Advances in Cancer Research Volume 38

ADVANCES IN CANCER RESEARCH VOLUME 38

Contributors to This Volume Benjamin Bonavida

Toshiko Kodama

Krystyna Frenkel

William L. McGuire

Motoo Hozumi

George W. Sledge, Jr.

Eli Kedar

George W. Teebor

Mitsuo Kodarna

David W. Weiss

G. Yogeeswaran

ADVANCES IN CANCERRESEARCH Edited by

GEORGE KLEIN Department of Tumor Biology Karolinska InStitUtet Stockholm, Sweden

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

Volume 3 8 4 9 8 3

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9 8 7 6 5 4 3 2 1

CONTENTS CONTRIBLITORS TO VOLUME 38 . . . . . . . . . . . . . . . .

ix

The SJL/J Spontaneous Reticulum Cell Sarcoma: New Insights in the Fields of Neoantigens. Host-Tumor Interactions. and Regulation of Tumor Growth BENJAMIN BONAVIDA 1 I . Introdnction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 11. Histopathology and Characteristics of SJWJ Rcticriliiin Cell Sarcomas . . . . . 7 III . Developnient of RCS in Vicu and Host Cell Infiltration . . . . . . . . . . . 8 I\’. Immune Coinpetence of Normal and Tumor-Bearing SJLiJ Mice . . . . . . . 9 I ! Transplantation Resistance of RCS i j i Wco . . . . . . . . . . . . . . . . . 10 VI . Thr Host Iminune Respnnse against SJL/J HCS Tumors . . . . . . . . . . . \JIl. Natiirr of Tiimor-Associated Antigens of RCX . . . . . . . . . . . . . . . 13 VIII . I t i Viim Regulation of Tiimor Growth . . . . . . . . . . . . . . . . . . . 17 IX . Ccllular Intcractions between RCS Tuniors and Host Cclls . . . . . . . . . . 18 20 X . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

The Initiation of DNA Excision-Repair GEORGE W. TEEBOR A N D KRYSTYNA FRENKEL Introcliietion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uackgronncl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . h’-Gly~wsylasesand O”-AlkylAcceptor Protein . . . . . . . . . . . . . . Repair of U\’-lnducrd Pyrimidine Diiners . . . . . . . . . . . . . . . . lic Aromatic Hydrocarbons \: Rqmir of Modifications of DNA Caused by Pol . . . . \‘I . Repair of Modifications of DNA Caused I)? M-Ar.rtyl-2-ainiiiofluoreiic VII . CIiromatin Structure and DNA Rrpair . . . . . . . . . . . . . . . . . \Jill. Inhibition of D N A Repair . . . . . . . . . . . . . . . . . . . . . . . . 1. I1 . 111. I\‘.

Refrrences

23 25

. . . . .

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

27 36 38 13

49 50 52

Steroid Hormone Receptors in Human Breast Cancer

.

GEORGE W. SLEDGE. JR. A N D WILLIAM L MCGUIRE I . I lit roduct ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . “Sul)tle and Mysterious Infliiriic.es”-Tlic Ilistorical Backgroinid . . . . . . . V

ti 1 62

vi

CONTENTS

I11. Measurement of Steroid Receptors . . . . . . . . . . . . IV. Physiology of Steroid Receptors in Breast Cancer . . . . . V. Pathology of Steroid Receptors . . . . . . . . . . . . . . VI . Steriod Receptors and Prognosis . . . . . . . . . . . . . VII . Steriod Receptors in the Treatment of Breast Cancer . . .

. . . . . . . .

62 63 66 68 69 72 72

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

. . . . . . . . .

VIII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Relation between Steroid Metabolism of the Host and Genesis of Cancers of the Breast. Uterine Cervix. and Endometrium MITSUOKODAMA A N D TOSHIKO KODAMA I . Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Epidemiological Aspects of Cancers of the Breast. Uterine Cervix. 78 and Entlometriiim . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 111. IIornional Aspects of Cancers of the Breast. Iltcriiie Cervix. and Endometrium. I\! New Trends in the Biology and Molecular Biology of Steroid Hormones . . . . 9.5 \’. Synthesis of a Unifying Theory . . . . . . . . . . . . . . . . . . . . . . 101 VI . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 VII . Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 11.

Fundamentals of Chemotherap of Myeloid Leukemia by Induction of Leukemia Eel1 Differentiation Moroo HOZUMI I . Introdiiction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 I1 . Myeloid I, eukeiiiia Cells Used for Experinicnts . . . . . . . . . . . . . . 122 111. Iiiduction of I)iVc~reiitiationof Cultiired Mouse Myeloid Lriikeinia Cells . . . 123 I\: In Vico Induction of Diffcrcntiation of Mouse Myeloid Leiikemia Cells a i i t l Therapy of Animals Iiroculated with Mvcloid Leukemia Cr.lls . . . . . . . . 148 . Induction of Differentiatioil of Cultured Hiiman Illyeloid Leiikeiiiia Cells . . . 1.52 VI . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 Heferrllces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 J\

The in Vitro Generation of Effector Lymphocytes and Their Employment in Tumor lmmunotherapy ELIKEDARA N D DAVIDW. WEISS I . Introductioii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Generation in Culture of Lyinplioid Effector Cells . . . . . . . . . . . . I11 . T-CeIl C h d i Factor: IL2 . . . . . . . . . . . . . . . . . . . . . . . I\! Atloptive l~iimiinotl~rrapy with Effector Lymphoid Cells Generated i n Culture Refkrences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

171

. 181

.

237 244 263

vii

CONTENTS

Cell Surface Glycolipids and Glycoproteins in Malignant Transformation G . YOGEESWARAN I . Iiitrodriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . arid Glycopr-oteins . . . . I1 . Structiirc and Fiiiictioii 01 Gl~eosp1iin~~:olipids and S1align;mcy . . . . . . . . . . . . . . . . . . 111. ~lyc.os~~Iiin~!olipitls I\! 61ycoprotcins a i i d Slaligiiaiicy . . . . . . . . . . . . . . . . . . . . \.. Sialofi1ycoru)ii~irRates aiid \laligi)anc~- . . . . . . . . . . . . . . . . el.;is~.s-.i.i.ii.sti. \:due and \'I . Serum (:l!,coc.oiijiigatrs and Gl~eosyltr-~iiisf

. .

2x9

. . . 291 299 :313 . . . 321 . .

. .

Patliopliysiological Significance . . . . . . . . . . . . . . . . . . . . . . \'I1 . Summary and Prospects. . . . . . . . . . . . . . . . . . . . . . . . .

332 336

Refcw?nces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

:3:3x

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

CONTENTS OF PREVIOUS VOLUMES. . . . . . . . . . . . . . . . . . . .

351 355

This Page Intentionally Left Blank

CONTRIBUTORS TO VOLUME 38 Numbers in parentheses indicate the pages on which the authors' coatri1)utions begin.

B E N J A M IBONAVIDA, N Department of Microbiology and Iininunology, University of Cali,foriiia School of Medicine, Los Angeles, Calgornia 00024 (1)

KRYSTYNA FRENKEL, Department of Pathology, New York University Medical Center, New York, New York 10016 (23) MOTOOHOZUMI, Department of Cheinotherapy, Saitaina Cancer Center Research Institute, Saitaina 362, Japan (121) ELI KEDAR,The Lautenberg Center f o r General and Tumor lininunology, The Hebrew University-Hadassah Medical School, Jerusalem, lsrael (171) MITSUOKODAMA, Laboratory of Clieinotliercqq, Aichi Cancer Center Research Institute, Nagoya, J a p n (77) TOSHIKO KODAMA, Laborutory of Chemotherqxy, Aichi Cancer Center Research lnstittite, Nagoya, Japan (77) WILLIAM L. MCGUIRE,Departtnent of Medicine, Univerqsityc.fTesas Health Sciences Center, San Antonio, Texas 78284 (61) GEORGEW. SLEDGE, JR., Department of Medicine, Unicersity oj-Terns Health Sciences Center, San Antonio, Tesas 78284 (61) GEORGE W. TEEBOR, Department of Pathologiy, New York Unitiersity Medical Center, New York, New York 10016 (23) DAVID W. WEISS,The Lautenberg Center for General and Ttrinor linniunology, The Hebrew University-Haclnsfa12 Medical School, Jerusalein, Israel (171) G. YOGEESWARAN, Department c$ Microbiology and Hubert H . Humphrey Cancer Research Center, Boston Unicersity School of Medicine, Boston, Massachusetts 02215 (289)

ix

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THE SJL/J SPONTANEOUS RETICULUM CELL SARCOMA: NEW INSIGHTS IN THE FIELDS OF NEOANTIGENS, HOST-TUMOR INTERACTIONS, AND REGULATION OF TUMOR GROWTH Benjamin Bonavida Department of Microbiologyand Immunology,University of California Schwl of Medicine Los Angeles, California

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

I

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

11. Histopathologyand Characteristicsof SJL/J Reticulum Cell Sarcomas..................

111. IV. V. VI.

VII. VIII.

IX. X.

A. Pathogenesis........................................................................................................... B. Origin ..................................................................................................................... C. Etiology.................................................................................................................. D. Maintenance of Tumor Lines in Vivo and in V i m ............................................. E. Relationship between SJL/J RCS and Hodgkin’s Disease in Man ..................... Development of RCS in Vivo and Host Cell Infiltration .......................................... Immune Competence of Normal and Tumor-Bearing SJL/J Mice ......................... Transplantation Resistance of RCS in Vivo............................................................... The Host Immune Response against SJL/J RCS Tumors ........................................ A. The Syngeneic Mixed Lymphocyte Tumor Interaction as Measured by Proliferation....................................................................................................... B. The Syngeneic Anti-RCS Cytotoxic T-cell Response......................................... Nature of Tumor-Associated Antigens of RCS ...................................................... ... A. Expression of Alien H-2 Antigens ..................... ..............* ..... B. Expression of Inappropriate Ia Hybrid IE/C Antigens on RCS.......................... I n Vivo Regulation of Tumor Growth ....................................................................... A. Antigen-Reactive Cell Opsonization .................................................................... B. Dependence of Tumor Growth on Host Response ............................................. Cellular Interactions between RCS Tumors and Host Cells ..................................... Concluding Remarks................................................................................................... References.................................... ...................................................................,..........

__

1 3 3 3 5 5 7 7 8

9 10 10 13 13 13 14 17 17 18 18

20 21

I. Introduction

The biology of cancer has been an area of intensive investigation for several years. Cancer remains one of the most challenging fields in the twentieth century and the most enigmatic. Research in cancer has attracted scientists from numerous disciplines and has provided the impetus for investigations in molecular biology, biochemistry, and immunology. In the field of immunology, interest in cancer research was awakened by the introduction of the concept of “immunological surveillance” by Burnet (197 1). This concept suggests that, normally,tumor cells arise spontaneously but are eliminated by the host immune response. However, tumors would 1 ADVANCES IN CANCER RESEARCH, VOL. 38

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0- 12-006638-6

2

BENJAMIN BONAVIDA

develop and grow when there is a failure of the host immune response. The presence of host immune response would suggest that tumor cells must express neoantigens which are recognized by the host as foreign. Thus, tumor immunology has developed into an intensivearea of research investigations in an attempt to provide evidence for the Immune Surveillance Theory. In addition, several studieshave been done to implement these concepts in the diagnosis and treatment of cancer. Convincingevidence of immune surveillance has been provided in areas of neoantigens, host immune response, and regulation. However, the evidence was primarily derived from experimental studies in animals using virally induced tumors or long-term transplantable or chemically induced tumors. Little information was available using primary tumors of spontaneous origin. Therefore, one important challenge in tumor immunology is to investigate primary spontaneous tumors and to determine whether they behave similarly to or differently from experimental tumors. Such studies would provide the means to investigate several fundamental questions of clinical significance in the field of cancer immunology. The spontaneouslyarising reticulum cell sarcoma (RCS)of SJL/J mice is a good experimental tumor system for investigation for the following reasons: ( 1)the tumor is spontaneousand resemblesHodgkin’slymphoma and is thus of clinical importance; (2) the majority of mice develop RCS by the age of 8 - 12 months, although a small percentage fails to develop the tumor, which suggeststhat possibly, with some of the mice, the host immune system may play a role in tumor arrest; (3)the role of an antitumor immune response was corroborated in studies showing resistance to transplantable RCS in mice immune to the tumor (Murphy, 1969); these studies suggested that RCS tumor cells may express neoantigens; (4) the exact histological nature of the tumor is not well defined and has been classified as a type B reticulum cell sarcoma (Dunn, 1954). The heterogeneity of the tumor with an unknown cell origin resembles several ill-defined spontaneous tumors in man. Since the RCS tumor system delineated above offers several unique features, several studies have provided experimental evidence for new concepts in the biology of SL / J RCS. For instance, the strong proliferative response induced by the tumor in the syngeneic host, the expression of inappropriate alien major histocompatibilitycomplex (MHC) antigens by the tumor, and the strong dependency of host cells for tumor growth are only a few examples that have emerged with this tumor system. This article will attempt to summarize present findings (published and unpublished) and establish a possible model@)of host -tumor interrelationship which takes into account the available information at hand.

THE SJL/J SPONTANEOUS RETICULUM CELL SARCOMA

3

II. Histopathology and Characteristicsof SJL/J Reticulum Cell Sarcomas

A. PATHOGENESIS

Murphy ( 1963)found that a new inbred strain, SJL/J, had a high incidence (up to 9 1%) of reticulum cell neoplasms at a mean age of 13.3 months. The basic histologicalpattern of the neoplasms was that of type B as described by Dunn (1954). Haran-Ghera et al. (1967) have reported that the incidence of RCS did not vary greatly between males and females(7 1 -78%,respectively), with an averagelatent period of 348 - 380 days. Lesions were restricted to the mesenteric and cervical lymph nodes, Peyer’s patches, and white pulp of spleens. More advanced lesions involved other lymph nodes, the liver, kidney, and ovaries. Twenty-five percent of the animals had marked thymus enlargement due to spontaneous reticulum cell invasion. Thymectomy, splenectomy,and castration had no effect on the incidenceoftumor or latent period. Siegler and Rich (1 968) have studied the pathogenesis of RCS in SJL/J mice. They found that the neoplasm arises in the mesentericlymph node and in Peyer’s patches. The neoplastic reticulum cells grow in clusters that resemble the normal germinal centers of lymph nodes. Following a period of cell proliferation, most of the neoplastic cells die and are replaced by fibrous scar tissue. They observed that a progression of tissue changes takes place at sites previously occupied by neoplastic cells. The pleiomorphic histologic picture characteristic of RCS seems to result from the juxtaposition of clusters of proliferating cells and giant cells, and fibrous tissue products.

B. ORIGIN The origin of the RCS tumor has not been convincingly delineated, although several suggestionshave been made. Based on the enlargement of the mesenteric lymph node at times, when all other tissues of the body were normal, Siegler and Rich ( 1968)suggested that the tumor is first observed in this location. It was not possible to determine whether tumors at other sites arose from metastasis by in situ neoplastic change. In addition, Siegler and Rich have reported that the cells that comprise the neoplasm mimic in appearance and growth characteristics the reticulum cells of the normal germinal follicles. Like the reticulum cells of normal germinal follicle cells, the tumor cells are associated with macrophages that contain cell fragments and, in some instances, nuclear fragments. Using histological criteria alone, it is relevant to speak only of neoplastic tissue rather than neoplastic cells,

4

BENJAMIN BONAVIDA

because cell for cell, there is as much variation between normal reticulum cells as between normal versus neoplastic cells. This close association is likely due to the fact that both cells are premature, relatively undifferentiated cells of the mesenchyme and have the most rudimentary developmental structure. The histologic appearance of the tumors suggests that RCS may be a tumor of the “germinal follicle cells” themselves. This is of interest because these cells are regarded by many as a site of antibody production, which means that this tumor could be a neoplasm of the antibody-producing apparatus itself. The findings of altered serum globulins early in this neoplasm by Wanebo et al. (1966) are of interest in this regard. Although the cellular identificationof RCS in culture was not possible,the identification of the RCS tumor in vivo is much more complex. Several observations lend supporting evidence to a presumptive B-cell origin for these tumors. The tumor is first detected in germinal centers, including lymph nodes and Peyer’s patches (Murphy, 1969). These misshapen large cells might be histiocytes or they might be early stages of B cells (Lukes and Collins, 1975). Homing experiments with labeled SJL/J tumor cells by Carswell et al. ( 1976)showed typical B migration to lymph node folliclesand splenic white pulp. However, these cells might have been normal contaminants present in the mixed tumor cell populations used for inoculation. In addition, studies by Katz et al. (1980b) have shown that neonatal mice treated with anti-IgM serum did not developspontaneous RCS. These results suggested that RCS may be of B-cell origin. Alternatively, RCS may require a cell of B-cell origin for its development. Last, the ability of these tumors to stimulate a strong proliferative response by syngeneic T lymphocytes (Lerman et al., 1974),and the presumptivepresence of Ia antigenson SJL/J RCS (Wilbur and Bonavida, 1981) also imply that it may be of B-cell origin but does not exclude a macrophage-like cell. Recently, Ford et al. (198 1)showed that when tumor-bearinglymph nodes were placed in cell culture, colonies of adherent cells grew slowly to confluence and exhibited morphologic and functional properties of macrophages. The “tumor cells” were also grown in soft agar where clusters and colonies of large binucleated cells predominated. These were nonspecific esterase positive, suggesting a macrophage origin. Although these studies were interesting in delineating the origin of the SJL/J neoplasms, the critical experimentswere not done (i.e., to demonstrate that the colonies obtained in vitro are tumorigenic and can induce a disease in vivo). Two reports have also suggested that RCS may be derived from clones of natural killer (NK) cells (Chang and Log, 1980;Fitzgerald and Ponzio, 1979, 1981). Ofinterest, only established transplantable tumors showed cytotoxic activity against NKsensitivetargets, whereas primary tumors showed no activity. Since NK cells and the tumor cells are both null-like cells, but the RCS tumor is Ia+whereas

THE SJL/J SPONTANEOUS RETICULUM CELL SARCOMA

5

NKcells are Ia-, the difficultyin identifyingthe tumor cell raisesthe question as to whether the NK activity is a contaminant cell of host cell origin (Chang, 1980). In addition, Ponzio et al. ( 1980)have suggested that SJL/J RCS can produce interferon known to enhance natural killer activity. Our studies with different transplantable lines and with in vitro tumor lines showed no significant NK activity accounted for by tumor size (unpublished). Therefore, the exact cellular origin of RCS remains unanswered and awaits the development of specifictumor cell markers for identification and characterization. C. ETIOLOGY Studies to demonstrate a viral etiology for RCS have not been successful (Chang et al., 1974,1975).Yumoto and Dmochowski (1967)reported virus of the murine leukemia virus (MuLV)type in cases of primary SJL/J disease, but virus of this morphological and antigenic type is widespread and ubiquitousin mice, so there is no firm ground to implicate it in SJL/Jdisease. Haran-Ghera et al. (1967) reported transmission by filtrates inoculated into subcapsular renal implants ofthymus, which suggestsa viral etiology. In their experience, RCS lines have not shown MuLV particles and they lack MuLV (Gross) viral antigens (Wanebo et al., 1966). Although intracisternal A particles occur in these lines, their significance is obscure. Thus, the viral etiology of RCS has not been established. OF TUMOR LINESin Vivo AND in Vitro D. MAINTENANCE

a. In Viva Reticulum cell sarcomas show unusual transplantation behavior. There is limited initial transplantability and continual instability of transplant lines (Murphy, 1969; Haran-Ghera et al., 1967). With passages, the latent period of tumor growth decreases and transplantable lines can be obtained with a relatively short latent period. In general, the tumor during serial passages retains the histological morphology of the original RCS. b. In Vitro. Although several spontaneous RCS lines have been transplanted in vivo and maintained transplantability,the fact that the tumors are pleiomorphic raises logistic questions for their analyses. In addition, the concomitant presence of normal blast cells confoundscharacterizationof the tumor cells. Although a homogeneous populationof RCS cells would aid in the study of this neoplasm, tissue culture.lines of spontaneous SJL/J neoplasms have been difficult to establish. However, three transplantable in vivo lines derived by us from spontaneous RCS were passaged 2 to 25 times and then established in culture (Owens and Bonavida, 1977). The initial growth of the tumor in culture was absolutely dependent upon glutathione. Two of the lines, RCS-LA6 and RCS-LA8, after 25 passages in

6

BENJAMIN BONAVIDA

TABLE I CHARACTERISTICS OF SJL/J RCS ESTABLISHED IN CULTURFP Reticulumcell sarcoma tumor line Characteristics

LA- 1

LA-6

Blast

Blast

+ +-

+/-

LA-8

1. Surface markers

H-21 Thy-1.2 Fc receptor Surface Ig Complementreceptor CytoplasmicIg 2. Histochemistry Wright’s stain &Glucoronidase Methyl-greenpyronin Lipase PAS Peroxidase 3. Phagocytosis 4. Transplantability a

+

-

+ + -

+

Owens and Bonavida (1977).

v i m , lost their sulfhydryl dependence although RCS-LA 1 retained the requirement for over 9 months. All three cultures were ofblast-like morphology by light and electron microscopy and produced the type B neoplasms (Dunn Classification, 1954) when injected into young SJL/J mice. The identity of these cultured tumors was investigated by surface markers, histochemical staining, and cytotoxic function. The results are summarized in Table I. The tumors could not be classified as typical T, B, or monocytes, but rather null-like cells. Electron micrographssuggestedthat the tumors are blast cells with euchromatic nuclei, aggregated polyribosomes, and high nuclear - cytoplasmic ratios. It was concluded from the blast morphology, swollen mitochondria, and detectable endoplasmic reticulumsthat the cells were metabolically very active which is expected from cells growing in culture. The in vivo administrationof the cultured RCS led to classical type B neoplasia of heterogeneity similar to spontaneous RCS. These observations are interesting since they raise the question as to whether there is a single or multiple malignant cell type($ in this neoplasm. Conceivably, the observed in vivo heterogeneity may be due to (a) host response to the tumor, (b) pluripotentiality ofRCS tumor cells, and (c) nonclonality of lines. Because of the lack ofa suitable marker specificfor RCS, these hypotheses have not been

THE SJL/J SPONTANEOUS RETICULUM CELL SARCOMA

7

tested experimentally. In addition, the in vivo transplantability of the RCS lines was lost and new lines must be generated for further studies. E. RELATIONSHIP BETWEEN SJL/J RCS AND HODGKIN’S DISEASE IN MAN The SJL/J tumor system closely resembles Hodgkin’s disease in man. This includes the classical Reed-Steinberg type cells. Eosinophils are not common in type B neoplasms of old mice of other strains, but they can be very prominent in some tumors of SJL/J mice. Extensive fibrosis does occur in the mouse in spontaneous type B tumors, and particularly in regressing transplants (Murphy, 1969). Siegler and Rich ( 1968) have described several common features between RCS and Hodgkin’s. Both are neoplasms of lymph tissues with a complex histologicpattern of growth and pleiomorphic cell characters. Following tumor tissue necrosis, plasma cell and giant cell granulomatosis ensues. The bulky tumor size represents only a few proliferatingtumor cells. Rarely, the neoplastic cells disseminateto the bloodstream. Significant perturbation of several immunological responses is frequently seen in both SJL/J RCS and Hodgkin’s disease. Even though such similaritiesexist, the complexity of Hodgkin’s tumors and SJL/J RCS tumors may reveal differencesbetween these two neoplasms. Thus, selection of SJL/S RCS tumor as a model for Hodgkin’s disease must be considered cautiously. 111. Development of RCS in Vivo and Host Cell Infiltration

The kinetics in vivo of tumor growth and the characteristics of the cells infiltrating the tumor were analyzed by estimating the relative frequency of various cell types in the neoplastic organs (Rand and Bonavida, unpublished). The demonstration that RCS tumors are null-like cells lacking detectable T and B surface markers with the concurrent demonstration of antigens that immunologicallycross-react with alloantigens (seeSection VII) allowed the identificationof tumor cells admixed with other cells. The in vivo kinetics of development of RCS LA-6 with other host cells was examined using density gradient centrifugation to separate cells on the basis of their specificgravities (Table 11). Clearly, following transplantation, the number of null tumor cells increases steadily and by day 10 more than 90%of the cells are tumor cells. In contrast, the number of B cells declines with time. The percentage of T cells, however, remains constant initially, but declines by days 7 and 10. The size of the tumor-bearingspleensincreasesmore than 10times, and therefore the T cells must have proliferated significantly. The majority of T cells are blasts. Such cells were not observed in normal spleen cells. It may be that

8

BENJAMIN BONAVIDA

TABLE 11 I N E T I C S OF DEVELOPMENT OF RCS-LA6 AND CHARACTERIZATION OF HOST CELLSIN

TUMOR-BEARING SPLEENS~ Percentage cells in fraction of specific gravityc Day 3 5

7

10

Cellb

Unfractionated

1.070- 1.080

I .065 - I .070

< 1.065

B

56 42 2 30 43 21 13 29 58 25 0 75

48 42 14 34 46 20

68 31 1 18 46 36

45 44 II 16 38 46 9 19 68 8 3 89

T Null B T Null B T Null B

T

Null

11

9

26 63 24 2 14

30 61 23 8 69

SJL/J mice 6-8 weeks old were injected intraperitoneally with lo8LA6 cells/mouse. Mice were sacrificed at varioustime intervalsand then spleenswere prepared for separation on density gradients. Indirect immunofluorescence was used to characterize the cell tumor phenotype. B cells were identified by anti-lg serum, T cells with anti-Thy-1.2, and null cells by C57BL/6 anti-BALB/c alloantiserum (anti-H-2dantibodies cross-reactivewith RCS). Low- and high-density stock solutions were prepared using Ficoll400 (Pharmacia), sodium metrozoate, and Hanks’ balanced salt solution with some modification to account for increased osmolarity of mouse cells. Gradients ofconstant pH were used. Three fractionswere selectedfor studies of different specific gravities.

these blast T cells represent an amplified immune response against the neoplasm, consistent with findings of the strong proliferative T-cell response induced by RCS cells in vitro. IV. Immune Competence of Normal and Turnor-Bearing SJL/J Mice

In many stems, tumor-bearing mice have been shown to have a generalized defect in immune functions and such immunodeficiencies appear to accentuate tumor growth in the compromised host. The immune competence of normal and tumor-bearing SJL/J mice was investigated in an effort to delineate whether RCS tumor growth is the result of a generalized immune deficiency (Table 111). The result of such studies indicated that the T-cell-mediated and the B-cell responses are not impaired in normal or tumor-bearing mice of different ages. In addition, there seems to exist an age-associated decrease in immunological regulation as indicated by an enhanced immune response in old mice,

9

THE SJL/J SPONTANEOUS RETICULUM CELL SARCOMA TABLE 111 IMMUNECOMPETENCE OF NORMAL AND TUMOR-BEARING MICE=

Normal Immune responses T-cell response Syngeneic MLTI Syngeneic cytotoxic T cells to RCS Allogeneic CMC (T) T helper (for IgG antibody) PHA, Con A

k e l l response Cytotoxic antibody to alloantigens (I& and IgM) Tolerance induction to HGG Natural Ab to poly(1-C)

Tumor bearing

2-5 months

6-10

11-16

months

months

6-10 months

months

+-

+-

+-

+-

+-

+ +

+ + +

+ + +

+ + +

+

+

+ + + +

-

-

-

-

+

+

+

+

11-16

+ +

~~

0

From Owens and Bonavida ( 1976) and Owens (1 977).

the inability of these mice to be rendered immunologically tolerant to soluble antigens (human y-globulin) after 2 months of age, and their unusual reactivity to endogenousdsRNA (Owens and Bonavida, 1976). While these studies showed that SJL/J mice have a good response to alloantigens, other investigators have reported that SJL/J mice have deficienciesin graft-versushost reactions, delayed type hypersensitivity, and skin allograft rejection (Haran-Ghera et al., 1973). Therefore, it seems that there might be a dichotomy in the ability of SJL/J tumor-bearing mice to mount certain immune responses but not others. The inability to induce tolerance to human y-globulin (HGG) in SJL/J mice is an interesting phenomenon previously seen with the NZB/W strain but not others. The results are consistent with reports by Fujiwara and Cinader ( I 974) using normal SJL/J mice less than 4 months old. The mechanism of this phenomenon is not clear, though eitherthe loss of regulatory cells or amplificationof the helper T cells may be involved. One might predict a high incidence of autoimmunity to be consistent with loss of regulation, but SJL/J mice do not show any classical symptoms of autoimmune disorders. V. Transplantation Resistance of RCS in Vivo

The SJL/J RCS tumors have shown unusual transplantation behavior. Murphy (1 969) has observed that RCS tumors have limited initial transplantability and there was continued instability of the transplanted lines. He

10

BENJAMIN BONAVIDA

suggested that this may be explained by a host reaction to a tumor-specific antigen. A host reaction has been demonstrated histologically. In addition, evidence for the existence of tumor-specific antigen has been demonstrated by the reinoculation of the tumors in animals in which the tumors had previously failed to grow. Carswell et al. (1970) have also examined the presence of tumor-specific antigens in RCS. Irradiated cells of each of five established RCS lines protected syngeneic recipients against transplants of the sameRCS. Resistance was limited to the RCS used for immunizationand was not elicited by inoculation of similarly irradiated normal tissues, SJL/J leukemias, or a different RCS. Transplantation resistance was adoptively transferred to syngeneicrecipients by viable lymph nodes, spleen, or peritoneal cells from resistant immunized donors. The mechanism underlying the tumor-specific resistance has not been delineated.

VI. The Host Immune Response against SJL/J RCS Tumors

Table 111summarizes the findings discussed in more detail below. A. THESYNGENEIC MIXEDLYMPHOCYTE TUMOR INTERACTION AS

MEASURED BY PROLIFERATION 1. In Vitro Studies

SJL/J RCS from different sources, spontaneously occumng, transplantable in vivo, and cultured in vitro have been shown to stimulate the proliferation of syngeneic lymphocytes in mixed lymphocyte tumor interaction (MLTI). The MLTI profile appears to be different from the primary allogeneic mixed lymphocyte reaction (MLR) (Owens, 1977). The kinetics of MLTI are similar to allogeneicreactions only in that peak responses occur at 96 hr of culture. However, the magnitude of [3H]thymidineincorporation is generallygreater than that found in MLR at all incubation times up to 120 hr (Fig. 1). Furthermore,the syngeneicMLTI requires one-tenth the number of stimulators as compared to the MLR. These results then suggest that in vivo priming might have taken place in the normal SJL/J mice and the results observed would represent a memory response. However, a decrease in the number of stimulatingcells required for secondary proliferationhas not been observed in other allogeneic systems although an accelerated response is seen. As indicated above, the high proliferation seen with normal SJL/J lymphocytes may represent an anamnesticresponse. This was tested by priming the mice with tumors before rechallenge in vitro. These studies showed that the syngeneic proliferation follows kinetics similar to a secondary allogeneic

THE SJL/J SPONTANEOUS RETICULUM CELL SARCOMA

SYNGENEIC RCS-LA1 TUMOR CELLS

11

ALLOGENEIC C3H SPLEEN CELLS

96 hr

72 hr 48 hr

,24hr 5 x 405 5 x 104 5 x 404 403

105

NUMBER OF STIMULATING CELLS

FIG. 1. Mixed lymphocytetumor interaction of SJL/J splenic lymphocytesagainstsyngeneic RCS as compared to mixed lymphocyte reaction of SJL/J against allogeneic C3H spleen cells. Respondingspleen cells(5 X 10') from young SJL/J mice were circulated with varying numbers of mitomycin C-treated RCSLAl or C3H spleen cells and harvested at daily intervals after a 6-hr pulse with ['Hlthymidine. Each data point represents the mean +SD of triplicate determinations in the same assay. The pattern shown here is representative of five separate kinetic experiments.

response. In addition, there was cross-priming among various tumor cell lines (Owens, 1977; Owens and Bonavida, unpublished). The mechanism underlying the MLTI will be further discussed in Sections VII and VIII. Furthermore, the SJL/J mice show a remarkably high autologousproliferation by mitomycin C-treated SJL/J stimulators. Isogenic stimulation of lymph node cells by mitomycin C-treated spleen cells has been reported to be caused by murine B-cell differentiation antigens in CBA mice (Ponzio et al., 1975). However, the response of SJL/J thymocytes to autologous lymph nodes reported by Lerman el al. (1974) and of SJL/J spleen to autologous spleen (Owens, 1977) may represent subsets of lymphocytes different from those responding to RCS or may be due to loss of regulation. 2. In Vivo Response to RCS The above findings have shown clearly that normal T cells from SJL/J mice respond to syngeneic RCS and proliferate. In addition, the studies of RCS separation by density gradient showed clearly that a large number of T-cell blasts constituted the host response to the tumor. A more direct approach to test the in vivo response to the tumor was to demonstrate host cell proliferation following antigenic stimulation. Such studies involved giving mice [*251]iododeoxyuridine (1251UdR) after tumor transplantation.

12

BENJAMIN BONAVIDA

These studies revealed that a large fraction of the radiolabel incorporated into the cells was found in the light-density fraction (T-cell blasts and tumor cells) as early as 3 days posttumor transplant. Furthermore, in mice injected with mitomycin C-treated RCS so as to avoid tumor cell growth, 12SIUdR incorporation was detected in the T-cell region as well as the Ball-rich region. In contrast, the response of SJL/J mice to allogenicBALB/c or CBA spleen cells resulted in radioactive incorporation of the T-cell but not the B-cell region at day 7. These results suggested that the host response to the tumor includes both T and B cells, whereas the allogeneic response is predominantly T cells (Bonavida et al., unpublished).

3. Immunoregulation of the MLTI The nature of antitumor B-cell response and tumor growth will be discussed in Section VII1,A. Although RCS tumors (primary and transplantable) stimulate a strong proliferative response, both in vivo and in vitro, they fail to stimulate a cytotoxic response. However, the biological function of the responding cells in vivo is not known. Since the tumor cells continue to grow in vivo in the presence of a proliferative response, one may argue that these cells have no immunologicalrole or may represent immunoregulatory cells. We have examined whether the antitumor responding cells exert a suppressive effect on the antitumor response of nonsensitized SJL/J lymphocytes. Thus, lymphocytes generated from an in vitro anti-RCS response were used as regulatory cells and cocultured with normal SJL/J lymphocytes stimulated by RCS or allogeneiccells. The lymph node proliferative response was measured by radiolabeled thymidine incorporation. The in vitro-cultured lymphocytes were found to suppressthe antitumor response specifically but the allogeneic response weakly (Wilbur and Bonavida, 1982). However, the exact mechanism of suppression is not yet known. Since the proliferating cells are primarily Lyt-l+ cells, it appears that the suppressor cells or the suppressor inducer are Lyt- 1+2-3-. If confirmed by the use of Lyt antibody reagents, this will be the first demonstration of the existence of Lyt-l+ suppressor cells or inducers of suppressor.In addition, preliminarydata have shown that the suppressor cell population produces a factor which suppresses the response of normal syngeneic SJL/J lymphocyte response. Using a double Marbrook chamber separated by a nucleopore membrane, suppressor cells alone or with stimulators in the upper chamber inhibited the response of SJL/J cells cocultured with RCS tumor cells in the lower chamber (Wilbur et al., unpublished). Further analysis of the suppressor cells, the role of soluble factors on tumor responses, and tumor growth will provide additional information on the biological role of the proliferative response to the RCS tumor. In addition, the possible role of lymphokinesin immunoregulation will be discussed in Sections VIII and IX.

THE SJL/J SPONTANEOUS RETICULUM CELL SARCOMA

13

B. THESYNGENEIC ANTI-RCSCYTOTOXIC T-CELLRESPONSE The induction of cytotoxic T cells specific to RCS has been difficult to demonstrate. With transplantable lines, it has not been possible to generate CTL in vitro in MLTI (Roman and Bonavida, 1980a).Ponzio et al. ( 1977b) have reported that followingrepeated in vivu inoculationsof irradiated RCS spleen and, to a lesser degree, lymph node cells acquired the ability to give moderate secondary cytotoxic responses in vitru upon coculture with irradiated RCS. However, the specificityofthe response was not well established in these studies. Reticulum cell sarcoma tumors maintained in vitro stimulate a strong syngeneic cell-mediated cytotoxicity response (Roman and Bonavida, 1980a; Bonavida and Roman, 1981). In contrast, in vivo transplantable or spontaneous tumors fail to generate a cytotoxic response although they stimulate a strong proliferative response. Since the tumor-associated antigens appear to be associated with la antigens, the generation of cytotoxic T cell directed against Ia may not easily be achieved. However, reports in the literature have shown that under certain circumstances Ia antigenscan serve as cytotoxic target antigens, and recently Dennert and Raschke (1977) reported the establishment of cytotoxic clones specific for Ia-associated antigens. Thus, it is conceivable that under optimal conditions, cytotoxic cells may be generated against RCS antigens, and the failure to elicit CTL might have been due to the effector of suppressorcells or factorsgenerated in the culture. VII. Nature of Tumor-Associated Antigens of RCS

Several studies have been done to delineatethe nature of tumor-associated antigens (TAA) of RCS. Our preliminary studies have suggested the expression of cryptic H-2 antigens on RCS which are not normally expressed on host H-2s cells but which cross-react with antigens present on cells of other haplotypes (Roman and Bonavida, 1980a).These results are reminiscent of alien antigens on chemically induced leukemias of SJL/J and C57BL/6, and a spontaneouslymphoma from D B A / 2 , which have been shown to be cryptic on normal syngeneiclymphoid cells by serologicaland biochemical methods (Pratt et al., 1978;Flaherty and Reinchik, 1978;Robinson and Schirmacher, 1979). A. EXPRESSION OF ALIENH-2 ANTIGENS

The demonstration of alien H-2 specificities on RCS tumor cells of spontaneous,transplantable,and cultured origin has been reported (Roman

14

BENJAMIN BONAVIDA

and Bonavida, 1980b; Bonavida and Roman, 1981). The reactivity of sera derived from mice bearing spontaneous tumors or transplantable tumors was also examined. The sera reacted by immunofluorescence with both the RCS tumor cells and with allogeneiccells but not with normal SJL cells. The reactivity was absorbed out with RCS tumor cells but not with normal SJL/J lymphoid cells, These results show that alien specificities on tumor cells are immunogenic and elicit antibodies in vivo (Hutchinson and Bonavida, unpublished). In addition, preliminary studies were performed to characterize the alien H-2 antigens on RCS tumor cells. Due to the possible presence of antiviral antibodies in the various available sources of anti-H-2 sera, analysis of the H-2d cross-reactive molecules of SJL/J tumors was performed by two-dimensional gel electrophoresis. This procedure permits the identification of molecules by their charge as well as molecular weight. It also allows the molecules of 45,000 molecular weight to be distinguished from actin and provides characteristic fingerprints which are used to distinguish haplotypespecific H-2 molecules. These studies revealed that there are 45,000 MW molecules present on SJL RCS tumors which are precipitatedwith a11ti-H-2~ alloantisera but not with normal serum (Roman and Bonavida, 1980a). The patterns obtained are identical to those obtained from normal BALB/c lymphocytes. Unexpectedly, however, anti-H-2dsera also precipitated similar patterns of molecules from normal SJL/J lymphocytes even though by serological and cellular criteria, normal SJL/J cells were unreactive with these sera. This finding is particularly surprising since both antisera used are produced in H-2' recipients [SJL/J anti-BALB/c serum and (B1OA.IR X ASW)F, anti-BlOA]. These results suggested that the immunization may have induced an autoimmune reaction in SJL/J mice, and the expression of alien molecules on SJL/J tumor cells cannot be termed inappropriate H-2 antigens but rather a previously undetected public antigenic specificity. Thus, the above studies strongly suggested the presence of alien-like H-2 antigenson tumor cells and indicated that they may represent the expression of cryptic antigens on host cells. Because the serological reagents used were conventional alloantisera, the results described above necessitate confirmation with monoclonal antibodiesand control non-RCS tumors ofSJL/J mice and mice of other haplotypes. Such studiesarebeing currently investigated in our laboratory. B. EXPRESSION OF INAPPROPRIATE Ia HYBRIDIE/C ANTIGENS ON RCS Ponzio et al. ( 1977a)have investigated the possibility that an ecotropic or xenotropicvirus on RCS induced the stimulation ofsyngeneic T cellswith no positive results. However, they have subsequently demonstrated by cytotoxicity assays and blocking antibody that Ia antigens are expressed on the RCS

15

THE SIL/J SPONTANEOUS RETICULUM CELL SARCOMA

TABLE IV

SUMMARYOF TUMOR-ASSOCIATED ANTIGENEXPRESSION ON RCS OF sJL/J MICE' Cell-mediated response Cell-mediated cytotoxicity

Sensitized T cells binding to

~

Origin of spontaneous RCS

Syngeneic MLTI (proliferation)

H-2s

+++

-

Primary Transplantable Culture

-

+++ t+c

RCS

-

+

All@ antigen

-

-

+

RCS

Ia-7 cell

++ ++

++

nd

nd

++

Serologicalanalysis Alloantibody Cytotoxic Origin of spontaneous RCS Primary Transplantable Culture

H-2'

Alien H-2

Immunofluorescence

H-2'

Alien H-2

+++ f +++ + + + + + + + + +

+ + + + + + + +

Monoclonal Ia-7 absorption

['2SI]protein A binding by a Ia-7

++

++

++ ++

nd

nd

From Owens ( I977), Roman and Bonavida ( I98 1a), Wilbur and Bonavida (1 98 1 ), and Wilbur efal. (1983).

population in the assay. These results suggested that Ias on tumor cells is involved in the stimulation of syngeneic T cells (see Table IV). Katz ef al. ( 1980a)have studied the ability of F, hybrids of SJL/J mice to support RCS growth and the responsiveness of their lymph node cells to irradiated RCS. They observed that changing the I region of the F, parent from H-25 to H-2' or H-2d, but not H-2b or H - ~ Pvirtually , abolished the ability of the F, mice to support tumor growth. The effect appeared to be partially due to the change in the IE/C antigen and partly a result of the change in ZA (B)region. The effect was not overcomeby higher tumor dose or larger intervals after injection. In addition, there was a striking degree of correlation between the proliferative responsiveness of F, cells to RCS and the ability of the F, mice to support tumor growth. These authors postulated that the RCS may express a normally repressed IE/C gene product on RCS cells that is absent from normal SJL/J cells. This hypothesis was reached independently from studies in our laboratory using a different experimental approach (Wilbur and Bonavida, 1981).

16

BENJAMIN BONAVIDA

During our analysis of antigens on SJL/J RCS that stimulate a syngeneic response, we have observed that RCS express Ia antigens not detectable on the parental SJL/J (H-29 mice. These Ia antigens have been shown to correspond to hybrid IE/C antigens which are not normally expressed on H-2aand H-2bstrains of mice (Wilbur and Bonavida, 198 1;Bonavida et al., 198 1). The findings that Ia antigens expressed on RCS stimulate syngeneic responses suggest that the activated lymphocytes bear receptors which recognize Ia antigens. This was assessed directly by binding of lymphocytes to targets. Such experiments showed that lymphocytes which respond to RCS tumors recognize and bind specificallyto both the tumor cells (primary or transplantable) and to target cells carrying H-2d antigens but not H-2k antigens. Further genetic analysis of the antigens involved in binding using intra-H-2 recombinant strains of mice mapped the antigens to the I region. The Ia molecules involved were hybrid Ia molecules. The results suggested that the RCS expresses a hybrid Ia molecule containing a p chain of the H-2* haplotype. Recognition of this hybrid molecule by the host resulted in a cross-reactive recognition of H-2dspecificities. Further analysis showed that a chain of the hybrid IE/C molecule expressed by RCS is involved in host antitumor recognition. Preincubation ofthe RCS with monoclonalantibody directed against the Ia-7 specificity on the a chain could block binding of the lymphocytes to tumor cells. These data suggest that the hybrid Ia molecules expressed on the RCS and recognized by tumor-primed syngeneiclymphocytes are composed of a syngeneic ( p ) and an alien Ia-7-bearingE a chain. In the RCS, the synthesisof alien (a) chain permits the expression on the surface membrane of the syngeneic( p )chain in the form of a hybrid IE/C molecule. One of the important findings regarding the expression of hybrid Ia antigenson RCS is the demonstrationthat primary spontaneousRCS tumor cells as well as transplantable lines express these antigens. In addition, isoenzyme markers on RCS-LA 16 line demonstrated its H-21origin thereby ruling out any possible contamination with H-2dtumor. In addition, preliminary findings indicate that the syngeneic SJL/J antiRCS proliferative response is inhibited by the addition of monoclonal anti-Ia-7 antibodies in the culture (Wilbur, Wayner, and Bonavida, unpublished). In summary, Table IV summarizes the data on neoantigen expression on RCS tumor cells. The expression of la antigens on RCS which are recognized as foreign by effector T lymphocytes, therefore, provides a molecular basis for understanding the mechanism of tumor- host interaction and resulting immunological manifestations. The studies also show that neoantigens may be the result of the derepression of geneswhich are not expressed in the normal host and how such antigens behave like alien MHC products on tumor cells (Festenstein and Schmidt, 1981; Parmiani et al., 1979).

THE SJL/J. SPONTANEOUS RETICULUM CELL SARCOMA

17

The exact mechanism by which Ia hybrid antigen becomes expressed on RCS tumor cells is not known. However, several recent reports have shown that tumor cells or normal macrophages can be induced to express Ia antigens, both IA and IE/C antigens by soluble factors (Warner et al., 1982). Thus, it is conceivable that a regulatory circuit exists by which the syngeneic MLTI results in the production of lymphokines which in turn may activate normal or tumor cells to express IE/C hybrid antigens. Such a model is being presently verified experimentally. VIII. In Vivo Regulation of Tumor Growth

A. ANTIGEN-REACTIVE CELLOPSONIZATION

The studies thus far have shown that RCS tumor cells are immunogenic, are susceptible to both antibody and cell-mediated recognition, and yet the tumor cells are not rejected in v i v a Several possible mechanisms may be involved independently or concomitantly. For instance, a specific suppressor system may be operative in tumor-bearing mice. Indeed, classical suppressor T cells which inhibit the MLTI have been observed and these may lead to immunological enhancement. Another observation is the tumor dependency on host response for growth as discussed in the previous section. A third mechanism is antibody-mediated enhancement by circulating antitumor antibodies and the reticuloendothelial system. SJL/J mice bearing RCS tumors have in their circulation antibodies that can recognize RCS tumor cells. The possible involvement of immunological enhancement in permitting tumor growth has been examined. Antigenreactive cell opsonization (ARCO), a mechanism of immunological enhancement, has been described and studied in both xenogeneic and allogeneic model systems (Hutchinson and Zola, 1978; Hutchinson et al., 1976). According to the ARCO hypothesis, specific antigen-reactive lymphocytes (both B and T cells) can bind free antigenic determinants in antigen-antibody complexes while the Fc portion of the antibody in the complexes is bound by the Fc receptor in macrophages. Thus, antitumor antigen-reactive lymphocytes can be specificallyopsonized in a manner entirely analogousto opsonization of an antibody-coated cell or particle. According to this model, tumor-specific antigen-reactive T and B cells may be diverted from the tumor sites and eliminated by the RCS. If such antigen-reactive cells (ARC) are responsible for regulating tumor growth, their elimination will influence the rate of tumor growth in vivo. Several experimental designs were used to investigate the possible role of ARCO in the survival of RCS tumors in SJL/J mice (Hutchinson et a/., 1980; Hutchinson and Bonavida, 1982). The results show that radiolabeled tumor-reactive T lymphocytes injected

18

BENJAMIN BONAVIDA

into tumor-bearing SJL/J mice have an abnormal homing pattern and are diverted to the liver. This is true whether the mice have been injected with transplantable or primary spontaneousRCS. The liver diversion of immune cells is tumor specific. These results also suggested that serum from tumorbearing mice contain “ARCO factors” which can cause opsonization of antitumor *ARC in normal mice. Complexes between antigens shed from the growing tumor cells and endogenously produced antibody have been shown to act as “ARCO factors” provided free antigenic determinants are exposed in these complexes. In addition, the in vivo experiments suggested that immune complexes could regulate antitumor immunity by the specific removal of antitumor-reactive T cells by ARCO. Therefore, such studies provide the first report of antigen-reactive cell opsonization in a syngeneic tumor system. Thus, ARCO could account for the survival of antigenic and immunogenic RCS tumors in SJL/J

B. DEPENDENCE OF TUMOR GROWTH ON HOSTRESPONSE Transplantable RCS have been shown to grow poorly in X-irradiated or cyclophosphamide-treated SJL/J mice. Reconstitution of irradiated hosts with normal spleen cells favored tumor growth, suggesting tumor cell dependency of a host cell for growth (Lerman et al., 1976).Recently, Katz et al. (1 98 1) have suggested that T cells promote RCS growth in vivo implicating a dependent RCS growth on host T cells. Since the tumor cells stimulatea syngeneicresponse, and since this response is under regulatory influences, it may be deducedthat some sort ofcircuitryofcell-cell interaction takes place between tumor cells and both helper and suppressorcells (Fig. 2). IX. Cellular Interactions between RCS Tumors and Host Cells

There are several findings in the SJL/J tumor system which collectively explain the host -tumor interrelationship. Clearly, the MLTI results in the generation of high quantities of lymphokines-in particular, interferon (1FN)-and interleukins 1 (IL-1) and 2 (IL-2). Since these lymphokinesare important regulatory factors, they lead to proliferation of host cells with extensive amplification of the response. A model is presented in Fig. 2. Sincethe tumorsexpressboth IA and alien IA/E antigensboth ofwhich are strongly stimulatory antigens, abnormally high LAF (IL- 1) production with subsequent IL-2 production would be expected within the lymph node. Indeed, our preliminary studies show that during the MLTI, high quantities of IL-2 are produced which are sufficient to trigger the T-cell response leading to a strong proliferative response (Wayner et al., 1982).Thus, macrophages or tumor cells and T cells interact with each other in a cyclical amplification

THE SJL/J SPONTANEOUS RETICULUM CELL SARCOMA

19

A

B GROWTH

__*

PROLIFERATION

Interleukin-2

+PROLIFERATION

polvclanol and Manodonal

Ig Synthesis

LYMPHOKINES

FIG.2. (A) Regulation of RCS tumor growth by responding T helper and T suppressor cells. (B)Model describing the cell-cell interaction that may take place between RCS tumors and respondinghost cells. The stimulation of host cells by RCS tumor to initiate a cascade of events leading to lymphokine production, cell proliferation, and induction of Ia antigens on the putative tumor cell. Thesecell-cell interactionsfom acircuitry which may explain the intricate dependency of tumors on host cells for growth.

