-20. Charpentier, B., Guttman, R. D., Shuster, J., and Gold, P. (1977). J. Immutzol. 119, 897-900. Chisari, F. V. (1977).J.Immunol. 119, 2129-2136. Clamp, J. R. (1975). I n “The Plasma Proteins 11” (F. W. Putnam, ed.), pp. 163-211. Academic Press, New York. Colquhoun, B., Gold, P., Barzell, W., Oakes, D. D., and Shuster, J. (1974).Cancer Res. 34,2296-2299. Coordinating Group for the Research on Liver Cancer (1974).The People’s Republic of China, pp..3-7. Danijanov, I., Skreb, N., and Sell, S. (1977).Int. J . Cnticer 19, 526-530. Dathvyler, R. J., and Tomasi, T. B. (1975).Int. J . Cancer 16, 942-945. Dathvyler, R. J., Murgita, R. A., and Tomasi, T. B., Jr. (1975).Nature (London) 256, 656-657. DeBruijn, H. W. A., and Huisjes, H. J . (1975).Lancet 1,525-526. de Nbchaud, B., Fromont, S., and Berges, J. (1977).Biochem. Biophys. Res. Commun. 79,789-795. de Nechaud, B., and Uriel, J. (1971). Znt. J . Cancer 8, 71-80. Dommergues, J. P., Odievre, M., Buffe, DI, and Nocton, F. (1972).Arch. Frcrnc. Ped. 29, 737-744. Elgort, D. A., Abelev, G. I., and O’Conor, G. T. (1972).Int. J . Cuncer 10, 331-337. Endo, Y., Kanai, K., Iino, S., and Oda, T. (1974). I n “C.R. Conf. Int. L’Alpha-foetoprot6ine” (R. Masseyeff, ed.), pp. 47-54. INSERM, Paris. Engelhardt, N. V., Shipova, L. J., Goussev, A. I., Yazova, A. K., and Ter-Grigorova, E. N. (1969).Riull. E k s p . B i d . Med. (USSR) 12, 62-64. Engelhardt, N. V., Goussev, A. I., Shipova, L. J., and Abelev, G. I. (1971).Int. J . Cancer 7, 198-206. Engelhardt, N. V., Poltoranina, V. S., and Yazova, A. K. (1973). I n t . J . Cancer 11, 448459. Engelhardt, N. V., Lazareva, M. N., Abelev, G. I., Uryvaeva, I. V., Factor, V. M., and Brodsky, V. Ya. (1976a).Nature (London) 263, 146-148. Engelhardt, N. V., Lazareva, M. N., Uryvaeva, I. V., Factor, V. M., Poltoranina, V. S., Gleiberman, A. S., Brodsky, V. Ya., and Abelev, G. I. (19761)). I n “OncoDevelopmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 533-539. Academic Press, New York. Engvall, E., and Perlmann, P. (1971). Imniuriochemi.stry 8, 871-874. Engvall, E., Pihko, H., Jalanko, H., and Ruoslahti, E. (1977).J.N a t l . Cancer Zn.Pt. 59, 277-280.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

339

Engvall, E., Shively, J., and Wrann, M. (1978).Proc. Nut/. Acud. Sci. USA 75, 16701674. Eraizer, T. L., Elgort, D. A., Abelev, G. I. (1977). Biul/.Eksp. B i d . Med. (USSR) 83, 711-713. Farber, E. (1976).I n “Liver Cell Cancer” ( H . M. Cameron, D. A. Linsell, and G. P. Warwick, eds.), Vol. 1, pp. 243-277. Elsevier, Amsterdam. Fisher, R. L., Sheuer, P. J., and Sherlock, S . (1976).Coricer 38, 901-905. Florin-Christensen, A,, and Arana, R. M. (1973).Br. Med. J. 2, 94-95. Garotf, L., and Sepplla, M. (1973).J . Ohstet. Gynuecol. Br. Commonw. 80, 695-700. Gitlin, D. (1975).Ann. N . Y . Acud. Sci. 259, 7-16. Gitlin, D., and Boesman, M. (1966).J.Clin. Znuest. 45, 1826-1838. Gitlin, D., and Boesman, M. (1967).Comp. Biochem. Physiol. 21, 327-336. Gitlin, D., and Gitlin, J. D. (1975).In “The Plasma Proteins” (F. W. Putnam, ed.), 2nd ed., Vol. 11, pp. 263-319. Academic Press, New York. Gitlin, D., and Perricelli, A. (1970).Nature (London)228, 995-997. Gitlin, D., Perricelli, A,, and Gitlin, G. M. (1972).Cancer Res. 32, 979-982. Gitlin, D., Perricelli, A., and Gitlin, J. D. (1973).C o m p . Biochem. Physiol. 46B, 207215. Coeken, N. E., and Thompson, J. S. (1977).J . Zmmunol. 119, 139-142. Gold, P., Labitan, A., Wong, H. C. C., Freedman, S. O., Krupey, J., and Shuster, J. (1978). Cuncer Res. 38, 6-12. Goussev, A. I., and Yazova, A. K. (1970).Biokhimin 35, 172-176. Goussev, A. I., and Yazova, A. K. (1974).I n “L,’Alpha-foetroproteine” (R. Masseyeff, ed.), pp. 255-270. INSERM, Paris. Goussev, A. I., Engelhardt, N. V., Masseyeff, R., Camain, R., and Basteris, B. (1971a). Znt. J . Cancer 7, 207-217. Goussev, A. I., Yazova, A. K., and Polyakova, E. V. (1971b). Onkologiyu 3,69-72. Grenier, A., Bklanger, L., and Laberge, C. (1976).Clin. C h m 22, ~ 1001-1004. Grigor, K. M., Detre, S. I., Kohn, J., and Neville, A. M. (1977).Br. J. Cuncer 35,52-58. Crigorova, A.-M., Cittanova, N., and Jayle, M.-F. (1977).Biochimie 59, 217-220. Grudzinskas, J. G., Kitau, M. J., and Clarke, P. C. (1977).Luncet 2, 1088. Gupta, S., and Good, R. A. (1977).J.Zrnmunol. 118, 405-408. Hallman, N., Norio, R., and Rapola, J. (1973).Nephron 11, 101-110. Hassoux, R., Berges, J., and Uriel, J. (1977).J.Steroid Biochern. 8, 127-132. Hevey, R., Bonacker, L. H., and Sparacio, R. (1976).I n “Immunoenzymatic Techniques, Proceedings of the First International Symposium on Immunoenzymatic Techniques, INSERM Symposium 2” (G. Feldinann rt [ I / . , eds.). North Holland P u l ~ lishing, Amsterdam. Hirai, H., Nishi, S., Watabe, H., and Tsukada, Y. (1973).Gann. Monogr. Cancer Res. 14, 19-34. Iio, T., and Tamaoki, T. (1976).Cun. J. Biochem. 54, 408-412. Inaoka, Y. (1967).G u n n 58, 355-366. Innis, M.A,, and Miller, D. L. (1977).J . Biol. Cheni.252, 8469-8475. Irunberry, J., Abbadi, M., Khati, B., Benabadji, M., and Rocha, E. (1971).Rev. E u r o ) ~ . Etudes C / i n . Biol. XVI, 372-379. Isaka, H., and Hirai, H. (1979).Scutid. J. Ztnniurio/., S z c l ~ p l .8, in press. Isaka, H., Umehara, S., Yoshii, H., Tsukada, Y., and Hirai, H. (1976).Gunn 67, 131-135. Ishiguro, T. (1975).Am. J. Ohstet. Gynecol. 121, 539-541. Jalanko, H., Virtanen, I., Engvall, E., and Ruoslahti, E. (1978a). Znt. J . Cuncer 21, 453-459. Jalanko, H., Engvall, E., and Ruoslahti, E. (197813).Znimunol. Cornmuti. 7, 209-222.

340

ERKKI RUOSLAHTI AND MARKKU SEPPALA

Johansson, S. G. O., Sherman, M. S., Hellsing, K., and Kjessler, B. (1974). Lancet 2, 839-84 1. Johnston, D. I., Mowat, A. P., Orr, H., and Kohn, J. (1976)-Actn Paedintr. Scatid. 65, 623-629. Jonsson, S., and Kronwall, G. (1974).Eur. J . Zmmunol. 4, 29-33. Karvountzis, G. G., and Redecker, A. G. (1974).Ann. Intern. Med. 80, 156-160. Kekomaki, M., Seppala, M., Enholm, C., Schwartz, A. L., and Raivio, K. (1971).I n t . J . Cancer 8, 250-258. Keller, R. H., and Tomasi, T. B., Jr. (1976).J.Exp. Med. 143, 1140-1153. Keller, R. H., Calvanico, N. J., and Tomasi, T. B., Jr. (1976).I n “Onco-Developmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 287-295. Academic Press, New York. Kerckaert, J. P., Bayard, B., Dehray, H., Sautiere, P., and Biserte, G. (1977).Riochim. B i o p h l s . Act“ 493, 293-303. Kessler, M. J., Shively, J. E., Pritchard, D. G., and Todd, C. W. (1978). Cancer Res. 38, 1041-1048. Kirsch, J. A. W., Wise, R. W., and Oliver, I. T. (1967).Biocheni. J . 102, 763-766. Kitagawa, T., Yokochi, T., and Sugano, H. (1972).Znt. J . Cnncer 10, 368-381. Kithier, K., and Poulik, M. D. (1972).Biochini. B i o p h y s . Acta 278, 505-516. Kjessler, B., and Johansson, S. G. 0. (eds.) (1977).Acta Obstet. Gynecol. Scand., S u p p l . 69. Kjessler, B., Johansson, S. G. O., Sherman, M., Gustavson, K.-H., and Hultquist, G. (1975).Luncet 1, 432-433. Kjessler, B., Johansson, S. G. O., Lidhjiirk, G., and Sherman, M. S. (1977).Actcr O h t e t . Gynecol. Scnnd. S u p p l . 69, 25-44. Koga, K., and Tamaoki, T. (1974).Biochemistq 13, 3024-3028. Kohn, J., Orr, A. H., McElwain, T. J., Bentall, M., and Peckham, M. J. (1976).Lancet 2, 433-436. Kroes, R., Williams, G. M., and Weisburger, J. H. (1972).Cancer Res. 32, 1526-1532. Kroes, R., Sontag, J. M., Sell, S., Williams, G. M., and Weisburger, J. H. (1975).Cancer R ~ s 35, . 1214-1217. Kuhlmann, W. D. (1978).I t i t . J . Cancer 21, 368-380. Kurman, R. J.. Scardino, P. T., McIntire, K. R., Waldmann, T. A., and Javadpour, N. (1977).Cancer 40, 2136-2151. h i , P. C. W., Hay, D. M., Peters, E. H., and Lorscheider, F. L. (1977).Riochim. Bioph!ys. Acts 493, 201-209. Lange, P. H., McIntire, K. R., Waldmann, T. A., Hakala, T. R., and Fraley, E. E. (1977).J. Urol. 118,593-596. Larouzi., B., Blumberg, B. S., London, W. T., Lustbader, E. D., Sankali., M., and Payet, M. (1977).J . Natl. Cuiacer Inst. 58, 1557-1561. Leek, A. E., and Chard, T. (1974).Laticet 1, 876. Leek, A. E., Kitau, M. J., and Chard, T. (1974).Lancet 2, 1088. Leffert, H. L., and Sell, S. (1974).J . Cell. Biol. 61, 823-829. Leffert, H. L., Moran, T., Sell, S., Skelly, H., Ibsen, K., Mueller, M., and Arias, I. (1978). Proc. N o t l . Acad. Sci. USA 75, 1834-1838. Lehmann, F.-G. (1976).In “Onco-Developmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 407-415. Academic Press, New York. Lester, E. P., Miller, J. B., and Yachnin, S. (1976). Proc. Natl. Acad. Sci. U S A 73, 4645-4648. Leung, C. C., Watahe, H., and Brent, R. L. (1977).Arner. J . Anat. 148, 457-462.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

341

Lindahl, G., Olsson, M.,and Ruoslahti, E. (1979).Scc1tid.J. I t i i n i u t i o l . , S u p p l . 8, i n press. Linder, E., and Seppali, M. (1968).Acta Puthol. Microbiol. Scond. 73, 565-571. Lindgren, J. (1976). In “Protides of the Biological Fluids, (H. Peeters, ed.), Vol. 24, pp. 277-279. Pergamon Press, Oxford. Lindgren, J., Vaheri, A., and Ruoslahti, E. (1974). Diferentiotion 2, 233-236. Littman, B. H., Alpert, E., and Rocklin, R. E. (1977).Cell. Inimuno/. 30, 35-42. MacMahon, B., and Yen, S. (1971).Loticet 1, 31-33. Marchalonis, J. J., and Weltman, J. K. (1971).Conip. Biochem. Physiol. 38B, 609-625. Marti, J., Aliau, S . , m d Moretti, J. (1976).Zti“Protidesofthe Biological Fluids” (H.Peeters, ed.), Vol. 24, pp. 259-262. Pergamon Press, Oxford. Masopust, J., Kithier, K., Radl, J., Kouteckj., J., and Kotil, L. (1968).I n t . J . Ctriicer 3, 364-373. Masseyeff, R., GilIi, J., Krebs, B., Bonet, C., and Zrihen, H. (1974). Bionaerlicine 21, 353-357. Masseyeff, R., Gilli, J., Krehs, B., Calluaud, A., and Bonet, C. (1975).Ann.N.Y. Accid. Sci. 259, 17-28. Masseyeff, R., Maiolini, R., Ferrua, B., and Ragimbeau-Gilli, J. (1976). I n “Protides of the Biological Fluids” (11. Peeters, ed.),Vol. 24, pp. 605-612. Pergamon Press, Oxford. Mawas, C., Kohen, M., Lemerle, J., Buffe, D., Schweisguth, O., and Burtin, P. (1969). I i i t . J . Cnricet- 4, 76-79. McEwen, B. S., Plapinger, L., Chaptal, C., Gerlach, J., and Wallach, G. (1975). Hroiri Res. 96, 400-406. McIntire, K. R., Vogel, C. L., Princler, G. L., and Patel, I. R. (1972). Cancer Res. 32, 1941- 1946. McIntire, K. R., Waldinann, T. A,, Moertel, C. G., and Go, V. L. W. (1975). C o w e r Res. 35, 991-996. McMahon,J.B., Kelleher, P. C., ;md Smith, C. J. (1977).Riochetn.Rioph!/r..Res. Cortttttuti. 76, 1144-1 150. Milunsky, A., and Alpert, E. (1974).J.Pedicitr. 84, 889-893. Milunsky, A., Alpert, E., Frigoletto, F. D., Driscoll, S. G., McCluskey, R. T., and Colvin, R. B. (1977).Pediutrics 59, 770-773. Mizejewski, G. J., and Allen, R. P. (1974).Nnttrre (London) 250, 50-52. Mizejewski, G. J.. Young, S. R., and Allen, R. P. (1975).J.Notl. Cnncer I n s t . 54, 13611367. Mukojima, T., Hattori, N., Nakayama, N., Hasegawa, H., Ohkura, H., and Kitaoka, H. (1973). Tumor Res. 8, 194-197. Murgita, R. A. (1976). Sccititl. /. Zinirmtio/. 5, 1003-1014. Murgita, R. A,, and Tomasi, T. B., Jr. (19754.J . E s p . Med. 141, 269-286. Murgita, R. A,, and Tomasi, T. B., Jr. (19751)).J . E s p . J4ed. 141, 440-452. Murgita, R. A , , and Wigzell, H. (1976). Scrrnd. J . Initnuiio/. 5, 1215-1220. Murgita, R. A., Goidl, E. A,, Kontiainen, S., and Wigzell, H. (1977).Nufur-e (Lotidoti) 267, 257-259. Nature, editorial (by T. B. Tang) (1977).Noture (London) 269, 378. Nayak, N. C., and Mital, I. (1977).Am. J . Pathol. 86, 359-374. Nishi, S. (1970). Cancer Res. 30, 2507-2513. Nishi, S., and Hirai, H. (1972). Riochiin. R i o p h y s . Actu 278, 293-298. Nishi, S., and Hirai, H. (1973). Cnnti Xlonogr. Caticer Res. 14, 79-87. Nishi, S., Watabe, H., and Hirai, H. (1972).]. Itnttiunol. 109, 957-960. Nishi, S., Watabe, H., and Hirai, H. (1973). Tumor Re.s. 8, 17-22. Nishi, S., Watabe, H., and Hirai, H. (1975).Atin. N.Y. Accid. Sci. 259, 109-118.

342

ERKKI RUOSLAHTI AND MAFXKU SEPPALA

Nishi, S., Katsuno, Y., and Hirai, H. (1976).In “Onco-Developmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 679-683. Academic Press, New York. Nishi, S., Fujiyania, Y., and Hirai, H. (1979).Scand. J. Zmmunol., S u p p l . 8, in press. Nishioka, M., Ibata, T., Okita, K., Harada, T., and Fujita, T. (1972). Cancer Res. 32, 162-166. Nishioka, K., Hirayama, T., Sekine, T., Okochi, K., Mayumi, M., Juei-Low, S., Chen-Hui, L., and Tong-Min, L. (1973). Gann Monogr. Cancer Res. 14, 167-175. Norgaard-Pedersen, B. (1976).Scand. J. Zmmunol. 4, 7-45. Norgaard-Pedersen, B., and Axelsen, N. H. (1976).Clin. Chim. Acta 71, 343-347. Ngrgaard-Pedersen, B., and Gaede, P. (1975).In “Quantitative Immunoelecttophoresis” (N. H. Axelsen, ed.), Scand. J. Zmmunol., S u p p l . 2, pp. 19-24. Universitetsforlaget, Oslo. Norgaard-Pedersen, B., Albrechtsen, R., and Teilum, G. (1975).Acta Pathol. Microbiol. Scand. 83A, 573-589. Norgaard-Pedersen, B., Lindholm, J., Albrechtsen, R., Arends, J., Diemer, N. H., and Riishede, J. (1978).Cancer 41,2315-2320. Nunez, E., Savu, L., Engelmann, F., Benassayag, C., CrBpy, O., and Jayle, M. F. (1971). C.R. Acad. Sc. Paris 273,242-245. Nunez, E. A., Benassayag, C., Savu, L., Vallette, G., and Jayle, M. F. (1976).Ann. Biol. Anim. Biochem. Biophys. 16,491-501. Okita, K., Gruenstein, M., Klaiber, M., and Farber, E. (1974).Cancer Res. 34, 27582763. Okon, E., Rosenmann, E., Dishon, T., and Boss, J. H. (1973). B r . J . Cancer 27,362-369. Okuda, K., Kotoda, K., Obata, H., Hayashi, N., Hisamitsu, T., Tamiya, M., Kubo, Y., Yakushiji, F., Nagata, E., Jinnouchi, S., and Shimokawa, Y. (1975).Gastroenterology 69,226-234. Olsson, M., Lindahl, G., and Ruoslahti, E. (1977).J.Exp. Med. 145,819-827. OnoB, T., Kaneko, A., Dempo, K., Ogawa, K., and Minase, T. (1975).Ann.N.Y. Acad. Sci. 259, 168-180. PagB, M. (1973). Can. J. Biochem. 51, 1213-1215. Parmelee, D. C., Evenson, M. A,, and Deutsch, H. F. (1978).J.Biol. Chem. 253,21142 119. Parmely, M. J., and Hsu, H. F. (1973).Fed. Proc. (Abstr.) 32, 979. Parmely, M. J., and Thompson, J. S. (1976).J.Zmmunol. 117, 1832-1837. Peck, A. B., Murgita, R. A., and Wigzell, H. (1978).J.E x p . Med. 147,667-683. Perova, S . D., Elgort, D. A., and Abelev, G. I. (1971). Bull. Exp. Biol. Med. (USSR) 71, 45-47. Peters, T., Jr. (1975).In “The Plasma Proteins” (F. W. Putnam, ed.), Vol. 1, pp. 133-181. Academic Press, New York. Peters, E. H., Nishi, S., and Tamaoki, T. (1978). Biochern. Biophys. Res. Commun.,83, 75-82. Peters, E. H., h i , P. C. W., Hay, D. M., and Lorscheider, F. L. (1979). Scand. J. Zmmunol., S u p p l . 8, in press. Pihko, H. (1976).Scand. J. Immunol. 5,281-285. Pihko, H., and Ruoslahti, E. (1973).Znt. J . Cancer 12, 354-360. Pihko, H., and Ruoslahti, E. (1974). In “C.R. Conf. Int. L’Alpha-foetoproteine” (R. Masseyeff, ed.), pp. 333-336. INSERM, Paris. Pihko, H., Lindgren, J., and Ruoslahti, E. (1973). Zmrnunochemistry 10, 381-385. Purves, L. R. (1976). I n “Liver Cell Cancer” (H. M. Cameron, D. A. Linsell, and G. P. Wanvick, eds.), pp. 61-79. Elsevier, Amsterdam. Purves, L. R., van der Merwe, E., and Bersohn, I. (1970). Lancet 2, 464-465.

a-FETOPROTEIN IN CANCER AND FETAL DEVELOPMENT

343

Purves, L. R., Branch, W. R., Geddes, E. W., Manso, C., and Portugal, M. (1973).Cancer 31,578-587. Radanyi, C., Mercier-Bodard, C., Secco-Millet, C., Baulieu, E.-E., and Richard-Foy, H. (1977).Proc. Natl. Acad. Sci. U S A 74,2269-2272. Raynaud, J. P., Mercier-Bodard, C., and Baulieu, E.-E. (1971).Steroids 18, 767-787. Report of the U.K. Collaborative Study on Alpha-Fetoprotein in Relation to Neural Tube Defects. (1977).Lancet 1, 1323-1332. Ruoslahti, E., ed. (1976). “Immunoadsorbents in Protein Purification.” Scand. J. Imniunol., Suppl. 3. Ruoslahti, E. (1978).J. Immunol. 121, 1687-1690. Ruoslahti, E., and Engvall, E. (1976).Proc. Natl. Acad. Sci. USA 73, 4641-4644. Ruoslahti, E., and Engvall, E. (1978).Scand. J. Zrnrnunol. Suppl. 6, 1-17. Ruoslahti, E., and Seppala, M. (1971a). Znt. J. Cancer 7, 218-225. Ruoslahti, E., and Seppala, M. (1971b). Znt. J . Cancer 8,374-383. Ruoslahti, E., and Terry, W. D. (1976).Nature (London) 260,804-805. , M., Pihko, H., and Vuopio, P. (1971).Int. J. Cancer 8,283-288. Ruoslahti, E., Seppali, M., Vuopio, P., Saksela, E., and Peltokallio, P. (1972).J. Natl. Cancer Inst. 49, 623-630. Ruoslahti, E., Seppala, M., Rasanen, J. A., Vuopio, P., and Helske, T. (1973).Scund. J. Gastroenterol. 8, 197-202. Ruoslahti, E., Pihko, H., and Seppala, M. (1974a).Transplant, Reo. 20, 38-60. Ruoslahti, E., Pihko, H., Vaheri, A., Seppala, M., Virolainen, M., and Konttinen, A. (1974b).In “The Role of Immunological Factors in Viral and Oncogenic Processes” (R. F. Beers, Jr., C. Tilghman, and E. G . Basset, eds.), Suppl. 3, pp. 249-255. The Johns Hopkins Univ. Press, Baltimore. . Ruoslahti, E., Salaspuro, M., Pihko, H., Andersson, L., and Seppala, M. ( 1 9 7 4 ~ )Br. Med. J . 393,527-529. Ruoslahti, E., Pihko, H., Becker, M., and Makela, 0.(1975). Ear. J. Zrnmunol. 5,7-10. Ruoslahti, E., Engvall, E., Jalanko, H., and Comings, D. E. (1977).J. E x p . Med. 146, 1054-1067. Ruoslahti, E., Engvall, E., Pekkala, A., and Seppila, M. (197th).Z n t . J . Cancer 22, 515520. Ruoslahti, E., Estes, T., and Seppala, M. (1978b). Riochim. Biophtjs. Actu, in press. Sakashita, S., Tsukada, Y.,Nakamura, K., Tsuji, I., and Hirai, H. (1977).Int.]. Cancer 20, 83-86. Sarcione, E. J., and Smalley, J. R. (1976).Cancer Res. 36, 3203-3206. Savu, L., Vallette, G., Nunez, E., Azria, M., and Jayle, M. F. (1974). I n “C.R. Conf. Int. L‘Alpha-foetoproteine” (R. Masseyeff, ed.), pp. 75-83. INSERM, Paris. Scardino, P. T., Cox, H. D., Waldmann, T. A., McIntire, K. R., Mittemeyer, B., and Javadpour, N. (1977).J. Urol. 118, 994-999. Schmid, J., and Miihlethaler, J. P. Hurnangenetik 26, 353-354. Schwartz, S . K., and Soloff, M. S. (1975).J.Cliii. Endocrinol. Metah. 39, 589-591. Searle, F., Leake, B. A., Bagshawe, K. D., and Dent, J. (1978) Lancet 1, 579-581. Sell, A., S@gaard,H., and Ncgaard-Pedersen, B. (1976).Znt. J. Cancer 18, 574-580. Sell, S. (1973).J.Natl. Cancer Ztist. 51, 699-701. Sell, S. (1974). 111 “C.R. Conf. Int. L’Alpha-foeto-proteine” (R. Masseyeff, ed.), pp. 365-381. INSERM, Paris. Sell, S., and Becker, F. F. (1978).J. Nntl. Cnricer Inst. 60, 19-26. Sell, S., and Gord, D. (1973). Irnrnunochemistry 10, 439-442. Sell, S., and Skelly, H. (1976).J.Natl. Cancer Inst. 56. 645-648. Sell, S., Jalowayski, I., Bellone, C., and Wepsic, H. T. (1972).Cancer Res. 32,1184-1189.

344

ERKKI RUOSLAHTI AND MARKKU SEPPALA

Sell, S., Nichols, M., Becker, F. F., and Leffert, H. L. (1974).Cancer Res. 34,865-871. Sell, S., Sheppard, H. W., Jr., Nickel, R., Stillman, D., and Michaelsen, M. (1976a). Cuiicer Res. 36, 476-480. Sell, S., Stillman, D., and Gochman, N. (1976b).A m . .I. Clin. Puthol. 66, 847-853. Sell, S., Sheppard, H. W., Jr., and Poler, M. (1977).J.Zmntunol. 119, 98-103. Seller, M. J. (1975).J.Med. Genet. 12, 109-110. Seller, M. J., and Berry, A. C. (1978).Lancet 1,660. Seppili, M. (1975).Ann. N.Y. Acud. Sci. 259, 59-73. Seppili, M. (1977). Cliti. Ohstet. Gynecol. 20, 737-757. Seppiili, M., and Ruoslahti, E . (1972a).Am. J. Obst et . Gynecol. 112, 208-212. Seppili, M., and Ruoslahti, E. (1972b). Luttcet 1, 375-376. Seppili, M., and Ruoslahti, E. ( 1 9 7 2 ~ Am. ) . J . Obstet. Gynecol. 114, 595-598. Seppalk, M., and Ruoslahti, E. (19724. Br. Med. J. 4, 769-771. Seppila, M., and Ruoslahti, E. (1973).Ant. J. Ohstet. Gynecol. 115, 48-52. Seppiili, M . , and Sepplli, I. J. T. (1969).Anti. Chir. G!piueco/. Fetirt. 58, 76-79. Seppali, M., and Unkrus, H. A. (1974).Am.J . Ohsf. Gyn. 119, 270-272. Seppala, M., Tallberg, T., and Ehnholm, C. (1967).Ann. Med. E s p . B i d . Fentt. 45, 16-19. Seppili, M., Bagshawe, K. D., and Ruoslahti, E. (1972).I n t . J. Cancer 10, 478-481. Seppiili, M., Pihko, H., and Ruoslahti, E. (1975).Cattcer 35, 1377-1381. Seppila, M., Ada, P., Rapola, J., Kajalainen, O., Huttunen, N.-P., and Ruoslahti, E. (1976).Lattcet 2, 123-128. Seppila, M., Rutanen, E.-M., Heikinheimo, M., Jalanko, H., and Engvall, E. (1978).Znt. J. Cancer 21, 265-267. Sheppard, H. W., Jr., Sell, S., Trefts, P., and Bahu, R. (1977).J.Zmmunol. 119,91-103. Shinozuka, H., and Lombardi, B., Sell, S., and Iammarino, R. M. (1978).Cuncer Res. 38, 1092- 1098. Silver, H. K. B., Deneault, J., Gold, P., Thompson, W. G., Shuster, J., and Freedman, S. 0. (1974).Cuticer Res. 34, 244-247. Sippel, A. E., Kurtz, D. T., Morris, H. P., and Feigelson, P. (1976). Caitcer Res. 36, 3588-3593. Sizaret, P., Breslow, N., and Anderson, S. G. (1975a).J.B i d . Stund. 3, 201-223. Sizaret, P., Tuyns, A., Martel, N., Jouvenceaux, A., Levin, A., Ong, Y. W., and Rive, J. (1975b).Ann. N.Y.Accid. Sci. 259, 136-155. Slade, B. (1973).Nature (London) 246, 493-494. Smith, C. J., and Kelleher, P. C. (1973). Biochim. Biophys. Acta 317, 231-235. Smith, C. J., Morris, H. P., and Kelleher, P. C. (1977).Cancer Res. 37, 2651-2656. Smith, J. B. (1971).I n “Proceedings of the First Workshop on Embryonic and Fetal Antigens in Cancer” (N. G. Anderson and J. H. Coggin, eds.), pp. 305-312. Molecular Anatomy Program, Oak Ridge, Tennessee. Smuckler, E. A., Koplitz, R. M., and Sell, S. (1976).Z i t “Onco-Developmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 253-257. Academic Press, New York. Soloff, M . S., Creange, J. E., Potts, G. 0. (1971).Eiidocrittology 88, 427-432. Soloff, M. S., Swartz, S. K., Pearlmutter, F., and Kithier, K. (1976). Biochim. Biophys. ACtU 427,644-65 1. Sutcliffe, R. G. (1975).Biol. Rev. 50, 1-33. Tabor, E., Gerety, R. J., Vogel, C. L., Bayley, A. C., Anthony, P. P., Chen, C. H., and Barker, L. F. (1977).J.Nutl. Crriicer Znst. 58, 1197-1200. Taketa, K., Watanabe, A., and Kosaka, K. (1973).Tumor Res. 8, 108-113.

n-FETOPROTEIN IX CANCER AND FETAL DEVELOPhlENT

345

Taketa, K., Watanahe, A., and Kosaka, K. (1976). Z t r “Onco-Developmental Gene Expression” (W. H. Fishman and S. Sell, eds.), pp. 219-226. Academic Press, New York. Talerman, A. (1977). Z t t t . J . Concer- 19, 741-746. Talerinan, A,, and Haije, W. C . (1974). Coiicer 34, 1722-1726. Tatarinov, Y. S. (1964). Vop. Jfetl. K h i r n . 10, 584-589. Tatarinov, Y. s. (1965). \’O)J. ,\led. k d t i i t i . 11, 20-24. Teiluin, G. (1965). Actri Pnthol. Microbid. Sctrttd. 64, 407-429. Teiluin, G., Albrechtsen, R., and N0rgaarcl-Pederseii, B. (1974).Actci Puthol. Jficrobiol. Scuitcl. A. 82, 586-588. Tshipysheva, T. A , , Guelstein, V. I., and Bannikov, G . A. (1977). Zitt. J . Cniicer 20, 388-393. Tsuchida, Y., Endo, Y., Urano, Y., and Ishida, M . (1975). A n n . N.)’. A c d . Sci. 259, 22 1-233. Tsukada, Y., and Hirai, H. (1975). Aitii. N.1’. Acacf. Sci. 259, 37-44. Tsukada, Y., Mikuni, M., Watahe, H., Nishi, S., and Hirai, H. (1974). Zut. J. Coiicer 13, 187-195. Tumyan, B. G., Svet-Moldavsky, G. G., and Kannanova, N . V. (1975).Nature (Lo~idott) 255,244-245. Uriel, J., d e Nkchaud, B., and Dupiers, M. (1972).Biocherti. B i o l ~ h ! / sRes. . C o ~ n m ~ r i46, t. 1175-1180. LJriel, J., Arissel, C . , Bouillon, D., tk N&chaud, B., antl Loisillier, F. (1973). Nirtrrrc (Loridoil) Neic H i o l . 244, 190-192. Uriel, J., Bouillon, D., and Dupiers, M. (1975). FEBS Lett. 53, 305-308. Uriel, J., Bouillon, D., Aussel, C., and Dupiers, M. (1976). Proc. Nritl. Accrd. Sci. USA 73, 1452-1456. Vaheri, A,, Ruoslahti, E., Hovi, T., and Nordling, S. (1973). 111 “Biology of Filxoblast” (E. Kulonen and J. Pikilarainen, eds.), pp. 267-272. Academic Press, London. Vallette, G., Benassayag, C., BPlanger, L., Nunez, E. A,, and Jayle, M. F. (1977). Steroich 29, 277-289. Vandeputte, M., Sobis, H., Billiau, A,, van d e Maele, B., and Leyten, R. (1973). Znf. 1. Cn ticer 11, 536-542. van Furth, R., and Adinolfi, M . (1969). Nature ( L m d o i t ) 222, 1296-1299. Vogel, C . , Anthony, P. P., Sadikali, F., Barker, L. F., and Peterson, M. R. (1972).J.N o t / . Cnitcer Z i t s t . 48, 1583-1588. Wahren, B., and Edsmyr, F. (1974). I t i f . J . Cancer 14, 207-214. Waltl, N., Cuckle, h l . , Stirrat, G. M., Bennett, bl. J., and Turnhull, A. C . (1977).Ltrncet 2, 268-270. Waldmann, T. A,, and McIntire, K. R. (1972). Lnricet 2, 1112-1115. Watabe, H. (1971). Caitcer Res. 31, 1192-1194. Watabe, H. (1974). Itit. J. Cuttcer 13, 377-388. Watannbe, A,, Miyazaki, hl., and Taketa, K. (1976a). Crrricer Res. 36, 2171-2175. Watanal,e, A,, Miyazaki, hl., antl Taketa, K. (197611).I t i t . J . Cnitcer 17, 518-524. Wiggelinkhuizen, J , , Nelson, M. M., Berger, G. M . B., and Kaschula, R. 0. C. (1976).J. Pedicitr. 89, 452-455. Williains, R. R., hlcIntire, K. R., Waldmann, T. A,, Feinleih, ha., Go, V. L. W.,Kannel, W. B., Dawl)er, T. R., Castelli, W. P., and McNaniara, P. M . (1977).J. N o t / . CattccvZ t t , y f . 58, 1547-1551. Yachnin, S. (1976). Proc. Not/. Acod. Sci. USA 73, 2857-2861. Yachnin, S., and Lester, E. (1976). C / i i i . E x p . Z~timtrnol.26, 484-490.

346

ERKKI RUOSLAHTI AND MARKKU SEPPALA

Yachnin, S., and Lester, E. P. (1977).J.Zmmunol. 119, 555-557. Yachnin, S., Hsu, R., Heinrikson, R. L., and Miller, J. B. (1977).Biochim.Biophys. Acta 493, 418-428. Young, J. L., Reid, R. G., and Crawford, J. W. (1976). Clin. Chim. Actn 69, 11-20. Zeltzer, P. M., Neerhout, R. C., Fonkalsrud, E. W., and Stiehm, E. R. (1974).Lnncet 1, 373-375. Ziegler, J. L., Adamson, R. H., Barker, L. F., Fraumeni, J. F., Jr., Gerin, J., and Purcell, R. H. (1978).J.N a t l . Caricer Znst. 60, 717. Zimmerman, E. F., and Wilson, J. R. (1976).In “Onco-Developmental Gene Expression’’ (W. H. Fishman and S. Sell, eds.), pp. 139-145. Academic Press, New York. Zimmerman, E. F., Bowen, D., Wilson,, J. R., and Madappally, M. M. (1976).Biochemist r y 15, 5534-5543. Zimmennan, E. F., Voorting-Hawking, M., and Michael, J. G. (1977).Nature (London) 265, 354-356.

MAMMARY TUMOR VIRUSES Dan H. Moore, Carole A. Long, and Akhil B. Vaidya Department of Microbiology and Immunology, Hahnemann Medical College and Hospital Philadelphia. Pennsylvania

Joel B. Sheffield Department of Biology, Temple University. Philadelphia, Pennsylvania

Arnold S. Dion and Etienne Y. Lasfargues Institute for Medical Research. Camden. New Jersey

I. Introduction. . . . . . . .......................... 347 11. Morphology ............................ ......................... 350 111. Structural Proteins and RNA-D DNA Polymerase of MuMTV . . . . . 356 A. MuMTV-Associated Protein .............................. B. Characterization and Locali f MuMTV Structural Protein C. RNA-Directed DNA Polymerase of MLIMTV. . . . . . . . . . . . . . . . . . IV. Synthesis and Assembly of MuMTV Proteins .................... V. Genome of MuMTV ................................ VI. Biology of MuMTV ...........................

364

. . . . . . . . . . . . . . . . . . . 370

. . . . . . . . . . . . 386 ............................. 386

VII. Antigens of MuMTV ...........................

. . . . . . . . . . . 395 . . . . . . . . . . . 396

A. Humoral Immune Responses to MuMTV . . .

C. Modulation of the Immunologic Interaction between MuMTV and ................................. Its Host IX. Comments on the Question of a hluMTV-Related Virus X. Concluding Remarks . . . . . . . . ..............................