20

BENJAMIN BONAVIDA

pathway in which T cells produce immune colony-stimulatingfactor (CSF) or IL-3 which augments the production of IL- 1 which in turn stimulatesthe production of additional CSF. Interleukin 1 also serves to provide a second signal to antigen-stimulated helper T cells to produce a variety of other immunoenhancing lymphokines such as IL-2. Interleukin 2 also provides a second signal to an antigen-stimulated T cells to produce IFNy. This cascading sequence provides the bare bones of antigen nonspecific lymphokines necessary to alter, sustain, and promote host T-cell responses and tumor growth (Fig. 2B). The development of suppressor cells concomitantly with the antitumor response may be regulating the host response. However, this regulation may not be efficient and overcome by the helper response. In addition, the anti-RCS response may not be totally switched off because the activating antigenic stimulus is inappropriately expressed on the tumor cells and/or within the reticuloendothelial system, and no negative feedback system exists to signal either the disappearance of the alien antigen or the suppression of lymphokine production. The underlying mechanism for alien antigen expression is not clear, but an explanation may be that it is due to specific induction of such antigens by lymphokines(Ia-inducingfactor)derived from other cells. The importance of this model is that it containstwo components, antigen specificand lymphokine dependent, which may be ultimately related or entirely independent (Fig. 2B). Also this model could account for (1) the low transplantabilityrate of spontaneousRCS, (2) the apparent dependence of in vivo RCS growth on the immune competence of the host, and (3) difficulty in isolating putative tumor cells (a strong proliferative response). The importance ofthis model is the ease with which severalofthe predictions could be tested experimentally. If verified, the studies provide new concepts in tumor immunology and biology. X. Concluding Remarks

Studies of RCS in SJL/J mice permits us to examine basic questions in cancer research, namely, identification of the primary malignant cell, the expression and characterization of tumor-specific neoantigen(s) that are absent on host cells, the close cooperationbetween tumor cells and host cells, and the intricate complexity of the regulatory cells or factors that participate in the overall response. As has been discussed above, this tumor system is unusual when compared to other well-studied classical experimental tumors. The unique attributes and features of this tumor model thus provide several new and intriguing areas of research in the field of cancer biology and immunology. Clearly, the tumor system is attractive to scientistsof different fields. Thus, the developmental biologist will be interested in the differentia-

THE SJL/J SPONTANEOUS RETICULUM CELL SARCOMA

21

tion and maturation behavior of RCS tumors. The geneticist may be interested in the expression of gene products coding for IA/E antigens or other TAA which are not expressed on host cells. For the membrane biochemist, there is interest in the characterization of several lymphokines that are produced in response to the tumor and characterization of signals and receptors involved. For the cellular immunologist, it provides a novel system to look at cell - cell interactions and collaboration between tumor cells and lymphocytes analogous to the well-studied cell -cell collaboration between normal lymphocytes and other cells, such as T-B cell cooperation. In addition, the RCS tumor system also resembles several human neoplastic diseases such as Hodgkin’s and other undifferentiated lymphomas and therefore can be used as a model system to investigate human neoplasia. ACKNOWLEDGMENTS 1 wish to acknowledge all my colleagues (graduate students and postdoctoral fellows) who havegenerated all the data presented in thisarticle. In addition, they have provided several ofthe ideas, principles, and concepts developed in this manuscript. The following colleagues have actively worked on the SJL/J RCS subject and will be listed chronologically: Doctors Marilyn Owens-Hescox, Ian Hutchinson, Janet Roman, Lina De,Stan Wilbur, and Liz Wayner. I am also thankful to Ms. Ann Kristy and Bette Tang for their helpful secretarial assistance and patience. This work has been supported by National Cancer Institute Grants CA 19753and CA 243 14 awarded to Doctor Benjamin Bonavida and in part by grants from CICR and CRCC of the University of California.

REFERENCES Bonavida, B., and Roman, J. M. (198 1). Cancer Immunol. Immunuther. 11, 115- 123. Bonavida, B., Wilbur, S. M., and Marelli, 0. (I98 I). Transplant Proc., 13th p. 1833. Burnet, F. M. (1971). Transplant Rev. I, 3-25. Carswell, E. A., Wanebo, H. J., Old, L. W., and Boyse, E. A. (1970). J. Nafl. Cancer Inst. 44, 1281. Carswell, E. A.. Lerman, S. P., and Thorbecke, G. J. (1976). Cell. Immunol. 23, 39-52. Chang, K. S. S. (1977). I n “Advances in Comparative Leukemia Research” (P. Bentvelzan, J. Hilgers, and D. S. John, eds.), pp. 327-330. Elsevier, Amsterdam. Chang, K. S. S., and Log, T. ( 1980). Int. J. Cancer 25,405 - 4 16. Chang, K. S. S., Law, L. W., and Aoki, T. (1974). J. Natl. Cancer Inst. 52,777-784. Chang, K. S. S., Aoki, T., and Law, L. W. (1975). J. Natl. Cancer Inst. 54,83-84. Dennert, G., and Raschke, W. (1977). Eur. J. Immunol. I, 352. Dunn, T. B. (1954). J. Nafl. Cancerlnst. 14, 1281 - 1433. Festenstein, H., and Schmidt, W. (198 I). Immunol. Rev. 60.85- 127. Fitzgerald, K. L., and Ponzio, N. M. ( 1 979). CeN. Immunol. 43, 185- 191. Fitzgerald, K. L., and Ponzio, N. M. (1981). Int. J. Cancer 28,635. Flaherty. L., and Reinchik, R. (1978). Nature(L.ondon)273,52. Ford, R. J., Ruppert, B., and Mizel, A. L. ( 1981). Lab. Invest. 45, 1 1 1 - 119. Fujiwara. M., and Cinader, B. (1974). Cell. Immunol. 12,205. Haran-Ghera, N . , Kotler, M., and Meshorer, A. (1967). J. Natl. Cancer Inst. 39,653-66 I.

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Haran-Ghera,N., Ben-Yacov, M., Peled, A., and Bentwich, Z. (1973). J. Nal. Cancer Inst. SO, 1227. Hutchinson, I. V., and Bonavida, B. (1982). Cancer Immunol. Immunother. 13, 176- 181. Hutchinson, I. V., and Zola, H. (1978). Transplantation23,464. Hutchinson, I. V., Zola, H., and Batchelor, J. R.(1976). Transplantation22,273. Hutchinson, I. V., Roman, J. M., and Bonavida, B. (1980). Adv. Exp. Biol. Med, 121B, 553. Katz,I., Lerman, S. P., Ponzio, N. M., Schrefiler, D. C., and Thorbecke, G. J. (1980a). J. Exp. Med. 151,347-361. Katz,I. R., Asofsky, R., and Thorbecke, G.J. (1980b). J. Immunol. 125, 1355- 1359. Katz, I. R., Chapman-Alexander,J., Jacobson, E. B., Lerman, S. P., and Thorbecke, G. J. ( 1981). Cell. Immunol. 65,84. Lerman, S.P., Chapman, J. M., Carswell, E. A., and Thorbecke,G. J. (1974).In?.J. Cancer 14, 808. Lerman,S . P., Carswell, E. A., Chapman, J., and Thorbecke,G. J. (1976). Cell.Immunol.23,53. Lerman, S. P., Chapman-Alexander, J., Umetsu, D., and Thorbecke, G. J. (1979). Cell. Immunol. 43,209 - 2 13. Lukes, R.J., and Collins, R. D. (1975). In “The Recticuloendothelial System” (J. W.Rebuck, C. W. Berard, and M. R.Abell, eds.), pp. 2 13-242. Murphy, E. D. (1963). Proc. Am. Assoc. Cancer Res. 4,46. Murphy, E. D. (1969). J. Natl. Cancer Inst. 42,797 - 8 14. Owens, M. H. (1977). Ph.D. dissertation,UCLA Microfilms. Owens, M. H., and Bonavida, B. ( I 976). Cancer Res. 36,1077- 1083. Owens, M. H., and Bonavida, B. (1977). Cancer Res. 37,4439-4448. Parmiani, G., Carbone, G., Invernizzi, M. A., Pierotti, M. A., Seresi, M. G., Rogers, M. T., and Apella, E. (1 979). Immunogenetics 9, 1. Ponzio, N. M., Finke, J. H., and Battisto, J. R. (1975). J. Imrnunol. 164,97 1. Ponzio, N. M., Chapman-Alexander, J., Umetsu, D., and Thorbecke, G. J. (1977a). Cell. Immunol. 32,lO - 22. Ponzio, N. M., David, C. S.,Schreffler, D.C., and Thorbecke, G. J. (1977b). J. Exp. Med. 145, 132- 145. Ponzio, N. M., Fitzgerald, K. L., Vilcek, J., and Thorbecke, G. J. (1980). Ann. N . Y.Acad. Sci. 350, 157. Pratt, M., Rogers, M. J., and Appela, E. (1978). J. Nail. Cancer Inst. Robinson, P. J., and Schinnacher, V. ( 1979) Eur. J. Immunol. 9,6 I. Roman, J. M., and Bonavida, B. (1980a). J. Immunogenetics 7 , 6 I. Roman, J. M.,and Bonavida, B. (1980b). Transplant Prm. 12th p. 59. Siegler, R., and Rich, M. A. (1968). J. Natl. Cancer Inst. 41, 125- 143. Walker, E., Lanier, L., and Warner, N. (1982).J. Exp. Med. 155,629. Wanebo, H. J., Gallmeier, W. M., Boyse, E. A., and Old, L. J. (1966). Science 154,90 1 - 903. Wayner, E. A., Wilbur, S.,and Bonavida, B. (1982). J. Cell.Biochem.Suppl. 6,36 (Abstr. 099). Wilbur,S. M.,andBonavida, B.(1981).J. Exp. Med 153,501-513. Wilbur, S . M., and Bonavida, B. (1983). Submitted. Yumoto, T.,and Dmochowski, L. I. (1967). Cancer Res. 27,2098

THE INITIATION OF DNA EXCISION-REPAIR George W. Teebor and Krystyna Frenkel Department of Pathology, New York University Medical Center, New Yo&. New York

1. Introduction ................................................................................................................. 11. Background..................................................................................................................

Ill. N-Glycosylases and 06-Alkyl Acceptor Protein .............. .............. ......... A. Uracil and Hypoxanthine N-GIycosylases ........................................................... B. 3-Methyladenine, 7-Methylguanine, and Formamidopyrimidine N-Glycosylases ........................................................ C. Thymine Glycol, Urea, and Pyrimidine Dimer N-Glycosylases ........................ D. Inducibility of N-Glycosylases and 06-Alkyl Acceptor Protein ....... ......... IV. Repair of UV-Induced Pyrimidine Dimers ............................................ ......... V. Repair of Modifications of DNA Caused by Polycyclic Aromatic Hydrocarbons .. VI. Repair of Modificationsof DNA Caused by N-Acetyl-2-aminofluorene................. VII. Chromatin Structure and DNA Repair...................................................................... VIII. Inhibition of DNA Repair ...................................................... ......... References....................................................................................................................

23 25 21 27 29 31 33 36 38 43 49 50 52

I. Introduction

Excision - repair of DNA is an enzymatically mediated process by which modified bases or fragments of bases are removed from cellular DNA together with adjoining normal nucleotides. This segment of excised DNA is then resynthesized through the action of a DNA polymerase using the opposite strand as a template. The last phosphodiester bond is sealed through the action of DNA ligase (Setlow and Camer, 1964; Boyce and HowardFlanders, 1964; Cleaver, 1968). The importance of this process to human well being through the prevention of cancer was emphasized by the demonstration that in contrast to normal cells, cells of individuals with xeroderma pigmentosum (XP),’ an hereditary disease, did not remove ultraviolet (UV) I Abbreviations: XP, xeroderma pigmentosum; AT, ataxia telangectasia; FA, Fanconi’s anemia; UDS, unscheduled DNA synthesis; AP, apurinic and/or apyrimidinic site; 3-MeA, 3-EtA, 3-rnethyIadenine,3-ethyladenine;7-MeG,7-EtG, 7-methylguanine,7-ethylguanine;06MeG,QEtG, @-methylguanine,06-ethylguanine; PAH, polycyclic aromatic hydrocarbon: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene diolepoxide; AAF, N-acetyl-2-aminofluorene; BA, benz[a]anthracene; DBA, dibenz[a]anthracene; DMBA, 7,12-dimethylbenz[a]anthracene; 7-BMBA, 7-bromomethylbenz[a]anthracene;MMS, methylmethane sulfonate; EMS, ethylmethane sulfonate; PpL,&propiolactone; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; 4NQO,4-nitroquinoline 1-oxide;FAPY, formamidopyrimidine; FPU, K-formyl-N-pyruvylurea; DMN, N-dimethyl-N-nitrosamine; MNU, N-methyl-N-nitrosourea.

23 ADVANCES IN CANCER RESEARCH. VOL. 38

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12406638-6

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radiation-induced pyrimidine dimers from their nuclear DNA (Cleaver, 1968, 1969; Setlow et al., 1969). Since individuals afflicted with XP suffer multiple skin cancers at an early age only on exposed surfaces, a link between genetic damage (the pyrimidine dimer), defective repair of the damage, and cancer in the exposed tissue (skin) was established. The insight that repair of DNA damage was enzymatically mediated stemmed from several observations and lines of investigation. The isolation of UV-sensitive Escherichia coli mutants (Witkin, 1946)first suggested that metabolic factors influenced resistance to genetic damage. The identification of a specific photoproduct in DNA induced by 254-nm radiation, the cyclobutane pyrimidine dimer (Beukers and Berends, 196I), supported the idea that UV mutagenesis was related to chemical modification of DNA. Strauss (1962) was the first to note that alkylated and UV-irradiated DNA was more susceptible to nuclease attack than was unmodified DNA. Subsequently it was found that the pyrimidine dimers were actually removed from cellular DNA and liberated in growth medium as low-molecular-weight fragments (Setlow and Carrier, 1964; Boyce and Howard-Flanders, 1964). These same investigatorsdemonstrated that the UV-sensitiveE. coli mutants did not effect removal of dimers. This work was complemented by that of Rasmussen and Painter (1964) and Pettijohn and Hanawalt (1964) who showed that a nonsemiconservative form of DNA synthesis, referred to as unscheduled DNA synthesis (UDS) or repair replication, occurred immediately after exposure of cells to UV radiation. These experiments were first performed in bacteria, then repeated in mammalian cells (Rasmussen and Painter, 1966; Regan et al., 1968), including human, and led to the demonstration that cells of cancer-prone individuals with XP were the apparent human counterparts of the UV-sensitive E. coli mutants (Cleaver, 1968). The importance of the DNA repair system was further emphasized by the demonstration that alkylating agents caused the onset of DNA repair synthesis when applied to human (HeLa) cells in culture (Roberts et al., 1968). Subsequently it became evident that the DNA repair system could effect the removal of many types of modified bases in addition to pyrimidine dimers and that DNA repair constituted ageneral defense mechanism against a wide variety of potentially mutagenic and/or carcinogenic DNA modifications. The first step of the DNA excision - repair system is endonucleolytic hydrolysis of the DNA backbone at or near the modified site (Setlow and Carrier, 1964). The capacity of the repair enzymes to “recognize” and thereby effect removal of modified bases determines whether such modifications persist in DNA to exert their effect on genomic function or whether they are removed and the integrity of the genome restored (Setlow, 1978;Cerutti, 1978). This is the aspect of excision- repair that this article will address.

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II. Background

The original concept that repair endonucleaseswere monomeric proteins with relatively broad recognition capacity acting on many substrates(Grossman et al., 1975)has been modified as a result of the identification of novel repair enzymes with limited substrate range (Lindahl, 1976). These recently characterized repair enzymes are termed N-glycosylases. They effect hydrolysis of N-glycosylbonds between specificmodified bases or base residues and the deoxyribose moiety. This hydrolytic action liberates the altered bases or base residues from the intact DNA backbone leaving abasic sites. These abasic sites, either apurinic or apyrimidinic (AP), are subject to attack at either the 3’ or 5’ phosphodiester bond by endonucleasestermed AP endonucleases. Hydrolysis of the phosphodiester bond is then followed by controlled exonucleolyticdegradation of a portion of the DNA adjacent to the AP site, resynthesis, and ligation of the resynthesizedpart with the remaining DNA. Repair of damage caused by alkylating agents, now known to be primarily mediated by concerted N-glycosylaselAP endonuclease action, is ofthe so-called “short patch” type and is associated with the insertion of three to four nucleotides for each repaired base (Regan and Setlow, 1973). The defect in XP, originally thought to reside in a monomeric repair endonuclease, probably lies in a more complex endonucleolytic system which may recognize conformational changes in the DNA backbone rather than the specific chemical modification of the DNA bases which are recognized by the N-glycosylases. In E. coli, the endonucleolyticsystem consists of three proteins coded for by the uvrA, B, and Cgenes (Seeberg, 1981). In XP, seven distinct complementation groups have so far been identified indicating that in human cells, even more proteins may participate in the endonucleolytic process (Robbinset al., 1974;Bootsma, 1978;Takebe et al., 1978).This endonucleolytic repair system seems to be the one which initiates the removal of pyrimidine dimers from the DNA of mammalian cells and most lower organisms. Its recognition capacity extendsto DNA adducts formed by the interaction of relatively large molecules such as N-acetyl-2-aminofluorene (AAF) and benzo[a]pyrene (B[a]P) with DNA bases. The repair ofthese adducts and of pyrimidine dimers is associated with the insertion of 35 - 100 nucleotides for each excised adduct. This type of repair has been called “long patch” repair (Regan and Setlow, 1973). Why repair initiated by N-glycosylaseis “short patch” and that initiated by the complex endonucleolyticsystem is “long patch” is not known. Smith and Hanawalt (1978) expanding on the experiments of Tanaka et a/. (1975) introduced the T4 E. coli dimer endonucleaseactivity into XP cells and then exposed them to UV radiation. [This enzyme has since been shown to be a

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GEORGE W. TEEBOR A N D KRYSTYNA FRENKEL

composite N-glycosylase and AP endonuclease activity (Radany and Friedberg, 1980; Nakebeppu and Sekiguchi, 198 l).] Xeroderma pigmentosum cells treated in this way performed normal levels of repair synthesis even though incision was mediated by an exogenous N-glycosylaselAP endonuclease rather than by the endogenous endonucleolytic complex which is defective in XP. The length of the repaired patch of DNA in these XP cells was the same as that in normal human fibroblasts. This experiment suggests that the length of the repaired patch is not a function of the incision mechanism. Both types of endonucleolytic incision mechanisms must have evolved in response to naturally occurring modifications of DNA which were potentially or directly injurious to the host. Yet, many modifications of DNA induced by physical and chemical carcinogens are removed from cellular DNA and their removal associated with repair synthesis. This suggests that these modifications are either identical to or sufficiently similar to those modifications which are natural substrates for N-glycosylases and repair endonucleases of the cell. In the case of N-glycosylase-mediatedrepair, the modifications introduced by exogenous agents (chemicals, ionizing radiation) and recognized by these enzymes are identical (or nearly identical) to naturally occurring modifications. They include methylation of purines, and ring opening and fragmentation of both purines and pyrimidines. The modifications recognized by the complex endonucleolytic repair system include relatively large adducts formed by the interaction of activated carcinogens such as AAF and B[a]P with the DNA bases. Since it is the UV radiation-induced pyrimidine dimer which appears to be the natural substrate for the complex endonucleolytic repair system, it is thought that these “bulky” carcinogen adducts cause conformational changes in DNA similar to those effected by pyrimidine dimers. It is possible that B[a]P represents a natural substrate as well since polycyclic aromatic hydrocarbons (PAHs) are universal products of the combustion of organic matter particularly under conditions of relative oxygen deficiency (Baum, 1978). There are three other mechanisms for the restoration of genomic integrity. AP endonucleases, acting alone, can incise the phosphodiester bond at any abasic site resulting from the loss of purines or pyrimidines from the DNA backbone (Lindahl, 1979). It has been estimated that the spontaneous hydrolysis of purines is an event of significant enough frequency to account for the presence of an enzymatic mechanism to effect repair of abasic sites. Since the chemical modification of bases in DNA frequently renders the glycosylic bonds unstable, base loss is a relatively frequent consequence of DNA modification and the AP endonucleases can effect initiation of repair of such damage. Base loss may also be repaired through the action of a protein

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which binds to apurinic sites and is able to effect insertion of G and A into apurinic sites of high-molecular-weight DNA. Thus, repair of apurinic sites may be effected without endonucleolytic action (Deutsch and Linn, 1979; Livneh et al., 1979). However, pyrimidines are not reinserted into apyrimidink sites. The subject of base loss and AP endonucleases has been extensively reviewed by Lindahl (1979) and more recently by Friedberg et al. (198 1) and will not be dealt with in further detail in this article. Another nonendonucleolytic repair system acts by removal of methyl and ethyl groups from the 0 6 position ofguanine and their transfer to an acceptor site on an alkyl acceptor protein which mediates this process (Samson and Cairns, 1977; Olsson and Lindahl, 1980; Mehta et al., 1981). 111. N-Glycosylases and Os-AlkylAcceptor Protein

A.

URACIL A N D HYPOXANTHINE N-GLYCOSYLASES

All N-glycosylases effect removal of damaged bases predominantly in double-stranded DNA and synthetic deoxypolymers. The sole exception is uracil N-glycosylase which acts preferentially on single-stranded substrates. In fact, the rate ofremoval of uracil from single-stranded DNA was twice that from native DNA when the E. coli enzyme was used (Lindahl et al., 1977). This may relate to the physiologic function of the enzyme. It is thought that uracil may appear in DNA as the result of the temperature-dependent hydrolytic deamination of cytosine. The actual incidence of this event in native cellular DNA is difficult to estimate but it apparently occurs at 0.3 0.5%of the rate observed with single-stranded DNA (Lindahl, 1979). Therefore, deamination of cytosine may be a significant event in cellular DNA when the DNA is replicating or when the DNA “breathes.” What evidence is there that such a hydrolytic deamination indeed takes place? The most convincing data derive from bacterial genetics in which mutational “hot spots” have been identified to contain a high content of 5-methylcytosine. This compound deaminates at four times the rate of native dCMP (Lindahl and Nyberg, 1974). The deamination of the 5-methylcytosine moiety in DNA causes formation of the thymine moiety for which there is no corrective enzymatic mechanism available. The conversion of 5-methylcytosine into T in DNA results in point mutations and accounts for the “hot spot” nature ofthese sites (Coulondre et al., 1978).Bacterial mutants (Ung-)which lack the enzyme and therefore cannot excise uracil from their DNA have an increased rate of spontaneous transition at cytosine residues similar to the “hot spot” rate at 5-methylcytosine residues (Duncan and Miller, 1980). Thus, it is likely that the spontaneous deamination of C to U constitutes a potentially significant mutagenic event and this is why a corrective mecha-

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GEORGE W. TEEBOR AND KRYSTYNA FRENKEL

nism for its removal has developed (Lindahl, 1979). It has been demonstrated by Gupta and Sirover ( 1980) that uracil N-glycosylase activity rises significantlyin mammalian cellsjust prior to the S phase (DNA synthesis)of the cell cycle. This finding suggests that the enzyme monitors DNA for base alterations during DNA replication when the cytosine in DNA is most susceptible to hydrolyticdeamination. This enzyme is absolutely specificfor U in DNA and does not remove U from RNA. Its substrate must be a deoxyoligonucleotideat least four bases in length (Cone et al., 1977) and the rate of release is much greater if the uracil moiety is central rather than terminal (Krokan and Wittwer, 198 1). Two exogenousenvironmentalagents may contribute to the deamination of cytosine in DNA. The first is the bisulfite ion which is formed as the result of sulfur dioxide dissolving in aqueous solutions at neutral pH. The bisulfite ion reacts with cytosine to form an unstable intermediatewhich deaminates yielding uracil (Shapiro et al., 1970; Hayatsu et al., 1970). A similar type of addition-elimination reaction is caused by UV radiation in which water is added to the 5,6-ethylenic bond of cytosine and, after deamination takes place, water is removed, the ethylenicbond restored, and the transition from C to U completed (Fisherand Johns, 1976). This may be another cause of UV mutagenesis. The spontaneous deamination of cytosine in DNA occurs as the result of water addition in the absence of UV light and follows the same reaction mechanism (Shapiro, 1982). The purification of prokaryotic uracil N-glycosylase and its properties have been extensively reviewed by Lindahl (1979). The enzyme has been purified from a variety of mammalian sources with differing molecular weight estimatesfrom 18,000 to 52,000. Like the prokaryoticenzyme, it was inhibited by uracil and several uracil analogs (Taelpert-BorlC et al., 1979; Caradonnaand Cheng, 1980; Krokan and Wittwer, 198 1). The enzyme from calf thymus was also inhibited by thymine and thymidine (Taelpert-BorlCet al., 1979). The apparent K , was two orders of magnitude greater than that of the prokaryotic enzyme. The HeLa cell enzyme released uracil three times faster from single-stranded DNA than from double-stranded DNA (Krokan and Wittwer, 198 1) similarly to the E. coli enzyme. In contrast, the enzyme from calf thymus released uracil almost one order of magnitude more rapidly from a double-stranded synthetic copolymer of poly(dT-dU),, poly(dA),, than from the single-stranded oligo(dT),,.poly(dU),, (Taelpert-BorlCet al., 1979). Caradonna and Cheng ( 1980) reported that their preparation of uracil N-glycosylase isolated from human leukemic cells (MW 30,000) was homogeneous with no detectable nuclease activity. Talpaert-BorlC et al., ( 1982) reported a MW of 28,000 for the calf thymus preparation with no accompanying nuclease activity. Two reports indicate that there mav be two forms of the uracil N-glycosy-

T H E INITIATION OF DNA EXCISION- REPAIR

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lase in mammalian cells. Sirover ( 1979)identified two chromatographically distinct enzymatic activities, one of which increased after stimulation of lymphocytes with a mitogen. Anderson and Friedberg ( 1980) separated the nuclear uracil N-glycosylase activity from mitochondria1 with the latter constituting about 5% of total enzyme activity in human KB cells. No deficiency of activity of uracil N-glycosylase has been demonstrated in any mammalian cell line nor in any cells of individuals with repair-deficient syndromes (Sekiguchi et al., 1976; Kuhnlein et al., 1978). Spontaneous deamination occurs also in purines containing amino groups. The mechanism of deamination of purines has not yet been elucidated. However, it cannot be the same as in pyrimidines. Deamination of guanine yields xanthine which should not lead to altered base pairing (Lindahl et al., 1978). Deamination of adenine yields hypoxanthine, which may pair as guanine rather than adenine, thus constituting a potentially mutagenic event. It has been estimated that this deamination occurs at about 290 of the rate of deamination of cytosine (Lindahl, 1979). In fact, an N-glycosylase activity directed against hypoxanthine was identified in E. coli and in calf thymus (Lindahl et al., 1978; Karran and Lindahl, 1978, 1980). The existence of an enzyme activity directed against hypoxanthine further strengthens the hypothesis that deamination ofbases is a significant naturally occurring potentially mutagenic event in response to which repair enzymes have evolved. In crude fractions of calf thymus, the activity of hypoxanthine N-glycosylase is one-thousandth that of uracil N-glycosylase (Karran, 1981).

B. 3-METHYLADENINE,7-METHYLGUANINE, A N D FORMAMIDOPYRYMIDINE N-GLYCOSYLASES A second group of glycosylase activities acts upon different types of modified bases in DNA and may be relevant to the protection of humans against carcinogens. These N-glycosylases remove methylated or ethylated purine residues from DNA and are specific for unique sites of alkylation on either G or A, e.g., 7-methylguanine (7-MeG) and 3-methyladenine (3MeA). Methylation of DNA is thought to be used by the cell for regulating gene expression and differentiation. Specific DNA methyltransferases transfer the methyl groups from S-adenosylmethionine to the 5 position of cytosine and the N6 position of adenine. In contrast, chemical alkylating agents react primarily with the N7 position of G and the N3 position of A which are the most chemically reactive sites of these heterocyclic bases (Lawley, 1966). Lindahl et al. ( 1982)have proposed that S-adenosylmethionine, acting as an endogenous alkylating agent in the absence of methyltransferase control, could alkylate adenine and guanine in DNA at a level of 3000 methylated

30

GEORGE W. TEEBOR AND KRYSTYNA FRENKEL

purine residues per day in the genome of a mammalian cell. Thus, these events may be of a mutagenic significance equal to deamination reactions and may have led to the evolution ofN-glycosylase activitiesdirected against these modified bases. Recent reports described a particular cause of aberrant methylation in mammalian systems which may be of relevance to carcinogenesis. The administration of toxic doses of hydrazine to rats resulted in the appearance of 7-MeG and @-methylguanine (WMeG) in the DNA of rat hepatocytes (Barrows and Shank, 1981; Becker et al., 1981). Aberrant methylation also occurred after administration of ethanol and carbon tetrachloride. The N-glycosylase directed against 3-MeA was isolated from human lymphoblasts, partially purified, and characterized by Brent (1979). The activity has been identified in rat and hamster liver by Margison and Pegg (198 1). The enzyme from human lymphoblasts characterized by Brent (1979) had a MW of 34,000 with a pH optimum of 7.5-8.5. An enzyme activity from mouse L cells has been purified 800-foldwith an apparent MW of 27,000 and additional enzyme activity at 47,000 MW and 68,000 MW which could reflect either aggregation of a monomeric species or more than one enzyme activity for the release of 3-MeA. This activity was inhibited 60% by the product 3-MeA (Male et al., 1981). An activity which released both 3-MeA and 7-MeG was partially purified from rat liver nuclei with an apparent MW of 24,000 (Cathcart and Goldthwait, 1981). All of these enzymes released 3-MeA more rapidly from double-stranded alkylated DNA than from single-stranded substrate DNA. The enzyme purified from E. coli had a MW of 20,000, similar pH range, and no obligatory cofactor requirements (Riazuddin and Lindahl, 1978). The removal of 7-MeG is also mediated by N-glycosylase activity and has been identified in both E. coli and Micrococcus luteus (Laval et al., 1981), human lymphoblasts (Singer and Brent, 198l), and rodent liver extracts (Margison and Pegg, 1981). This activity has not yet been well characterized but apparently it is inducible (see below). The enzymes removing 3-MeA and 7-MeG also remove the ethylated bases 3-ethyladenine (3-EtA) and 7-ethylguanine (7-EtG) (Singer and Brent, 1981). An interesting N-glycosylase activity has been identified in bacteria by Chetsanga and Lindahl(l979) and in rat and hamster liver by Margison and Peg%( 1981). This activity has been named formamidopyrimidine (FAPY) N-glycosylase. The enzyme has been partially purified from E. coli (Chetsanga et al., 1981) and, like other N-glycosylases, it has a relatively low molecular weight (30,000), does not have obligatory cofactor requirements, and acts more effectively on double-stranded than on single-stranded DNA as substrate. This enzyme removes guanine and adenine moieties in which the imidazole ring is open. The imidazole ring of adenine or adenosine is

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readily cleaved at alkaline pH because the C8 position is an electrophilic center attracting hydroxyl ions. In addition to alkali, radiogenically induced hydroxyl radicals facilitate opening of the imidazole ring of the adenine moiety yielding the C6 amino-substituted pyrimidine derivative (van Hemmen and Bleichrodt, 1971). The mechanism of opening of the imidazole ring of guanine is different. It does not open at alkaline pH because such conditions cause a removal ofthe N I proton and thus render the C8 position less electrophilic than at neutral pH (Jones et al., 1966; Shapiro, 1969). However, when the N7 position of guanine is alkylated, the electrophilicity of the C8 position markedly increases,which in turn facilitatesopening ofthe imidazole ring. The imidazole rings of 7-alkylguanines or guanosines open even at near neutral pH. However, in the case ofthe 7-alkyldeoxyguanosines, not ring opening but glycosyl bond cleavage predominates below pH 8.5 (Haines et a!., 1962; Kriek and Emmelot, 1963, 1964; Brookes and Lawley, 1963; Brookes et al., 1968). It should be pointed out the ring opening of the alkylated residues at neutral pH has not been demonstrated in DNA in vitro (Kriek and Emmelot, 1964). Yet the presence of this unusual N-glycosylase activity suggests that whatever the mechanism, imidazole ring opening of purines in cellular DNA must be an event of significant frequency. C. THYMINE GLYCOL,UREA,AND PYRIMIDINE DIMERN-GLYCOSYLASES Ionizing radiation is mutagenic and carcinogenic. It exerts these actions through the formation of free radicals and solvated electrons, which in turn may react with any portion of the DNA backbone and the DNA bases. It has been suggested that base modification may be responsible for the long-term mutagenic and carcinogenic effects of radiation (Little, 1978). In Section III,B it was pointed out that free radical attack on adenine may lead to imidazole ring opening and that this modified base is recognized and removed by FAPY-N-glycosylase. Pyrimidine modification in DNA is quantitatively even more significant than is the modification of purines. Attack on the 5,6-ethylenic bond of thymine in DNA leads to the formation of 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), which is the end stable product of hydroperoxide intermediates (Scholes, 1976). This compound has been identified as the most readily formed product of ionizing radiation in solutions of irradiated DNA (Teoule et al., 1974, 1977). It has also been unambiguously identified by high-pressure liquid chromatography (HPLC) analysis in irradiated cellular DNA together with other thymine derivatives (Frenkei el al., 198I b). Some of these other derivatives are probably formed through the oxidative ring opening of thymine glycol, yielding among others N’-formyl-N-pyruvylurea (FPU). This compound cyclizes quite easily with the elimination of the C6 atom and the formation of 5-hydroxy-5-methylhy-

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GEORGE W. TEEBOR AND KRYSTYNA F'RENKEL

dantoin. The relative occurrence and stability of these compounds in irradiated cellular DNA are under investigation (Teebor et al., 1982a,b Frenkel and Teebor, unpublished results). The mechanism of formation of these compounds has been described in solutions of irradiated thymine, thymidine, and DNA (Teoule et al., 1970, 1974, 1977; Cadet and Teoule, 1975).That some or all of these compounds are enzymatically removed from cellular DNA was demonstrated both in bacteria and mammalian cells (Hariharan and Cerutti, 1971, 1972; Painter and Young, 1972; Mattern et al., 1973; Mattern and Welch, 1979). Through the use of an assay which measured all types of ring-saturated thymines, it was shown that such modified thymines were rapidly released from irradiated cellular DNA. These modified thymines appeared in the acid-soluble fraction where the ratio of modified to unmodified thymines was high, indicating that this was not the result of nonspecificdegradation of high-molecular-weight DNA but selective enzyme-mediated removal (Hariharan and Cerutti, 1974). It is also possible that modified thymines were released from the backbone of irradiated DNA by nonenzymatic hydrolytic processes (Ward and Kuo, 1976; Dunlap and Cerutti, 1975). However, it was shown that cells of individuals with Fanconi's anemia (FA) did not release modified thymines from their DNA as well as did control fibroblasts (Remsen and Cerutti, 1976). If the main cause of base release from irradiated DNA was nonenzymatically mediated hydrolysis, then there should be no apparent difference between the cell lines. It has been shown that DNA exposed to ionizing radiation in vitro became susceptible to attack by a variety of endonuclease activities from both bacterial and mammalian cell preparations (Paterson and Setlow, 1972; Brent, 1973; Bacchetti and Benne, 1975; Arne1 et al., 1977; Gates and Linn, 1977; Schafer et al., 1980). The substrate for these enzymes was presumed to be a modification in the DNA introduced by the radiation. That the substrate might be thymine glycol was suggested by the finding that DNA oxidized by OsO, and KMnO,, agents which introduce thymine glycol into DNA (Burton and Riley, 1966; Beer et al., 1966;Iida and Hayatsu, 197 l; Frenkel et al., 1981a,b), was also susceptible to attack by endonuclease activities from both mammalian and bacterial cells (Gates and Linn, 1977; Hariharan and Cerutti, 1974, 1977;Nes and Nissen-Meyer, 1978).Demple and Linn ( 1980) reported that indeed there was an N-glycosylase activity in E. coli endonuclease 111 which specifically released thymine glycol from DNA which had been oxidized with OsO,. An additional evolutionary rationale for the existence of such an activity stems from the report by Hariharan and Cerutti (1977) that UV irradiation of HeLa cells also introduced thymine glycol into cellular DNA. They particularly emphasized that this was not a photohydrate and showed that the formation of the glycol at wavelengths which approached those of solar

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radiation was of the same order of magnitude as the formation of cyclobutane pyrimidine dimers. They suggested that these derivatives might be as important to the etiology of actinic skin cancer as the dimer. When DNA is heavily irradiated at 254 nm, it becomes susceptible to attack by endonuclease activities both in mammalian cells and bacteria which are not directed against pyrimidine dimers. It has been suggestedthat these activities are directed against thymine glycol or a related compound introduced into DNA by the high dose of 254 nm radiation (Duker and Teebor, 1975; Gates and Linn, 1977; Teebor et al., 1978; Hecht and Thielmann, 1978). Of considerable interest is the report by Breimer and Lindahl (1980) detailing the identification of an N-glycosylase activity directed against the urea residue in DNA. Urea was introduced into a poly(dA-dT) copolymer by prolonged oxidation with KMnO,. Thymine glycol is formed first, followed by ring opening and then fragmentation leaving the N1 -C2-N3 moiety (urea) attached to deoxyribose as the stable end product. In theory, this residue could be formed by any oxidative attack on thymine. The activity directed against urea has been identified in E. coli but not in mammalian cells as yet. Thus there exist in E. coli two distinct N-glycosylase activities which act on ionizing radiation-induced modifications and their counterparts may be the endonuclase activities of mammalian cells which act on ?-irradiated, chemically oxidized, and heavily UV-irradiated DNA. The repair endonuclease activity of M. luteus directed against pyrimidine dimers has been shown to be a composite N-glycosylaselAP endonuclease activity (Haseltine et al., 1980).This has also been shown to be the case for the T4 pyrimidine dimer endonuclease (Friedberg et al., I98 I). The enzyme first catalyzes the hydrolysis of the 5’-glycosyl bond of the pyrimidine dimer which yields an apyrimidinic site, which in turn is subject to attack by an AP endonuclease. Studies using the T4 phage indicate that there is an actual physical association between the glycosylase activity and the AP endonuclease activity and that these activities cannot be separated (Nakabeppu and Sekiguchi, 1981). Whether this will prove to be a characteristic of all N-glycosylases is not yet certain. Most purifications have not succeeded in separating AP endonuclease activity from the accompanying N-glycosylase action and where there has been a report of no endonuclease activity it may be a function of the sensitivity or conditions of the assay. D. INDUCIBILITYOF N-GLYCOSYLASES AND @-ALKYL ACCEPTOR PROTEIN The physiologic control of DNA repair has been studied by Gupta and Sirover (1980, 1981a,b). They found that uracil N-glycosylase activity in creased fivefold during cell proliferation preceding the induction of both

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GEORGE W. TEEBOR AND KRYSTYNA FRENKEL

DNA polymerase activity and the incorporation of precursor nucleotides into cellular DNA. Using methylmethane sulfonate(MMS)to alkylateDNA and bisulfite to deaminate deoxycytosine to deoxyuridine, they measured UDS and repair replication and found that UDS and repair replication were highestjust prior to the onset of DNA replication (Gupta and Sirover, 1980). This work was extended to the study of repair of the damage induced by either UV radiation or N-acetoxy-AAF in both normal and repair-deficient XP (group D) cells. Both UDS and repair replication were maximal just prior to DNA replication in control WI-38 cells in response to both agents. However, in XP cells, repair replication and UDS rose only in response to MMS (Gupta and Sirover, 1981a). Nuclear uracil N-glycosylase activity increased during the replicative phase of the cell cycle while mitochondria1 uracil N-glycosylaseactivity remained constant (Gupta and Sirover, 1981b). Finally, it was shown that both 3-MeA N-glycosylase and uracil N-glycosylase activities increased two- to threefold prior to the replication of hepatocytes in regenerating rat liver and returned to basal levels within 48 hr after partial hepatectomy (Gombar et al., 1981). An interesting selective removal of adducts during the cell cycle was demonstrated by Smith et al. (198 1) who used proliferating mouse 10T1/2 cells. The application of N-methyl-N-nitro-N-nitrosoguanidine (MNNG)to these cells resulted in the formation of several DNA adducts including 3-MeA, 7-MeG, and 06-MeG. 3-Methyladenine was rapidly removed from high-molecular-weightDNA during both GI and S phases of the cell cycle. 7-Methylguanineand 06-MeG were removed duringGl but not during Sand were further removed during the succeeding GI period. This explained, in part, earlier observationsby this group (Grisham et al., 1980) that cytotoxicity and transformation frequency decreased during G I , reached a maximum during early S, decreased during the next G I ,and increased during the second S when MNNG was applied during different phases of the cell cycle. This selective removal of alkyl adducts may be correlated with data obtained from the study of bacteria. In E. coli, the main 3-MeA N-glycosylase activity has been found to be constitutive and 3-MeA residuesrapidly disappear from cellular DNA. In contrast, the removal of 7-MeG is sluggish. The removal of 7-MeG is mediated through the action of an N-glycosylase activity which is inducible through exposure to low levels of alkylating agent. The basal levels of this N-glycosylase are low. This enzyme also has activity against 3-MeA and carboxylated purines (Thomas et al., 1982; Lindahl et al., 1982). In mammalian cells, it may be that the main 3-MeA N-glycosylase activity is also constituitive and present in amounts sufficient to remove 3-MeA residues throughout the cell cycle. Although the enzymatic removal of 7-MeG has been described in mammalian cells (see above), the low basal

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levelsof 7-MeGN-glycosylase activity may drop further during S or it may be that this enzyme is not synthesized at all during DNA synthesis, and, therefore, 7-MeG residues cannot be effectively removed. In E. culi, the removal of 06-MeG is not mediated through the action ofan N-glycosylase but through the action of a methyl acceptor protein (MW 17,000)(Olsson and Lindahl, 1980)which binds the methyl ofthe Q-MeG to an acceptor sulfhydrylgroup on a cysteine residue. This binding saturates the protein and renders it inactive. Thus the capacity ofa cell to remove 06-MeG is a function of the number of acceptor protein molecules available. This activity is inducible in E. culi by prior exposure to low levels of alkylating agents (Samson and Cairns, 1977; Olsson and Lindahl, 1980). A similar mechanism for the removal of 06-EtG has been described in rat liver chromatin (Mehta et al., 1981). In nonadapted mammalian cells, such as those studied by Grisham et al. (1980), the small amounts of protein being synthesized at basal levels may have been consumed during G, ,not synthesized during S, and then resynthesized again during the next G, period. The inducibility of this alkyl acceptor protein activity was demonstrated by Montesano tcl al. (1980) by pretreating rats with nonradioactive N-dimethylN-nitrosamine (DMN) at different dosages and times and the determination of the formation and disappearance of radioactive 3-MeA, 7-MeG, and 06-MeG in rat liver DNA after administration of radioactively labeled DMN. Pretreatment with DMN lowered the level of 06-MeG but not 3-MeA or 7-MeG. Extracts of livers of pretreated rats were also able to effect removal of G.S-MeG more rapidly than extracts of livers from untreated rats. The inducibility of the alkyl acceptor protein was limited to liver. Lung and kidney activities did not differ from those of nonpretreated rats. This result is consistent with the demonstration by others (Goth and Rajewsky, 1974; Kleihues and Margison, 1974;Nicoll et al., 1975;Margison et al., 1976;Cox and Irving, 1977; Kleihues and Bucheler, 1977; Frei el al., 1978) that the formation of @-MeG varies in different organs of animals exposed to alkylating agents and that the levels of 06-MeG correlate with the incidence of tumors. The more 06-MeG is formed in the DNA of cells of a particular organ, the higher the incidence of cancer in that organ. Medcalf and Lawley ( 1981) followed the time course of the removal of 3-MeA, 7-MeG, and 06-MeG in human fibroblasts in culture including XP cells and ataxia telangectasia (AT) cells. Both XP cells and AT cells showed normal rates of removal of all three adducts which differed from an earlier report indicating XP cells to be defective in 06-MeG removal (Goth-Goldstein, 1977). Medcalf and Lawley (198 1) suggested that this difference might be due to the fact that the earlier observation was made in virally transformed cells and perhaps was an acquired defect. Day et al. (1980) had shown that

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GEORGE W. TEEBOR AND KRYSTYNA FRENKEL

cells capable of supporting the growth of MNNG-treated adenovirus 5 often lost this capacity when transformed with simian virus 40 (SV40). These phenotypes were referred to as Mer+and Mer- and were assoCiated with the capacity ofthe cells to remove (Mer+)or not to remove (Mer-) 06-MeG from their cellular DNA. The removal of 06-MeG was found to be nonlinear in human fibroblasts, showing a much more rapid rate at low levels of alkylation. This was consistent with the mechanism of a “quasi-enzyme” which was consumed during the reaction. In contrast to the report of apparent inducibilityof rat liver 06-MeG removal (Montesano et al.. 1980),inducibility was not demonstrated in cells in culture. IV. Repair of UV-Induced Pyrimidine Dimers

It is generally thought that the removal of pyrimidine dimers from the DNA of UV-irradiatedcells is initiated by an endonucleolyticactivitywhich, in E. coli, consists of the three products of the uvr genes, A, B, and C, and which is probably even more complex in mammalian cells. The E. culigene productshave a combined molecular weight of 250,000 and require ATP for endonucleolytic activity (for details, see Seeberg, 198I). Lindahl (1979) offered a rationale for the existence of such a complex enzymatic activity by pointing out that it was more “versatile” than N-glycosylase-mediatedrepair since, in addition to dimers, the enzymecould initiatethe removal ofadducts from cellular DNA resulting from the reaction of the DNA bases with carcinogens such as B[a]P, N-acetyl-2-aminofluorene (AAF), 7-bromomethylbenz[a]anthracene (7-BMBA), 4-nitroquinoline 1-oxide (4NQO), and crosslinking agents. It is thought that the endonucleolytic complex recognizes helical distortions in the DNA caused by the dimer and/or the adducts rather than the chemical structure of the adducts themselves (Murray, 1979). The most convincing argument for the broad substrate range of this endonucleolytic activity stems from experiments which have measured repair replication and removal of adducts from the DNA of wild-type E. coli in comparison to uvr mutants, and normal human fibroblastsin comparison to excision-defectiveXP fibroblasts. Repair of adducts caused by the aforementioned carcinogens is reduced or absent in uvr mutants and XP cells. Furthermore, in normal cells, the repair of modifications caused by these carcinogens is of the “long patch” type and is protracted, over a period of up to 24 hr, just like the repair of pyrimidine dimers. Thus, these results suggest that both the uvr gene products and the human enzyme activity which is defective in XP participate in the initiation step of the repair of a heterogeneous group of modifications ofwhich the pyrimidinedimer is the prototype.

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The complexity of the endonucleolytic system in human cells (reviewed by Friedberg el al., 1979)is manifest by the existence ofseven complementation groups within the clinical syndrome of XP, all showing differing degrees of repair capacity as measured in cell culture. Levels of repair synthesis range from 0 to 60%of normal after UV radiation. All XP cell lines studied have shown increased sensitivity to the killing effects of UV as well as increased mutagenesis but it has not always been possible to correlate the severity ofthe apparent defect in repair synthesis with the other two parameters. Furthermore, within each complementation group there is sufficient variation in repair capacity from individual to individual to preclude assigning an individual case to a complementation group on the basis of levels of repair synthesis after UV damage. No endonucleolytic activity analogous to the uvrABCendonuclease has been reconstituted from mammalian cell extracts. A low-molecular-weight DNA endonuclease activity directed against UV-irradiated double-stranded DNA has been described in human and monkey tissues (Tomura and van Lancker, 1980) with characteristics similar to a rodent enzyme previously described by this group. These enzyme activities had a low molecular weight and were also active against AAF-modified DNA. Such activities are perhaps analogous to the M. luteus and T4-phagedirected endonuclease activities which have been shown to be a composite of an N-glycosylase and an AP endonuclease. If it is true that the enzyme complex indeed recognizes helical distortion and that the pyrimidine dimer is the prototype substrate, it is appropriate to examine the nature of the distortion caused by the dimer and describe, in subsequent sections, conformational changes induced by AAF adducts and B[a]P adducts in DNA and correlate these with the reparability of these modifications. The effect of thymine dimers on the melting temperature ofUV-irradiated oligo(dT-dA) was examined by Hayes et al. (1971). The results led to the conclusion that each dimer caused the rupture of4.3 base pairs. Two ofthese were the bonds of the dimer itself and the other two the adjacent base pairs, An additional 15% weakening of the second distal pair was also found. Broyde et a/. ( 1980)proposed a model for the conformation of the cis-syn pyrimidine dimer based on minimum energy computations. In order to evaluate how this dimer might alter the normal B-form of DNA, the computed minimum-energy conformation was incorporated into a model of single-stranded B-DNA. The results of these calculations led to the conclusion that the dimer causes a severe bend in the helix similar to the kink described by Crick and H u g (1975). In this model (Broyde et al., 1980), rupture ofadjacent base pairs is not a prerequisite; therefore, in principle, the severe bend can be formed without significant denaturation of the helix

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GEORGE W. TEEBOR A N D KRYSTYNA FRENKEL

beyond the lesion. This model is consistent with data that indicate that dimers act as blocks to replicative DNA synthesis (Lehmann, 1979; Moore and Strauss, 1979). V. Repair of Modifications of DNA Caused by Polycyclic Aromatic Hydrocarbons

The mechanism of repair of cellular DNA modified by PAHs is not as well understood as the repair of alkylating agent-induced damage, which is apparently primarily mediated through the action of N-glycosylases and 06-alkyl acceptor protein. At best, there are data pertaining to the number of PAH adducts which persist in DNA for differing time periods. One of the difficulties encountered in studying repair of PAH-modified DNA is that PAH(s) are metabolized by cells to many active intermediates capable of interacting with different bases ofDNA and thus form many types ofadducts which may or may not be substrates for repair enzymes. As a class, PAH(s) have one feature in common-the absence of functional groups which precludes their direct chemical interaction with cellular macromolecules. However, they are metabolized by cells to a number of reactive intermediates. Among the metabolites identified are K- and non-Kregion epoxides and diolepoxides, phenols, dihydrodiols, phenoxyl radicals, and PAH(s) with methyl groups oxidized to various degrees (Sims, 1970; Heidelberger, 1973;Grandjean and Cavalieri, 1974;Gentil et al., 1974;Yang and Dower, 1975; Selkirk et al., 1976; Selkirk, 1977). Metabolized PAH(s) covalently bound to nucleic acids have been identified in both animal tissues and cells in culture (Heidelberger, 1973). Some of the DNA adducts were released from cellular DNA spontaneously or through enzymatic action. Others remained bound to DNA for relatively long periods of time. As with alkylating agents, the formation and persistence of some of the PAH adducts in cellular DNA are well correlated with carcinogenicity. The first indications of the possible deleterious effects of a compound are cytotoxicity and mutagenicity. The relationships of these properties to DNA repair have been studied by comparison of the effects of the chemical on normal human cells and on repair-deficienthuman cell lines such as XP, FA, and AT (Setlow, 1978). It was found (Maher et al.. 1977) that the cytotoxic effects of the K-region epoxides of B(a]P, benz[a]anthracene, (BA), dibenz[a]anthracene (DBA), and 7,12-dimethylbenz[a]anthracene (DMBA) and of 7-bromomethyl derivatives of MBA and DMBA were two to three times higher on XP2BE cells than on normal human skin fibroblasts. All of these compounds exhibited the greatest cytotoxic effect on the XP12BE strain cells, which are known to perform less than 1% of normal cell repair of UV damage. Similarly, Heflich et al.(1977) showed that 7-BMBA, B[a]P 4,5-epoxide, UV (254 nm), and benzo[a]pyrene diolepoxide (BPDE) I and I1

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39

were much more cytotoxic for two XP strains studied than for normal cells. Xeroderma pigmentosum cells were also two to three times more susceptible to mutations induced by the K-region epoxides of B[a]P, DBA, and DMBA, as measured by the frequency of induced mutations to 8-azaguanine resistance (Maher et al., 1977). Xeroderma pigmentosum lymphocytes treated with 7-BMBA showed deficiency in UDS (Slor, 1973). The loss of cloning ability was greatest in the most repair-deficient XP strain and intermediate in the XP strain which demonstrated only a moderate deficiency of repair. The same XP strains were not affected by MNNG or X irradiation (Cleaver, 1971a,b; Heflich et af., 1977) indicating that the repair deficiency was manifest only in response to damage by specific agents. The binding to DNA and subsequent removal of 7-BMBA adducts was extensively studied by Dipple et al. ( 197l), Lieberman and Dipple ( 1972), and Dipple and Roberts (1977). When nondividing human lymphocytes were treated with this compound, two types of adducts were formed (Lieberman and Dipple, 1972). The major one was the product of the interaction with the amino group of guanine and the minor one with the amino group of adenine. The removal of these adducts from nondividing cells was correlated with UDS. Thirty percent of the adenine adducts and 15% of guanine adducts were removed from DNA while UDS was completed within 12 hr. Since both adducts were stable in DNA, the differential removal of the adenine and guanine adducts suggested that it was enzymatically mediated. Other cell lines studied (replicating HeLa S3 and Chinese Hamster V-79) showed that 7-BMBA adducts of adenine were excised more rapidly than those of guanine (Dipple and Roberts, 1977) as in the DNA of human lymphocytes (Lieberman and Dipple, 1972).The half-life ofguanine adducts in DNA was about twice that ofadenine in V-79 cells and about fourfold that in HeLa S3. Another adduct, possibly a cytosine derivative, was also partially removed. The percentage of total products excised decreased with increasing concentration of 7-BMBA and finally ceased when toxicity supervened. When low concentrations of this carcinogen were used, replication of V-79 cells was not affected although 50% of the initial damage was present. HeLa cells were two- to threefold more sensitiveto the toxic effect of 7-BMBA than were V-79 cells. Since human fibroblasts, both normal and XP, cannot metabolize PAHs(s) (Maher et af., 1977), XP cells were cocultivated (Aust et al., 1980) with lethally X-irradiated epithelial cell lines which retained the capacity to metabolize B[a]P. The B[a]P adducts formed in the DNA of XP cells were the same as those identified after treating XP cells with BPDE I and 11, the active intermediates of B[a]P metabolism. The validity of this technique was corroborated by the demonstration that the frequency of 6-thioguanine-resistant XP colonies increased with increasing concentrations of B[a]P which

40

GEORGE W. TEEBOR A N D KRYSTYNA FRENKEL

caused the formation of an increased number of B[a]P adducts in the DNA of the cocultivated XP cells. Many experiments indicate that BPDE is repaired by the cellular excision-repair system. Damage to DNA induced by BPDE was repaired in normal human skin fibroblastsby a long-patch mechanism (50-55 nucleotides per repaired region) (Regan et al., 1978).However, XP cells, which were deficient in excision of UV damage, were not able to repair BPDE-induced damage. Xeroderma pigmentosum variant cells known to be proficient in excision - repair of UV damage excised BPDE adducts as well as did normal cells. Day et al. (1978) demonstrated that XPl2BE cells were deficient in the excision of BPDE adducts and in the reactivation of adenovirus 5 which had been exposed to BPDE prior to infection. The survival of BPDE I-treated adenovirus 5 was 30 times greater when infecting normal cells as compared to XPl2BE. These results were similar to those obtained with UV-irradiated adenovirus. One BPDE moiety bound to each viral genome correlated well with one lethal hit, as determined by use of these XP cells. These XPl2BE fibroblasts(complementation group A) were totally deficient in the repair of BPDE-damaged adenovirus 5 . Normal cells, proficient in dimer excision, showed a linear increase in repair synthesis with an increase in UV dose or concentration of BPDE I. The variant XP 12BE(complementation group A) showed very little repair synthesisafter treatment with either UV or BPDE I, while XP5BE (group D) showed intermediate levels of repair synthesis in response to UV or BPDE I. When human cells were treated with BPDE I, normal cells removed 85% of adducts from their DNA while XP12BE (group A) removed only 2OYo. These results suggested that the removal of BPDE I adducts was enzymatically mediated and proceeded through a similar, if not identical, enzymatic pathway, which controls the removal of UV-induced pyrimidine dimers. An interesting correlation between repair of B[a]P-induced damage and carcinogenicity was made by Feldman et al. (1978) and Cerutti et al. (1978) studying the excisability and repair of B[a]P adducts in human epithelioid lung cells. These cells were of interest, since they were considered as representative of target cells for PAH(s) which were carcinogenic to human lung. A549 alveolar tumor cells showed persistence of dG-BPDE I and dG-BPDE I1 adducts over several generations. The total amount of DNA synthesized within a 30-min period after treatment ofthese cells with BPDE I or I1 decreased to 10- 25% of untreated controls. Parental DNA strands were fragmented within 3 hr after treatment and slowly elongated until they reached the length of control cells at 30 hr of incubation. Treatment with BPDE caused formation of short daughter strands within the first 3 hr which reached control levels within 15 hr. During the 30-hr posttreatment incubation, only 30% of dG-BPDE I and 50% of dG-BPDE I1 were excised by the

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cells. The authors concluded that this sluggish removal of BPDE I and I1 adducts may be related to the ultimate carcinogenic transformation of human epithelial lung cells. To show the progression of the excision-repair process, Eastman et al. (1981) studied the removal of B[a]P adducts between 2 and 48 hr after treatment of hamster tracheal epithelial cells with this carcinogen. They found that the formation of adducts was rapid reaching a maximum at 8 hr. Removal of adducts was biphasic with a rapid excision between 4 and 8 hr followed by a slow excision, with 50Yo of the adducts still remaining after 48 hr. Analysis by HPLC ofB[a]P-DNA adducts revealed that B[a]P-adenine adducts were rapidly removed within 20 hr, while others remained practically unchanged. These results were similar to the finding that 7-BMBA adenine residues disappeared four times as rapidly from high-molecularweight DNA as did guanine adducts (Dipple and Roberts, 1977). There is strong evidence that the major mutagenic and carcinogenic metabolites of B[a]P are the bay region diastereomeric 9,lO-epoxides of the (Sims et enantiomeric (+) trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene a/., 1974; Huberman et al., 1976; Thakker et al., 1976; Wood et a/., 1976; Yang er al., 1976; Moore er al., 1977; Yagi er al., 1977a,b).It was found that among these four derivatives of B[a]P there is a marked difference in carcinogenic activity with (+)BPDE I being, by far, the most carcinogenic and mutagenic, and (+)BPDE I1 being a distant second (Kapitulnik er al., 1978). Each diolepoxide interacts with nucleic acids both in vizro and in vivo (Daudel et al., 1975; King et al., 1976)with the formation ofa bond between the C 10 position of the diol epoxide and an amino group of the bases in DNA. These include the N2 position of guanine (Jeffrey et al., 1976, 1977; Weinstein et al., 1976; Moore et al., 1977), the N6 position of adenine, and the N4 position ofcytosine (Straub et al., 1977; Grunberger and Weinstein, 1979). All of these are stable adducts. Addition of the amino group to the diolepoxide may be either cis or trans but it is known that trans predominates (Meehan er al., 1977). It hasbeen shown that the N7 position ofguanine may also be modified by BPDE I (Osborne et al., 1978). This adduct was formed upon prolonged incubation of DNA with BPDE I and then the glycosylic bond of the N7-modified guanine residue rapidly hydrolyzed leaving an apurinic site (Gamper el al., 1980). In cellular DNA, these sites may be repaired through the action of AP endonucleases (Lindahl, 1979). Benzo[a]pyrene metabolized by human or bovine bronchial explants or by mouse skin formed the same guanine derivatives in DNA as did the diolepoxides (Moore et a/., 1977; Jeffrey et a/., 1977). The predominant guanine product was the same as that obtained in the reaction of DNA with BPDEI(Jeffrey er al., 1977;ShinoharaandCerutti,1977;Cerutti et al., 1978; Feldman et af.,1978).The major guanine derivative constituted over 90% of

42

GEORGE W. TEEBOR A N D KRYSTYNA FRENKEL

the stable adducts. Other adducts included those of adenine (5%) and possibly cytosine (3%)(Meehan et al., 1977).Since BPDE I is a mixture of(+) and (-) enantiomers, one would expect that the two trans-guanine and two trans-adenine derivatives would be formed in equal amounts. This was true when diolepoxide interacted with single-stranded DNA. However, when double-stranded DNA was used, there was a 9 : 1 predominance of the (+) adenine adduct. When a microsomal activating system was used to metabolize B[a]P, only one (+) guanine adduct was formed and no adenine derivatives were detected. This was explained, in part, by the fact that metabolic activation is stereoselective yielding only the (+) enantiomer of BPDE I (Yang et al., 1976). It has been suggested that the binding of BPDE to nucleic acids changes their native conformation. If the reaction with the diolepoxide was carried out under nondenaturing conditions, superhelicity of supercoiled DNA changed, and supercoiling relaxed to the open circular form followed by rewinding of the coil to the opposite direction (Drinkwater et al., 1978). It was proposed that BPDE first binds covalently to the N2 position of the guanine moiety in nucleic acids. Then, the guanine rotates about 45" from the anti toward the syn conformation. This opens the space allowing the pyrene moiety to slide below the guanine residue and thus facilitates a stacking interaction of BPDE with the neighboring base (Frenkel et al., 1978). This model is in agreement with the finding that in order for any intercalating agent to enter between bases in a dinucleoside monophosphate, the glycosylic bond should assume a high anti conformation (Berman et al., 1978). In deoxydinucleoside monophosphates, rotation of the guanine residue about the glycosylic bond to the high anti also permits the entry of an intercalating agent into the interior. This may occur with or without change in the pattern of sugar puckering (Sobell et al., 1977; Shieh et al., 1980). These changescause an unwinding ofthe DNA helix by 20- 30" (Drinkwater et al., 1978). Recently, Hogan et al. (198 1) proposed a model in which covalently bound BPDE I is intercalated within the helix of double-stranded DNA forming a wedge-shaped complex which bends the helix. The bound carcinogen is located within the helix rather than at its surface and is relatively inaccessible to solvent. Molecules of bound BPDE do not lie in a plane parallel to the DNA bases. Individual segments of the modified DNA are tipped 29" relative to the helix axis ofunmodified DNA. The region ofaltered DNA extends beyond the immediate site of covalent modification and involves a tipping of at least four base pairs with respect to the helix axis causing a bend in the backbone. BPDE may bind on the 3' or 5' side of guanine. In either case the wedge-shaped complex would be formed. The 3' complex is less distorted than the 5' and base pairing could be maintained. Other workers (Geacintov et al., 1978; Beland, 1978; Prusik et al., 1979)

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43

have proposed that after binding to DNA, the BPDE moiety remains outside of the helix lying in the minor groove. According to them, such a conformation may possibly cause destabilization of the helix by interfering with G-C base pairing which would lead to increased S 1 nuclease sensitivitywhich was, in fact, observed by Pulkrabek et af.(1977). Hogan et af.(198 1) showed that BPDE I binds preferentially to preexisting single-strand regions in DNA and suggested that the model of Geacintov et al. ( 1978)might have derived as the result of BPDE I binding to these regions. Therefore, it seems that there are two ways in which BPDE bound covalently to guanine in DNA may be positioned with respect to the helix, one in which the BPDE moiety is outside the helix (Geacintov et al., 1978;Beland, 1978; Prusik ef al., 1979)and one in which it is intercalated within the helix (Drinkwater et al., 1978; Frenkel et af.,1978; Hogan et af.,1981). Since cellular DNA may contain both singleand double-stranded regions simultaneously as a function of the cell cycle (Painter and Schaefer, 1969;Scudiero el al., 1975;Stewartet al., 1979),both models may be representative of events within cellular DNA. An interesting correlation of repair data with conformational studies can be made from the work of Kakefuda and Yamamoto (1978). These workers showed that BPDE binding to adenine in DNA caused localized denaturation whereas the more than 10-fold greater binding to guanine did not. No conformational model to explain this difference has been proposed but the much more rapid disappearance of BPDE-modified adenine residues from DNA described by Eastman et al. (1981) suggests that these apparent con formational differences between modified adenine residuesand modified guanine residues may influence their reparability. VI. Repair of Modifications of DNA Caused by N-Acetyl-2-aminofluotene

The covalent binding of AAF to DNA was demonstrated by Irving and Veazey (1969). The persistenceofbound carcinogen in the DNA of organs in which tumors eventually developed was described by several investigators (Szafarz and Weisburger, 1969; Irving, 1973; Kriek, 1974). N-Acetyl-2aminofluorene, its N-hydroxy metabolite (N-OH-AAF), and N-acetoxy or N-O-sulfate esters reacted with the DNA of rat liver cells in vivo and with DNA from other cells lines in vitro (Kriek, 1969, 1972; Westra et al., 1976; Yamasaki et al., 1977a,b; Andrews et al., 1979; Visser and Westra, 1981; Meerman et al., 1981; Howard et af., 1981). Three products resulting from such interactions have been identified. The two main derivatives are the adducts at the C8 position of guanine [dG(8)-AAFand dG(8)-AF], while the minor adduct is at the N2 position of guanine [dG(NZ)-AAFl(Kriek, 1972; Westra et al., 1976;Westra and Visser, 1979;Kriek and Westra, 1980;Visser and Westra, 1981). Some of these adducts were removed from cellular DNA in vivo quite

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rapidly while others remained for a long time. Maximum binding of N-OHAAF to liver DNA occurred at 16- 18 hr after a single injection (Kriek, 1972).The major adduct (go%), identified as dG(8)-AAF, disappeared from DNA with a half-life of about 7 days. The minor adduct (20%), identified subsequently by Westra et a1. (1976) as dG(N2)-AAF,remained on cellular DNA for a period of up to 8 weeks. A recent study (Visser and Westra, 1981) showed that a single ip injection of AAF to male rats caused the formation of 2 to 2.5 times more of the AF derivative than the AAF derivative. Both C8 adducts were rapidly removed during the first 2 weeks. The removal rate slowed down considerably during the following 2 weeks with 15% still present after 4 weeks. Primary rat culture hepatocytes exposed to N-OH-AAF formed dG(8)AAF as the major product (Howard et al., 1981). However, in contrast to the similar rates of removal ofboth C8 (AF and AAF) adducts by rat liver in vivo (Visser and Westra, 1981), hepatocytes in culture removed the C8-AAF adduct much more readily than the C8-AF. The enzymatic mechanism accounting for the differences in removal of these adducts has not been elucidated as yet. Recently, Tang et al. (M.-S. Tang, C. King, and M. W. Lieberman, 1982, personal communication) showed in bacterial transfection experiments that uvrC mutants of E. coli were not able to “repair” the dG(8)-AF-containing phage DNA while uvrA and uvrB mutants and wildtype E. coli were capable of effecting such repair. All three gene products (uvrA,B,C)were needed for repair of the dG(8)-AAF adducts. This was the first demonstration of a unique function of the uvrCgene in excision- repair manifest by an apparent failure to repair the C8-AF adduct. Studies using repair-deficient XP cells indicated that cells deficient in UV repair were much more susceptibleto the cytotoxic effects ofN-acetoxy-AAF and other related aromatic amides than were normal fibroblasts. Heflich et al. ( 1980)measured repair synthesis, removal of bound radioactive carcinogen from DNA, and cytotoxicity and found that one-tenth the number of AAF residues bound to the DNA of XP cells as compared to normal cells was needed to reduce survival to 37%. Similar results were obtained with two other aromatic amides. The XP cells were incapable of excision - repair of DNA damage induced by these compounds nor could they recover from the damage. It is not clear whether the removal of aromatic amide residues from the DNA of XP cells is mediated by the same enzymatic complex which removes pyrimidine dimers. Setlow and Regan (1972) had first demonstrated that XP cells were defective in the repair of N-acetoxy-AAF-induced damage. Later studies by Ahmed and Setlow (1977) and Amacher et al. (1977) indicated that different rate-limiting steps were operative in the removal of UV dimers and AAF residues. Amacher et al. (1977) found that AAF residues are

THE INITIATION OF DNA EXCISION- REPAIR

45

removed from DNA at a slower rate and less completely than dimers, and that mouse cells, which did not excise dimers, removed AAF residues from their DNA. Ahmed and Setlow ( 1977)came to their conclusion by showing that the levels of repair synthesis induced by combined doses of UV and N-acetoxy-AAF were additive even at saturating doses of both agents. However, a different result was obtained by Brown et al. ( 1979)who did not find additivity of repair synthesis of N-acetoxy-AAF and UV-induced damage and therefore concluded that the repair mechanisms were probably identical. In a more recent publication, Ahmed and Setlow (198 1) reaffirmed their finding of the additivity of N-acetoxy-AAF and UV-induced repair synthesis and suggested that differences in cell culture techniques between the two laboratories might be responsible for the discrepancies in the repair data. The relationship of the endonucleolytic defect in XP cells to these two putatively independent pathways of repair is not clear. Ahmed and Setlow ( 1 98 1) were able to demonstrate additivity of repair synthesis of 4NQ0 and N-acetoxy-AAF-induced damage in XP cells but saw inhibition of repair synthesis when UV and N-acetoxy-AAF were administered to the same XP cells. As yet, no model to explain these findings has been proposed. Of the three adducts of the guanine moiety of DNA-dG(E)-AAF, dG(8)-AF, and dG(N2)-AAF- the most extensively studied has been dG(8)-AAF. Calculations indicate that this compound can adopt at least two types of conformation (Hingerty and Broyde, 1982a) in random sequence DNA. The predominant one, about 82%, termed the fluorene-intrastrand stack model, is consistent with the insertion -denaturation model (Fuchs and Daune, 1972, 1974) and the base displacement model deduced from studying modified oligonucleotides (Grunberger et al., 1970; Levine et al., 1974; Weinstein and Grunberger, 1974; Grunberger and Weinstein, 1976). In this model (Broyde et al., 198I; Broyde and Hingerty, 1982; Hingerty and Broyde, 1982a),the plane of guanine which is in the syn rather than the anti conformation is twisted nearly perpendicular to the plane of fluorene. The fluorene moiety is stacked approximately coplanar with the neighboring base. This causes such distortion at the site of the modification that a severe change in the shape of the helix occurs. This model is in agreement with experimental findings reporting substantial base pair rupturing near the regions of the adduct (Fuchs and Daune, 1974, 1974; Yamasaki et al., 1977a,b). A suggestion was made (Hingerty and Broyde, 1982a) that it is these severely altered fluorene-intrastrand base-stacked forms which are recognized and repaired by the cellular repair systems. This hypothesis was suggested by the experimental findings of Visser and Westra (198 1) who demonstrated the rapid removal of 85% ofdG(8)-AAF from rat liver DNA in vivo while the remainder persisted. Hingerty and Broyde ( 1982a) suggested that the remaining 15% of the dG(8)-AAF adducts were in a different

46

GEORGE W. TEEBOR A N D KRYSTYNA FRENKEL

conformationwhich they call the internal fluorene model. According to their calculations, this low-energy conformer constitutes about 18% of probable conformations in random sequence DNA. In this model, the modified guanine rotates to the helix exterior in the syn conformation but is still coplanar with the neighboring base, which allows for stacking interactions between them. The plane of the fluorene is again nearly perpendicular to the plane of guanine. However, the AAF moiety is located inside the helix. Although the AAF obstructs the two base pairs of the altered dinucleoside monophosphateand probably the base pair above, this conformation causes only a gentle bend in the axis of the helix, and thus causes little distortion in the helix direction. An interesting third possible conformation preserves the coplanarity of syn-guanine and cytidine but has the Z DNA-type backbone and sugar pucker. In this conformation, fluorene is placed outside of the left-handed (Z) double helix and induces little strain or distortion (Broyde et af., 1981; Santella et af., 1981a; Broyde and Hingerty, 1982). This conformation is possible for alternating purine - pyrimidine sequences which permit the Z form. Experiments of Sage and Leng (1980) and Santella et af. (1981b) showed that the covalent binding of AAF residues to the C8 position of guanine favors the transition of the modified poly(dG-dC). poly(dG-dC) from the B form to the Z form or to a Z-like form. They also suggested that such a sequenceconformationalchange (Zor Z-like form)can occur in DNA containing alternating G-C tracts. Santella et af. (1981a,b) found that AAF-modified poly(dG-dC) poly(dG-dC) was resistant to digestion by S1 nuclease and did not interact with anticytidine antibodies, thus showingthat base pairing remained intact which is consistent with location of AAF at the exterior of the helix. It has been found that certain naturally occurring organismscontain some oftheir DNA in the Z form (Nordheimet al., 198I). These include Drosophila and NBZ mice with the lupus syndrome. It has also been suggested that it is possible for both the B and Z forms to coexist with about an 1 1 bp interface between them (Neidle, 1981). In the stretches of Z form, the C8 position of the guanine moiety is more exposed than in B DNA (Wang et al., 1979)and thus would be more prone to attack by AAF or other carcinogens (Santella et af., 198la). The second derivative, dG(8)-AF,is the major adduct formed in DNA in vivoconstitutingup to 80%ofthe C8 adduct (Irving, 1966;King and Phillips, 1969; Visser and Westra, 1981;Meerman et af.,1981). It has been proposed that in this adduct, guanosine remains in the anti conformation and is stabilizedby the intramolecularhydrogen bond between the NH group of the aminofluoreneresidue and the 5’-OH group or the 5’ phosphate group of the deoxyribose (Sage and Leng, 1980;Leng el af.,1980;Evans eta!., 1980).This is in contrast to the dG(8)-AAF adduct which has a bulky acetyl group +

THE INITIATION OF DNA EXCISION-REPAIR

47

instead of a proton attached to the N of aminofluorene forcing the guanosine to assume the syn conformation (Leng et al., 1980). Santella et al. (1980) found that in modified dApdG there is less stacking interaction between A F moieties and the neighboring base than in the case of AAF. Evans et al. ( 1980)proposed that the AF ring is attached to the guanine moiety of DNA in such a way that the hydrogen bonds with the complementary cytosine base on the other strand remain intact. Thus, AF bound to the C8 position of guanine should not significantly distort the conformation of DNA. The model of Evans et al. (1980) which places the AF moiety outside of the helix is consistent with the finding that AF-modified DNA is in fact only slightly susceptible to digestion by single-strand-specific nucleases (Santella et al., 1979; Kriek and Spelt, 1979), again in contrast to AAF-modified DNA (Fuchs, 1975; Yamasaki et al., 1977b). The computer-generated model of the third derivative [dG(N2)-AAF] which is formed in vivo shows the AAF moiety buried entirely within the minor groove (Beland, 1978). It has also been proposed that attachment of AAF causes rotation of the guanine moiety from the anti to the syn conformation in such a way that its N7 and 0 6 atoms are in the base pairing region (Kadlubar, 1980). The N7 and 0 6 of syn-guanine may then, during replication, mispair with the NI and N2 of guanine and, therefore, cause transversion mutation, i.e., insertion of guanine instead of cytosine. In summary, it has been established that the binding of AAF to the C8 position of the guanine moiety causes conformational changes (Grunberger et al., 1970, 1974; Nelson et a/., 1971; Levine et al., 1974; Grunberger and Weinstein, 1976;Hingerty and Broyde, 1982a)leadingto localized regionsof denaturation of the double-stranded DNA (Fuchs and Daune, 1974; Fuchs, 1975;Fuchs et al., 1976).It has been found that about 85% ofthe dG(8)-AAF is removed from the DNA in vivo (Visser and Westra, 1981). This coincides with a similar proportion of this adduct computed to occur in a fluorene intrastrand stack conformation where fluorene stacks with the adjacent base on the same strand (Hingerty and Broyde, 1982a). This model by giving conformational detail substantiates earlier concepts of the insertion - denaturation and base displacement models (Grunberger et a!., 1970 Fuchs and Daune, 1972, 1974; Weinstein and Grunberger, 1974; Grunberger and Weinstein, 1976). The 15% of the dG(8)-AAF adduct which is not removed from the DNA in vivo for up to 4 weeks (Visser and Westra, 1981) may correspond to the internal fluorene model (Hingerty and Broyde, 1982a) which does not cause as extensive distortion of the DNA double helix as the fluorene-intrastrand stack model. The dG(8)-AF adduct was removed from DNA in vivo at a rate similar to that of dG(8)-AAF (Visser and Westra, 1981). This is interesting since it has been shown that in AF adducts, the deoxyguanosine moiety is in the normal

48

GEORGE W. TEEBOR AND KRYSTYNA FRENKEL

anti position rather than in the syn position of the AAF adducts. This anti conformation of the deoxyguanosine moiety in dG(8)-AF does not greatly distort the DNA helix as shown by relative resistance to S 1 nuclease (Kriek and Spelt, 1979) and by physicochemical measurements such as NMR and CD(Lengetal., 1980SageandLeng, 1980;Evansetal., 1980;Santellaetal., 1980). However, even though the deoxyguanosine moiety is in the anti configuration the calculations of Hingerty and Broyde ( 1982b) indicate that a base displacement conformation is also possible. If the enzymatic removal of adducts is related to specificconformation, the fact that 15% of AF residues also persist in hepatocyte DNA, both in vivo and in tissue culture (Visser and Westra, 1981; Howard et al., 1981), may again be an indication of the existence of an additional conformation. It is interesting to note that the defect in excision of AAF-induced damage was demonstrated in XP cells after the administration of N-acetoxy-AAF (Setlow and Regan, 1972). It has since been shown that the majority of adducts (85%) formed in DNA by this compound are in the deacetylated form (AF) (Poirier et al., 1979; Kaneko and Cerutti, 1980). It is not yet known whether the defect in XP cells also includes failure to remove acetylated derivatives. The data of M. S. Tang et al. (personal communication) indicating that AF excision is related to uvrC gene function in E. coli suggest the possibility that in mammalian cells, different gene products may function in the excision of AAF and AF residues from cellular DNA. The third derivative formed in vivo,dG(N2)-AAF,was found to persist in DNA for periods ofup to 8 weeks (JSriek, 1972).Beland (1 978) suggestedthat the AAF moiety of this derivative is located in a minor groove in such a way that it does not distort the DNA helix. If, as suggestedby Kadlubar( 1980),the guanine moiety is in the syn conformation, the AAF moiety, being attached to the amino group, does not distort the DNA helix significantly either. Therefore, in either case, the AAF moiety attached to the N2 position of guanine might not be recognized and excised by DNA repair enzymes. It seems that of the three main derivatives, only AF adducts are known to retain the normal anti configuration of the deoxyguanosine moiety. It is possible that the presence of the acetyl group of AAF forces guanine into the syn position regardless of whether the AF moiety is on C8 or N2. However, distortion of the DNA helix seems to depend on the spatial orientation of the AF moiety and not of the guanine. When either AAF or AF intercalates into the helix in the base displacement models, distortion of the helix may be recognized by repair enzymes. When the conformation is of the internal fluorene type, the degree of helical distortion may not be sufficient to be a substrate for the repair enzymes. Finally, ifthe AAF adducts are present in Z or Z-like forms of DNA, such regions of DNA may not be recognized by repair enzymes at all because these regions are left handed, and therefore, adducts would persist in cellular DNA.

-

THE INITIATION OF DNA EXCISION REPAIR

49

VII. Chromatin Structure and DNA Repair

Nuclear DNA is complexed with histones to form a structure known as the nucleosome. The nucleosomal unit of human fibroblastsconsists of I92 base pairsofwhich about 145are wrappedaround inner histones(H2A, H2B, H3, H4) and are referred to as “core” DNA. The remaining 47 base pairs are in “linker” DNA (Kornberg,1977;Chambon, 1977;Felsenfeld, 1978). Several investigators have addressed themselves to the problem of determining whether the rate and extent of repair of damage caused by physical and chemical carcinogens may be influenced by chromatin structure. It was thought that “linker” DNA may be more prone to interaction with activated carcinogens because of its relatively greater accessibility, and that adducts in this DNA might also be more readily repaired. Feldman et al. (1978) found that when human epithelioid cells were treated with B[a]P for 48 hr the initial amounts of the two BPDE adducts (dG-BPDE I and dG-BPDE 11) were the same in linker and core DNA. However, treatment of the cells with BPDE I resulted in the formation of twice as many adducts in linker as in core DNA. It was suggested that the complexities of metabolic activation ofB[a]P might lead to more uniform distribution ofadducts while the relative chemical reactivity of DNA in different parts of chromatin was the determining factor for the distribution of adducts when an ultimate carcinogen was applied to the cells. When normal human fibroblasts were treated with N-acetoxy-AAF, the concentration of adducts was found to be higher in linker DNA than in core DNA (Kaneko and Cerutti, 1980). These adducts were also fourfold more efficiently removed from linker than from core DNA during the 8 hr immediately after treatment. After 24 hr, the rate of removal slowed, leaving 50%of the adduct unexcised. Of these, 65% were located in the nucleosomal “core” DNA. This study emphasized that since treatment of the cells with N-acetoxy-AAF or BPDE I yielded a uniform type of modification [dG(8)AF or dG-BPDE I], the differential removal of adducts from linker and core DNA may be a function of their accessibility to repair enzymes. The importance of accessibility of the adduct to the repair enzymes was shown in vitro by Ishiwata and Oikawa (1979) who reported that purified 3-MeA N-glycosylasewas able to effect removal of only 50%of 3-MeA residues from a chromatin preparation while the enzyme was able to remove all 3-MeA residues from naked DNA. The significance of this finding for DNA repair was first emphasized in a study which showed that extracts of XP cells from complementation group A could not perform thymine dimer excision from DNA in UV-irradiated chromatin but could effect dimer excision from purified irradiated DNA, be it from E. culi or from the XP cells themselves (Mortelmans et al., 1976). A process OY which residues in modified nucleosomal DNA become

50

GEORGE W. TEEBOR AND KRYSTYNA FRENKEL

accessible to repair enzymes was studied by utilizing relative nuclease sensitivity of nucleosomal DNA (core and linker) (Sollner-Webb et af., 1978). It was found that when human fibroblasts were exposed to UV radiation, N-acetoxy-AAF, or 7-BMBA, nucleotides which were newly incorporated during repair synthesiswere initially nuclease sensitive (Smerdon et al., 1978; Smerdon and Lieberman, 1978). This was observed during a period of 24 hr after the insult to the cells. With time, the repaired (initially nuclease sensitive) segments of DNA became relatively nuclease resistant, indicatingthat some sort of rearrangement had taken place. Even after 24 hr, this rearrangement was not entirely completed. It has been suggested that chromatin undergoes conformationalchanges during excision-repair (Lieberman et d., 1979; Oleson et af., 1979; Williams and Friedberg, 1979; Bodell and Cleaver, 1981; Lieberman, 1982). The proposed model calls for “unfolding” of the damaged region of DNA by dissociation from the inner histones. This renders the DNA nuclease sensitive. After repair is complete, the newly synthesized DNA refolds itself around histones and becomes relatively nuclease resistant. The nuclease sensitivity of newly repaired DNA was also obtained after treatment with alkylating agents. Bodell ( 1977; Bodell and Banerjee, 1979) showed that after treatment with either MMS or N-methyl-N-nitrosourea(MNU), newly repaired DNA of mouse mammary gland tissue was more sensitive to nuclear digestion than bulk DNA. Stewart et af. (1979) and Stewart (198 I ) studied differences between repair of alkylatingagents and carcinogenssuch as B[a]P and N-OH-AAF by quantitation of the amount of single-stranded regions present in DNA as a result of UDS induced by such treatment. Single-stranded regions of DNA bound to benzoylated- naphthoylated DEAE cellulose (Strauss et af., 1979; Roberts, 1980) and their amount was determined by elution with caffeine solutions. Repair intermediateswere present only for 24 hr after exposure ofthe cells to MMS, ethylmethane sulfonate (EMS), and P-propiolactone (PPL), while they were detectable for up to 3 days after exposure to B[a]P or N-OH-AAF. The latter finding is consistent with that of Smerdon et af. (1978) and Smerdon and Lieberman (1978) who detected nuclease sensitivity of repaired chromatin even at 24 hr after UV radiation and exposure to N-acetoxy-AAF or 7-BMBA. VIII. Inhibition of DNA Repair

The capacity of cells to perform DNA excision -repair can be saturated as shown by Ahmed and Setlow ( 1977, 198 I) and Brown et af. (1 979). Therefore, the simultaneous application of different compounds which are repaired by the same pathways may saturate the repair system and this may lead to an effect equivalentto saturation by a singlecompound (Brown et af., 1979). Thus, in theory, subcarcinogenicdoses of different substances which

T H E INITIATION OF DNA EXCISION-REPAIR

51

share the same repair pathway may have the same biologic effect as a carcinogenic dose of a single compound. It would be of interest to determine whether one DNA damaging agent can inhibit the removal ofdamage caused by a second agent. Such a result would increase the effectivenessof a low dose of a substance through the inhibition of its repair. Since most agents which inhibit repair also inhibit semiconservative replication, this effect has been viewed as being the result of toxicity rather than a specific effect on the DNA repair system (Cleaver and Painter, 1975). However, the results of the following studies point to some degree of specificity of the inhibition of repair. Rasmussen et al. (1981) studied the effect of cigarette smoke on UDS of lung tissue explants exposed to either MMS or 4NQO. Exposure of lung tissue to cigarette smoke resulted in a diminished (50%)response to the applied chemical as compared to control. Replicative function returned to normal long before repair function which remained depressed for as long as 5 months after the last exposure to cigarette smoke. The use ofa radioactively labeled alkylatingagent indicated that both control and experimental tissue had the same amount of initial DNA modification, but, after 24 hr, the control tissue had 60% less radioactivity bound to DNA than did the tissue which had been exposed to smoke. Park et al. (1981) studied the effects of MMS and UV radiation on fibroblaststo determine whether additivity of repair could be demonstrated. Since the damage caused by these two agents was presumably repaired by different pathways, additivity was the expected result. However, these investigators found that repair of UV-induced damage was inhibited by exposure to MMS while repair of MMS damage was not inhibited by exposure to UV radiation. It was concluded that the alkylating agent might act on the repair system by alkylating the repair enzymes directed against UV damage, thereby rendering these enzymes inactive. A most interesting mechanism ofinhibition of repair was demonstrated by Duker et al. (1982). They introduced modifications into the DNA isolated from PBS 2 phage which normally contains uracil instead of thymine (Takahashi and Marmur, 1963) and determined the effect the modification had on V , , of purified bacterial uracil N-glycosylase. The introduction of 3.8%of 7-MeG into PBS DNA did not change V,, for uracil N-glycosylase. When AP sites were introduced into the DNA by heating the 7-MeG-containing DNA, V,,, was lowered 2.5-fold. Modification of the C8 position of guanine and adenine by photoalkylation (isopropanoland UV) lowered V,,, more than threefold. V,,, was also lowered by the uracil cyclobutane dimers introduced by 254 nm radiation (Duker et al., 1981). These investigators found uracil N-glycosylase to be a processive enzyme which, like other enzymes of this kind, was inhibited by modification at the C8 position of

52

GEORGE W. TEEBOR AND KRYSTYNA FRENKEL

purine moieties and by AP sites. The activity of calf thymus uracil N-glycosylase was also inhibited by apyrimidinic sites in DNA (Talpaert-Borl6et al., 1982). It had previously been shown that C8-AAF adducts of guanine acted as blocks to E. coli polymerase I which is known to be a processive enzyme (Moore and Strauss, 1979). The action of other polymeraseswas terminated by AAF residues on sequenced DNA segments as well (Moore et al., 1981). The processive movement of rat brain cytosine 5-methyltransferase was also inhibited by AAF residues in DNA (Pfohl-Leszkowiczet al., 198 1). Based on their results, Duker et al. (1982) suggested that care should be exercised in drawing conclusions about the apparent number of repair pathways from experiments measuring additivity of repair synthesis caused by more than one DNA-modifying agent. ACKNOWLEDGMENTS We thank Suse Broyde, Nahum Duker, David Goldthwaite, Michael Lieberman, Veronica Maher, Robert Shapiro, and Michael Sirover for reprints and preprints of publications. We greatly appreciate the critical comments and invaluable suggestions of Suse Broyde, Nahum Duker, and Walter Troll. We greatly appreciate the secretarial assistance of Jon Hart. Supported by USPHS grants CA 16669 and ES 02234.

REFERENCES Ahmed, F. E., andsetlow, R. B. (1977). CuncerRes. 37,3414-3419. Ahmed, F. E.,andSetlow, R. G . (1981). Biophys. J. 35, 17-22. Amacher, D. E., Elliott, J. A., and Lieberman, M. W. (1977). Proc. Nut/. Acud. Sci. U.S.A. 74, 1553-1557. Anderson, C. T. M., and Friedberg, E. C. (1980). Nucleic Acids Res. 8,875-888. Andrews, L. S., Fysh, J. M., Hinson, J. A., and Gillette, J. R. (1979). Life Sci. 24, 59-64. Arnel, P. R., Strniste, G. F., and Wallace, S. S. (1977). Radial. Res. 69,328-338. Aust, A. E., Falahee, K. J., Maher, V. M., and McCormick, J. J. (1980). Cuncer Res. 40, 4070- 4075. Bacchetti, S., and Benne, R. (1975). Biochim. Biophys. Acta 390,285-297. Barrows, L. R., and Shank, R. C. (1981). Toxicol. Appl. Pharmucol. 60,334-345. Baum, E. J. ( 1978).I n “Polycyclic Hydrocarbons and Cancer” (H. V. Gelboin and P. 0. Ts’o, eds.), Vol. I , pp. 45- 70. Academic Press, New York. Becker, R. A., Barrows, L.R., and Shank, R. C. (1981). Curcinogenesis2, I181 - 1188. Beer, M., Stern, S., Cannalt, D., and Mohlhenrich, K. H. (1966).Biochemistry 5,2283-2288. Beland, F. A. (1978). Chem. Biol. Interact. 22,329-339. Berman, H. M., Neidle,S.,andStodola,R. K.( 1978).Proc.Nutl. Acud. Sci.U.S.A.75,828-832. Beukers, R., and Berends, W. (1961). Biochim. Biophys. Actu 49, 181 - 189. Bodell, W. J. (1977). NucleicAcidsRes. 4, 2619-2628. Bodell, W. J., and Banejee, M. R. (1979). NucleicAcids Res. 6, 359-370. Bodell, W. J., and Cleaver, J. E. ( 198 1 ). Nucleic Acids Res. 9,203 - 2 13. Bootsma, D. (1978). In “DNA Repair Mechanisms” (P. C. Hanawdt, E.C. Friedberg, and C. F. Fox, eds.), pp. 589-601. Academic Press, New York. Boyce, R. P., and Howard-Flanders, P. (1964). Proc. Natl. Acad. Sci. U.S.A.51,293-300.

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STEROID HORMONE RECEPTORS IN HUMAN BREAST CANCER

George W. Sledge, Jr. and William L. McGuire

University of Texas Health Science Center, Department of Medicine. San Antonio, Texas

Introduction ................................................................................................................. “Subtle and Mysterious Influences”-The Historical Background Measurement of Steroid Receptors ............................................................................ Physiology of Steroid Receptors in Breast Cancer ................. Pathology of Steroid Receptors ......................................................................... A. Cell Morphology .................................................................................................... B. Cell Kinetics. VI. Steroid Receptor VII. Steroid Receptors in the Treatment of Breast Cancer ............................................... VIII. Conclusion ........... ........ ........ ......... ........ References.. .................................................................................................................. 1. 11. 111. IV. V.

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I. Introduction Neither the naked hand nor the understanding left to itself can effect much. It is by instrumentsand helps that the work isdone, which areas much wanted for the understanding as for the hand. (Francis Bacon, Novztm Organum)

The relation between human breast cancer and the endocrine organs, long suspected and frequently (if imperfectly) manipulated, lacked a systematic biological basis until the development of steroid receptor assays. These “instruments and helps” truly have been “as much wanted for the understanding as for the hand.” They have revolutionized our knowledge of the cell biology of breast cancer, and have guided the hand ofthe internist and the surgeon. The purpose of this article will be to explore both the foundations and the superstructure of this revolution. Briefly, we will explore the historical background to the development of steroid receptors, the current methodology, the physiology and cell biology of steroid receptors, and their pathologic, prognostic, and therapeutic implications. Steroid receptors have been with us a relatively short time. It is a tribute to the foresight and ingenuity of collaborating basic scientists and clinicians that progress in this field has been so informative, rapid, and exciting. 61 ADVANCES IN CANCER RESEARCH. VOL. 18

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISBN 0-12-006638-6

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II. “Subtle and Mysterious Influences’’-The Historical Background

That human breast cancer can be manipulated indirectly, through the endocrine organs, is not a recent observation. In 1896, the English surgeon Beatson produced clinical remissions of advanced breast cancer with oophorectomy. Commenting on his success, Beatson perceptively wrote that “we must look in the female to the ovaries as the seat of the exciting cause of carcinoma, certainly of the mamma, in all probability of the female organs generally. . . .I am satisfiedthat in the ovary of the female and the testicleof the male we have organs that send out influences more subtle it may be and more mysterious than those emanating from the nervous system, but possibly much more potent than the latter for good or ill.” Systematic exploitation of Beatson’s early surgical therapy did not occur until the 1950s. Huggins and Dao (1954), performing 100 ovariectoniies for palliation of advanced breast cancer, obtained clinical remissions in 38. The best responses seemed to occur in patients over the age of 40, and in those with a prolonged interval between radical mastectomy and onset of metastases. Huggins and Bergenstal ( 1952) similarly reported remissions in approximately a third of patients receiving adrenalectomy. Shortly after Huggins’ pioneering surgical treatments of hormone-dependent breast cancer, Folca et al. (196 1) discovered that patients responding to adrenalectomy concentrated [3H]hexestrolin their metastases to a greater degree than nonresponders. King et al. (1966) and Jensen et al. (1967) reported the existence of specificmacromolecular substances, later identified as proteins, that acted as estrogen receptors in malignant tumors of the breast. Jensen proposed that these receptors might be useful in predicting response to surgical adrenalectomy. In the late 1960s and early 1970s evidence accumulated in favor of this idea. Early methods and clinical studies of estrogen receptor (ER) were summarized at a 1974 international symposium (McGuire et al., 1975). Estrogen receptor was found to occur more commonly in postmenopausal than premenopausal patients. Lack of estrogen receptor generally denoted inability to respond to hormonal manipulation, and presence of ERthough it did not clearly define those tumors which were hormone dependent -was found to be necessary for response. This article deals primarily with subsequent advances in steroid receptor research. 111. Measurement of Steroid Receptors

The methodology of steroid receptor assays has been reviewed extensively in a previous publication from our laboratory (Chamness and McGuire, 1979), and will be discussed only briefly here, Steroid receptor assays

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commonly used are based on binding ofa radiolabeled steroid to either tissue slices or soluble cytosol proteins. Assays are performed on either fresh or frozen surgical specimens, and are reported in terms of receptor concentration per milligram of tissue or cytosol protein. Estrogen receptor- the most widely used steroid receptor -is measured usually either by sucrose density gradient centrifugation (pioneered by Jensen in breast cancer tissue determinations) or by the dextran-coated charcoal technique. Both methods are in common use, and provide reproducible results in trained hands. Other methods of assay have included gel filtration,agar gel electrophoresis,protamine sulfateprecipitation,hydroxylapatite adsorption, isoelectric focusing, and antisteroid antibodies. Immunochemical and cytochemical methods, though promising, have not to date been shown to reveal estrogen receptor (Chamness et al., 1980). Estrogen receptor determinations may be quantitative as well as qualitative in nature. Quantitative determinations have proved to be of clinical usefulness, as will be discussed later in this article. Measurements of estrogen receptor translocated to the cell nucleus (ERN) have been pursued by a variety of methods, and have proved to be of both conceptual and clinical interest. Progesterone receptor (PgR), the only other steroid receptor commonly assayed clinically, is found in the cytosol of breast tumors. Its presence in the nuclei of breast tumors has not been well documented to date. At present the most reliable measure of PgR appears to be that using sucrose density gradients. Androgen and glucocorticoid receptors, present in many human and animal breast cancer cell lines, have not yet been shown conclusively to be of clinical value, though they are of considerable experimental interest. In the near future, the rapidly developing technology of monoclonal antibodies should allow for rapid, reliable determination of steroid receptor levels using radioimmunoassay techniques. Several groups at present are investigating monoclonal antibodies directed against steroid receptors.