408

. . . . . . . . . . . 410

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

At the time when the influence of milk on the development of mammary tumors in mice was discovered (Bittner, 1936),means were 347 Copyright U,VAV('ES

I> C * ( X H

RESE*H('H.

VIL.

29

0 I979

h ~ i d i ~ i i i I'rvw, ic Inc.

~ l rixhtr l OS rc*prodttction in

,111)

timi w w r v t d .

IS RN 0- 12-006629-7

348

DAN H. MOORE

et nl.

not available for associating the entity carrying the influence with any particular structure. It was only after analyses of the agent-containing milk and mammary tumors by ultracentrifugation, electron microscopy, and laborious bioassays that Bittner's agent was eventually identified as a virus with distinct physical and chemical properties. Murine mammary tumor virus (MuMTV), as the agent is now called, belongs to genus Oncomavirus B in the viral family Retraviridae (Dalton et a1 ., 1975). MuMTV enjoys a unique position among oncomaviruses since it is the only virus whose main pathological manifestation is a carcinoma; all other oncomaviruses are involved in neoplasms of inesodermal cells. The effects that genetic, endocrinologic, and immunologic status of mice have on MuMTV synthesis and mammary tumorigenesis make this virus still more attractive by providing systems to investigate two of the major problems of modem biology, viz., the mechanisms for the emergence of cancer cells and the control of eukaryotic gene expression. Strains of MuMTV have been recognized for some time. The classification of these strains proposed by Bentvelzen (1974)was based TABLE I MAMMARY TUMORINCIllENCE, TUMORAGE, AND VIRION CONTENT OF MILKOF VARIOUS MOUSESTRAINS Mouse strain (Moore's strain)

Trim or incidence ( 70)

Average tumor age (months)

MuMTV virions/ ml milk"

A AfC57BU6 BALBIcfC3H BALBIc C3H C3NfC57BU6 C57BLfRIII C57BLi6 DBN2 DD DDfCS7BLJ6 GR CRfCS7BU6 RIIl RIIIfC57BU6

98.6 39.0 98.1 24.0 99.2 40.0 92.6 0.3 92.0 90.9 5.9 97.6 97.0 95.0 9.7

7.7 18.8 7.3 19.6 6.4 19.0 11.5 21.5 11.0 12.4 19.0 7.7 7.3 8.5 17.3

1.4 x 10" 7.5 x 10'" 1.8 x 10" Not detectable 1.3 x 10" 1.3 x 10" 3.6 x 10" Not detectable 1.0 x 101" 9.0 x 10'" 1.2 x 10'2 4.2 x 1011 2.8 x 10%"

'I Low parity milks from the strains with a high mammary tumor incidence were used and high parity (sixth to tenth lactation) milks from strains with a low mammary tumor incidence were used. Estimates of virion content are based on molecular hybridization assays.

349

MAMMARY TUMOR VIRUSES

on tumorigenic potential, host range, histological type of the tumor produced, and the mode of transmission of MuMTVs obtained from different strains of mice. However, due to the lack of proper immunological and biochemical characterizations of various MuMTVs and because such diverse biological criteria are applied singly or in various combinations, the classification proposed by Bentvelzen has been difficult to use. Recent advances in infection of cells with MuMTV in vitro and the application of molecular biological and immunological techniques in understanding the biology of MuMTV will eventually make it possible to develop a more satisfactory classification. At present, we prefer to denote a strain of MuMTV b y referring to its source, e.g., MuMTV from RIII strain will be called MuMTV(RII1). MuMTVs can be broadly divided into two categories: (a) viruses that are transmitted through both low and high parity milks and are found in early occurring tumors and which are responsible for high mammary tumor incidence in their strains of origin; (b) endogenous, genetically transmitted MuMTVs which are usually expressed only in high parity milk or late occurring tumors and may be minimally tumorigenic in their strains of origin but are highly infectious and tuinorigenic in another strain. The two categories of viruses can usually be separated by foster nursing a newborn litter of a high mammary

I

1

2

3

4

5

6

7

8

9

PARITY

FIG. 1. MuhlTV antigen expression in milks a s a function ofparity for indicated inouse strains and snlxtrains. T h e substrains Af, CBHf, m d RIIIfwere started by foster nursing newl)orn litters A, C3H, and RIII on C57BL to remove the so-called tirilk-tr;unsmitted virus. hlany o f t h e mice o f t h e foster-nursed sub1 ines, however, secrete another hluMT\’, ntid its prevalence increases with parity. Tumor incidences and the avelage age at which they occ11r are indicated on eaclr curve.

350

DAN H. MOORE

et ul.

tumor strain on a mother such as C57BL, whose milk is virus free. The foster-nursed substrain (e.g., RIIIfC57BL, or simply RIIIf) thus started (and proliferated by sibling mating) is left with only the endogenous virus, which is transmitted genetically. The host range of the two MuMTVs from the same strain may differ. A large number of mouse strains have been used in mammary tumor virus research. For the convenience of the reader, characteristics of a few of the most common ones are given in Table I and in Fig. 1. The interaction between viral and genetic factors in murine mammary cancer has recently been reviewed by Hilgers and Bentvelzen (1978). The purpose of this chapter, therefore, is to review the current understanding of structure, biochemistry, infectivity, and immunology of the genus B Oncornaviruses and their association with mammary carcinoma in mice. A discussion of the possible involvement of a MuMTV-like agent in human breast cancer is also included. II. Morphology

The morphology of the MuMTV virion has been studied in detail. Its size and shape depend on the conditions and methods of measurement. In its natural environment, it is believed to be spherical with diameters ranging from approximately 50 to 140 nm (Sarkar et ul., 1975). In comparison, the size of the leukemia-sarcoma C-type particles is more uniform, 110to 140 nm. MuMTV appears to be bimodal in size with peaks at 60 and 120 nm. Bioactivity has also been associated with particles of two sizes (Moore et al., 1959, 1962). The virion contains a nucleoid, usually eccentrically located, about 40 nm in diameter, surrounded by a “shell” (Fig. 2a). Negatively stained (phosphotungstate) virions usually have head and tail forms (Fig. 2b); this is a drying artifact that can be prevented by prior fixation in glutaraldehyde or other stabilizers. The MuMTV virion can be disrupted by Tween 80-ether treatment with the release of three structural components: nucleoids, large fragments of membranes, and small pieces of membrane that tend to form rosettes (Sarkar et al., 1971a). The buoyant densities in sucrose or potassium citrate or nitrate are about 1.18 gm/cm3 for whole virions and 1.24 gm/cm3 for isolated nucleoids, and the whole virions contain about 1.9% RNA on a dry weight basis, compared with 4.4% for the isolated nucleoids. Isolated nucleoids after Tween 80-ether treatment sometimes reveal the nucleocapsid as a coiled ropelike structure consisting of strands (Fig. 3). Two distinctive nucleocapsid structures have been seen (Sarkar and Moore, 1968; Sarkar et al., 1971a,b): (a)

MAMMARY TUMOR VIRUSES

351

FIG.2. (A) MuMTV B particle profiles in thin section showing eccentric nucleoids and nucleoidal shell. ~90,000. (B) Negatively stained (sodium phosphotungstate) MuMTV B particle. ~ 2 5 0 , 0 0 0 .

single strands 30-50 A in diameter, and (b)paired strands 79 to 90 A in diameter; the paired strands are often connected by periodic bridges 63 A apart or the two strands are twisted around each other to make a double helix with a pitch of 126 A. These paired strands are seen at the periphery of the nucleoid (in the equatorial plane) only in particles oriented so that they are seen from a polar view (Figs. 4a and b).

352

DAN H. MOORE

et (11.

FIG.3. (A) Negatively stained virion showing internal structure: segments of nuclear protein structure (arrows). Spikes with knobs at distal elids can be seen at periphery of particle. From Sarkar et d.,1971h. (B) Negiitively stained virion showing outer membrane (large white ring), spikes (outer white ring), and apparently helical strands ofthe nucleocapsid (arrow). A drawing ofthe helical structure is shown opposite arrow. Froin Sarkar et a / . , 19711).

When a new MuMTV virion separates from the cell membrane, it has a large nucleoid, centrally located (Figs. 5 and 6). Apparently, the structural integrity of the supercoiled nucleocapsid is maintained by interactions between the protein subunits of the neighboring strands. These interactions within the virion are under the influence of the

FIG.4. (A) Concentric rings of double helical components show how the helical structures are coiled together to form a nucleocapsid. ~ 4 6 2 , 0 0 0(B) . A model (from Home and Wildy, 1961)showing possible arrangeiuent ofcomponents foniiing inyxovirus particle. A vertical view ofthe core ofthis model would give a profile similar to that ofthe structure presented in Fig. 4a. From Sarkar and Moore, 1968.

FIG.5. Section ofRIII mammary tumor showing profiles ofcell border and acinarlriinen with budding and released MuMTV B particles. Cytoplasmic A particles may be seen in the cytoplasm (a) and at the plasma membrane (h). Sometimes the B particle nucleoid is assembled at the cell membrane, as at (c)(and in the lower micrograph).Newly released B particles have large, centrally located nucleoids (d), which later shrink aiid become eccentrically located (e). Occasionally, B particles contain two nucleoids (t). Upper ; micrograph, x 90,000. From Sarkar and Moore, 1974. micrograph, ~ 4 5 , 0 0 0lower

354

DAN H. MOORE

et al.

FIG.6. Partially or completely damaged particles showing reticular structure on the viral membrane. N in (a) possibly represents the viral nucleoid, and the detached membrane is shown at the left. The arrows in (c),(d),and (h) indicate the membrane reticulum. The thickened corners suggest that projections were attached at those points. Arrows in (e-g) point to clusters of six projections where the central ones were missing. The projections appear to be linked with each other by some structures, probably the reticulums. A reticulum having 5-fold symmetry is shown in (h) (short arrow). (e-g) were obtained with glutawldehyde-fixed virus and stained with UAC. (a-d) ~215,600;(e) x238,700; (f and g) ~312,620;(h) ~346,500.From Sarkar and Moore, 1974.

MAMMARY TUMOR VIRUSES

355

environment, which is essentially intracellular. As soon as the virion leaves the cell and enters the extracellular lumen, the microenvironment within the particle is changed, resulting in a collapse of the hollow nucleocapsid. This is a random process, since in thin section there are variations in the appearance and location of the shrunken nucleoid (Figs. 2a and 5). The outer coat of the MuMTV virion has somewhat the same appearance as the outer coat of the myxoviruses (Moore, 1962; Sarkar and Moore, 1974). The surface of the outer membranes of both MuMTV and myxoviruses is covered with projections or spikes, but the spikes of MuMTV have knobs at their distal ends and are longer than those of the myxoviruses (Moore et al., 196913). The MuMTV spikes (Fig. 2b) are approximately 95 A long, are spaced about 74 A apart, and the diameter of the knobs is about 54 A (Sarkar and Moore, 1974). The membrane seems to have a reticular structure composed mainly of hexagons, but with a few pentagons (Fig. 6), and the spikes are arranged in a regular pattern corresponding to the reticulum. Examination of RIII virions by the freeze-etch technique (Sheffield, 1973) or by freeze drying (Sarkar and Moore, 1974) did not reveal the spikes but presented a pattern of pits in the surface, spaced at about 250 A in a hexagonal array (Fig. 7). In recent studies, Sheffield et (12. (1976a) and Sarkar and Demsey (1977) have found that this pattern

FIG.7. A freeze-etch preparation of a typical virion isolated from RIII milk. Spikes are masked by an overcoat showing an array of pits. ~360,000.

356

DAN H. MOORE et (11.

appears only in virus derived from milk, and that virus derived from tissue culture lacks this material and has the expected pattern of spikes. Conceivably, this pattern is due to the presence of some milk proteins which are adsorbed onto the virion. The virion membrane itself appears as a typical bilaminar structure, although in some micrographs it appears that the inner lamina is less densely stained than the outer. In freeze-fracture studies (Sheffield, 1973),the membrane was found to be relatively free of intramembranous particles. Sarkar and Moore (1974) have suggested that the spikes are anchored to a hexagonal lattice within the membrane.

111. Structural Proteins and RNA-Directed DNA Polymerase of MuMTV

A. MuMTV-ASSOCIATEDPROTEINS MuMTV is composed of60 to 70% protein which, regardless of host mouse strain or whether purified from mouse milk or tissue culture supernatants, shows a remarkable degree of similarity when analyzed by polyacrylamide gel electrophoresis (Dion and Moore, 1977). For the designation of RNA tumor virus polypeptides, the nomenclature of August et al. (1974) is employed, i.e., glycoproteins and proteins are specified b y the prefixes g p and p, respectively, followed by the molecular weight times Different investigators have reported various values for the molecular weights of viral polypeptides, and these determinations are summarized in Table 11. The variations observed within each molecular weight range may be reconciled within the 2 5 1 0 % accuracy of molecular weight estimations based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and it should be emphasized that the determinations for the viral glycoproteins are overestimates because of the decreased charge density imposed by the carbohydrate moiety. Implicit in this consideration is the possibility of variable molecular weights of glycoproteins resulting from differences in glycosylation, a host cell function. Finally, molecular weight estimates for low molecular weight proteins b y gel electrophoresis are usually unreliable and probably account for the relatively large variations observed for the p8-pl2 protein(s). An SDS-PAGE profile of the polypeptides from MuMTVs prepared from supernatants of cultured cells and milk of two mouse strains is shown in Fig. 8. It is apparent that there are differences in the polypeptide patterns of tissue culture-produced and milk-produced viruses. For convenience, we shall refer to the major proteins of

TABLE I1 THE MAJOR STRUCTURAL PROTEINS REFQRTEDTO BE ASSOCIATEDWITH MuMTV PURIFIED FROM MOUSE MILK OR TISSUECULTURE SUPERNATANTS Reference

Source

MuMTV polypeptide designation (MW x Tissue culture

Dicksoii and Skehel (1974) Teramoto et (11. (1974) Sarkar et (11. (1977) Yagi et 01. (1977)

BALBlcfC3H BALBlcfC3H BALB/cfC3H MJY-Alpha

>70 100 90 83

70 67 68

60

58 60

52 49 55 52

46 42.5

38 37.5

46

37

36 33.5 34 33

30 29 30

28 24 28 24

22 17 18 22

14 13.5 14 17

10 9 12 13

8

23

18 15

14 13 15

12 7 12

8

Milk Nowinski et (il. (1971) Smith and Wive1 (1973) Witte et (11. (1973) Sarkar and Dion (1975) Tanaka (1977) Karaiide et (11. (1978)

C3H RIII BALBIcfC3H RIII RIII ICRC

90 90 85 90 90 90

70 69 68 78 72

62 62

52 55 52.5 55 50 49

33 37 43 42 43

37

32 34 32 34

30

23 27.5 26 28 25 24

5

358

DAN H. MOORE

et al.

4

100

4- 90

52 + 42 +

4

68

4

52

4

38

36 -+

t34

28 +

+ 28

18 +

4

18

4

14 12

f

12 f

A

B

C

FIG.8. Polyacrylamide gel electrophoretic patterns of sodium dodecyl sulfate-treated MuMTV virions from: (A) tissue culture supernatant of BALB/cfC3H mammary tumor cells; (B) BALB/cfC3H milk; (C) RIII milk.

MuMTV from milk as follows: gp68, gp52, gp34, p28, p24, p18, p14, and p12. Figure 9 shows an SDS-PAGE pattern of virus-free C57BU6 milk subjected to the same virus purification procedure that was used for MuMTV(RII1) virus. The p14 band and most of the polypeptide bands with molecular weights greater than gp52 found in milkderived viruses are not detectable in virus produced in tissue culture. However, bands with the same apparent molecular weights are found in gels of vesicles from virus-free C57BU6 milk. This points out the difficulties in distinguishing polypeptides actually coded by the viral genome from those contributed by the host.

MAMMARY TUMOR VIRUSES

-

359

* 100 90

,*5 2 * 42

A

B

FIG.9. SDS-PAGE patterns of: (A) mock vinis preparation from C57BLvirus-free milk; (B) RIII milk virus.

B. CHARACTERIZATION AND LOCALIZATION OF MuMTV STRUCTURAL PROTEINS g p 6 8 is a glycosylated protein by the criteria of staining with periodic acid-Schiff (PAS) reagent (Teramoto et al., 1974; Parks et al., 1974a; Sarkar and Dion, 1975; Karande et al., 1978), glucosamine labeling (Teramoto et al., 1974; Sarkar and Dion, 1975; Dickson and Skehel, 1974; Sarkar et al., 1977; Yagi and Compans, 1977), and extrinsic labeling by the galactose oxidase-tritiated borohydride (GOB) technique (Sheffield and Daly, 1976). The oligosaccharide moiety is

360

DAN H. MOORE

et d .

also sulfated (Yagi and Compans, 1977) and is readily sialylated within intact virions (Schloemer et ul., 1976).The latter observation, together with its susceptibility to protease digestion (Yagi and Compans, 1977; Sheffield et al., 1976b), lactoperoxidase iodination (Parks et ul., 1974a; Sarkar and Dion, 1975; Witte et al., 1973), and presence in rosettes [MuMTV membranes containing projections (Sarkar et ul., 1976)], strongly complement the conclusion that this glycoprotein is membrane associated and is quite accessible to the external milieu. g p 5 2 is the most prominent structural protein of MuMTV and is glycosylated by the criteria given above. The accessibility of gp52 to various enzymatic treatments as with gp68 and its presence in rosettes but not in projectionless virions (Sarkar et nl., 1976; Cardiff et al., 1974) support the conclusion that this structural protein is located on the external membrane of MuMTV. This conclusion was further corroborated by the localization of gp52 on the viral surface by immunoelectron microscopy (Sarkar and Dion, 1975). The gp52s from virions of two different mouse strains, RIII and A, have been exten1976a). sively studied (Dion et d . , unpublished data; Black et d., These glycoproteins from the two sources have routinely displayed differences in molecular weights as determined by polyacrylamide gel electrophoresis and gel permeation chromatography and have been designated gp50(A) and gp55(RIII). However, they have been found to be closely related immunologically, as determined by competition radioimmunoassays and immunodiffusion (Dion et a1., unpublished data). Biochemical evidence for this relatedness has been obtained by quantitative amino acid analyses (Table 111),indicating a high degree of similarity between these two proteins. Some differences are observed: Black et (11. (1976a) have found gp50(A) to be different from gp55(RIII), as determined by human leukocyte reactivity (see Section VIII). Moreover, the content of half-cystine is remarkably dissimilar and smaller differences in the number of threonine, serine, proline, and isoleucine residues have been found (Table 111).Tryptic peptide maps of the major glycoproteins (gp52) of MuMTVs isolated from the milks of RIII, A, and GR mouse strains are similar, if not identical. However, it cannot be concluded from these results that these proteins are identical because gp52 is relatively insensitive to trypsin digestion, yielding too few peptides to observe possible differences in amino acid sequences. In addition to investigating the protein moiety of gp55(RIII) and gp50(A), quantitative analyses of the oligosaccharide chains have also been performed. The latter has indicated only minor differences in neutral sugar or sialic acid content; however, gp50(A) contains considerably more amino sugar than gp55(RIII). Data on amino acid sequencing and oligosaccharide composition of

36 1

SIAMSIARY TUMOR VIRUSES

TABLE I11 AMINOACID ANALYSIS OF A GLYCOPOLYPEPT~DE FROSI TWO DIFFERENT STRAINSOF MtiMTV Residuesimole of protein Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phen ylalanine

gp55(RIII)

gp50(A)

27 10 16 43 30 37 30 37 31 24 8 22 3-4 23 36 11 23

27 10 17 43 24 32 28 42 28 22 17 22 4 16 34 9 22

gp52s from different sources will be required to account for typespecific antigenic determinants on these molecules (Teranioto, 1977b,c; see also Section VI1,B). g p 3 4 is the second most prominent glycoprotein of MuMTV, containing sulfated oligosaccharicle moieties composed of glucosamine, galactose and/or galactosaniine, and sialic acid, as determined by the techniques described for gp68. However, the localization of gp34 within the viral structure differs significantly from gp68 or gp52. Enzyme-catalyzed reactions leading to iodination, sialylation, or protein digestion require detergent disruption of the virus, suggesting that this protein is located within the viral membrane. Confirmatory evidence for this localization has been provided by the observation of gp34 in rosettes (Sarkar et al., 1976) and projectionless particles (Sarkar et al., 1976; Cardiff et ol., 1974),and its depletion in viral nucleoid or core preparations (Teramotoet a1 ., 1977a). Marcus et a l . (1978) have recently shown that gp34 is strongly hydrophobic, while gp52 is only moderately hydrophobic. From these results, it is plausible to conclude that the protein moiety of gp34 is embedded within the viral membrane. However, the labeling of gp34 of undisrupted MuMTV b y

362

DAN H. MOORE

et al.

the GOB technique (Sheffield and Daly, 1976) and the absence of labeling by sialyl transferase (Schloemer et al., 1976)would mean that complex, sialic acid-containing oligosaccharide chains are relatively inaccessible and the galactose-containing chains protrude to the exterior. Such an arrangement of oligosaccharides seems unlikely and further studies are required to resolve this question. p28 is the major nonglycosylated protein of MuMTV. Investigations of its localization within the viral structure have consistently confirmed that p28 is an internal viral protein. This conclusion is based on the following evidence: (1)p28 of undisrupted virus is not digested b y proteases (Yagi and Compans, 1977; Sheffield et al., 1976b) or iodinated by lactoperoxidase (Parks et al., 1974a; Sarkar and Dion, 1975; Witte et nl., 1973); (2) p28 has been demonstrated in viral cores (Teramoto et al., 1977a) and projectionless virus particles (Sarkar et al., 1976; Cardiff et nl., 1974); (3) it is absent in rosette preparations (Sarkar et uZ., 1976); (4) inimunoelectron microscopic studies disclosed that p28 was not associated with the viral surface (Sarkar and Dion, 1975). 1718, p 1 4 , and p 1 2 are internal viral polypeptides. From crosslinking studies, most of the p18 was found to form a large disulfide cross-linked aggregate (Dion and Pomenti, 1979); however, at present the precise location of p18 within the virus is unknown. p18, p12, and possibly p14 are phosphorylated viral proteins by [32P]phosphate labeling which was confirmed by quantitative amino acid analyses (Dion et al., J. Gen. Virol., submitted). From the determination of isoelectric points of these proteins and their ion-exchange chromatographic behavior, it is likely that one or both of these proteins binds to RNA to form the ribonucleoprotein complex of MuMTV. The characterization of polypeptides present in isolated core preparations revealed that p28 and p14 were the major proteins; smaller quantities ofpl2 were also observed (Teramoto et nl., 1977a). However, other data indicate that p12 is a major protein of MuMTV cores (Sheffield and Daly, unpublished data; see Figs. 8 and 9). OF MuMTV C. RNA-DIRECTEDDNA POLYMERASE

All reported isolations and characterizations of RNA-directed DNA polymerase (RDDP) to date have been confined to milk-borne MuMTV from the RIII mouse strain (Howk et ul., 1973; Dion et al., 1976; Dion et al., 1977b). The DNA polymerase of MuMTV is a zinc nietalloenzyme (Marcus et al., 1976) possessing a molecular weight of approximately 100,000 by glycerol sedimentation analyses (Dion et

MAMMARY TUMOR VIRUSES

363

al., 197413; Marcus et al., 1976; Dion et al., 197%) and by SDS-PAGE (Dion et al., 1974b; Dion et nl., 197%). By the latter technique, molecular weights of 1251-labeledRDDP (lactoperoxidase-mediated iodination) were also estimated to be 85,000, 50,000, and other minor molecular weight (Marcus et al., 1976). However, it is possible that this result reflects polymerase fragmentation induced by labeling. Also, since lactoperoxidase was not removed after iodination, selfiodination by the enzyme could have affected the radioautographs of the gels. Following purification, the RDDP of MuMTV has an absolute requirement for an exogenous template/primer for DNA polymerase activity, and its enodgenous viral RNA, cellular DNA, and various synthetic templates (Howk et al., 1973; Dion et al., 1974b; Marcus et al., 1976; Dion et al., 1977b) are active in directing DNA synthesis. A unique feature of the RDDP of MuMTV vis-h-uis mammalian type C viruses is the Mg2+ preference of type B viruses for a maximal DNA polymerase reaction, regardless of the template/primer employed (Howk et al., 1973; Dion et al., 1974a; Marcus et al., 1976; Dion et al., 1977b, Dion et af., 1974b);type C retraviruses require Mn*+.A notable exception to this observation is that poly rCm oligo dG directed DNA synthesis by the polymerase of MuMTV prefers Mn2+ (Dion and Moore, 1977; Marcus et al., 1976), and this preference is completely stringent when the polymerase is purified to homogeneity (Dion and Williams, unpublished data); i.e., no polymerase activity is observed in the presence of Mg2+.Another aspect relevant to the isolation and storage of MuMTV polymerase relates to its differential lability as a function of the template/primer utilized to monitor the DNA polymerase reaction (Dion et d., 1974b; Marcus et al., 1976). In addition to DNA polymerase activity templated by RNA or DNA templates, a ribonuclease H (RNase H) enzymatic activity copurifies with the RDDP of MuMTV (Dion et al., 197713). Digestion studies of various substrates demonstrated that only the RNA strand of an RNA . DNA hybrid was cleaved, corroborating the conclusion that RNase H was a true hybridase. Both RNase H and RDDP possess a Mg2+preference for enzymatic activity and probably occupy different sites on the same polypeptide chain, which would account for the differential labilities of RNase H and RDDP activities. Ashley et nl. (1977) reported that MuMTV also had an associated terminal deoxynucleotidyl transferase activity which was distinct from RDDP by various criteria, the most noteworthy being its ability to catalyze terminal addition to primer in the absence of template. That MuMTV does not possess a terminal addition enzyme was subse-

364

DAN H. MOORE

et

(11.

quently reported (Marcus and Sarkar, 1978), and it was clearly demonstrated that the putative terminal transferase activity reported by Ashley et al. (1977) was a consequence of incomplete viral FWA removal. Under these conditions, the added primer, oligo dT, would bind to the polyadenylate tract of MuMTV RNA, resulting in template-directed DNA polymerase activity catalyzed by the RDDP of MuMTV. IV. Synthesis and Assembly of MuMTV Proteins

The process of viral biogenesis is complex, involving: (a) synthesis of virus-specific messenger RNA; (b) synthesis and glycosylation of the virus proteins; (c) the assembly of these proteins into characteristic structures and their incorporation into the virion; and (d) the subsequent release of the virions from the cell surface. The study of the synthesis and assembly of MuMTV is in its early stages, since reliable in vitro systems for controlled growth of the virus have been developed only within the past 5 years. A major problem in the study of MuMTV protein synthesis is that the virus is produced asynchronously by host cells in the presence of continuous host protein synthesis. Despite this restriction, pulse-chase experiments have been successfully combined with immunoprecipitation of cytoplasmic extracts to yield some information on precursorproduct relationships in this system. Dickson et nl. (1975, 1976) extracted virus-producing cells from primary cultures ofmammary tumors with a mixture of Nonidet P-40 and deoxycholate, and precipitated the extract with antiserum to whole MuMTV. The precipitates were found to contain a protein (73,000 daltons) which could be converted by trypsinization to gp52. Tryptic maps of the precursor contained many spots in common with purified gp52. However, they were unable to detect a precursor to the nucleoid proteins. More recently, Racevskis and Sarkar (1978), using monospecific antisera to gp52 and p28, have demonstrated distinct precursors to both envelope and nucleoid proteins. In their system, the p28 precursor is identified as Pr75, and the glycoprotein precursor as gPr7O. It was found that the rate of processing of Pr75 was considerably slower than that of the glycoprotein precursor. In fact, a considerable amount of label remained in the large molecular weight form after a chase as long as 180 minutes. Schochetman et (11. (1978) obtained similar results and were further able to isolate Pr75 by its affinity to columns of single-stranded DNA. Schochetman et al. (1977) provided evidence that the gp52 portion of gPr70 lies close to the amino terminal end of the polypeptide and that the gp34 portion lies close to the carboxy terminal end.

MAMMARY TUhlOR VIRUSES

365

Two other approaches have been used to study MuMTV protein synthesis. Schochetman and Schlom (1976) reported that when cells were cultured in the presence of high concentrations of salt, glycoprotein synthesis was inhibited. This indicated that the processes leading to nucleoid synthesis and assembly were separable from those leading to envelope formation. Scolnick et aZ. ( 1976), using radioimmunoassay for gp52 and p12, reported that intracellular levels of both antigens rose at the same rate after hormonal stimulation of increased virus synthesis, indicating that the synthesis of these proteins, one from the envelope and one from the core, inight be coordinate. This does not necessarily imply that the relationship is obligate. A curious fact noted by several groups (Kimball et d . , 1976; Sheffield et al., 1977; Racevskis and Sarkar, 1978) is that a significant amount of soluble viral glycoprotein not associated with intact virus is released into the medium by cells in culture and by mammary glands in situ. It is not known if soluble gp52 is released directly from the host cell or is eluted from virions. At the present time, the bulk of the information about virus assembly comes from morphological studies. It should be kept in mind that the following description is based primarily on electron micrographs and represents only a logical hypothesis based on the images obtained with various processing procedures for electron microscopy. The mature virion is produced b y budding at the cell membrane or, iiiore commonly, from the tips of microvilli. Initially, a small amount of dense material appears under the plasma membrane and rapidly develops into a spherical nucleoid within a membrane bud. The initial stages of this process are not seen frequently, and their rarity has led some observers to state that MuMTV, as the archetypal B-type particle, is assembled with preexisting complete nucleoids, while C-type particle nucleoids are assembled during the budding process. In fact, incomplete nucleoids can be found during the synthesis of both types of viruses. Concomitant with the appearance of internal structures, the external side of the membrane develops a spiked coat, and viral envelope antigens can be detected in the bud by ininiunoelectron microscopy (Tanaka and Moore, 1967; Sarkar and Dion, 1975). Freeze-fracture studies (Sheffield, 1973, 1974) have indicated that cellular intranieinbranous particles are excluded from the part of the microvillus that contains the developing virion. Using ininiune electron microscopy, Calafkt et (11. (1974, 1976) have found that the virus spike protein, gp52, is not restricted to the developing virus bud but is found in small amounts randonily distributed along the plasina membrane. It is possible that the viral glycoproteins are randomly inserted into the cell

366

DAN H. MOORE

et d .

membrane and move laterally within the plane of the membrane until they are stabilized by some undefined interaction within the budding virion. In many, but not all, virus-producing cells, cytoplasmic structures having the appearance of viral nucleoids are seen either in association with Golgi vesicles or in clusters around large cytoplasmic vacuoles. They were first described by Bernhard et al. (1955) and were called intracytoplasmic A particles. They are distinct from the intracisternal particles of similar morphology seen in many cells carrying virions of the leukemia-sarcoma group. The particles of about 100-nm diameter appear to contain two concentric shells with uncharacterized electron-dense bodies located eccentrically within the particles, Studies by Sarkar and Dion (1975); Smith and Lee (1975) and Tanaka (1977) have indicated that these particles show antigenic crossreactivity with the viral core protein, p28 (see Section VI1,B). However, SDS-PAGE analysis of isolated A particles reveals no polypeptide of corresponding molecular weight. Tanaka (1977) and Smith and Lee (1975) reported that treatment of isolated A particles with trypsin or other proteases converts the A particle proteins to polypeptides which are apparently identical to p28 and some of the smaller MuMTV polypeptides. It has been generally assumed that the intracytoplasmic A particles represent precursor forms of the virion core and that the virus is formed by the envelopment of A particles by the membranes of the host cell. However, it has not been ruled out that A-particle aggregates represent an accumulation of excess or unused viral nucleoproteins. It would appear that the processes leading to virus release must be rate limiting, since virus-producing cells tend to have substantial accumulations of unreleased virions in microvilli. In discussions about enveloped viruses in general (Alison and Davis, 1974; Blough and Tiffany, 1975; Lenard and Compans, 1974), it has been assumed without substantial evidence that the interaction between virion nucleoid and the envelope produces a sufficient alteration in the configuration of the membrane to facilitate pinching off of the virion. Recently, it has been demonstrated that MuMTV (Damsky et al., 1976a,b; Raceveskis and Sarkar, 1978), as well as other enveloped viruses (Wang et .al., 1977), contains the contractile protein, actin, within the virion. Morphological studies (Damsky et d.,1977) have suggested that virus extrusion is associated with filament-containing microvilli and that these filaments bind the subfragment S1 of myosin. Furthermore, it is conceivable that a similar contractile system functions in pinching off of the assembled virion. Residual contractile elements in the released

MAMMARY TUMOR VIRUSES

367

virion may be responsible for the characteristic “head and tail” configuration seen under hyperosmotic conditions (Sarkar et nl., 1975). V. Genome of MuMTV

As with other oncornaviruses, MuMTV contains a high molecular weight RNA with a sedimentation coefficient between 60 and 70s. I n addition to the 60-70s RNA, there are several low molecular weight RNA molecules of probable cellular origin present in the virion. The 60-70s RNA is an aggregate of two or more subunits of 30 to 35s RNA molecules. The subunit RNA molecules are held together by hydrogen bonds to form a 60-70s RNA molecule. It is now quite well established that 60-70s RNA molecules of many, if not all, C-type retroviruses are dimers held together by linkages formed between 5’-ends 1978). It is not known if such dimer of two subunits (Bender et d., linkages are present in 60-70s RNA of MuMTV; neither has it been shown that the 60-70s RNA of MuMTV is indeed a dimer. The subunits of MuMTV RNA have a molecular weight of about 3.1 x 106, as estimated from electron microscopic examination (Dion et a1., 1977a). PAGE of‘denatured MuMTV RNA also supports this estimate. Analysis of oligonucleotides generated from MuMTV RNA by digestion with ribonuclease T, indicates that the complexity of the MuMTV genome is about MW 3 x lo6 (Friedrich et al., 1970). Therefore, it is most likely that, as with C-type retraviruses, the genome of MuMTV is polyploid and consists of two or more identical RNA molecules of about MW 3 x lo6. Molecular hybridization experiments have shown that the MuMTV genome is unique among retroviruses, since no nucleic acid homology can be demonstrated between MuMTV RNA and the genomes of any other retraviruses. This would indicate that the B-type and the C-type retroviruses do not share a common evolutionary progenitor. However, this is not surprising, since pathogenesis of these two groups of viruses is so distinct. But it is possible that MuMTV might share some of its genetic information with another mammalian carcinomainducing retrovirus if such a virus were to be discovered. All mouse cells contain. DNA sequences related to MuMTV RNA. This fact was first directly demonstrated by Varmus et al. (1972), whose estimate of the number of MuMTV-related genes per cell has now been revised to a lower number (Michalides and Schlom, 1975; Michalides et al., 1976; Morris et al., 1977). Normal organs of inbred strains of mice with a high mammary tumor incidence (e.g., RIII, C3H, A) as well as a low mammary tumor incidence (e.g., BALB/c) contain

368

DAN H. MOORE

et

(11.

from 5 to 10 copies of MuMTV genes per cell; cells from GR strain may contain a few more copies of MuMTV genes (Michalides et al., 1976; Morris et al., 1977).Morris et al. (1977) reported that feral mice and a subspecies of M u s musculus ( M . musculus molossimus) contain half the number of MuMTV DNA copies found in laboratory mice, and no significant differences seem to exist between the viral DNAs from these mice as judged by thermal denaturation experiments. Two Asian species of mice, M. caroli and M . ceruicolor, which diverged from M. musculus three to five million years ago, contain limited portions of MuMTV genome in their DNA. In M . ceruicolor, this fraction of the viral genome is reiterated 30 to 50 times; M. cciroli contains a much smaller fraction of MuMTV genome than M. ceruicolor, and it is reiterated to a lesser extent. These results suggest that MuMTV-related DNA sequences have been present in the genome of M u s for at least three to five million years. In strains of mice which derive their high mammary tumor incidence through milk-transmitted MuMTV, mammary tumor cells seem to contain an increased number of MuMTV DNA copies (Michalides et al., 1976; Morris et cd., 1977). In contrast, mammary tumors of GR mice, which have germinal transmission of virulent MuMTV, contain about the same number of MuMTV proviral genes as normal organs (Michalides et al., 1976; Morris et al., 1977). It is conceivable that in GR mice MuMTV that is expressed in milk is the progeny of a virulent germinal provirus, and the virus that is transmitted to the newborn via milk is not capable of reinfecting the target mammary epithelial cells. In other strains of mice (e.g., RIII, C3H), milk transmission of virulent MuMTV may led to infection of only the target cells, giving these cells additional MLIMTV proviral sequences while other cells may contain only the endogenous provirus which, at least in its strain of origin, is much less virulent. Alternatively, the increased number of MuMTV proviral sequences observed in mammary tumor cells of some mouse strains may be the result of “gene amplification” mediated through reinfection of transformed, MuMTV-expressing mammary epithelial cells. It is interesting to note that in nonmurine cells chronically infected with MuMTV, hormonal stimulation leads to accumulation of unintegrated MuMTV DNA (Ringold et al., 1978; Vaidya, unpublished observations; also see Section VI,E and F). Although a similar increase in MuMTV DNA does not seem to occur in cultured mammary tumor cells (Ringold et nl., 1978) or in in vitro infected mouse mammary epithelial cells (Vaidya et d.,1978), it is possible that, during the growth of mammary tumors, cells may have accumulated such unintegrated proviral DNA sequences, some of which may