IV. Physiology of Steroid Receptors in Breast Cancer

The actions of steroid hormones are mediated by cytoplasmic steroid receptors. These receptors are translocated to the cell nucleus, where they participate in the regulation of DNA replication. Though there are many assayable steroid receptors in human breast cancer cell lines, the one for estrogen necessarily has attracted the most interest. Cytoplasmic estrogen receptor (ERC) has high affinity for 17P-estradiol(EJ and lesser affinity for other steroid hormones ( 17a-estradiol, androgens) and for antiestrogens

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such as tamoxifen (Edwardsand McGuire, 1980; Zava and McGuire, 1978; Horwitz and McGuire, 1979). In patients with low circulating levels of E2, the patients’ tumors may synthesize the hormone either by aromatization of testosterone (Adams and Li, 1975) and androstenedione (Varela and Dao, 1978) or by conversion from estrone (Wilking et al., 1980). The significance of these processes is uncertain. Varela and Dao (1 978) found no correlationbetween the ability of a tumor to aromatize androstenedione and steroid receptor levels, whereas Wilking et al. ( 1980)claim that a tumor’s ability to convert estrone to E, may determine its ER status. While the presence or absence of ERC probably is a function of tumor differentiation (see below), the relative level of ER in ER-positive tumors may be under several steroidal and nonsteroidal influences. Zava and McGuire ( 1977) have shown that testosterone-treated DMBA-induced rat mammary tumors have decreased cytoplasmic estrogen receptor, an observation which may help explain the therapeutic benefit of androgens in breast cancer. Whether progesterone has any effect on ERC level in human breast cancer is uncertain, though it is known to decrease the level, and inhibit replenishment, of ERC in the rat uterus (Hsueh et al., 1976). Estradiol may control its own receptor levels. Edery et al. ( 1981), using an isotope dilution-mass fragmentography technique, have demonstrated increased intracytoplasmic E2 levels in ER-positive breast cancers. Such an example of positive feedback (if this indeed is what it represents) requires further confirmation. Nonsteroidal factors may also play a role in regulation of ERC. Insulin (Butleret al., 1981;Moore, 1981) appears to have an effect on ERC similar to that of testosterone, by decreasingERC levelswithout changing ERC affinity for E,. A curious and unexplained observation from a large French study (Martinet al., 1979a) is that ER content vanes with the time ofyear in which surgery was performed. A fascinating series of recent experiments from the National Cancer Institute suggests the existence of a previously unknown, complex control mechanism in breast cancer cells. This mechanism, among its other functions, may regulate ER content. Increasing levels of cyclic adenosine 3’3’monophosphate (CAMP)arrest cell growth in both human and rat breast tumors (Cho-Changet al., 1981a,b). This action, mediated through a cAMP receptor protein, occurs only in hormone-dependenttumors. There appears to be an inverse relationshipbetween CAMP binding and ERC (Bodwin el al., 1978).L-Arginine,which acts synergisticallywith cAMP to arrest cell growth, decreases ERC content (Cho-Chung et al., 198la). Whatever the factors that regulate ERC content, the receptor protein clearly undergoes significant changes upon binding with estradiol. It has been suggested that the effect of this interaction is twofold first, an increase in

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“nucleotropy,” or the ability of the cytoplasmic receptor to translocate to the nucleus; second, “activation” of the receptor, a process enhancing translocated estrogen receptor’s (ERN’S) ability to induce DNA transcription (Jungblat et al., 1979). The mechanism by which ERC becomes ERN and acts on DNA is not well elucidated. It probably involves an estrogen-induced allosteric change, in which binding alters the conformation of the receptor molecule in a type-specific fashion (Bullock et al., 1978). Rochefort and Borgna (1981) have proposed that allosteric interactions, separate from the previously documented differences in receptor affinity, explain differences in the actions of estrogens and antiestrogens. They note differences in dissociation rates of receptor - steroid complexes between E, and 4-hydroxytamoxifen. Similar differences in nuclear binding of ER between estradiol and antiestrogens have been reported by Katzenellenbogen et al. (1980). A related observation by Sat0 et al. ( 198 1) is the product ofexperience with ER-positive, hormone-resistance tumors. Lookingat in v i m binding ofERC to DNA, this group found a temperature-dependent difference in binding between E,-sensitive and E,-resistant tumors, suggesting the existence of a DNA binding site in the latter that is destroyed or inactivated by heat. The same authors offered evidence that there is a cytoplasmic factor in ER-positive, E,-resistant tumors that inactivates ER’s DNA binding capability. The translocated receptor (ERN) interacts with the DNA- histone complex to stimulate DNA transcription of messenger RNA. The results of this interaction are many and varied, affecting both the function of the DNA itself and the production of a multitude of proteins. Estrogen stimulation induces production of DNA polymerase a,a highmolecular-weight cytosolic enzyme implicated in eukaryote cell proliferation (Edwards et al., 1980a).It also induces thymidine kinase, an enzyme in the salvage pathway of nucleotide biosynthesis closely related to DNA synthesis (Bronzert ef al., 1981). Thymidine kinase stimulation (and thymidine incorporation by breast cancer cells) is low in cell lines lacking ER. Estradiol-stimulated cells preferentially incorporate exogenous thymidine into their nuclei, in preference to thymidine derived from intracellular salvage pathways, thereby increasing intracellular thymidine pools (Lippman and Aitken, 1980). Of all the protein products of estrogen-regulated DNA transcription, the best known (and to date, most useful clinically) is PgR. The function of progesterone receptor in mammary tissue is unknown, but its usefulnessas a marker of EJnduced DNA transcription (first proposed by Horwitz et a[. in 1975) is now well established. The presence of PgR in an ER-positive breast cancer is evidence that the tumor has a functioning ER pathway, and argues for a certain minimal degree of tumor differentiation. Numerous other end products ofestrogen regulation in breast cancer have

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now joined the ranks with progesterone receptor. These include the 24,000 dalton (24K) and 54,000 dalton (54K) proteins discovered by researchersin our laboratory (Edwards et al., 1980b; Adams et al., 1980), the so-called estrogen-induced protein (IP) (Kage el al., 1980), endogenous peroxidase (Anderson et al., 1979),lactalbumin (Woods et al., 1977),and plasminogen activator (Butler et al, 1979).The exact function of these various proteins in breast tumors, and their usefulness as tumor markers in breast cancer, awaits further investigation. The discovery of severalestrogen-inducedor estrogenregulated proteins, however, suggests a complicated control system centered around estradiol, and has profound implications regarding the biology of the malignant mammary cell. It is of interest that one estrogen-regulated product, plasminogen activator, may increase the “tumorigenicity” of breast cancer cells through its fibrolytic activity. V. Pathology of Steroid Receptors

Early attempts at relating steroid receptor status (particularly estrogen receptor status)to histological type were generally disappointing or inconsistent (reviewedby Rosen et a/.(1975).Recent pathologic studies, using more sophisticated measures, indicate that there are fundamental qualitative differences between steroid receptor-negative and steroid receptor-positive tumors. These studies have focused on differences in cell morphology and cell kinetics. A. CELLMORPHOLOGY

Studies on steroid receptors in breast cancer have focused on prognostic variables related to cellular differentiation. A general observation, independent of steroid receptor status, is that survival in breast neoplasms is better in well-differentiated,and worse in poorly differentiated,tumors. Severalclassification schemes have exploitedthis observation.All rely to agreater or lesser degree on light microscopic evaluation of tubule formation, nuclear and cellular atypia, and number of mitoses. Other histologic features of prognostic value, such as tumor fibrosis or elastosis, tumor necrosis, lymphoid infiltration,and epithelialcellularity, have been given differingweights in the various classifications. A consistent finding in these studies has been that estrogen receptor-negative tumors tend to be less well differentiatedthan estrogen receptor-positive tumors, and that other negative prognostic characteristics tend to clump in ER-negative tumor populations(Antoniadesand Spector, 1979;E. R. Fisher et al., 1980, 1981; Hartveit et al., 1981; McCarty et al., 1980; Martin et al., 1979b; Maynard et al., 1978; Millis, 1980; Rich et al., 1978; Rolland et af.,

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1980; Russo et al., 198I ; Silversward et al., 1980).Few studies have considered cell morphology in light of steroid receptors other than ER. One that did (Wurz et al., 1980) found that only 8%of ER-negative tumors were well or moderately differentiated, as opposed to 59% of tumors with multiple assayable steroid receptors. An intriguing examination of human breast tumors by Borjesson and Sarfaty ( 1 98 I ) found that tritiated estradiol is taken up only by large (10.1 - 18.5 Fm) tumor cells, and that the proportion of these cells vaned from tumor to tumor. This study has two interesting implications: first, from a morphologic standpoint ER status may be related to cell size in breast tumors; second, individual breast tumors may be heterogeneous in terms of presence of estrogen receptor. The existence of heterogeneity of estrogen receptor in a population of tumor cells, and its relation to cell morphology, would be of great importance. Studies on the biology of metastasis in other neoplasms (reviewed by Poste and Fuller, 1980) suggest that primary tumors are heterogeneous rather than homogeneous, in nature, and that only cells with certain specific morphologic and functional characteristics are capable of establishing metastatic foci. Does this observation hold for breast cancer, and are ER-negative (or E,-resistant) cells in generally ER-positive primaries the culprits as regards metastasis? Definitive evidence for such heterogeneity (and its importance) is lacking, and should be a target of further investigation. Lee et a/. ( 198l), using the DMBA-induced rat mammary tumor system as a model for metastatic disease, showed that in serially transplanted tumors in ovariectomized animals, tissues of early passages were a mix of epithelial and spindle cells. Late passage tumors were primarily spindle cell in nature, and had lower ER levels than early-passage tumors. Are estrogen receptors positive because tumors are better differentiated, or are the tumors better differentiated because they are under steroid receptor regulation? This is a chicken-and-the-eggsort ofquestion, and is not entirely amenable to logical or experimental proof. As has been shown earlier in this article, estrogen receptor, the DNA - nuclear histone complex, and the end-products of steroid regulation are intricately and intimately related, and not readily divisible. Recent studies of cellular morphology in experimental breast cancer systems suggest that the end products of steroid receptor regulation may indeed affect cellular morphology. Yates et al. (1980), working with the androgen receptor-bearing Shionigi 1 15 mouse mammary tumor, showed distinct phenotypic characteristics (loss of anchorage dependence, fibroblastic morphology, changes in cell surface fibronectin) in testosterone-stimulated cells. Flow cytometry studies have been used to evaluate the relation of estrogen

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receptor status to cellular aneuploidy. While Muss el al. (1980) and Olszewski et al. (1981a) were able to show an increase in aneuploidy in ER-negative patients, Raber et al. (1980) were unable to confirm such a relationship. Undoubtedly further investigation will clear up this point. B. CELLKINETICS

Kinetic studies of breast tumors have employed either the thymidine labeling index (TLI) (Cooke et al., 1980; Gioanni et al., 1979; Meyer et al., 1977) or flow cytometry-derived estimation of the percentage of cells in S phase(Kuteetal., 198l;Mussetal., 1380;Raberetal., 1980;Olszewskietal, 1981b). In general these investigations are consistent with the cell morphology studies. As befits a well-differentiated population of tumor cells, ER-positive tumors tend to be slowly dividing. Estrogen receptor-negative tumors tend to have higher proliferative activity, with higher TLIsand more cells in S phase. VI. Steroid Receptors and Prognosis

Cell morphology and cell kinetic studies suggest that human breast cancers can be divided by steroid receptor analysis into two separate camps. On the one hand, ER-positive cells appear relatively well differentiated and unaggressive; on the other, ER-negative tumors seem to be less well differentiated and kinetically aggressive. One then might expect these laboratory parameters to have clinical counterparts. Gratifyingly, this indeed seems to be the case. Knight et al. of our group first demonstrated in 1977 a statistically significant difference in disease-free interval in postmastectomy patients. This difference, with ERpositive patients recurring less frequently than ER-negative patients, was independent of primary size, location, number of involved axillary nodes, menopausal status, age, or adjuvant therapy. When one combined ER negativity with axillary node positivity, agroup with a 50%recurrence rate at 18 months postmastectomy was defined. Subsequent studies (Allegra and Lippman, 1980; Bertuzzi et al., 1981; Hartveit el al., 1980; Leake et al., 1981; Rich et a]., 1978; Saez et al., 1981) have confirmed and extended these observations. As might be expected, difference in disease-free recurrence translates into differences in survival (Bishop et al., 1979; Gapinski and Donegan, 1980; Hahnel et al., 1979; Kinne et al., 1981; Samaan et al., 1981; Stewart et al., 1981). The recent literature also suggests that the presence of the PgR in a breast tumor may be of positive prognostic value. Saez et al. ( 1981) reported that in a group of 50 ER-positive, PgR-positive patients, only one had recurred by 23 months

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follow-up, as opposed to 10 of 57 ER-negative, PgR-negative patients. Pichon et uf. (1980) have reported similar results, with a 3.6 times greater likelihood of metastases in PgR-negative than PgR-positive patients. Though not all investigators looking at PgR have come to this conclusion, some (Bertuzzi eta/.,198I ) feel that PgR alone is a more sensitive prognostic factor than either ER or ER and PgR. The better prognosis of steroid receptor-positive patients may be related to site of metastasis as well as disease-free interval. Though hardly a uniform finding (Hahnel et a/., 1979), there is evidence (Singhakowinta et a/., 1976; Stewart et af., 198 1) that ER-negative tumors are more likely to metastasize to visceral organs such as the brain and the liver, there to do maximum damage. Estrogen receptor-positive tumors would seem not only to metastasize less readily, but are more likely to spread to bone than viscera. Prognostic studies have important therapeutic implications. Estrogen receptor-negative patients, given their high rate of early metastases, may be the most important target for adjuvant chemotherapy, particularly if steroid receptor status is combined with axillary node positivity. On the other hand, ER-positive, node-negative patients, given their low likelihood of recurrence after mastectomy, might well be spared the rigors of adjuvant chemotherapy. Trials are underway in the Southwest Oncology Group employing receptor status in the treatment algorithm of adjuvant therapy. Investigations of the prognostic factors in breast cancer (or any other neoplasm) are in reality investigations of the natural history of the disease. Summarizing the pathologic and prognostic implications of steroid receptors, we now can see that estrogen receptor status defines (albeit broadly) two biological types of breast cancer: in effect, two diseases. Analyses of breast cancer that fail to take estrogen receptor status into account neglect an important aspect of the tumor, and must therefore be considered flawed in a significant sense. VII. Steroid Receptors in the Treatment of Breast Cancer

Jensen et ul. ( 1967) concluded their seminal 1967 paper on the estrogen receptor with this little ditty: The surgeon who strives for perfection Needs some basis for patient selection. He would like to be sure There’s a good chance for cure Before he begins the resection.

They offered the hope that “it should be possible to predict in advance which patients will respond to adrenalectomy, and thus enable the surgeon to select

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for this operation those individuals who have a chance of receiving benefit.” This hope was largely realized by the time an international symposium met in 1974 to review progress in the estrogen receptor field (McGuire et al., 1975). Estrogen receptor-negative patients rarely respond to surgical endocrine manipulation for metastatic disease. A less encouraging conclusion derived from early studies was that ER-positive patients, while more likely to respond to surgical endocrine manipulation, did not uniformly respond. In fact, only about one-half of ER-positive patients underwent demonstrable clinical remission of their disease after endocrine manipulation. Horwitz et al. (1975), in an attempt to explain this paradoxical finding, proposed the progesterone receptor hypothesis in 1975. The progesterone receptor hypothesis, in simple terms, stated that “when progesterone receptors (PgR) are present, tumors will be endocrine responsive; when absent, tumors will be resistant to endocrine manipulation.” The basis for this hypothesis was the realization that progesterone receptor was an estrogendependent protein; its presence in a tumor suggested the presence of functioning ER-regulated DNA transcription, necessary for E, action. Since its proposal, this hypothesis has received considerable experimental support. ER-positive, PgR-positive tumors are more responsive to endocrine therapy for advanced breast cancer (77% in one large combined series) than either ER-positive, PgR-negative tumors (27O/o), ER-negative, PgR-positive tumors (46%),or ER-negative, PgR-negative tumors (1 1Yo) (Osborne et al., 1980). It would appear that a biologically intact estrogen-receptor pathway is either a necessary or important part of determining tumor response to external manipulation. However, as with ER data, PgR determinationswhether alone or combined with ER data-do not completely pinpoint hormonally dependent tumors, nor entirely exclude hormone-resistant or autonomous tumors. This may be due to difficulties in assaying steroid receptors, or to lack of close linkage between PgR and the tumor growth mechanism in all tumors. Other promising measurements have been offered in an attempt to improve the predictability ofendocrine manipulation. Quantitative estrogen receptor measurements have been employed, on the assumption that tumors with a high ER level might contain a higher proportion of ER-positive cells responding to endocrine therapy (McGuire et al., 1979). Data from our laboratory (Osborne et al., 1980)and other institutions (Allegra et al., 1980; DeSombre and Jensen, 1980; Paridaens et al., 1980; Young et al., 1980) support this concept. The presence of ERN in breast tumors may also help predict for response to endocrine manipulation in metastatic breast cancer. Like PgR, ERN

STEROID HORMONE RECEPTORS IN BREAST CANCER

71

presumably indicates a more or less functional estrogen pathway in the tumor cell. Fazekas and MacFarlane ( I 98 1 ) have suggested that the presence of ERN is a powerful tool in predicting response to endocrine manipulation. Nonsurgical endocrine manipulations of metastatic breast cancer, in the form of androgen therapy, diethylstilbestrol, aminoglutethimide, and, most recently, nonsteroid estrogen antagonists (such as tamoxifen), have been used increasingly, particularly in postmenopausal patients. Steroid receptor studies have contributed significantly to our understanding of these agents. The action of androgens or the estrogen receptor has been discussed previously. Androgens compete with E2for ERC binding (though with low affinity) and are (at least in experimental models) capable oftranslocating ER to the nucleus with subsequent increase in PgR (Zava and McGuire, 1978). This finding may explain the occasional increase in tumor size found in patients treated with androgens. Presumably the positive therapeutic benefit of androgen therapy is due either to competition with E, for binding, or to allosteric changes in the ER molecule preventing proper gene expression. The antiestrogen tamoxifen also competes with E, for ERC. As mentioned previously, E,-ERC and tamoxifen - ERC complexes have different functional characteristics. Antiestrogens also appear to decrease levels of cytoplasmic ER (Katzenellenbogen et al., 1979). Antiestrogen- receptor complexes also show differences in salt extractability from normal ERN, and in nuclear processing of ER (Katzenellenbogen et al., 1980). Tamoxifen, in contradistinction to E, , decreases intracellular thymidine pools and forces the cells to rely on extracellular sources of thymidine for DNA synthesis (Lippman and Aitken, 1980).A provocative new study suggeststhat there isa high-affinity antiestrogen binding site separate from ERC (Sutherland el al., 1980). The existence of this receptor awaits independent confirmation, as does an exploration of its biological purpose (it is difficult to postulate a natural receptor with tamoxifen as its sole ligand). In recent years, new treatment modalities for both primary and metastatic breast cancer have been developed. Adjuvant therapy in particular seems to have increased survival and decreased recurrence in primary breast cancer. Two recently reported trials suggest that the use of combined hormonal and chemotherapy is superior to chemotherapy alone in an adjuvant setting. Hubay et al. ( 1980) used CMF f T (cyclophosphamide, methotrexate, 5fluorouracil, f tamoxifen), and the National Surgical Adjuvant Breast Project (Fisher el al., 1981) trial compared PF with PIT (L-phenylalanine mustard, 5-fluorouracil, tamoxifen). In both groups the benefit of tamoxifen therapy in an adjuvant setting was confined to ER-positive patients, particularly postmenopausal patients. Combination chemotherapy has been used successfully for the palliation of metastatic breast cancer. Steroid receptor assays, by targeting a group of

72

GEORGE W.

SLEDGE, JR.

AND WILLIAM L. MCGUIRE

patients unlikely to respond to hormonal manipulation, have spread the use of palliative chemotherapy and reduced time-wasting surgical and other hormonal manipulations. It is now standard therapy in our and other institutions to use combination chemotherapy as the first treatment in metastatic ER-negative breast malignancy. Cell kinetic studies, by suggesting an increased proliferative potential of ER-negative tumors, have naturally raised the important question of whether these tumors are more sensitive to cytotoxic agents acting against replicating cells. Kaufmann et af. (1980) determined in vitro sensitivity of breast tumors to adriamycin in 90 cases; ER-poor, PgR-poor tumors were significantly less chemoresistant than tumors rich in either ER or PgR. Allegra et al. (1 978), reporting on the National Cancer Institute experience, found ER-negative patients to have significantly better responses to chemotherapy then ER-positive patients. Promising as this lead was, it has not been generally confirmed in other studies (Kiang et af.,1978; Hilf et al., 1980; Mortimer et al., 1981; Paore et al., 198 I; Young et af.,1980). Rapidly dividing, ER-negative tumor cells may simply be too aggressive, or current therapy not sufficiently effective. The management of ER-negative breast cancer patients continues to be a major therapeutic dilemma. VIII. Conclusion

Steroid receptors, originally used for the prediction of response to surgical endocrine manipulation, have proven a powerful tool in other basic and clinical investigations.Our knowledge of the biology and treatment of breast cancer has increased dramatically through the selective use of steroid receptor assays. If past results are any promise of future performance, steroid receptor analysis should prove a useful key in unlocking the secret doors of the breast cancer cell. Much of what has come to pass in the last decade or so was unexpected and not a little perplexing. It is a mark of good research that it poses as many new questions as it gives answers. This article began with a quotation from Bacon’s Novum Organum, the sentinel gun of our current scientificrevolution. Perhaps it would be fittingto end with a passage from the same source: The human understandingis unquiet; it cannot stop or rest, and still presses onward, but

in vain. Therefore it is said that we cannot conceive of any end or limit to the world; but always as of necessity it occurs to us that there is something beyond.

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Paore, J. F., Abeloff, M. D., Ettinger, D. S., Arnold, E. A., and Baker, R. R. (1981). Surg. Gynecul. Obstet. 152.70- 14. Pandaens, R., Sylvester, R. J., Ferrazi, E., Legros, N., Leclerq, G., and Heuson, J. C. (1980). Cancer46,2889-2895. Pichon, M. F., Pallud, C., Brunet, M., and Milgram, E. (1980). Cancer Res. 40,3357-3360. Poste, G., and Fuller, I. J. (1980).Nature (London)283, 139- 146. Raber, M., Barlogie, B., Latreille, J., Fu, C. T., and Fritsche, H. ( 1 980). Cell Tissue Kinet. 13, 682. Rich, M. A.. Furmenski, P., Brooks, S. C., and the Breast Cancer Prognostic Study Surgery and Pathology Associates. (1978). Cancer Rex 38,4296-4298. Rochefort, H., and Borgna, J.-L. (1981).Nature 292,257-259. Rolland, P. H., Jacquemier, J., and Martin. P. M. (1980). Cancer Chemofher. Phrmnacol. 5, 73-77. Rosen, P. P., Menendez-Botet, C. J., Nisselbaum, J. S., Urgen, J. A., Mike, V., Fracchia, A., and Schweltz, M. K. ( 1975). Cancer Res. 35,3 187 - 3 194. Russo, J., Fine, G., Husain, H., Krickstein, H., Robbins, T., Rosenberg, B.,Brooks, S., Ownby, H., Roi, L., Miller, J., Furmonski, P., Brennan. M. J., and Rich, M. A. (1981).Proc. AACR/ASCO 22, 146. Saez. S., Chouvet, C., Mayer. M., and Cheix, F. (198 1). Proc. AACR/ASCO 21, 139. Samaan, N. A., Buzdar, A. U., Aldinger, K. A., Schultz. P. N., Yang, K. P., Romsdahl, M. M., and Markin, R. (1981).Cancer47,554-560. Sato, B., Nomura, Y., NaKao. K., Ochi, H., and Matsumato, K. ( I 98 1). J. Steroid Biochem. 14, 295-303. Silfversward, C., Gustafsson, J.-A., Gustafsson, S. A., Humla, S., Nordenskjold, B.. Wollgren, A., and Wrarge, 0. (1980). Cancer45,2001-2005. Singhakowinta, A., Potter, H. G., Buroker, T. R., Samal. B., Brooks, S. C., and Vaitkeviuus, V. K . (1976).Ann. Surg. 183,84-88. Stewart, J. F., King, R. J. B., and Rubens, R. D. (1981). Proc AACR/ASCO 22. 148. Sutherland, R. L., Murphy, L. C., Foo, M. S., Green, M. D., Whybourne, A. M.,and Krozowski, Z. S. (1980).Nature(Lundun) 288,273-275. Varela, R. M., and Dao, T. L. (1978).Cancer Res. 38,2429-2433. Wilking, N., Carlstrom, K., Gustafsson, S. A., Skoldefors, H.. and Tollbovn, 0.( 1980). Eur. J . Cancer 16, 1339- 1344. Woods, K. L., Cove, D. H., and Howell, A. (1977).Lance1 2, 14- 16. Wurz, H., Citoler, P., Schulz, K. D., Roos, B., and Kaiser, R. (1980).Klin. Wochenschr.58,643. Yates. J., Couchman, J. R., and King, R. J. B. ( 1 980). Prog. Cancer Res. Thrr. 14, 3 I -39. Young, P. C. M., Ehrlick, C. E., and Einhorn, L. (1980). Proc. AACR/ASCO 21, 142. Zava, D. T., and McGuire, W. L. (1977). Cancer Res. 37, 1608- 1610. Zava. D. T., and Mcguire, W. L. (1978).Science 199,787-788.

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RELATION BETWEEN STEROID METABOLISMOF THE HOST AND GENESIS OF CANCERS OF THE BREAST, UTERINE CERVIX, AND ENDOMETRIUM Mitsuo Kodama and Toshiko Kodama Laboratoryof Chemotherapy. Achi Cancer Center Research Institute. Nagoya. Japan

1. Prologue ........................................................................................................................ 11. EpidemiologicalAspects of Cancers of the Breast, Uterine Cervix,

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

and Endometrium

ervix, and Endometrium ....

111. Hormonal Aspects

IV.

V.

VI. VII.

A. General Considerations..................................................... B. Breast Cancer .......................................................................................................... C. Cervical Cancer ....................................................................................................... D. Endometrial Cancer... .......................................... New Trends in the Biolog roid Hormones ................ A. General Considerations.......................................................................................... B. Chick Oviduct ......................................................................................................... C. Insects in Growth .................................................................................... D. Tumor Virus ........................................................................................................... E. Vaginal Epithelium................................................................................................. Synthesis of a Unifying Theory .......... ......................... A. Junction between Epidemiology and Endocrinology ........................................... B. Genesis of Breast Cancer ........................................................................................ C. Genesis of Cervical Cancer..................................................................................... D. Genesis of Endometrial Cancer ............................................................................. Epilogue........... ........ ......... .............................................................. Addendum .................................................................................................................... References .....................................................................................................................

71 78 86 86 87 91 92 95 95 96 96 91 100 101 101 104 I07

I10 113 115 115

I. Prologue

Our knowledge about the nature of human cancer increased to a great extent due to recent progress in molecular biology (Anderson, 1980). We also owe very much to the enthusiasticeffort ofepidemiologistsin optimizing our life style for cancer prevention (Reddyet a!., 1980).It seems that a lack of coordination between micro and macro approaches prevents full exploitation of our knowledge for the conquest of this malignant disease. Recently, the incidence of cancers of the stomach and uterine cervix has been decreasing in Japan, whereas the incidence of cancers of the breast and ovary has been increasing (Kodama and Kodama, 1981). There are also differential cancer risks between the United States and Japan. No unifylng theory is available to cover all these phenomena without contradiction. 71 ADVANCES 1N CANCER RESEARCH, VOL. 38

Copyright 0 1983 by Academic Press. he. All rights of reproduction in any form reserved. ISBN 0-12-006638-6

78

MITSUO KODAMA AND TOSHIKO KODAMA

This article will describe the relation between the steroid metabolism ofthe host and the genesis of three female cancers from the breast and uterus, organs which are known to be subject to steroidal influences during their developmental stage (adolescence). Relevant information from epidemiology, endocrinology, and molecular biology is exploited in an effort to extract common principles operating in the production of these three cancers. II. EpidemiologicalAspects of Cancers of the Breast, Uterine Cervix, and Endometrium

More than 100 years ago, Rigoni-Stern noted that Catholic Sisters of Verona, Italy, were at reduced risk for uterine (cervical) cancer and at increased risk for breast cancer as compared with the nonmonastic Veronese. He was smart enough to add that all four deaths from mammary cancer in male patients, as registered in Verona between 1760 and 1839, occurred in priests (Clemmesen, 195 1). Though he himself was not aware of the fact that the above bias of cancer incidence in the monastic people was related to their abstension from sexual experience, his discovery of the rarity of cervical cancer in the monastic women gave rise to a number of investigations on the sex life of cancer patients including two confirmatory reports on the substantial lack of cervical cancer in nuns (Gagnon et al., 1950; Taylor et al., 1959). Clemmesen also noted differential effects of marital life on the morbidity rates of cancers of the breast and uterine cervix in the cancer registry of Danish women, 1943 - 1947 (Clemmesen, 195 1). On the basis of the epidemiologicaldata, he expressed a hope that a hormonal study ofbreast cancer could be performed in parallel with that of cervical cancer. From the same epidemiologicalstandpoint, Stern anticipated a hormonal implication in the genesisofcervical cancer (Stern et al., 1967), and Wynder suggestedthe possible usefulness of a steroid study to elucidate the etiology of endometrial cancer (Wynder et al., 1966). Based on past literature on cancer epidemiology, a patient with breast cancer seems to share much the same background as a patient with endometrial cancer, whereas a sharp contrast existsbetween cervical and breast cancers concerning some background factors of the patients, as shown in Table I. The above differences in the background of cancer patients may well provide a clue in the search for carcinogenic agents of the three cancers. Another clue came from the consideration ofthe secular trend of cancer incidence in Japan. Figure 1 indicates that the mortality rates from cancers of the stomach and uterine cervix are on the decline whereas those from cancers of the breast and ovary are on the rise. In view of the current status as well as the chronological transition of cancer incidence in the United States (Wynder et al., 198 l), it is apparent that Japan, under the influence of westernization in life style, is following the United States in the

TABLE I THEEPIDEMIOLOGICAL BACKGROUND OF THREE FEMALE CANCERS Epidemiologic discriminator Geographic distribution Marital life Panty Socioeconomic Diet

Breast cancer Frigid zone Late, often unmamed Oligoparous Above average Rich in fat and animal Drotein

Cervical cancer

Endometrial cancer

Reference

Torrid zone Early

Indifferent

Haenszel and Hillhouse (1959); Kodama and Kodama (1981)

Indifferent

Polyparous

Oligoparous

Below average

Above average Rich in fat and animal Drotein

Haenszel and Hillhouse (1959); Wynder et a/. (1966); Rotkin (1973); Hirayama (1 979) Clemmesen (195 1); MacMahon et al. (1973); Elwood et al. (1977); Hirayama ( 1979) Clemmesen (1951); Stocks (1955); Elwood el al. (1977)

?

Drasar and Irving (1973);Armstrong and Doll ( 1975);Reddy ef a/. (1980)

80

MITSUO KODAMA A N D TOSHIKO KODAMA

?Ovarian cancer .Ii

I I

i i

2501

I

1’

i

200

r01

i’

/’

/

a,\

L”*xx Uterine cancer

L

1950

1960

1970 Calendar

1980 Years

FIG. I. Secular trend of cancer mortality in Japanese women. For comparison among various cancers, the relative mortality rate for each year was expressed as a percentage of the figure for 1950. The weight ofendometrial cancer is probably less than 10% in the total uterine cancer. (Reproduced from Kodama and Kodama, 198 I .)

constitution of various cancer risks. Interestingly, the mortality rate from cervical cancer in Japan dropped to one-third, and the intake of dietary fat tripled during the same time range of 1950to 1975. The increase ofdietary fat and animal protein was compensated for by a decrease of vegetable protein and carbohydrate so that the total caloric intake was kept fairly constant during the same period (Fig. 2a). As a consequence of dietary improvements, the process of physical growth was accelerated, and final height reached was also increased from year to year (Fig. 2b). Apparently, an increase ofdietary fat and animal protein resulted in an acceleration of adolescent growth. It should be recalledthat an acceleration of menarche, as is in progressin Japan

HOST STEROID METABOLISM AND GENESIS OF CANCER

81

300

,PAnirnal protein ._ 1:'

Carbohydrate

1950 '60 '70 year

u 0 20 10 Age (years)

FIG. 2. Relation between per capita diet consumption and physical growth ofJapanese girls. For comparison, the secular trend of various dietary elements was illustrated as a percentage of the figure for 1950. Based on the annual report from the Ministry of Health and Welfare (a) and also on that from the Ministry of Education and Culture (b), Japan.

(unpublished data), may herald an increased risk for breast cancer (Staszewski, I97 1). The fact that the effects of westernization are equally evident in our diet, physical growth, and constitution of various cancer incidences may not be a mere coincidence. The data so far obtained indicate that patients with cancers of the breast, uterine cervix, and endometrium are distinguishable from one another by three discriminant factors- climate, diet, and reproductive activity. All these discriminators are more or less involved in the establishment of the hormonal environment ofthe host. Thus, one may bejustified in speculating that the genesis of the three female cancers could be related to specified hormonal environments of the host. Hormonal implication in the genesis ofbreast cancer was first suggestedby Laccasagne who observed the development of breast cancer in male mice injected with estrone benzoate (Laccasagne, 1932). There was a long-lasting belief that the genesis of breast cancer was somehow related to the presence of the ovary (Hayward, 1970). In support of this, Lilienfeld reported that the risk for breast cancer was reduced by an early artificial menopause (Lilienfeld, 1956). De Waard emphasized the role ofthe adrenal in postmenopausal patients who were often associated with adrenal-related diseases such as obesity, hypertension, and diabetes (De Waard et al., 1964).An association

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of the breast cancer risk with a delay in first birth (MacMahon et al., 1973)or with relative sterility (Shapiro et al., 1968) may indicate that the ovary of a patient is prone to anovularcycle.Actually, the predispositionofa patient for ovulation failure was confirmed by endometrial biopsy (Grattarola, 1964) and by urinary (Kodama et al., 1977, 1979a) and plasma steroid analysis (Bulbrook et al., 1978). Articles in support of a hormonal implication in the genesis of cervical cancer are rather limited, mainly because this epidemiological suggestion (Clemmesen, 1951; Stern et al., 1967) was not supported by clinical researchers, who observed no benefits from hormone treatment of cervical cancer. Furthermore, the association of early coitus (or sexual instability) with a high risk for cervical cancer, a most prominent trait of the patient, was often discussed in favor of the virus theory (Rotkin, 1973)or of the smegma theory (Wynder et ul., 1954). Accumulation of further data along that line, however, disproved rather than confirmed the exogenous contamination theories (Kodama et al., 1977). The pioneering work of Laccasagne was followed by a number of papers on hormone-inducedtumors in mice: Allen and Gardner observed an increase of cervical cancer in (C57 X CBA)F, mice following long-continued administration of estrogen (Allen and Gardner, 1941). Taki and his associates noted a promoting effect of estradiol for methylcholanthrene carcinogenesis in both the cervix and corpus uteri of mice (Taki and Iijima, 1963;Iijima et al., 1964). Interestingly,progesterone and testosterone, given at a level which inhibits the biological effect of estrogen, markedly suppressed carcinogenesis in such cases (Taki, 1967). Cowdry remarked that Bantu women, living on nutritionally poor diets, suffer from a high incidence of both hepatic disorder and cervical cancer. He explained the above association in terms of the depressed estrogen-detoxifying function of the liver: many Bantu women with hepatic disturbances might have been exposed to excessive concentrations of estrogen, and the development of cervical cancer, once triggered by the carcinogenicaction of the endogenous estrogen, was not stopped in spite of the general practice of circumcisingtheir husbands (Cowdry, 1968). Further evidence in support of estrogen carcinogenesis was presented by Herbst, who reported that the daughters of women given large amounts of synthetic estrogen during their pregnancies have a substantially increased risk of developing clear cell cancers of the vagina and cervix (Herbst et al., 1972, 1974). This report clearly indicates that the genital epithelium of a female fetus is extremely sensitive to the carcinogenic action of estrogen. The recent increase of endometrial cancer in some areas of the United States was discussed in relation to the effect of exogenous estrogen (Mack, 1978), but the etiological relevancy is still far from being well established (Nisker et al., 1978; Feinstein and Horwitz, 1978). The correlation of the

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intake of dietary fat to the risk for endometrial cancer suggeststhe possibility that fat-rich diets could initiate or promote endometrial carcinogenesisvia excessive endogenous production of estrogens (Armstrong, 1977). The trouble is that there are only small, if any, case-control differences in the production, metabolism, and excretion of estrogens, and Poortman and his associate, on the basis of their urinary androgen data, proposed the hypothesis that the hyperestrogenic status of a cancer candidate could be produced by a deficiency of endogenous androgen rather than by an excess of endogenous estrogen (Poortman and Thijssen, 1978). Additional descriptionsof the incidence of cancer in the Japanesewill be of help in understanding the relationship between the epidemiological and hormonal aspects of the three female cancers, since the life style of the Japanese, even under the strong influence of westernization, still conserves much of the traditional habit, and Japanese women with the three cancers have their own epidemiological traits which are distinctly different from those of Westerners. Among cancer epidemiologists,Japan has been known to have a notoriously high risk for stomach cancer and an incredibly low risk for cancers of the breast, corpus uteri, colon, and prostate, as compared to the figures from Western countries (Reddy ef al., 1980; Armstrong and Doll, 1975). The incidence of breast cancer among postmenopausal patients in Japan is low (De Waard, 1969). Figure 3 illustrates the relation between the age-adjusted death rate of female breast cancer patients and per capita daily fat (and oil) consumption in 18 countries. The correlation is statisticallysignificant, and Japan is at the lowest level in death rate and fat intake. Figure4 presents the

c

5

'Chi

100 I

0

-

10 20 30 A.A.RR. of cancer of the breast

FIG. 3. Correlation between age-adjusteddeath rates (A.A.D.R.)from female breast cancer and per capita consumption of fat and oil. (Reproduced from Segi er al., 1977.)

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0 10 20 30 40 50 60 70 Age (years)

FIG. 4. Age-specific death rate for breast cancer among Caucasians in the United States (1959- 1961) and native Japanese (1970) women: thin solid line, single Caucasians in the United States;thin dashed line, mamed Caucasians in the United States;thick solid line, single native Japanese; thick dashed line, mamed native Japanese. (Reproduced with some modification from Kodama and Kodama, 198 I .)

age-specific death rates from breast cancer, as compared between the United States and Japan, and between married women and single ones: the death rate for the mamed Japanese slows down at later stagesoflife, but that for the single Japanese increases with age throughout life, like that for Whites in the United States. It is noteworthy that mamed women had a markedly depressed mortality rate from breast cancer in Japan, but not in the United States. Conceivably, the risk for breast cancer is determined by a synthesis of multiple factors including fat intake (Reddy et al., 1980) and reproductive activity (Clemmesen, 195 I), and the promoting effect of celibacy is eclipsed in the United States by a predominance of other risk factors (probably a fatty diet). In the international comparison of the morbidity or mortality rates from cervical cancer, Japan could be classified as an intermediate, neither high nor low. Early mamage, one of the most prominent risk factors for cervical cancer, was not clearly demonstrable in Japanese patients (Kodama et al., 1978). This lack of the sexual discriminator in Japanese patients with cervical cancer contrasts sharply with the lack ofanother sexual discriminator in the United States, Whites with breast cancer (Fig. 4). These paradoxes could somehow be related to the different constitutions of dietary elements between the two countries. Another feature of Japanese cancer patients is the biases in geographic distribution: as shown in Fig. 5, the high-risk areas for cervical cancer are distributed mainly in the southwestern corner of Japan, whereas those for breast cancer are in the northeastern or metropolitan areas.

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FIG. 5. Geographic distributions ofhigh-risk areas in Japan for two female cancers. The dark areas represent the 10 prefectures most at risk for each cancer. The borderline passingacross the Japanese mainland divides the whole population of Japan into two equal parts ( 5 . 5 X lo7each). Tokyo, Osaka, and Nagoya, the three largest cities of Japan, are all in the high-risk areas for breast cancer. (Reproduced with some modification from Kodama and Kodama, 1981 .)

As previously mentioned, Japan is classified as a country with the lowest incidence of endometrial cancer (Armstrong and Doll, 1975). It might be interestingto see whether or not the above characteristicsofJapanese cancer incidence are still preserved in Japanese emigrants in Hawaii and in the United States. There are many reports on the secular trend of cancer incidences in Japanese emigrants abroad, and our discussion will be restricted to a comparison between mainland Japan and Hawaii regarding the incidence of the three female cancers. Figure 6 compares the age-adjusted cancer incidence between the Japanese mainlanders (open bars) and the Japanese Hawaiians (solid bars). Emigration to Hawaii increased the morbidity rates from cancers of the breast and endometrium, and reduced the incidence of cancer of the uterine cervix. In support of this finding, an

&east Cancer

Cervical Endometrial Cancer Cancer

FIG. 6. Comparison between native Japanese (open bars) and Hawaiian Japanese (solid bars) regardingthe age-adjusted incidence of three female cancers. The data on native Japanese are based on the Cancer Registry in Miyagi Prefecture, Japan. (Plotted from data in Waterhouse ct a/., 1976.)

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increase in the death rate from breast cancer and a decrease in the death rate from cervical cancer are evidenced in mainland Japan where the life style is becoming more and more westernized. The possible relevancies of all these epidemiological data to the genesis of the three female cancers will be discussed in detail in Section V. 111. Hormonal Aspects of Cancers of the Breast, Uterine Cervix, and Endometrium

A. GENERAL CONSIDERATIONS

Until recently, advances in steroid studies in cancer patients were rather slow and stagnant, mainly because of the technical difficulties in steroid estimation. In 1955- 1957, Brown and his associates established the standard method for measuring urinary estrogen (Brown, 1955; Brown et a!., 1957). Even with the high sensitivity of fluorometry, the content of urinary estrogen in women with quiescent ovaries was so low, and the amount of interfering material so large, as to throw considerable doubt on the validity of the results obtained (Fotherby and James, 1972). Competitive proteinbinding methods were not completed before 1967 (Murphy, 1967), and the radioimmunoassay system became available as late as 197 1 (Caldwell, 1971). Because of the technical and economic restrictions, one had to be satisfied with information on a limited number of steroidsor steroid families. It should be mentioned that the assessment of the hormonal state of an organism whose function is maintained by concerted effects of multiple hormones will not be feasible without acquisition of multihormonal information. This requirement was fulfilled in 1971 by Horning and his associate (Sakauchi and Horning, 1971), who succeeded in developing a profile of urinary steroids by temperature-programmed gas- liquid chromatography. The profile consists of more than 20 identified steroid peaks from 3 steroid families so that the activities of endogenous androgen, progestin, and corticosteroid could each be assessed on the basis of metabolic considerations of individual steroid data. The data skew due to aging and the menstrual cycle is another nuisance to be corrected for (Kodama and Kodama, 1975).The normal range (mean and standard deviation) should be settled for each steroid using the new technique. In the case-control study, one must extract a specific “face” of the disease from the complexity of multihormonal changes. To accomplish all of this, a researcher should be armed with a sufficient knowledge on both endocrinology and biostatistics. Evidence in support of the hormonal data (and their interpretation) could be found either in the epidemiological information stock or in the data of molecular biology. Nothing is more important than the recognition that the etiology of human cancer is beyond the scope of one specified branch of science.

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Reviewing the past literature on the hormonal aspects of human cancer, one may notice that an association between the presence of tumor and an increase in the activity of endogenous estrogen is lacking or very weak (Zumoff et al., 1975; Reddy et ul., 1980). Although the interaction between the estrogen -receptor complex and the cell nucleus has been extensively studied (Higgins and Gehring, 1978), we were left with much the same ignorance as before concerning the etiology of hormone-related cancers. Quite unexpectedly, case-control differenceswere often found in the levelsof nonphenolic steroids (androgen, progestin, and corticosteroids) which are supposed to be modifiers of estrogen carcinogenesis (Taki, 1967). From the above chaotic situationemergedthe new concept of hormone balance- that a hyperestrogenic state could be produced either by an excess of estrogen or by a deficiency of escort steroids (Poortman and Thijssen, 1978). The above change in the stream of research will be described in more detail for each of the three female cancers together with data from our laboratory. B. BREASTCANCER

The review by Zumoff and his associates briefly surveyed some problematic points on steroid hormones and breast cancer (Zumoff et al., 1975). There are two sets of hormonal parameters that might be of use in discriminating a patient (or population with a high risk) from a healthy control (or population with a low risk): urinary androgen metabolites and urinary estrogens. More specifically, a breast cancer patient was associated with a decrease of urinary androgen (androsteroneand etiocholanolone) or with a reduction of the ratio estriol/(estrone estradiol-178) as compared with a normal control. Zumoff remarked in his review that, in order to implicate a causal relationship, it is essential to have the same direction of differences between “within-population” studies (i.e., cancer patients versus normal controls) and “between-population” studies (i.e., high-risk versus low-risk populations). The androgen theory, originally derived from the retrospective case-control study in England, found further support from a prospective case-control study in Guernsey (Bulbrook ef ul.. 197 1). Unfortunately the between-population study by the same research group did not confirm the results of the within-population studies: a Japanese woman with a low risk for breast cancer should have excreted more androgen than a British woman with a high risk. In fact, the former was found to have a much lower excretion of urinary androgen than the latter (Bulbrook et al., 1967). The investigation in our laboratory also failed to find an association of reduced androgen excretion with the presence of breast tumor or with an increased risk for breast cancer (Kodama et al., 1975). The relation between the results ofthe British group and ours will be detailed later in this section.

+

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The estrogen theory, which is not necessarily incompatible with the androgen theory, found support in the Harvard School. Subsequently, a number of investigators have tested the validity of the theory. The association between an increased risk for breast cancer and a reduction in the estriol ratio, as observed in earlier studies, was not reproducible in later studies, and Zumoff et al. denounced both the androgen theory and the estrogentheory as dubious (Zumoff et al., 1975). Wynder and his associates indicated a possible role of prolactin in breast carcinogenesis(Wynder et al., 1981). They speculated that the prolactin/estradiol ratio (P/E,) may reflect the risk for breast cancer. Their proposal might have been based on the fact that the level of plasma prolactin was increased under the influence of excessive dietary fat, which was known to promote breast cancinogenesisin animals (Chan et al., 1977)and in humans (Wynder et al., 1981), and that plasma estradiol drops at menopause, a finding which suggests an inverse relation between the level of plasma estradiol and the risk of breast cancer (Reddy et al., 1980). The relation between plasma prolactin and breast carcinogenesis as tested by either the within-population study or the between-population study is as yet obscure (Hayward et al., 1978; Nagasawa, 1979). The proposition that a breast cancer patient (or a subject with an increased cancer risk) is associated with reduced androgenicity and/or with increased estrogenicity as compared with the corresponding control was not substantiated by laboratory data: both the between-population and within-population studies revealed no consistent differences regarding (1) urinary andro-

TABLE I1 NEUTRAL STEROIDS IN URINE Name of steroid (abbreviation)

Physiological classification

Androsterone (A) Etiocholanolone (E) Dehydroepiandrosterone(D) 1 I-Ketoandrosterone(KA) 1 1-Hydroxypregnanolone(HPn) Pregnanolone (Pn) Pregnanediol (Pd) Pregnanetriol (Pt) Tetrahydrocortisone (THE) Tetrahydrocorticosterone (THB) Tetrahydrocortisol(THO Cortolone (Cn) p-Cortolone and p-Cortol cp) Cortol (Cl)

Androgen Androgen Androgen Androgen Progestin Progestin Progestin Progestin Corticoid Corticoid Corticoid Corticoid Corticoid Corticoid

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gen, (2) urinary estrogen, (3) plasma androgen, (4) plasma estrogen, and (5) plasma prolactin. The reader is referred to recent publications for details of the data for and against an hormonal implication (Hill et al., 1976a,b); Henderson et al., 1977;Fishman et al., 1978;Moneal et al., 1979;Nagasawa, 1979; Reddy et al., 1980). Our steroid profile analysis, originally based on the Homing method (Horning et al., 1967; Sakauchi and Homing, 1971), permits the separate estimation of 14 identified steroids of urine with one run. Table I1 presents the names of the steroids together with their hormonal classification. The recognition that all the steroid members in the table are directly or indirectly linked with each other in the network of steroid metabolism (to be shown later) is essential for an understanding of intersteroidalrelations: in either the group data or the follow-up data of one subject, the excretion of one steroid often exhibits a positive correlation with that of another steroid (Kodama and Kodama, 1975). A low correlation coefficient indicates that the paired steroids are remotely located in the metabolic map, or are stagnant in the metabolic interconversion. Thus one can test the presence or absence of ovulation failure in the group data or in the follow-up data of a subject by investigating the extent of correlation among the four pregnane steroids (Kodama et al., 1977). In establishing the regular association of a specified steroidal change with a defined hormonal disorder, we often employed a clinically proved case as a “Rosetta stone” after the strategy of a genius Egyptologist (Kodama et al., 1979a, 1982). Figure 7 presents the deviation of 14 urinary steroids in premenopausal patients with breast cancer (Kodama and Kodama, 1981). The depressed

+5 c

-5

t

E. :

KA HPn Pn Pd Pt [THETHBTHF Cn I3 CI

A

E

O

FIG. 7. Deviations of premenopausalpatients with breast cancer from normal controls in the excretion of 14 urinary steroids. Comparison was made between 81 patients and 135 healthy women of correspondingages. The deviations ofthe case group were expressed by t values of the Student’s f test. A minus sign means reduction from the normal level. Refer to Table I1 for the abbreviations of urinary steroids. (Reproduced from Kodama and Kodama, 198 I .)