XIAhlhIARY TUhlOR VIRUSES

369

have subsequently become integrated. However, these are inaiiily speculations, and a better understanding of the events leading to increased MuMTV proviral DNA sequences in mammary tumors and their significance to tumorigeiiesis must await direct experimentation. Competition molecular hybridization experiments have indicated that geiioines of MuMTVs obtained from most of the high mammary tumor incidence strains are very similar (Michalides aiid Schloin, 1975; Ringold et d., 1976). However, when Friedrich et (11. (1976) studied fiiigeiprints of gligonucleotides produced b y RNase T, digestion of MuMTV RNAs from cultured BALB/cfC3H and GR mammary tumors, they found that there were some differences between the geiiomes of these two strains of MuMTVS. Since fingerprint analysis of RNA involves only a sinall fraction of the genome, these results are not necessarily in disagreement with competition hybridization experimeiits but only reflect the limitations of the techniques used. Michalides and Schloiii ( 1975) reported that RNA of MuMTV derived from foster-nursed low mammary tumor C3Hf mice was able to compete up to oiily 75% with MuMTV(C3H) RNA in hybridization reactions. Drohaii et (11. (1977) obtained fragments of radiolabeled MuMTV(RII1) and MuMTV(C3H) RNA by hybridizing the viral RNA to a vast excess of DNA obtained from the liver of RIII and C3H mice and separating unhybridized RNA by hydroxyapatite chromatography. These so-called recycled viral RNA fragments, believed to comprise about 25% of the MuMTV (RIII and C3H) genome, hyh i d i z e d efficiently to the DNAs from RIII and C3H mammary tumors, but not to DNAs from apparently normal organs of tunior-bearing RIII and C3H mice nor to DNAs from mammary tumors of RIIIf and C3Hf 1977). These results indicate that M~iMTv(R111) mice (Drohan et d., aiid MuMTV(C3H) derived from milk or maminary tumors contain sequences of RNA that are not germinally transmitted and are therefore absent from the normal organs of these mice. Since the size of MuMTV(C3Hf) or MuMTV(R1IIf) RNA does not appear to be significantly siiialler than that of MuMTV(C3H or RIII), one may assume that the sequences present in milk-transmitted MuMTV are not deleted but are substituted in endogenous MuMTV. The findings of Drohaii et (11. (1977) are of great significance to the biology of MuMTV, and they should be confimied in other laboratories. As mentioned earlier, noninurine cells infected in vitro with MuMTV accumulate unintegrated viral DNA in linear as well as closed circular forms when stimulated with glucocorticoids (Ringold et al., 1977c, 1978; Vaidya, unpublished results). Shank et aZ. (1978) have taken advantage of this phenomenon and generated a physical

370

DAN H. MOORE

et al.

map of restriction endonuclease cleavage sites on unintegrated viral DNA isolated from a clone of rat hepatoma cells chronically infected with MuMTV(GR). A curious observation of Shank et al. (1978) was that although the linear unintegrated MuMTV DNA had a molecular weight of 5.9 x lo6, covalently closed circular forms had molecular weights of 5.9 x lo6and 5.1 x lo6.This would indicate that during the process of circularization, some of the viral DNA molecules underwent a deletion of about 1.2 kilobase pairs of DNA. Evidence was provided that this deletion occurred at the end of the linear DNA molecule that corresponded with the 3’-end of MuMTV RNA (Shank et al., 1978). VI. Biology of MuMTV

A. In Vivo INFECTIONAND TUMORIGENESIS

The process of MuMTV infection in mice is not yet understood. Three procedures have been used to detect infection, and they all require many weeks or months of lapsed time between introduction of virus and detection of replication. The procedures are: (1) detection of tumors, (2) detection of hyperplastic alveolar nodules (HAN) (Pullinger, 1947; Nandi, 1963), and (3) detection of viral antigen in milk (Nowinski et ul., 1967; Blair, 1969; Chamey et al., 1969). The infective dose seems to depend on the route of inoculation. In order to obtain a high incidence of mammary tumors by nursing, it is necessary to nurse for an appreciable period of time. On the other hand, if administered intraperitoneally (i.p.), a large dose usually results in a lower tumor incidence than more diluted doses (Bittner, 1945; Moore et al., 1969a, 1974; Arthur et al., 1978). In foster-nursing experiments, Andervont and McEleney (1939) noted that allowing newborn C3H mice to be nursed by their mothers 17 hours or less before transferring them to C57BL foster mothers resulted in a low tumor incidence which increased progressively with each day they remained with their own mothers, until 100% was reached after the third day. In other experiments, Bentvelzen (1968b) foster-nursed groups of reciprocal hybrids of BALB/c and C57BL on high mammary tumor strain mothers, BALB/cfC3H, for either 1day or for the whole nursing period and obtained tumor incidences of 28 to 36% for the l-day foster-nursed groups and 53 to 73% for the full-term foster-nursed groups. In still other experiments, Moore et al. (1974) found that mice of the substrain RIIIfC57BL foster nursed on RIII mothers were not all infected unless they received the RIII milk for at least 4 days. Determination of

MAMMARY TUMOR VIRUSES

371

MuMTV RNA content of RIII milks taken on consecutive days after parturition indicated that during the first few days, milk was not deficient in virus, but contained about 1OI2 viriondm1 (Vaidya and Moore, unpublished data). In contrast to these foster-nursing results, when RIIIfC57BL mice were inoculated i.p. at weanling age with RIII skim milk at dilutions lo-’ to lop6,only dilutions of lop3or higher were infective (Moore et ul., 1974), and when dilutions of the same sample were inoculated i.p. into C57BL weanlings, maximum infectivities occurred at dilutions lop3to Similar results have been obtained when saline or water extracts of mammary tumor tissue titrations were used. Bittner (1952) wrote: “In general, the highest incidences occurred in the progeny of the mice that received a single injection of the fraction with the lowest concentration of the agent.” Again he stated (Bittner, 1962): “A dilution of or will give us more tumors in probably 90% of the experiments than a dilution of lo-’ or 2 x lo+.’’ The reason for the high dose by the i.p. route resulting in a lower incidence of infection is not understood. Immune responses of the host, first suggested by Bittner (1945), have been considered, and attempts have been made to mix live and formalin-inactivated virus in varying proportions to simulate a large virus inoculum (Charney and Long, unpublished data). The results of several such experiments suggested that increasing the dose by adding killed virus does indeed decrease the incidence of infection, but the magnitude of the decrease was not so great as might be expected from the many experiments where high doses of milk or tumor extract were used. The large amounts of nonviral and possibly inhibitory (Moore et al., 1959, 1962) constituents of skim milk or tumor extract may have been more effective in preventing infection than the highly purified inactivated virus used in the mixing experiments. Perhaps nonspecific clearing mechanisms are more efficiently activated by the large amounts of foreign material introduced into the peritoneal cavity. Another possibility is that milk and tumors contain defective virions that interfere with infection in a manner that has been found for other viruses (Huang and Baltimore, 1977). When administered via the gastrointestinal route, most of the virus may be digested and denatured before reaching a replication site; thus several days of nursing may be required to introduce an infecting dose. This slow introduction may be far less arousing to both the cellular and humoral immune responses. The gradual daily increase of virus intake as the mouse grows may eventually infect and start viral replication without sufficient reaction to cause suppression.

372

DAN H. MOORE

et (11.

Another important factor in the MuMTV infection process is the change in the susceptibility of the host with age. In C57BL mice inoculated with RIII virus, it has been repeatedly shown that i.p. inoculation of neonates results in a lower incidenck of infection than 1970).This may in part account inoculation at later ages (Moore et d., for the low infectivity of mice nursed on infected mothers for only the first few days of life. In nine separate experiments (Moore, unpublished data) between 1968 and 1976 where the susceptibilities of newborns (under 24 hours) were compared directly with 3- to 10week-old C57BL mice injected i.p. with RIII skim milk dilutions lo-' to the incidence (measured by MuMTV antigen in third lactation milks) in the neonatally inoculated mice was consistently lower. In summation, the results were 73/193 or 38% of the newborns were infected, and 124165 or 75% of the older mice were infected. It is possible that during this very early period of rapid host growth and differentiation, appropriate target cells or transport mechanisms are not available to the virus. The acceptance of the B particle as the carrier of MuMTV activity is almost universal, but it may not be the only particle that carries this activity. Bioassay results for RIII milk fractions separated according to sedimentation rates (rate zonal fractionation) in Ficoll gradients often indicated considerable tumorigenic activity at levels in the centrifuge tube above and below the main particle light-scattering zone, where few B particles could be found (Miroff and Magdoff-Fairchild, 1965; Moore, unpublished data). In many experiments, titrations of bioactivity of fractions recovered from levels below the principal B particle zone gave tumorigenic activities which increased with dilution, soinetimes even to a dilution of lo-'. There is a great deal of evidence that bioactivity is also carried by particles smaller (Moore, 1962) and of different density than that of B particles. Another enigmatic problem in understanding infectivity and tumorigenesis by MuMTV has been the apparent resistance of MuMTV to extremely high doses of ionizing radiations. Ionizing radiation inactivates virions by causing breaks in the nucleic acids and degradation or proteins of the viruses. The dose of ionizing radiation required to inactivate a virus increases as the size of the target decreases. Latarjet (1970) found that y radiation of 6 x lop5R was capable of reducing the infectivity of avian sarcoma virus and Gross leukemia virus to one-tenth of its original value. However, Ardashnikov and Spasskaia (1949) and Moore et n l . (1962) were not able to reduce tumorigenic activity of MuMTV by high doses of ionizing radiation. Recently, Gorka and Mouriquand (1975) reported that samples of

MAMMARY TUMOR VIRUSES

373

MuMTV irradiated with up to 4 x 106 R of y rays did not show any decline in their tumorigenicity, even when diluted to lop5.These authors reported that there was extensive morphological damage to the virions after irradiation and that MuMTV antigenicity was destroyed. They also showed that irradiated virions treated with Pronase, RNase, or DNase were still capable of inducing mammary tumors in mice. These findings are difficult to explain and need corroboration. One would assume that the ability of MuMTV to infect would be severely diminished by such high doses of radiation, but the tumors produced by irradiated MuMTV contain large quantities of virions of type-B morphology. It is possible that a radio-resistant component of irradiated MuMTV preparations is capable of activating the endogenous MuMTV DNA sequences of the host, which in turn are responsible for the eventual appearance of the tumor.

B. INFECTIVITY OF ENDOGENOUS MuMTVs Before 1933, mammary tumorigenesis in mice had been established as resulting from a genetic trait, but studies with reciprocal hybrids of high and low mammary tumor strains indicated the existence of an extrachromosomal influence (Staff of Jackson Laboratory, 1933; Korteweg, 1934), which was shown by Bittner (1936) to be transmitted through the milk. Heston et al. (1945) showed that transmission of the milk influence was under genetic control and later (Heston et al., 1960) obtained evidence that there were possible differences in the “quality” as well as the quantity of milk agent produced by different mice. However, it was generally accepted that foster nursing of neonates on agent-free mothers precluded the transfer of the mammary tumor agent to offspring, even though the offspring and its progeny often demonstrated an appreciable mammary tumor incidence. Possible integration of the viral genome into the host cell was suggested by Moore (1963), and, after the development of strain GR by Miihlbock (1965), a clear demonstration of both milk and genetic transmission of 1970a) the mammary tumor influence was provided (Bentvelzen et d., Virus in milk could be demonstrated by infection in other mouse strains, and genetic transmission was indicated because foster iiursing on agent-free C57BL mothers did not diminish the tumor incidence. In other mouse strains, the genetic transmission of an endogenous virus was not easily accepted because foster nursing C3H new-born mice on agent-free C57BL mothers to provide the subline C3HfC57BL changed the tumor incidence from very high to relatively

374

DAN H. MOORE

et (11.

low and the tumor age from early to late; furthermore, there was no clustering of tumor development in progeny of mothers that bore tumors (Heston, 1958), and tumor extracts, although they contained typical B particles, did not seem to infect and cause tumor development in inoculated test mice (Heston et al., 1950; Boot and Muhlbock, 1956; Pullinger, 1960; Pitelka et al., 1964). Even reciprocal crosses between C3Hf and C57BL (Heston and Deringer, 1952) gave no evidence for a maternal influence. It was therefore concluded that' the late occurring tumors resulted mainly from a combination of genetic and hormonal influences, a conclusion that may have been correct, although MuMTV, active when transferred to certain other strains, is present in the tumors and is secreted in high parity milk. Despite the belief widely held at that time that C3Hf mice were free of a transmissible mammary tumor agent, Dmochowski and Gray (1957) and Dmochowski et ul. (1963) reported tumorigenic activity in C3Hf tumor extracts when tested in (C57BL x RIIIQFl and (C57BL x AQF1 hybrids. Recently, Moore et al. (1976) have reported bioactivity in high parity C3Hf milk when inoculated at several dilutions into weanling Af, BALB/c, C57BL, and RIIIf mice. The reasons for the wide discrepancies found in the literature are not readily apparent, but several factors should be considered: (1) size of dose; when inoculated i.p., a large dose may be noneffective, whereas a more diluted one will, be (see Section V1,A); (2) MuMTV(C3HQ is probably very poorly infectious in C3Hf mice, since we now know that MuMTV(AQand MuMTV(RI1If) are weakly infectious in their strains or origin; (3) the specific genetic characteristics of the test mice as well as that of the virus seem to be very important; nominal test strains or viruses may not be identical in different laboratories; (4) there are also variations in results from one experiment to another in the same laboratory; (5) when BALB/c mice were foster nursed on CSHf, the foster mothers were probably of low panty and did not secrete infectious virus (K. B. DeOme, personal communication). We have recently tested two pools of high parity C3Hf milk in BALB/c, C57BL, and RIIIf weanlings. Activity was determined by tumor development as well as by secretion of MuMTV antigen in milk after the third litter. As in the earlier data (Moore et al., 1976),all of the test strains responded to the inoculations of high parity C3Hf milk at several dilutions (inoculations ranged from lo-' to Although there was variation between the results obtained in the two experiments and the tumor incidences are not yet complete, it appears that BALB/c and RIIIf are most susceptible to MuMTV(C3HQ and C57BL

MAMMARY TUMOR VIRUSES

375

are least susceptible. In one experiment, tumors occurred in BALB/c mice before the age of 8 months, and, in the other, they developed at an average age of 13.3months. Bentvelzen (1968a, 1972) was first to postulate that MuMTV information could be transmitted as a chromosomally integrated provirus under the control of one regulator gene. Determination of the number of genes involved in the regulation of MuMTV expression has received a great deal of attention and has been discussed thoroughly by Hilgers and Bentvelzen (1978). Only a few recent reports, therefore, will be mentioned here. Much of the work on gene regulation has been done on GR-C57BL hybrids and backcrosses. van Nie et al. (1977) and van Nie and de Moes (1977) have described a single gene (Mtu-2) which regulates expression of viral information. Experiments by Heston et al. (1976) and Heston and Parks (1977) suggested that multiple genetic influences controlled virus expression and that the genesis of mammary tumors was largely controlled by the regulation of chromosomally integrated and inherited viral genes, rather than b y horizontal transmission of virus. It must be recognized, however, that this conclusion applies to a specific system (GR-C57BL), which may not be universally representative. Unlike the GR strain, which seems to harbor only one virus, other high mammary tumor strains carry two viruses, one that can be removed by foster nursing and the other that cannot be removed. For example, in RIII mice the high percentage of tumors that occur early in life must be largely controlled by a virus that is not chromosomally integrated and inherited. On the other hand, an appreciable number of mammary tumors may occur in BALB/c 1974, 1976) without any detectable expression of mice (Moore et d,, virus or viral antigen. Extensive unpublished studies (Sarkar and Sheffield) of BALB/c tumors and of milk have failed to find either B particles or MuMTV antigen, although traces of antigen have been reported by others (Hageman et al., 1972; Bentvelzen, 1975). In the C3Hf-BALB/c system, van Nie and Verstraeten (1975) concluded that the release of virus was controlled b y a single gene (Mtu-l ) located on chromosome 7. However, it is not likely that we now have enough information to make generalized statements about the genetic control of MuMTV expression or the genesis of mammary tumors. In the GR-C57BL system, Heston and Parks (1977), in their analysis of the second backcross generation, were unable to prove a single gene hypothesis and favored “the view that mammary tumorigenesis in these crosses is not a single gene influence but [is] a threshold expression of multifactoral genetic inheritance from the parental phenotypes.”

376

DAN H. MOORE et (11.

C. DEPENDENCE OF MuMTV INFECTIVITY ON STRAIN OF TEST MOUSE It now appears that each mouse strain, except GR, that has a high incidence of mammary tumors carries two different MuMTVs, one of which is endogenous. The other is milk-transmitted only. All of them can be distinguished by their differences in incidences of infectivity when assayed in several mouse strains (Moore et al., 1976). The occurrence of MuMTV in milks of six mouse strains and substrains as a function of parity are presented in Fig. 1, and the relative activities of various MuMTVs when tested in several mouse strains are shown in Table IV. Immunological and biochemical specificity for some of the various MuMTVs have also been demonstrated (Charney et al., 1975; Teramoto, 1977b,c; Section 111,B). The infectivities of MuMTV(Af) and MuMTV(RIIIf), in the respective high parity skim milks, have been compared in F, hybrid mice derived from several mouse strains (Moore et al., 1978). In addition, the infectivities of MuMTV(A) and MuMTV(RIII) in low parity A and RIII skim milks were tested in the same F, hybrids. The results showed a very low susceptibility of (C57BL9 x BALB/cd)F, hybrid mice to both MuMTV(A) and MuMTV(Af) (Table V) and a very low susceptibility of (RIIIf? x C57BLd)F, hybrids to MuMTV(RIII~), but a very high susceptibility of (RIIIf? x BALB/cd)F, hybrids to MuMTV(R1IIf). The results demonstrate the high tumorigenic activity of MuMTV(RII1f)in a host of suitable genetic constitution. MuMTV(Af) is highly active when introduced into RIIIf mice but almost inactive in BALB/c (Moore et aZ., 1976). TABLE IV DEGREEOF SUSCEPTIBILITY OF SEVERAL MOUSE STRAINSTO MuMTVs FOUNDIN MILKS OF VARIOUSSTRAINSAND FOSTER-NURSED SUBSTRAINS Source of milk

Recipient strain

Af BALB/c C57BL C3Hf RIIIf

High

High

Low panty

parity

Low parity

parity

Low parity

parity

A

Af

C3H

C3Hf

RIII

RIIIf

+++ ++++ +

+ + ++ +++

++++ ++ +

++++

+++

+++ + +++ + +

++++ ++++ + ++

++

+++ + + +++

High

377

MAhlbIARY TUMOR VIRUSES TABLE V COhiPARISON OF SUSCEPTIBILITIES OF hfOUSE STRAINS AND F, HYBRIDS TO SEVERAL MIIMTVS Recipient strain or hybrid

BALBIc C57BL (C57BL x BAiBIc)F, BALB/c C57BL (C57BL x BALB/c)F, C57BL RIIIf (RIIIf x C57BL)F, BALBIc RIIIf (RIIIf x BALB/c)F, C57BL RIIIf (RIIIf x C57BL)F, BALBIc RIIIf (RIIIf x BALB/c)F,

Source of milk

A (low parity)

Af (high parity)

RIII (low parity) RIII (low parity) RIIIf (high parity) RIIIf (high parity)

Degree of susceptibility

++++

+ + + + -t +++ + +++ + + +++ + ++ +

++++ ++ ++++

D. TRANSMISSION OF MuMTV THROUGH CONTACT

The question of contact transmission of MuMTV between mice living in the same cage or in the same laboratory has recently been reviewed (Hilgers and Bentvelzen, 1978) with the conclusion that there is little evidence for such a transmission. Additional new data briefly presented here further support this conclusion. In six experiments where weanling C57BW6 females lived for 5 weeks in cages with RIII or GR multiparous females or where weanling BALB/c females lived with C3H or BALB/cfC3H inultiparous females (Moore and Holben, 1978), there was no significant increase in the mammary tumor incidence in the C57BL or BALB/c experimental cagemate mice, but there was a significant elevation in MuMTV antigen incidence in third lactation milks as determined by microimmunodiffusion (Table VI). Thus, there was an unusual lack of correlation between viral antigen expression and tumor incidence. However, antigen, but not tumor, incidences in C57BL and BALB/c mice inoculated i.p. with phosphate-buffered salt solution (PBS) were also ele-

ANTIGEN h V D

TABLE VI TUMORINCIDENCES Ih' BALB/c AND C57BL MICE AFTER LIVINGAS WEANLINGS FOR 5 WEEKS IN CAGES WITH MuMTV-INFECTEDFEMALES" Control and experimental susceptible mice Mean tumorage and range

Antigen incidence

Tumor incidence

Test strain

Control

Experimental

Control

Experimental

Control (months)

C57BL BALB/c C57BL C57BL BALBlc BALB/c

1r/189(0.5)d 1'/86( 1) 4/333(1) 4/333(1) 2/ 103(2) 2/103(2)

4/32( 12.5) 5/28( 18) 8/51(16) 15/48(31) 14/51(27) 15/48(31)

lf/312(O.3) 10/52(19) 1/353(0.3) 1/353(0.3) 1/34(3) 1/34(3)

1/21(5) 3/23(13) 1/34(3) 0/42(0) 2/47(4) 1/38(3)

23(-) 19(12-24) 22(-) 21(-)

W-1 2

4

~

Experimental (months)

2.U-l 19(17-22) 22(-) 22(20-24) 23(-)

Infected cagemate carrier strainsb RIII BALB/cfC3H GR RIII BALB/cfC3H C3H

" The first two lines of data are for experiments started in 1973 and the bottom four lines of data are for experiments started in 1975. Two different sublines of BALBlc mice were used; in the earlier experiment (line 2) the mammary tumor incidence in the foundation stock was 19% at an average of 19 months; in the later experiment (lines 5 and 6) the incidence was 3% at an average age of 24 months. Both sublines were descendants 9f the breeding pair we received in 1965 from the Cancer Research Genetics Laboratory, Berkeley, California. Ir All of the carrier strain cagemate mice had MLIMTVantigen in their second lactation milks and developed mammary tumors at average ages 7 to 10 months. Positive at sixth lactation, lived 23.5 months with no manimary tumor. Numbers in parentheses in incidence columns denotes percentage positive. Positive at ninth lactation, lived 21 months with no mammary tumor. Milk from this mouse was antigen-negative at both third and fourth lactations.

379

MAMMARY TUMOR VIRUSES

vated. Some of the positive milks were tested for the presence of MuMTV RNA by molecular hybridization, but none was detected (Vaidya and Long, unpublished data). Additionally, BALB/c mice inoculated as weanlings with MuMTV-free C57BL milk expressed MuMTV antigen in third lactation milks (Table VII). Even the i.p. insertion of a sterile needle with no inoculum into the C57BL weanlings resulted in an elevation of MuMTV antigen incidence in third lactation milks. At later lactations, however, the antigen incidences were less (Table VII). In fact, all of the positive readings on shaminoculated (sterile needle only) mice turned negative at later lactations, and no tumors developed by 24 months of age. Many of the immunodiffusion tests were checked by radioimmunoassay for MuMTV gp52 (Sheffield et al., 1977), and the results from the two procedures were in agreement. Blair et nl. (1974) found that spleen cells of BALB/c females over 14 weeks of age were reactive against primary mammary tumor cells of the syngeneic BALB/cfC3H subline in a microcytotoxicity assay, and Blair and Lane (1974b) reported that in contrast to the laboratoryraised mice, spleen cells of the BALB/c mice raised in isolation away TABLE VII IN MILKS OF C57BL SUMMATION DATAON MuMTV ANTIGEN INCIDENCES BALBIc MICE AFTER VARIOUS TREATMENTS

AND

Antigen incidence Third lactation

Fourth or fifth lactation

Description of mice

Number of experiments

C57BL foundation stock (no treatment) PBSb inoculated Sham inoculated (needle only) Cage inates (from Table VI)

13 2 3

4333 (1.2)" 171145 (11.7) 5/31 (16.1) 271131 (20.6)

0132 (0) 31128 (2.3) 0125 (0) 8/98 (8.2)

9 4 3

W103 (1.9) 211105 (20) 9149 (18.4) 34127 (26.7)

1/39 (2.6) 8/92 (8.7) 1/49 (2.1) 11/100 (11)

BALBlc foundation stock (no treatment) PBS" inoculated Experimental' Cage mates (from Table VI)

Numbers in Parentheses are percentages. PBS, phosphate-buffered salt solution (0.02 M phosphate, pH 7.4, 0.15 Sf NaCI; 0.1% bovine serum albumin). and Inoculated with C57BL skim milk (at dilutions lo-',

380

DAN H. MOORE

et al.

from the laboratory were not reactive. Lopez et uZ. (1976) reported cellular immunity to MuMTV-specific antigen in young adult females of their BALB/c colony. It is possible that the viral antigen expression in milk or the cellular immune response may not result from viral infections but may reflect partial expression of the endogenous MuMTV genes. The induction of viral antigen expression may result from some form of stress (Riley, 1975) or other factors. Varmus et ul. (1973) showed that lactating mammary gland tissues of C57BL and BALB/c contain MuMTV RNA. However, the RNA molecules are not normally translated to the extent that viral proteins are detected in milk. The presence of two viral proteins (gp52 and p28) in mammary tissues and tumors of low mammary tumor strains after hormonal stimulation has been examined by a sensitive competition radioimmune assay procedure (R. Nusse and R. Michalides, personal communication). In C3Hf, RIIIf, and BALB/c animals, high levels of viral proteins were found in both the mammary glands and the tumors. In the C57BL and 020 strains, the hormonal treatment resulted in elevated levels of these viral proteins in the mammary glands, while the tumors contained very low, hardly detectable amounts. If tumors were induced in BALB/c mice by a combination of urethane and X rays, there was a marked increase in viral proteins in the mammary glands but not in the tumors. It may be concluded from these findings that the nonviral promotion of mammary tumor development in the low cancer strains is accompanied by an elevated expression of the genome of MuMTV in the mammary glands. Sometimes the tumors obtained in this manner contain little or no detectable virus, suggesting the possibility that MuMTV antigen expression in mammary gland cells is transient or that cell types which do not contain MuMTV proteins proliferate to form the tumors. Apparently, there can be transient expression of viral proteins without tumor development and also tumor development without viral expression, e.g., BALB/c mice with a mammary tumor incidence of 27% with no detectable expression of MuMTV antigen (Moore et nl., 1974).

E. MuMTV INFECTIONOF CELLSi l l Vitro A major block in the investigations on MuMTV has been the inability to infect and transforni cultured cells. Although attempts to infect cells in vitro with MuMTV have been made for a long time, it was not until recently that successful infection was achieved. Lasfargues et ul. ( 1974) presented electron microscopic evidence showing that a feline

MAMMARY TUMOR VIRUSES

381

kidney cell line, when experimentally infected with MuMTV, contained B-type particles budding at the cell membrane. In the past few years, a number of other nonmurine cell lines have been found to be susceptible to MuMTV infection; these are mink lung cell line CCL64 (Vaidya et al., 1976; Lasfargues et al., 1976b; Howard et al., 1977),rat hepatoma cell line HTC (Ringold et al., 1977a), and human breast tumor cell line BT-474 (Vaidya and Lasfargues, manuscript in preparation).Recently, an epithelial cell line from C57BL/6 mouse mammary glands has also been found to be susceptible to MuMTV (Vaidyaet al., 1978). In all cases, an unusually high multiplicity of infection (m.0.i.)-about lo5 virions/cell-was required for optimum infection. However, it appears that epithelioid cells from a number of species can support replication of MuMTV. Fibroblastic cells, on the other hand, have not yet been reported to be susceptible to infection. A number of attempts to infect fibroblasts from various species and some human lymphoid cell lines were unsuccessful (Lasfargues and Vaidya, unpublished observations). The reasons for the apparent resistance of mesothelial cell cultures to MuMTV infection are not known. Following infection, MuMTV proviral DNA can be detected within a few hours (Vaidya et al., 1976). Since the infection is not very efficient, however, and since a very high m.0.i. is required, a more detailed analysis of MuMTV proviral DNA synthesis has not been possible. Infected cells carry stable MuMTV provirus in multiple copies (Vaidya et al., 1976; Ringold et al., 1977a,c; Howard et al., 1977). In addition, chronically infected nonniurine cells seem to contain unintegrated MuMTV proviral DNA, and these molecules are present in linear as well as covalently closed circular forms, the latter being located predominantly in the nucleus (Ringold et al., 1 9 7 7 ~ )The . linear form of unintegrated MuMTV DNA has a genomic length “minus” strand and a subgenomic “plus” strand, indicating that these molecules are perhaps the product of reverse transcription of the viral RNA (Ringold et al., 1977~). The presence of unintegrated viral DNA niolecules in chronically infected nonmurine cells appears to be dependent upon stimulation of MLIMTVRNA synthesis by glucocorticoids (Ringold et al., 1978). Mouse cell lines producing MuMTV, on the other hand, do not seem to accumulate unintegrated viral DNA, even after the glucocorticoid-induced increase in the amount of intracellular MuMTV RNA (Ringold et al., 1978). Vaidya et (11. (1978) reported that in vitro infected mouse epithelial cells also do not seem to accumulate unintegrated MuMTV DNA when treated with dexamethasone. These results are comparable to the avian system in which quail and duck cells chronically infected with avian sarcoma virus (ASV) carry unin-

382

DAN H. MOORE

et

NI.

tegrated proviral DNA but ASV-infected homologous chicken cells do not (Guntaka et al., 1976). MuMTV-infected cells in culture are engaged in the synthesis of viral RNA and proteins, and mature virions are released by the cells (Vaidya et al., 1976; Ringold et ul., 1977a; Howard et al., 1977). Infected feline and mink cell lines release large numbers (50-300 virions/cell/day) of MuMTV into the culture medium (Vaid'ya et al., 1976; Howard et ul., 1977). Infected rat hepatoma cells, however, release very few virions (Ringold et aZ., 1977a). In all these cells, the amount of intracellular MuMTV RNA increases dramatically with the addition of the synthetic glucocorticoid dexamethasone (Vaidya et al., 1976; Ringold et ul., 1977a), just as it does in MuMTV-producing mouse mammary tumor cells (Ringold et al., 1975a,b; Parks et al., 1975a,b). This phenomenon has important implications which will be described in Section V1,F. MuMTV infection of heterologous cells does not seem to impart any morphologic or growth characteristic changes to these cells. However, since MuMTV is able to produce neoplasms of only a specialized organ, the lack of such changes in heterologous cells is not surprising. Recently, an epithelial cell line established from the mammary glands of a C57BL/6 mouse (designated C57MG cell line) was found to be susceptible to infection with MuMTV(RII1) (Vaidya et d.,1978). C57MG cells do carry endogenous MuMTV DNA, but this information is not expressed, since no viral RNA can be detected. Following infection with MuMTV, C57MG cells acquire additional proviral sequences and viral RNA, proteins, and mature virions are synthesized by chronically infected cells (Vaidya et al., 1978). This productive in vitro infection of a putative in vivo target cell for MuMTV does not appear to alter growth or morphologic characteristics of the cells. However, such in vitro correlates for malignant transformation, which were established for cultured fibroblastic cells, need not always apply to epithelial cells. Other means to detect transformation of epithelial cells must be developed and evaluated to study the effects of MuMTV infection on mammary cells in culture. Finally, the infection process of MuMTV in homologous (Vaidya et al., 1978) as well as heterologous cells (Vaidya et aZ., 1976; Lasfargues et al., 1976a; Ringold et ul., 1977a; Howard et al., 1977) appears to be very inefficient. Extremely high m.0.i.s are required to achieve optimum level of infection. The reasons for this inefficiency are unknown. Recently, ZLvada et al. (1977) obtained phenotypically mixed vesicular stomatitis virus (VSV) b y infecting MuMTV-producing cells

MAMMARY TUMOR VIRUSES

383

with VSV. These pseudotypes, carrying the VSV genome surrounded by MuMTV envelope glycoproteins, were able to induce typical VSV plaques in mink lung and normal mouse mammary gland cells (epithelial) but not in Vero or BALB3T3 (fibroblastic) cells. The findings of Zavada et (11. (1977) indicate that penetration of cells by a viruscarrying MuMTV envelope proteins is relatively efficient in MuMTVsusceptible cells. Therefore, the inefficiency observed for MuMTV infection of cells in uitro may be due to a postpenetration restriction in the life cycle of the virus.

F. CONTROLOF MuMTV GENEEXPRESSION Mammary tumors of mice are limited mainly to the females of high mammary tumor strains, although infected males treated with feminizing steroids may also develop mammary tumors. In some strains of mice, such as the A strain, virgin mice have a low incidence of tumors, while breeders develop mammary tumors with high frequency. Mice from strains such as GR show fluctuations in the growth patterns of their mammary tumors dependent upon changes in hormonal milieuthe growth of tumors being greatest during pregnancy, while they often regress between pregnancies. The interplay of hypophysial and ovarian hormones regulating the development and growth of mammary tumors in mice has been reviewed previously (Nandi and McGrath, 1973);therefore in this section, only the control of MuMTV gene expression will be discussed. Although all mouse cells carry DNA sequences for MuMTV, the main site for MuMTV production is the mammary glands of high mammary tumor strains. Varmus et al., (1973), using molecular hybridization techniques, conducted a quantitative survey of MuMTV RNA in various tissues and tumors from high and low tumor incidence strains. They found that large but variable amounts of MuMTV RNA were present in lactating mammary glands and mammary tumors of mice of strains RIII, C3H, GR, and DBAIB. Mammary tumors and lactating mammary glands of BALB/c mice, on the other hand, contained extremely small amounts of MuMTV RNA. The endogenous MuMTV information of low mammary tumor strains such as C57BL/6, 129, and C58 was at least partially transcribed into RNA since MuMTV-related RNA could be detected in the lactating mammary glands of these mice; however, viral antigens or assembled virions are not observed in these tissues. In organs other than the mammary glands or tumors, e.g., liver and spleen, viral RNA is detected in ex-

384

DAN H. MOOHE

et nl.

treniely small amounts. In murine neoplasms of nonmammary gland origin, MuMTV antigens, virions, and RNA are occasionally detected. Most notable among these are DBAI2 lymphomas and Leydig cell tumors (Noweinski et d.,1967, 1971; Hilgers et nl., 1972; Varmus et d.,1973). However, there has not been any indication of an etiologic link between MuMTV and these tumors. From the above description, it is obvious that in mice, MuMTV gene expression is controlled at both the transcriptional and translational levels. Both hormonal and genetic factors play important roles in the control of MuMTV gene expression. The systems to investigate MuMTV gene expression have become available only in the past few years. In vitro cultivation of MuMTV-producing cells provided means to study viral replication under more defined and reproducible conditions. This, combined with molecular hybridization and radioimmunoassay techniques to detect and quantitate viral nucleic acids and proteins, has given important information about MuMTV gene expression. McGrath and co-workers (McGrath, 1971; McGrath et al., 1972) reported that inclusion of hydrocortisone and insulin in the growth medium resulted in increased MuMTV production in primary explants of mammary tumors. Such an increase in MuMTV production by primary tumor explants, as judged by electron microscopy, immunofluorescence, RNA-directed DNA polymerase measurements, and radioinimunoassay, was also reported b y others (Dickson et al., 1974; Shyamala and Dickson, 1976; Young et ul., 1975). Several continuous cell lines established from mammary tumors of mice are now available and virus production by these cell lines can be increased by glucocorticoids, particularly the synthetic steroid dexamethasone (Fine et al., 1974; Parks et al., 1974b; Ringold et al., 1975a,b). Hormones such as estrogen, progesterone, and insulin may influence differentiation of cultured mammary cells and may be involved in primary induction of MuMTV genes. But dexamethasone has a dramatic effect on MuMTV production of virus-producing cells, causing an increase ranging from 5 to 50-fold. Because of this, the effect of glucocorticoids on MuMTV gene expression in cultured cells has been more extensively investigated. Two reviews have appeared on this subject (Yamamoto and Ringold, 1977; Varmus et al., 1979) giving a more detailed description of the phenomenon. The intracellular concentrations of MuMTV RNA and proteins are stimulated by glucocorticoids, and the extent of stimulation is correlated fairly well with the glucocorticoid-mediated increase in virus production (Parks et al., 197417, 1975a,b; Ringold et al., 1975a,b; Scolnick et d., 1976). The increase in intracellular viral RNA can be de-

MA hlMA R Y T U h 1 0 R VIRUSES

385

tected within 30 minutes after glucocorticoid treatment (Ringold et

al., 1975b; Scolnick et al., 1976). Evidence has been presented showing that the higher level of MuMTV RNA is mainly due to the enhanced rate at which viral genes are transcribed rather than the possible steroid-mediated retardation in the degradation of constitutively produced MuMTV RNA (Young et d . , 1977; Ringold et d., 197711). In MuMTV-infected mink and cat cells, the intracellular viral RNA concentration is increased by dexamethasone (Vaidya et a1., 1976). This has also been observed for infected rat hepatoma cells (Ringold et nl., 1977a).Vaidya et (11. (1978)reported that, although the endogenous MuMTV genes of an epithelial cell line from C57BL/6 mammary glands were unexpressed and not inducible by dexamethasone, the same cells when infected with MuMTV showed increased concentrations of viral RNA after treatment with dexamethasone. Ringold e t (11. (1978) have shown that in infected rat hepatoma cells, the increased amount of MuMTV RNA is due to an enhanced rate of viral RNA synthesis. The increased synthesis of MuMTV RNA by cells treated with glucocorticoids occurs without any significant effect on total cellular RNA synthesis (Young et ul., 1977; Ringold et al., 1977b), indicating specificity of the effect. Steroids are believed to act through their allosteric effect on specific cytoplasmic receptor proteins. The steroidreceptor complex then interacts with steroid-responsive sites on chromatin, modulating the genes present therein. Mouse mammary gland cells contain glucocorticoid receptor proteins having a high 1975).The extent binding affinity (Shyamala, 1974, 1975; Young et d., to which a glucocorticoid is able to stimulate MuMTV synthesis correlates well with its affinity for cytoplasmic receptors (Shyamala and Dickson, 1976; Young et al., 1975), with dexamethasone having maximum stimulatory effect while aldosterone has minimal activity. The effect of glucocorticoids on MuMTV gene expression has been observed in a variety of cells, including infected heterologous cells. This indicates that either the MuMTV provirus integrates at a site(s) adjacent to DNA sequences that are recognized by the glucocorticoidreceptor complex, or that such recognition sequences are present in the proviral DNA itself. It is possible to study the specific interaction between glucocorticoid receptors and their target sequences using the MuMTV system, since the proviral DNA and its adjacent sequences can be isolated. It is hoped that studies using this system will provide important information about hormone-mediated control of gene expression.