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MITSUO KODAMA A N D TOSHIKO KODAMA

excretions of four pregnane steroids [4-hydroxypregnanolone(HPn), pregnanolone (Pn), pregnanediol(Pd), and pregnanetriol (Pt)]suggestan association of ovulation failure with breast cancer patients. The proposed disorder of ovarian function was tested by both intersteroidal correlationanalysisand a case-control comparison of the reproductive activity. As anticipated, our patients were associatedwith a disruption of two key steroidlinkages (Pn - Pd and HPn-Pt) which was not detectable in the normal controls. It was indicated that the patients had a disturbance in progesterone synthesis. The patients were also found to have significantlyless live births than the normal controls (Kodama et al.. 1977), and the age at first birth was significantly higher in the patient group than in the control group (Kodama et al., 1982). Another feature of the steroid deviation is an increased excretion of tetrahydrocortisol (THF) in the patient group as compared with the control group (Fig. 7). In the postmenopausal comparison,the depressionof four pregnane steroids is not detectable, but the increased excretion of THF still persists together with additional increases of other corticosteroids (Kodama et al., 1975). Evidence is available to suggest that ovulation failure in a premenopausal patient is a secondary product of the preexisting hypercorticoidism (Kodama et al., 1979a). The association of a breast cancer patient with the dual steroidal disorders (hypercorticoidism and depression of endogenous progesterone)was lately confirmed by another research group (Drafta et al., 1980). Bulbrook and his associatesreported that the risk for breast cancer in premenopausal women inversely correlated with the level of plasma progesterone at the luteal phase, and also with the excretion of urinary androgen metabolites (Bulbrook et al., 1978). Plasma estradiol did not correlate with the risk. Their results on plasma progesteroneare in good agreement with our urinary steroid data. Although there was no reduced excretion of urinary androgens in Japanese patients (Fig. 7), some patients with a relative deficiency of urinary androgen are more likely to be nonresponders to hormonal therapy (ovariectomy) than other patients with euandrogenic excretion (Kodama et al., 1975, 1979a). It is possible that the activity of endogenous androgen plays a role in forwardingtumor malignancy but not in initiating breast carcinogenesis. The reported inverse relationship of the discriminant function to cancer risk (Bulbrook et al., 1962) could be reconciled with our results, provided that a reduction ofthediscriminant in a patient reflects an increase of urinary corticoid rather than a reduction of urinary androgen. In support of this interpretation, the same British group reported that British women with a high risk for breast cancer excreted more urinary corticosteroids than Japanese women with a low cancer risk. The level of urinary androgen was also higher in British women than in Japanese women (Hayward et al., 1978). Likewise, the excretion of many steroids including androgen and corticoid was higher in urban Japanese women

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(high-risk group) than in rural Japanese women (low-riskgroup) (Kodama et al., 1975). It may be that only a part of the geographic influences (increase of THF) is responsible for an increased risk for breast cancer. C. CERVICAL CANCER The hormonal aspects ofcervical cancer do not seem to be a very incentive thesis to either gynecologists or endocrinologists. Quite a few investigators ventured clinical studies along the line of hormonal carcinogenesis(Jones et al., 1958). Studies were limited to the activity of endogenous estrogen, and the case-control difference was inconsistent (Fracer et al., 1967). Thus, it might be fair to say that, in a state of substantial ignorance, one should refrain from drawing any conclusion about the thesis. In starting a hormonal study of cervical cancer, we were motivated by the confident anticipation that the cervical epithelium in growth and maintenance is under the concerted actions of various steroids, and that cervical carcinogenesiscould likewise be conditioned under some unbalanced actions of the same steroids. As in patients with breast cancer, the case-control difference was calculated for each of 14 steroid excretions, and the results are illustrated in Fig. 8: a premenopausal patient with cervical cancer is associated with a significant reduction of three androgens and two corticosteroids (Kodama and Kodama, 1981). The direction of deviation and the combination of affected steroids in cervical cancer were different from those in breast cancer. The extent of the steroidal deviations was independent of the progress of the disease (Kodama et al., 1977) and was not affected by radical surgery (unpublished data). These findings indicate that the observed deviations of urinary steroids in cervical cancer could be of etiological relevancy to the

"I 10

@B

c

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I

A

E

D KAiHPnPn Pd Pt iTHETHBTHFCn 0 CI

1 FIG. 8. Deviations of premenopausal patients with cervical cancer from normal controls in the excretion of 14 urinary steroids. Comparison was made between 59 patientsand 60 healthy women of correspondingages. (Reproduced from Kodama et al., 198 1.)

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--Normal women x-xCcrvical cancer patients

;

0

0

1

10 20 30

40 50 60

70

Age (years)

FIG. 9. Age dependency of androsterone excretion in women with and without cervical cancer. (Reproduced from Kodama and Kodama, 198 I .)

tumor. It is noteworthy that the affected steroids in cervical cancer are all of adrenal origin, and that their excretions in a normal woman were distin guished from those of other steroids by a remarkable age dependency (Kodama et al., 1978). Figure 9 illustrates the change with age in the excretion of androsterone (A) in normal women as compared to patients with cervical cancer: in the former, the steroid excretion exhibits a steep adolescent rise, followed by a gradual decline during middle to old age. In the latter, the excretion level always remains at the level of the postmenopausal stage irrespective of the host age. This finding, combined with the indifference of the steroidal deviations to the progress of disease, leads one to the notion that a patient with cervical cancer would have missed (by any reason) the adolescent rise of adrenal steroids (adrenarche)during her youth. In this context, it should be recalled that, on the basis of abundant epidemiological evidence, adolescence plays a crucial role in establishing the predisposition for both cervical and breast cancers, and that a depression of adrenal steroids in cervical cancer contrasts sharply with an increase of adrenal steroids plus ovarian dysfunction in breast cancer.

D. ENDOMETRIAL CANCER The notion that estrogen is the criminal in the production of endometrial cancer was not conceivedwithout reason: endometrial hyperplasia, an entity that has been considered as the precursor of endometrial cancer from its morphological traits, was induced by long-term administration of estrogen (Kistner, I 972). Epidemiologically, endometrial cancer is often associated with signs of endocrinological disorders (Wynder el al., 1966).Patients with

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endometrial cancerwere often found to have a history ofestrogen use (Mack, 1978). However, it could be noted that endometrial hyperplasia is not neoplasia. There is a long distance to go between hyperplasia and cancer. Reported signs of hormonal disorder may not necessarily be related to a state of hyperestrogenism. In addition, cancer patients with signs of hormonal disorder represent just a minority group (Lucas and Yen, 1979). The association of the neoplasia with the history of estrogen use could be protopathic (Feinstein and Horwitz, 1978): a woman with both climacteric disorder and incipient cancer (a highly probable combination, if the cancer is hormone related) may have more access to remedial estrogen. In this case, the production of endometrial cancer should not be accounted for by the effect of exogenous estrogen. There is no evidence to prove that long-term administration of estrogen causes endometrial cancer (Kistner, 1972;Segaroff, 1975). Lucas and Yen Fonducted a case-control study regarding the plasma levels of seven hormones: (1) growth hormone, (2) insulin, (3) prolactin, (4) follicle-stimulating hormone, ( 5 ) luteinizing hormone (LH), (6) estrone, and (7) estradiol. No significant difference was detected with all items between 16 nonobese postmenopausal patients with endometrial cancer and 16 controls matched for age and weight. It should be noted that their hormonal survey failed to cover the realm of neutral (nonphenolic) steroids, Again, we applied the same method, steroid-profile analysis, to endometrial cancer. Figure 10illustrates the deviation diagram of 14 urinary steroids in 28 postmenopausal patients as compared with 55 postmenopausal controls (Kodama et al., 1982). To our satisfaction, the combination of affected steroids and the direction of deviation in endometrial cancer were different from those in breast and cervical cancers. Again, the steroidal

'

A

E

D KA'HPnPn Pd Pt .THE THBTHF Cn

D

CI

FIG. 10. Deviations of postmenopausal patients with endometrial cancer from normal controlsin the excretion of 14 urinary steroids. Comparison was made between 28 patients and 55 healthy women of corresponding ages. (Plotted from the data of Kodama el a!., 1982.)

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2 Pregnenolone ,

1

17u-Hydroxyprogesterone

1

1

1

/

I

,',(Estrone) 11-Deoxyhndrostenecor ticosterone 3.17-dione Cortisol + Cortisone 1' 'kAndrosteneJ. L. ll-ol-3,17-dione Corticosterone (THF) (THE) Testosterone 1 ,/ +/ ( A f $1 (TH B) (CI) (fl-CI) (Cn) (R-Cn) A4Androstene3,11,17-trioneI or Adrenosterone (KSA)

I

1

FIG. I 1. Metabolic relation between endogenous and urinary steroids. A urinary steroid is placed in parentheses. Some endogenous steroids (such as D) are excreted as such in urine. (Reproduced from Kodama ef al., 1982.)

changes of the patients were not affected by radical surgery (Kodama et al., 1982).The most prominent trait that distinguished endometrial cancer from the other two cancers was the increase of urinary 1 1-ketoandrosterone(KA). On the basis ofa parallel study on urinary steroids(Rosettastone operation!), we reached the conclusionthat urinary KA was ofandrogen origin (Kodama et al., 1982). The metabolic relation between our 14 urinary steroids and endogenous steroids is summarized in Fig. 1 1. In the figure, a reduction of urinary A and etiocholanolone (E), as observed in endometrial cancer, indicates that there is a stagnation in the metabolic pathway from androstenedione to testosterone. Likewise, the observed increase of urinary KA indicates an acceleration of the metabolic flow from androstenedione to androstenetrione (adrenosterone). This interpretation is in good agreement with the report of Calanog and his associatesthat the conversion of testosterone to androstenedionein patients with endometrial cancer was significantly decreased (Calanog et al., 1976). The sequence of events in an endometrial cancer patient could be described as follows: she is primarily defective in the production of endogenous testosterone [decrease of urinary A, E, and dehydroepiandrosterone(D)] and suffers from an excess of 3-A4-androstene compounds (increase of urinary KA)that are known to possess antigonadotropic activity. In consequence,the secretions ofpituitary LH and ofits target steroid, progesterone, will drop (decrease of urinary HPn, Pn, Pd, and Pt). Depression of endogenoustestosteronewill automaticallylead to a reduction of endogenous corticosteroids [decrease of urinary tetrahydrocortisone (THE), tetrahydrocorticosterone (THB), THF,cortolone (Cn), P-cortolone

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u),

and 8-cortol and cortol (Cl)] since there exists a homeostatic balancing power operating between protein-anabolic testosterone and protein-catabolic cortisol via the hypothalamus-pituitary system. The problem of what mechanisms underlie the production of three specific steroidal disorders in three female cancers will be discussed in Section V together with their possible relevancy to their epidemiological traits. IV. New Trends in the Biology and Molecular Biology of Steroid Hormones

A. GENERAL CONSIDERATIONS

Before constructing a unifying theory about hormonal carcinogenesis,it is essential to have a better understanding of the mode of action of various steroids in the target tissue. Recently, we greatly increased our knowledge about the molecular mechanism of steroid action and experienced some conceptual modification about the readiness of the receptor site. There used to be a belief that cervical cancer is irrelevant in its genesis to the hormonal milieu of the host, since the tumor is not responsive to hormonal therapy. In fact, the epithelium of the vagina and cervix changes its morphology in response to the actions of various steroids (Wied and Bibbo, 1970). It is also well known that the administration of corticosteroids leads to adaptive formation of many hepatic enzymes without appreciable change of cell population (Higgins and Gehring, 1978). Lippman reported that a tissue culture cell line MCF-7 derived from human mammary cancer had four distinct receptors which were each found to bind specifically estrogen, androgen, glucocorticoids, and progesterone to exert steroid-specificactions on cell growth (Lippman et a[., 1976). Evidence indicates that the development of the mammary tissue as well as its neoplasia is promoted by the concertedeffectsof multiple hormones(Topper, 1970;Koyama et al., 1972). It is intriguing and pertinent to explore the problem of how multiple hormonal actions could be organized to trigger a predestined sequence of events in a target cell. This section will deal with some specific aspects of multihormonal actions together with the interaction of a specified steroid hormone with its target gene. Another feature of steroid action to be discussed is the duration of the hormonal effect: for example, the carcinogenic action of diethylstilbestrol was demonstrated in a fetus (Herbst et a!., 1972, 1974) but not in an adult (Segaloff, 1975). It was indicated that administration of the hormone produced an irreversible change in the immature target cell whereas, in the well-differentiatedtarget cell, the same treatment induced just a transitional change which disappeared upon withdrawal of the hormone. The above problems will be reviewed in subsequent sections using five experimental models, which we find appropriate to give further insight into the molecular mechanism of hormonal carcinogenesis.

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B. CHICKOVIDUCT The response of chick oviduct to estrogen is of a transient nature, the synthesis of egg white proteins falling rapidly upon withdrawal of the hormone (Schimke et af.,1975). It is worth noting that the proportion of ovalbumin, conalbumin, and ovomucoid synthesized in the oviduct can be altered by the combination of estrogen, progesterone, and testosterone (Palmiter, 1972). There is evidence to suggest that these hormones may not be acting by the same mechanism involving binding at the same site (Schimke et af., 1975). The action of estrogen on chick oviduct is largely explained by an increased transcription of egg-protein mRNA (Schimke et al., 1975). It should be mentioned, however, that there are a variety of findings which do not fit a simple model assuming an unconditioned interaction between one steroid - receptor complex and one specified gene (Higginsand Gehring, 1978):for example, testosterone administrationper se has little effect on the growth of the oviduct in immature chicks, whereas, in combination with estrogen administration, it results in a synergisticstimulation of the oviduct (Schimke et af.,1975). Probably, estrogen prepares the receptor site for testosterone stimulation.

C. INSECTS

I N GROWTH

The process of molt and metamorphosisprovides a valuable model system for the study of developmental endocrinology. For example, the silkworm Bombix mori, like other homometabolous insects, molts four times during its larval stages, and then starts spinning a silken cocoon within which the first metamorphic molt occurs, i.e., larva to pupa. After an appropriate time interval, a nonfeeding adult moth emerges from the old pupal cuticle and cocoon (second metamorphic molt) and mates. Throughout the whole growth period from the first larval instar until the emergence of the adult moth, two insect hormones are involved in the induction of molt and metamorphosis: juvenile hormone (JH) from the corpora allata and ecdysone (steroid hormone) from the prothoracic gland (Gilbert, 1974). The terminal metamorphic stages of an insect could be compared to the adolescence of a human being. Ecdysone stimulatesboth molt and metamorphosis, whereas JH suppresses metamorphosis in the presence of ecdysone. Otherwise, JH does not affect the developmentalprocess (Gilbert, 1974).Thus, the molt, as induced by ecdysone, will be larval-larval (high JH titer), larvalpupal (lowJH titer), or pupal -adult (no JH) dependingon the activity ofJH. The above-mentioned four regular molts and two metamorphic molts are expected to occur in the regular order in nature. Exposure to an inappropriate temperature, for example, during the larval stages often resulted in the formation of larvae with varying degrees of pupal characteristics(prothetely

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or metathetely). Such admixture larvae invariably failed to emit silk fibers from their spinneret. Careful dissection of these larvae revealed variable deformities in the structure of their silk glands. The production of these abnormalities in the outer and inner structures of the larval body is explainable in terms of an imbalance between ecdysone and JH during the preceding instar, a state which could be induced under unfavorable culture conditions (mainly in diet and temperature; Fukuda, 1952, 1956). In contrast to the response of chick oviduct, the hormone-induced changes of the insect are permanent and irreversible. Another interesting feature of ecdysone is found in the hormone-gene interaction: ecdysone causes puffing in salivary gland polytene chromosomes of the blow fly, Chironomus tentans (Karlson and Sekeris, 1966). The phenomenon takes place at specified regions of the chromosomes and in a specified time sequence (Ashburner et al., 1974). There is evidence that suggests that the puffed region of the chromosome is the visual manifestation of mRNA synthesis (Gilbert, 1974). In another blow fly, Calliphora erythrocephala, the rise of endogenous ecdysone chronologically coincided with the rise of DOPA-decarboxylase activity which is known to mediate the sclerotization of the larval cuticle (Shaaya and Sekeris, 1965). Furthermore the content of DOPA-decarboxylase mRNA in the epidermis so increased at the stage of white prepupae as to match the increase in the ecdysone content as well as in the activity of DOPA-decarboxylaseitself (Fragoulis and Sekeris, 1975). All these findings seem to suggest that the sequence of events proceeds along the line of one hormone -one gene -one enzyme. Unfortunately, another surge of ecdysone that appeared in the middle ofthe pupal stage was not accompanied by a corresponding rise of DOPA-decarboxylase activity (Shaaya and Sekeris, 1965). It was indicated that the expression of ecdysone action was subject to a change depending on the degree of differention of the receptor cells. VIRUS D. TUMOR

It is well established that genetics, hormones, and viruses play etiological roles in the production of mouse mammary cancer. For a long time, the interrelation among them in mammary carcinogenesis remained imperfectly understood. Furthermore, there was no unequivocal evidence to indicate that viral infection is implicated in the genesis of human breast cancer. Recently, the concept of viral oncogenesis underwent a drastic change with the discovery of cellular oncogenes which reside within the chromosomes of a normal cell as a part of the constitutional gene assembly (Stehelin et al., 1976). Possible implications of endogenous oncogenes in leukemogenesis were suggested on the basis ofchromosome analysis (Klein, 198 1) (referred to later

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in this section). Independently of the emergence of the oncogene theory, influences of various hormones on the production of mouse mammary tumor virus (MMTV) have been attracting the interest of experimental oncologists. Of the several groups of mammotropic hormones tested, a glucocorticoid was found to have the most dramatic inductive effect on MMTV production in the tissue culture system (McGrath and Jones, 1978). There may be some relevancy to the fact that a glucocorticoid plays an essential role in the differentiation of secretory precursor cells in normal mammary glands (Topper, 1970). Of prime importance is the fact that uninfected normal mouse cells contain by nature multiple copies of MMTV DNA covalently integrated into the genome (Vermus et al., 1972). The inductive effect of a glucocorticoid is detectable in the tissue culture of premalignant BALB/cfC3H and malignant BALB/c or BALB/cfC3H cells, but not in that of BALB/c normal mammary epithelial cells (McGrath and Jones, 1978). The same hormonal stimulation on virus production was detectable in cultured normal cells which had been pretreated with 5-iOdO2’-deoxyuridine (MacGrath and Jones, 1978). These findings indicate that the MMTV cDNA in the normal cells is in a locked state, and that the halogenated nucleotide relaxed repressive controls over specific MMTV genes. Like the actions of other steroids, the stimulative effect of a glucocorticoid on viral RNA production could be explained in terms of an acceleration of specific gene transcription (Yamamoto and Ringold, 1978;McGrath and Jones, 1978). Yamamoto and his associates succeeded in infecting rat hepatoma (HTC) cell line with MMTV. Interestingly, the infected rat cells were so stimulated by a glucocorticoid as to increase the production of MMTV: in the case of one infected cell line, M 1.19, the basal intracellular content of MTV RNA without hormonal induction turned out to be about 25 transcripts per cell, whereas it increased about 500-fold after induction with 1O+ Mdexamethasone (Yamamoto and Ringold, 1978).Furthermore, the inducibility of tyrosine aminotransferase (TAT) and glutamine synthetase (GS), other reflections of glucocorticoid-responsive genes of HTC cells, underwent a change after virus infection (Yamamoto and Ringold, 1978). These findings suggest that the same hormone-responsive promoter gene controls the transcriptions of both newly integrated MMTV cDNA and constitutional hepatic genes that are responsible for the induction of TAT and GS. In the classical dogma of viral carcinogenesis, an oncogenic virus was considered to be a carrier of a viral oncogene that is quite foreign to the natural hosts. Following viral infection, the oncogene is integrated into chromosomes ofthe host cell to induce malignant transformation. Biological properties that are specific for a tumor cell are the phenotypic expressions of the integrated viral oncogene itself. The whole process of malignant trans-

--

HOST STEROID METABOLISM AND GENESIS OF CANCER

LTR

Cell’”*-

Integrated Provirus

gag

pol

99

LTR Cell DNA

env

(c-onc)

U,,&bQZDZ=

RNA containing viral

v

and cellural information-

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Normal viral RNA gag

u3

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FIG. 12. Structureand transcriptional productsofthe integrated avian leukosisvirus(ALV). LTR, Long terminal repeats; U,, unique sequence derived from the 3’ end of ALV genomic RNA; U,, unique sequence derived from the 5’end ofgenomic RNA; c-onc, cellular oncogenes: gag, gene specifying a series of internal virion; pol. gene encoding the reverse transcriptase; m v , gene coding for the envelope glycoprotein: “c,” putative promoter for eukaryotic RNA polymerase 11. (Reproduced with some modification from Hayward ef a/., 1981.)

formation was classified as mutation, since the genetics of the host cell underwent a dramatic change due to the introduction of an exogenous oncogene. The situation is quite different as far as so-called “retroviruses” are concerned: an oncogenic RNA virus is mostly a composite of viral structure genomes (gag,pol, and env) and a transforming genome v-onc, as shown in Fig. 12 (Hayward et al., 1981). The noninfected cells of the natural hosts possess a nuclear DNA sequence which is complementary to the viral RNA genome (Fig. 12). The complementary DNA sequences, under appropriate conditions, could be activated to produce transforming protein and render the host cell malignant (Klein, 1981). The transforming sequence of one retrovirus may not be the same as that of another retrovirus. Evidence indicates that the complementary DNA sequences have been conserved in the noninfected natural host animals for several million years (Morris et al.. 1977). Therefore, it is quite conceivable that the retrovirus-related transforming genes in a well-controlled state play a role in the maintenance of normal cell function (Klein, 1981). The conclusion derived from the above observations is that all the normal cells with the constitutional oncogenes are capable of becoming malignant under any provocative circumstances, and that an “oncogenic”(?) virus plays a role in just switching on the machinery of cellular oncogenes (Klein, 1981). Recently, Hayward et al. produced lymphomas in chickens by inoculating nontransforming avian leukosis virus (ALV). This virus was known to lack a discrete “transforming gene.” They suggested that the production of lymphoma was just an enhanced transcription of a constitutional oncogene (c-oncin Fig. 12),and that ALV played the role of promoter in leukemogenesistaking over a locus adjacent to the c-onc (Nee1et al., 1981). The same neoplastic transformation could be induced by the introduction ofrandomly sheared DNA from normal cells (Cooper et al.,

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1980) or by chromosome translocations (Klein, 1981). The new oncogene theory is revolutionaryin that neoplastic transformation does not require the participation of specified mutagenes to be introduced from the outside environment. The production of a tumor that isjust an increased expression of normal cellular genes (Klein, 1981) could be triggered as a sequence of genetic events by nonspecificincentives such as radiation, chemicals, and an unbalanced set of endogenous hormones. E. VAGINAL EPITHELIUM

Since 1963, Takasugi and his associates have presented a series of investigations on the influence of neonatal steroid administration on the genital tract of mice. The most interesting finding is that persistent proliferative cornification of vaginas was induced in mice by five consecutive injections of estradiol starting on the first day of birth (Takasugi, 1963). In spite of a persistent estrous state of the vaginal epithelium, the uteri were mostly at the state of threadlike atrophy (Takasugi and Bern, 1964). Ovariectomy performed at 55 -65 days of age reduced the incidence of hyperplastic vaginal lesions, but did not completely eradicate the hormonal effect (Takasugi, 1979).The estrogen treatment starting later than 8 days ofage failed to induce any persistent vaginal changes (Takasugi, 1979). Hyperplastic vaginas, as induced by neonatal injections of estradiol, were found to be transplantable vaginal carcinomas in syngeneic mice (Takasugi, 1972). Similar neonatal administration of testosterone (Takasugi, 1979) or progesterone (Jones and Bern, 1977) also induced persistent vaginal cornification in mice. Concurrent use of progesterone and estradiol at the neonatal stage rather decreased the incidence of persistent vaginal cornification as compared with the single use of estradiol (Jones and Bern, 1977). From these observations, one could draw a few important conclusions together with some speculative comments about the genesis of the genital tract cancer: (1) estrogen could induce in the neonatal vagina irreversible lesions which are considered to be a precursor ofgenuine neoplasia; (2) the above sensitivity of the vaginal epithelium to the carcinogenic action of estrogen vanishes not later than 8 days after birth; (3) the vaginal lesions, once induced by exogenous estrogen, become independent in its maintenance of the hormonal environment of the host; (4) for the production of preneoplastic changes in the mouse vagina, the predominance of a single hormone rather than the choice of a specified steroid is crucial. It is worth noting that earlier reports from Takasugi’s laboratory appeared prior to the reported appearance ofvaginal cancer following fetal exposure to a synthetic estrogen (Herbst et al., 1972, 1974). Reading through Section IVYone may notice that steroid hormones can induce irreversible (or neoplastic)changes

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only in those tissues that are undergoing differentiation (a larva in metamorphosis and vagina at the neonatal stage). In this context, it is pertinent to recall the fact that the squamocolumnar junction of the uterine cervix is the locus of cellular differentiation during adolescence, and that the lesions of early cervical neoplasia often occur near that transition zone (Wilbanks, 1973). V. Synthesis of a Unifying Theory

A. JUNCTION BETWEEN EPIDEMIOLOGY AND ENDOCRINOLOGY Epidemiologicalcharacteristicsthat discriminate among patients with the three female cancers could be related to three factors: climate, diet, and sex life (Table I). They are each supposed to play a role in establishing the hormonal constitution of the patients. In consonance with the above reasoning, steroid analysis in our laboratory revealed that patients with cancers of the breast, uterine cervix, and endometrium were each associated with specified different deviations of urinary steroids in the combination of affected steroids and the direction of deviations (Figs. 7,8, and 10). Further insight into the nature of those hormonal disorders suggested that the observed changes of steroid metabolism in cervical cancer patients could have been produced during adolescence(Fig. 9). The notion that adolescence plays a crucial role in the genesis of those hormone-related cancers found further support from epidemiological studies (detailed later in this section) for each of the three female cancers. Experience with the rat vagina (Section IV,E) suggests that some specified hormonal imbalances may induce irreversible changes in the breast and uterus of a young girl that are destined to grow and differentiate during adolescence. The above notion was conceived on the basis of the understanding that the progress of all these changes was mediated by the concerted effects of steroids from the maturing ovary and adrenal. Along a similar line is the observation on insect metamorphosis that unfavorable conditions in diet or in culture room temperature may lead to an aberration from the normal course to produce morphological deformities (Fukuda, 1956). Some of the defective individuals are regarded as premature pupae with adult characters (prothetely) and others as adults with pupal characters (metathetely). The partial inhibition or promotion of metamorphosis, as mentioned above, could be explained by an imbalance in the ecdysone-JH interaction (Wigglesworth, 1934; Fukuda, 1952). In Sections I1 and I11 we demonstrated that there is a sharp contrast between cancers of the breast and uterine cervix in regard to the epidemiological backgrounds of patients, and that the hormonal disorder in cervical

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cancer, a steroidal expression of arrested adrenal maturation, could have been produced during adolescence. Thus, one is tempted to speculate that the steroidal deviations in cancers of the breast and uterine cervix may each be related to two extremities of imbalance in ovarian -adrenal maturation. More specifically, an arrest of adrenal maturation combined with a promotion of ovarian maturation will lead to the production of cervical cancer, whereas an overrun of adrenal maturation at the sacrificeof ovarian maturation will induce breast cancer (Kodama and Kodama, 198 1). Before testing the validity of the above hypothesis, it is imperative to have more information about conditions which may accelerate or retard the maturations oftwo puberty promoters, the ovary and adrenal. For that purpose, a number of epidemiological parameters that were found useful in cancer research were divided into two groups on the basis of the developmentaldependencyon the two endocrine organs: (1) height, adrenal indicator; the physical growth is stimulated by adrenal androgen; (2) weight, adrenal indicator; (3) age at menarche, adrenal indicator; the menstruation itself is related to the ovarian function, but the puberty onset undergoes a permissive influence from the maturing adrenal (Gorski-Firlitand Lawton, 1974; Gelato et al., 1978); (4) age at first birth, ovarian indicator; and ( 5 ) number of live births, ovarian indicator. It has long been known that Japanese children of primary school age are taller and heavier in the northeastern districts than in the southwestern districts. Figure 13 illustrates the north -south difference in the growth of 1 1-year-old Japanese girls: in the figure, each point represents the mean value for one prefecture. Apparently,girls in the northeastern Tohoku- Hokkaido district (solid circles) grow faster than those in the southwestern Kyushu

6 .L

'C

145144143142I

I

I

I

I

3 4 35 36 37 38 WQight

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FIG. 13. Differential growths of I 1-year-old girls among 16 prefectures of Japan. Each figure represents the mean value for each prefecture. The seven solid triangles are derived from seven prefectures of the Kyushu district (the southwesterncomer of Japan); the seven solid circles are the figures from Seven prefecturesoftheTohoku-Hokkaidodistricts(the northeasterncomer of Japan); the open circle at the right represents the figure for the Tokyo Prefecture, and the one at the left for the Osaka Prefecture. (Reproduced with some modification from Kodama and Kodama, 198 I .)

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district (open triangles). Girls in the metropolitan areas (Tokyo and Osaka) of central Japan (open circles) were situated just in between. Again, girls living in the northern city, Tokyo (at right), are heavier than those in the southern city, Osaka (at left). The above geographicdifferencein the growth of Japanese girls is in accord with the famous Bergmann’s Rule that the size of a mammal tends to increase progressively with an increase of latitude. It may be that an individual living in a cold climate needs more fat than the one in a warm climate in order to minimize thermal loss from the body surface. The increased intake ofa fatty diet should be met with an increased secretion of corticosteroids that are involved in fat metabolism (Meier, 1977;Fain, 1979).Thus, the adrenal maturation of a northern girl could have been so accelerated under the influence of a fat-rich diet as to surpass a southern girl in height and weight. In support ofthe above explanation, intake ofa fat-rich diet increased the excretion of 17-hydroxycorticosteroidsin humans (Simset

al., 1973). There is general agreement about an associationbetween an increased risk for cervical cancer and early onset of sexual life (Rotkin, 1973). Our steroid analysis indicated that in patients with cervical cancer there is an arrest of adrenal maturation. One may wonder whether or not there is any causal relationship among early coitus, arrested adrenal maturation, and cervical carcinogenesis. To our knowledge, no clinical investigation has ever been attempted to prove that premature start of sexual life can induce an endocrinologic disorder. Hence, the answer to the above question should be sought in studies of experimental endocrinology. It is well known that the practice of vaginal stimulation, a substitutefor copulation,induces a release of luteinizing hormone in the pituitary gland and ovulation in the ovary of a rabbit (Dufy et al., 1974).The same manipulation in virgin rats gave rise to a surge of prolactin release (Alonso and Deis, 1973-74). Thus, one can produce a state of pseudopregnancy in rats by mechanical stimulation of the vagina and cervix uteri. More importantly, virgin rats in a state ofman-made pseudopregnancy exhibited a reduced corticosterone production of the adrenal, and also a reduced pituitary response to stress, as measured in terms of the release of prolactin and adrenocorticotropic hormon (ACTH) (Vasquez and Kitay, 1980).These findings indicate that mechanical stimulation of the vagina exerts a stimulativeaction on the ovary and an inhibitive action on the adrenal via the hypothalamus- pituitary axis. As shown in Section 11, relative sterility is a common feature of patients with cancers of the breast and endometrium. Can the association be accounted for by one and the same mechanism? Comparison of two cancer incidences revealed discrepanciesof cancer risks in time and space which will be described later in this section. The steroidal deviations in cancer patients are also different. While the excretions of progesterone derivatives (four

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pregnane compounds) were reduced in patients with either breast or endometrial cancer, the excretion of urinary androgen was intact in breast cancer patients, and markedly reduced in endometrial cancer patients. The excretion of urinary corticosteroid was increased in the former and reduced in the latter. On the basis ofthe urinary steroid findings, we suggestedthe possibility that the process ofadrenal maturation (adrenarche) in a breast cancer patient could have been promoted at the sacrifice of ovarian maturation (gonadarche), and that a specified combination of climate, diet, and sexual activity is responsible for an unbalanced maturation of the ovarian - adrenal axis. It seems implausible that the same mechanism is operative in reducing the reproductive activity of an endometrial cancer patient, since there are distinct differences between two cancers in the epidemiological as well as endocrinological features. Is there any evidence available to indicate that the reproductive activity of an animal could be suppressed by factors other than diet and sanitation? Calhoun’s report might be relevant to this question: the productivity of experimental animals in a confined space decreased in proportion to the increase of population density in spite of enough dietary supply and sanitation (Calhoun, 1962).Sometimes, the confined population ends with complete extinction after experiencing a temporary population explosion (Machida, 1978). It was indicated that the observed reduction of reproductive activity in the aging society should be accounted for by psychological stress ensuing after the collapse of the social structure rather then the pressure of population density itself (Christian and Lemunyan, 1958).It was interesting to note that the crowding effect on fertility was more marked in the progeny females than in the precedent females (Christian and Lemunyan, 1958), probably because the aged animals born early in the history of a population are likely to be dominant over animals born later (Christian, 1965). Although ample evidence was presented to suggest adrenal implication in self-regulation of the mammalian populations, the nature of the regulation mechanism still remains to be clarified (Christian et al., 1965). In conclusion, four factors were proposed as subjects that would deserve consideration in elucidating the genesis of the three hormone-related cancers: (1) climate, (2) diet, (3) sexual life, and (4) social stress.

B. GENESIS OF BREAST CANCER Reading through the preceding discussion, one may raise a few questions concerning the genesis ofbreast cancer. (1) Is adolescencealso critical for the establishment of cancer risk in the breast as it is in the uterine cervix? (2) Is there any feasible explanation as to why mammary carcinogenesisshould be conditioned by a specified hormonal disorder (Fig. 7)? (3) What is the situation of hormones in mammary carcinogenesis? Initiation or promo-

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tion? Land and his associates calculated the risk for various cancers on the basis ofthe cancer registry ofpatients from the radiation clinicsin the United States, and of atomic bomb survivors in Hiroshima and Nagasaki, Japan: they reached the conclusion that, irrespective of the source of radiation, the risk for breast cancer was highest in those women who were exposed to that hazard in adolescence(Land et al., 1980). Lee and his associatesreported that the rate of increase with age in female cancer incidence was maximal at age 25 years, the lowest limit in this study. It was indicated that the postulated precursor of breast cancer is already prevalent by that age (Lee et al., 1976). Huggins and his associates investigated the relation between the timing of carcinogen administration and the incidence of mammary cancer in rats: a maximal cancer incidence was observed in those rats that underwent hydrocarbon treatment at 50 days of age, the terminal stage of adrenarche (Morii and Huggins, 1962). A shift in any direction resulted in a reduction of cancer incidence (Huggins et al., 196 1 ;Dao, 1969). Another interesting feature of mammocarcinogenic hydrocarbons [3methylcholanthrene (3-MC) and dimethylbenz[a]anthracene (DMBA)] is that the steric structure ofthese compoundsresemblesthat ofhydrocortisone (Yang et al., 196 1 ;Wong et al., 1962), and that the administration of those carcinogens inhibits the synthesis of corticosteroids (Dao et al., 1963). DMBA, a powerful mammocarcinogen, was found to induce adrenal necrosis (Morii and Huggins, 1962). The optimal timing of DMBA administration for the production of adrenal apoplexy again coincided with the terminal stage of adrenal development. We found that DMBA administration failed to induce mammary cancer in those rats that had excreted subnormal amounts of 17-deoxycorticosteroidsduring the latent period, as shown in Fig. 14 (Nakamura et al., 198 1). In accord with the above findings,

40 80 120 160 Age (days)

FIG. 14. Secular change of urinary I7-deoxycorticosteroid (17-DOCS) in Sprague- Dawley female rats with and without DMBA (mammocarcinogen) challenge. Group 1 (solid circle), 9 normal rats without DMBA challenge; group 2 (solid triangle), 6 rats which, despite DMBA feedings, failed to develop mammary cancer during an observation period of 200 days; group 3 (open circle),29 rats which developed mammary cancer within 200 days afterDMBA challenge. Each figure representsthe group mean ofthe logarithm of steroid excretion (microgram per day per rat). The difference between groups 2 and 3 was significant(p < 0.05)at 80 and 120 days of age, but not at 160 days of age. (Reproduced with some modification from Nakamura et al., 1981.)

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breast cancer was associated with an increase of urinary (Kodama et af., 1975)and plasma corticosteroid (Drah et al., 1980). Nagasawa pointed out that the increased sensitivityof rats to DMBA at 50 days ofage was matched by a corresponding rise of DNA synthesisor mitotic rate of their mammary cells (Nagasawa, 1977, 1981). All these findings indicate that the risk for breast cancer is conditioned at the terminal stage of adolescence, and that the activity of endogenous corticosteroid plays a crucial role in the genesis of breast cancer. Probably, the action of corticosteroid is to accelerate the transcriptional process of host-derived oncogenes in humans, and that of MMTV-cDNA plus murine oncogenes in mice so as to induce mammary cancer. Progesterone is known to promote lobuloalveolar differentiation of mouse mammary glands either in vivo (Traurigand Morgan, 1964)or in vitro (Koyama et al., 1972). Combined use of progesterone and estradiol-17P strongly suppressed the appearance of DMBA-induced mammary cancer in rats (Hugginset al., 1961).There was, however, no case-controldifference in the excretion of urinary pregnanes at the postmenopausal stage (Kodama et af., 1975), where the difference in breast cancer risk between western countriesand Japan becomes prominent (De Waard etal., 1964).Evidence is also available to suggest that ovulation failure in a breast cancer patient is a secondary response of the ovary to a primary increase of endogenous corticosteroid (Kodama et al., 1979a).It is quite conceivable that ovulation failure (lack of ovarian progesterone) may intensify the carcinogenic action of endogenous estrogen on the mammary tissue, but the extent of its contribution to mammocarcinogenesisis to be assessed in combination with the increased activity of endogenous corticosteroid in the pre- and postmenopausal patients with breast cancer (Kodama et af.,1975).It is almost certain that endogenousestrogen also plays some role in breast carcinogenesis, since artificial menopause at early stages of life reduces the risk for breast cancer. Yet the fact that there is no case-control difference in estrogen metabolism could be taken as evidence in support of a permissive role of estrogen in mammary carcinogenesis. Our knowledge on breast carcinogenesis is still insufficient to answer the third question. One thing is clear: a cancer initiator, if present, will not be very powerful in its effect, because the influence of a promoter would not have been detectable, ifa carcinogenhad been present in excess (Reddy et al., 1980). In reality, the breast cancer risk varies tremendously from one population to another depending on the conditions of climate, diet, and marital life. On the basis of the discussion so far presented, one can describe a candidate at risk for breast cancer: she may grow up very fast and experience early menarche under the influence of a fatty diet and in a cold climate, and/or stay single too long to ensure an integral gonadarche. If married, she may be infertile because of her predisposition for ovulation failure. In old

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age, she may suffer from adrenal-oriented diseases because of her constitutional hypercorticoidism: central obesity, hypertension, diabetes, and osteoporosis are prone to occur in such a woman. These descriptions are in good agreement with the epidemiological findings on breast cancer patients. C.

GENESIS OF CERVICAL CANCER

Western epidemiologists unanimously agree that early coitus is strongly associated with an increased risk for cervical cancer (Rotkin, 1973). This phenomenon was often referred to as supportive evidence for the venereal nature ofthis disease. Some exceptional examples were presented to disprove the validity of exogenouscontamination theories: ( 1) a reduced cancer risk of Navajo women, who start marital life prior to menarche with uncircumcised consorts (Jordan et al., 1969); (2) an increased cancer risk of Bantu women in South Africa, where circumcision is a common practice among the male natives (Cowdry, 1968); (3) no case-control difference in the onset of marriage in Japanese women (Kodama et al., 1978); and (4)a continual decline of cervical cancer mortality in Japan without any corresponding changes in the onset of marriage as well as in sanitation (Fig. 1). Incidentally, all these exceptions were derived from countries outside the western world. The epidemiological data including the above results could better be explained in terms of hormonal implication rather than of contagion (virus or penile smegma). Before proceeding to a discussion of the human problem, experience with insect metamorphosis will be pertinent to explain the above exceptions along the line of hormonal carcinogenesis. Fukuda reported that exposure to higher-than-optimal temperature (28°C)during the larval stages often gave rise to the production of defective silkworms that failed to spin their cocoons at the expiration of fifth instar [they are called “Gorotsuki” (rascals)by Japanese farmers]. The highest incidence of the above defectiveness was observed in those larvae that were exposed to high temperatures at the third or fourth instar. To investigate the relation between the timing of thermal stress and the production of silk gland defectiveness further, the third and the fourths instars were each divided into four and five stagesso that one stage in each of the two instars covered a period of 18 hr (the third and fourth instars lasted for 72 and 92 hr, respectively). Then, the effect of thermal stress (higher-than-optimal temperature) was tested for each of nine stages covering two larval instars. The highest incidence of cocoonless larvae occurred with thermal stress at the second stage of the third instar ( 17.1%), and with the one at the fourth stage of the fourth instar (20.0%), while the incidence in the control larvae cultured throughout at the optimum temperature (24°C) was 1.9%(Fukuda, 1956).Thermal stress at the fifth instar did not produce any significant change in the course of silk gland development (the

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incidence of cocoonless larvae was around 2%). These findings indicate that thermal stressexerts some deterioratinginfluenceon the function of corpora allata to produce deformitiesin the structureof maturingsilkglands,and that the magnitude of the effect varies depending on the progress of insect growth or, more specifically, on the extent of regression of corpora allata (Fukuda, 1956). Likewise, hormonal impact ensuing from the first sexual experience may well vary depending on the progress of adolescent maturation: in a premenarchial girl, the relation between adrenarche and gonadarche might not be very competitive, and the start of marital life will not produce any imbalance between the two processes, as suggestedin the case ofNavajogirls, in whom premature experiences did not increase cancer risk (Jordan et af., 1969). Girls in western countries, living on a fat-rich diet, experience menarche as early as 12 years of age, and continue to grow in stature until 17 or 18 years of age (adrenarche in progress). The onset of marriage during that period may accelerate gonadarche at the sacrifice of adrenarche to increase the risk for cervical cancer. Feldman and his associates reported that mild to severe cervical dysplasia was noted in 188 of 2655 sexually active teenagers, of whom about one-fourth were classified as having severe dysplasia or carcinoma in situ (Feldman et al., 1976, 1978). In contrast, the incidence of cervical dysplasia in Taiwan prostitutes was only 8 of 750 subjects (Sebastian et al., 1978).It is evident that the risk for cervical cancer is conditioned by the premature start of sexual life, but not by the multiplicityof sex partners. Also worth noting is that a critical period conditioning the risk for cervical cancer ranges approximately from menarche until termination of stature growth, neither earlier nor later. In that sense, a human being at adolescence with respect to hormonal similarity has much in common with an insect at metamorphosis. Japanese women of old age, having lived on a fat-deficient diet during adolescence, mostly experienced menarche at 15- 17 years of age, and did not cease to grow before 20 years of age. The start of marital life was estimated to fall between 18 and 25. Consequently, many girls at the prewar time had a chance to catch an arrest ofadrenarcheafter marriage. The recent increase of dietary fat, as is in progress in Japan (Fig. 2a), is likely to have accelerated adrenarche, and the age range at risk was shifted toward the younger side, As a result, the younger generations are going to have an increasingly reduced risk for cervical cancer (less chance for premature marriage) in Japan without any change in the age of marriage. The appearance of specified deviations of urinary steroids in a cervical cancer patient (Fig. 8) led to the emergence of a new concept on cervical carcinogenesis, i.e., an arrest of adrenarche, as evidenced in Japanese patients, accounts for all features of patients with cervical cancer, and is supposed to have etiological relevancy to neoplasia. The activity of endogenous estrogen on the maturing cervical epithelium will be intensified in the

HOST STEROID METABOLISM AND GENESIS OF CANCER

I09

absence of escort steroids, adrenal androgen. The hyperestrogenic milieu thus produced will further induce irreversible hyperplasia and cornification in the adolescent uterine cervix as in the fetal (Herbst et al., 1972, 1974) and neonatal (Takasugi, 1963)vagina. It is very likely that discrete dysplasia and carcinoma in situ could emerge from that lesion under the influence of the unopposed action of ovarian estrogen. The above conjecture found support in the report of Henson and Tarone, who noted that the incidence of carcinoma in situ of the uterine cervix reached its maximum at age 35, and the incidence after menopause became negligibly low (Henson and Tarone, 1977). Apparently, the presence of active ovaries was indispensable for the initiation and maintenance of cervical dysplasia. As in the case of breast cancer, one can depict the features of a woman who is most likely to get cervical cancer: she might have grown up under the influence of a fat-deficient diet and in a warm climate. She might also have been mamed before the termination ofgrowth stage. Because ofthe induced arrest of adrenarche, she may suffer from androgen deficiency:she is smaller and thinner than average (Kessleret al., 1974), has a feeling ofdislike for sex because of reduced libido, and tends to be unstable in marital life (Stern et al., 1967; Stephenson and Grace, 1954), but is as reproductive as a normal woman (Kodama et al., 1977). One may ask whether readiness for getting cervical cancer is completed by the end of adolescence? A clue to the answer will be found in the rarity of cervical cancer in Jewish women, a fact that is contrasted with the relatively high incidence of cervical cancer in other Caucasian and non-Caucasian women. It cannot be explained by either the effect ofa fatty diet or the practice of circumcision (Rotkin, 1973). The most plausible explanation is Breast Normal Cervical Cancer Cancer

CLIMATE

frigid

moderate torrid

DI E T high fat MARRIAGE late or timely never '8

low fat early

FIG. 15. Schematic representation of the genesis of two female cancers, as related to two imbalances of ovarian-adrenal maturation at adolescence.

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MITSUO KODAMA AND TOSHIKO KODAMA

that Jewish ritual plays a role in reducing the risk for cervical cancer, for it obligates abstention from intercourse during most of the first half of the ovulatory cycle (Kennaway, 1948), which protects women from untimely hormonal impact throughout life (Kodama and Kodama, 1981). In this context, one should recall the fact that cervical cancer is a rather rare phenomenon in nonhuman mammals (Allen and Gardner, 1941) whose mating period is strictly defined by the rise and fall of the pheromone. Finally, the relation between the epidemiological and hormonal aspects of breast and cervical cancer is illustrated in Fig. 15, showing the contrasting situations of the two cancers in adolescent endocrinology. D. GENESIS OF END~METRIAL CANCER No doubt the incidence of endometrialcancer exhibitsa positive correlation with the intake of dietary fat (Armstrong and Doll, 1975). It is also true that an integral amplitude of difference in the risk of a cancer has seldom been covered as a function of one single variable, as explained in the preceding discussion, i.e., dietary habit and marital life were found to be factorsin the genesisof two female cancers, each actingindependentlyand in different directions. There is a distinct differencein the deviationsof urinary steroids between breast and endometrial cancers (Figs. 7 and 10). One may wonder whether any epidemiological discriminator is available to separate the former from the latter. Japanese patients with cancer of either the breast or endometrium were associated with reduced reproductive activity as compared with age-matched normal controls (Kodama et al., 1982). There was no significant difference between the two groups of cancer patients in regard to height, weight, age of menarche, and number of live births (Kodamaet al., 1982). A surveyofrecent statisticaldatain the fivecontinents gives an impression that some factor other than diet seemsto be operativefor Alamcda:Whitc

r

Hawaii:Hawaiian HawaiKawasian

Newvork State Hawaii: Filipino

UK: Birmingham

Japan:Miyagi

0

20

40

Ratclloqooo POP

FIG. 16. Comparison ofthe ageadjusted incidenceofendometrialcanceramongfour ethnic

groups from five Cancer Registration areas.(Plotted from data in Waterhouse et al., 1976.)