386

DAN H. MOORE

et ul.

VII. Antigens of MuMTV

A. IMMUNOLOGIC TECHNIQUES Immunodiffusion, passive hemagglutination, immunofluorescence, immunoelectron microscopy, and radioimmunoassay (RIA) have been the principal immunologic techniques used to detect MuMTV antigens or antibody to these antigens. Once it became evident that MuMTV was immunogenic in heterologous species, antisera to the virus were prepared for use .in these tests. One difficulty with such sera is that the viral antigen preparations were often contaminated with nonviral host components yielding antisera requiring absorption. Moreover, these antisera are polyvalent and may or may not contain antibodies to the internal virion components, depending on the extent of disruption of the antigen preparation. Many anomalous results in immunologic studies with MuMTV have probably been due to the complex antigen-antibody systems employed and the lack of specificity of the reagents. These difficulties have been partially resolved by the purification of MuMTV polypeptides and the preparation of monospecific antibody against them. However, the specificity of these antisera must still be demonstrated. Many investigators have used immunodiffusion to detect MuMTV antigens by precipitation in gels (Blair, 1965, 1969; Nowinski et ul., 1967, 1968; Charney et ul., 1969). To obtain semiquantitative data, several workers have developed passive hemagglutination assays by coupling MuMTV to erythrocytes. This allows measurement of specific anti-MuMTV antibody (Fink et al., 1968) or MuMTV antigen by inhibition of hemagglutination (Sibal et al., 1969; Gillette and Junker, 1973). However, these techniques have not been extensively used for MuMTV research. In contrast, immunofluorescence has been more widely applied to the detection of MuMTV antigens in tissues and cells (McGrath and Blair, 1970; Daams et ul., 1970; Daams, 1970; Hilgers et ul., 1971a, 1972, 1973). Progress in the development of quantitative assays for MuMTV antigens and antibodies to these antigens awaited the application of RIA procedures and the concomitant purification and characterization of MuMTV polypeptides. Radioimmune precipitation techniques have been applied to labeled whole virions (Cardiff, 1973; Lo Gerfo et ul., 1974; Ihle et al., 1976), iodinated virions which were subsequently disrupted (Verstraeten et al., 1973, 1975), and labeled purified viral polypeptides (Parks and Scolnick, 1973; Parks et al., 1974a; Noon et ul., 1975; Ritzi et al., 1976a; Sheffield et ul., 1977; Zangerle et ul.,

MAMMARY TUMOR VIRUSES

387

1977). Most of the investigators cited have established that their assays do not measure C-type antigens, thus confirming the antigenic uniqueness of the mouse mammary tumor virus. Those RIAs measuring whole virions have generally used purified virus labeled externally with lZ5Iusing lactoperoxidase. Several investigators (Witte et al., 1973; Cardiff, 1973; Teramoto et al., 1974) have reported that this procedure labels predominantly gp52. It is likely that the use of intact virions preserves the antigenic configuration of the viral surface, a fact which may be significant in the search for type-specific antigenic determinants (Teramoto et al., 1977c) or autogenous antibody responses to MuMTV antigens (Ihle et al., 1976). Other investigators (Verstraeten et a1., 1973, 1975) iodinated MuMTV by the chloramine T procedure and subsequently extracted it with ether, yielding radiolabeled antigens in two phases. However, since the viral antigens thus fractionated have not been completely characterized, the specificity of RIAs using these antigens is undefined. RIAs with labeled purified polypeptides offer the advantages of defined antigens so that the specificity of antibody responses to MuMTV can be tested and the identity of unknown viral antigens can be determined by competition assays. The major external glycoprotein has been primarily selected for quantitative assays (Parks et al., 1974a; Ritzi et ul., 1976a; Sheffield et al., 1977; Zangerle et al., 1977), but p28 (Parks et al., 1974a) and p12 (Noon et al., 1975) have also been labeled. Using competition assays, 0.05 to 1.5 ng of gp52 can be detected. Parks et (11. (19744 experienced problems with their gp52 assay, since biologic materials containing the antigen were unable to completely displace all of the antibody from labeled antigen. They attributed this to confomiational shifts in the preparation of the antigen. However, others who have labeled gp52 either with chloramine T or the Bolton-Hunter reagent have not had similar difficulties. In the same report, Parks and co-workers also indicated that assays with purified p28 appeared to be of minimal applicability, since the iodinated protein rapidly lost immunologic activity.

B. ANTIGENICCHARACTEFUZATION OF MuMTV AND COMFQNENTS 1. M u M T V und Its Polypeptides

All immunologic techniques tested-precipitation, fluorescence, RIA, inhibition of RDDP activity-have confirmed molecular biological evidence that MuMTV virions, MuMTV polypeptides, including the reverse transcriptase, and intracytoplasmic A particles are distinct

388

DAN H. MOORE

et a1.

from the C-type oncomaviruses. Information from RIA has also shown that the Mason-Pfizer monkey virus (MPMV) does not bear antigenic similarity to MuMTV (Yeh et ul., 1975). Finally, antigenic analysis has demonstrated that the intracytoplasmic A particles of mice are a different population than the intracisternal A particles often found in plasmacytomas, neuroblastomas, and other mouse tumors (Kuff et ul., 1972; Tanaka et al., 1972). It has been apparent for some time that MuMTV contains a number of antigenic components as illustrated by the multiple precipitation lines observed in Ouchterlony gels with disrupted virions and polyvalent antisera. A combination of many techniques has since clarified the number of MuMTV polypeptides, their location within the virion, and their immunologic relationship. The first distinct subunit of the mammary tumor virus to be described was a soluble antigen designated by Nowinski et al. (1967) as s l . As demonstrated by immune precipitation techniques, the s l antigen occurred in ether-treated virions and in milk and mammary tumors of mouse strains recognized to be MuMTV-positive. Since it could be found in all infected strains, they termed it a group-specific antigen (Nowinski et ul., 1968). It has subsequently been shown that the s l antigen is the ,same as the gp52 glycoprotein isolated by biochemical techniques (Nowinski et al., 1971; Sarkar and Dion, 1975). The other major virion polypeptide is p28, a component common to MuMTV virions and A particles from a number of strains. Virions treated either with proteases or nonionic detergents lose their glycoproteins but retain p28, which seems to be an internal component (see Section 111,B). This protein is not antigenically related to gp52 by immunodiffusion (Parks et al., 1974a; Sarkar and Dion, 1975; Zotter et al., 1976a). Some minor cross-reactivity between these proteins has been reported using RIA techniques (Parks et al., 1974a; Zangerle et al., 1977), but this is most likely due to cross-contamination. The lower molecular weight polypeptides of MuMTV are not as well characterized. However, Noon et al. (1975) found that gp52 and p28 are unrelated to the small proteins in an RIA measuring primarily p12. Using a polyvalent antiserum to A particles, Tanaka (1977) obtained reactions of nonidentity in immunodiffusion tests between p28, p18, and p12 eluted from SDS-PAGE gels. Zangerle et al. (1977) stated that p12 did not compete in an RIA for gp52. Serologic information of another sort has come from studies on leukemias of mice. Using cytotoxicity and cytotoxic absorption tests, Stiick et ul. (1964) described an antigen present on several leukemias of DBN2 mice and in mammary tissue and mammary tumors of

MAMMARY TUMOR VIRUSES

389

MuMTV-infected C3H, A, and DBA/2 strains. This antigen was detected with a mouse antiserum and was denoted ML, for mammary leukemia. Subsequent studies by Nowinski et al. (1967, 1971) confirmed that the ML antigen was indeed related to the mammary tumor virus, in particular the s l antigen. Recently, Zak-Nejmark and colleagues (1978) have reported the isolation of M L antigen from L1210 leukemia cells and its purification both by PAGE and by affinity chromatography. In both cases, the protein thus isolated migrated as a 73,500-dalton band on SDS-PAGE and may possibly represent an uncleaved glycoprotein precursor. In 1975, Hilgers et al. suggested that the ML antigen might be identical to MuMTV gp52.

2 . Type-Speci.cit!lStudies of MuRITVs front Vcirious Mouse Strciiiis As discussed in Section I, attempts to distinguish MuMTVs from different sources and thus develop a classification scheme have primarily relied upon biological functions of the virus. Because of the exquisite sensitivity and specificity of immunological reactions, a variety of assays have been employed to search for antigenic variants of ML~MTV.Early investigations used neutralization, cross-imniunization, or imniunodiffiision to compare viruses from different mouse strains. Blair ( 1970) reported some evidence for antigenic variation by the formation of spurs in immunodiffusion when different mouse milks were tested against a series of rabbit antisera. To control for host differences, all test viruses were obtained from BALB/c mice which had been foster nursed on a variety of other MuMTV-carrying strains. It is therefore possible that the infecting virus recombined with the endogenous virus of the BALB/c host or that infection induced the expression of these endogenous genes. While viruses from most strains showed considerable antigenic similarity, Blair reported that the MuMTVs derived from wild mice and from C3Hf mice lacked an antigenic determinant present on MuMTV(C3H). She extended these studies (Blair, 1971a) by absorbing a series of antisera with several MLIMTVantigens and testing the absorbed sera against MuMTV from four different mouse strains. Type-specific antigens were described for MuMTV(C3Hf) and MuMTV(fera1 mice), as well as other antigens which were common to some but not all of the MuMTV types she tested. However, detection of such differences by spur formation in immunodifision is, as Blair acknowledged, “a hazardous procedure,” especially when the whole virion is diffusing through the agar, and other investigators have not identified such differences. A more sensitive, quantitative, and refined search for type-specific differences awaited the development of techniques such as RIA.

390

DAN H. MOORE

et

(11.

However, most studies using these assays have revealed only groupspecific antigenic reactivity common to all the MuMTVs tested (Cardiff, 1973; Verstraeten et d., 1973, 1975; Parks et d., 1974a; Noon et (zl,, 1975; Kimball et ul,, 1976; Sheffield et al., 1977). In contrast, Teramoto et al. (1977b,c) have reported that both group- and typespecific antigenic determinants are present on gp52. These workers compared three different types of MuMTV (GR, C3H, and RIII) obtained either from virus-producing mammary tumor cells in culture or from infected heterologous feline kidney cells in a competition RIA with labeled whole virions. Although anti-MuMTV(RII1) sera was able to precipitate more than 90% of 1251-labeledMuMTV(RIII) or 1251-labeledMuMTV(C3H) virions, unlabeled MuMTV(C3H) virus or gp52(C3H) competed with lZ5I-labeledMuMTV(RII1) with a slope different from that of the homologous RIII virion or glycoprotein. In contrast, when the same anti-MuMTV(RII1) serum was tested against 1251-labeledMuMTV(C3H), a system presumably measuring groupspecific determinants, both types of virions and their glycoproteins were indistinguishable in competition assays. Similar results were obtained with MuMTV(GR) virus in tests with the anti-MuMTV(RII1) sera, indicating that more GR protein than RIII protein was required to achieve a comparable level of displacement of 1251-labeled MuMTV(RII1) derived from homologous or heterologous cells. In reciprocal experiments using anti-MuMTV(C3H) sera, similar differences were noted between the three viruses. In fact, MuMTV(GR) was incapable of completely inhibiting the binding of the antibody to labeled MuMTV(C3H),even at the highest concentration of virus tested. This pattern of reactivity was amplified b y absorption of the anti-MuMTV(C3H) serum with MuMTV(GR), making it unreactive with MuMTV(GR) but still able to bind MuMTV(C3H). Teramoto and colleagues (1977~) have also reported that they are able to distinguish endogenous and milk-transmitted MuMTVs from C3H mice, using similar techniques. Ideally, the viruses to be tested by competition assays should all be derived from a heterologous cell to eliminate any possible effects of endogenous MuMTV information or host cell modification of virus determinants. Recent progress in infecting heterologous cells in vitro with MuMTV from several strains has made such experiments possible (see Section V1,E). Teramoto and co-workers have incorporated such techniques into their type-specificity studies and reported that similar differences in competition assays exist whether the viruses are derived from murine cells or from feline kidney cells. In an attempt to account for the fact that other investigators have not found such type-

MAMMARY TUMOR VIRUSES

391

specific differences, Teramoto et c i l . ( 1 9 7 7 ~have ) suggested that assays with the labeled whole virion rather than the isolated glycoprotein magnify type-specific differences, although they can be identified in both assay systems. Moreover, serum antibodies recognizing typespecific determinants may be rather low in titer, since type-specific differences apparent at higher concentrations of antibody disappear with further dilution, although group-specific antibodies can still be detected. The dilution of antiserum selected for an assay may therefore be important. They also have noted that type-specific differences can be magnified by absorbing these sera with cross-reacting virions to remove the group-reactive antibodies, leaving only the ones recognizing type-specific determinants. Further progress in this area depends upon the confirmation and extension of these interesting results as well as on a more detailed biochemical characterization of the polypeptides from different MuMTVs.

3. Ant igeriicit y of 1tit rclcy t op l usmic A Part icles Based on electron microscopic studies of cells producing mammary tumor virus, Bernhard (1958) and others have suggested that intracytoplasniic A particles represent the precursors of MuMTV B particles. Since then, considerable progress has been made in characterizing intracytoplasmic A particles using biochemical and imniunochemical techniques. Nowinski et (11. (1967, 1971) studied ML-positive leukemias and Leydig cell tumors which did not produce B-type particles but had large accumulations of intracytoplasmic A particles. As judged by immunofluorescence and immunodiffusion, such cells shared antigenic determinants ( s l and s2) with B particles. In retrospect, it appears likely that the antigen detected was the major virion glycoprotein, which is unrelated to antigens of purified A particles. Direct evidence for an antigenic relationship was obtained by Tanaka and colleagues ( 1972), who isolated and purified intracytoplasmic A particles from spontaneous leukemias and mammary tumors of DBN2 mice and prepared antisera in rabbits to them. Immunodifhsion tests revealed an antigen common to B particles from mammary tumors of several, possibly all, mouse strains and A particles either from mammary tumors or leukemias. These investigators designated the antigen as MTV-a and suggested that it is an internal component of B particles. While A particles formed only a single precipitin arc in immunoelectrophoresis using the anti-A serum, B particles split into two arcs, suggesting that the MTV-a antigen of A particles appears in the mature virion as two components. Other investigators have also verified an immunologic

392

DAN H. MOORE et

d.

relationship between A particles and B virions (Smith and Wivel, 1973; Zotter et al., 1976~). To complement these studies, various investigators have contributed to an understanding of the polypeptide composition and maturation of A particles. Sarkar and Dion (1975) compared the antigenic properties of the two major virion proteins gp52 and p28 with A particles from leukemias by immunodiffusion. Antiserum to A particles gave a reaction of identity between A particles and p28 from RIII milk virus while anti-gp52 or anti-sl gave no reaction with A particles. Other investigators have verified this antigenic relationship between intracytoplasmic A particles obtained either from Leydig cell tumors or mammary tumors and p28 of B particles, using immunodiffusion (Smith and Lee, 1975) or indirect immunofluorescence (Zotter et ul., 1976a). However, since Smith and Lee (1975) could not find any polypeptide in A particles from Leydig cell tumors with the molecular weight of p28 using SDS-PAGE, they treated these A particles with trypsin and produced a polypeptide which comigrated with p28. Tanaka (1977) has extensively studied the maturation of A particles by comparing their polypeptide composition to the internal proteins of B particles. These studies indicate that A particles contain a large precursor (A-p70) which is cleaved enzymatically to yield the three internal, nonglycosylated proteins of B particles. Using a combination of SDS-PAGE and immunodiffusion, Tanaka showed that the A-p70 which was eluted from gels of A particles contained all antigenic determinants present in the three major internal proteins of B particles-p28, p18, and p12. Moreover, he showed that the polypeptides A-p28 and A-p12 ofA particles from the DBN2 leukemic cells are antigenically identical to the polypeptides of the same molecular weight of MuMTV(RII1) B particles. Sarkar and Whittington (1977) have also confirmed an immunologic relationship between intracytoplasmic A particles and internal B polypeptides. Rabbit antisera to A particles from Leydig cell tumors precipitated iodinated p28, p18, and p12 of disrupted MuMTV B particles. Such evidence that A particles contain internal proteins of the MuMTV virion supports the thesis that intracytoplasmic A particles are the pronucleocapsids of B particles.

c. OTHER APPLICATIONS OF IMMUNOLOGIC TECHNIQUES 1. Detection of Viral Antigen in Milk and Plasma of Mice As might be expected, immunodiffusion using specific antisera to the mammary tumor virus was applied to the detection of virus or viral

hlAhlhlARY TUMOR VIRUSES

393

antigen in the milks of infected mice. Blair (1969) described a gel diffusion assay in which intact virions diffused through the agar. She reported that the number of BALB/cfC3H mice expressing virus in their milk increased up to the third lactation and that nearly all of the MuMTV-positive mice later developed mammary tumors. As discussed above, Nowinski et (11. (1967, 1968) used an Ouchterlony assay to identif). the s l antigen in milks of mice. Charney et al. (1969) used a similar microimmunodiffusion technique to obtain a dose-response infectivity curve for C57BLJ6 mice inoculated with various dilutions of MuMTV(RII1). These authors further showed that the infectivity could be abolished by neutralization of the inoculum with specific rabbit anti-MuMTV(RII1). Since then, various RIA techniques have been applied to assay MuMTV antigens in mouse milks (Verstraeten et ul., 1973, 1975; Parks et ul., 1974a; Lo Gerfo et ul., 1974; Noon et al., 1975; Ritzi et al., 197613; Sheffield et ul., 1977). While concentrations of viral antigens in milk appear to vary considerably between individual mice of the same strain (Verstraeten et al., 1975; Sheffield et d . , 1977), there seems to be a correlation between expression of gp52 and p12 (Noon et al., 1975). It should be noted that quantitation of gp52 in milks may be subject to variability contributed by a number of factors, including parity; number of days after parturition that the milk is collected; different amounts of gp52 present in virions obtained from different strains; and detachment of gp52 from the virion envelope. Several investigators have made use of both immunodiffusion and RIA to follow the genetic regulation of MuMTV expression in several mouse strains (for a review see Hilgers and Bentvelzen, 1978). Van Nie and Verstraeten (1975)took advantage of the sensitivity of RIA to measure viral antigen in the milks of individual low-mammary tumor C3Hf mice as well as the progeny of hybrids and backcrosses with BALB/c mice. They report that their antigen incidence data are consistent with a single gene, denoted ( M t v - I ) , being responsible for milk expression of MuMTV(C3Hf). They used a similar approach to study the genetic transmission of MuMTV(GR) and described a gene (Mtu-2) responsible for the early expression of MuMTV in the milk of GR mice (van Nie et n l . , 1977; van Nie and de Moes, 1977). However, similar data obtained by Heston and Parks (1975, 1977) and Hester et al. (1976) are not compatible with a simple Mendelian transmission scheme, and they suggest that multiple genetic factors regulate expression of the MuMTV genome in GR mice. Recently, RIAs for MuMTV have been used to quantitate plasma levels of viral antigen in mice and to correlate such data with the presence of a tumor (Verstraeten et al., 1975; Ritzi et al., 197613;

394

DAN H. MOOFW et

al.

Zangerle et al., 1977). Only low levels of gp52 (2-10 ng/ml) were found by Ritzi et al. (1976b) in males and females of both low and high tumor mouse strains, even when the animals were lactating and producing large quantities of virus in their milks. In contrast, plasma concentrations of gp52 were markedly elevated (100-1000 ng/ml) in tumor-bearing mice. However, such elevated levels of plasma antigen did not appear before a palpable tumor. A similar correlation had been observed earlier by Verstraeten et al. (1973), using an incompletely characterized RIA for MuMTV. However, they reported that in certain foster-nursed hybrid mice not tested by Ritzi et al. (1976b), some tumor-bearing females did not have detectable concentrations of circulating viral antigens. Using an RIA for gp52, Zangerle et (11. (1977) found differences between the serum levels of tumor-bearing females and isogenic males, although the titers varied widely and no tumorfree females from high tumor strains were tested. 2. Identijication of MuMTV Antigens in Cultured Cells and Tissues Immunologic reagents have also been employed in the identification of MuMTV antigens in cells in culture. Such antigens were detected by indirect immunofluorescence in the cytoplasm of in vitro BALB/cfC3H mammary tumor cells by McGrath and Blair (1970),who observed that the viral antigens were associated primarily with epithelioid mounds of cells. Cytoplasmic as opposed to nuclear production of MuMTV was verified by Hilgers et al. (1971a, 1972), who were able to correlate the presence of MuMTV-fluorescence in a number of cell lines with production of B particles by electron microscopy. More recently, RIAs have proved useful in quantitating viral antigen produced by cells in culture, including the characterization of various clones of tumor cells with differential virus production, the verification of molecular biological evidence that glucocorticoids stimulate virus production in vitro and the demonstration of coordinate expression of gp52 and p12 in cultured producer cells (Parks and Scolnick, 1973; Parks et al., 1975a; Noon et al., 1975; Kimball et al., 1976; Fine et al., 1976). As discussed above, immunofluorescence has also been used to determine whether cells in culture have been infected in vitro by MuMTV, as judged by MuMTV antigen expression (Lasfargues et al., 1976a,b). In some cases, virions produced by cells infected in vitro have been characterized by RIAs (Howard et al., 1977; Vaidya et al., 1978). Immunologic techniques have been used to study the distribution of MuMTV antigens on various murine tissues and cells. By immunodiffusion, immunofluorescence, and RIA, MuMTV was shown to be pres-

M.421bIARY TUMOR VIRUSES

395

ent in mammary gland cells and mammary tumors of MTV-infected, high tumor strains (Blair, 1965; Nowinski et al., 1967; Daams et al., 1970; Daams, 1970; Hilgers et aZ., 1972, 1973; Parks et nl., 1974a; Gillette et al., 1974; Noon et d.,1975; Verstraeten et al., 1975; Zangerle et al., 1977). Older reports in the literature of MuMTV in seminal vesicles have been confirmed by the finding of MuMTV virions or antigens in the epididymis (Daams, 1970; Verstraeten et al., 1975); in the seminal vesicles (Zangerle et al., 1977); and in Leydig cell tumors (Hilgers et nl., 1972). Moore et (11. ( 1 9 6 9 ~found ) MuMTV antigens in a transplantable ependymoblastoma. Others have reported MuMTV antigens in the spleens of some, but not all, high mammary tumor strains (Daams et al., 1970; Daams, 1970; Hilgers et aZ., 1972; Zangerle, 1977). Gillette et aZ. (1974) examined MuMTV antigens on niouse lymphoid cells measured by immunofluorescence and by a 51Crrelease cytotoxicity assay. These investigators found greatest MuMTV expression in the spleen, lymph nodes, and peritoneal cells, but very little in the thymus or fetal liver. Enrichment ofspleen.cel1 preparations for B cells increased the percentage of fluroescing cells, suggested that MuMTV antigen expression is primarily in B cells. Furtheimore, the frequency of antigen-positive cells in the spleen increased with age. In contrast to the results of Daams, Hilgers, and Zangerle (cited above), all mouse strains, including low mammary himor C57BL and BALB/c, possessed positive cells. This raises the possibility that some of the antigens detected may be host related, and such discrepancies must be resolved by more defined and sensitive techniques. VIII. Interaction of MuMTV with the Host's Immune System

MuMTV-induced mammary tumors have long been recognized to be immunogenic in syngeneic virus-free hosts and to possess crossreactive antigens detectable in transplantation-challenge experiments. Transplantation immunity to MuMTV antigens has been much more difficult to demonstrate in mice neonatally infected with the virus. These results gave rise to the concept of MuMTV-specific tolerance and have been reviewed by Blair (1968b, 1971b). Data illustrating this point have been obtained by Vaage and Medina (1974), using C3H and syngeneic C3Hf mice. All of the MuMTV-containing C3H tumors tested in this study were immunogenic in C3Hf hosts, since implantation and excision of the tumors resulted in resistance to a subsequent challenge. In contrast, only about 30% of these spontaneous tumors affected the progressive growth of a subsequent tumor

396

DAN H. MOORE

et al.

challenge in virus-burdened C3H mice. In a further survey of 100 spontaneously arising tumors in C3H mice, Vaage (1978)reported that perimmunization with 57 of the tumors elicitecf some transplantation resistance in C3H hosts. In the few cases tested, these tumors did not cross-immunize. This implies that tumor transplantation antigens not related to MuMTV may also be recognized by the host, a subject which will not be discussed in this review. Of the remaining 43 tumors, 21 had no significant effect on the growth rate of a subsequent challenge, and implantation and excision of 22 tumors actually promoted growth of the challenge. The in uivo interaction of an oncornavirus with the immune system of its host is extremely complex, and most of the recent studies have consequently focused on newer in vitro techniques, many of which have developed as part of the rapid growth of cellular immunology. Therefore, the humoral and cellular immune responses of mice to viral antigens as detected by a variety of in vitro techniques will be considered in this section. Such studies have uncovered a whole spectrum of responses to MuMTV in hosts with both low and high mammary tumor incidences. The interaction of these responses in, vivo and their relationship to the overall response of the animal to MuMTV and tumors induced by the virus are not yet completely understood. The effect of MuMTV infection on the immune responses of the host to heterologous antigens will also be reported, and a summary of attempts to immunize mice with the virus is included.

A. HUMORALIMMUNERESPONSES TO MuMTV Although early investigations of the immune response of neonatally infected mice suggested that MuMTV was not antigenic, subsequent studies revealed that precipitating or neutralizing antibodies to MuMTV antigens could be induced in mice by various immunization procedures (Blairet al., 1966; Nowinski et al., 1967; Blair, 1968a; Fink et al., 1968; Holmes and Morton, 1969; Hilgers et al., 1971b). Moreover, the lack of tolerance to these antigens has been further established by the finding that infected mice often develop spontaneous antiviral antibodies. Bentvelzen et al. (1970b) described such humoral reactivity in mammary tumor-bearing C3H mice. Spontaneously occurring antibodies to MuMTV were also found by immunoprecipitation techniques in nontumor-bearing multiparous females of several mouse strains, including fostered substrains (Muller et al., 1971). In latter studies, Muller and Zotter (1973) tested mouse serum for anti-

MAMMARY T U M O R VIRUSES

397

body using an indirect immunofluorescence technique and reported antibody to MuMTV in both males and females of virus-infected CBA and SVIIfCBA mice but not in uninfected syngeneic mice. This reactivity appeared at about the fourth week in life in CBA mice and increased with age in both virgins and breeders (Zotter et ul., 1973). Other investigators have also described autogenous antibodies in tumor-bearing and some tumor-free infected mice either by immunoferritin- or immunoperoxidase-labeling techniques (Hoshino and Dniochowski, 1973; Bowen et ( i l . , 1976; Miller et ul., 1977).The most extensive quantitative study of autogenous immunity to MuMTV has been done by Ihle et al. (1976), using an RIA technique with labeled whole virions. Antibodies to B particles were detected in females of several high mammary tumor strains (C3H, GR, BALB/ cfC3H). In C3H mice, the titer increased with age, and maximum levels were found in tumor-bearing females. Low mammary tumor strains (AKR, BALB/c, C57BIJ6, and virus-negative C3H/HeN) did not possess such antibodies unless they were actively immunized with the virus. The viral antigenic determinants recognized by the immunoglobulins described in all these studies have not been characterized. Such identification would be especially interesting in light of the finding that these antibodies presumably coexist with gp52 in the sera of tumor-bearing mice (see Section VI1,C). It has been suggested that the humoral immunity is directed toward external viral antigens (Muller et d.,1971), but Zotter and Muller (1977)have recently reported that serum antibody in mice also reacts with intracytoplasmic A particles as determined by immunofluorescence. Unlike the antibodies to B particles, this anti-A particle reactivity appeared in both high mammary tumor (CBA) and low tumor (XVII and CBABVII) strains. The origin of this reactivity and its specificity have not been completely defined. Deposits of immunoglobulin, Complement, and antigenic material reacting with heterologous antiserum to MuMTV have been reported in the glomeruli of tumor-bearing RIII mice (Pascal et ul., 1975).It has been suggested on the basis of immunofluorescence tests with immunoglobulin-containing eluates of these kidneys that some of the antibodies recognize tumor antigens but not viral antigens (Slovin et al., 1977). Another study described immunoglobulin, complement, and MuMTV antigen deposits in the glomeruli of tumor-free females of several mouse strains (Zotter et ul., 197611). Although many mice, including low mammary tumor strains, possessed renal inimunoglobu-

398

DAN H. MOORE

et (11.

lin and complement, there was an age-dependent increase in the percentage of mice from high mammary tumor strains having renal MuMTV antigen deposits. The origin and in vivo biological role of all of these humoral antibodies remains to be elucidated. Although in vitro studies of murine cell-mediated immunity to MuMTV have described a number of humoral “blocking factors” (see Section VIII,B), it has not been established that these factors are identical to the autogenous humoral immunity described above. Definition of the antibody populations involved in both systems and their specificity will be required. Nevertheless, it is clear that even mice neonatally infected with MuMTV do develop humoral immunity to the virus and are thus not tolerant.

B. CELLULAR IMMUNE RESPONSES TO MuMTV An indication that virus-infected mice are also capable of cellular immune responses to MuMTV antigens came from the report of Miiller and Zotter (1972) that the migration of peritoneal cells from infected but not uninfected mice was inhibited by preparations from tumors containing MuMTV. Although the presence of serum antibodies to MuMTV did correlate with resistance to tumors containing MuMTV, the migration inhibition observed appeared to be unrelated. Blair and colleagues have carried out an extensive series of experiments with spleen cells from BALB/c and BALB/cfC3H mice, using a microcytotoxicity assay. Their assay involves incubation of excess splenic effector cells with a small number of primary BALBIcfC3H tumor target cells for various lengths of time and then the enumeration of the remaining targets by staining and direct counting. With this technique, they found that spleen cells from both tumor-bearing and tumor-free BALB/cfC3H females older than 3 weeks of age had a significant effect on the survival and growth of target cells in culture (Blair et al., 1974). Moreover, cells from tumor bearers were active against autochthonous and isogenic MuMTV-positive tumor cells, suggesting that a common virus-related antigen was being recognized. The investigators were surprised to find that uninfected BALB/c spleen cells from mice older than 14 weeks also were positive in this assay. In contrast, BALB/c mice raised in isolation remote from the rest of the colony did not exhibit this reactivity, and it only appeared when they were returned to the colony (Blair and Lane, 1974b). To further demonstrate the specificity of these reactions, they showed that (1) neither BALB/c nor BALB/cfC3H spleen cells were active against

hfAhlMARY TUMOR VIRUSES

399

mammary tumor cells induced by carcinogens or mammary tumor cells producing only C-type virus particles (Blair et uZ., 1974); ( 2 ) spleen cell cytotoxicity could be partially or completely blocked by pretreatment of the effector population with MuMTV but not muriiie leukemia virus (MuLV) (Blair et ul., 1975); (3) blocking factors (discussed below) interfering with spleen cell cytotoxicity could be absorbed from the sera of BALB/c and BALB/cfC3H mice by cells producing MuMTV but not MuLV (Blair et d.,1974). Time course studies of the niicrocytotoxicity reaction revealed qualitative differences among the cell types tested (Blair and Lane, 1975a). When target cell survival was determined at different times of incubation, it appeared that both cytostasis and cytotoxicity were involved in the activity observed with some of the effector populations. Activity of spleen cells from virgin BALB/cfC3H mice was detected late in the assay period. In parous females, an additional cytostatic activity was found early in the assay period; this reactivity disappears in mice with large mammary tumors. Correlation of the time course of these responses with studies on the cell populations involved indicated that the reactivity of virgin BALB/cfC3H females is due to thymusdependent (T) lymphocytes (Lane et al., 1975). Multiparous BALB/ cfC3H females exhibit an additional non-T-cell reactivity effective early in the culture period which is lost in those females with large tumors. Spleen cell activity of BALB/c mice was found to be T independent. These non-T-cell effector cells were apparently not bone marrow-derived (B) lymphocytes, but “null” cells with Fc receptors which could be recruited by a few B cells or supernatants from populations enriched for B cells (Lane et al., 1975; Blair and Lane, 1975b). Studies with serum-blocking factors in this system have proved to be extremely complex. Initially, it was observed that serum from BALB/cfC3H donors could block activity of BALB/cfC3H spleen cells but not that of BALB/c spleen cells; the converse was also true, i.e., BALB/c serum inhibited only BALB/c reactivity, suggesting differences between the two effector populations (Blair et al., 1974). However, although progressor sera from tunlor-bearing BALB/cfC3H mice inhibited the activity of spleen cells against homologous target cells, the opposite effect was noted when the serum and target cells were obtained from the same donor (Blair and Lane, 1974a).These “recruiting factors” appeared in the 19s fraction of serum, while the “blocking factors” appeared in the 7s fraction (Blair et al., 1976a). A plethora of other “factors” were also described in fractionated sera (Blair et al., 1976), including (1)antibodies in both BALB/cfC3H and BALB/c mice

400

DAN H. MOORE et

al.

mediating complement-dependent lysis of target cells; (2) nonspecific “ recruiting factors” in both BALB/cfC3H and BALBlc mice effective against homologous as opposed to autologous targets. Data obtained from studies of other in vitro correlates of cellular immunity also have suggested that cells from BALB/c mice are responsive to MuMTV antigens (Lopez et al., 1976). In contrast to the results of Blair et al. summarized above, BALB/cfC3H lymphocytes were negative in blast transformation and migration inhibition assays when whole virions were used as antigen. However, the BALB/cfC3H cells became responsive after implantation of an MuMTV-positive tumor. Although immune responses to MuMTV antigens in neonatally infected C3H mice are difficult to demonstrate by in vivo transplantation studies, Stutman (1976) has shown that these mice do display cellmediated responses to MuMTV antigens in a microcytotoxicity assay. This technique employs a series of radioactively labeled tumor target cells and lymph node cells as effectors. Both C3H and C3Hf mice immunized with either tumor cells or soluble antigen mixtures derived from them displayed T-cell-dependent responses against target tumor antigens. Although the magnitude of these responses was less in the infected C3H mice, both strains responded to antigens common to a number of syngeneic mammary tumor cells. The degree of crossreactivity appeared to correlate with the expression of MuMTV antigens on the targets, which in turn was inversely proportional to the H-2 antigen content of the tumors. Some spontaneous reactivity to MuMTV also appeared with increasing age in mice from these two strains. Stutman has pointed out that such reactivity may be significantly influenced by environmental factors such as bacterial or viral infection or helminth infestation, and these variables must be monitored in a mouse colony. He has also investigated the role of the major histocompatibility complex in the generation of effector cells against MuMTV antigens (Stutman, 1977). In these studies, cells from C3H mice immunized with syngeneic or allogeneic MuMTV-positive tumor cells displayed cytotoxic responses against MuMTV antigens on syngeneic targets. This observation is of particular interest in view of the many studies on H-2 compatibility requirements in T-cell cytotoxicity assays against viral antigens. In fact, in Stutman’s system, the allogeneic antigens appeared to be acting as “carriers” since they were much more effective in immunizing C3H mice against viral antigens than syngeneic tumor cells. In support of the importance of H-2 in immunization, mice tolerant to the specific allogeneic H-2 antigens

hlAMblAHY TUMOR VIRUSES

40 1

used for immunization were not effectively immunized against MuMTV antigens when tested on syngeneic cells. Other indications of cell-mediated immunity to MuMTV-related antigens have also been reported. Gillette and Lowery (1976) have described a reactivity occurring naturally in all mouse strains detectable in a cytostasis assay against mammary tumor target cells. This activity was more evident in high mammary tumor strains, was absent from newborn and T-deficient nude mice (although the actual effector cells did not seem to be T lymphocytes), and could be blocked by purified, disrupted MuMTV. Creemers and Bentvelzen ( 197711)have found cellular immunity to MuMTV but not MuLV antigens in mice bearing virus-induced mammary tumors as measured by blast transfonnation and by leukocyte adherence inhibition. It was reported that the sensitized cells in the blast transformation assay were T cells (Creemers and Bentvelzen, 1977a), while the adherence inhibition test was more complex and involved both T cells and adherent cells (Creemers, 1977). Cellular reactivity declined with the progressive growth of the tumor due in part to adherent suppressor cells (Creemers and Bentvelzen, 1977a), and also due to other suppressive factors which were thought to be antigen, antibody, or complexes of the two (Creemers and Brinkhof, 1977). A recent report has described a glassadherent cell in the spleens of tumor-bearing C3H mice which nonspecifically suppressed mitogen responses of normal T lyniphocytes (Rudczynski and Mortensen, 1978). It is apparent that a variety of cell-mediated immunologic reactivitics to MuMTV has been described. Although some question remains as to the existence of autogenous cellular responses, it is nevertheless clear that mice are not tolerant to MuMTV antigens when their cells are tested in vitro. A characterization of the cells reactive in the various assays and an elucidation of their interaction with the humoral component of the immune response may contribute to an understanding of the responses of the animal.

c. MODULATIONOF THE IMMUNOLOGIC INTERACTION BETWEEN MuMTV AND ITS HOST

1. Eflect to MuMTV Infection on Host Responses to Heterologous Antigens Several reports have appeared on the alteration of immunocompetence of the host as a result of MuMTV infection. Blair et al. (1971)

402

DAN H. MOORE

et

(11.

have reported that BALB/c mice infected with MuMTV (C3H) developed lower hemagglutinating titers to sheep red blood cells and rejected skin grafts more slowly than uninfected controls. Such differences were primarily evident in older animals. However, some of these experiments employed fostered strains derived generations earlier, allowing for possible subline divergence. Griswold et a1. (1973) found that young mice from the low mammary tumor strain C3HeB/ FeJ, when directly infected by foster nursing, had a depressed hypersensitivity response to methylated bovine serum albumin, but there was no decrease in the number of plaque-forming cells to sheep red blood cells. Other manifestations of T-cell mediated immunity in infected mice were either unaffected (oxzaolone-induced contact hypersensitivity) or increased (lymphocyte stimulation by phytohemagglutinin) (Griswold et a/., 1975).

2. Modijication of Host lmmune System MuMTV-induced tumorigenesis in mice has been modified by many factors including stress (Riley, 1975), hormone treatment (Nandi and McGrath, 1973), and calorie restriction (Tannenbaum and Silverstone, 1953), some of which may alter the complex balance between the virus and the host’s immune system. More direct studies involving manipulation of the immunologic system have included thymectomy, splenectomy, and immunosuppression. After the initial observations of Martinez (1964), several investigators confirmed his finding that neonatal thymectomy of females from some strains carrying MuMTV decreases or delays mammary tumors. This interesting effect could be abrogated by syngeneic thymus grafts in one case (Yunis et al., 1969) but not another (Roubinian and Blair, 1977).It has also been reported that restoration of thymectomized C3H mice with syngeneic spleen cells derived from C3Hf mice resulted in a higher tumor incidence and a shorter latent period than restoration with spleen cells obtained from C3H mice (Yunis et al., 1969).It has been hypothesized that the effects of thymectomy on mammary tumorigenesis might be due to either endocrine functions of the thymus, alteration of the immune system, or involvement of the thymus in MuMTV replication. More recent studies by Roubinian et al. (1976)and Roubinian and Blair ( 1977) have suggested that delayed tumorigenesis only occurs when surgical thymectomy is incomplete. Complete thymectomy of BALB/cfC3H mice had no effect on mammary tumor development and in addition abrogated the ability of spleen cells to respond to MuMTV antigens in a microcytotoxicity assay. Tests with spleen cells from incompletely thymectomized mice revealed altered patterns of cell-

MAMMARY TUMOR VIRUSES

403

mediated cytotoxicity and serum blocking activities. In another strain-(C57BL x I)F1fC3H-the age at which thymectomy was performed was shown to be an important factor in the time of tumor onset. Thus, there appears to be a delicate balance between the virus and the host’s thymic functions. Other investigators have attempted to modify the niurine immune system by splenectomy or immunosuppressive treatment. I n contrast to thymectomy, splenectomy has been reported to decrease the incidence but accelerate the appearance of the tumors that do arise in virgin BALBIcfC3H but not in RIII female mice (Squartini and Bolis, 1970).Brief immunosuppressive treatment with antithymocyte serum (ATS) alters both humoral (Blair, 1972) and cellular (Blair and Lane, 1977) immune responses to viral antigens and decreases the incidence of mammary tumors (Lappi, and Blair, 1970). Precipitating antibodies to MuMTV were much more frequently detected in ATS-treated BALB/cfC3H and C3H mice. After a short period of immunosuppression, ATS-treated mice also displayed an altered pattern of reactivity to tumor cell targets in a microcytotoxicity assay, as evidenced by the appearance of non-T-cell reactivity and changes in serum blocking activity. The relationship of these immunologic observations to changes in tuniorigenesis has not been documented.

3. linmunizution Studies Since the early observations that mammary tumor cells from one tumor immunized mice against challenge with another tumor, evidence has accumulated that the antigen common to this group of mammary tumors is in fact the mammary tumor virus. After demonstrations that extracts of mammary tissues containing MuMTV were also protective against challenge with isogenic tumor cells, Burton et (11. (1969) reported that purified MuMTV preparations could achieve the same result. However, immunization with irradiated tumor cells gave more effective protection than this live virus vaccine. Peritoneal cells from immunized donors were reported to inactivate tumor cells when injected into noninimune hosts along with the challenge cells. Virus preparations inactivated by fornialin have also been shown to protect C57BL mice against a subsequent virus challenge (Charney and Moore, 1972). More recent experiments giving the vaccine at various times relative to the live virus have revealed that protection OCcurs only when the vaccine is given prior to or shortly after challenge, suggesting that the immune response affects an early event in infection or replication (Charney and Moore, unpublished observations). This is supported by the finding that virus expression in the milk and

404

DAN H. MOORE

et al.

spontaneous tumorigenesis are not reduced in similarly vaccinated but unchallenged RIII mice exposed to MuMTV since birth. In preliminary studies, Stutman (1976) has found that virus-burdened C3HfC3H mice are also relatively unaffected by vaccination with formalinized intact MuMTV. Further immunization studies with the low mammary tumor foster-nursed strains revealed that the occurrence of tumors was reduced or delayed in Af and RIIIf strains vaccinated with MuMTV(RII1) virus (Chamey et al., 1976). However, no effect was noted in inoculated C3Hf mice (Charney and Moore, unpublished observations). Additional studies using inactivated MuMTV(C3H) as a vaccine in BALBlc mice susceptible to a challenge with this virus have not indicated any differences in viral antigen expression or tumor incidence between the immunized and control groups. Because of the multiplicity of antigenic determinants involved when whole virions are used as immunogens, several attempts have been made to determine which polypeptide is responsible for protection. Sarkar and Moore (1978) compared the efficacy of a formalinized whole virus vaccine with that of purified gp52 from MuMTV(RII1) virus, both emulsified with complete Freund’s adjuvant. At a dose of 10 pg, both preparations protected C57BL mice against a challenge of MuMTV(RII1) measured either by virus expression in the milk or mammary tumor incidence. In contrast, an acid-solubilized fraction of the virus enriched for gp52 was not protective, suggesting that the method of preparation of the antigen affected immunogenicity. A similar experiment has been done by Long and Dion (unpublished results), who have also prevented tumors by immunization with gp52, although the suppression of viral antigen was not as complete. The route of immunization and adjuvant appeared to be important since intradermal inoculation in the absence of complete Freund’s adjuvant was ineffective. As in the studies with the whole virus vaccine, gp52 immunization had no significant effect on viral antigen expression in high tumor RIII mice. Finally, when p28 from MuMTV(RII1) was tested as an immunogen in C57BL/6 subsequently challenged with MuMTV(RIII), only minimal reductions were observed in viral antigen expression and tumor incidence. A recent report (Creemers et a1., 1977) described the immunization of BABL/c and DBAf mice with a gp52 preparation followed by challenge with MuMTV-positive isogenic tumor cells. Both acceleration and retardation of tumor growth were observed, depending on the dose of antigen and the type and amount of adjuvant used. However, only about 60% of the protein preparation used as an immunogen in these studies was gp52, and there was probably contamination by

MAMMARY T U M O R VIRUSES

405

protein material from the Con A-Sepharose used for purification. Stutman ( 1976) has reported experiments in which immunization of C3H and C3Hf mice with soluble tumor antigens prepared by KCI extraction accelerated mammary tumor development. Such solubilized mixtures have many components in addition to the specific viral antigens, and results obtained with these iminunogens are difficult to interpret. The immune responses of mice to inoculation with whole virions or viral components have not been extensively studied. Charney (personal communication) has stated that he was unable to find specific antibody in C57BL mice immunized with fomialinized MuMTV(RII1). However, Ihle et al. (1976)have reported that BALB/c mice inoculated i.p. with MuMTV(C3H) produced high titers of antibody when tneasured in an RIA employing labeled whole virions. Antibody to MuMTV prepared in a heterologous species and administered passively in multiple doses to neonatal RIII mice has reduced viral antigen expression and possibly tumorigenesis (Frensdorf, personal communication). The effect of this antibody in these high tumor mice may be to neutralize the infectious virus coming from the mother’s milk. Creemers et (11. (1977) have used an in vitro leukocyte adherence inhibition system to measure cellular immune responses to their complex viral antigen. They described serum blocking factors which were inimical to protection. As in the C-type oncomavinis system, the complex interactions between the endogenous virus, the milk-transmitted virus, and the host’s immune system complicate immunization experiments. Furthermore, different results may be obtained depending on whether the mouse is already infected with MuMTV, challenged with live virus, or challenged with MuMTV-positive tumor cells. More extensive experiments are required measuring specific cellular and humoral responses of mice to defined viral antigens. IX. Comments on the Question of a MuMTV-related Virus in Humans

Since a viral etiology of mouse mammary tumors appears to be well established, searches for a siinilar virus in human breast cancer have been conducted recently in numerous laboratories. Although there are major discrepancies which make a MuMTV-related etiological agent in man improbable, suggestive results implicating a MuMTV-like virus in human beings have been obtained (Moore, 1974). These include:

(1) Particles n~orphologicallysimilar to B particles have been seen by electron microscopists in some human breast tumors and milk sam-

406

DAN H. MOORE et

nl.

ples (Senian et al., 1969; Moore et al., 1971). Extensive studies using negative-stained preparations of particulate material isolated from human milk have indicated that only a very small proportion of particles observed have membrane characteristics identical to B particles (Sarkar and Moore, 1972a), and that there is no apparent correlation with breast cancer risks of the milk donors (Sarkar and Moore, 1972b). (2) Particulate fractions from some human milks contain a DNA polymerase activity that has some characteristics of RDDP (Schlom et al., 1971, 1973). When DNA synthesized by such particles in the absence of exogenous template-primer is examined by rate zonal centrifugation, it is found to be associated with high molecular weight RNA (Schlom and Spiegelman, 1971; Spiegelman and Schlom, 1972; Schloni et al., 1973). The RDDP activity of human milk, like that of MuMTV, prefers Mg2+ as a divalent cation when synthesizing DNA using oligo (dG).poly (rC) as a primer-template (Dion et al., 19744. (3) Some human breast tumors have RNA sequences that are hoinologous to the MuMTV genome. Using CsS04 gradients to detect hybrid fomiation, 69% of human breast tumors were found to contain RNA sequences that hybridized to MuMTV cDNA (Axel et d , ,1972). When more stringent conditions for hybridization and hybrid detection were employed, a much lower proportion of human tumors was found to contain MuMTV-related RNA (Vaidya et al., 1974). (4) Numerous investigators have sought MuMTV-related antigens in human materials or evidence of immune responses to MuMTV in breast cancer patients. In 1972, Miiller and Grossman reported an antigen in some sera of breast cancer patients precipitable with antiMuMTV. Recently, Mesa-Tejada et ul. (1978) have reported that immunoperoxidase staining of tumor tissue sections using antibody to MuMTV yielded positive results in patients with breast carcinomas, and that this staining was specifically blocked by gp52. Other studies have described antibodies in human sera capable of binding to murine cells producing MuMTV (Priori et al., 1972; Hoshino and Dniochowski, 1973; Bowen et ul., 1976; Miller et ul., 1976). In one case, evidence was presented that antibodies found with greater frequency in sera of women with breast cancer are directed toward intracytoplasmic A particles (Muller et al., 1974).With respect to cellmediated immunity, migration of leukocytes from approximately onethird of breast cancer patients is inhibited by MuMTV(RII1) or gp52(RIII), and this response correlates with patients’ reactivity to a component present in some breast tumors (Black et ul., 1974, 1975, 1976a,b; Black, 1977; Zachrau et ul., 1976, 1978).

MAMMARY TUMOR VIRUSES

407

As can be seen, the results linking a MuMTV-like virus with human breast cancer have been obtained from investigations using electron microscopy, molecular hybridization, RDDP detection, and immune reactivity. However, none of these studies have provided sufficient evidence to arrive at any conclusion with reference to a viral role in human breast cancer. Electron microscopic detection of particles resembling MuMTV in human milk and breast cancer has, at best, been infrequent and equivocal. The DNA polymerase in human milk having some characteristics of RDDP has neither been well characterized nor has been shown to be of viral origin. Although MuMTVrelated RNA can be detected under stringent hybridization conditions in some human breast tumors and there appears to be a correlation with the histopathological level of differentiation of the tumors (Vaidya et al., 1974 and unpublished data), no MuMTV-related DNA can be detected in human tumors (Bishop et d . , 1974). In fact, when some of the human tumors that showed MuMTV-related RNA were tested for DNA viral sequences, none were detectable even under conditions that could detect 0.3 copies of MuMTV DNA per cell (Vaidya, unpublished results). Moreover, the presence of MuMTV-related RNA was not correlated with the cellular immune response of patients to MuMTV antigens. The specificity of most of the immunologic reactions observed has not been adequately defined (Newgaard et al., 1976). The results obtained by Black and his colleagues and by Mesa-Tejada et (11. (1978) suggest that some human breast cancer patients have a component in their tumors that is immunologically related to gp52 of MuMTV and that some of these patients have cellular immune responses to gp52. However, there are indications that the gp52-related material in human breast cancer may be cross-reactive due more to its carbohydrate moiety than its amino acid sequence. First, gp52 isolated from MuMTV(A) that shows some differences in migration in SDS-PAGE when compared to gp52 from MuMTV(RII1) is found to be unreactive in the leukocyte migration inhibition tests (Black et nl., 1976a; Zachrau et nl., 1978), even though gp52(A) is completely cross-reactive with gp52(RIII) by RIA (Sheffield and Dion, unpublished observations): Second, digestion of gp52(RIII) with proteinase K does not appear to remove the ability of the resultant glycopeptides to elicit LMI response (Black and Dion, unpublished data). Third, eluates from human tumors that have LMI-eliciting activity do not seem to contain gp52 when tested by RIA (Sheffield, unpublished results). Furthermore, unlike the transmission of MuMTV in mice, there is no evidence for milk transmission of a breast cancer influence in humans.

408

DAN H. MOORE et

fd.

As pointed out by Fraumeni and Miller (1971), all considerations seem to negate the possibility of milk transmission. In countries where breast feeding is common, breast cancer rates are relatively low. In communities (e.g., rural, immigrant) in the United States where breast feeding is most prevalent, breast cancer rates are low. During the twentieth century, breast feeding in the United States and other Weste m countries has decreased while breast cancer incidence has risen. Moreover, in a study of 1232 offspring of 551 women with breast cancer, Tokuhata (1969) found no difference in the breast cancer incidence in breast-fed and nonbreast-fed daughters. Anderson ( 1975)also found no difference in risk for women who were ever breast fed and those who were never breast fed. From the above, we conclude that if an MuMTV-related virus is involved in human breast cancer, the biology of such a virus must be very different from that of MuMTV. Each of the experiments showing a link between MuMTV and the human disease has been complicated by results that are difficult to explain. Many factors are involved in carcinogenesis of the mammary gland. In the mouse, the viral factor is prominent. In other mammals studied, including man, it is not so prominent. We believe, however, that new investigative approaches developed during the past few years, coupled with comparative studies of etiological factors involved in mammary tuniorigenesis in different species will, in time, lead to an understanding of the human disease. X. Concluding Remarks

This review has summarized much of the recent literature on the structure, biochemistry, biology, and immunology of the murine mammary tumor viruses. Since the influence of milk on mouse mammary tumorigenesis was discovered 40 years ago, a considerable amount of information has accumulated identifying the B particle as the biologically active agent. However, because of the lack of in vitro systems and the lengthy bioassays required, advances in the understanding of this virus have lagged behind studies with the C-type retraviruses. Recently, the application of improved biochemical and immunological techniques coupled with progress in infecting cells in vitro with MuMTV have caused a resurgence of interest in this virus. Many investigators have recognized its potential not only as a system for studying the transformation of epithelial as opposed to mesothelial cells, but also as a means of approaching problems in eukaryotic gene regulation. Evidence from a variety of biological studies and some

MAXfhlARY TUMOR VIRUSES

409

recent immunological results have suggested that MuMTV is, in fact, a group of closely related viruses. However, the information available is not yet adequate to develop a reasonable classification scheme. Additional biochemical, immunologic, and biologic data will eventually make it possible to classify the MuMTVs. Very little is known about the in uiuo process of infection. The route b y which MuMTV reaches its target tissue and the consequences of this infection for the cell are not understood. The multistage sequence of events following infection which result in the appearance of a mammary tumor many months later is not known. Host genetic factors which influence virus expression or susceptibility to tumorigenesis have been recognized and their number and mechanism of action are being investigated. After many years, successful infection of cells in culture has been possible, due in part to the newer molecular biological techniques for detecting specific viral information within the cell. Of particular interest is the observation that the presence of hormones in uitro influences the expression of viral RNA and viral proteins, even in infected heterologous cells. This provides a model system for the study of hormonal control of genes. Molecular hybridization techniques have also directly established that all mice, even those that do not develop inamniary tumors, contain MuMTV genetic information. The role of these endogenous viral genes and the regulation of their expression by the cell remain to be elucidated. Most of the viral polypeptides have been identified and purified in the last few years, and infonnatioii regarding their function and their location in the virion is being accumulated. Application of radioimmunoassay techniques to immunologic studies of MuMTV have resulted in the development of better defined and quantitative assays for the virus and its components. These assays allow detection of sinall amounts of vinis in biological materials. Application of serological techniques and others adopted from cellular immunology have revealed both huinoral and cellular responses to MuMTV antigens in mice neonatally infected with MuMTV. The antigenic specificity of autogenous immune responses and those induced by immunization are currently being studied. The in uiuo interactions of these' responses and their significance to the host are areas for future research. In conclusion, MuMTV, being a unique retrovirus that is etiologically involved in a hormone-influenced carcinoma, provides an attractive system for investigators from many disciplines. In the coming few years, it is likely that much more will be known about this intriguing virus.

410

DAN H. MOORE

et al.

ACKNOWLEDGMENTS The preparation of this review and some of the results reported therein have been supported by Grant Nos. 5ROlCA22413-02, 5ROlCA22720-02, and 5ROlCA08515-12 and by Contract Nos. N01-CP-81007, N01-CP-81003, N01-CP-74201, and NO1-CP801007 awarded by the National Cancer Institute, D.H.E.W. We are also indebted to Dr. Nurul Sarkar for providing some ofthe electron micrographs and especially to Ms. Alice Smith for her patience and competence in the typing of the manuscript.

REFERENCES Allison, A. C., and Davis, P. (1974). Symp. Soc. E x p . Biol. 28, 419-446. Anderson, D. E. (1975). In “Early Breast Cancer: Detection and Treatment.” (H. S. Gallager, ed.), pp. 41-49. John Wiley, New York. Andervont, H. B., and McEleney, W. J. (1939). Public Heulth Rep. 54, 1597-1603. Ardashnikov, S. V., and Spasskaia, I. G. (1949).J. Microbiol. Epideniiol. Irnniuiiol?iol. U.S.S.R.(English transl.) 9, 44-48. Arthur, L. O., Fine, D. L., and Bentvelzen, P. (1978).J. Natl. Cancer Inst. 60,461-464. Ashley, R. L., Cardiff, R. D., and Manning, J. S. (1977). Virology 77, 367-375. August, J. T., Bolognesi, D. P., Fleissner, E., Gilden, R. V., and Nowinski, R. C. (1974). Virology 60, 595-600. Axel, R., Schlom, J., and Spiegelman, S. (1972). Nature (London) 235, 32-36. Bender, W., Chien, Y. H., Chattopadyay, S., Vogt, P. K., Gardener, M. R., and Davidson, N. (1978)J. Virol. 25, 888-896. Bentvelzen, P. (1968a). Ph.D. Thesis, University of Leiden, Hollandia, Amsterdam. Bentvelzen, P. (1968b).J. N a t l . Cancer Inst. 41, 757-765. Bentvelzen, P. (1972). I n t . Rev. E x p . Pathol. 11, 259-297. Bentvelzen, P. (1974).Biochiin. Biophys. Acta 355, 236-259. Bentvelzen, P. (1975). Cold Spring Harbor S ! y m p . Quant. Biol. 39, 1145-1150. Bentvelzen, P., Daams, J. H., Hageman, P., and Calafat, J. (1970a).Proc. Natl. Accid. Sci. USA 67, 377-384. Bentvelzen, P., Van der Gugten, A., Hilgers, J., and Daams, J. H. (1970b). I n “Immunity and Tolerance in Oncogenesis” (L. Severi, ed.), pp. 525-539. Perugia, Italy. Bemhard, W. (1958). Concer Res. 18,491-509. Bernhard, W., Bauer, A., Gubrin, M., and Oberling, C. (1955).Bull. Cancer 42,163-178. Bishop, J. M., Quintrell, N., Medeiros, E., and Varmns, H. E. (1974). Crrticer 34, 14211426. Bittner, J. J. (1936). Science 84, 162. Bittner, J. J. (1939). U.S. Public Health Rep. 54, 1590-1597. Bittner, J. J. (1945). Proc. SOC. E x p . Biol. Med. 59, 43-44. Bittner, J. J. (1952). Cancer Res. 12, 510-515. Bittner, J. J. (1962). I n “Tumor Viruses of Muririe Origin” (G. E. W. Wolstenholme and M. O’Connor, eds.), pp. 56-81. Churchill, London. Black, M. M. (1977). In “Contemporary Topics in Immunobiology,” (M. G. Hanna, Jr. and F. Rapp, eds.), Vol. 6 pp. 239-262. Plenum, New York. Black, M . M., Moore, D. H., Shore, B., Zachrau, R. E., and Leis, H. P., Jr. (1974). Cancer Res. 34, 1054-1060. Black, M. M., Zachrau, R. E., Shore, B., Moore, D. H., and Leis, H. P., Jr. (1975).Cancer 35, 121-128.

MAMMARY TUMOR VIRUSES

41 1

Black, M. M., Zachrau, R. E., Dion, A. S., Shore, B., Fine, D. L., Leis, H. P., Jr., and Williams, C. J. (1976a). Cancer Res. 36, 4137-4142. Black, M. M., Zachrau, R. E., and Shore, B. (197611).111 “Bibliotheca Haematologica,” No. 43 (J. Clemmesen and D. S. Yohn, eds.), Comparative Leukemia Research 1975, pp. 559-564. Karger, Basel. Blair, P. B. (1965). Nature (London) 208, 165-168. Blair, P. B. (1968a). Cancer Res. 28, 148-149. Blair, P. B. (196811).C u r t . Top. Microhiol. Z?nniutio/.45, 1-69. Blair, P. B. (1969).Cancer Res. 29, 745-748. Blair, P. B. (1970). Cancer Res. 30,625-631. Blair, P. B. (1971a). Cancer Res. 31, 1473-1477. Blair, P. B. (1971b).Israel J . Med. Sci. 7 , 161-186. Blair, P. B. (1972).Cancer Res. 32, 356-359. Blair, P. B. (1976).Cancer Res. 36, 734-738. Blair, P. B., and Lane, M. A. (1974a).J. Inimtttrol. 112, 439443. Blair, P. B., and Lane, M. A. (1974b).J. Irnmrrnol. 113, 1446-1449. Blair, P. B., and Lane, M. A. (1975a).J. Inimzrnol. 114, 17-23. Blair, P. B., and Lane, M. A. (1975b).J. Inznzunol. 115, 184-189. Blair, P. B., and Lane, M. A. (1977).J. Natl. Cancer I n s t . 59, 251-257. Blair, P. B., Lavrin, D. H., Dezfulian, M., and Weiss, D. W. (1966). Cancer Re.p. 26, 647-651. Blair, P. B., Kripke, M. L., Lapp&, M. A., Bonhag, R. S . , and Young, L. (1971).J . I n i nruiiol. 106, 364-370. Blair, P. B., Lane, M. A., and Yagi, M. J. (1974).J. I ~ n m u n o /112, . 693-705. Blair, P. B., Lane, M. A., and Yagi, M. J. (1975).J. Irnrnunol. 115, 190-194. Blair, P. B., Lane, M. A., and Mar, P. (1976a).J. Inzmutiol. 116, 606-609. Blair, P. B., Lane, M. A., and Mar, P. (1976b).J. Imnzritiol. 116, 610-614. Blough, H. A., and Tiffany, J. M. (1975). Crtrr. Top. Microbial. Imnzunol. 70, 1-30. Boot, L. M., and Miihlbock, 0. (1956). Actn Utrio I t i f . Cancrum 12, 569-581. Bowen, J. M., Dmochowski, L., Miller, M. F., Priori, E. S., Seman, G., Dodson, M. L., and Maruyama, K. (1976).C m c e r Re.9. 36, 759-764. Burton, D. S., Blair, P. B., and Weiss, D. W. (1969). Cancer Res. 29, 971-973. Calafat, J., Buijs, F., Hageman, P. C., Links, J., Hilgers, J., and Hekman, A. (1974).J. N o t l . Cancer I n s t . 53, 977-991. Calafat, J., Hilgers, J., Van Blitterswijk, W. J., Verbeet, M., and Hageman, P. C. (1976).J. N o t / . Cancer I t i s t . 56, 1019-1023. Cardiff, R. D. (1973).J. Inimttnol. 111, 1722-1729. Cardiff, R. D., Blair, P. B., and DeOme, K. B. (1968).Virology 36, 313-317. Cardiff, R. D., Puentes, M. J , , Teramoto, Y. A., and Lund, J. K. (1974).J . Virol. 14, 1293-1303. Charney, J., and Moore, D. H. (1972).J. Natl. Caticer Itist. 48, 1125-1129. Charney, J., Pullinger, B. D., and Moore, D. H. (1969).J. Notl. Cancer I n s t . 43, 12891296. Charney, J., Moore, D. H., Holben, J. A,, and Cody, C. M. (1975).111 “Multiple Primary Malignant Tumors” (L. Severi, ed.), pp. 1125-1 133. Div. Cancer Research, Perugia (Monteluce), Italy. Chamey, J., Hollien, J. A,, Cody, C. hl., and Moore, D. H. (1976). C m c e r Res. 36, 777-780. Creemers, P. (1977).Eur. J . Imtnztriol. 7, 48-53. Creemers, P., and Bentvelzen, P. (1977a). Ezrr. J. Cancer 13, 261-267.

412

DAN H. MOORE

et nl.

Creemers, P., and Bentvelzen, P. (197%). Eur. J. Cancer 13, 503-510. Creemers, P., and Brinkhof, J. (1977). Znt. J. Cancer 20,441447. Creemers, P., Ouwehand, J., and Bentvelzen, P. (1977)J.Natl. Cancer Inst. 59,895-903. Daams, J. H. (1970). In “Immunity and Tolerance in Oncogenesis” (L. Severi, ed.), IV Perugia Conference on Cancer, p. 463. Daams, J. H., Calafat, J., Lasfargues, E. Y., Kramarsky, B., and Bentvelzen, P. (1970). Virology 41, 184-186. Dalton, A. J., Heine, U. I., and Melnick, J. L. (1975).J.Natl. Cancer Inst. 55,941-942. Damsky, C. H., Sheffield, J. B., and Warren, L. (1976a). Zst Int. Cong. Cell Biol. 117a (Abstr.) Damsky, C. H., Tuszynski, G. P., Sheffield, J. B., and Warren, L. (1976b).Bi0phy.s.J.16, 116a (Abstr.) Damsky, C. H., Sheffield, J. B., Tuszynski, G. P., and Warren, L. (1977).J.Cell B i d . 75, 593-605. Dickson, C., and Skehel, J. J. (1974).Virology 58, 387-395. Dickson, C., Haslam, S., and Nandi, S. (1974).Virology 62, 242-252. Dickson, C., Puma, J. P., and Nandi, S. (1975).J.Virol. 16, 250-258. Dickson, C., Puma, J. P., and Nandi, S. (1976).J.Virol. 17, 275-282. Dion, A. S., and Moore, D. H. (1977).In “Recent Advances in Cancer Research: Cell Biology, Molecular Biology, and Tumor Virology, Vol. 11” (R. C. Gallo, ed.), pp. 69-87. CRC Press, Cleveland, Ohio. Dion, A. S., and Pomenti, A. A. (1979),Virology (in press). Dion, A. S., Vaidya, A. B., and Fout, G. S. (1974a).Cancer Res. 34,3509-3515. Dion, A. S., Vaidya, A. B., Fout, G. S., and Moore, D. H. (1974b).J.Virol. 1 4 , 4 0 4 6 . Dion, A. S., Heine, U. I., Pomenti, A. A., Korb, J., and Weber, G. H. (1977a).J.Virol. 22, 822-825. Dion, A. S., Williams, C. J., and Moore, D. H. (1977b).J.Virol. 22, 187-193. Dmochowski, L., and Grey, C. E. (1957).Ann. N.Y. Acad. Sci. 68,559-615. Dmochowski, L., Grey, C. E., and Sykes, J. A. (1963). Acta Unio Znt. Cancrum 19, 276-279. Drohan, W., Kettman, R., Colcher, D., and Schlom, J. (1977).J. Virol. 21, 986-995. Fine, D. L., Plowman, J. K., Kelley, S. P., Arthur, L. O., and Hillman, E. A. (1974).J. Natl. Cancer Znst. 52, 1881-1886. Fine, D. L., Arthur, L. O., and Young, L. J. T. (1976).In Vitro 12,693. Fink, M. A., Feller, W. F., and Sibal, L. R. (1968).J. Natl. Cancer Znst. 41, 1395-1400. Fraunieni, J. F., Jr., and Miller, R. W. (1971).Lancet 2, 1196-1197. Friedrich, R., Morris, V., Goodman, H. M., Bishop, J. M., and Varmus, H. E. (1976). Virology 72, 330-340. Gillette, R. W., and Junker, D. (1973).A p p l . Microhiol. 26, 63-65. Gillette, R. W., and Lowery, L. T. (1976).Cancer Res. 36, 40084014. Gillette, R. W., Robertson, S., Brown, R., and Blackman, K. E. (1974).J.Natl. Cancer Znst. 53, 499-505. Gorka, C., and Mouriquand, J. (1975).Eur. J. Cuncer 11, 397-402. Griswold, D. E., Heppner, G. H., and Calabresi, P. (1973).J. Nut. Cancer Znst. 50, 1035-1038. Griswold, D. E., Kopp, J. S., Manning, J. S., and Heppner, G. H. (1975).Cancer Res. 35, 2670-2673. Guntaka, R. R., Richards, 0. C., Shank, P. R., Kung, H. J., Davidson, N., Fritsch, E., Bishop, J. M., and Varmus, H. E. (1976).J.h4ol. Biol. 106, 337-357. Hageman, P., Calafat, J.. and Daams, J. H. (1972).I n “RNA Viruses and Host Genome in

MAMMARY TUMOR VIRUSES

413

Oncogenesis” (P. Einmelot and P. Bentvelzen, eds.), pp. 283-300. North-Holland, Amsterdam. Heston, W. E. (1958).Ann. N.1’. Acad. Sci. 71, 931-942. Heston, W. E., and Deringer, M. K. (1952).J.Natl. Cancer Inst. 13, 167-175. Heston, W. E., and Parks, W. P. (1975). Can. J. Genet. Cytol. 17, 493-502. Heston, W. E., and Parks, W. P. (1977).J.E x p . Med. 146, 1206-1220. Heston, W. E., Deringer, M. K., and Andervont, H. B. (1945).J. Natl. Concer Inst. 5, 289-307. Heston, W. E., Deringer, M. K., Dann, T. B., and Levillain, W. D. (1950).J . N u t / . Cniicer l i i s t . 10, 1139-1155. Heston, W. E., Vlahakis, G., and Deringer, M. K. (1960).J.Natl. Cancer I n s t . 24, 721731. Heston, W. E., Smith, B., and Parks, W. P. (1976).J.E x p . Med. 144, 1022-1030. Hilgers, J., and Bentvelzen, P. (1978).I n “Advances in Cancer Research” ( G . Klein and S. Weinhouse, eds.), Vol. 26, pp. 143-195. Academic Press, New York. Hilgers, J.. Williams, W. C., Myers, B., and Dmochowski, L. (1971a).Virology 45, 470483. Hilgers, J., Daanis, J. H., and Bentvelzen, P. (197111).Isr. J. Med. Sci. 7, 154-160. Hilgers, J., Nowinski, R. C., Geering, G., and Hardy, W. (1972). Cuncer Res. 32, 98106. Hilgers, J., Theuns, G . J., and van Nie, R. (1973).Int. J. Cancer 12, 568-576. Hilgers, J., Haverman, J., Nusse, R., van Blitterswijk, W. J., Cleton, F. J., Hageman, P. C., van Nie, R., and Calafat, J. (1975).J.Nut. Cancer I n s t . 54, 1323-1333. Holnies, E. C., and Morton, D. L. (1969).J.N u t / . Cuncer Inst. 42, 733-738. Home, R. W., and Wilde, P. (1961).Virology 15, 348-373. Hoshino, M., and Dmochowski, L. (1973).Cancer Res. 33, 2551-2561. Howard, D. K., Colcher, D., Teramoto, Y. A,, Young, J. M., and Schloni, J. (1977).Coticer Res. 37, 2696-2704. Howk, R. S., Rye, L. A,, Killeen, L. A., Scolnick, E. M., and Parks, W. P. (1973).Proc. N o t l . Acud. Sci. USA 70, 2117-2121. Huang, A. S., and Baltimore, D. (1977).I i i “Comprehensive Virology 10, Regulation and Genetics, Viral Gene Expression and Integration” (H. Fraenkel-Conrat and R. R. Wagner, eds.), pp. 73-116. Plenum Press, New York and London. Ihle, J. N., Arthur, L. O., and Fine, D. L. (1976).Cancer Res. 36, 2840-2844. Karande, K. A., Joshi, B. J., Talageri, \i R., Dumaswala, R. V., and Ranadive, K. J. (1978). Eur. J. Cancer 14, 251-261. Kimball, P. C., Boehm-Truitt, M., Schochehnan, G., and Schlom, J. (1976).J. N a t l . Cancer I t i s t . 56, 111-117. Korteweg, R. (1934).N e d . Tjdschr. Ceniesk. 78, 240-245. Kuff, E. L., Leuders, K. K., Ozer, H. L., and Wivel, N. A. (1972).Proc. N n t l . Acad. Sci. USA 69,218-222. Lane, M . A., Roubinian, J., Slomich, M., Trefts, P., and Blair, P. B. (1975).J.Imnint~ol. 114, 24-33. Lappi‘. M . A,, and Blair, P. B. (1970).Proc. A m . Assoc. C m . Res. 2, 47. Lasfargues, E. Y., Kraniarsky, B., Lasfargues, J. C., and Moore, D. H. (1974).J , N u t [ . Cniicer I n s t . 53, 1831-1833. Lasfargues, E. Y., Lasfargues, J. C., Dion, A. S., Greene, A. E., and Moore, D. H. (1976a). Coticer Res. 36, 67-72. Lasfargues, E. Y., Vaidya, A. B., Lasfargues, J. C., and Moore, D. H. (19761,).J. N u t / . Concer I n s t . 57, 447-449.

414

DAN H. MOORE

et

(11.