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111

producing ethnic or geographic differences in the incidence of endometrial cancer. Figure 16 comparesthe age-adjusted cancer incidences of four ethnic groups from five selected places of the world (see Waterhouse ef al., 1976).A high incidence among Caucasians in Hawaii and the San Francisco Bay area (Alameda county) contrasts sharply with a low incidence among Japanese mainlanders (Miyagi prefecture). The intraethnic cancer incidences in Caucasian and Japanese women varied from one place to another (Figs. 3 and 16).The incidence ofendometrialcancer is generallyon the rise in the United States (Weiss ef al., 1976), but the amplitude of increase again varied from one place to another, e.g., a sharp increase of cancer incidence was observed in West Coast areas (Szekelyef al., 1978;Austin and Roe, 1979),whereas the cancer incidence in New Orleans always remained at a level as low as that of other states in 1962 (see Doll ef al., 1966; Waterhouse et al., 1976). Geographically, this chronological increase of endometrial cancer incidence was more marked in West Coast areas (San Francisco) than in the rest of the United States or Europe. Comparison of the age-specific cancer incidence revealed that a markedly increased risk for endometrial cancer was detectable in specified population groups in the age range of 50 to 70 years: (1) Hawaiians (Polynesians)and Caucasians in Hawaii, together with Caucasians in the San Francisco Bay area, are ranked as highest in cancer risk; (2) Caucasians in New York State are ranked as intermediate; and (3)Japanese women in mainland Japan are ranked as lowest throughout all age ranges (Waterhouse et al., 1976). It is worth noting that the age-adjusted incidence rate of endometrial cancer for Caucasiansin Alameda County has increased from 19.1 per 100,000 population during the period of 1960- 1961 to 47.5 per 100,000 population during 1974- 1975 (Hirayama, 1978), whereas the cancer risk for Polynesians in Hawaii has been persistently high since 1960 (see Doll er al., 1966; Waterhouse et al., 1976).In West Coast areas, the risk for endometrialcancer tends to increasewith the elevation of socioeconomic status (Mack, 1978; Austin and Roe, 1979). The constant elevation of endometrial cancer risk in the natives of Hawaii might have some historical relevancy to the fact that the population of Polynesians continually decreased from about 300,000 in 1778 to 10,502 in 1960. It is strongly suggested that Polynesian women suffer from some endocrinological disorder which may be related to the genesis of both infertility and infertile neoplasia (endometrial cancer). There is an indication that the reproductive activity of female natives could have been remarkably reduced during that period due to a lack of adaptability to the newly introduced civilization [Hawaii (Encyclopaedia Britannica)]. Selye remarked that the activity of gonadotropic hormones in humans could be reduced in a stressful environment such as a concentration camp (Selye, 1976). Christian stated that crowding may lead to the reduction (or even collapse) of population in

I12

MITSUO KODAMA A N D TOSHIKO KODAMA

various rodents, and that the animals of both sexes in the declining population often exhibit many manifestations of gonadal depression (Christian et al., 1965). It may not be a mere coincidence that the endometrial cancer incidence in West Coast areas seems to have increased in parallel with the progress of economic recession after the oil crisis. The proposed role of exogenous estrogen (Mack, 1978)does not explain either differential cancer risks of the United States in time and space, or persistently high risks among Polynesians in Hawaii. The deviation profile of urinary steroids in endometrial cancer patients remarkably resembles that in women who were taking oral contraceptives (Fig. 10, and Kodama et al., 1979b), i.e., in both groups of women, a remarkable increase of urinary KA was associated with a significantdecrease of I I-deoxy-17-ketosteroids(A, E, and D), and excretion of pregnanes and corticosteroids was generally depressed. It is conceivable that quite a similar sequence of events was triggered in the steroid metabolism by two different inducers (a set of two synthetic steroids and unidentified producer of endometrial cancer). The case with a pill user will serve as a “Rosetta stone” to investigate the nature of steroidal disorders. Oral contraceptives mostly consist of one synthetic 19-norandrogen and one synthetic estrogen. The former, being an analog of testosterone, is expected to suppress testosterone synthesis (this also explains the reason for the reduced libido of pill users). The above suppression automatically leads to an overflow of A4-(2-19 compounds(increase of urinary KA), steroidswith antigonadotropic activity (Christian, 1965), which in turn suppresses the production of ovarian progesterone (reduction of urinary HPn, Pn, Pd, and Pt) via the hypothalamus- pituitary axis. The reduction of 2 protein-anabolic steroids, testosterone and progesterone, also leads to a matched reduction of protein-catabolic corticosteroids(reduction of urinary corticosteroids)to ensure a homeostatic balance. The nature of the testosterone suppressor in endometrial cancer patients still remains unidentified. It is, however, certain that the steroidal disorders in a cancer patient are adrenal oriented, since hysterectoovariectomy did not affect the steroidal deviations of urine (Kodama et al., 1982). This conclusion makes all the more plausible our speculation that stress may play a role in establishing a predisposition for endometrial cancer. The proposed association of adrenal testosteronedeficiency with an endometrial cancer patient found new support in the epidemiological finding that Japanese patients with that neoplasm were distinguishable from age-matched controlsby a reduced stature,despitea lack of case-control differencein body weight (Kodama et al., 1982). Probably, the hormonal disorder was already present in those patients before the termination of adrenarche. It might be pertinent for our discussion to pose a final question: Is a “pill effect” detectable in those women who are placed in a stressful environment? Recently, we had a chance to investigate the excretion of urinary steroids of

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normal women livingin a village near Toyotacity, Japan. One half the village was mountainous and isolated, and the other half was flat and open. When compared with the inhabitants of the mountain area, those of the flat area were more urbanized in their life style, and many of the housewives were working during the day in near-by Toyota city, a center of the automobile industry. Comparison between the mountain area and the plain area revealed that an apparent “pill effect” was detectable in the urinary steroid excretions of women from the latter area as compared with those from the former (unpublished data). Probably, living in an industrialized society is going to be stressful for rural women who have been accustomed to a traditional life style. It should be emphasized that, although information is still not sufficient regarding the nature of endometrial carcinogenesis, our effort to unify the epidemiological and hormonal data has encountered no contradiction as far as the presently available materials are concerned. Basically, the carcinogenic action of endogenous estrogen, as manifested indirectly by aberrations of escort steroids, may account for the genesis of endometrial cancer, as of the other two cancers. Missing links that are now preventing us from drawing a final conclusion about the genesis of endometrial cancer will hopefully be found from future studies using humans as well as experimental animals. VI. Epilogue

An attempt was made to investigate comparatively the epidemiological and endocrinological aspects of cancers of the breast, uterine cervix, and endometrium in order to construct a unifying theory about the genesis of three sex-related neoplasias. Recent information on the biology and molecular biology of steroid hormones was exploited in an effort to breach the gap between these two branches of science. Epidemiologicalstudiesdemonstrated that the risks for breast and cervical cancers were conditioned by specified combinations of three factors-climate, diet, and sex life: an increased risk for breast cancer was associated with a cold climate, a fatty diet, and a delay of, or abstention from, sexual experience, whereas an increased risk for cervical cancer was accompanied by just the reverse in each of the three factors. Reduced reproductiveactivity was detected in patients with cancers of the breast and endometrium, but not in those with cervical cancer. Evidence was presented to indicate that a patient with endometrial cancer is smaller in stature, less fertile, and more disposed to a fatty diet than a healthy control. Evidencealso suggeststhat the risk of endometrial cancer could be augmented in a stressful environment. Migration studies indicated that the risks for each of the three cancers are subject to a change through emigration (conversion of life style). Case-control studies of urinary steroid excretion revealed that patients

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with three sex-linked neoplasias were associated with specified deviations of urinary steroids as compared with the corresponding normal controls: a patient with breast cancer, as judged from urinary steroid findings, was associated with hypercorticoidism and an increased frequency of ovulation failure,whereas a patient with cervical cancer was suffering from a deficiency of both androgen and corticosteroid of adrenal origin. A patient with endometrial cancer was found to have some defectiveness in testosterone synthesis which led to an overproduction of A4-androgen and a general depression of endogenous progestin and corticosteroid. Biological and biochemical studies indicated the possibility that an irreversible change (or carcinogenesis) could be induced by an imbalance of multiple steroid actions in those tissues in which differentiation was still in progress, and that the action of a steroid was brought to its expression through acceleration of specified gene transcription. Other evidence suggested that the neoplastic transformation of a cell is to be explained in terms of increased expression of endogenous (host cell-derived) oncogenes rather than of random mutation following the introduction of exogenous mutagens. In view of the complexity of the data, some concluding remarks will be of help in promoting the reader’s comprehension about our discussion. 1. Epidemiologicaland hormonal data on breast and cervical cancers are in good agreement with our proposed hypothesis that the risk for breast cancer could be conditioned by an early adrenarche at the sacrifice of gonadarche,whereas that for cervical cancer could be defined as a product of arrested adrenarche. 2. The above bidirectional imbalances of two endocrine maturation processes is to be induced during adolescenceunder the influenceof specified combinations of climate, diet, and sexual activity. 3. Our interdisciplinarystudies also indicate that the risk for endometrial cancer could be related to a defectiveness in the synthesis of adrenal testosterone which may lead to infertility and a delay in stature growth. 4. Evidence suggeststhat intake of a fatty diet and long-lastinganxiety can be responsible for increased endometrial cancer risk. 5. Throughout the studieson the three female cancers,the possible impact of adolescent maturation was emphasized, but there is also evidence to indicate that the environmental conditions after the termination of adolescence may also be worth consideration for assessingcancer risks, especiallyin the case of endometrial cancer. 6. Both the concept of cellular oncogene activation emerging from the study of retroviruses and our steroid study on three female cancers support the recognition that anyone can get cancer under certain provocative

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conditions, and that the only way to avoid it is to optimize our life style including diet, sexual life, and peace of mind. 7. Looking back at the history of scientific revolutions, one may notice that Darwin made a breakthrough in biology by taking out the barrier of species. Einstein rescued physical science from its chaotic state by removing a conceptual septum regarding mass, energy, and time. It is our hopeful anticipation that more advancement will be made in cancer research by removing a psychological barrier that is now preventing us from unifying an abundance of knowledge on various cancers from various species of animals, including humans. VII. Addendum

In Sections IV,C and E, we indicated that one can induce a permanent change of genetic expression by imposing a hormonal turbulance on differentiating cells, but not on fully differentiated cells. One may ask: Why is the impact of hormonal imbalance to be restricted to a stage of differentiation? The report by Tonegawa and his associates might shed light on the nature of cell differentiation: a pre-B cell, the forerunner of the B-cell lineage, contains only freep class heavy chains in the cytoplasm. The integral synthesisof IgM and IgD starts at the stage of B cell, but the release of immunoreactive IgM and IgG will not become feasible before entering the stage of plasma cell. Evidence indicates that these switch-overs in the expression of immunoglobulin genes should be explained in terms of differential mRNA splicings, and also in terms of differential DNA recombinations at the gene level (Tone1981). It is quite conceivable that a drastic change of gene gawa et d., expression could be induced by a hormonal impact on a differentiating cell, within which recombination of various gene units is still in progress.

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Lippman, M., Bolan, G., and Huff, K. (1976). Cancer Res. 36,4595-4618. Lucas, W.E.,andYen. S . S . C. (1979).Am. J. Obstet. Gynecol. 134, 180-186. McGrath, C. M.,and Jones, R.F. (1978). Cancer Rex 38,4112-4125. Machida, T. (1978). Exp. Anim. 27,138- 146 (in Japanese). Mack, T. M. (1978). I n “Endometrial Cancer” (M. G. Brush, R. J. B. King, and R. W. Taylor, eds.), pp. 17-28. Baillibre, London. MacMahon, B., Cole, P., and Brown, J. B.(1973). J. Nad Cancer Inst. 50,21-42. Meier, A. H. (1977). In “Comparative Endocrinology of Prolactin” (H. D. Dellmann, J. A. Johnson, and D. M. Klachko, eds.), pp. 153- 171. Plenum, New York. Morii, S., and Huggins, C. (1962). Endocrinology 71,972-976. Morreal, C. E., Dao, T. L., Nemoto, T.,and Lonergan, P. A. (1979). Cancer Res. 63, 1 171- I 174. Moms, V. L., Medeiros, E., Ringold, G. M., Bishop, J. M., and Vennus, H. E. (1977). J. Mol. Biol. 114.73-91. Murphy, B. E. P. (1967).J. Clin. Endocrinol. 27,973-990. Nagasawa, H. (1977). IRCS Med. Sci. 5,405-408. Nagasawa, H.(1979). Eur. J. Cancer 15,267-279. Nagasawa, H. (1981). Biomedicine 34,9 - I 1. Nakamura, Y., Kodama, M.,and Kodama, T. (1981). Gann72,679-683. Neel, B. G., Hayward, W. S., Robinson, H. L., Fang, J., and Astrin, S. M.(1981). Cell 23, 323- 334. Nisker, J. A., Ramzy, I., and Collins, J. A. (1978).Am. J. Obstet. Gynecol. 130,546-550. Palmiter, R. D. (1972). J. Biol. Chem. 247,6450-6461. Poortman, J., and Thijssen, J. H.H.(I 978). In “Endometrial Cancer (M. G. Brush, J. B. King, and R. W. Taylor, eds.), pp. 375-382. Baillitre, London. Reddy, B. S.,Cohen, L. A., McCoy, G .D., Hill, P., Weisburger,J. H., and Wynder, E. L. (1980). Adv. Cancer Res. 32,238 - 345. Rotkin, D. (1973). Cancer Res. 33, 1353- 1367. Sakauchi, N., and Homing, E. C. (1971).Anal. Lett. 4,41-52. Schimke, R. T., McKnight, G. S., Shapiro, D. J., Sullivan, D., and Palacios, R. (1975).Recent Prog. Horm. Res. 31,175-21 1. Sebastian, J. A,, Leeb, B. O., and See,R. (1978).Am. J. Obstet. Gynecol. 131,620-623. Segaloff, A. ( I 975). J. SteroidBiochem. 6, 17I - 175. Segi, M., Noye, H.,and Segi, R.(1977). Report from Segi Institute ofCancer Epidemiology,pp. 1 - 16. Segi Institute of Cancer Epidemiology, Nagoya. Selye, H. (1976).“Stress in Health and Disease,” pp. 258-277. Butterworth, London. Shaaya, E., and Sekeris, C. E. (1965). Gen. Comp. Endocrinol. 5.35-39. Shapiro, S., Strax, P.,Venet, L., and Fink, R.(1968). Am. J. Publ. Health SS, 820-835. Sims, E. A. H., Danforth, E., Jr., Horton, E. S., Bray, G. A,, Glennon, J. A., and Salans, L. B. (1973).Recent Prog. Horm. Res. 29,457-487. Stamwski, J.(1971). J. Nafl.CanCerInst.47,935-940. Stehelin, D., Vermus, H. E., Bishop, J. M., and Vogt, P. K. (1976). Nafure (London) 260, 170-173. Stephenson, J. H., and Grace, W. J. (1954). Psychosom. Med. 16,287-293. Stem, E.,Lachenbruch, P. A., and a x o n , W.J. (1967). Cancer 2’0,190-201. Stocks, P. (1955). Br. J. Cancer9,487-494. Szekely, D. R., Weiss, N. S., and Schweid, A. I. (1978).J. Natl. Cancer Insf. 60,985-989. Takasugi, N. (1963). EndocrinorOgY72,607-619. Takasugi, N. (1972). Gunn 63,73-77. Takasugi, N. (1979). J. Nafl. Cancer Insf.Monogr. 51,57-66.

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Takasugi, N., and Bern, H. A. (1964).J. Natl. Cancer Znst. 33,855-865. Taki, I. (1967).J. Jpn. Obstet. Gynecol. Soc. 19,855-866 (in Japanese). Taki, I., and Iijima, H. (1963).Am. J. Obstet. Gynecol.87,926-934. Taylor, R. S.,Carroll, B. E., and Lloyd, J. W. (1959). Cancer 12, 1207- 1223. Tonegawa, W., Sakano, H., Maki, R., Traunecker, A., Heinrich, G., Roeder, W.,and Kurosawa, Y. (1981). Coldspring HarborSymp. Quant. Biol. 45 (Pt. 2), 839-858. Topper, Y. J. (1970).Recent Prog. Horm. Res. 25,287-308. Traurig, H. H., and Morgan, C. F. (1964).Proc. Soc. Exp. Bid. Med. 115,1076- 1080. Vasquez, S. B., and Kitay, J. 1. (1980).NeuruendOcrinology30,159-163. Vermus, H . E.,Bishop, J. M.,Nowinski, R.C., and Sarkar, N. H. (1972).Nature(L0ndon) 238, 189-191. Waterhouse, J., Muir, C., Correa, P., and Powell, J., eds. (1976).“Cancer Incidence in Five Continents 111” IARC Publ. No. 15, Lyon. Weiss, N. S., Szekely, D. R., and Austin, D. F. ( 1976). N. Engl. J. Med. 294, 1259 - 1262. Wied, G. L., and Bibbo, M.(1970). In “Steroid Biochemistry and Pharmacology 1” (M.H. Briggs, ed.), pp. 453-476. Academic Press, New York. Wigglesworth, V. B. (1934). Q. J. Microsc. Sci. 77, 191 -222. Wilbanks,G. D. (1973).CuncerRes.33, 1379-1381. Wong, T.-W., Warner, N. E., and Yang, N. C. (1962).Cancer Res. 22, 1053- 1057. Wynder, E. L., Cornfield, J., Shroff, P. D., and Doraiswami, K. R. (1954). Am. J. Obstet. Gynecof.68, 1016- 1052. Wynder, E. L., Escher, G. C., and Mantel, N. (1966).Cancer 19,489-520. Wynder, E. L., McCoy, G. D., Reddy, B. S., Cohen, L., Hill, P., Spingarn,N. E., and Weisburger, J. H. ( I 98 I). In “Nutrition and Cancer: Etiologyand Treatment” (G. R. Newell and N. M. Ellison, eds.), pp. I 1 -48. Raven, New York. Yamamoto, K. B., and Ringold, G. M. (1978). In “Receptors and Hormone Action” (B. W. OMalley and L. Birnbaumer, eds.), Vol. 2, pp. 297-322. Academic Press, New York. Yang, N. C., Castro, A. J., Lewis, M., and Wong, T.-W. (1961).Science134,386-387. Zumoff, B., Fishman, J., Bradlow, H. L., and Hellman, L. (1975).CancerRes. 35,3365-3373.

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FUNDAMENTALS OF CHEMOTHERAPY OF MYELOID LEUKEMIA BY INDUCTION OF LEUKEMIA CELL DIFFERENTIATION Motoo Hozumi Department of Chemotherapy, Saitama Cancer Center Research Institute, Saitama. Japan

I. Introduction ....................... ............................................ 11. Myeloid Leukemia Cells U A. Animal Cells............................................................................................................. B. Human Cells............................................................................................................. 111. Induction of Differentiation of Cultured Mouse Myeloid Leukemia Cells ................ A. Inducers of Cell Differentiation............................................................................... B. Stimulators of Production by Leukocytes of D-Factor for Myeloid Leukemia Cells ................................................................................... C. Biochemical Phenotypic Changes Associated with Myeloid Leukemia Cell Differentiation................................................................................ D. Changes in Proliferation Potential during Differentiation of Myeloid Leukemia Cells..................................................................................... E. Positive Feedback Control Mechanisms of Cell Differentiation by Protein Inducer Produced by Differentiating Myeloid Leukemia Cells............................. F. Inhibitors and Their Mechanisms of Inhibition of Differentiation ............................................................... of Myeloid Leukemia Cells..... G. Properties of Myeloid Leukemia Cells Resistant to Inducers of Cell Differentiation .............................................................................................. H. Sensitization of Myeloid Leukemia Cells Resistant to Inducers of Cell Differentiation.............................................................................................. IV. In Vivo Induction of Differentiation of Mouse Myeloid Leukemia Cells and Therapy of Animals Inoculated with Myeloid Leukemia Cells................................... A. In Vrvo Induction of Differentiation of Mouse Myeloid Leukemia Cells............. B. Therapy of Mice Inoculated with Myeloid Leukemia Cells by Induction of Differentiation............................................................................... V. Induction of Differentiation of Cultured Human Myeloid Leukemia Cells............... A. Cultured Myeloid Leukemia Cell Lines.................................................................. B. Primary Cultured Myeloid Leukemia Cells............................................................ VI. Summary ........................................................................................................................ References....................................................................................................................... I.

121 I22 123 123 I23 I23

132 I34 138 141

142 145 I47 148 148 I49 152 153 16 I 162 164

Introduction

There is accumulating evidence that various myeloid leukemia cells can be induced to differentiate into cells with the normal characteristics of macrophages, granulocytes, or erythrocytes. On differentiation the cells cease to proliferate and lose their transplantability into animals. These findings suggest that induction of terminal cell differentiation by certain inducers is 121 ADVANCES IN CANCER RESEARCH, VOL. 38

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-006638-6

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another approach to therapy of myeloid leukemia and that some myeloid leukemia cells are formed by impairment of a certain stage of differentiation of the normal hematopoietic process (Anderson et af.,1979; Bodner et af., 1981;Breitman and Gallo, 1981;Breitman et al., 1981; Burgess and Metcalf, 1980b; Collins et af.,1980; Gallo et af., 1979; Honma et af., 1982a, 1983; Hozumi et af.,1979a,b; Hozumi, 1982; Ichikawa, 1969, 1970; Ichikawa et al., 1976; Koeffler and Gold, 1980; Koeffler et al., 1981;Lozzio and Lozzio, 1975; Lozzio et af., 1981; Marks and RiWnd, 1978; Marks et af., 1978; Metcalf, 1979, 1980 PalC et af., 1979a,b; Rutherford et al., 1979; Sachs, 1978a,b, 1980, 1981; Sugiyama el af.,1979a; Takeda et af.,1982). We have been trying to establish a new method ofcancer chemotherapy by controlling the malignancy of tumor cells by induction of terminal cell differentiation. As a model for this purpose, we examined the in vitro and in vivo effects of various compounds on induction of differentiation of the mouse myeloid leukemia cell line, MI, which was established from a spontaneous myeloid leukemia in an SL strain mouse and which can be induced to differentiate into macrophages and granulocytes with loss of leukemogenicityto syngeneic mice (Hozumi et af.,1979a,b;Hozumi, 1982). Recent results of our experiments and those of others on induction of differentiation of M 1 cells are described in this article. During studies on differentiation of M 1 cells, other myeloid leukemia cell lines from mice (R453, Ichikawa et af.,1976; WEHI-3B, Metcalf, 1979)and humans (HL-60, Collins et af.,1977;K562, Lozzio and Lozzio, 1975;KG- 1, Koeffler and Gold, 1978; ML-1 and ML-3, Minowada, 1981) were also found to be induced to differentiate into macrophages, granulocytes, or erythrocytes by various compounds. Furthermore, the effects of inducers of differentiation of leukemia cell lines were examined on leukemic cells in primary culture from patients with myeloid leukemia to develop a method of therapy by induction of terminal cell differentiation. The compounds and endogenous factors affecting differentiation of these leukemic cells, mechanisms of induction of differentiation of the cells, and perspectives of therapy of myeloid leukemia by induction of terminal cell differentiation are described in this article. II. Myeloid Leukemia Cells Used for Experiments

Most studies on induction of differentiation of myeloid leukemia cells have been conducted with the cultured cell lines described in this section. Primary cultured human leukemic cells began to be employed only recently to obtain information on the differentiation of cultured leukemic cell lines for therapy of human leukemia.

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A. ANIMALCELLS A myeloid leukemia cell line, M 1, was established from a spontaneous myeloid leukemia in an SL mouse (Ichikawa, 1969) and another leukemia cell line, R453, was established from a C57BL/6 mouse with a Rauscher virus-induced leukemia (Ichikawa et al., 1976). WEHI-3B myelomonocytic leukemia was induced in a BALB/c mouse injected with mineral oil (Warner et al., 1969).The myelomonocytic leukemia cell line WEHI-3B was found to form colonies in semisolid agar culture (Metcalf et al., 1969) and it was established in 1972 by Dr. C. Wyss (unpublished data) as a cloned continuous cell line in liquid culture (Metcalf, 1979). B. HUMANCELLS A human promyelocytic cell line, HL-60, was established from peripheral blood leukocytes of a patient with acute promyelocytic leukemia (Collins et al., 1977). Lozzio and Lozzio (1 975) established a human myelogenous leukemia cell line, K562, from the pleural effusion of a patient with chronic myelogenous leukemia in terminal blast crisis. Koeffler and Gold (1978) established a human myelogenous leukemia cell line, KG- 1, from a patient with acute myelogenous leukemia. KG-1 cells are at the myeloblast and promyelocyte stages of differentiation and retain the morphological and cytochemical characteristics of acute myelogenous leukemia cells. The human myeloid leukemia cell lines ML- 1 and ML-3 were established from a patient with acute myelogenous leukemia by Minowada ( 1982). Leukemic cells from patients with myeloid leukemia were obtained from peripheral blood or bone marrow of the patients. Leukemia cells in primary culture were used for experiments. 111. Induction of Differentiationof Cultured Mouse Myeloid Leukemia Cells

A. INDUCERS OF CELLDIFFERENTIATION

Mouse myeloid leukemia cell lines such as M 1, R453, and WEHI-3B can be induced to differentiate both in vitro and in vivo into macrophages and granulocytes by treatment with various compounds. Differentiated cells mainly express properties similar to normal macrophages and granulocytes. These properties or markers of differentiated cells are Fc, C, receptors, motility, phagocytosis, lysosomal enzymes, and morphological changes to macrophages and granulocytes (Fig. 1). Furthermore, differentiated M 1 cells express glycoprotein with a molecular weight of 180,000 in their plasma

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FIG. 1. Typical morphology of differentiated M 1 cells (Okabeet a/., 1979). (a) A subclone of M 1 cells (MI -R 1) cultured in a diffusion chamber in an syngeneic SL mouse for 4 days. (b-d) MI-Rl cells were treated with actinomycin D (5 ng/ml) for 2 days in vitro. Then the cells were cultured in diffusion chambers in SL mice. May-Griinwald-Giemsa stain.

membrane, and lose proliferating capacity in vitro and leukemogenicity to syngeneic mice. The main properties of these inducers are described below. 1. Proteins

a. D-Factor and Colony-Stimulating Factor. Induction of differentiation of M1 cells into macrophages and granulocytes was first demonstrated by Ichikawa (1 969)withconditioned medium (CM) ofmouse embryo cells. The factods) stimulating differentiation of MI cells (D-factor) in the CM of embryo cells was found to be a glycoprotein with a molecular weight of 40,000 to 50,000, differing in nature from the colony-stimulating factor (CSF). The D-factor was detected in the CM of embryo cells of various animals and in the CM of established lines of various cells. One of the D-factors in the CM of the established cell line E 1 of mouse embryo cells was

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named MGI, macrophage-granulocyte inducer, by Guez and Sachs ( 1973). It had a molecular weight of 68,000, CSF activity, and no detectable sugar component. Later, Lotem and Sachs (1978a) reported that CSF purified from mouse lung CM could induce differentiation of M 1 cells. We found that a clone, YS-T22, of cells from the Yoshsida sarcoma cell line YSSF-2 12 grown in serum-free culture medium could produce D-factor (Hozumi et al., 1979c)with or without CSF activity. The D-factor and CSFin CM of spleen cells stimulated with mitogens or copolymer of polyinosinic and polycytidilic acids, poly(1) - poly(C), could be separated by gel filtration (Yamamoto et al., 1979). Additional evidence that the D-factor and CSF are distinct substances was obtained by experiments with CM of the mouse mammary carcinoma cell line, FM3A, (Ayusawa et al., 1979;Hozumi et al., 1981) and mouse fibroblast L929 cells (Yamamoto et al., 198Ib). We examined the molecular sizes of the CSF and the D-factor produced from FM3A cells by gel filtration. The CSF consisted of a major and a minor component with apparent molecular weights of 80,000 and 30,000, respectively, while the D-factor was a single component with an apparent molecular weight of 60,000 to 70,000. Although the molecular size of the D-factor from FM3A cells was scarcely affected by treatment of the cells with tunicamycin, a specific inhibitor of asparagine-linked glycosylation, the CSF produced by the cells had a lower molecular weight of 30,000 and was homogeneous with noaffinity toconcanavalin A (Con A)-Sepharose(Hozumi etal., I98 1). The cells with or without properties of CSF and D-factor produced by b29 tunicamycin were also examined by gel filtration (Yamamoto et al., 1981b). Although D-factor produced by untreated L929 cellsgave a single peak with an apparent molecular weight of 67,000, D-factor produced in the presence of tunicamycin had an apparent molecular weight of 25,000. In contrast, most of the CSF was eluted in the void volume, even when it was produced in the presence of tunicamycin. The D-factor produced from tunicamycin-treated L929cells was more sensitive to trypsin or heat treatment than the D-factor from untreated L929 cells, but the CSF produced in the presence of tunicamycin was resistant to these treatments. These results show that the D-factor is distinct from CSF and that carbohydrate is not essential for production of the activity of the D-factor, although it may stabilize the D-factor. Although Guez and Sachs ( I 973) and Lotem and Sachs ( 1978a) reported previously that CSF from CM of a mouse fibroblast cell line, E 1, and mouse lung had the activities of both D-factor and CSF, Lotem et al. ( 1980)recently demonstrated that D-factor (MGI-2) and CSF (MGI-1) present in serum of mice injected with endotoxin and in CM of lung or macrophages were separable by gel filtration. Furthermore, Lipton and Sachs ( 198 1) reported that CM of serum-free cultures of Krebs ascites tumor cells contained two different molecular species of CSF (MGI- 1) and D-factor (MGI-2). The CM

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of the Krebs ascites tumor cells contained one species of MGI-1 and two species of MGI-2, MGI-2A, and MGI-2B. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the MGI- 1 and MGI-2A activities were associated with a substance of similar molecular weight and each activity gave two bands, one of 23,000 and the other of 25,000. MGI-2B activity was associated with one band with a molecular weight of45,OOO. The production of MGI- 1 and MGI-2 did not appearto involveglywsylation and these compounds did not bind to lectins such as Con A, soybean agglutinin, or wheat germ agglutinin (Lipton and Sachs, 1981). MGI-2 was more readily inactivated by proteases and was more labile at high temperature and low pH than MGI-1 (Lipton and Sachs, 1981). These findings suggest that the general properties of MGI- 1 (CSF) and MGI-2 (D-factor) are similar to those of CSF and D-factor produced by L,,, cells (Yamamoto et al., 1981b). Furthermore, the results suggest that D-factor is not always associated with CSF and that there are at least two types of CSF with or without D-factor activity. However, the chemical properties of D-factor without CSF activity remain to be determined since the D-factor has not yet been purified. D-Factors for M 1 cells were also found in various body fluids containing CSF activity such as ascitic fluid (Hozumi et al., 1979a,b), serum of mice injected with bacterial endotoxin2 (Sachs, 1978a), saliva (Nakayasu et al., 1978),urine(Nakayasuetal., 1978),andamnioticfluid(Nagataetal., 1977). The presence of the D-factor in these body fluids is of special interest in connection with in vivo regulation of proliferation and differentiation of leukemia cells. Therefore, we examined the properties and origin of the D-factor in ascitic fluids of animals bearing tumors. Results showed that the D-factor is a heat-labile protein of heterogeneous molecular size ranging from about 10,000to 400,000, but about halfthe total activity was recovered in the fraction with a molecular weight of less than 50,000 containing a subfraction with an apparent molecular weight of 20,000 to 25,000 and a specific activity 300 times that of the original ascitic fluid (Hozumi et al., 1979a).We examined the mechanisms of production of D-factors in ascites and found that macrophages and granulocytes in the ascites, but not lymphocytes, were mainly responsible for production of the D-factor (Hozumi et al., 1979a,b). Other mouse myeloid leukemia cells, such as R453 cells and WEHI-3B cells, were also found to be induced to differentiate into macrophages and granulocytes by CM of various cells or body fluids containing CSF and D-factors for M1 cells (Ichikawa et al., 1976; Sugiyama et al., 1979a; Metcalf, 1980; Burgess and Metcalf, 1980a,b). The D-factors for R453 cells in ascitic fluid of rats were also proteinous substanceswith properties similar to those of the D-factors for M 1 cells (Sugiyamaet al.,1979a).However, it is still unknown whether the factors from the two sources are identical.

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Although differentiation of WEHI3B cells was induced by purified CSF from CM of lung (Metcalf, 1979), an active factor stimulating differentiation of WEHI3B cells in serum of mice injected with endotoxin had an apparent molecular weight of 23,000 and it was separated from most other components of CSF by several fractionation procedures (Burgess and Metcalf, 1980a;Nicolaand Metcalf, 1981).Part ofthe activefactor, however, was found to be associated with a subset of residual CSF molecules with a selective capacity to stimulate granulocyte colony formation by normal hematopoietic stem cells (Burgess and Metcalf, 1980a,b; Nicola and Metcalf, 1981). Sera from patients with acute myeloid leukemia were recently found to have activity to induce differentiation of WEHI3B cells (Metcalf, 1981). Although the activity was higher in infected patients, the activity was also detected in sera from uninfected patients. 6. Arginase and Histones. During investigations of the mechanisms of induction of differentiation of MI cells by D-factor, we found that two proteins with known chemical structures, arginase and a fraction of histone, H1, were inducers of MI cells (Okabe et al., 1979,1981). The effect of arginase on the induction ofdifferentiation of M 1 cells was found to be due to arginine depletion of the culture media: the leukemia cells did not differentiate in cultcre media containing arginine, but did differentiate into macrophages and granulocytes in arginine-deficient culture media (Okabe et al., 1979).Growth of M 1 cells was significantlyinhibited by treatingthe cells with arginase or culturing them in arginine-deficient medium, but the cells could not be induced to differentiate simply by inhibition of their growth with an inhibitor such as 5-fluorodeoxyuridine (FUdR) (Okabe et al., 1979). We found that M 1 cells could be induced to differentiate into macrophages and granulocytes by lysine-rich, histone H 1 fractions ( 10 to 100pg/ml) isolated from calf thymus, rat liver, and M 1 cells (Okabe et al., 1981). Histone H2A and H2B fractions did not induce differentiation of M 1 cells at concentrations of 10 to 100pg/ml but did induce differentiation at a high concentration (200pg/ml). Other basic polypeptides, such as the histone H3 fraction, poly-L-lysine, and poly-L-arginine, significantly inhibited induction of differentiation of M 1 cells (Okabeet al., 1981). The mechanismsof these effits of histones on induction of differentiationof M 1 cells are unknown, but may be due to the complex structural features of the histones, because, for instance, lysine-rich histone H 1 is an inducer of M 1 cell differentiation, but poly-L-lysine is not. 2. Lipids Both lipopolysaccharide (LPS)and lipid A from various bacteria could inducedifferentiationof M 1 cellsinto macrophagesand granulocytes(Sachs, 1978a). Although their mechanisms of induction were unclear, these com-

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TABLE I INDUCTION OF DIFFERENTIATION OF M 1 CELLS AND

HL-60 CELLS BY SYNTHETIC ALKYLLYSOPHOSPHOLIPIDS~

Differentiation Alkyllysophospholipid chemical name ~

~~

MI cells

HL-60 cells

ST-024

+ + + +

ST-040

NTb

ST-04 1

NT

+ + + + + +

Abbreviation ~

(3-Tetradecyloxy-2-methoxy)propyl-2trimethylammonioethylphosphate (3-Octadecyl-2-methoxy)propyl-2trimethylammonioethylphosphate 3-Octadecyloxypropyl-2trimethylammonioethylphosphate (3-Tetradecyloxy-2-methoxy)propyl-2aminoethylphosphate (3-Tridecyloxy-2-methoxy)propyl-2trimethylammonioethylphosphate 3-Tetradecyloxypropy1-2trimethylammonioethylphosphate

ST-023 ST-008 ST-00 I

" From Honma el a/. ( I98 I b). NT, not tested.

pounds were found to induce differentiation of M1 cells indirectly by inducing protein inducer, MGI, in the cells (Sachs, 1978a). We examined the effects of alkyllysophospholipids, synthetic analogs of naturally occurring lysophospholipids,on induction of differentiation of M 1 cells and HL-60 cells. Both types of cells were induced to differentiate into morphologicallyand functionally mature granulocytes and macrophages by treatment with a wide variety of these compounds, as shown in Table I (Honma et al., 198 1b). Among these compounds, (3-tetradecycloxy-2methoxy)propyl-2-trimethylammonioethylphosphate(ST-023) was the most effective for induction of differentiation of both M 1 cells and HL-60 cells. This compound and some other alkyllysophospholipids induced differentiation of both M1 and HL-60 cells at concentration of 1 pg/ml. However, these compounds did not affect colony formation of normal mouse bone marrow cells even at a higher concentration of 20pg/ml. These results suggest that alkyllysophospholipids induce cell differentiation of myeloid leukemia cells without affecting growth or differentiation of normal bone marrow cells. Other lysophospholipids such as acyllysophospholipidshad no influence on cell growth or differentiation at the effective concentrations of their alkyllysophospholipid analogs (Honma et al., 198lb). On the other hand, 0-alkyllysophosphatidylethanolamineanalogs were as active as O-alkyllysophosphatidylcholine analogs on M 1 cells, but less active than the latter

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compounds on HL60 cells. Therefore, 0-alkyllysophosphatidylcholineanalogs are suggested to be the most suitable for inhibition of cell growth and induction of cell differentiationof myeloid leukemia cells. 0-Alkyllysophosphatidylcholineanalogs with a short aliphaticside chain had weak inhibitory effects on cell proliferation, but 0-alkyllysophospholipid with a C,,H,, aliphatic side chain had a marked effect on cell proliferation. These results show that the cytotoxic and growth-inhibitoryeffects of alkyllysophospholipids are separable from their effects in inducing cell differentiation. 3. Glucocorticoid Hormones, Prostaglandins,and CAMP Some glucocorticoid hormones also induced differentiation of M 1 cells into macrophages and granulocytes (Sachs, 1978a; Hozumi et al., 1979a). Dexamethasone, prednisolone, and hydrocortisone (optimal inducers) M, whereas another group caused maximal induction at 5 X lO-'-2 X of steroid suboptimal inducers including corticosterone, aldosterone, 1 1jlhydroxyprogesterone, 1 I -deoxycortisol,and 1 1-deoxycorticosteronedid not cause maximum induction, even when added at very high concentrations, but each induced a submaximal level (Hozumi et al., 1979a). All steroids with optimal inducer activity had three functional groups (1 ljl-OH, 17aOH, 2 1-OH), and the simplest optimal inducer was hydrocortisone. The inducibility of cell differentiationof the steroidswas closely associated with their effects on the specific cell-free cytoplasmic binding of ['Hldexamethasone (Hozumi et al., 1979a). Dexamethasone also induced differentiation of mouse myeloid leukemia R453 cells into mainly granulocytes (Sugiyama et al., 1979a), but not differentiation of human promyelocytic leukemia HL-60 cells (Y.Honma, K. Kasukabe, and M.Hozumi, unpublished data). On the other hand, glucocorticoid hormones such as hydrocortisone and dexamethasone (Scher et al., 1978, 1980; Santoro et al., 1978) and, to a lesser extent, aldosterone, corticosterone,and deoxycorticosterone inhibited induction of differentiation of Friend erythroleukemiacells (Scher et al., 1978, 1980). Analysis of glucocorticoid-mediatedinhibition revealed that both hemoglobin and globin mRNA synthesis were markedly inhibited without any cytotoxic effect (Scher et al., 1978). These results suggest that responses to glucocorticoid hormones differ in different types of leukemia cells. ProstaglandinE, ,D, ,and F, were all produced from [14C]arachidonatein an early stage of differentiation of M1 cells, but mature cells produced predominantly prostaglandin E, (Honma er al., 1980d).Indomethacincompletely inhibited the productions of these prostaglandins and markedly inhibited the induction of differentiation of M 1 cells by dexamethasone or D-factor from ascitic fluid of rats (Honma et al., 1980d). The indomethacinmediated inhibitionofcell differentiationwas counteracted by prostaglandin

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E, or E2,but not by prostaglandin F, or Fk (Hozumi et al., 1979a). Prostaglandin E stimulated differentiation of MI cells induced by a suboptimal concentration of dexamethasone, but prostaglandin F did not. Moreover, prostaglandins such as E, and E2and, to a lesser extent, A, and A2when added alone induced lysozyme activity in M1 cells (Hozumi et a/., 1979a). These findings show that the induction of prostaglandin synthesisis involved in the mechanism of differentiation of M 1 cells. Although cAMP markedly induced lysozyme activity in MI cells, dbcGMP or butyric acid did not (Hozumi et al., 1979b). cAMP or prostaglandin E alone had no effect on other differentiation-associated properties of M 1 cells, such as phagocytosis, migrating activity, and the morphology of the cells. Prostaglandin E stimulated phagocytosis, migrating activity, and changes in morphology induced by dexamethasone, but cAMP did not (Hozumi et al., 1979b). 4. Vitamins

The active form of vitamin D,, la,25-dihydroxyvitamin D,,was recently found to induce differentiation of M1 cells into macrophages (Abe et af., 1981) and HL-60 cells into granulocytes (Miyaura et al.. 1981). In MI cells, 1a,25-dihydroxyvitamin D, also induced several markers of differentiated cells, such as phagocytic, migrating, and lysozyme activities. la-Hydroxyvitamin D, had similar inducing activity, but 25-hydroxyvitamin D, and 24R,25-dihydroxyvitamin D, showed very weak activity (Abe et al., 1981). The relations between the mechanisms involved in induction of leukemia cells by these vitamin D, analogs and their well-known biological activities in enhancing intestinal calcium transport and bone mineral mobilization are unknown. Other vitamins, such as vitamin A and vitamin A analogs (retinoids) (Takenaga et al., 1980, 1981a), vitamin C (Takenaga et al., 1981a), and vitamin E (Sakagami ef al., 1981; Takenaga et al., 1981a) did not induce differentiation of M 1 cells but rather inhibited cell differentiation. 5 . Synthetic Polyribonucleotides and Interferons Some synthetic polyribonucleotides, such as poly(1) and poly(C), but not poly(A) and poly(U), induced differentiation of MI cells (Hozumi et al., 1979b).The inducing effects of these single-stranded RNAs were influenced not only by the specificity of nucleotides but also by their chain length. ,~ of 10 or 12 was active as an inducer, but poly(1) of Poly(1) with an s ~ , value 6.9 S was less active (Hozumi et al., 1979b). Double-stranded RNAs such as poly(1) poly(C) or poly(A) * poly(U) could not alone induce differentiation of M1 cells, but markedly enhanced induction of differentiation of M 1 cells by low concentrations of D-factor from mouse peritoneal macrophages and induced a significant amount

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of interferon in M1 cells (Hozumi et al., 1979b, 1982). Although slight interferon activity was detected in medium of poly(1)-treated M1 cells, no activity was induced by other single-stranded RNAs (Yamamoto et al., 1979). Simultaneous treatment of M 1 cells with anti-interferon serum, poly(1) poly(C), and D-factor abolished the enhancing effect of poly(1) * poly(C) on the action of the D-factor (Yamamoto et al., 1979). These results suggest that interferon produced from MI cells mediated the effect of the polynucleotide. We have confirmed that interferon, which was prepared from M1 cells treated with poly(1) poly(C) and purified by anti-interferon antibody column chromatography, did not itself induce differentiation of M 1 cells, but enhanced the induction ofdifferentiation by D-factor from mouse peritoneal macrophages, LPS, or poly(1) (Tomida et al., 1980a). Interferon did not stimulate differentiation of the cells by dexamethasone. Interferon alone could induce lysozyme activity in M 1 cells and the effects of interferon and D-factor or dexamethasone on induction of the lysozyme activity were synergistic (Tomida et al., 1980a). On the other hand, sera from mice given injections of poly(1) poly(C), containing interferon and D-factor, induced differentiation of all M 1 clone cells tested, including resistant clones (R-4and DR-3) that could not be induced to differentiate by D-factor alone (Tomida et al., 1980a). Lotem and Sachs ( 1978b)reported that interferon enhanced the induction of lysozyme in MI cells by dexamethasone or protein inducer, but had no effect on induction of C, rosettes, immune phagocytosis, or differentiation to mature macrophages or granulocytes. In contrast, we observed that interferon stimulated induction ofdifferentiation of our various clones of M 1 cells with different sensitivities to inducers. The reasons for this discrepancy between our results and those of Lotem and Sachs (1978a) are unknown, but may be mainly due to differences in the cell clones used. A polymer of adenosine diphosphate ribose, poly(ADP-ribose), could induce differentiation of M 1 cells into macrophages and granulocytes (Yamada et al., 1978). Although the mechanism of induction of differentiation of the cells by poly(ADP-ribose)is unknown, the polymer may change some physiological functions of the nuclei of the cells, since it has been detected in the nuclei and nuclear membranes ofthe cells by autoradiography (Yamada et al., 1978).

-

6 . Tunicamycin, Chloroquine,Bacillus Calmette-Guirin (BCG),and

Bacterial Cell Wall Skeletons Tunicamycin, a specific inhibitor of asparagine-linked glycosylation,was found by Nakayasu et al. (1 980) to induce functional and morphological differentiation of both MI cells and HL-60 cells. The inducing effect of tunicamycin on MI cells, however, may vary in different clones since

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tunicamycin could not induce differentiationof clone T-22 of M 1 cells even at a concentration of 1 ,ug/ml (Yamamoto et al., 1981b) although Nakayasu et al. ( 1980)reported that it was effectiveon M 1 cells at a concentration ofO. 1 to 1.Opg/ml. Chloroquine [7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline] has antiinflammatory activity, forms complexes with DNA, and inhibits DNA-dependent nucleic acid polymerase reactions. We found that chloroquine was an inducer of differentiation of M1 cells (Takenaga and Hozumi, 1980). Immunostimulants such as Mycobacterium bovis BCG, but not Corynebacterium parvum, and cell wall skeletons from Mycobacterium bovis and Nocardia rubra,but not from Propionibacteriumacnes,were also inducersof differentiation of M 1 cells (Maeda et al., 1980). 7. Other Compounds The effects of various other compoundson the induction ofdifferentiation of M1 cells have been examined by many investigators. Sachs (1978a) reported that dimethyl sulfoxide (DMSO), lectins, various compounds interacting with DNA, and X-ray irradiation induced some phenotypes of macrophages or granulocytes in some clones of M1 cells. Among the compounds tested, the protein inducer MGI was the only compound that induced all the changes to mature macrophages and granulocytes. Sachs and his collegues isolated MI cell clones responding differently to different inducersand showed that they had different cellular sites for MGI and other inducers (Sachs, 1978a). Ichikawa et al. (1975) and Nagata and Ichikawa ( 1979)detected no effect of various inhibitorsof DNA synthesison induction of differentiation of M 1 cells. We observed that W i n s (Con A), wheat germ agglutinin, Ricinus communis agglutinin, phytohemagglutinin (Yamamoto et al., 1980), DMSO (Hozumi et al., 1979b;Hozumi, 1982),and some chemicalsinteracting with DNA or inhibiting synthesis of DNA (Hozumi et al., 1979b;Hozumi, 1982) did not induce significantdifferentiation of some clones of M 1 cells that were sensitive to protein inducers. However, DMSO and some chemicals interacting with DNA (Hozumi et al., 1979b Hozumi, 1982) sensitized some clonesof M 1 cells that were resistantto inducers, causingdifferentiationwith these inducers. The compounds and the mechanisms of sensitization of the resistant M 1 cells will be described later. B. STIMULATORS OF PRODUCTION BY LEUKOCYTES OF D-FACTOR FOR MYELOID LEUKEMIA CELLS For induction of differentiation of myeloid leukemia cells in vivo, compounds stimulating production of D-factors and other modifiers of differen-

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tiation of leukemia cells seem of potential value as indirect inducers of cell differentiation in vivo. Some compounds were found to stimulate production of D-factor from leukocytes, although alone they could not induce differentiation of M 1 cells. Mouse peritoneal macrophages produced D-factor as described previously (Hozumi et al., 1979a) and treatment of the macrophages with either poly(1) * poly(C) or poly(A) * poly(U) enhanced their release of D-factor into the culture medium significantly, but treatment with poly(1) or poly(C) did not (Hozumi et al., 1979b; Hozumi, 1982). Synthetic N-acetylmuramyldipeptide(MDP), with the minimal structure required for adjuvant activity of bacterial cell peptidoglycan for increasing both humoral and cell-mediated immune responses (Azuma et al., 1976; Ellouz et a/., 1974; Taniyama and Holden, 1979), also enhanced the production of D-factor by a macrophage-like cell line, 5774.1, and differentiation of M 1 cells morphologically similar to macrophages, although MDP alone did not induce differentiation of M 1 cells (Akagawa and Tokunaga, 1980). We examined the effects of various compounds on production of D-factor from spleen cells, which are mostly lymphocytes and macrophages (Yamamot0 ef al., 1980,198la). Lectins (Con A, pokeweed mitogen, and phytohemagglutinin) stimulated spleen lymphocytes, but not spleen macrophages, to produce a D-factor with an apparent molecular weight of 40,000 to 50,000. In contrast, LPS and poly(1) poly(C) stimulated both spleen macrophages and spleen lymphocytes to produce D-factors. Although spleen macrophages produced D-factors with apparent molecular weights of both 40,000 to 50,000 and 20,000 to 25,000, spleen lymphocytes produced only the larger molecules. Immunostimulants such as the cell wall skeletons of Nocardia rubru and Propionibacterium acnes C7 and Corynebacterium parvum CN6 134 stimulated production of both spleen lymphocytes and spleen macrophages but none of these immunostimulants alone except Nocardia rubra and Mycobacterium bovis BCG could induce differentiation of M 1 cells, as described previously (Yamamoto et al., 198la). Furthermore, synthetic derivatives of N-acetylmuramyldipeptide (N-acetylmuramyl-L-valyl-D-isoglutamine and benzoquinon yl derivatives of N-acetylmuramyldipeptide), the minimal subunit of the bacterial cell wall with adjuvant activity, had no direct inducing effect on the differentiation of M1 cells and only slightly stimulated the production of D-factor by spleen cells (Yamamoto et al., 198la). The compounds that stimulated production of D-factor from spleen lymphocytes and spleen macrophages were also reported to induce production of various substances including interferon, CSF, and macrophage-activating factor (MAF) which might affect proliferation and differentiation of M 1 cells. We found that spleen lymphocytes treated with poly(1) poly(C) or Con A produced D-factor, CSF, and interferon (Yamamoto et a!., 1980). Although the activity of D-factor overlapped that of interferon with the same

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molecular size of about 40,000 to 50,000 on Sephadex G-75 or G-100 gel filtration, most of the CSF activity did not. The activity of D-factor was separated from that of interferon by treatment of the CM of lymphocytes with acid at pH 2.0, a treatment that reduced the activity of interferon only (Yamamoto el al., 1980). C.