Latarjet, R. (1970).I t i t . J . Concer 6, 31-39. Lenard, J., and Compans, R. W. (1974).Biochim. Biophys. Actcl 344, 51-94. Lo Gerfo, P., Silverstein, G., and Charney, J. (1974). Surgery 76, 16-22. Lopez, D. M., Ortiz-Muniz, G., and Sigel, M. M. (1976).Proc. SOC.E . Y ~B. i d . Med. 151, 225-230. Marcus, S. L., and Sarkar, N. H. (1978). Virology 84,257-259. Marcus, S. L., Sarkar, N. H., and Modak, M. J. (1976). Virology 71, 242-254. hiarcus, S. L., Smith, S. W., Racevskis, J., and Sarkar, N. H. (1978). Virology 86, 183-197. Martinez, C. (1964).Nature (London) 203, 1188. McGrath, C. M. (1971).J.Nrttl. Cancer Ztist. 47, 455-467. McCrath, C. M., and Blair, P. B. (1970). Cancer Res. 30, 1963-1968. McGrath, C. M., Nandi, S., and Young, L. (1972).J. Virol. 9, 367-376. Mesa-Tejada, R., Keydar, I., Ramanarayanan, M., Ohno, T., Fenoglio, C., and Spiegelman, S. (1978).Proc. Natl. Acad. Sci. USA 75, 1529-1533. Michalides, R., and Schlom, J. (1975).Proc. Nntl. Acctd. Sci., USA 72, 46354639. Michalides, R., Vlahakis, G . , and Schlom, J. (1976). Znt. J. Cancer 18, 105-115. Miller, M. F., Dmochowski, L., and Bowen, J . M. (1977).Cancer Res. 37, 2086-2091. Miroff, G., and Magdoff-Fairchild, B. S. (1965).Nature (London) 206, 519-520. Moore, D. H. (1962). In “Ciba Foundation Symposium on Tumour Viruses of Murine Origin” (G. E. W. Wolstenholme and M. O’Connor, eds.), pp. 107-129. Churchill, London. Moore, D. H. (1963).Nature (London) 198, 429433. Moore, D. H. (1974). Cuncer Res,. 34, 2322-2329. Moore, D. H., and Holben, J. A. (1978). Cuncer Res. 38, 2455-2457. Moore, D. H., Lasfargues, E. Y., Murray, M. R., Haagensen, C. D., and Pollard, E. C. (1959).J. Biophys. Biochetn. Cytol. 5,85-92. Moore, D. H., Pollard, E. C., and Haagensen, C. D. (1962).Fed. Proc. 21, 942-946. Moore, D. H., Pillsbury, N., and Pullinger, B. D. (1969a).J , Natl. Cancer Znst. 43, 1263-1273. Moore, D. H., Sarkar, N. H., Kelly, C. E., Piillsbury, N., and Chamey, J. (1969b). Tex. Rep. Biol. Med. 27, 1027-1039. Moore, D. H., Charney, J., Lasfargues, E. Y., Sarkar, N. H., Rubin, R. C., and Anies, R. P. Proc. SOC.E x p . Biol. Med. 132, 125-127. (1969~). Moore, D. H., Charney, J., and Pullinger, B. D. (1970).J.Nutl. Cancer Inst. 45,561-565. Moore, D. H., Chamey, J., Kramarsky, B., Lasfargues, E. Y., Sarkar, N. H., Brennan, M. J., Burrows, J. H., Sirsat, S. M., Paymaster, J. C., and Vaidya, A. B. (1971).Nature (London) 229, 611-614. Moore, D. H., Charney, J., and Holben, J. A. (19741.J.Natl. CancerInst. 52,1757-1761. Moore, D. H., Holben, J. A., and Chamey, J. (1976).J. Natl. Cancer Inst. 57,889-896. Moore, D. H., Merryman, C. F., Maurer, P. H., and Holben, J. A. (1978). Cancer Res. 38,3871-3878. Morris, V. L., Medeiros, E., Ringold, G. M., Bishop, M. J., and Varmus, H. E. (1977).J . Mol. Biol. 114, 73-92. Muhlbock, 0. (1965).Eur. J. Cuncer 1, 123-124. Miiller, M., and Grossmann, H. (1972).Nature N e w B i d . 237, 116-117. Miiller, M., and Zotter, S. (1972). Eur. J. Cancer 8,495-500. Miiller, M., and Zotter, S. (1973).J. Natl. Cancer Inst. 50, 713-717. Muller, M., Hageman, P. C., and Daams, 3. H. (1971).J. Natl. Cancer Inst. 47,801-805. Nandi, S. (1963).J. Natl. Cancer Znst. 31, 57-73.

MAMMARY TUMOR VIRUSES

415

Nandi, S., and McGrath, C. M. (1973).Ado. Cancer Res. 17, 353-414. Newgaard, K. W., Cardiff, R. D., and Blair, P. B. (1976).Cnricer Res. 36, 765-768. Noon, M . C., Wolford, R. G., and Parks, W. P. (1975).J.Zmtnunol. 115, 653-658. Nowinski, R. C., Old, L. J., Moore, D. H., Geering, G., and Boyse, E. A. (1967).Virology 31, 1-14. Nowinski, R. C., Old, L. J., Boyse, E. A,, d e Harven, E., and Geering, G. (1968).Virology 34,617-629. Nowinski, R. C., Sarkar, N. H., Old, L. J., Moore, D. H., Scheer, D. I., and Hilgers, J. (1971).Virolog!] 46, 21-38. Parks, W. P., and Scolnick, E. M. (1973).Virolog!! 55, 163-173. Parks, W. P., Howk, R. S., Scolnick, E. M., Oroszlan, S., and Gilden, R. V. (1974a).J . Virol. 13, 1200-1210. Parks, W. P., Scolnick, E. M., and Kozikowski, E. H. (197411).Science 184, 158-160. Parks, W. P., Ransom, J. C., Young, H. A,, and Scolnick, E. M. (1975a).J.B i d . Chem. 250, 3330-3336. Parks, W. P., Scolnick, E. M., and Ransom, J. C. (1975b). Cold Spring Harhor Sytnp. Quotit. B i d . 39, 1151-1158. Pascal, R. R., Rollwagen, F. M., Harding, T. A., and Schiavone, W. A. (1975).Ctrticer Res. 35, 302-304. Pitelka, D. R., Bern, H. A,, Nandi, S., and DeOme, K. B. (1964).J.Nrrtl. Cnncer Inst. 33, 867-885. Priori, E. S., Anderson, D. E., Williams, W. C., and Dmochowski, L. (1972).J.N o t / . Cancer I n s t . 48, 1131-1135. Pullinger, B. D. (1947).Rr. J . Concer 1, 177-191. Pullinger, B. D. (1960).Rr. J . Cnncer 14, 279-284. Racevskis, J., and Sarkar, N. H. (1978).J.Virol. 25, 374-383. Riley, V. (1975).Science 189, 465467. Ringold, G. M., Lasfargues, E. Y., Bishop, J. M., and Varmus, H. E. ( 1975a).Virology 65, 135- 147. Ringold, G. M., Yamamoto, K. R., Tomkins, G. M., Bishop, J. M., and Varmus, H. E. (197511).Cell 6, 299-305. Ringold, G. M., Blair, P. B., Bishop, J. M., and Vannus, H. E . (1976). Virology 70, 550-553. Ringold, G. M., Cardiff, R. D., Vaniius, H. E., and Yamamoto, K. R. (1977a). Cell 10, 11-18. Ringold, G. M., Yaniamoto, K. R., Bishop, J. M., and Varmus, H. E. (197711).Proc. N a t l . Acad. Sci. U S A 74, 2879-2883. Ringold, G. M., Yamamoto, K. R., Shank, P. R., and Varmus, H. E . ( 1 9 7 7 ~ )Cell . 10, 19-26. Ringold, G. M., Shank, P. R., and Yaniamoto, K. R. (1978).J.Virol. 26, 93-101. Ritzi, E., Baldi, A., and Spiegelman, S. (1976a). Virology 75, 188-197. Ritzi, E., Martin, D. S., Stolfi, R. L., and Spiegelman, S. (197613).Proc. N a t l . Accid. Sci. USA 73,41904194. Roubinian, J . R., and Blair, P. B. (1977).]. Natl. Cancer Inst. 58, 727-734. Roubinian, J. R., Lane, M. A,, Slomich, M., and Blair, P. B. (1976).J . Zrnmunol. 117, 1767- 1773. Rudczynski, A. B., and Mortensen, R. F. (1978).J.N a t l . Cancer Inst. 60, 205-211. Sarkar, N. H., and Denisey, A. (1977).Proc. Elec. Micros. Soc. A m . , 382. Sarkar, N. H., and Dion, A. S. (1975).Virology 64, 471-491. Sarkar, N. H., and Moore, D. H. (1968).J . Microsc. 7, 539-548.

416

DAN H. MOORE

et al.

Sarkar, N. H., and Moore, D. H. (19724.J. Natl. Cancer Inst. 48, 1051. Sarkar, N. H., and Moore, D. H. (197213).Nature (London)236, 103-106. Sarkar, N. H., and Moore, D. H. (1974).Virology 61, 38-55. Sarkar, N. H., and Moore, D. H. (1978). Cancer Res. 38, 1468-1472. Sarkar, N. H., and Whittington, E. S. (1977).Virology 81, 91-106. Sarkar, N. H., Nowinski, R. C., and Moore, D. H. (1971a).Virology 46, 1-20. Sarkar, N. H., Nowinski, R. C., and Moore, D. H. (1971b).J. Virol. 8, 564-572. Sarkar, N. H., Manthey, W. J., and Sheffield, J. B. (1975).Concer Res. 35, 740-749. Sarkar, N. H., Taraschi, N. E., Pomenti, A. A., and Dion, A. S. (1976). Virology 69, 677-690. Sarkar, N. H., Pomenti, A. A., and Dion, A. S. (1977).Virology 77, 12-30. Schloemer, R. H., Schlom, J., Schochetman, G., Kimball, P., and Wagner, R. R. (1976).J. Virol. 18, 806-808. Schloni, J., and Spiegelman, S. (1971). Science 174, 840-843. Schlom, J., and Spiegelman, S. (1972). Science 175, 542-544. Schlom, J., Spiegelman, S., and Moore, D. H. (1971).Nature (London)231,97-100. Schlom, J., Colcher, D., Spiegelman, S., Gillespie, S., and Gillespie, D. (1973).Science 179,696-698. Schochetman, G., and Schlom, J. (1976).Virology 73,431-441. Schochetman, G., Oroszlan, S., Arthur, L., and Fine, D. (1977). Virology 83, 72-83. Schochetman, G., Long, C. W., Oroszlan, S., Arthur, L., and Fine, D. L. (1978).Virology 85, 168-174. Scolnick, E. M., Young, H. A., and Parks, W. P. (1976).Virology 69, 148-156. Seman, G., Myers, B., Williams, W. C., Gallager, H. S., and Dmochowski, L. (1969).Tex. Rep. Biol. Med. 27, 839-866. Shank, P. R., Cohen, J. C., Varmus, H. E., Yamamoto, K. R., and Ringold, G. M. (1978). Proc. Nritl. Accid. Sci. USA 75, 2112-2116. Sheffield, J. B. (1973).J.Virol. 12, 616-624. Sheffield, J. B. (1974).Virology 57, 287-290. Sheffield, J. B., and Daly, T. M. (1976).Virology 70,247-250. Sheffield, J . B., Daly, T. M., and Taraschi, N. (19764. Proc. Am. Assoc. Cancer Res. 17, 560. Sheffield, J. B., Zacharchuk, C. M., Taraschi, N., and Daly, T. M. (197613).J . Virol. 19, 255-266. Sheffield, J. B., Daly, T., Dion, A. S., and Taraschi, N. (1977). C o w e r Res. 37, 14801485. Shyamala, G. (1974).J. B i d . Chem. 249, 2160-2163. Shyamala, G. (1975). Biochemistry 14, 437-444. Shyamala, G., and Dickson, C. (1976).Nature (London)262, 107-112. Sihal, L. R., Feller, W. F., Fink, M. A,, Kohler, B. E., Hall, W. T., and Bond, H. E. (1969). Science 164, 76-78. Slovin, S. F., Bennett, S. J., and Pascal, R. R. (1977).J.Natl. Cancer Inst. 59,1499-1501. Smith, G. H., and Lee, B. K. (1975).J. Natl. Cancer Inst. 55,493496. Smith, G. H., and Wivel, N. A. (1973).J. Virol. 11, 575-584. Soini, I. (1977). Int. 1.Epiderniol. 6, 365-373. Squartini, F., and Bolis, G. B. (1970).Tex. Rep. Biol. Med. 28, 115-121. Staff of Roscoe B. Jackson Memorial Laboratory (1933). Science 78,465-466. Stuck, B., Boyse, E. A., Old, L. J., and Carswell, E. A. (1964). Nature (London) 203, 1033-1034. Stutman, 0. (1976). Cancer Res. 36, 739-747.

hlAhlhlARY TUMOR VIRUSES

417

Sttitman, 0 . (1977). I ’ r ~ / i r . ~ ~ dProc. u / i t 9, 1153-1155. Taiiaka, H. (1977). Virology 76, 835-850. Tanaka, H., and Moore, D. H. (1967). Virology 33, 197. Tanaka, H., Tamura, A., and Tsujimura, D. (1972). Virology 49, 61-78. Tannenbaum, A., and Silverstone, H. (1953). Ado. Cniicer Res. 1, 451-501. Teramoto, Y. A., Puentes, M. J., Young, L. J. T., and Cardiff, R. D. (1974).J. Virol. 13, 4 11-4 18. Teramoto, Y. A., Cardiff, R. D., and Lund, J. K. (19774. Virology 77, 135-148. Teramoto, Y. A., Kufe, D., and Schlom, J. (197711). Proc. N o t / . Acud. Sci. USA 74, 35643568. Teramoto, Y. A., Kufe, D., and Schlom, J. (1977c).J. Virol. 24, 525-533. Tokuhata, G. K. (1969).Am. J. Epicleniiol. 89, 139-153. Vaage, J. (1978). Caticer Res. 38, 331-338. Vaage, J., a n d Medina, D. (1974). Ctrticer Re.7. 34, 1319-1324. Vaidya, A. B., Black, M .M., Dion, A. S., and hloore, D. H. (1974).Nnttire (Loridotr)249, 565-567. Vaidyn, A. B., Lasfhgues, E. Y., Heubel, G., Lasfargues, J. C., and Moore, D. H. (1976). J . Virol. 18, 911-917. Vaitlya, A. B., Lasfargues, E. Y., Sheffield, J. B., and Coutiiiho, W. G. (1978). Virolog!/ 90, 12-22. van Nie, R., and de Moes, J. (1977). I t i t . J. Cnticer 20, 588-594. van Nie, R., and Verskaeten, A. A. (1975). I t i t , J . Ctrticer 16, 922-931. van Nie, R., Verstraeten, A. A,, and de Moes, J. (1977). Itif. J . Caticer 19, 383-390. Vamrus, H. E., Bishop, J. M., Nowinski, R. C., and Sarkar, N. H. (1972). Ntrttire Nctc B i d . 238, 189-191. Varmus, H. E., Quintrell, N., Medeiros, E., Bishop, J. M., Nowinski, R. C., and Sarkar, N. H. (1973). J . Xlol. Biol. 79, 663-679. Varmus, H. E., Ringold, G., and Yanramoto, K. R. (1979). Z I I “Glucocorticoid Honnone Action” (J. D. Baxter and G. G . Rousseau, eds.), Springer-Verlag, New York (in press). Verstraeten, A. A,, Hageman, P. C., and Kwn, H. G. (1973). Eur. J. Cotrcer 9, 155-157. Verstraeten, A. A , , van Nie, R., Kwa, H. G ., and Hageinan, P. C . (1975). I t 1 t . J . Catrcer 15, 270-281. Wang, E., Wolf, B. A,, Lamb, R. A., Choppin, P. W., and Goldberg, A. R. (1976). I I I “Cell Motility” (R. Goldman, T. Pollard, and J. Rosenl)aum, etls.), 1111. 589-600. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Witte, 0. N., Weissnian, I. L., aiid Kaplan, H. S. (1973). Proc. Natl. Acrid. Sci. USA 70, 36-40. Yagi, M. J. (1973).J. N o t / . C ~ t i c e rIti.vt. 51, 1849-1860. Yamanroto, K. R., and Ringold, G. M. (1977). It1 “Homione Receptors: Steroid Hormones” (B. W. O’Malley and L. Biriibaumer, eds.), Vol. I, pp. 297-322. Academic Press, New York. Yeh, J., Ahmed, M., Lyles, J., Larson, D., and Mayyasi, S. A. (1975). I n t . J. Cancer 15, 632-639. Young, H. A,, Scolnick, E. M., and Parks, W. P. (197S).J. B i o l . Clzem. 250, 3337-3343. Young, H. A., Shih, T. Y., Scohiick, E. M., and Parks, W. P. (1977). J . Virol. 21, 139146. Yunis, E. J., Martinez, C., Smith, J., Shitnian, 0. (1969). C m c e r Res. 29, 174-178. Zachrau, R. E., Black, M. M., Dion, A. S., Shore, B., lsac, M., Andrade, A. M., and Williams, C. J. (1976). Cancer Res. 36, 3143-3146.

418

DAN H. MOORE

et

(11.

Zachrau, R. E., Black, M. M., Dion, A. S., Shore, B., Williams, C. J., and Leis, H. P. (1978).Cancer Res. (submitted). Zak-Nejmark, T., Steuden, J., and Radzikowski, C. (1978).Znt. /. Coiicer 21, 490-495. Zangerle, P. F., Calberg-Bacq, C. M., Colin, C., Franchimont, P., Francois, C., Gosselin, L., Kozma, S., Osterrieth, P. M. (1977).Cancer Res. 37, 4326-4331. Zivada, J., Dickson, C., and Weiss, R. (1977).Virology 82, 221-231. Zotter, S., and Muller, M. (1977)./. N a t l . Cancer Z i i s t . 58, 967-975. Zotter, S., Miiller, M., and Grossmann, H. (1973). Ewp. Pathol. 8, 115-121. Zotter, S., Kryukowa, I. N., Bukrinskaya, A. G., Lezhnewa, 0. M., Ilyink, K. V., Miller, G. G., Muller, M. (1976a). Arch. Gesch. 46, 621-629. Zotter, S., Lossnitzer, A., and Klihr, G . (197613).Acta Biol. Med. Ger. 35, 253-257. Zotter, S., Miiller, M., Kemmer, C. H., Johannsen, B. A,, and Grossmann, H. ( 1 9 7 6 ~ ) . E x p . Pathol. Uena) 12, 46-54.

ROLE OF SELENIUM IN THE CHEMOPREVENTION OF CANCER A. Clark Griffin Department of Biochemistry The University of Texas System Cancer Center M D Anderson Hospital and Tumor Institute. Houston. Texas

.... ............................ .................................... ........................................ IV. Selenium and Cancer.. ........................ I. Introduction

11. Selenium Tox 111. Nutritional Aspects of Selenium

A. Selenium as a Causative Factor or C B. Selenium Effects on Cancer Growth C. Effect of Selenium on Carcinogenesis., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Biological Functions of Selenium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Possible Mechanisms of Action of Selenium in the Inhibition of Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Mutagenic Aspec ...................... B. Effects on Carcin C. Protection agains

VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... References . . .

419 420 421

424 429 432 432

438 440

I. Introduction

The element selenium (Se) was discovered in 1818 by Joiis Jacob Berzelius in lead chamber deposits in a sulfuric acid plant. A close similarity was observed between sulfur and Se by Berzelius and later by chemists who synthesized Se compounds and the corresponding sulfur analogs. The periodic table shows the IV B group as oxygen, sulfur, selenium, tellurium, and polonium. Se, atomic number 34 and atomic weight 78.96, is between arsenic and bromine. Se comprises of the Earth’s crust and ranks seventeenth in order of abundance. It is widely distributed in igneous-type rocks but the major commercial sources are as by-products of the mining of copper, nickel, silver, and zinc. The geological occurrences of Se, the seleniferous areas in the United States, plants and foods, water and air, and the cycling of Se in nature have been summarized in a publication entitled, Selenium, by a Committee on Medical and Biological Effects of Environmental Pollutants, National Academy of Sciences, Washington, D.C., 1976. Many of the properties of Se are covered in this publication and also b y Crystal (1973). Zingaro and Cooper (1974) 419

420

A. CLARK GRIFFIN

have published a comprehensive treatise on Se. The electrical properties of Se have led to its application in many commercial devices including photocells and xerography. Se is also used in the manufacture of drugs, cosmetics including shampoos, pigments, paints, colored glass, rectifiers, transistors, lasers, and as a catalyst in the production of rubber and plastics. It is estimated that approximately one million pounds of Se per year are consumed in the United States. An excellent and comprehensive coverage of the entire field of organic selenium compounds has been edited by Klayman and Gunther

(1973). This chapter will be directed toward the role Se compounds may have on the inhibition or slowing of the induction of cancer by chemicals and other agents. Actually, this constitutes a relatively new field with most of the publications appearing during the last 7-8 years. However, Se has been implicated in many aspects of agriculture, nutrition, toxicology, and medicine for much of this century. At least a part of this extensive literature will require some review in order to provide a rationale and understanding of the possible mechanism(s) that Se may have in the newer findings related to the inhibition of carcinogenesis. It should be mentioned that Se has been suspected of being a carcinogen and that Se, both from a nutritional consideration as well as the administration of selenium compounds, may effect the rate of growth of established tumors. These aspects will be briefly reviewed in this chapter. II. Selenium Toxicology and Pathology

There remains little doubt that higher forms of life are adversely affected by the ingestion of excess quantities of Se compounds. A most interesting review of some of the early observations of diseases resulting from the ingestion of seleniferous plants is covered by Martin (1973). It was noted that Marco Polo in 1295 recorded from his travels to the Orient that beasts of burden were affected by a certain plant which grew in Western China near the border of Turkistan and Tibet. Hooves of these affected animals dropped off and this disorder has been associated with chronic selenosis. A fatal disease in horses grazing in certain areas of South Dakota with loss of hair and with tenderness and inflammation of the feet was ascribed to Se toxicosis. Sheep and cattle in certain regions of Wyoming developed central nervous disturbance (blind staggers), probably from excess Se intake. Workers at the South Dakota experiment station (Franke, Moxen, Potter, Olson, Fihian, Halverson, Petersen, and others) established that grains and

ROLE OF SELENIUM IN CANCER CHEMOPREVENTION

42 1

grasses grown in certain areas contained several parts per million Se and were toxic to animals. Clinical symptoms in horses, cattle, and swine also included retarded growth, emaciation, arthritis, and eventual death. The liver and kidneys of animals exposed to the seleniferous grains and plants exhibited the highest levels of Se followed by the spleen and lungs. These and many other investigators in other laboratories studied all phases of Se toxicology and pathology and ascertained that the animal species and age and the chemical forms of the seleniuni consumed must all be considered. Selenate and selenite had about the same toxicity as equivalent amounts of Se in natural grains, or as Se-methylselenocysteine and selenocystathione. Hydrogen selenide appears to be the most toxic and irritating of the Se compounds known. Apparently the toxicities of organic Se compounds may be related to the extent of involvement of their sulfur analogs in metabolism (Moxen et al., 1938), i.e., relatively low toxicities of ti-propylseleninic acid, P,P’-diselenodipropionic acid. Correspondingly, Schwarz and Pathak (1975) reporting upon the biological essentiality of seleniuni listed several organic Se compounds (selenouracil, phenyl selenide, etc.) that are biologically active in this respect but of minimal toxicity. There are reports in the literature concerning Se toxicity in man. Levels of Se within the soil, water supplies, plants, some industrial processes, and other varied sources have been reported (Shapiro, 1973).It is of interest to point out that the mean concentrations of Se in whole blood of individuals in 19 locations in the United States ranged from approximately 15 pg/lOO ml in Lima, Ohio, to 25.6 pg/lOO ml in Rapid City, North Dakota (collections froin blood banks) (Shaniberger et (11 ,, 1976). Se compounds have some liniited clinical applications. Further aspects of Se levels in tissues as well as Se intake will be considered later. Ill. Nutritional Aspects of Selenium

As noted by Frost and Lish (1975) at least 40 animal species have been shown to demonstrate selenium-responsive diseases. These include liver, kidney and heart necrosis, muscular dystrophy, growth depression, and exudative diathesis. It was further noted that Se in early times had not been recognized as an essential nutrient for humans. However, later studies were quoted emphasizing the need for Se in children and most likely all humans. In addition to the above mentioned organs these authors included the findings of cataracts in rats with advanced Se deficiency, and a possible involvement of Se in

422

A. CLARK GRIFFIN

sperm motility. Selenium was reported by McKeehan et d.(1976) to be essential for clonal growth of diploid fibroblasts from human fetal lung (WI-38) in media containing small amounts of serum proteins. Maximum growth was obtained when 30 nM neutralized selenious acid was added to the synthetic medium. Se was also required by a Chinese hamster cell line that was grown in a protein free synthetic culture medium. A tabulation of the daily dietary intake of Se of selected populations within the United States and also several other countries has been compiled by Zabel and co-workers (1978). The daily Se intake of United States adults ranges from 62-169 pg of Se per day which compares favorably with a desirable daily intake for humans of 60-120 p g Se that has been proposed. It was pointed out however that individuals in certain regions of the world as well as infants on some formulas, vegetarians, and patients on restricted long-term diets may have low Se intakes. Space requirements do not make it possible to cover the elaborate studies from many laboratories that have contributed to our present knowledge of selenium involvement in nutrition. Certainly the requirements of this trace or micronutrient are quite low and under ordinary conditions are supplied by the minute quantities of ubiquitous selenium in our foodstuffs and water. Several factors have influenced the overall efforts directed to the study of the Se requirements for human nutrition and health. One of these concerns the high toxicity and the corresponding fears and regulatory restraints that have been placed upon Se. A related factor concerns the previous lack of knowledge as to what constitutes adequate body or tissue levels of Se and a safe margin of intake of Se compounds. There are major differences in the toxicity, and, most likely, in the biological activities among the inorganic and organic Se compounds that have been adequately discussed by Frost (1976). Also, it was noted that a safety margin of 100 times has been assigned between the safe and toxic doses of inorganic selenite or selenate. It will be of interest to ascertain whether the organic selenicals with reportedly greatly lowered toxicities are also nutritionally and therapeutically adequate. Other complicating aspects of the nutrition of Se are the interrelations with other minerals, many of the vitamins, proteins, and other nutrients. This is best illustrated by the “saving effects” exerted by vitamin E and Se which have been the most extensively studied. An interesting presentation on the interrelationships of Se with other trace elements was given by Hill (1975). Briefly, it was noted that

ROLE OF SELENIUM IN CANCER CHEMOPREVENTION

423

arsenic, mercury, cadmium, and copper render selenium much less toxic than when it is present alone. The presence of arsenic also shifts the excretion of selenium to the bile. This aspect will be covered in more detail in Section VI.

IV. Selenium and Cancer

A.

SELENIUM AS A CAUSATIVE

FACTOR OR

CARCINOGEN

Several of the early studies reported that Se may be a carcinogen. Groups of male Osborne-Mendel rats were fed diets containing either seleniferous grain or a mixture of ammonium potassium sulfate and ammonium potassium selenide. The Se levels were 5, 7, and 10 ppm. Of 53 animals that survived for 18 months it was reported that liver tumors were present in 11 animals although the precise nature ofthese lesions was not indicated (Nelson et a1., 1943). Subsequently, Seifter et (11. (1946) observed multiple thyroid adenomas in rats fed bis-4acetylaniinophenyl selenium dehydroxide (0.05%)for 105 days. Some liver hyperplasia was also noted. In subsequent studies, Tscherkes and associates (1963)reported neoplastic growths in male rats fed 12% protein diets containing 4.3 ppm Se. They concluded that the neoplastic changes were attributable to Se and probably related to an antagonistic effect between Se and methionine. However, both the nature of the growths reported as well as the methionine antagonism of Se have not been further confirmed. Based largely upon some of these early studies, Se was included as a carcinogen in the Delaney Clause of the Food Additive amendment of 1958. As reviewed by Shapiro (1973) several authors have concluded that there exists only incomplete knowledge on which to base the assumption that selenium is carcinogenic. It is noteworthy to indicate that the appearance of tumors has not been reported along with the other multiple pathological conditions associated with Se deficiencies. In the IARC Monographs on the “Evaluation of Carcinogenic Risk of Chemicals to Man” (Vol. 9, 1975, p. 245), Se and Se compounds have been evaluated. This report essentially confirms the above findings with the conclusion: “The available data provide no suggestion that selenium is carcinogenic in man, and the evidence for a negative correlation between regional cancer deaths and selenium is not convincing.”

424

B.

SELENIUM

A. CLARK GRIFFIN

EFFECTSON

CANCER

GROWTH

Klayman has reviewed the role of Se compounds as potential chemotherapeutic agents, including cancer therapy (Klayman, 1973). One rationale in this respect is the replacement of sulfur-containing nutrients by Se analogs. Selenocysteine has been administered in leukemic patients and animals without success. Selenopurine was found to inhibit mouse leukemia L1210 as effectively as 6-mercaptopurine (6MP). When employed in other mouse leukemia however, the seleno compound was less effective and more toxic than the 6MP. Shapiro (1973) has also covered some of the clinical use of selenium-containing compounds. [75Se]-labeledmethionine has been quite widely used as an agent for external organ scanning since it is concentrated by tissues that actively synthesize proteins. Pancreas, liver, and parathyroid have been the most studied. A brief review of these procedures is included in the above reference. However, Kuikka and Nordman (1978) have concluded that better results may be obtained in tumor diagnosis with other radiophannaceuticals. By and large, Se compounds have not found any widespread application in the treatment of established tumors.

c. EFFECTOF SELENIUM ON CARCINOGENESIS 1. Epidemiology und Historicul Shamberger and Frost (1969) were among the first to report that an inverse relationship existed between cancer death rates and the Se levels in forage plants. Further epidemiological studies appeared to relate an enhanced incidence of colon, breast, and other forms of cancer in humans in geographic regions where selenium is deficient and a lowered incidence with higher selenium levels (Shamberger and Willis, 1971; Shamberger et ul., 1976; Schrauzer et al., 1976; Jansson et nl., 1977, 1978; Schrauzer and Ishmael, 1974). Attempts have been made to correlate blood and tissue levels of selenium with the presence of cancer Broghamer et (11. (1976, 1978). Whether Se levels within the body, diet, and environment are major factors in terms of human cancer is indeed a complex issue and in the writer’s opinion a largely unresolved one. Se, as already indicated, is highly ubiquitious in distribution and is present in many chemical forms. These factors along with the long latency period in terms of carcinogenesis and the present trends of individuals in locale, occupation, and travel, as well as medical records, diagnosis, and autopsy reports, etc., make it indeed difficult for precise correlations between

ROLE O F SELENIUM IN CANCER CHEMOPREVENTION

425

Se and cancer causation and/or prevention. With an increasing knowledge of the biological functions of Se such as blood levels of glutathione peroxidase, etc., it may be possible to obtain more meaningful data in this respect (see Section V). Virgin female mice, @,H Strong, were given 2 ppm Se in the fomi of SeO, in the drinking water for 15 months. This exposure reportedly lowered the incidence of spontaneous mammary tumors from 82 (in untreated controls) to 10% (Schrauzer and Ishmael, 1974). In a subsequent study the spontaneous tumor incidence was observed in the C,H St mice that were given 2,5, and 15 ppm selenium, arsenic at the 10 and 80 ppin level, or a Se/Zn supplement at the ratio 5/200 ppm. The percentage tumor incidences in the 2, 5, and 15 ppm Se groups were 10,36, and 33%, respectively, the 10 and 80 ppm arsenic-treated groups were 27 and 40%, respectively, while the SelZn group was listed as 94% mammary tumor incidence, close to the 82% observed in the iiontreated control groups (Schrauzer et ul., 1976). The higher percentages of the spontaneous tumors observed at the two enhanced levels of Se certainly require some further elucidation. Arsenic and zinc may function as Se antagonists thus accounting for the higher tumor incidences found in these studies. The complex interrelations that exist among the trace or minor minimal as well as other micronutrieiits has already been mentioned and will be discussed in more detail in Section V. An early study involving the role of Se in carcinogenesis was carried out by Clayton and Baumann (1949). In these studies albino rats were maintained on semisynthetic diets and were given 0.064% 3’methyl-4-dimethylaminoazobenzene (3’-MeDAB) for 2 weeks. At this time the animals were fed the dye-free diet supplemented with 5 ppm Se (sodium selenite) for 4 weeks. Finally, the animals were given the dye-containing diet for an additional 4 weeks. In two separate experiments there was a reduction of approximately 50% in the liver tumor incidence compared with the control animals that received the basal diet during the intemiediate period. The authors concluded that there was indeed an inhibiting effect of Se on tumor development under the experimental conditions employed. 2. Further Studies on Chemical Curcinogenesis An inhibition of dimethylbenzanthracene-induced tumors in mice by selenium administration was noted by Shamberger (1970).A series of classical studies carried out by Wattenberg and his associates have firmly established that several phenolic antioxidants, ethoxyquin, etc., inhibit the formation of tumors resulting from exposure to the polycy-

426

A. CLARK GRIFFIN

clic hydrocarbons. The inhibition of chemical carcinogenesis, in general, has been covered by Wattenberg (1978). A specific study b y this investigator (Wattenberg, 1975) will be quoted since this provided the basis for many of the Se studies that have been carried out in this laboratory. Disulfiram added to the diet of female CF, mice prevented neoplasia of the large bowel in mice given repeated subcutaneous injections of dimethylhydrazine. Subsequently, a comparable study was carried out in this laboratory to determine the effects of Se on colon carcinogenesis. Sprague-Dawley rats were injected weekly with either 1,2-dimethylhydrazine (DMH) or methylazoxymethanol acetate (MAM). Selenium was added to the drinking water at the level of 4 ppm (sodium selenite). The duration of the study was 17 weeks at which time the colons were evaluated with the results shown in Table I (Jacobs et ul., 1977a). Fewer tumors were present in the seleniumtreated animals with each of the carcinogens. The incidence of MAMinduced tumors was 93% (14/15) with the Se additive and 100% (14/ 14) without the Se supplement. However, Se reduced the incidence of DMH-induced colon tumors by more than 50% (Jacobs et (11 ., 1977a). In a subsequent study the effects of Se on 3’-methyl-4dimethylaminoazobenzene (3’MeDAB)-induced hepatocarcinogenesis was ascertained (Griffin and Jacobs, 1977). Three groups of male Sprague-Dawley rats were maintained as follows : (I) Laboratory chow plus 0.05% 3’-MeDAB; (11) Same as I plus 6 ppm Se (Na,SeO,) in the drinking water; (111) Same as I plus 6 ppm Se added to the diet in the form of a high-Se yeast. The azo compound was incorporated in the diet for 8 weeks and then removed and the Se supplements in Groups I1 and I11 were continued for an additional 4 weeks. Evaluation at this time revealed a 92% tumor incidence (11/12 animals) in the Group I controls while the Se injection reduced the incidence to 46% (7/15) in Group I1 and the dietary supplementation to 64% (9/14) in Group 111. Harr et al. (1972) reported that Se supplementation reduced the number of tumors in rats given 2-acetylaminofluorene (AAF).Similar findings were noted when male rats given diets containing 0.03% 2-acetylaniinofluorene (AAF) were given 4 ppm Se (N&jeO,) in water for 14 weeks. A final tumor incidence of 9/13 rats was observed in the group given the basal diet alone while 4/14 rats developed hepatoinas when given the Se supplement. No tumors or liver changes were noted in the control animals fed the carcinogen-free basal diet but maintained on the Se supplement for the entire period (Marshall et uZ., 1978a,b). These findings are generally in agreement with those reported by Harr et a l . (1972). However, their experimental design

TABLE I SELENIUM EFFECTSON BODYWEIGHT,TUMORINCIDENCE, AND LOCATION

IN THE

PRESENCE OF DMH OR MAMa

Colonic tunior

Group DMH

+ Se

205 2 8 206

DMH MAM

Weight gain in grams (k-SD)

+ Se

2

6

P 184 2 5

P MAM

" Jacobs et

185 ? 7

=

=

Total number

Average number tumodrat

11

0.7

Colonic tumor location Incidence*

39

2.6

6'15\ P = 0.025 13/15

42

2.8

14/15

0.001

0.8 73

5.2

1

14/14

P

=

Proximal

Transverse

Distal

1

8

2

11

22

6

6

22

14

22

35

16

1.0

( I / . (19774. Number of rats with tumorsltotal number surviving rats per group. One animal in the MAM group died after 5 weeks.

428

A. CLARK GRIFFIN

differed considerably from our own. These investigators employed young female OSU-Brown rats that were fed a low Se formula ration that was supplemented with vitamin E at the level of 60 ppm. These investigators added AAF to the diets of 0.015% acd four groups were given Se at the levels of 2.5,0.5,0.10,and 0 ppm. They noted signs of Se deficiency in the latter groups and “toxic hepatitis” in most of the livers of rats in the high Se groups (2.5 ppm Se). These were not observed in the present study and as noted the control animals given 4 ppm in the water did not manifest toxic hepatitis. Harr et al. saw a relatively large number of mammary tumors and the beginning of hepatic neoplasms in the first two groups after approximately 100 days of exposure to AAF. After 200 days there was an accumulated neoplasm incidence of 60%in these two low Se groups and less than 10% in the Se-supplemented groups. It is of interest to note, however, that all of the groups exhibited quite high accumulative percentage incidence of tumors (SO-100%) after 300 days of exposure to AAF. Harr et nl. noted that “this study did not determine whether the observed effects of added selenite represented a prevention of carcinogenesis or a modification of the rate of induction.” Since we observed a major decrease in the actual numbers of hepatic tumors with the Se supplementation, it appeared to be the result of a modification of the rate of induction. A study recently completed included groups of Sprague-Dawley male rats all fed diets (ground Wayne Lab-Blox, Allied Mills, Inc., Chicago, IL) containing 0.05% 3’-MeDAB for 9 weeks (Table 11). TABLE I1 SELENIUM

Group (15 rats)

I

I1 111

IV V

VI VII

EFFECTSON AZO DYEHEPATOCARCINOGENESIS

Treatment Controls 4 ppm Se in drinking water, entire period 4 ppin Se in drinking water, initial 3 weeks 4 ppm Se in drinking water, final 4 weeks 2 ppm Se in drinking water, entire period 1% Sorbic acid added to diet, entire period 0.5% Butylated hydroxytoluene added to diet, entire period

Liver tumor incidence at termination of study 12/15 2/14 7/13 5/13 6/15 15/15

0115

ROLE OF SELENIUM IN CANCER CHEMOPREVENTLON

429

Selenium was added to water as Na,SeO,. Following the 9-week period on the carcinogenic diet, the animals were maintained for 1 month on basal diet plus the supplement as indicated in the treatment (A. C. Griffin and A. H. Daoud, unpublished data). The above findings confirm the finding of the inhibition of selenium upon azo dye hepatocarcinogenesis (Griffin and Jacobs, 1977). Also of interest was the findings in Group IV that a reduction was observed in hepatocarcinogenesis when the Se was given during the later stages of the study. This exciting possibility of counteracting or inhibiting carcinogenesis in animals already exposed to cancer-inducing agents will be discussed further in Section VI,D.

V. Biological Functions of Selenium

The requirement for selenium as a micronutrient in man and animals was partially covered in Section 111. Attention also is directed to the publication “Selenium, Medical and Biological Effects of Environmental Pollutants” from the National Research Council, National Academy of Sciences, Washington, D.C. (1976). Stadtman (1977), in a recent review pointed out that there are three presently known selenoenzymes : Fonnate dehydrogenase catalyzing the reaction: HCOOH

+ A + AH, + CO,

(Pinsent, 1954) in Escherichin coli and various anaerobic bacteria of the genus Clo~tridium. Glycine reductase catalyzing:

(Turner and Stadtman, 1973) in Clostridium. Glutathione peroxidase which catalyzes the important reaction: BGSH

+ HZOp + GSSG + 2HZO

Reference was also made to a selenoprotein of about 10,000 daltons that is missing from heart and other muscles of animals suffering from a Se syndrome known as white muscle disease (Stadtman, 1977). Since glutathione peroxidase is widely distributed in mammalian tissues and with a possible involvement in tuinorigeilesis this will be considered in further detail. The essentiality of Se for animals was first noted by Schwarz and Foltz (1957),who observed that liver necrosis in rats was

430

A. CLARK GRIFFIN

prevented by vitamin E or “factor 3” an organic form of selenium. Other organic forms of Se as well as selenite and selenate also exhibited preventive activity but to a considerably lesser degree than “factor 3.” The nutritional interrelations between Se and vitamin E have been adequately reviewed by Hoekstra (1975) and the evidence leading to the essentiality of each was presented. For example, it was pointed out that in chicks the pancreatic necrosis occurring with diets low in Se was not alleviated with vitamin E. This vitamin was also shown to be essential since many of its deficiency defects and specifically those aggravated by diets high in unsaturated fats were not prevented by Se. Biological antioxidant properties have been ascribed to Se and vitamin E. Since selenite per se is not an antioxidant as noted b y Hoekstra (1975-4, Se must be a precursor of a compound or complex capable of carrying out antioxidant functions. This function of Se was elucidated with the discovery by Hoekstra and associates that erythrocyte glutathione peroxidase is a selenoenzyme (see Hoekstra, 1974, for a review of these important contributions). Mills (1957) found that cattle erythrocytes contained in addition to catalase, another enzyme which catalyzed the breakdown of H202.This latter enzyme, along with reduced glutathione, would prevent the oxidative denaturation of hemoglobin by H202.Mills later showed that this enzyme glutathione peroxidase, was present in liver, lung, kidney, and other tissues ( 1960).

Glutathione peroxidase from ovine or bovine erythrocytes contains four Se atoms per mole of protein of 88,000 daltons or an average of one Se per protein subunit of 22,000 (Oh et al., 1974). As reported by Hoekstra (1975) Se deficiency in the rat, chick, and sheep caused remarkable decreases in the activity of this enzyme in liver, red blood cells, kidney, heart, adrenal, lung, testis, brain, and lens. Decreases in the glutathione peroxidase correlated with lesions caused by selenium deficiency. This constitutes an important enzyme in the breakdown of H,O, and organic hydroperoxides such as those generated from the metabolism of lipids. One of its important functions is the prevention of oxidative damage to cellular membranes and other sensitive cellular sites or components. According to Hoekstra (1975) the Sedependent enzyme destroys lipid hydroperoxides and other peroxides. Vitamin E also protects against oxidant damage to membranes by preventing the formation of the lipid hydroperoxides. Hoekstra has proposed a scheme as shown in Fig. 1which provides at least a partial explanation of the interactions among Se, vitamin E, unsaturated lipids, sulfur-containing amino acids, and cell-damaging agents [see also Combs et al. (1975); Burk et al. (1978)l.

ROLE OF SELENIUM IN CANCER CHEMOPREVENTION

43 1

CHEMICAL DAMAGE TO CRITICAL SH-PROTEIN

ENZYME SYSTEMS AS

2Hz0

OXIDANT STRESSORS

ROH t H20

LIPIDS

+

LIPID PEROXIDATION MALONIC DIALDEHYDE, ETC CELL DAMAGE VITAMIN E "BLOCKS" REACTION SE, AS A COMPONENT OF GSH-PEROXIDASE, CATALYZES REACTION

0 2

FIG. 1. Scheme showing the postulated mechaiiisrns of interaction between Se and vitamin E in preventing oxidant damage. See text for detailed explanation (Hoekstra, 1975).

Selenium appears to play an invaluable role as a metabolic antidote to heavy metal and other metal toxicities including arsenic, cadmium, mercury, methyl mercury, copper, silver, and lead (Rastogi et d., 1976) [see also Frost and Lish (1975); Prohaska et al. (1977); Sifri and Hoekstra (1978);Jensen et al. (1977); Yaniane et al. (1977)l.It should also be pointed out that compounds of some of the above metals also have been reported to detoxify or counteract toxic levels of selenium. The mechAnism(s) involved in these reciprocal detoxifications are not fully understood. Other functions of selenium have been reviewed by Frost and Lish (1975) and Frost (1976). These include an apparent need for Se for transferance of ions across cell membranes, ubiquinone (Coenzyme Q) biosynthesis, antibody formation (see also Burton et al., 1977),biosynthesis of certain prostaglandins, and counteraction of toxicity of chlorinated hydrocarbons. Oniaye et (11. (1978) reported an enhanced lung toxicity of the herbicide paraquat (1,l'-dimethyl-4,4'bipyridylium dichloride) in selenium-deficient rats. It was suggested that lipid peroxide formation was a factor in the enhanced toxicity. A radiation protective effect of some Se components has been reported b y Badiello and co-workers (1977).

432

A. CLARK GHIFFIN

Saelinger et nl. (1972) found in E . coli grown in a medium containing [75Se]selenite,the presence of selenobases in transfer RNA, probably a transfer RNA tyrosine. It was suggested that Se may be a commonly occurring minor constituent of tRNA, probably through the same pathways of the thiolation of the tRNAs. Later, it was shown that the Se in the tRNAs was in the form of 4-selenouridine (Hoffman and McConnell, 1974). Finally, the hormonal regulation of Se metabolism was studied by Behne et al. (1976) by determining the element level in the blood serum of rats after the administration of sex hormones and during pregnancy. The main finding was a decrease in the Se serum level the tenth and fifteenth days of gestation. VI. Possible Mechanisms of Action of Selenium in the Inhibition of Carcinogenesis

A comprehensive coverage of the inhibition of chemical carcinogenesis recently has been made by Wattenberg (1978). Accordingly, many of the concepts of the mechanisms involved have been presented and the reader is directed to this excellent review. The effectiveness of a relatively large number of the inhibitory compounds may be related to their antioxidant properties. However, other characteristics are undoubtedly involved since all “antioxidants” that have been studied have not been shown to be effective inhibitors of carcinogenesis. In fact, the selenium (sodium selenite) that has been quite effective in many studies in carcinogenesis is not an antioxidant per se. The possibility does exist that the Se may be functioning through glutathione peroxidase which will be considered later in this section. A. MUTAGENICASPECTS Selenium decreased the mutagenicity of 2-acetylaminofluorene (AAF),N-hydroxyacetylaminofluorene (N-OHAAF), and N-hydroxyaminofluorene (N-OHAF) in the Salnzonella typhimurium TA 1538 bacterial tester system (Table 111). It may be noted that decreases in mutagenicity accompanied increasing Se concentrations. Se decreased the mutagenicity of AAF, N-OHAAF, and N-OHAF to 65,68, and 61% of their respective controls with mutagen alone (molar ratios of Se to mutagen: Se : AAF, 10 : 1, Se :N-OHAAF, 10 : 1, and Se :N-OHAF, 300 : 1). Shamberger et al. (1978) have reported that malonaldehyde, a peroxidative breakdown product of unsaturated lipid, was a carcinogenic initiator on

ROLE OF SELENIUM IN CANCER CHEMOPREVENTION

SELENIUhl

Carcinogen

TABLE 111 REDUCTIONOF MUTAGENICITYOF AAF, N-OHAAF, AND N-OHAF"

(tiiA\f)

in .If Se len i i t 111 (Na2Se0,,)

His+ revertants per plate (fSD)

-

1768 149 1411 f 41 1336 f 69 1148 f 71 2353 f 12 1891 f 210 1598 f 71 1247 53 655 43 1628 2 41 1280 f 88 1233 f 31 999 f 26

AAF (4.5) AAF (4.5) AAF (4.5) AAF (4.5) N-OHAAF (0.45) N-OHAAF (0.45) N-OHAAF (0.45) N-OHAAF (0.45) N-OHAAF (0.45) N-OHAF (0.065) N-OHAF (0.065) N-OHAF (0.06S) N-OHAF (0.065) "

433

4 10 40 -

0.4 4.0 10 40 -

0.1 10 20

*

* *

Activity ( %')

100 80 76 65 100 80 68 53 28 100 79 76 61

Jwobs et n l . (19771)).

mouse skin and was shown to be mutagenic in the Aiiies tester system. Malonaldehyde was mixed in separate studies with Se, ascorbic acid, or vitaniiii E and the mutant strain was poured into the top agar which was applied to histidine-deficient media. Se and the antioxidant compounds prevented niutagenesis in the strains which have been employed to detect various types of frameshift mutagens. Contrastingly, Lo et (11. (1978)found that at doses froin 8 x lop5to 3 x 1OP3A4 sodium selenite induced DNA fragmentation, DNA-repair synthesis, chromosome aberrations, and mitotic inhibition in cultured human fibroblasts. Comparable results were obtained when xerodemia pigmentosuni fibroblasts were exposed to selenite. Incubation with mouse liver S-9 microsomal fraction increased the capacity of selenite to induce chromosomal aberrations. Sodium selenite at doses from 8 x lop5 to 3 x M could not be activated by incubation with the S-9 preparation. The authors pointed out that there was a good correlation between the carcinogenic properties of a compound and its response with the DNA-repair assay. Also, they reiterated that most chemical carcinogens induced chromosome aberrations in mammalian cells and that the current findings do support the claim that Se is a carcinogen. Generally, these findings are in agreement with those of Nakamura et al. (1976) who listed the cytogenetic effects and the reactivity with DNA of a series of Se compounds in cultured human lymphocytes.

434

A. CLARK GRIFFIN

Chromosome breaking activity was noted in decreasing order: H,SeO,, N*SeO,, SeO,, H2Se04,and Na2Se04(concentration ranges from 13to 53 x lop5M with the exception of Se02which was from 6.5 to 26 x lop5M ) . Chromosome breakage activity was higher for the compounds with four-valent Se than with the six-valent compounds. Selenites produced greater damage to the DNA than selenates and a significant loss of transformation of tryptophan markers of Bacillus subtilis DNA treated with H2Se0, and SeO,. Thus there is a need for further studies in order to clarify the complex interrelations that exist in terms of the requirements and functions of Se, toxicological effects, possible carcinogenic involvement or inhibition that are coupled with the many different Se compounds. €3. EFFECTSON CARCINOGEN METABOLISM

Early studies in this laboratory (Rasco et al., 1977) provided indication that Se may effect cellular aryl hydrocarbon hydroxylase activities. Human lymphocytes were cultured in the presence and absence of inducers of aryl hydrocarbon hydroxylase (AHH). The presence of M Se (NhSeO,) in the culture medium had no effect on noninduced or induced levels of AHH. The presence of Se in the actual AHH assay inhibited the AHH activity by more than 50%. This level of inhibition was observed at concentrations of the substrate, benzo(a)pyrene of 1, 3, 10, and 100 pA4 and Se concentrations of 0.1, 0.3,1, and 10 mM, respectively. Liver microsomes were isolated from 3-methylcholanthrene-induced and noninduced rats. AHH activity was ascertained with different ratios of Se and benzo(a)pyrene (BP) in the assay mixture. Chromatographic analysis revealed a decrease in the BP metabolites in the presence of Se (Marshall et al., 1 9 7 8 ~ ) . A study is now in progress to ascertain the i n vivo and in vitro effects of Se on 2-acetylaminofluorene. An i n vitro assay system utilizing microsomes from Se-supplemented or nonsupplemented 3-methylcholanthrene-induced rats was used to determine the effects of dietary Se in the metabolism of AAF. Se did not decrease the induction of mixed function oxidase activity following 3-methylcholanthrene administration. A decrease in the formation of N-OHAAF was observed along with an approximate 33% increase in the appearance of ring OHAAF. From these early studies it would appear that the effect of selenium in the slowing or inhibition of carcinogenesis may be attributed to an increase in ring hydroxylation along with a decrease in the N-hydroxylation of the AAF (Marshall et al., 1978a, 1978b).

ROLE OF SELENIUM IN CANCER CHEMOPREVENTION

435

In another study male albino rats were given 4 ppni Se (as N%SeO,) in the drinking water for 3 days. Control (no Se) and the above animals were given 17 mg [14C]AAF-9 or [l4C]N-0HAAF-9 per kg body weight. Administration of Se enhanced glucuronyl transferase activity b y 100% and inhibited sulfotransferase activity by 50%. Pretreatment with the Se in these short-term studies resulted in a lowering of the liver tissue levels of both AAF and N-OHAAF by approximately 30%. These studies also provided indication that Se may enhance the detoxification of AAF and also affect the enzymes that may be involved in the metabolic activation (Daoud and Griffin, 1978).

c. PROTECTION AGAINST OXIDATIVE DAMAGE Selenium, as well as several other trace minerals and nutrients, are required to protect cells from aberrant oxidations. The superoxide disinutases which require manganese, zinc, or copper are involved in which is a common the protection against the superoxide radical, 02-, intermediate in many biological oxidoreductions. Copper-zincsuperoxide dismutase is a highly ubiquitous enzyme appearing in all cells of eukaryotic species that have been studied. This enzyme catalyzes the change in the superoxide radical in order that it may be further reduced by the glutathione peroxidase. The important role of Se, as well as vitamin E, in the activation and function of glutathione peroxidase has been presented in Section V. At the current time there is no direct evidence linking glutathione peroxidase activities with carcinogenesis. This indeed constitutes an area in which there is and will continue to be intensive investigation. Pinto and Bartley (1973) have ascertained the glutathione oxidase and peroxidase activities in the livers of rats treated with diethylnitrosamine and observed a decrease in the glutathione peroxidase. York et al. (1976) observed a 34% increase in the lung glutathione peroxidase activity in rats that were exposed to cigarette smoke for several days. It is possible that a selenium deficiency may have altered this activity; however, this still remains to be ascertained. Burk et nl. (1978) have shown that the presence of a Se-independent glutathione peroxidase removes organic hydroperoxides in the intact rat liver. Peskin et (11. (1977) reported low levels of superoxide dismutase and glutathione peroxidase in tumors that were investigated. Evidence has been presented in this review that an excess of many of the minerals may deplete the body of Se and also reduce the glutathione peroxidase levels. Such prolonged effects may provide at least a partial explanation and rationale for the carcinogenic effect of

436

A. CLARK GRIFFIN

metals. Blot and Fraumeni (1975) have reported an increased mortality from lung cancer in men and women living in counties of the United States where lead, copper, and zinc refineries are located. An excellent review of the carcinogenic effects of metals in man and animals was recently compiled by Sunderman (1978). There appears to be a general consensus that a high dietary intake of fat may be related to an increased incidence of large bowel, breast, prostate, and other forms of cancer. Since lipid oxidation may be affected by glutathione peroxidase levels it may be assumed that deficiencies in Se and/or vitamin E may be related to the origin of these tumors. It would be worthwhile to conduct careful experimentation in animals to ascertain if the enhancement of chemical carcinogenesis by high lipid intakes could be modified by the Se levels of the food or water. Also, the findings should be correlated with tissue levels of the food or water and with tissue levels of both Se and glutathione peroxidase. While many studies have been carried out in terms of the interrelations among selenium deficiency diseases, effects of dietary intake of selenium, blood, tissue levels of glutathione peroxidase, etc., it would appear that further comprehensive studies should be conducted to ascertain whether this would lead to a better assessment of the actual requirements for Se in human nutrition. Recently, Schrauzer and White (1978) reported findings suggesting that glutathione peroxidase levels did not correlate with the blood Se concentration. In their survey it was reported that most individuals had Se intakes of 90-168 pg per day. Supplementation with 150 p g Se per day resulted in increased blood levels to approximately 0.2 pg/ml and greater supplementation to 0.35 p g Se/ml. However, the blood levels of glutathione peroxidase remained relatively constant following the enhanced Se supplementation. Anderson et al. (1978) found a highly significant correlation between erythrocyte and glutathione peroxidase activity and whole blood Se in cattle and sheep. Low Se intakes caused a steady decline in the blood cell glutathione peroxidase; later, the muscle activity of this enzyme declined. The distribution of Se and glutathione peroxidases in various tissue cytosols in lambs has been investigated (Black et al., 1978). Finally, it appears to this writer that one of the major unanswered questions in this respect is whether the optimal levels of dietary Se for the postulated nutritional requirement and for cancer prevention are the same. In the inhibition of chemical carcinogenesis studies reported in Section IV we employed Se levels in most instances at the level of 4 ppni Se in the food or water, which is considerably higher than the reported levels for adequate nutrition. Our only justification was that of at-

ROLE OF SELENIUM IN CANCER CHEMOPREVENTION

437

tempting to ascertain Se effects, and, if a little Se inhibited or reduced carcinogenesis, more Se would be even better. Control animals in these studies (mostly young adult rats) given the same basal diet and this same level of Se, but without the carcinogen, did not exhibit adverse effects in ternis of weight loss, appetite, or gross liver or organ change. However, the addition of 4 ppm Se to the water of newborn rats (and mother) resulted in a major weight decrease in both the males and fernales of 4 weeks of age. At this time the animals were weaned but continued on the same Se level in the water. At 8 weeks of age the weights of the males and females were about 50% that of comparable control animals not given Se (unpublished data, A. H. Daoud and A. C. Griffin). This severe weight loss indic.‘I t es sonie extent of toxicity in these young animals. Further studies with varying levels of Se in young animals, as well as prenatal levels of Se, are in progress. A definite inhibition of azo dye-induced hepatomas was seen in rats given 2 ppm in the water (Table 11, Group V). However, additional studies are required utilizing highly purified diets of known Se content in order to establish the optimal Se requirements for the inhibition of chemical carcinogenesis.

D. STAGES OF CARCINOGENESIS AFFECTED BY SELENIUM Retinoids, analogs of vitamin A, appear at this time to be the most promising compounds for cheniopreveiition of carcinogenesis [Workshop on Chemoprevention of Cancer, National Institutes of Health, February 2-3,1978, Bethesda, Md. See also Sporn et u1. (1976) and Spom 1977)l. These compounds have been shown to prevent cancer of the skin, lung, bladder, and breast in experimental animals and are involved in controlling cell differentiation. Results from several studies indicate that the retinoids are involved in the promotional aspects of carcinogenesis and have little effect on initiation. Boutwell and associates (Boutwell, 1977) reported the induction of ornithine decarboxylase and other enzymes in mouse epidermis by various classes of tumor-promoting agents. However, only the induction of oi-nithine decarboxylase appeared to be specific for tumor promotion in this system. The experimental protocol was a s follows: mice (skin) were treated with a low initiating dose of dimethylbenzanthracene (DMBA),followed in a few days by the weekly application ofthe potent tumor-promoting phorbol ester ( 12-O-tetradecanoyl-phorbol-I3-acetate). There was an almost immediate several hundred-fold rise in the ornithine decarboxylase. This induced enzyme has a short half-life.

438

A. CLARK GRIFFIN

Papillomas appeared in a few weeks, followed by a high incidence of skin tumors. When the retinoids (p-retinoic acid, a-retinoic acid, 13-cis-retinoic acid and other compounds tested) were given simultaneously with the initiator DMBA or before the phorbol, there was still a major increase in the enzyme followed by the appearance of papillomas and cancerous growth. However, if the retinoids were given after the phorbol esters there was a marked inhibition of the enzyme induction and tumor formation (Verma et nl., 1978). As this important approach proceeds it should be possible to pinpoint the precise stage(s) in the promotion or progression of carcinogenesis in which the retinoids or other inhibitors are most effective. A most intriguing paper recently appeared (Grubbs et ul., 1977), wherein transitional cell carcinoma was induced in the bladders of male rats given 12 oral doses of N-l~utyl-N-(4-hydroxybutyl)nitrosamine. Feeding of 13cis-retinoic acid after completion of the carcinogen treatment reduced the number by approximately 50% of the cancers in the bladder. If it is possible to inhibit or reverse the effects of previous exposure to carcinogens at one of the stages of tumor promotion the effectiveness of this chemopreventive approach will become more promising. At the present time little is known as to the effect of selenium supplementation at various stages or intervals with relation to when the carcinogens are given. Some preliminary approaches have been made in this direction and other studies, especially the Boutwell model as described above, are in progress. Clayton and Baunian (1949)reported a decrease in azo dye-induced hepatomas when the diet was supplemented with Se during a period when the carcinogen was not fed. In quite preliminary studies it was observed (Table 11) that administration of Se in the drinking water during the final 4 weeks of the feeding of diets containing 3-methyl4-dimethylaminoasobenzene for a total of 9 weeks resulted in an approximate 50% decrease in liver tumor formation. These time and dose interrelations between the exposure to the carcinogen and the chemopreventive compound constitute the critical factors in determining the effectiveness of this overall approach. VII. Summary

Selenium compounds in the diet or water have now been shown to be effective inhibitors of carcinogenesis in experimental animals exposed to increasing numbers of chemical carcinogens includdimethylbenzanthracene, ing: 3-methyl-4-dimethylaminoazobenzene, 2-acetylaminofluorene, 172-dimethylhydrazine, and methylazoxymethanol acetate. In addition, female C3Hmice given 2 ppm Se in the

ROLE OF SELENIUM IN CANCER CHEMOPREVENTION

439

drinking water had a 10% spontaneous inanimary tumor incidence compared to an 82% tumor incidence in the untreated control series, There are several epidemiological studies suggesting an increased incidence of colon, mammary, and perhaps other forms of cancer associated with low levels of environmental Se or with Se deficiencies. Or, the inverse relationship of a lowered incidence of human tumors with adequate or enhanced Se intake has been implied. As noted in this review, Se may be involved in the conjugation and detoxification of heavy metals and the resulting lowered body and tissue levels of Se has been proposed as a factor in the higher levels of cancer associated with some mining and smelting operations. It would appear that additional studies will be required in order to establish that selenium levels may be related to the incidence of clinical cancer. Postulated roles or mechanisms that may be involved in the inhibition of carciiiogenesis by Se are: (a) a reduction in the mutagenicity of cancer causing chemical as was shown to be the case with 2acetylaminofluorene and N-hydroxyacetylaminofluorene when Se was added in the Salmonella typhirnuriuiri bacterial tester system; (b) effects on carcinogen metabolism. The addition of Se to cultured human lymphocytes resulted in a major inhibition of aryl hydrocarbon hydroxylase activity. In studies to ascertain the effects of Se on 2-acetylaminofluorene metabolism, a decrease in the formation of N-hydroxyacetylaminofluorene was observed along with a 33% or higher increase in the appearance of the less carcinogenic ring OH metabolites. Subsequent studies have revealed that Se enhances the activation of enzymes involved in carcinogen metabolism and decreases in enzyme activities that play a role in the metabolic activation of chemical carcinogens. (c) Protection against oxidative damages. Se a s a component of one of the glutathione peroxidases may protect cells and, more specifically, cellular membranes from aberrant oxidations. At least 40 animal species have been reported that deinonstrate selenium-responsive diseases and the evidence indicates that humans are no exceptions. In view of the ubiquitous nature of Se, it has been difficult to arrive at precise requirements for humans. In fact, it appears that the toxic manifestations, many admittedly unfounded, have produced legislative regulations on allowable Se levels in foods and water that may be too low to meet the nutritional needs in many instances. The important discovery by Hoekstra and associates that Se is a component of one of the glutathione peroxidases opens new avenues for assessment of nutritional adequacy or deficiency of Se by determination of blood or other tissue levels of this enzyme. It is not unlikely that other biochemical functions of Se will be found. One of the major unanswered questions at this time is whether the

440

A. CLARK GRIFFIN

optimal Se levels or intakes for nutrition and for the inhibition of carcinogenesis are the same. It will be difficult to answer such a question until the mechanism(s) of the Se inhibition have been elaborated. As mentioned in the text, the requirements for Se are complicated by factors including age, the chemical form of selenium, and the levels of metals such as arsenic, cadmium, zinc, copper, etc. Also, it was reported that Se may be involved in counteracting the toxicity of chlorinated hydrocarbons. It is quite possible that many environmental components, including pesticides and even potential carcinogens, may enhance the requirements for selenium. Admittedly, the levels of Se employed in the carcinogenesis inhibition studies that have been reported are quite high and further studies are now in progress to ascertain if lower levels will be effective. The relatively high toxicity of Se still constitutes a major problem and must be given serious consideration. It is possible that some of the organic selenium compounds may have greatly lowered toxicities but still provide the Se required for nutritional and biological activities. The effect of such compounds on the inhibition of carcinogenesis will undoubtedly be investigated in the near future. While there are some proponents for the supplemention of Se in humans it is the firm opinion of this reviewer that this is not warranted at this time. In fact, there are reasonable doubts that Se may have any practical value in the prevention of cancer in humans. The concept of the chemoprevention of cancer is of major importance and should be given every serious consideration. Perhaps a better understanding of the mechanisms involved in the inhibition or slowing of carcinogenesis by administration of Se will lead to safer and even more effective methods of prevention.

ACKNOWLEDGMENTS The author is an American Cancer Society Professor of Biochemistry. Part of the studies reported were supported by research grants from the Robert A. Welch Foundation, Houston, Texas. Acknowledgment with great appreciation is made to colleagues involved in our own studies reported herein, including Maryce M. Jacobs, Thoinas K. Matney, Marilyn Arnott, Milton Marshall, and A. H. Daoud.

REFERENCES Anderson, P. H., Berrett, S., and Patterson, D. S. P. (1978)./. C o m p o r . Prrthol. 88, 181. Badiello, R., Solenghi, M. D., and Tmnba, M. (1978). Ztul. /. Biochetn. 26, 255. Behne, D., Elger, W., Schmelzer, W., and Witte, M. (1976). Bioinorg. Clwm.5, 199. . Black, R. S., Tripp, M. J., Whanger, P. D., and Weswig, P. H. (1978). Bioinorg.C h e t ~ i8, 161. Blot, W. J., and Fraumeni, J. F., Jr. (1975). Lancet 2, 142.

ROLE O F SELENIUhf IN CANCER CHEIIOPHEVENTION

44 1

Boutwell, R. K. (1977). I I I “Origins of‘ Hitman Cancer, Book R” (H. €I. Hiatt, J. D. Watson, antl J. A. Winston, etls.), 11. 165. Cold Spring Harl)or, Lalmratory, Cold Spring Harbor, New York. Broghamer, W., Jr., McConnell, K., and Blotcky, A. (1976).Cuiicer 37, 1348. Broghamer, W. L., hlcConnell, K. P., Grimdtli, hl., and Blotcky, A. J. (1978).Cnricer 41, 1462. Burk, R. F., Nishiki, K , , Lawrence, R. A , , and Chance, B. (1978).J . H i o l . C ~ P I 253, J~. 43. Burton, R. hi., Higgins, P. J., and McConnell, K. P. (1977).Bioc/iim. Biolih!/.v. A c f n 493, 323. Clnyton, C. C., and Bauniahn, C . A. (1949).Cnricer Res. 9, 575. Cotnl,s, G. F., Jr., Noguchi, T., and Scott, M. L. (1975).Fed. Proc. 34, 2090. Crystal, R. G. (1973).I I I “Organic Seleniutn Compounds: Their Chemistry and Biology” (D. L. Klayman and W. H. H . Giinther, eds.), p. 13. Wiley, New York. Daoud, A. H., antl Griffin, A. C . (1978).Cciiicer- Lett. 5, 231. Frost, D. V. (1976).Feedstufi? 48, 55. Frost, D. V., and Lish, P. M. (1975).Aiiii. Rec. Phrrricicol. 15, 259. Griffin, A. C., and Jacolis, h1. M. (1977).Curicer Lett. 3, 177. Gruhbs, C. J., Moon, R. C., Squire, R. A , , Farrow, G. M., Stinson, S. F., Gootlman, D. G., Brown, C. C., and Sporn, M. G. (1977).Science 198, 743. Harr, J. 9.. Eson, J. €I., Whanger, P. D., nnd Weswig, P. H. (1972).C/iit. Tosico/. 5 ( 2 ) , 187. * Hill, C. H. (1975).Fed. Proc. 34, 2096. Hoekstra, W. G. (1974).111 “Trace Element hletabolism in Animals 2” (W. G. Hoekstra, J. W. S1tttie.H. E. Ganther, and W. Mertz, eds.), p. 61. University Park Press, Baltimore, Maryland. Hoekstra, W. G. (1975).Fed. Proc. 34, 20HO. Hothian, J. L., and McConnell, K. P. (1974).B i o p h y s . Bioc/iirii. Acto 366, 109. Jacohs, M. M . , Jansson, B., m d Griffin, A. C. (1977a).Caricer Lett. 2, 133. Jncolx, M. M., Matney, T. S., and Griffin, A. C. (197711).C o m e r Lett. 2, 319. Jansson, B., Jacol)s, hi. Il., and Griffin, A. C . (1978).Proc. Iiiciugunrl Corif. Ziit. A s . w J ~ . Bkiiiiorg. Sci., p. 305. Jansson, B., Malahy, M.A,, and Seiliett, C. B. (1977).Proc. .3rd Znt. S y n i ] i . o i i Detectioit cind Pretxritioit of Ctrricer, 1161. Jensen, L. S., Werho, D. S., and Leyden, D. E . (1977)./.N u t r . 107, 391. Klayman, D. L. (1973).111 “Organic Selenium Compounds: Their Chemistry and Biology” (D. L. Klayman and W. H. H. Ciinther, eds.), p. 727. Wiley, New York. Klayman, D. L., and Giinther, W. H. H. (1973).I n “Organic Selenium Cotnpounds: Their Chemistry and Biology” (D. L. Klaynian and W. H. H. Giinther, eds.), 1188 pp. Wiley, New York. Kuikka, J . and Nordman, E. (1978).I ~ i tJ. . Nucl. M e d . , R i d . 5, 30. Lo, L. W., Koropatnick, J., and Stich, H. F. (1978).Mut. Res. 49, 305. Marshall, M. V., Jacobs, hl. M., and Griffin, A. C. (19784.Proc. A m . Assoc. Cancer Res. 9, 75. Marshall, M. V., Rasco, hi. A,, and Griffin, A. C. (19781,).Fed. Proc. 37, 1383. Martin, J. L. (1973).111 “Organic Selenium Compounds: Their Chemistry and Biology” (D. L. Klayman and W. H. H. Giinther, eds.), p. 663. Wiley, New York. McKeehan, W. L., Hamilton, W. G., and Ham, R. G. (1976).Proc. N n t l . Acad. Sci. USA 73, 2023. Mills, G. C. (1957).J.B i d . Chent. 229, 189. Mills, G. C. (1960).Arch. Biochem. 86, 1.

442

A. CLARK GRIFFIN

Moxen, A. L., Anderson, H. D., and Painter, E. P. (1938).J . Phurmacol. Exp. Ther. 63, 357. Nakaniura, K. K., Yoshikawa, K., Sayato, Y., Kurata, H., Tonomura, M., and Tononiura, A. (1976).Mut. Res. 40, 177. Nelson, A. A,, Fitzhugh, 0. G., and Calvery, H. 0. (1943). Cancer Res. 3, 230. Oh, S. H., Ganther, H. E., and Hoekstra, W. G. (1974).Biochemistry 13, 1185. Omaye, S. T., Reddy, K. A,, and Cross, C. E. (1978). Toxicol. Appl. Pharmacol. 43,237. Peskin, A. V., Koen, Y. M., Zbarsky, I. B., and Konstantinov, A. A. (1977). FEBS Lett. 78, 41. Pinsent, J. (1954). Biochem. J. 57, 10. Pinto, R. E., and Bartley, W. (1973). FEBS Lett. 32, 307. Prohaska, J. R., Mowafy, M., and Ganther, H. E. (1977). Clzem. Biol. Interact. 18, 253. Rasco, M. A,, Jacobs, M. M., and Griffin, A. C. (1977).Cancer Lett. 3, 295. Rastogi, S. C., Clausen, J., and Srivastava, K. C. (1976). Toxicology 6, 377. Saelinger, D. A,, Hoffman, J. L., and McConnell, K. P. (1972).J. Mol. B i d . 69, 9. Schrauzer, G. N., and Ishmael, D. (1974). Anti. Clin. Lah. Sci. 4, 411. Schrauzer, G. N., and White, D. A. (1978). Bioitiorg. Chem. 8, 303. Schrauzer, G. N., White, D. A,, and Schneider, C. J. (1976). Bioinorg. Chem. 6, 265. Schwarz, K., and Foltz, C. M. (1957).J. Am. Chern. Soc. 79, 3292. Schwarz, K., and Pathak, K. D. (1975). Cheni. Scripta (Sweden) 81, 85. Seifter, J. W., Ehrich, E., Hudgma, G., aiid Mueller, G. (1946). Science 103, 762. Shamberger, R. J. (1970).J. Natl. Cuncer Inst. 44, 931. Shamberger, R. J., and Frost, D. V. (1969).Cnn. Rled. Assoc. 100, 682. Shamberger, R. J., and Willis, C. E. (1971).Clin. Lab. Sci. 2, 211. Shamberger, R. J., Tytko, S. A., aiid Willis, C. E. (1976).Arch. Enairon. Health 31, 231. Shamberger, R. I., Beanian, K. D., Corlett, C. L., and Kasten, B. L. (1978).Fed. Proc. 37, 261. Shapiro, J. R. (1973).Zri “Organic Selenium Compounds: Their Chemistry and Biology” (D. L. Klayman and W. H . H. Giinther, eds.), p. 693. Wiley, New York. Sifri, M., and Hoekstra, W. G. (1978). Fed. Proc. 37, 757. Spom, M. B. (1977). Nutr. Reo. 35, 65. Spom, M. B., Dunlop, N. M., Newton, D. L., and Smith, J. M. (1976). Fed. Proc. 35, 1332. Stadbnan, T. C. (1977). Nutr. Reo. 35, 161. Sunderman, W. F., Jr. (1978). Fed. Proc. 37, 40. Tscherkes, L. A., Volgarev, M. N., and Aptekar, S. G. (1963).Acto Utzio. Zritern. Cotttru Cuncrum 19, 632. Turner, D. C., and Stadtman, T. C. (1973).Arch. Biochem. B i o p h y s . 154, 366. Verina, A. K., Rice, H. M., Shapas, B. F., and Boutwell, R. K. (1978).Cuncer Res. 38,793. Wattenberg, L. W. (1975).J. Natl. Cancer Inst. 54, 1005. Wattenberg, L. W. (1978). Adu. Curicer Res. 26, 197. Yamane, Y., Fukino, H., Aida, Y., and Imagawa, M. (1977).Chenr. Phorm. Bull. Tokyo 25, 283 1. York, G. K., Pierce, T. H., Schwartz, L. W., and Cross, C. E. (1976). Arch. Enuiroti. Heulth 31, 286. Zabal, N. L., Harland, J., Gormican, A. T., and Ganther, H. E. (1978).An1.J. Clin. Nutr. 31, 850. Zingaro, R. A,, and Cooper, W. C. (1974). In “Selenium,” 835 pp. Van NostrandReinhold, New York.