BIOCHEMICAL PHENOTYPIC CHANGES ASSOCIATEDWITH MYELOID LEUKEMIA CELLDIFFERENTIATION 1. Membrane Components

During induction of differentiation of the mouse myeloid leukemia cell lines M 1, R453,and WEHI-3B, their morphological and functional phenotypes change into those of normal macrophages and granulocytes. Among these phenotypic changes in M 1 cells, changes such as migrating activity in soft agar, phagocytosis, adhesion to the substratum,agglutinabilityby Con A and Fc and C3receptors are suggested to be due to changes in membrane structure of the cells (Sachs, 1978a; Hozumi, 1982). We found that a glycoprotein with a molecular weight of 180,000(pl80) was induced in the cell surface of differentiated M 1 cells by protein inducers, dexamethasone, dbcAMP, or prostaglandin E, (Sugiyama et al., 1979b, 1980). The role ofp 180in the expressionofother phenotypesofdifferentiatedM 1 cells was examined. It was suggested that p 180 was involved in the mechanisms of cell-substrate adhesion and increase in agglutinability by Con A during differentiation of M 1 cells (Sugiyama et al., 1980). Pearlstein et al. (1978) found that an external membrane protein with a molecular weight of 195,000 on mouse peritoneal macrophages was related to cell-substrate adhesion. In addition, Trowbridge and Omary (198 1) reported that leukocyte surface glycoproteins bearing the macrophage differentiation antigen Mac- 1 could be detected on other murine hematopoietic cell types, including differentiated M 1 cells with Mac- 1 monoclonal antibody. They suggested that some of these surface glycoproteins were also involved in cell - cell or cell - substrate interactions. We examined the changes in the phospholipid compositions of two types of M 1 cells: sensitive M 1 cells that could be induced to differentiate into macrophages and granulocytesby various inducers, and resistant M 1 cells that could not differentiate even with high concentrations of the inducers (Honma et al., 1980~).Although there was no significant difference in the phospholipid compositions of these two types of cells, the percentage of phosphatidylcholine in total membrane phospholipids was less and the percentage of phosphatidylethanolamine was more in differentiated M 1 cells. Changes in the percentages of other phospholipids, such as lysophos-

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phatidylcholine and sphingomyelin,during differentiation were slight. The phospholipid composition of differentiated M 1 cells was similar to that of macrophages. Saito et al. (1980) reported that during differentiation of M 1 cells into macrophages induced by LPS, phospholipidssuch as sphingomyelin and phosphatidylserinedid not change significantly,but the amounts of phosphatidylcholine,phosphatidylethanolamine, and phosphatidylinositol increased markedly. We examined the change in phospholipid methylation during differentiation of M l cells (Honma ef al., 198l a). When M l cells were cultured with inducer, the incorporation of methyl groups into phosphatidylethanolamine decreased while the incorporation of choline into phosphatidylcholine increased significantly. The decrease of phospholipid methylation seemed to be partly due to a decrease of methyltransferase activity. These changes in phospholipid metabolism could alter the structure and function of the cell membrane (Hozumi, 1982). Saito et al. ( 1980) detected three major gangliosides in M 1 cells. During induction of differentiation of M 1 cells into macrophages with LPS, one ganglioside, a monosialoganglioside(GM 1b), increased markedly while the other two gangliosides, which were chromatographicallysimilar to GD3 and GD2 or GTla, decreased. The relative total amount of these major gangliosides remained unchanged. Akagawa et al. ( 1981) also reported that asialo GM 1, gangliotetraosylceramide, appeared on the cell surface of M 1 cells during differentiation induced by lymphokine-rich mouse sera. These changes in ganglioside composition of differentiated M1 cells might be related to some structural and functional alterations associated with cell differentiation.

2. Enzymes a. Lysosomal Enzymes. The activities of various lysosomal enzymes, such as lysozyme, acid protease, acid phosphatase, and 8-glucuronidase, were induced during induction of differentiation of M1 cells by various inducers (Sachs, 1978a; Kasukabe et al., 1979a; Hozumi, 1982). Of these lysosomal enzymes, lysozyme showed the most pronounced increase in activity. However, the activities of other lysosomal enzymes, such as acid DNase and acid RNase, in M1 cells were not induced significantly by dexamethasone (Kasukabe et al., 1977a). Although acid protease in M 1 cells was induced during differentiation,an alkaline protease, the main protease in untreated M 1 cells with maximum activity at pH 1 1.O, disappeared concomitantly (Oshima et al., 1979). This alkalineprotease could not be detected in a macrophage-likecell line (Mm-1) established from spontaneously differentiated macrophage-like cells from M 1 cells (Oshima et al., 1979).

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The biochemical properties of lysozyme produced by differentiated M 1 cells were compared with those of lysozymes produced by normal cells and tissues (Kasukabe et al., 1979a). Lysozyme purified from the culture medium of the macrophage-like cell line Mm- 1 cells had a molecular weight of 15,000 with an optimum pH of 6.6. The electrophoretic mobility of this lysozyme was distinctly lower than those of lysozymes from hen egg white and human urine. On the other hand, rabbit anti-Mm-1 lysozyme serum inhibited the activities of lysozyme preparations from peritoneal macrophages of normal mice and rats and dexamethasone-inducedM 1 cells, but did not inhibit the activities of hen egg white or human preparations (Kasukabe et al., 1979a).The biochemical and immunochemical properties of lysozyme purified from normal mouse lung, which is rich in alveolar macrophages,were also similar to those of the purified lysozyme from Mm- 1 cells (Kasukabe et al., 1979a). These results show that the molecular structure of the lysozyme induced in differentiatedM 1 cells is similar to that of the lysozyme produced by normal cells. Use of different inducers showed that the induction of lysozyme was under separate control from those of other phenotypes, such as Fc and C, receptors and morphological changes to mature macrophages and granulocytes, in mutant clones of M1 cells (Sachs, 1978a; Hozumi, 1982). b. Prostaglandin Synthetases. We examined the activity of prostaglandin synthetase in M1 cells with [14C]arachidonate(Honma et al., 1980d). Although untreated M l cells could not convert arachidonateto prostaglandins, dexamethasone-treatedM 1 cells produced prostaglandin E2,D, ,and F, in an early stage of differentiation, whereas mature cells produced mainly prostaglandin E, , In differentiated M 1 cells, prostaglandin F, isomerase activity was suggested to be repressed in the process of prostaglandin synthesis (Honma et al., 1980d). The conversion of arachidonate to prostaglandins might be catalyzed by fatty acid cyclooxygenasesince it was completely inhibited by incubating an homogenate of dexamethasone-treated M 1 cells with indomethacin. Homogenates of LPS-treated MI cells also converted arachidonate to prostaglandin F,, E,, and D, ,whereas the particulate fraction of dexamethasonetreated M 1 cells produced prostaglandin F, and E, . These results suggest that prostaglandin D isomerase is present as a cytosolic component and that induction of prostaglandin synthesis during differentiation of M 1 cells results from induction of the activities of prostaglandin synthetases and stimulation of arachidonate from cellular phospholipids. The pattern of prostaglandin production by differentiated M1 cells was similar to that by mouse macrophages, except that the macrophages also released a little 6-oxoprostaglandinF,, whereas differentiated M 1 cells did not. c. Reverse Transcriptase. Dexamethasone induced differentiation of

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some clones of M 1 cells, but it did not induce an increase in the amount of reverse transcriptase activityin the culture medium or in the virus fraction in the culture medium of the cells. Since the cells produced a detectable level of the enzyme activity continuously (Kasukabe et al., 1977b), virus production in M 1 cellsdoes not seem to be associated with induction ofdifferentiationby dexamethasone. Liebermann and Sachs (1977) found that some clones of MI cells that could be induced by MGI to differentiate into mature macrophages and granulocytes (MGI+D+cells) produced a higher activity of reverse transcriptase in the culture medium, indicatingproduction of a higher amount of type c virus than clones in which induction ofdifferentiationby MGI was partially (MGI+D-) or almost completely (MGI-D-) blocked. They further indicated a specific pattern for ecotropic virus production during MGI-induced differentiation of MGI+D+-typeM 1 cells, involving enhanced virus production at an early stage of differentiation and interruption of Virus production in mature cells (Lieberman and Sachs, 1978). They also showed that infection of normal myeloblasts with ecotropic virus from MGI+D+-typeMI cells promoted proliferation of these myeloblasts, which could then still be induced to differentiate normally (Liebermann and Sachs, 1979), d. Cytochrome Oxidase, Glucose-6-phosphatase, and Lipogenic Enzymes. In differentiatedM1 cells induced by CM from secondary embryo cells, or an LPS, the number of mitochondria increased markedly with increase in activity of cytochrome oxidase per cell, although the activity per mitochondrion remained unchanged (Hirai et al., 1979). The rough-surfaced endoplasmic reticulum elongated and the activity of a marker enzyme of the reticulum, glucose-6-phosphataseY also increased in differentiatedM 1 cells. Furthermore, primary lysosomes with histochemically demonstrable acid phosphatase activity were found to be newly formed in the M1 cells (Hirai et al., 1979). Okuma et al. ( 1976) examined the synthesis of phosphatidic acid, a key intermediate in the synthesis of phospholipids and triglycerides in most animal tissues, from sn-glycerol3-phosphatein M 1 cells. They found that the microsomal fraction of M1 cells could catalyze acylation of sn-glycerol 3-phosphate by long-chain fatty acyl-CoA thioesters and could produce phosphatidic acid. But since M 1 cells and macrophages differentiated from M 1 cells had similar levels of sn-glycerol3-phosphate-acylatingactivity and of acetyl-CoA carboxylase activity, differentiation of MI cells is not associated with changes in the activities of these lipogenic enzymes.

3. Cytoplasmic Proteins Liebermann et al. (1980) analyzed changes in cytoplasmic proteins of various clones of mouse myeloid leukemia cells, including M 1 cells, in total

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cell extracts pulse-labeled with [35S]methioninefrom 1 hr to 6 days after addition of a protein inducer, MGI. Results showed that the sequence of protein changeswas similar in MGI-inducednormal and MGI+D+leukemic cells. Many proteins decreased before the appearance of de novo-synthesized proteins and differentiation of the cells was suggested to involve multiple, parallel, separately programmed pathways of gene expression that could be induced separately. These findingssuggest that there is a relation between the constitutive expression of certain pathways of genes in myeloid leukemia cells and cell competence for growth and differentiation in myeloid leukemia cells (Sachs, 1980, 1981). D. CHANGES IN PROLIFERATION POTENTIAL DURING DIFFERENTIATION OF MYELOID LEUKEMIA CELLS 1. Relation between the Cell Cycle and Commitment to Cell Diferentiation

The relation between differentiation and the cell cycle of M1 cells was examined with a protein inducer, CM of hamster embryo cells (Hayashi et al., 1982). A clone of M1 cells, B24, was induced to differentiate into macrophages by the protein inducer. When the M 1 B24 cells were treated with the protein inducer, the cells traversed the S phase of the cell cycle at least once. Then a fraction of the cells lost the ability to enter the S phase and accumulated in the G , phase. Incorporation of [3H]thymidinein phagocytosis-induced cells decreased after treatment with the inducer for 12 - 18 hr and the morphology of the cells changed in association with a significant decreasein the nucleus - cell ratio (NCR)of individual cells during treatment with inducer for 24 hr. The NCR was determined in M 1 B24 cells that had been prelabeled with [3H]thymidine,chased for various periods, and treated with the protein inducer for 24 hr. No significant difference was found between the NCRs of labeled and unlabeled cells. These results suggest that M 1 B24 cells in any phase ofthe cell cycle can respond to the protein inducer and can be initiated to differentiate. M 1 B24 cells treated with the protein inducer showed a lag time of about one cell cycle before arrest in G, (Hayashi et al., 1982). The kinetics of decreasein proliferation of M 1 cells differs from that of HL-60 cells or Friend cells. When HL-60 cells were induced to differentiate into macrophages by phorbol ester, most ofthe cells that were not in the S phase never entered the S phase, and there was a slight increase in cell number shortly after treatment with inducer (Rovera el al., 1980). In contrast, Friend erythroleukemiacells, which also had limited ability to proliferate after inducer treatment, showed four or five additional mitoses after treatment with inducer (Gusella et al.,

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1976; Friedman and Schildkraut, 1977). The discrepancies in these results may be due to differences in the cells, inducers, and differentiation pathways, as well as to differences in variety in the states in which cellulardifferentiation is blocked during leukemic transformation. The finding that there is no specific phase of the cell cycle at which induction of M1 B24 cell differentiation occurs seems consistent with previous observations by Ichikawa et al. (1975) that M 1 cells could be induced to differentiate under conditions when DNA synthesiswas inhibited by FUdR. In the human myeloid leukemia cells HL-60 cells and KG- 1 cells DNA synthesis was also shown to be unnecessary for development of phenotypic properties of macrophages such as phagocytic activity and a-naphthyl acetate esterase and acid phosphatase activities (Rovera et al., 1980; Territo and Koeffler, 1981).

2. Kinetics of Changes in Proliferation and Differentiationof Populations of Myeloid Leukemia Cells Regulation by humoral factors of growth and differentiation of various human and myeloid leukemic cells has been studied by quantitative determinations of biochemical and functional properties during differentiation of the cells (Sachs, 1978a; Burgess and Metcalf, I980b; Hozumi, 1982). These studies showed that the proportion of differentiating cells among the total cells depended on the concentration of inducer and that the cell population did not differentiate synchronously. Similar asynchronous differentiation was also observed in colonies of normal hemopoietic cells in semisolid agar as well as in clonal leukemic cell populations (Metcalf, 1977).However, the details of the kinetics of proliferation and differentiation of the myeloid leukemia cells and the mechanisms of induction of this asynchronous differentiation in the cells remain to be examined. We investigated the mechanisms regulating the kinetics of proliferation and differentiation of M 1 B24 cells by quantitative determination of cellular morphology (Hayashi et al., 1981). Results showed that the process of differentiation of M 1 B24 cells was promoted by increasing the concentration of inducer, protein inducer in CM of embryo cells, and that the transition of M1 B24 cells from the undifferentiated state to the differentiated state occurred in a stochastic manner and the proportion of well-differentiated cells in the whole cell population WPF higher at higher concentration of the inducer. The proliferative activity of Individual M 1 B24 cells, the labeling index of the cells with [3H]thymidine,decreased at a specificstage of differentiation at which the NCR of the cells was between 50 and 30Y0,and this decrease was independent of the culture time of the cells and the concentration of the inducer. No proliferative activity was observed in cells

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day 0

day 1

r J

0

2 la.

20

day 2

day 4

- 5%CM *

60 50 40 30

60 50 40 30 20

60 50 40 30 20

Nucleus - cell ratio ( */.

FIG. 2. Decrease in synthesis of DNA in MI B24 cells during morphological differentiation (Hayashi ef al., 1981). MI B24 cells were cultured with 2.5%conditioned medium (CM) of hamster embryo cells (upper figures) or 5% CM (lower figures) for the indicated times. The ratio of the area of the nucleus to that of the cell was determined on 100 randomly selected [3H]thymidine-labeled (stippled histogram) and unlabeled (open histogram) cells in each specimen.

in which the NCR had decreased below 30% (Fig. 2). These results suggest that the production of differentiated cells is controlled by a balance between proliferation and differentiation of the cells that is dependent on the concentration of the inducer. Ichikawa et al. (1969,1975) previously reported that high concentrations of protein inducer in CM from embryo cells or spleen macrophages suppressed the formation of colonies of M1 cells in agar medium. Recently, Metcalf ( 1980)found that the fraction of colony-formingcells in a WEHI-3B cell population gradually decreased and completely disappeared during serial recloning in the continuous presence of a protein inducer, postendotoxin mouse serum. In principle, the mechanisms controlling the kinetics of proliferation and differentiation of this cell population of leukemic cells may be similar to those of M1 B24 cells. Postendotoxin serum or CM of embryo cells, however, contain various factors affecting growth and differentiation of WEHI-3B or MI cells, as described previously. Therefore, further detailed studies are needed on the mechanisms controlling the proliferation and differentiation of these leukemic cells with purified factors for proliferation and differentiation ofthe cells.

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E. POSITIVEFEEDBACK CONTROLMECHANISMS OF CELL DIFFERENTIATION BY PROTEIN INDUCER PRODUCED BY DIFFERENTIATING MYELOIDLEUKEMIA CELLS M 1 cells could be induced by glucocorticoidsto produce a glycoprotein(s) with a molecular weight of 20,000 to 40,000 that stimulated induction of differentiation of M 1 cells into macrophages and granulocytes (Hozumi, 1982). Although glucocorticoids induced production of the glycoprotein inducer in M 1 cells that could be induced to differentiate by glucocorticoids, they could not stimulate the production of the glycoprotein inducer in dexamethasone-resistant M 1 cells that could not differentiate even with a high concentration of dexamethasone (Hozumi, 1982).Thus, production of the glycoprotein inducer was associated with differentiation of M 1 cells. We recently found that dexamethasone could also induce CSF for mouse bone marrow cells (Okabe-Kado et al., 1982). Induction of differentiation of M 1 cells by glucocorticoidswas suppressed by treatment with actinomycin D or puromycin, but not with FUdR or cytosine arabinoside (Ara C) (Hozumi, 1982).These findings suggest that the synthesis of some species of RNA and proteins may be required for the induction of differentiation of M 1 cells, although it is unknown whether the proteins synthesized mediate glucocorticoid-induced differentiation of M 1 cells. Some clones of M 1 cells were also recently found to produce a factor(s) inducing differentiation of M 1 cells, MGI, during differentiation of the cells by treatment of the cells with various compounds such as LPS (Sachs, 1978a), phorbol esters (Lotem and Sachs, 1979), and N-methyl-N-nitro& nitrosoguanidine (NMNG) (Falk and Sachs, 1980). The regulation of induction of two activities, MGI- I (CSF) and MGI-2 (D-factor), during differentiation ofM 1 cells induced by NMNG or LPS was examined (Falk and Sachs, 1980). Experiments on the time courses of induction of the activities of MGI-1 and MGI-2 by NMNG or LPS showed that MGI-1 was induced before MGI-2. Dexamethasone, however, did not induce either MGI- 1 or MGI-2, contrary to our findings (Honma ez al., 1982a; Hozumi, 1982), although a clonal variation in the inductions of MGI-1 and MGI-2 was found. These results show that the regulations of the inductions of MGI- 1 and MGI-2 in M 1 cellsare different and that the inducibilities ofthe two MGI activities also vary in different clones of M 1 cells. Maeda and Ichikawa ( 1980) also reported that CSF was produced by M 1 cells during differentiation induced by bacterial LPS, although no significant amount of D-factor was produced. Furthermore, they showed that the continued presence of LPS was necessary to stimulate the differentiated M 1 cells, macrophages, to release CSF, whereas a macrophage-like cell line

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(Mm-1) derived from the M1 line produced CSF without stimulation by LPS. F. INHIBITORS AND THEIR MECHANISMS OF INHIBITION OF DIFFERENTIATION OF MYELOID LEUKEMIA CELLS

1. ThymidineAnalogs, Actinomycin D, and Puromycin 5-Bromodeoxyuridine(BUdR) was reported to induce some phenotypes of a certainclone of M 1cells (Sachs, 1978a),but Nagata and Ichikawa (1979) showed that BUdR, 5-bromodeoxycytidine, and 5-iododeoxyuridine blocked the induction of phagocytosis and motility of M1 cells without affectingthe induction of Fc receptor. The blocking effect of BUdR on the induction of cellular phagocytosis and motility was prevented by the addition of excess thymidine and BUdR had no effect in a BUdR-resistant cell line (Nagata and Ichikawa, 1979). Therefore, the inducibilitiesof phagocytosis and motility of M 1 cells, but not the induction of the Fc receptor, are suggested to be controlled genetically. Actinomycin D at 30- 50 pg/ml markedly inhibited the induction of phagocytosis, but not that of the Fc receptor in M 1 cells, while puromycin at a concentrationof more than 2.5 X 10-6Mmarkedlyinhibited the induction of phagocytosis but slightly enhanced the induction of Fc receptor (Nagata and Ichikawa, 1979). These results suggest that new syntheses of messenger RNA and protein are required for induction of new phenotypic markers in M 1 cells and that the mechanisms controlling induction of Fc receptor are different from those controlling induction of phagocytosis. 2. Cytochalasin3 Cytochalasin B reversibly inhibited the induction by CM from mouse embryo cells of phagocytosisand motility of M1 cells (Ichikawa el al., 1975). This suggests that cytochalasin B-sensitive proteins containing actin, such as microfilaments,may be involved in the mechanisms of differentiation of M 1 cells. Nagata et al. ( 1980) found that polymerization of G-actin in the M 1 cells is closely associated with differentiation of the cells. Hoffman-Lieberman and Sachs (1978) also reported that there was a marked increase in the content of actin during differentiation of M 1 cells, although both untreated MI cells and mature macrophages and granulocytes contained actin as a major protein component.

3. Nonsteroidal Antiinfammatory Agents and B- and F-Type Prostaglandins Synthesis of E-type prostaglandins is involved in the mechanisms of differentiation of M 1 cells and nonsteroidalantiinflammatory agents such as

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salicylate, phenylbutazone, and indomethacin, that inhibit synthesis of these prostaglandins, blocked the induction of differentiation of M 1 cells by various inducers (Hozumi et al., 1979a). The inhibition was unrelated to cytotoxicity and was reversible. The inhibition by indomethacin of dexamethasone-induced differentiation was observed only when indomethacin w'as added before the time of commitment of the cells to differentiation. Although prostaglandins E, , E,, and D, induced lysozyme activity and stimulated differentiation of M 1 cells induced by a suboptimal concentraF inhibited the tion of inducer (Honma et al., 1979,1980d),prostaglandin , induction by dexamethasone of phagocytic and lysozyme activities in the F stimulated production of cells (Takenaga et al., 1982). Prostaglandin , differentiation-inhibiting activity (I-activity) in M 1 cells (Takenaga et al., 1982).The I-activity was found to be due to a heat-labile, trypsin-sensitive proteinous substance(s). B-Type prostaglandins also stimulated I-activity production, whereas A-, E-, and D-type ones did not. The induction of I-activity by prostaglandin F, was suppressed by simultaneous treatment with prostaglandin E, (Takenaga et af., 1982). 4. Retinoids and Phorbol Esters

Various retinoids other than the pyridyl analog of retinoic acid induced lysosomal activities in M1 cells (Takenaga et al., 1980). However, the retinoids did not induce phagocytic or migrating activity or morphological changes of MI cells and they reversibly inhibited the induction of these properties by various inducers (Takenaga et al., 1980).These findings suggest that there are distinct mechanisms for control of induction of lysosomal enzyme activities and of other differentiation-associated properties of M 1 cells. The retinoids could induce prostaglandins D, , E,, and F, in M 1 cells and the induction of prostaglandin E, was a prerequisite for increase in lysozyme activity (Takenaga, 198 l), whereas retinoic acid stimulated the productions of prostaglandin F,, and I-activity in M 1 cells, and indomethacin inhibited the I-activity by retinoic acid (Takenaga et al., 1981 b, 1982).It is , alone unknown whether I-activity is actually produced by prostaglandin F in M 1 cells treated with retinoic acid, since prostaglandin E,, which counteracts the production of I-activity in the cells, is also produced by retinoic acid. Furthermore, it is unknown whether the I-activity induced by retinoic acid is actually identical with that induced by prostaglandin F,, although this I-activity is heat labile and protease sensitive (Takenaga et al., 198 1 b). Tumor-promoting phorbol esters such as 12-0-tetradecanoylphorbol- 13acetate (TPA) have been reported to modify differentiation of various types of normal and tumor cells and cellular modification by phorbol esters is suggested to be involved in mechanisms of the promotion phase of carcinogenesis(Weinstein etal., 1979;Yamasaki, 1980;Hecker, 198 1). Although we found that TPA and other tumor-promoting plant diterpenes could inhibit

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induction of differentiationof M 1 cells into macrophages and granulocytes by dexamethasone or protein inducer (Kasukabe et al., 1979b), Lotem and Sachs (1979) and Nakayasu et al. (1979) reported that tumor promoters scarcely affected,or rather enhanced the induction of cell differentiation by some inducers. On examination ofthese conflictingresults, we found that the response of M 1 cells to TPA was affected by culture of the cells with different sera (Kasukabe et af., 1981; Hozumi et af., 1982): TPA inhibited both functional and morphological differentiation of MI cells cultured in medium containing calf serum or horse serum, as we reported (Kasukabe et al., 1979b), but it enhanced these inductions in medium containing fetal calf serum, as reported by Nakayasu et al. ( 1979)and Lotem and Sachs ( 1979). Therefore, these discrepant results on responses of the cells to TPA observed in previous studies were partly due to differences in the sera used: other workers used fetal calf serum but we used calf serum. The factor@)in the serum affecting the differentiationof M 1 cells with TPA was a nondialyzable macromolecule. On Sephadex G-200 gel filtration, much more inhibitory activity was found in calf serum than in fetal calf serum and stimulatory activity was found only in fetal calf serum (Hozumi et al., 1982). The modification by TPA of synthesis of prostaglandinE, was found to be closely associated with the effects of different sera on differentiation of M1 cells (Hozumi et al., 1982). Lotem and Sachs ( 1979) reported clonal differences in susceptibility to induction of differentiation by TPA. Hoffman-Liebermann et al. ( 1981) showed that TPA could complement changes in gene expression induced by MGI, and that TPA could regulate gene expression at the level of both mRNA production and mRNA translation. TPA and phorbol 12,13-didecanoateinhibited the induction of lysozyme activityby retinoic acid in M 1 cells, but 4a-phorbol didecanoateand phorbol did not. TPA and phorbol 12,13-didecanoate,but not 4cu-phorbol didecanoate, also inhibited the stimuIation of prostaglandin E, production by retinoic acid, suggestingthat stimulation by retinoic acid of prostaglandin E, production in M 1 cells is a prerequisite for induction of lysozyme activity (Takenaga, 1981). Both TPA and retinoic acid synergistically inhibited the induction of phagocytic activity in M 1 cells by dexamethasone (Takenaga, 1981).

5. Antioxidants Antioxidants, such as phenolic antioxidants, sulfur-containing compounds, a-tocopherol, retinoids, ascorbate, and selenium, have been reported to inhibit chemical carcinogenesis(Wattenberg, 1979;Griffin, 1979). The cellular mechanisms of inhibition of chemical carcinogenesis by these antioxidants are unknown, but have been suggested to involve modification

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of mutation (Batzingeret al., 1978;Calle et al., 1978; Rosin and Stich, 1979) and cellular differentiation (Hozumi, 1982). Therefore, we examined the effects of the antioxidants on differentiation of M1 cells. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and a-tocopherol significantly inhibited differentiation of the cells by dexamethasone or D-factor from ascitic fluid, but other antioxidants had little or no inhibitory activity. Of the antioxidants that were not cytotoxic, BHA was the most potent inhibitor (Takenaga et al., 198la). The inhibition ofdifferentiation of M 1 cells by BHA was reversible and was suggested to be due to inhibition of synthesis of E-type prostaglandins(Takenaga et al., 1981b). Several antioxidants were reported to inhibit prostaglandin synthesis and lipid peroxides formed by lipooxygenase were shown to activate prostaglandin synthesis (Hozumi, 1982). Of the antioxidants tested, hydrophobic antioxidants (BHA, BHT, and a-tocopherol) had more effect than hydrophilic ones (cysteamine, selenite, and glutathione) in inhibiting differentiation of the cells (Takenaga et al., 1981a). These findings suggest that the sites of action of the antioxidants on the cells may be related to their hydrophobicities.

6. Histone H3. Poly-L-lysine. and poly-L-arginine Although histone H 1 could induce differentiationof M 1 cells into macrophages and granulocytes, histone H2A and H2B could not (Okabe-Kado et al., 1981). On the contrary, the histone H3 fraction, poly-L-lysine, and poly-L-arginine markedly inhibited induction of differentiation of M 1 cells by dexamethasone (Okabe-Kado et al., 198I). The differencesin the mechanisms of effects of histone H1 and histone H3 as well as poly-L-lysine and poly-L-arginine require investigation. G. PROPERTIES OF MYELOID LEUKEMIA CELLSRESISTANT TO INDUCERS OF CELLDIFFERENTIATION Some populations of myeloid leukemia cell lines spontaneously become resistant to inducers of cell differentiation during long-term culture of the cells. These resistant cells were isolated by cloning in soft agar medium and their properties were examined. The isolation and characterization of these spontaneous resistant clones from M 1 cells and those ofresistant clones from myeloid leukemiasin X-ray irradiated SJL/J mice were reviewed extensively by Sachs (1978a, 1980, 1981). Results with these resistant clones show that there can be blocks at different stages of differentiation by various inducers and that there are separate controls for the induction of each phenotype of differentiation (Sachs, 1978a). Furthermore, studies on changes in the synthesis of specific proteins in normal myeloblasts, and various resistant

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clones of M 1 cells at different times after treatment with MGI suggested that the constitutive expression of some pathways of gene expression results in leukemia, whereas the constitutive expression of other pathways results in decreased competence for induction of differentiation of the leukemia cells (Sachs, 1980, 198I). Ichikawa er al. ( 1975)also isolated aclone (D-) that was resistant to protein inducer in CM of mouse embryo cells and his group examined its properties. Nagata er al. ( 1980) showed that actin harvested from CM-treated differentiating M 1 cells could polymerize, but could not polymerize with the actin from the D- clone. Moreover, Mae& and Ichikawa (1980) found that bacterial LPS could stimulate production of CSF from differentiation-sensitive M 1 cells but not from the D- clone of M 1 cells. We isolated several variant clones that were resistant to either dexamethasone (DR-1 to DR-6, six clones) or protein inducer (D-factor) in ascitic fluid ofratsbearinghepatoma(R,R1, R2, R15, andRl8)(Hozumier al., 1979a). The dexamethasone-resistant clones did not have a defect in penetration of glucocorticoidsor in cytoplasmic receptor binding in the cells. However, the number of nuclear receptor binding sites of [3H]dexamethasone in the resistant clones was markedly less than in sensitive clones of MI cells (Hozumi et al., 1979a). Sachs and his co-worker also islated dexamethasone-resistant clones of M 1 cells, but found that the lack of response of these resistant clones to dexamethasone was not due to any defects in the binding of dexamethasone to cytoplasmic or nuclear receptor sites (Sachs, 1978a). We examined differences in sensitivitiesof the resistant clones of M 1 cells to glucocorticoids and protein inducer in ascitic fluid from rats (Hozumi et al., 1979a). Although all the dexamethasone-resistant clones showed less response than the sensitivecells to the protein inducer, some clones that were resistant to induction with the protein inducer were induced to differentiate by dexamethasone. Therefore, the steroid and the protein inducer have different targets in the cells. The M 1 cell clones that were resistant to the protein inducer or dexamethasone, but not the sensitive clones, were found to produce an inhibitory activity (I-activity)for induction of differentiation of M 1 cells (Okabe er al., 1978). The I-activity was due to a nondialyzable, heat-labile, and proteasesensitive substance(s) that was precipitated at 30- 50% saturation of ammonium sulfate (Okabe er al., 1978). The I-activity in the culture medium of the resistant cells was decreased by treatment of the cells with a low concentration (5 - 10 ng/ml) of actinomycin D (Okabe er al., 1978). During decrease of their I-activity, the resistant cells became sensitive to the protein inducer in ascitic fluid. Therefore, production of I-activity in the resistant cells was closely associated with resistance of M 1 cells to the D-factor.

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The CM from resistant M1 cells was recently found to inhibit colony formation of bone marrow cells of normal mice by CSF. This inhibitory activity ofthe CM also decreased on treatment ofthe resistant cells with a low concentration of actinomycin D (Okabe-Kado et al., 1982). It is unknown, however, whether the inhibitory activity against normal bone marrow cells is identical with that against leukemia M 1 cells. Normal hematopoiesis has been found to be negatively regulated by inhibitors from various sources (Broxmeyer and Moore, 1978; Broxmeyer et al., 1981;Moore, 1979).It will be interesting to examine the relation between the actions of these inhibitors and that of the I-activities from resistant M 1 cells.

H. SENSITIZATION OF MYELOID LEUKEMIA CELLSRESISTANT TO INDUCERS OF CELLDIFFERENTIATION 1. A41 Cells

We examined the effects of various compounds on sensitization of resistant clones of M1 cells to inducers of cell differentiation. Inhibitors of RNA or protein synthesis were found to induce differentiation of resistant cells in the presence of the protein inducer in ascitic fluid, although they had no effect alone. In contrast, inhibitors of DNA synthesis, such as Ara C and FUdR, had no effect (Hozumi et al., 1979a). Inhibitors of RNA synthesis (actinomycin D, chromomycin A3, nogalamycin, and cordycepin) were effective at low concentrations that scarcely affected cell viability. Of these inhibitors, actinomycin D was the most effective. The sensitizing effect of actinomycin D on the resistant cells (R1 or R2) was found to be roughly parallel to the extent of its inhibition of RNA synthesis in the cells without affecting synthesis of DNA (Hozumi et al., 1979a). We found that the I-activity, protein(s), in CM of the resistant clone of M 1 cells decreased with development of sensitivity to the inducer, suggesting that production of the I-activity in the resistant cells was associated with resistance of the M 1 cells to the inducer (Okabe et a!., 1978). Some cancer chemotherapeutic drugs (adriamycin, daunomycin, mitomycin C, hydroxyurea, 5-fluorouracil, and bleomycin), which might also inhibit the production of I-activity in the resistant M 1 cells, induced differentiation of the resistant cells in the presence of D-factor in ascitic fluid (Hozumi et al., 1979b). The drugs or D-factor alone had no effect. In combination with the D-factor, 6-mercaptopurine, amethopterin, or aminopterin could not induce differentiation of resistant cells. On the other hand, Sachs (1978a) reported that several cytotoxic chemicals, including some anticancer drugs, and X irradiation alone could induce

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some phenotypic markers in differentiated cells from some sensitive clones of M 1 cells. These results suggest that some of the cytotoxic anticancer drugs alone or in the presence of an inducer of cell differentiation control tumor growth not only by their cytotoxic effect, but also by their ability to induce differentiation of the cells. Poly(1) poly(C), which could induce interferon in M1 cells, markedly stimulated differentiationof a resistant clone of M 1 cells, R4,in the presence of CM of macrophages, although CM or poly(1) - poly(C) alone induced scarcely any differentiation of the cells (Yamamoto et al., 1979). Sera obtained from SL strain mice injected with poly(1) poly(C) could induce differentiation of both sensitive and resistant clones of MI cells, since the sera contained both D-factor and interferon that could sensitizethe resistant M 1 clone cells to the D-factor (Tomida et al., 1980a,b).

-

2. R453 Cells R453 cells were induced to differentiate into macrophages and granulocytes by protein inducer in ascitic fluids, CM from various cell lines, and glucocorticoid hormone (Sugiyama et al., 1979a). This induction of differentiation of R453 cells was markedly enhanced by addition of inhibitors of the synthesis of RNA (actinomycin D, chromomycin A3, nogalamycin), protein (puromycin),or DNA (Ara C, FUdR, methotrexate, hydroxyurea)in the presence of ascitic fluid (Sugiyama et al., 1979a). Of the inhibitors, actinomycin D was the most effective, although the inhibitors alone had no effect. IV. In Vivo Induction of Differentiation of Mouse Myeloid Leukemia Cells and Therapy of Animals Inoculated with Myeloid Leukemia Cells

A. In Vivo INDUCTION OF DIFFERENTIATION OF MOUSE MYELOID LEUKEMIA CELLS

Based on the results of in vitro experiments on induction of differentiation of myeloid leukemia cells, we tried to induce in vivo differentiationof mouse myeloid leukemia M 1 cells. We used two types of clones of M 1 cells, clones that were sensitive and resistant to inducers of differentiation, to investigate the relationshipbetween cell competencefor induction ofdifferentiationand changes in leukemogenicity (Honma et al., 1978). Although no detectable difference in the saturation densities or growth rates ofsensitive and resistant cells in culture was observed in vitro, the sensitivecellsgrew more slowly than the resistant cells in diffusion chambers in syngeneic SL mice. The sensitive cells were induced by some endogenous factors to differentiate into mature macrophages and granulocytes losing their proliferation potentials while the resistant cells in the diffusion chambers remained undifferentiated (Honma et al., 1978).

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The leukemogenicities of resistant clones and sensitive clones of M 1 cells were examined in syngeneic SL mice. All the resistant clones were much more leukemogenic than the sensitive cells, and the survival times of syngeneic mice inoculated with them were less than those of mice inoculated with the sensitiveclones (Honma et al., 1978).These findings suggest that the leukemogenicityof M 1 cells in syngeneicmice is related to in vitro and in vivo inducibility of differentiation of the cells. Lotem and Sachs (1978a) also examined the in vivo inducibility of differentiation of several clones of mouse myeloid leukemia cells that differed in their competence to differentiate into macrophages and granulocytes with protein inducer. A sensitive clone, MGI+D+,as well as a resistant clone, MGI+D-, that did not form mature cells with protein inducer in vitro were both induced to differentiate into mature cells in diffusion chambers in normal syngeneic or allogeneic mice. Another resistant clone, MGI-D-, which could not be induced by the protein inducer to develop any differentiation-associated properties in vitro, was also induced in vivo to show C3and Fc receptors, lysozyme, and intermediate stages of morphologically differentiated cells, but not to differentiate into mature cells. The reasons for these differences between the inducibilities of cell differentiation in vitro and in vivo remain to be examined. Although diffusion chambers are useful in studies on differentiation in vivo, they prevent cell-to-cell interaction of inoculated leukemia cells with host immunocompetent cells. To examine the relation between leukemogenicity and in vivo inducibility of differentiation of the cells under natural conditions without any artificial bamers we labeled MI cells in vitro with [3H]thymidine and injected them into the peritoneal cavity of a syngeneic mouse. After several days the peritoneal cells were harvested and the 3H-labeledcells were determined by autoradiography. Results showed that the inoculated isotope-labeled M 1 cells differentiated into macrophages and granulocytes in the peritoneal cavity of the syngeneic mouse (Honma et al., 1982b). An inducer of cell differentiation, LPS, significantly stimulated differentiation of the isotope-labeled cells inoculated into the peritoneal cavity. These results provide direct evidence that M 1 cells can be induced to differentiate in vivo under conditions in which leukemia can develop.

B. THERAPY OF MICEINOCULATED WITH MYELOID LEUKEMIA CELLS BY INDUCTION OF DIFFERENTIATION I . Sensitive M I Cells The effects of LPS and glucocorticoids,two potent inducers of differentiation of M l cells, on the leukemogenicities of sensitive or resistant M l cells were examined (Honma et al., 1978, 1979).The inducers of cell differentia-

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TABLE I1 RELATIONSHIP BETWEEN in Vivo INDUCTION OF DIFFERENTIATION OF MI CELLS AND PROLONGATION OF SURVIVAL TIMES OF SYNGENEIC SL MICE INOCULATED WITH M 1 CELW

M I cell clone

Differentiation

Prolongation of survival times of mice

Protein inducer (MGI)

Clones sensitive to inducer DS4 DS4 T-22 MGI+D+

+ + + +

+ + + +

Lipopolysaccharide Lipopolysaccharide Actinomycin D POlY(1) * PolY (C)

Clones resistant to inducer DR3 R- 1 R- I R-4

-

-

+

+

Compound

Lipopolysaccharide Dexamethasone POlY(1)

*

POIY (C)

+

+

,,FromHonmaelal. (1978,1979,1980b),LotemandSachs(1978,198l),Okabeefal.(1979), and Tomida et al. ( i980b).

tion significantly enhanced the survival times of mice inoculated with sensitivecells, but scarcely affectedthe survivaltimes of mice inoculated with resistant cells (Table 11). These effects are consistent with the effects of the inducers on induction of differentiation of sensitiveand resistant cells in vivo and in vitro, suggesting that prolongation of the survival time may be associated with stimulation by the inducers of in vivo cell differentiation. The sensitive and resistant M1 clone cells contained similar common tumor-related surface antigens (Honma et al., 1978, 1979). LPS enhanced the survival times of immunodeficient newborn syngeneic SL mice and athymic nude mice inoculated with the sensitive M 1 cells to similar extents (Honma et al., 1978, 1979). Therefore, T-lymphocyte-mediated immune mechanisms may not be directly involved in the effect of LPS on prolongation of the survival time. The survival times of syngeneic SL mice were prolonged by dexamethasone at an optimal dose (20pg/mouse) but shortened by dexamethasone at a high dose (400 &mouse) (Table 11). Similar results were obtained with prednisolone, indicating that glucocorticoids, at an optimal dose, prolonged the survival times of syngeneic SL mice inoculated with sensitive M 1 cells (Honma et al., 1978, 1979).

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Intraperitoneal injections of protein inducers from various CMs or cells producing protein inducers into syngeneic SL mice were found to induce differentiation of sensitive M 1 cells, MGI+D+clone 9, in diffusion chambers in the mice (Lotem and Sachs, 1978a).On the other hand, Lotem and Sachs ( 1981) showed that CM from Krebs ascites tumor cells, which contains both inducer for differentiation of M 1 cells and inducer for colony formation of normal myeloblasts, CSF, significantly inhibited the development of myeloid leukemia by M 1 cells and stimulated normal myelopoiesis (Table 11). The CM could enhance the antitumor effect of cyclophosphamide (Lotem and Sachs, 1981)suggestingthat the protein inducer and cytotoxic antitumor drug had synergistic therapeutic effects on myeloid leukemia. 2. Resistant MI Cells Although sensitive M1 cells could be induced to differentiate in vivo by inducer alone, resistant M 1 cells could not. Therefore, we examined the in vivo effect of actinomycin D on differentiation of the resistant M 1 cells since actinomycin D was most effective in vitro in sensitizing the resistant cells to inducers of differentiation. Resistant clone R- 1 cells were treated in vitro with actinomycin D, and then washed and introduced into diffusion chambers (Okabe ef af.,1979). When the chambers were implanted into syngeneic SL mice, the untreated resistant cells remained undifferentiated, whereas the actinomycin D-treated cells differentiated into macrophages and granulocytes (Fig. 1 and Table 11). Then, we examined the effect of actinomycin D on differentiation of resistant M 1 cells in diffusion chambers implanted into syngeneic SL mice (Okabe et al., 1979). As expected, the cells in the chambers in mice treated with low doses of actinomycin D were induced to differentiate, but most of the resistant cells in the chambers in untreated mice remained undifferentiated (Table 11). These findings show that treatment with actinomycin D either in vitro or in vivo sensitizes resistant cells to endogenous inducers. Next, we examined the in vivo effects of actinomycin D (sensitizer) and LPS (inducer) on differentiation of resistant M 1 cells in diffusion chambers in syngeneic SL mice (Honma et al., 1980b). The resistant M 1 cells showed marked induction of differentiation on these treatments. Lipopolysaccharide alone had scarcely any effect on the survival of syngeneic SL mice inoculated with resistant cells, but LPS plus actinomycin D significantly prolonged their survival time (Honma et al., 1980b).Administration of LPS plus actinomycin D also prolonged the survival of athymic nude mice inoculated with resistant M1 cells (Honma et al., 1980b). These findings suggest that the effects of the drugs may not be directly related to T-lymphocyte-mediated immune responses and that combination therapy with the

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inducer and its sensitizer for cell differentiation is definitely more effective than therapy with the inducer alone. Double-stranded RNA, poly(1) poly(C), not only enhanced the sensitivity of M1 cells to D-factor (Yamamoto et al., 1979), but also stimulated production of D-factor by mouse peritoneal macrophages (Hozumi et al., 1979a). We examined the effects of poly(1) poly(C) and other doublestranded RNAs on the survival times of syngeneic SL mice inoculated with sensitive and resistant clones of MI cells (Tomida et a)., 1980b).A11 the mice inoculated with one sensitive (T-22)and two resistant clones (R-4, DR-3) of M1 cells died 20 to 40 days after inoculation. However, treatment with poly(1) poly(C)greatly increased the survival times of mice inoculated with these three clones ofMl cells (Table 11). Treatment with poly(A) poly(U) or poly(1) was far less effective than treatment with poly(1) poly(C) in suppressing leukemia but slightly prolonged the survival time of mice inoculated with the sensitive M1 cells (Tomida et al., 1980b). On injection of poly(1) poly(C) into mice, their serum levels of interferon activity and D-factor activity increased markedly within 3 hr. Interferon disappeared more rapidly than D-factor activity, which remained at a significant level for 72 hr after treatment. The effect of poly(1) poly(C) on in vivo induction of differentiation of MI cells was examined by the diffusion chamber method. Although the induction of differentiation of the sensitive and the resistant clones of MI cells in untreated mice varied, all the clonal cells in mice treated with poly(1) poly(C) differentiated markedly into mature macrophages and granulocytes(Tomida et al., 1980b).These results suggest that stimulation of differentiation of M1 cells is one mechanism of inhibition of leukemogenicity of the cells, but that the cytotoxic activities of poly(1) poly(C) and interferon on leukemia cells and the immunopotentiating activity of poly(1) poly(C) may also be involved in the effect (Hozumi, 1982).

-

-

V. Induction of Differentiationof Cultured Human Myeloid Leukemia Cells

Several cultured human myeloid leukemia cell lines, such as HL-60, K562, KG- 1, ML-1, and ML-3 cells, were recently found to be induced by various compounds to differentiate into macrophages, granulocytes, or erythroid cells. Some primary cultured leukemic cells from patients with myeloid leukemia were also induced by some compounds to differentiate into mature leukocytes. The inducers, phenotypes of the differentiated leukemic cells, and mechanisms of differentiation of the leukemic cells are described below.

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A. CULTURED MYELOID LEUKEMIA CELLLINES 1. HL-60 Cells

a. Inducers of Cell Diflerentiation. i. Proteins. The presence of protein inducers of differentiation of HL-60 cells and the relationship between the protein inducer and CSF were examined recently. Although HL-60 cells proliferated with CSFs from various sources, they were not induced to differentiate by CSFs (Gallo et al., 1979; Ruscetti et al., 1981). On the other hand, Olsson et al. (1981) reported that human mononuclear blood cells stimulated with various mitogens such as Con A, pokeweed mitogen, or protein A produced CSF and protein inducer@) for HL-60 cells with apparent molecular weights of 40,000 and 25,000. However, at least the protein inducer with the molecular weight of 40,000 was distinct from the CSF, which was produced simultaneously. The protein inducer could induce differentiation of HL-60 cells into cells with phagocytic activity, ability to reduce Nitro Blue Tetrazolium, and the morphologic characteristic of granulopoietic or myelomonocytic cells. Conditioned medium from the 2-mercaptoethanol-treated mononuclear leukocyte fraction of normal human peripheral blood (Elias et al., 1980)and CM from human allogeneic lymphocytes (Chiao et al., 1981) or phytohemagglutinin-stimulated lymphocytes (Lotem and Sachs, 1979; Chiao et al., 1981) also induced HL-60 cells to differentiate into macrophage-like cells. Although inducers for HL-60 cells have mainly been found in CM of human leukocytes, we recently found that CM of mouse myeloid leukemia M 1 cells treated with phytohernagglutinin and 2-mercaptoethanol could induce HL-60 cells to differentiate into monocytes or macrophages (Tomida et al., 1982).The factorsin the CM inducingdifferentiation have not yet been characterized. Conditioned medium of HL-60 cells has no activity to induce differentiation or stimulate growth of colonies ofthe same HL-60 cells in medium with agar or methylcellulose (Ruscetti et al,, 1981), but the CM from cultures of HL-60 cells at high cell density stimulates growth of the cell in liquid culture medium (Brennan et al., 1981). The material with this activity for autostimulation of growth of HL-60 cells gave a single peak on Ultrogel AcA.54 with an apparent molecular weight of 13,000. Arginase, a protein inducer for differentiation of MI cells, also induced HL-60 cells to differentiate into macrophages and granulocytes (Honma et al., 1980a). The induction of differentiation by arginase was significantly inhibited by excess arginine, but not by lysine or leucine, suggesting that the effect of argmase may be due to arginase-mediated arginine depletion.