SUBJECT INDEX A

2-Acetamido-4-chloromethylthiazole, glutathione conjugation with, 206207 Acrolein, glutathione conjugation with, 232, 247-248 Acrylamide, glutathione conjugation with, 231,234,252 Acrylate esters, glutathione conjugation with, 231 Acrylonitrile, glutathione conjugation with, 231, 252 Adriamycin, glutathione conjugation of, 250 Aflatoxin, glutathione conjugation with, 227-230 Alanine aminotransferase, isozymes of, in cancer, 142 Albumin, a-fetoprotein cross-reaction with, 278-279 Aldolases in hepatocarcinogenesis, 133-134 isozymes of, in cancer, 142, 144 Alicyclic halides, glutathione conjugation with, 216-217 Alkaline phosphatase, placental, see Regan isoenzyme Alkene halides, glutathione conjugation with, 213-216 Alkyl halides, glutathione conjugation with, 208-213 Alkyl methanesulfonates, glutathione conjugation with, 219 Alkyl sulfates, glutathione conjugation with 208-213 Ally1 compounds, glutathione conjugation with, 217-218 Amniotic fluid, a-fetoprotein in, 285-286 Aiigiosarcomas, vinyl chloride and, 213214 Aniline, glutathione conjugation with, 238 Antitumor resistance, effects of external intervention on, 52-60 Aralkyl esters, glutathione conjugation with, 207-208

Aralkyl halides, glutathione conjugation with, 205-207 Arecoline, glutathione conjugation with, 233 Arene oxides, glutathione conjugation with, 221-226 Arginase, in cancer, 140 Arylamines, glutathione conjugation with, 235-240 Arylhalide epoxides, glutathione conjugation with, 226-227 Arylhydroxylamines, glutathione coiijugation with, 235-240 Aryl nitrocompounds, glutathione conjugation with, 202-204 Ataxia telangiectasia, a-fetoprotein in, 327 ATS treatment, as immunosuppressive procedure, 55-59 Azathioprine, glutathione conjugation of, 245-246,252 B

B cell, as suppressor cell, 68-69, 81-83 Benz[a]anthracene, glutathione conjugation with, 221-222, 224-225 Benzothiazole-2-sulfonamide, glutathione conjugation of, 246-247 Benzyl chloride, glutathione conjugation with, 206, 252 BHT, glutathione conjugation with, 208 Biliary atresia, a-fetoprotein levels in, 327 Branched-chain amino acid transferase, isozymes of, in cancer, 144 Breast cancer, mammary tumor virus and, 405-408 Bromazepam, glutathione conjugation of, 247 Bromoalkanes, glutathione conjugation with, 209 Bromobenzene, glutathione conjugation with, 226-227, 252 Bromocycloheptane, glutathione conjugation with, 252 Bromvaletone, glutathione conjugation with, 210

443

444

SUBJECT INDEX

C Cadmium, glutathione complex of, 245 Cambendazole, glutathione conjugation of, 247 Cancer enzymes in, 138-141 fetal isozymic transitions in, 150-151 a-fetoprotein in, 333-334, 336 gene expression in, fetal patterns in, 127- 174 of liver, a-fetoprotein role in, 319 metabolic changes in, 138-141 morphologic and structure-dependent properties of, 146-147, 151-152 plasticity of differentiated state in, 152-155 retrodifferentiation in, 127-174 differentiation and, 152-155 summary, 166-169 selenium in prevention of, 419-442 spontaneous regression of, 156-157 tumor-associated antigens in, 130-138 Cancer cells differentiation of, 155-158 suppressor cells’ relationship to, 64-104 nonspecific cells, 65-92 Captan, glutathione conjugation with, 243-244 Carbamates, glutathione conjugation with, 235-240 Carbon tetrachloride, glutathione conjugation with, 216 Carboxylic esterases, isozymes of, in cancer, 144 Carcinoembryonic antigen (CEA), characteristics of, 135-136 Carcinogens glutathione conjugation with, 175-274 glutathione S-transferase rule in nietabolism of, 175-274 Catechols, glutathione conjugation with, 241-242 Cell injury fetal antigens in, 147-150 retrodifferentiation in, 158-162 Chloramphenicol, glutathione conjugation of, 250 Chloronlkyl ethers, glutathione conjugation with, 211

Chlorobenzene, glutathione conjugation with, 226-227 o-Chlorobenzylidenemalononitrile, glutathione conjugation with, 234 Chloroethanol, glutathione conjugation of, 252 Chloroform, glutathione conjugation with, 213,252 Chlorohydrins, glutathione conjugation with, 210 Chloroprene, glutathione conjugation with, 215 Chlorotoluron, glutathione conjugation of, 247 2-Chloro-S-triazines, glutathione conjugation with, 201-202 Cigarette smoke, carcinogens in, 221 Cinnamaldehyde, glutathione conjugation with, 231, 252 Cirrhosis, a-fetoprotein levels in, 322 Cobalt, glutathione complex of, 245 Cyanazine, biotransformation of, 202 Cycloheptene, glutathione conjugation of, 252 Cycloheptene epoxide, glutathione conjugation of, 252 Cyclohex-2-en- 1-one, glutathione conjugation of, 252 Cyclophosphamide, glutathione conjugation with, 232, 247-248 Cystic fibrosis, a-fetoprotein and, 327 Cytembena, glutathione conjugation of, 248-249 D

DDT, glutathione conjugation of, 249 Dehydroretronecine, glutathione conjugation with, 251 Delayed-type hypersensitivity, transfer of, 18 Deoxycytidylate deaminase, in cancer, 140 Diabetes, in pregnancy, a-fetoprotein increase in, 331 1,2-Dichloroethane, glutathione conjugation with, 211-212, 252 Dichloroethylene, glutathione conjugation of, 252

SUBJECT INDEX Dichloronitrobenzene, glutathione conjugation with, 252 1,3-Dichloropropene, glutathione conjugation with, 215 Dichlorvos, glutathione conjugation with, 220,252 Diethyl maleate, glutathione conjugation with, 230, 251-252 Diethyl sulfate, glutathione conjugation with, 208 Dihalomethanes, glutathione conjugation with, 212 1,2-Dihydronaphthalene, glutathione conjugation with, 222 E

Electrophilic agents, glutathione conjugation of, 175-274 Enzymes, in cancer, 138-141 Epoxides, glutathione conjugation with, 227-230 Esophageal atresia, a-fetoprotein increase in, 331 Estradiol-l7fl, glutathione conjugation with, 240-241 Estrogen, a-fetoprotein binding of, 306308 Estrone, glutathione conjugation with, 241 Ethacrynic acid, glutathione conjugation with, 232-233 Ethanol, glutathione conjugation of, 250 Ethyl acrylate, glutathione conjugation with, 231 S-Ethyl dipropylthiocarbainate, glutathione conjugation of, 253 Ethyl iodide glutathione conjugation of, 253 Ethyl methanesulfonate glutathione conjugation with, 219 Ethyl vinyl ketone, glutathione conjugation with, 231 Exomphalos, a-fetoprotein increase in, 331 Experimental allergic encephalomyelitis (EAE), induction of, 49 F

F9 antigen, T, cells for, 9 Fetal patterns in cancer, 127-174

445

in noncancerous growth, 147-152 a-Fetoprotein (AFP), 275-346 antibodies to, 276-277 biological half-life of, 287-288 biological properties of, 305-313 biosynthesis sites of, 282 in body fluids, 284-290 amniotic fluid, 285-286 fetal serum, 284-285 maternal serum, 286-287 newborn serum, 287 nonnal serum, 288-290 characteristics of, 134 chemical composition and primary structure of, 296-300 detection of, 276-282 by internal labeling, 281 in tissues, 281-282 estrogen binding of, 306-308 in fetal pathology diagnosis, 327-332 future research on, 335-336 in g e m cell tumors, 323-326 in hepatocarcinogenesis, 147-149, 315-323 in hereditary tyrosinemia, 327 imniunoregulatory role of, 308-313 iminunoassay of, 279-280 immunological cross-reactions to alhumin, 278-279 international standard for, 280-281 interspecies cross-reactions to, 277 microheterogeneity of, 300-305 moIecular weight of, 292-296 nonnal, chemical nature of, 305 purification and chemical properties of, 290-305 synthesis of, molecular basis of regulation of, 334-335 tolerance to, 332-333 in trophoblastic disease, 327 in tumor immunoprevention, 333-334 Fetus, pathology, a-fetoprotein levels in, 327-332 Fluroxene, glutathione conjugation with, 212 Folpet, glutathione conjugation with, 243-244 2-(2-Fonnylhydrazino)-4-(5-iiitro-2-furyl)-thi;~zole, glutathione conjugation with, 203

SUBJECT INDEX

446 G

Germ cell tumors, a-fetoprotein levels in, 323-326 Glucokinase, in cancer, 140, 141 Glucosamine-6-phosphate synthetase, isozymes of, in cancer, 144 Glucose-ATP phosphotransferase, isozymes of, in cancer, 141-143 Glutaininase, isozymes of, in cancer, 142, 144 a-Glutamyl transpeptidase, in cancer, 140 Glutathione (GSH) conjugation with carcinogens, 175-274 properties and cellular presence of, 176-177 Glutathione S-transferase assay and stability of, 186-187 in carcinogen metabolism, 175-274 induction of, 194-198 inhibition of, 198-199 intracehlar location of, 187 ligandin identity with, 183-184 maturation of, 193-194 mode of action of, 184-186 nature of, 180-183 properties of, 177-200 sex differences in, 199-200 species distribution of, 191-193 substrates for, 178-180 tissue distribution of, 187-191 Glycogen phosphorylase, isozymes of, in cancer, 142, 144 Growth, noncancerous, fetal patterns in, 147-152

Hepatitis, neonatal, a-fetoprotein levels in, 327 Hepatocarcinogenesis, a-fetoprotein role in, 147-149, 315-323 Hepatocytes, fetal and neoplastic, compairson of, 146-147 Hepatomas, enzyme activity in, 139-141 Hexachlorocyclohexane, glutathione conjugation with, 216, 253 Hexokinases, in cancer, 140, 141 Hexosaminidase, isozymes of, in cancer, 142 Horniones, ectopic, in cancer, 137 Hydrocephaly, a-fetoprotein increase in, 33 1 I Ia antigens, genetic control of, 3 Immune response, macrophage role in, 49 Immunological balance, scheme of, 113 Immunostimulation, suppressor cells as initiators of, 60-64 Immunosurveillance theory, of tumor resistance, 53-54 4-Ipomeanol, glutathione conjugation with, 230 Ir genes MHC-linked, 21-28 T-cell recognition and, 29-36 Isoniazid, glutathione conjugation of, 249 Isothiocyanates, glutathione conjugation with, 242-243 Isozymes, in cancer, 141-144

L H

H-2 restriction phenomena description of, 2-3 of major histocompatibility complex, 5-11 relationship to Zr genes tumor resistance and, 36-39 Haloforms, glutathione conjugation with, 213 Halogenonitrobenzenes, glutathione conjugation with, 200-201 Halothane, glutathione conjugation with, 212

Lactate dehydrogenase, isozymes of, i n cancer, 142, 144 Ligandin, glutathione S-transferase identity with, 183-184 Liver disease, a-fetoprotein role in, 313323

M Macrophages nonspecific suppression mediated by, 65-81 role in immune response, 49

447

SUBJECT INDEX

Major histocompatibility coiiiplex (MHC) description of, 2-3 effects on, T-cell activation, 1-44 gene products of, effects on T-cells, 4-5, 34-36 genetic organization of, 3 H-2 rescriction phenomena of, 5-11 altered self-model, 12-13 cytotoxicity, 6 delayed-type hypersensitivity, 6-7 exceptions, 9-11 helper functions, 7-8 intimacy model, 11-12 models, 11-21 suppression, 8-9 two-receptor model, 13-14 Malignancy, see Cancer Mammary tumor viruses (MuMTV),347418 antigens of, 386-395 characterization, 387-392 immunologic techniques, 386-387 in milk, 392-395 biology of, 370-385 control of gene expression by, 383-385 genome of, 367-370 glycopeptide from, amino acids in, 361 incidence of, 348 infectivity of, 373-375 by contact, 377-380 i n oitro, 380-383 strains in, 376-377 interaction with host’s immune system, 395-405 cellular response, 398-401 humoral response, 396-397 modulation, 401-405 intracytoplasmic A particles of, 391-392 in oiuo infection and tumorigenesis by, 370-373 morphology of, 350-356 polypeptides of, 387-389 proteins associated with, 356-358 synthesis and assembly, 364-367 relation to human viruses, 405-408 RNA-directed DNA polymerase of, 362-364 structural proteins of, 356-364 characterization and localization, 359 from various mouse strains, 389-391

Meckel syndrome, a-fetoprotein increase in, 331 1-Menaphthylsulfate, glutathione conjugation with, 207-208 Mercapturic acid, formation of, 178 Mercury, glutathione conjugation of, 244-245 Mersalyl, glutathione conjugation with, 244-245 Metals, glutathione conjugation with, 244-245 3-Methylfuran, glutathione conjugation with, 230 Methyl iodide, glutathione conjugation with, 208-209,253 Methylparathion, glutathione conjugation with, 219-220 Methylvinyl sulfone, glutathione conjugation with, 231 Milk, mammary tumor virus antigen in, 392-395 Mustard gas glutathione conjugation with, 212 Myleran, glutathione conjugation with, 219 N

Naphthalene, glutathione Conjugation with, 253 P-Naphthylamine, glutathione conjugation with, 238 a-Naphthylisothiocyanate, glutathione conjugation of, 250 Nephrosis, congenital, a-fetoprotein increase in, 328-330 Nephthalene, glutathione conjugation with , 22 1-222 Neuroblastomas, spontaneous regression of, 156-157 Neural tube defects, a-fetoprotein increase in, 328-329, 331 Nickel, glutathione complex of, 245 1-Nitrobutane, glutathione conjugation with, 253 Nitro compounds, glutathione conjugation with, 208-213 Nitrofurazome, glutathione conjugation with, 203 5-Nitrofurfural, glutathione conjugation with, 202-203

448

SUBJECT INDEX

4-Nitropyridine N-oxide, glutathione conjugation with, 203 4-Nitroquinoline N-oxide, glutathione conjugation with, 202 P-Nitrostyrene, glutathione conjugation with, 231

0 Organophosphorus compounds, glutathione conjugation with, 21922 1 P

Paracetamol, glutathione conjugation with, 235-238, 253 Paraquat, toxicity of, glutathione effects on, 251-252 Parasorbic acid, glutathione conjugation with, 231, 233, 253 Parathion, glutathione conjugation with, 220-22 1 Phenanthrene, glutathione conjugation with, 253 Phenoltetrabromphthaleins, glutathione conjugation with, 204-205 Phenacyl halide, glutathione conjugation with, 207 3-Phenyl-5-methyl- 1,2,4-oxadiazole, glutathione conjugation of, 250 Phosphofructokinase, isozymes of, in cancer, 142, 143 Platinum, glutathione complex of, 245 Polycyclic aromatic compounds, glutathione conjugation with, 221222 Pregnancies aberrant, a-fetoprotein levels in, 328 multiple, a-fetoprotein increase in, 331 Propachlor, glutathione conjugation with, 206-207 P-Propiolactone, glutathione conjugation of, 250 Prostaglandin Al, glutathione conjugation with, 223-234 Pyruvate kinase, isozymes of, in cancer, 140, 142-144

0 Quinones, glutathione conjugation with, 241-242

R

Regan isozyme, in retrodifferentiation, 135, 165 Regeneration, fetal antigens in, 147-150 Retrodifferentiation in cancer, 127-174 summary, 166-169 if1 tjitro in malignant cells, 164-166 in normal cells, 162-164 Retrorsine, glutathione conjugation of, 250 RH immunization, a-fetoprotein increase in, 331 Ribonucleotide reductase, in cancer, 139, 140 RNA-directed DNA polymerase, of mammary tumor virus, 362-364

S Sacrococcygeal teratoma, a-fetoprotein increase in, 331 Safrole, glutathione conjugation with, 2 17-2 18 Selenium biological functions of, 429-432 carcinogenesis and, 423 epidemiological, 424-425 mechanisms, 439-440 stages in, 437-439 in cancer prevention, 419-442 mutagenic aspects, 432-434 effects on cancer growth, 424 carcinogen metabolism, 434-435 oxidative damage, 435-437 nutritional aspects of, 421-423 properties of, 419-420 toxicology and pathology of, 420-421 Serum, a-fetoprotein in, 284-290 “Sneaking through” phenomena studies on, 62-63 suppressor cells as initiators of, 60-64 Spina bifida a-fetoprotein increase in, 328-329 Splenectomy, as immunosuppressive procedure, 55-59 Splenocytes, as suppressor cells, 76-77 Squamous cell carcinoma, differentiation in, 156

449

SUBJECT INDEX Steroids glutathione coiijugation with, 240-24 1 Styrene and styrene oxide, glutathione conjugation with, 228-229, 253 Sumithion, glutathione conjugation with,

253 Suppressor cells classification and activity of, 66-73 in human cancers, 119 induced hy nontumorigenic stimuli,

104- 106 inacrophages a s , 65-81, 114-115 malignant cells’ relationship to, 64-104 nonspecific cells, 65-92 specific cells, 92-101 unclassified cells, 98-101 undefined cells, 101-104 a s possible initiators of “immunostimulation,” 60-64 as possible permitters and promotors of malignancy, 45-125 properties of, comparison, 109-11 1

T

T cells as suppressor cells, 69-70, 72, 83-92 specific activity, 93-98 T,. cells cross-reactive lysis by, 9-10 for F9 antigen, 9 generation of repertoire of, 25-28 for I-region determinants, 9 models for recognition by, 29-36 Teratocarcinoma, differentiation of, 157 Thiocarbamates, glutathione conjugation with, 244 Thymectoiny, as immunosuppressive procedure, 55-59 Thymidylate synthetase, in cancer, 140 Thymus, T-cell differentiation in, 16-21 Tissue repair fetal antigens in, 147-150 retrodifferentiation in, 158-162 T-lymphocytes activation of major histocompatibility complex effects on, 1-44 priming in, 15-16 with anti-self-H-2 receptors, 29-32 differentiation of, 16-21

selection against, 32-34 subsets of, 3 4 s-Triazine herbicide, biotransfor~~lation of, 202 Trichloroethylene, glutathione conjugntion with, 215, 253 Trichloroinethylsulfenyls, glutathione conjugation with, 243-244 Trichloropropene oxide, glutathione conjugation with, 253 Trophoblastic disease, a-fetoprotein in,

327 Tumor-associated antigens in cell lineages, 134-137 ‘in c e l l u h elements of organism, 131-

137 in tumors, 130-138 Tumor-associated surface antigens (TASA), 130 Tumor-associated transplantation an tigens (TATA), 130 Cancer) Tumors (see (11.~o excision of, suppressor cells induced by,

104-106 resistance to H-2 restriction and, 36-39 imniunosurveillance theory of, 53-54 suppressor cells a s possible promoters

of, 45-125 Turner’s syndrome, a-fetoprotein increase in, 331 Tyrosinemia, hereditary, a-fetoprotein levels in. 327

U aP-Unsaturated compounds, glutathione conjugation with, 230-235 Urethane, glutathione conjugation with,

239 V

Vinyl chloride and vinylidene chloride, glutathione conjugation with, 213-

215, 253 X

Xanthine oxidase, in cancer, 139, 140 X-irradiation, as immunosuppressive procedure, 55-59

This Page Intentionally Left Blank

CONTENTS OF PREVIOUS VOLUMES

Carcinogenesis and Tumor Pathogenesis 1 . Berenblum Ionizing Radiations and Cancer

Volume 1 Electronic Configuration cinogenesis C. A. Coulson Epidermal Carcinogenesis

and

Car-

Arrstin M.Brues Survival and Preservation of Tumors i n the Frozen State Jatiles C ruigie Energy and Nitrogen Metabolism i n Cancer Leonard D. Fenninger rind G . Bur-

E. V. Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice

L. Dmochowski

roughs Mider

Hormonal Aspects of Experimental Tumorigenesis T. U . Gardner Properties of the Agent of Rous No. 1 Sarcoma R. J . C. Harris Applications of Radioisotopes to Studies of Carcinogenesis and Tumor Metabolism

Sonie Aspects of the Clinical Use of Nitrogen Mustards Caloin T. Klopp and Jeanne C . Bateman Genetic Studies in Experimental Cancer

L.w. hu: The Role of Viruses in the Production of Cancer C . Oberling and M. Guerin Experimental Cancer Chemotherapy C . Chester Stock

Charles Heidelberger The Carcinogenic Aminoazo Dyes James A . Miller und Elizuheth C . Miller The Chemistry o f Cytotoxic Alkylating Agents M . C. J . Ross Nutrition in Relation to Cancer

AUTHOR INDEX-SUBJECT INDEX

Volume 3 Etiology of Lung Cancer

Albert Tannenhuum und Herhert Silverstone

Richard Doll The

Plasma Proteins in Cancer

Richard J . Winder

Harold P. Morris

AUTHOR INDEX-SUBJECT INDEX

Volume 2 The Reactions of Carcinogens with Macromolecules

Electronic Structure and Carcinogenic Activity and Aromatic Molecules: New Developments A . Pullmun und B . Pullwian Some Aspects o f Carcinogenesis

P. Rondoni

Peter Alexander Cheniical Constitution and Carcinogenic Activity

G . M. Badger

Experimental Development and Metaboliqm ofThyroid Gland Tumors

Pulmonary Tumors in Experimental Animals Michael B . Shimkin

451

452

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic Tissues

Sidney Weinhouse AUTHOR INDEX-SUBJECT INDEX

The Newer Concept of Cancer Toxin War0 Nukahara and Fumiko Fukuoku Chemically Induced Tumors of Fowls

P. R. Peucock Anemia in Cancer

Volume 4 Advances in Chemotherapy of Cancer in ,Man

Sidney Farher, Rudolf Toch, Edwurd Manning Sears, and Donald Pinkel The Use of Myleran and Similar Agents in Chronic Leukemias D. A. G. Galton The Employment of Methods of Inhibition Analysis in the Normal and OrTumor-Bearing Mammalian gan is i n

Abraham Goldin Some Recent Work on Tumor Immunity P. A. Corer Inductive Tissue Interaction in Development

Clifiord Grobstein Lipids in Cancer

Frances L. Haven ond W. R. Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines

Vincent E. Price Greenfield

and

Robert

E.

Specific Tumor Antigens L. A. Zilber Chemistry, Carcinogenicity, and Metabbolism of 2-Fluorenamine and Related Compounds

Elizubeth K . Weishurger and John H. Weishurger AUTHOR INDEX-SUBJECT INDEX

Volume 6 Blood Enzymes in Cancer and Other Diseases Oscar

Bodansky

The Plant Tumor Problem Armin C . Braun and Henry N . Wood Cancer Chemotherapy by Perfusion Oscar Creech, Jr. and Edward T.Kre-

mentz Viral Etiology of Mouse Leukemia

Ludwick Gross Radiation Chimeras

A. Lacassagne, N. P. BuuHoi, R. Daudel, and F. Zajdela

P. C. Koller, A. J . S . Duvies, and Sheila M . A. Douk

The Hormonal Genesis of Mammary Cancer 0. Muhlhock

Etiology and Pathogenesis of Mouse Leukemia J . F. A . P. Miller Antagonists of Purine and Pyrimidine Metabolites and of Folic Acid G. M. Tinimis Behavior of Liver Enzymes in Hepatocarcinogenesis

AUTHOR INDEX-SUBJECT INDEX

Volume 5 Tumor-Host Relations R. W. Begg Primary Carcinoma of the Liver

Charles Bermun Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal P. N. Camphell.

George Weher AUTHOR INDEX-SUBJECT INDEX

Volume 7 Avian Virus Growths and Their Etiologic Agents J . W. Beard

CONTENTS OF PREVIOUS VOLUMES ,Mechanisms of Resistance to Anticancer Agents R. W. Brockman Cross Resistance and Collateral Sensitivity Studies in Cancer Chemoerapy

Dorris J . Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W. M. Court Brown und lshbel M.

Tough Ethionine Carcinogenesis

E m ma nu el Fa rber Atmospheric Factors in Pathogenesis of Lung Cancer

453

The Relation of the Immune Reaction to Cancer Louis V. Caso Amino Acid Transport in Tumor Cells R. M.Johnstone and P. C . Scholejeld Studies on the Development, Biocheinistry, and Biology of Experimental iIepatomas Harold P. Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes, and Bone Marrow Cells 1. F. Seitz AUTHOR INDEX-SUBJECT INDEX

Paul Kotin and Hans L. Falk Progress with Some Tumor Viruses of Chickens and Mammals: T h e Problem of Passenger Viruses G. Negroni AUTHOR INDEX-SUBJECT INDEX

Volume 8 T h e Structure of Tumor Viruses and Its Bearing on Their Relation to Viruses in General A. F. Howatson Nuclear Proteins o f Neoplastic Cells

Harris Busch and William J . Steele Nucleolar Chromosomes: Structures, Interactions, and Perspectives

M . J . Kopuc and Gladys M. Muteyko Carcinogenesis Related to Foods Contaminated by Processing and Fungal Metabolites H. F. Kraybill and M. B . Shimkin Experimental Tobacco Carcinogenesis

Ernest L. Wynder Hoffman

and

Harrt, and W . J . Stuckley, J r . AUTHOR INDEX-SUBJECT INDEX

Volume 11 T h e Carcinogenic Action and Metabolism of Urethran and N-Hydroxyurethan Sidney S. Miruish Runting Syndromes, Autoimmunity, and Neoplasia

Volume 9 Urinary Enzymes and Their Diagnostic Value in Human Cancer

and

Carcinogens, Enzyme Induction, and Gene Action H . V . Gelboin 111 Vitro Studies on Protein Synthesis by Malignant Cells A. Clark Crijin T h e Enzymatic Pattern of Neoplastic Tissue W. Eugene Knox Carcinogenic Nitroso Compounds P. N. Magee and J . M. Burnes The Sulfhydryl Group and Carcinogenesis J . S. Harrington T h e Treatment o f Plasma Cell Myeloma Duniel E. Bergsagel, K . M. Grijith, A .

Dietrich

AUTHOR INDEX-SUBJECT INDEX

Richard Stamhuugh Weinhou.se

Volume 10

Sidney

D. Keast Viral-Induced Enzymes and the Problem of Viral Oncogenesis

Saul Kit

454

CONTENTS OF PREVIOUS VOLUMES

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

The

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. 1. Deichtnan 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 L. Kessler and Abraham iM. Lilienfeld AUTHOR INDEX-SUBJECT INDEX

Volume 13 T h e 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 0 swaId Jarrett

T h e Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culhire Hairn Ginshurg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gajanan V. Sherhet T h e Characteristics of Animal Cells Transformed in Vitro I a n Macpherson Role of Cell Association in Virus Infection and Virus Rescue J . Svoboda and 1. Hlofanek Cancer of the Urinary Tract D. B . Cluyson and E . H . Cooper Aspects of the E B Virus M . A. Epstein AUTHOR INDEX-SUBJECT INDEX

Volume 14 Active Immunotherapy Georges Matht? 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 George 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 . 1. Abeleu Low Dose Radiation Cancers in Man Alice Stewart AUTHOR INDEX-SUBJECT INDEX

455

CONTENTS OF PREVIOUS VOLUMES

Volume 15 Oncogenicity and Cell Transformation by Papovavirus SV40: T h e Role of the Viral Genome 1.S . Butel, S . S. Tevethia, a n d ] . L. Me/-

nick Nasopharyngeal Carcinoma (NPC) I . H . C . Ho Transcriptional Regulation in Eukaryotic Cells A . 1. MacGillioray, I . Paul, and G.

Threlfall Atypical Transfer RNA’s and Their Origin in Neoplastic Cells

Ernest Borek and Sylvia]. Kerr Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females

Philip]. Fiulkou; Electron Spin Resonance Studies of Carcinogenesis

Harold M . Swurtz 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 Basergo AUTHOR INDEX-SUBJECT INDEX

1,3-Bis ( 2-Ch loroeth yl )- 1-N i trosourea (BCNU) mid Other Nitrosoureas in Cancer Treatment: A Review

Stephen K . Carter, Frunk M. Schahel, ]r., kiwrence E. Broder, nnd Thomas P. Johnston AUTHOR INDEX-SUBJECT INDEX

Volume 17 Polysaccharides in Cancer: GI ycoproteins and Glycolipids

Vijai N.Nigarn and Antonio Cuntero Some Aspects of the Epidemiology and Etiology of Esophageal Cancer with Particular Emphasis on the Transkei, South Africa Geruld P. Wurwick and John S .

Harington Genetic Control o f Leukemogenesis

,Murine

Viral

Frunk Lilly and Theodore Pincus Marek’s Disease: A Neoplastic Disease of Chickens Caused by a Herpesvirus K . Nazerian Mutation and Human Cancer Alfred G. Knudson, J r . Mammary Neoplasia in ,Mice S . Nundi and Charles M. McGrath AUTHOR INDEX-SUBJECT INDEX

Volume 16 Polysaccharides in Cancer Vijai N . Nigarn and Antonio Cantero Antitumor Effects of Interferon lon Gresser Transformation by Polyonia Virus and Simian Virus 40

]oe Samhrook Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing? Sir Alexander Haddow The Expression of Normal Histoconipatibility Antigens in Tumor Cells

Alena Lengerova

Volume 18 Immunological Aspects of Chemical Carcinogenesis

R. W. Baldwin Isozymes and Cancer

Fanny Schapira Physiological and Biochemical Reviews of Sex Differences and Carcinogenesis with Particular Reference to the Liver

Yee Chu Toh Immunodeficiency and Cancer John H . Kersey, Beatrice D. Spector,

and Robert A. Good

456

CONTENTS OF PREVIOUS VOLUMES

Recent Observations Related to the Chemotherapy and Immunology of Gestational Choriocarcinoma K . D. Bugshuve Glycolipids of Tumor Cell .Membrane

Sen-itiroh Hukomori Chemical Oncogenesis in Culture

Chur1e.y Heidelherger AUTHOR INDEX-SUBJECT INDEX

Principles of Immunological Tolerance d Immunocyte Receptor Blockade G. J . V. Nossul The Role of Macrophages in Defense against Neoplastic Disease

Michuel H . Levy und E . Frederick Wheelock Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis

P. Sims and P. L. Grover Volume 19

Virion and Tumor Cell Antigens ofC-Type RNA Tumor Viruses

Heinz Bauer Comparative Aspects of Mammary Tumors /. hi. Hamilton The Cellular and Molecular Biology of RNA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses and Their Relatives H o wurd M. Te mi n Cancer, Differentiation, and Embryonic Antigens: Some Central Problems J . H . Coggin, J r , nnd N . G. Anderson Simian Herpesviruses and Neoplasia

Addendum to “Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing?”

Fredrich W. Deinhurdt, Luwrence A . Falk, und k u r e n G . Wolfe

Cell Death in Normal and Malignant Tissues E . H . Cooper, A. J . Bedford, and T. E.

Cell-Mediated Inimunity to Tumor Cells

Ronuld B . Herherman Herpesviruses and Cancer

Fred R u ~ p Cyclic AMP and the Transformation of Fibroblasts Ira Pustun und George S . Johnson Tumor Angiogenesis

/uduh Folkmun SUBJECT INDEX

Volume 20 Tumor Cell Surfaces: General Alterations Detected by Agglutinins

Annette M . C. Rupin and Max M . Burger

Sir Alexunder Huddow SUBJECT INDEX

Volume 21 Lung Tumors in Mice: Application to Carcinogenesis Bioassay

Michael 8 . Shimkin and Gary D. Stoner

Kenny The Histocompatibility-Linked Immune Response Genes Buruj Benacerraf and David H . Kutz Horizontally and Vertically Transmitted Oncornaviruses of Cats

M . Essex Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms Keen A. Rufferty,]r. Selection of Biochemically Variant, in Some Cases Mutant, Mainmalian Cells in Culture G. B. Clements The Role of DNA Repair and Somatic Mutation in Carcinogenesis

James E. Trosko and Ernest H . Y. Chu SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

Volume 22

Volume 24

Renal Carcinogenesis J . iM. Hamilton Toxicity of Antineoplastic Agents in Man: Chromosomal Aberrations, Antifertility Effects, Congenital llalformations, and Carcino&mic Potential Susan M ,Sieber und Richard H . Adam-

The

son Interrelationships among RNA T~tttiot Viruses and Host Cells Raymond V . Gilden Proteolytic Enzymes, Cell Surfwe Changes, and Viral Transformation

Richard Rohlin, lih-Nun Chou, and Paul H. Black Imtnunodepression and Malignancy Osias S tu t mu n SUBJECT INDEX

Volume 23 The Genetic Aspects of Human Ciincer

W . E. Heston T h e Structure and Function of Intercellular Junctions in Cancer Rotiuld S. Weinstein, Frederick B . Merk,

457

Xlurine Sarconin Virus-Intluced Tumor: Exception or General Model in Tumor Iniinunology? J . P. Lecy ond J . C . Leclerc Organization of the Genonies of Polyotmi Virus and SV40 Mike Fried und B e ~ e r l yE . Griffin &-Microglobulin and the Major Histocompatibility Complex Per A. Peteraon, Lars Rusk, und k i r s

Ostberg Chromosomal Almorinalities and Their Specificity in Human Neoplasms: An Assessment of Recent Observations b y Banding Techniques Jouchiti1 M a r k Temperature-Sensitive Mutations in Animal Cells Claudio Basilico Current Concepts of the Biology of Human Cutaneous Maligniint Melanoina Wullace H . Clark, J r . , Michael J . M a s -

trangelo, Ann M.Ainsworth, David Berd, Rohert E . Bellet, und Euelinu A. Bernurdino SUBJECT INDEX

and Joseph Alroy Genetics of Adenoviruses Harold S. Ginsberg and C . S . H. Young Molecular Biology of the Cnrci nogen, 4-Nitroquinoline 1-Oxide

Minako Nugao and Takashi Sugimuru Epstein-Barr Virus and Nonhuman Primates: Natural and Experimental Infection A. Frank, W. A. Andiman, and G. Miller Tumor Progression and Homeostasis

Richmond T. Prehn Genetic Transformation of Animal Cells with Viral DNA or RNA Tumor Viruses Miroslav Hill und Jana Hillova SUBJECT INDEX

Volume 25 Biological Activity of Tumor Virus DNA

F. L. Grahawi Malignancy and Transformation: Expression in Somatic Cell Hybrids and Variants Hamey L. Ozer and Krishna K . ] h a Tumor- Bound Immunoglobulins : 1ti Si tu Expressions of Humoral Immunity

lsauc P. Witz T h e Ah Locus and the Metabolism of Chemical Carcinogens and Other Foreign Compounds Stiorri S . Thwgeirsson and Daniel W.

Nebert

458

CONTENTS OF PREVIOUS VOLUMES

Formation and Metabolism of Alkylated Nucleosides: Possible Role in Carcinogenesis by Nitroso Compounds and Alkylating Agents

Anthony E. Pegg I inmunosuppression and the Role of Suppressive Factors in Cancer I s m Kumo and Herman Friedman Passive Immunotherapy of Cancer in Animals and Man Steven A. Rosenherg and Willium D. Terry SUBJECT INDEX

Volume 26 Epidemiology of Large-Bowel Cancer Pelayo Correa und William Haenszel Interaction between Viral kind Genetic Factors in Murine Mammary Cancer J . Hilgers and P. Bentvelzen Inhibitors of Chemical Carcinogenesis

The

Lee W. Wattenherg Latent Characteristics of Selected Herpesviruses Jack G. Steoens Antitumor Activity of Corynehncterium

porvum Luka Milus and Martin T. Scott SUBJECT INDEX

SUBJECT INDEX

Volume 28 Cancer: Somatic-Genetic Considerations F. h4. Burnet Tumors Arising in Organ Transplant Recipients

lsruel Penn Structure and Morphogenesis of Type-C Retroviruses Ronald C. Montelaro and Datii P.

Bolognesi BCG in Tumor Immunotherapy

Ro1,ert W. Baldwin nnd A4cilcolin V. Pimrn The Biology of Cancer Invasion and

Volume 27

Metastasis

Translational Products of Type-C RNA Tumor Viruses

John R. Stephenson, Sushilkumar G . Devare, and Fred H. Reynolds, Jr. Quantitative Theories of Oncogenesis Alice S. Whitternore Gestational Trophoblastic of Choriocarcinoma, and Choriocarcinoma Hydatidiform Mole, inunologic Aspects

J . 1. Brewer, E. E . Torok, B. D. Kahatl, C . R. Stnnhope, and B. Halpern The Choice of Animal Tumors for Experimental Studies of Cancer Therapy Harold B. Hewitt Mass Spectrometry in Cancer Research John Roboz Marrow Transplantation in the Treahnent of Acute Leukemia E. Donna11 Thomas, C . Deun Bucktier, Alexander Fefer, Paul E . Neiman, and Ruiner Storb Susceptibility of Human Population Groups to Colon Cancer Martin Lipkin Natural Cell-Mediated Immunity Ronald B. Herberman and Howord T. Holden

Disease: Origin Invasive Mole Associated with and Some Im-

lsciiah J. Fidler, Dougln.~hl. Gersten, und l u n R . Hart Bovine Leukemia Virus Involvement in Enzootic Bovine Leukosis

A. Burny, F. Bea, H. Chantrenne, Y. Cleuter, D. Dekegel,J . Ghysdoel, R . Kettmann, M . Leclercq, J . Leunen, M . Mammerickx, and U . Portetelle Molecular Mechanisms of Steroid Hormone Action

Stephen J . Higgins und Ulrich Gehring SUBJECT INDEX