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Histone H 1, another protein inducer of M1 cells, could not induce differentiation of HL-60 cells (J. Okabe-Kado, Y. Honma, and M. Hozumi, unpublished data). ii. Inducers of diferentiation of murine leukemic cells and cancer chemotherapeutic compounds. The effects of various other inducers of differentiation of murine leukemia cells besides proteins on differentiation of HL-60 cells were examined. Collins et af.(1980) found that polar compounds, such as hexamethylene bisacetamide and DMSO, certain purines, particularly hypoxanthine, and actinomycin D were potent inducers of differentiation of HL-60 cells. However, no significant induction of differentiation of the leukemic cells was observed with hemin, ouabain, prostaglandin E, , X irradiation, dexamethasone, or some other antitumor drugs, such as adriamycin, daunomycin, Ara C, vincristine, and hydroxyurea. They suggested from these results that human HL-60 cells have common cellular target sites with Friend erythroleukemia cells for the inducing action of the polar planar compounds hypoxanthine and actinomycin D, and that the other compounds tested might be specific to murine leukemia cells such as Friend erythroleukemia cells or MI cells, but not to HL-60 cells (Collins et al., 1980). L-Ethionine, another inducer of differentiation of Friend erythroleukemia cells, was reported to induce differentiation of HL-60 cells into granulocytic cells (Mendelsohn et al., 1980). Lotem and Sachs (1980) also examined the effects of various cancer chemotherapeutic agents on induction of differentiation of HL-60 cells. They found that the properties of these compounds in inducing Fc and C, receptors on HL-60 cells were in the following order: actinomycin D > Ara C > mitomycin C > adriamycin > BUdR > hydroxyurea. Furthermore, all the compounds except BUdR induced lysozyme with the same order of effectiveness as Fc and C3 receptors, but only actinomycin D and BUdR induced differentiation of HL-60 cells into mature granulocytes. Bodner et al. (198 1) tested the abilities of various purine and pyrimidine analogs and methotrexate to induce differentiation of HL-60 cells. They found that during 6 days treatment, 3-deazauridine induced nearly all the cells to differentiate. Pyrazofurin, virazole, puromycin aminonucleoside, and the tricyclic nucleoside 3-amino- 1,5-dihydro-5-methyl-1-P-Dribofuranosyl1,4,5,6,8-~entaazaacenaphthylene induced differentiation of 44 - 64%of the cells, whereas 5-azacytidine, BUdR, 5-iododeoxyuridine, thymidine, and methotrexate induced differentiation of 28 - 36Yo of the cells. Of these inducers, methotrexate (IO-*M) was the most potent in terms of its effective concentration. After treatment with all ofthese compounds the predominant cell types were metamyelocytes and banded neutrophilic granulocytes. Inducers of differentiation of M 1 cells, such as tunicamycin (Nakayasu et al., 1980), alkyllysophospholipids (Honma et af., 198lb), and la,25-dihy-

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155

FIG. 3. Morphology of differentiatedHL-60 cells induced by treatment with alkyllysophospholipid (ST-023) (Honma et al., 1981b). (a) Untreated cell. (b-d) HL-60 cells treated with ST-023 (4 pg/ml) for 7 days. May-Griinwald-Giemsa stain.

droxyvitamin D, (Miyauraet al., 1981) also induced differentiationofHL-60 cells (Fig. 3 and Table I). Tunicamycin and la,25-dihydroxyvitamin D, induced the leukemia cells to differentiate mainly into mature granulocytes, but alkyllysophospholipidsinduced the cells to differentiate into both mature macrophages and granulocytes. Retinoids, which induced various lysosomal enzymes in M 1 cells and inhibited differentiation of M 1 cells by other inducers (Takenaga et al., 1980, 1981b), were found to be potent inducers of differentiation of HL-60 cells (Breitman et al., 1980; Honma et al., 1980e).Retinoic acid and related retinoids, but not the pyridyl analog of retinoic acid, induced HL-60 cells to phagocytize, reduce Nitro Blue Tetrazolium, and change into forms that were morphologically similar to mature granulocytes. iii. Tumor promoters. Huberman and Callahan (1 979) observed, on the basis of morphological and functional changes, that HL-60 cells were terminally differentiated by tumor-promoting phorbol esters. Rovera et al. ( 1979a,b, 1980)and Lotem and Sachs ( 1979)showed that TPA could induce HL-60 cells to differentiate into cells with macrophage-like morphology, ability to adhere to plastic, increased activities of NADase and a-naphthyl

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acetate esterase, decreased activities of peroxidase and chloroacetate esterase, and synthesis of acid phosphatase with the typical isozyme pattern of monocytes. Furthermore, Todd et al. (198 1) recently reported that TPA could induce normal monocyte -macrophage differentiation antigens (Mo 1 and Mo2) in HL-60 cells. In HL-60 cells, TPA was also found to induce several properties conimon to both granulocytes and macrophages, such as phagocytosis of IgG-coated erythrocytes, increased activity of lysozyme, and decreased activity of myeloperoxidase (Lotem and Sachs, 1979; Rovera et al., 1979a,b). Although TPA-induced HL-60 cells developed the morphological appearance and enzymatic characteristics of macrophages, they did not show increase in hexose monophosphate shunt activity, superoxide generation, Nitro Blue Tetrazolium reduction, bacterial ingestion, or complement secretion above the uninduced levels (Newberger et al., 1981). New tumor promoters, such as teleocidin from Streptomyces mediocidicus, its catalytically hydrogenated compound dihydroteleocidin B, and lyngbyatoxin A isolated from the marine blue-green alga Lyngbya majuscula, as well as its hydrogenated product, tetrahydrolyngbyatoxin A, were found to induce differentiation of HL-60 cells into macrophage-like cells (Nakayasu et al., 1981). As described before, the effect of TPA on differentiation of mouse myeloid leukemia M1 cells was affected significantly by the type of serum in the culture medium of the cells. Therefore, we examined the effect of serum on differentiation of HL-60 cells (Honma et al., 1982a).HL-60 cells which grew in serum-free synthetic medium supplemented with insulin, transferrin, and several trace elements were induced by TPA to differentiate into macrophage-like cells. Addition of serum inhibited the induction of differentiation of HL-60 cells that had been grown in serum-free medium. Calf serum was more inhibitory than fetal calf serum on TPA-induced differentiation, but it had an effect similar to the latter on the inductions by actinomycin D or arginase. These results suggest that different responses in media with different sera may be specific to TPA. iv. Enhancement of inducer activity on HL-60 cells by other compounds. Although inhibitors of prostaglandin synthesis such as indomethacin and aspirin had no inhibitory effect on retinoic acid-induced differentiation of HL-60 cells, addition of prostaglandin E2 or El was found to stimulate induction of differentiation of HL-60 cells by retinoic acid (Breitman, 1982). The prostaglandin E2and El alone did not induce differentiation of the cells, and other prostaglandins, either alone or in combination with retinoic acid, were much less active than E-type prostaglandins in inducing differentiation of HL-60 cells (Breitman, 1982). Since CAMP is reported to be involved in the mechanisms of various

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cellular actions by prostaglandin E, the effects of CAMP and theophylline, an inhibitor of CAMP phosphodiesterase, on differentiation of HL-60 cells were examined (Breitman, 1982). Addition of theophylline alone or in combination with retinoic acid or dbcAMP increased intracellular cAMP to a relatively small extent in HL-60 cells. Treatment ofthe cells with dbcAMP in combination with retinoic acid or prostaglandin E2 resulted in increase in differentiation of the cells that appeared to be synergistic in the case of retinoic acid and additive in that of prostaglandin E,. Furthermore, the intracellular level of cAMP in HL-60 cells was found to be markedly enhanced by addition of prostaglandin E. These findings suggest that cAMP is involved in the mechanisms of stimulation of differentiation ofHL-60 cells by retinoic acid and prostaglandin E. Interferon alone did not induce differentiation of M 1 cells, but it markedly enhanced induction of differentiation by various inducers (Tomida et al., 1980a). Therefore, we examined the effect of interferon on differentiation of HL-60 cells. We found that human interferon-a and interferon-/Icould enhance induction of cell differentiation by the CM of phytohemagglutinin-treated M1 cells, TPA, or retinoic acid, although the interferons alone did not induce differentiation of HL-60 cells (Tomida et af., 1982). Glucocorticoids did not induce differentiation of HL-60 cells (Collins et af., 1980; Brandt et af., 1981; Y . Honma, K. Kasukabe, and M. Hozumi, unpublished data), but glucocorticoids enhanced the number of N-formylated chemotactic peptide receptors on differentiatingHL-60 cells induced by DMSO (Brandt et al., 1981). This steroid effectwas dose dependent, proportional to glucocorticoid activity, and abolished by cycloheximide. Furthermore, this effect was scarcely observed unless the cells were first induced to differentiate by DMSO. b. Phenotypic ChangesAssociated with Cell Diflerentiation.As described before, HL-60 cells could be induced by various compounds to differentiate into macrophages, granulocytes, or macrophage-like cells and granulocytic cells although the type of differentiated cells differed with different inducers. The phenotypes of some of the differentiated cells were examined in detail. HL-60 cells, which were induced to differentiate into morphologically similar cells to granulocytes by various compounds (butyrate, hypoxanthine, actinomycin D, DMSO, and hexamethylene bisacetamide) also had many of the functional characteristics of normal peripheral blood granulocytes, such as phagocytic and chemotaxic activities, complement receptors, and the ability to reduce Nitro Blue Tetrazolium. Furthermore, the differentiated cells lost activities to synthesize DNA, proliferate in both liquid culture medium and sofi agar medium, and form tumors in nude mice (Breitman and Gallo, 1981). On the other hand, differentiated HL-60 cells induced by DMSO were

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found to express various membrane components like those of mature granulocytes, such as surface glycoprotein with a molecular weight of 130,000 (Gahmberg et al., 1979), surface antigens of mature granulocytes (Perussia et al., 198I), cytoskeletal elements with ability to cap fluorescent Con A (Brown et al., 1981), and sterol and phospholipids (Cooper et al., 1981). Several other biochemical changes were also associated with induction of differentiationof HL-60 cells into granulocytesby DMSO. The levels of a histone-2A related polypeptide with an apparent molecular weight of 12,500 (Pantazis et al., 1981),poly(ADP-ribose)(Kanai et al., 1982), and sialidase activity (Nojiri et al., 1982)were significantly increased in the differentiated cells. Although the differentiated HL-60 cells showed various normal morphological and functional characteristics, the compositions of the cytoplasmic granules of both undifferentiated and differentiatedcells were reported to be abnormal (Olsson and Olofsson, 1981).Untreated HL-60 cells had a higher content of myeloperoxidase than normal neutrophils, but had only a low content of other enzymes associated with primary granules formed in promyelocytes. Enzymes in the primary granules decreased, but the amount of lysozyme increased during differentiation of the cells induced by DMSO. Lactofemn was not produced during differentiationof the cells, indicating that the composition of the secondary granules was abnormal or that these granules were produced abnormally. Phenotypic changes associated with induction of differentiation of HL-60 cells into macrophage-like cells by phorbol esters have been investigated extensively. Phorbol esters induced various morphological and functional changes to macrophage-like cells, as described before, and on differentiation the cells were found to stop proliferationand synthesisofDNA (Rovera et al., 1979b). Furthermore, various membrane components and some biochemical characteristics were recently found to change during differentiation of HL-60 cells. These included binding of phorbol esters to specific membrane receptors (Solanski et al,, 198I ) and modifications of membrane phospholipid synthesis (Cabot et al., 1980; Cassileth et al., 1981) and glycoproteins (Cossu et al., 1982). Some specific cytoplasmic proteins that might be associated with the regulation of differentiation of HL-60 cells into macrophages were expressed in TPA-treated HL-60 cells (Liebermann et al., 1981). Polyamine levels were also increased in the differentiatedHL-60 cells by treatment with TPA (Huberman et al., 1981). Although these phenotypic changes in the differentiated HL-60 cells induced by phorbol estersshowedthat the cells acquiredthe characteristicsof macrophages, the macrophage-like cells induced by TPA did not show increases above the uninduced levels of hexose monophosphate shunt activity, superoxidegeneration, Nitro Blue Tetrazoliumreduction, bacterial

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ingestion, or complement secretion. This finding suggests that the cells did not meet several important functional criteria of macrophages (Newberger et al., 1981). The relationship between the cell cycle of HL-60 cells and induction by TPA of differentiation of the cells into macrophages was examined by Rovera et al. (1980). They found that TPA-induced macrophage differentiation was independent of a round of DNA synthesis, and that differentiating HL-60 cells accumulated in the G, phase.

2. K562 Cells a. Inducers of Cell Diferentiation into Erythroid Cells. K562 cells were reported to differentiate into erythroid cells that synthesize either adult (Anderson et al., 1979)or embryonic (Rutherford et al., 1979) hemoglobin on treatment with sodium butyrate and hemin, respectively. Fuhr et al. (198 1) also reported recently that the original K562 cell line established in Lozzio’s laboratory could be induced to synthesize hemoglobin by a low concentration of hemin (0.05 mM) although no indication of normal maturation or of terminal differentiation into reticulocytes or erythrocytes was observed. Rowley et al. (1 98 1) examined the effects of various compounds including many inducers of differentiation of Friend mouse erythroleukemia cells on induction of erythroid differentiation in K562 cells. They found by benzidine staining that 19 of 39 compounds tested were inducers of differentiation of K562 cells, and that most of the compounds inducing differentiation in medium containing fetal calf serum showed little activity in medium containing newborn calf serum. Among these inducers, actinomycin D had the lowest optimal concentration of 2.4 X 10-9M.The optimal concentration for induction of differentiation of K562 cells was higher than that for Friend erythroleukemia cells for Ara C , but lower for Gaminolevulinic acid, bleomycin, butyric acid, cycloheximide, mitomycin C , ouabain, and 6-thioguanine, and the same for actinomycin D, cadaverine, hemin, and 1,6-hexanediamine (Rowley et al., 1981). b. Modijication of Potential of Cell Diflerentiation. Although K562 cells did not differentiate spontaneously when cultured for 7-8 days in liquid medium or for 14- 16 days in soft agar medium, they were induced to differentiate into early precursors of monocytic, granulocytic, and erythrocytic cells by cultivation for 10- 1 1 days in liquid medium that was gradually depleted of the essential nutrients needed for cell proliferation (Lozzio et al., 1981 ). The peroxidase reaction for hemoglobin demonstrated benzidinepositive material only in the region of the Golgi apparatus and this hemoglobin was shown to be the embryonic type. Most cellsgave a strong reaction for a-naphthyl acetate esterase typical of monocytes, and other cells had abundant red cytoplasmic granules characteristic of naphthol AS-D chloroacetate

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esterasein granulocyticprecursors. Some cells had myeloperoxidaseactivity. These results suggest that K562 cells are multipotential hematopoietic malignant cells that differentiatespontaneously into progenitors of erythrocytes, monocytes, and granulocytes (Lozzio et a!., 1981). Inhibition of cell division in K562 cells by glutamine-deficient medium or hydroxyurea was also found to modify the potential of differentiation of the cells and reversibly enhance the amount of hemoglobin in hemin-induced K562 cells up to the level in normal human red cells (Erard et al., 1981). Markers of both granulopoietic (My- 1) and erythropoietic (spectrin) differentiation were detected together in some K562 cells using specificantibodies (Mane et al., 1981). We found that cultivation of K562 cells in serum-free medium for a long period (4 months)or addition ofserum factor could modify their potential of differentiation. Although K562 cells cultured in medium with serum could not be induced to differentiate by TPA, cells that had been grown in serum-free medium for 4 months were induced to differentiate into macrophages by TPA, arginase, or actinomycin D (Honma et al., 1982a).Addition of serum inhibited the induction of differentiation of the cells by TPA or actinomycin D (Honma et al., 1982a).Calf serum was more inhibitory than fetal calf serum on TPA-induced differentiation,but there was no significant difference in the effects of the two sera on induction by actinomycin D or arginase. These findings indicate that various factors in the cultures are involved in the mechanisms of induction of differentiation of K562 cells.

3. KG-I Cells A unique characteristicof KG- 1 cells is their almost complete dependence on CSF for growth in soft agar medium. However, CSF has no effect on differentiation of the cells (Koeffler and Golde, 1980). 12-0-Tetradecanoylphorbol- 13-acetate could induce differentiation of KG- 1 cells into macrophage-like cells. On differentiation the cells became adherent, developed pseudopodia, morphological characteristics of macrophages, phagocytic activity, nonspecific acid esterase, several lysosomal enzymes, and Fc receptors (Koeffleret al., 1979, 1981). Territo and Koeffler (1981) found that TPA-induced differentiation of KG-1 cells did not require DNA synthesis. Proliferation of KG- 1 cells was inhibited by various compounds such as E-type prostaglandins, dbcAMP, theophylline, epinephrine, and other agents known to increase cellular CAMP,although estradiol, thyroxine, and the polypeptide hormones insulin and growth hormone had no effect. Prostaglandins, cyclic nucleotides, thyroxine, and the polypeptide hormones could not induce morphological differentiation of K562 cells (Koeffler and Golde, 1980). KG- 1 cells in diffusion chambers were implanted into mice treated with cyclophosphamide, glucan, or endotoxin to examine the effects

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of humoral factor in vivo on growth and differentiation ofthe cells. Although no differentiation of the cells was observed under these conditions, their proliferation was significantly enhanced by these compounds (Niskanen ef at., 1980). 4. ML-I and ML-3 Cells Human myeloblastic leukemia ML- 1 cells were induced to differentiate into macrophage-like cells by TPA or Ara C, but into granulocytic cells by DMSO (Takeda et at., 1982). Induction of differentiation of the cells was maximal at drug concentrations that inhibited cell proliferation most effectively. Although ML- 1 cells were induced to differentiate into macrophagelike cells by TPA and into granulocytic cells by DMSO like other human leukemic cells, they nearly all differentiated into monocytic cells with Ara C, unlike HL-60 cells, which showed only slight differentiation into granulocytic intermediates with this drug. These results suggest that the process of differentiation may depend, in part, on the stage at which the process of maturation of leukemic cells is blocked (Takeda ef al., 1982). Another human myeloblastic leukemic cell line, ML-3, differentiated into macrophage-like cells with TPA (Koeffler ef al., 1981).

B. PRIMARY CULTURED MYELOID LEUKEMIA CELLS Differentiation of human leukemia cells was examined in primary cultures of cells from patients with various leukemias. Palfi et al. ( 1979a)reported that leukemic cells in primary culture from two patients with acute myelogenous leukemia (AML)differentiated spontaneously into macrophage- or granulocyte-like cells during culture concomitantly with cessation of proliferation. Furthermore, they found that during culture, cryopreserved human AML cells acquired the characteristics of macrophages and lost their tumorigenicity in nude mice, although undifferentiated leukemic cells gave rise to tumors in more than 90% of the animals inoculated (Paltj ef al., 1979b). These results suggest that some human myeloid leukemic cells have the potential to differentiate in some conditions such as in culture in vifro. On the other hand, some inducers of differentiation of myeloid leukemic cells were found also to be effective on human leukemic cells in primary culture. Leukemic cells in primary culture from patients with AML (Pegoraro ef al., 1980; Chang and McCulloch, 1981; Fibach and Rachmilewitz, 1981) or chronic myelogenous leukemia (CML) (Fibach and Rachmilewitz, 1981) were induced by TPA to differentiate into macrophage-like cells with various characteristics of normal macrophages. Breitman el al. ( 1981) examined the effect of retinoic acid on differentiation in primary culture of leukemic cells from 2 1 patients with various types of myelogenous leukemia.

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Of the 21 leukemic specimens, only cells from two patients with acute promyelocytic leukemia (APL) differentiated into granulocytic cells in response to retinoic acid. Although prostaglandin E, induced granulocytic differentiation of HL-60 cells synergistically with retinoic acid (Breitman, 1982), it had no effect on the differentiation of leukemic cells in primary culture either alone or in combination with retinoic acid (Breitman et al., 1981). In primary cultures of several leukemic cells, spontaneous morphological differentiation into monocyte- or macrophage-likecells was observed after a few days, but retinoic acid had essentially no effect on this spontaneous differentiation (Breitman et al., 1981). We examined the effects of various inducers of differentiation of HL-60 cells [actinomycin D, TPA, retinoic acid, arginase, alkyllysophospholipids (ST-023 and ST-OOS), butyrate, and DMSO] and two antitumor drugs (aclacinomycin A and behenoyl cytosine arabinoside) on induction of differentiation ofleukemic cells from 14 patients with acute nonlymphocytic Consistent with previous leukemia in primary culture (Honma et al., 1982~). findings, TPA induced differentiation of leukemic cells from patients with acute nonlymphocytic leukemia, especially those with AML(M2), acute myelomonocytic leukemia (AMMoL), and acute monocytic leukemia (AMoL) into monocyte- and macrophage-like cells. Retinoic acid induced differentiation of cells from three patients with APL into mature granulocytic cells, but had no effect on cells from one patient with APL. The leukemic cells from some patients with AMMoL and AMoL were also induced by retinoic acid to differentiate into monocyte- and macrophage-like cells. Some inducers of differentiation of HL-60 cells induced differentiation of leukemic cells from all the 14 patients tested, and in 12 of 14 cases, more than 50% of the treated cells showed the characteristics of more differentiated cells. Actinomycin D was effectiveon all the leukemia cells, but with some cells TPA or retinoic acid was more effective. These results suggest that most of the acute nonlymphocytic leukemia cells can be induced to differentiate into macrophage-like cells or granulocytic cells by treatment with an appropriate combination of inducers of differentiation of HL-60 cells including actinomycin D. VI. Summary

Various myeloid leukemia cells from both human and experimental animals have been shown to be induced by a variety of compounds to differentiate in vitro into macrophage-like cells, granulocytic cells, or erythroid cells. Some of the differentiated cells from humans and experimental animals were found to stop proliferating in vitro and lose their leukemogenicity in either nude mice or syngeneic mice. Furthermore, some inducers of differentiation were found to induce differentiation of myeloid leukemia

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cells such as M 1 cells in vivo and to prolong the survival of mice inoculated with these cells. Thus induction of terminal differentiation of the myeloid leukemia cells seems to be a possible new method of therapy of myeloid leukemia. Some compounds were found to induce differentiation of myeloid leukemia cells from both human and experimental animals, but differences were also found in the effectiveness of inducers of differentiation of different cell clones of human and murine leukemia, suggesting that the cellular sites responsible for the inducing actions of various inducers differ in different clones. Therefore, appropriate inducers are required to induce differentiation of particular leukemia cells since these cells differ in sensitivity to different inducers. Although different cultured leukemia cell lines and leukemia cells in primary culture from patients with myeloid leukemia also differ in sensitivity to inducers, several inducers are active on a variety of myeloid leukemia cells including leukemia cells in primary culture. These inducers are TPA, actinomycin D, arginase, retinoic acid, (3-tetradecyloxy-2-methoxy)propyl2-trimethylammonioethyl phosphate (ST-023),DMSO, and butyrate. To establish a suitable therapy for induction of terminal differentiation of leukemia cells, we should examine the mechanisms of induction of differentiation of leukemic cells by these inducers and develop inducers with more potent activity and a wider spectrum. Besides direct inducers of differentiation of human myeloid leukemia cells, some compounds, such as prostaglandin E and interferon, enhance the activity of inducers and some sensitize leukemic cells that are resistant to inducers. So far these compounds have mostly been examined with mouse leukemia M 1 cells. Further studies are required to develop compounds that are effective for induction of differentiation of human leukemia cells. Studies have suggested that leukemia cells can be induced or stimulated to differentiate by various biological response modifiers (BRM) that modify a biological host response to a tumor with resultant therapeutic benefit (Carter, 1980a,b). Biological response modifiers affecting differentiation of myeloid leukemia cells, such as immunopotentiators, interferons, hormones, vitamins, and cytokinesincluding protein inducers (D-factor), have mainly been examined with M1 cells, although some BRM have been shown to be involved in the induction of differentiation of human myeloid leukemia cells. Further studies are necessary on the effect on induction of differentiation of human myeloid leukemia cells of BRM affecting host responses to a wide spectrum of tumors. In this way it should be possible to find BRM inducing differentiation of the cells and develop a method of therapy of human myeloid leukemia by induction of terminal differentiation of the leukemia cells. Various cancer chemotherapeutic drugs stimulate differentiation of both

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human and myeloid leukemia cells, although their mechanisms of action are still unknown. Some drugs may control leukemia not only by their cytotoxic effects but also by their ability to induce differentiation ofleukemia cells. We should investigate the particular mechanisms of induction of differentiation of leukemia cells by these anticancer drugs and develop compounds with potent activity to induce terminal differentiation of the myeloid leukemia cells but with less side effects. In conclusion, it seems from available evidence reviewed in this article that it should be possible to establish a suitable therapy for myeloid leukemia by induction of terminal cell differentiation. For this purpose compounds with more potent effects in stimulating cell differentiation must be developed and the effects of various cancer therapeutic agents including BRM must be reevaluated on the basis of cell differentiation. ACKNOWLEDGMENTS The work of our group cited was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare, Japan.

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THE IN VlTRO GENERATION OF EFFECTOR LYMPHOCYTES AND THEIR EMPLOYMENT IN TUMOR IMMUNOTHERAPY Eli Kedar and David W. Weiss The Lautenberg Center tor General and Tumor Immunology The Hebrew University- Hadassah Medical School. Jerusalem, Israel

1. Introduction

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A. Tumor Immunotherapy: Theory, Expectations, Disappointment B. Considerations in Support of Continued Efforts at Immunologic in Neoplastic Disease ............... ................................................. C. Adoptive Immunotherapy with Effector Cells Produced in Culture (in Vitro Sensitization)- Experimental Back 11. Generation in Culture of Lymphoid Effector Cells A. Cytotoxic T Lymphocytes ................ B. Other Effector Lymphocytes .................................................................................... 111. T-cell Growth Factor: IL-2. ..... A. Production of 1L-2.................................................................................................... B. Amplification of Antitumor Cytotoxic Responsiveness in MLTC ........................ C. Propagation of Specifically and Nonspecifically Cytotoxic Lymphoid Cells 1V. Adoptive Immunoth A. General Considerations. B. Adoptive Immun C. The Prospects of Clinical Application ..................................................................... References. ..............................................

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I . Introduction

A. TUMORIMMUNOTHERAPY: THEORY, EXPECTATIONS, DISAPPOINTMENT The theory of immune surveillanceagainst progressive neoplasia has been widely contested in recent years. The doubts cast on its axioms and corollaries have undermined much ofthe conceptual framework for efforts at tumor immunotherapy, and place into serious question the validity of continued attempts in this direction. Advanced originally by Ehrlich and reformulated by Burnet and others, the theory proposes that neoplastic cells arise with (great) frequency in normal tissues of higher animals; that the neoplastic variants are ubiquitously distinguished by antigens capable of evoking protective immunologic reactions in the autochthonous host; that such immune reactivity normally succeeds in eliminating the neoplastic cells, or in decisively retarding their development; and that progressive neoplastic disease reflects the consequence(s) either of immune failure on the part of the organism or of 171 ADVANCES IN CANCER RESEARCH, VOL. 38

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exceptional adaptive and selective processes which lead populations of neoplastic cells to evade or neutralize immune defenses. The major corollaries of this hypothesis posit the centrality of immunologic resistance in the protection of higher animals against cancer, and the ready possibilities of therapeutic intervention in such diseases by correction of immune dyscrasias or the closing of tumor cell escape routes from surveillance. A large body of experimental findings, accruing rapidly after the early demonstrations by Foley, Prehn and Main, and the Kleins and their associates that chemically induced tumors of mice can incite specifically heightened resistance against reimplantation in the syngeneic and autochthonous host, appeared to lend credibility to the theory of immune surveillance. Most neoplasms caused in laboratory animals by oncogenic viruses and chemical or physical agents were found to differ in immunologic properties from corresponding normal cells at similar stages of ontogeny; although the true tumor specificityof tumor antigens came to be questioned already some time ago, the uniquely expressed epitopes of neoplastic cells, now designated tumor-associated antigens (TAA) or tumor-associated transplantation antigens (TATA), continued to be viewed as adequate stimulators and targets of immunologic reactivity. Adoptive transfer studies established cell-mediated immune responsiveness as the mechanisms of primary importance in the development of specificallyacquired resistance against both allogeneic normal and syngeneic neoplastic tissue, and subsequent investigations revealed that various families of reticuloendothelial - lymphoid cells can participate in antitumor defense. Efforts aimed at heightening cell-mediated immunologic reactivity have proven efficacious in preventing or retarding the growth of many experimental animal tumors, and in some test systems lead to cures of established disease. These observations generated large hopes for immunology in the treatment of human malignant disease. On the strength of extrapolation from animal models, the great majority of which have dealt with a limited spectrum of induced, transplantable neoplasms in inbred rodents, it was anticipated that cancer cells of man also express TATA, and that with the rapid advances in understanding of immunologic function and of the parameters of specific and nonspecific immunomodulation, immunotherapeutic measures of salient efficacy would become available. It was the signal failure ofcancer immunotherapy in patients that impelled a searching second look not only at the design and conduct of these trials but also at the veracity of the underlying conceptions. The reexamination betokened the theory of immune surveillance as simplistic and of very uncertain applicability to progressive neoplasia in nature. In contradistinction to tumors induced in the laboratory by selected, potent oncogenic viruses or by extreme chemical and physical manipulations, transformations that take place in nature tend to lead to full neoplastic

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potential of the cells only slowly, by multiple-step progressions. The relatively infrequent occurrence of human neoplasms, and of the majority of these late in life, may be inherent in the nature of human neoplasia and not, primarily, a consequence of idiosyncratic or age-associated immune decline. There are, accordingly, protracted opportunities in the genesis of such tumors for selection of neoplastic variants of low immunogenicity and high immunoresistance; on the other hand, there can be no evolutionary selection of individuals within a species for high resistance against a relatively infrequent challenge confronted, on the whole, past the peak of reproductive function. On theoretic reconsideration, then, the likelihood that most cancers of man are intrinsically immunoreactive, as are many ofthe growths produced at will by the investigator, would seem remote. The probable irrelevance of most investigational models in tumor immunology to the natural history of neoplastic disease (Hewitt, 1978; Weiss, 1978) has been accentuated by recent findings that signify the inability of neoplasms arising spontaneously(i.e., without the intentional interferenceof the investigator)in inbred experimental animals to evoke defensive immune reactions (Hewitt et al., 1976; Klein and Klein, 1977). These observations brought about a shift in analogies. Human cancers have come to be considered as analogous, immunologically, to spontaneous tumors of laboratory mice and rats rather than to those that have been the stock in trade of the experimental tumor immunologist. The growing belief that the expectations held only recently of immunology’s contribution to cancer control were little more than hopeful illusion has gained momentum from other directions. Immunologic reactions have been seen to expedite the growth of tumor cells on occasion, rather than to afford resistance; progressive malignant disease in man has not been found to be preceded or accompanied invariably, or even commonly, by massive immunologic abnormalities of the patient; attention has been called increasingly to a variety of nonimmunologic defense mechanisms against tumor cells; and firm identification of tumor-specific antigenic characteristics of human cancers has so far largely eluded intensive effort. At present, accordingly, the climate of opinion regarding the role of immunologic responsiveness in cancer defenses has become pervasively unfavorable. Some investigators still entertain the possibility that immune surveillance may be prominent in the protection against cancers that do not normally come to appearance in nature, but that for those that do, immunologic function is thought to be excluded -as absent or inadequate- from the confrontation. It is the essential, inherent immunologic neutrality ascribed to host -parasite relationships in progressive neoplasia that has come to be seen as responsible for the failures at immunotherapy, rather than a still-unmastered ability to correct putative host dyscrasiasor to close tumor cell escape routes.

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The now-prevailing disappointment in tumor immunology and immunotherapy is as unbalanced, however, as were the hopes at the advent of these disciplines, Tumor immunology has been put to the clinicaltest far too soon and far too carelessly to have warranted bright forecasts. Knowledge and even awareness of the vast complexity of immunologic reactivity and of the dynamics of host - tumor interactions were lacking, and many of the clinical studies mounted have been carried out in indifference or defiance of considerations for which persuasive grounds were already at hand. For instance, immunotherapy and powerful chemotherapy have often been administered simultaneously. Thus, reporting on the inefficacy of chemoimmunotherapy in advanced melanoma, Ramseur et al. (1 978) wrote that “MER-BCG (the immunologic arm) was given at the same time as chemotherapy in our study mainly for patient convenience because most of our patients drove 2 100 miles to be treated at our center. In a recent article . . . (it was) stated that these variables are still unknown. It is true from a theoretic viewpoint that immunotherapy administration might be more beneficial at a time other than when chemotherapy is given.” Or, in many of the larger scale, cooperative trials, immunologic treatment was maintained repeatedly and for long periods of time, according to initial protocol, even in the face of accruing information that such treatment is likely to lead, after a finite period of time, to marked suppressive influences on responsiveness (Weiss, 1983). It is not inconceivable that greater success might be met in future trials more carefully designed in light of gathering understanding of the immunologic mechanism. Already to date, a number of small, preliminary studies in patients, in which greater attention was given to immunologic parameters and to an individualization of therapeutic regimen, have yielded the impression of some, albeit inconsistent, limited, or transient, efficacy. Far more importantly, inclusive authenticity of the theory of immune surveillance as classically formulated, with its implied preeminence of immunologic reactions in tumor defenses, is not a necessary premise or condition for continued effort in tumor immunology and immunotherapy. There remains extant, independently, a legitimate conceptual foundation for these disciplines, although large changes in orientation are now required of the investigator. A number of considerations argue in favor of this perspective. These have been discussed recently in another article (Weiss, 1980), and will be cited only briefly here.

B. CONSIDERATIONS IN SUPPORT OF CONTINUED EFFORTSAT IMMUNOLOGIC INTERVENTION IN NEOPLASTIC DISEASE 1. The natural history of human neoplastic disease is, in fact, often suggestive of active resistance activities on the part of the host, rather than of his passivity, and of some participation of immunologic responses in the

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defense. The analogy inferred by some investigators between human cancer on the whole and the spontaneous tumors of inbred rodents against which the host appears to be incapable of mounting immunologic defensesmay not be correct. At least with regard to any such neoplasms as may arise partially in consequence of pronounced immunosuppressive influences on the person (Penn, 198l), the strengthening of immunologic defensive reactivity might be expected to have some prophylactic and even therapeutic value. 2. Some, and perhaps many, neoplastic cells manifest a preferential, nonspecific susceptibility to certain lymphokine-activated immunocytes; some lymphokines or lymphokine-like substances can promote cellular immunologic reactivity even against normal autochthonous tissue (Altman and Katz, 1980; Grimm et al., 1982a). The selective vulnerability of tumor cells to such injury may beg entirely the question of a specific target antigenicity, and might lend itself to therapeutic exploitation. The nonspecific consequences of immunologic responsiveness may thus represent the important effector arm in at least some circumstances, specific reactivity against any TAA (or against wholly incidental antigens in host tissues) serving to trigger the production of nonspecifically damaging reagents. 3. The immunologic potency of TAA may often lie below the threshold for elicitation of responses that contribute to host defense. Nonetheless, the occurrence of any antigenic deviation from normal on the part of neoplastic cells can provide a “handle” for immunologic intervention; the essential task of the immunologist would then be to bring “weak” or irrelevant (to resistance) tumor-associated antigenicity to potent immunogenicity (Sulitzeanu and Weiss, 1981). Even where TAA represent only quantitative or configurational changes from the antigenic profile of analogous normal cells, or displacements in the ontogenic appearance and tissue expression of normal epitopes, it is not unrealistic to expect that immune attack at such determinants can be facilitated. Persuasive evidence has come from tissue culture studies showing that human thymus-derived lymphocytes are responsiveto certain autochthonous cells, even normal and unaltered, and that this responsiveness may be manifested under appropriate conditions by cytotoxic effector action (Weksler et al., 1981); and, the intact organism is well capable of mounting cytotoxic immune reactions against some of its complement of normal antigens where these are errant in ontogenic time, place, or mode of expression, or where usual barriers-morphologic or physiologic-between organ compartments break down. The suggestion recently advanced that most human cancers result from genetic transpositions and gene amplification, rather than from mutations (Cairns, 1981; G. Klein, 1981), provides persuasive theoretical backing for anticipating correspondingly deviant, and not novel tumor-specific, antigenicities as characteristic of the neoplastic state. 4. A variety of avenues for the experimental potentiation of nonimmu-

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nogenic tumors to frank immunogenicity have already been elaborated in laboratory models. Reactivity induced by chemically and enzymatically modified tumor cells, and by such cells following viral and genetic heterogenization, has been shown to “spill over” at times toward the native cells. It may be possible, accordingly, to bring about defensive immune reactivity of patients by hyperimmunization with intentionally altered neoplastic cells, even where the native tumor is incapable of exciting resistance. 5. Apparently nonimmunogenic growths may in some instances owe their neutrality not to an absence of immunogenic determinants, but rather to a concurrent expression of similar epitopes that are presented in a manner favoring suppressive reactivity (B. Klein et al., 1981). This reactivity could then be imagined to mask, or prevent, any that could have been induced by the immunogenic configurations on their own. A conceptual context for such eventuality is afforded by the current view that the physiologic role of products coded by the major histocompatibility complex (MHC) locus is to accomplish differential immunologic recognition and reactivity to jointly presented antigens (“in some situations the particular antigen/H-2 combination is not recognized by T cells . . , in other situations helper T cells are stimulated but, at the same time, so are suppressor T cells” (J. Klein et al., 1981 ;see also Wagner et al., 1981). From this perspective, it can be imagined readily that the Same antigenic unit can stimulate responsivenessof different import as a function of its association with different MHC entities. The eventuality then presents itself for guiding immunologic responsiveness toward manifest immunity by hyperimmunization with immunogenic TAA -MHC constellations and/or by a selective interference with suppressor reactivity. 6. The approach of “nonspecific immune potentiation” of the cancer patient or cancer-prone individual also remains cogent, despite the massive failure so far to achieve clear-cut therapeutic successes. Persuasive evidence is at hand from numerous laboratory models, artifactual as these may be, that efficacy of such therapy is conditioned on the precise setting of relevant variables; felicitousintervention appears to hinge on the individualization of treatment, taking into account the type of tumor, the stage of its development, genetic and phenotypic characteristics of the host, and the parameters of agent application. Such individualization has not been effected in the clinic, and, as mentioned above, diaphanous errors of trial design have marked many of the studies. It must also be considered that the nonspecific biologic response modifiers (BRM) employed so far have largely been crude, of an unknown range of activities, and otherwise inadequate to the task of fine modulation of exceedingly complex biological responses. It is not impossible, accordingly, that agents ofdefined capacities may prove to be clinically useful in the future

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if they will be applied with better understanding of immunologic function and host - tumor interactions, and with attention to the particular circumstances of the individual patient at a given time. Nonspecific immune potentiation may be especially appropriate where immunologic abnormality of genetic, age-related, or acquired origin contributes, if only to a limited extent, to tumor etiology. If analogy to experimental tumor systems is valid, it might also be anticipated that such agents can be more effectivewhen used prophylactically in healthy persons, at high risk of neoplastic disease because of environmental exposure or otherwise, than when in cancer patients. Neither should there be ignored certain auxiliary benefits that might be effected by judicious utilization of nonspecific immunopotentiating substances. There have been some indications, for example, that these can elevate resistance broadly, in man as well asin experimental animals, against pathogenic microorganisms and viruses (Weiss, 1983); they may come to have a significant role, accordingly, in allowing for more intensive regimens of conventional antitumor therapy than otherwise permissible because of ensuant patient exposure to serious infection. 7. The focus of attempts at immunologic intervention has been placed so far on TAA located at the cell surface. Some evidence has been brought forward, however, that antigens within the cell interior can also elicit protective immunologic rejoinder upon liberation from disrupted cells, by facilitating inflammatory reactions that lead to mechanical restraints on tumor niduses (Vaage and Gandbhir, 1978). It is not inconceivable that manipulations aimed at giving prominence to such responses could contribUte to the immune defense of some patients. 8. Particular interest is attached to the avenue of passive immunotherapy’ with effector elements produced in vitro. The cancer patient may be incapable of generating a sufficient immunologic weaponry against the neoplastic cells as he encounters these in the course of the disease. In addition to the immunogenic paucity of human TAA, there must be taken into account the operation of homeostatic control mechanisms in vivo that are likely to restrict the magnitude and duration of immune responses against any determinants closely related to normal self

I In keeping with general current usage, the term “adoptive immunotherapy” is here employed to connote intervention effected by cells that have been stimulated to antitumor reactivity outside the body ofthe recipient, regardless ofwhether the cells are ofautochthonous, syngeneic,or allogeneic origin. The term “passive immunotherapy” refersto the introductionof noncellular immunologic products with direct or indirect antitumor action, and to the administration of preformed immunologic elements in an inclusive sense, where the nature of these is not specified.

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constituents. Anatomic and physiologic barriers may prevent effective contact between neoplastic and immunologically reactive tissues. Specific and nonspecific suppressor elements may be prominent in the tumor host (Broder et al., 1978; Naor, 1979), as may be blocking antigen-antibody complexes (Hellstrom and Hellstrom, 1976a)and TAA shed by the neoplastic cells (Doljanski, 1982). The tumor itself may be able to abort the formation of immune reagents and to neutralize those that have been made (Israel and Edelstein, 1978). In tissue culture, on the other hand, the conditions of immune excitation can be idealized, empirically; the immunogenic strength of tumor cells (and perhaps of purified TAA preparations) can be heightened and immunocyte responsiveness faceted and magnified; neither patient nor cell donor need be subjected to any possibly deleterious procedure; blocking activities by the neoplasm can be vitiated; and introduction of the effector reagents to the patient can be optimized in the light of gained experience. Adoptive intervention with selected populations of potent antitumor effector cells, of autochthonous or allogeneic source, that have been generated in culture could thus circumvent some of the salient difficulties inherent in active specific immunization and nonspecific immunomodulation, and furnish a focused, controlled, and in essence less-encumbered modality of enlisting immunologic reactivity to the defense of the patient. It is obvious that formidable obstacles must be overcome in bringing this approach to the clinic. Upon introduction to the host, cultured lymphoid cells may be rapidly entrapped in liver, lungs, and other sites, and thereby prevented from concentrating at disseminated tumor foci or from “homing” to lymphoid centers (Kedar et al., 1978b; Lotze et al., 1980a). Sensitization cultures may also favor the evocation of specific and nonspecific suppressor activity (Kedar et al., 1978d)capable of neutralizing the immune capacities of effector cells made in vitro or in the tissues of the host (Greenberg et af., 1979). An additional risk lies with the possible stimulation in culture of immunocytes with specific or broad cytotoxic reactivity against normal tissue constituents, and the induction of severe autoimmune pathology following their application (Cohen et al., 1971a; Orgad and Cohen, 1974; Tomonari, 1980). There is no assurance today that these and other conceivableimpediments can be resolved. Nonetheless, the endeavor of passive therapy with effector immunocytes and certain lymphokines- the subject of this article- holds sufficient promise to warrant intensive further exploration, and has indeed been given impetus recently (Fefer and Goldstein, 1982). Research in this direction can also be hoped to cast new light on immunologic concomitants of host - tumor relationships.

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c. ADOPTIVEIMMUNOTHERAPY WITH EFFECTORCELLS PRODUCED IN

CULTURE (in Vitro SENSITIZATION) -EXPERIMENTAL BACKGROUND AND SCOPEOF THE REVIEW

Recent developments in immunology pave the way for concerted attempts at adoptive immunologic intervention in neoplastic disease. The availability of monocloncal antibodies produced by rnurine hybridomas (Kohler and Milstein, 1975; Melchen et al., 1978; Moller, 1979; Kennett et al., 1980) is leading to refined delineation of the multiple, interlocking events that make for responsiveness to allografts and syngeneic neoplasms. Such antibodies are also facilitating disclosure of what may be truly unique, or uniquely expressed, antigens of human tumor cells (Hellstrom et al., 1980; Nadler et al., 1980; Old, 1981; Mazauric et al., 1982). Knowledge of the nature of the inciting antigens, and of the evolvement of differentialreactivity mounted in reponse to cells that express them, lies at the basis of intelligent immunotherapeutic engineering in the future. And advances in tissue culture technology are enabling the efficient in vitro manufacture of antitumor effector cells. Following the pioneering work of Ginsburg and colleagues on the induction and assessment of cell-mediated immune responses to xenogeneic cells in culture (Ginsburg and Sachs, 1965;Ginsburg, 1968;Berke et al., 1969)rat lymphocytes cultivated on mouse fibroblasts were shown to acquire cytotoxic reactivity against the monolayer cells- numerous other investigators demonstrated the generation of cytotoxic effector (or killer) cells in lymphoid tissue upon cocultivation with inactivated allogeneic leukocytesin mouse (Hayry and Defendi, 1970; Hodes and Svedmyr, 1970) and human (Hardy et al., 1970b; Solliday and Bach, 1970)unidirectional mixed leukocyte reaction (MLR) systems. The methodology was adapted by other groups for the in vitro production of cytotoxic effector cells [i.e., in mixed lymphocyte -tumor cell cultures (MLTC)] against allogeneic, syngeneic, and autochthonous neoplastic tissue of mice and humans (Hardy et al., 1970a; Wunderlich and Canty, 1970; McKhann and Jagarlamoody, 197 1;Golub et al., 1972; Lundak and Raidt, 1973; Wagner and Rollinghoff, 1973; Golub and Morton, 1974; Sharma and Terasaki, 1974a; Burton et al., 1975; Martin-Chandon et al., 1975; Plata et al., 1975;Kedar et al., 1976;Zarling et al., 1976), and it became apparent that such effector cells are capable of destroying the relevant tumor targets in vitro and, under appropriate circumstances, of protecting experimental hosts against their growth in vivo (McKhann and Jagarlamoody, 1971; Rollinghoff and Wagner, 1973;Treves etal., 1975;Cheeveretal., 1977; Kedaretal., 1977,1978b). Thediscoveryof IL-2 has presented a novel expedient for propagating and cloning T-cell

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populations with distinctive cytotoxic, helper, and suppressor function (Moller, 1980, 1981, 1982; Ruscetti and Gallo, 1981). It has already been shown that the level of antitumor cytotoxic reactivity that can be induced in thymus-derived lymphocytes in vitro may be considerably higher than that attainable in the intact organism, and that the in vitro sensitization (IVS) process is more rapid (Wagner et al., 1973; Plata et al., 1975; Kedar et al., 1977). The greater efficacy of culture sensitization &as demonstrated by resorting to a sensitive mixed leukocyte microculture assay (Miller et al., 1977; Ryser and MacDonald, 1979a;MacDonald et al., 1980; Lutz et al., 1981), in which limiting numbers of responder lymphoid cells are exposed to excess numbers of irradiated stimulating cells and optimal amounts of supernatant from a secondary MLR (as source of IL-2). After 4- 8 days, cells recovered from individual wells are tested for proliferative activity ([3H]thymidine uptake) and cytotoxicity ( W r release) toward the relevant targets. An estimation is thereby obtained of the precursor frequency of proliferating and cytotoxic T lymphoid cells (PTL-P and CTL-P, respectively)in the responding cell population. Working with C57BL/6 mice immunized in vivo with allogeneic (DBA/2) cells, Ryser and MacDonald (1979b) found that the proportion of corresponding CTL-P in the spleen increased three- to fourfold over that in spleen of unimmunized mice; following MLR stimulation with cells of the same strain combination, CTL-P frequency increased to a much greater extent (25- to 100-fold), 5 -20% of the surviving responder cells identifiable as such. Maryanski et al. ( 1980)observed a CTL-P representation of up to 50%in the fraction of large lymphoblastic cells harvested after MLR by velocity sedimentation on continuous fetal calf serum (FCS) gradients. The literature on in vitro generation of effector cells and lymphokines and their application in vivo has expanded rapidly over the past years, and is beyond inclusive survey here. The present article focuses, selectively, on certain recent findings pertaining to lymphoid effector cells (macrophage cytotoxic reactivity is not discussed) of mice and man, with emphasis on current views on the nature and interactions of cells and nonspecific soluble factors involved in the genesis of cytotoxic lymphoid cells, and on the provoking antigens; the methodology of in vitro induction of such immunocytes directed against neoplastic targets; the facilitation by IL-2 of lymphocyte propagation and cytotoxic reactivity;the immunotherapeutic capacities of lymphocytes sensitized and/or activated in culture; and the likely prospects and difficulties of adoptive immunotherapy in cancer patients. For other deliberations on concepts and methodology of IVS and immunologic intervention with effector cells so obtained, the reader is referred to other recent reviews (Engers and MacDonald, 1976; Rosenberg and Terry, 1977; Burton et al., 1978; Weiss, 1980; Mokyr and Dray, 1982).

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II. Generation in Culture of Lymphoid Effector Cells

A. CYTOTOXIC T LYMPHOCYTES 1. Reactivity to Allogeneic Major Histocompatibility Complex Antigens

a. Inciting Antigens and Responder Cell Interactions. Although correctness of the analogy awaits confirmation,it seems probable that the processes that lead to formation of cytotoxic T lymphocytes (CTL) against normal foreign cells are similar or related to those involved in reactivity against neoplastic self variants. Cellular responses to allogeneic, and to experimentally altered syngeneic, cells have been studied extensively during recent years (Bach et al., 1976, 1979a;Bach and van Rood, 1976;Zinkernagel and Doherty, 1979; Wagner et a!., 1980~;Klein and Nagy, 1981; Dutton and Swain, 1982),and a brief purview ofcurrent methodology and thought in this area is germane to the topic of this article. Early investigations on CMI responsiveness to alloantigens in the mouse pointed to the synergistic participation of two types of T lymphocyte. Studies by Cantor and Asofsky ( 1972)on graft-versus-host (GvH) responses in Fl neonatal mice revealed the collaboration between a subset of effector precursors (designatedby these investigators T 1) that are largely resident in thymus and spleen, short lived, noncirculating,and resistant to antithymocyte serum (ATS),and a subset of amplifier/helper cells (designated T2) that are long lived, recirculating,and concentrated in the blood and lymph nodes. Another group of investigators designated these T-cell subsets differently. Analysis of cytotoxic responsiveness generated in allogeneic MLR brought Wagner et al, (1 973)to conclude that both CTL precursors(denoted by them as T2) and nonkiller amplifiercells (T 1)are present, in different proportions, in peripheral lymphoid tissue and in the cortisone-resistant cell compartment ofthe thymus; the T2 cells were found to be prominent in the lymphoid periphery, and the T1 in the thymus. Further examination of responder lymphoid cells and inciting antigens in mouse and human MLR (Wagner et al., 1973; Bach et al., 1976; Bach and van Rood, 1976;Dupont et al., 1976; Eijsvoogel et al., 1976)led to the view that each of the two T subsets responds to one of two distinct classes of antigens that are expressed on stimulating cells under control of the genes that make up the chromosome segment known as the major histocompatibility complex: CTL progenitor cells are stimulated (become “poised”) by “cytotoxicity-defined”(CD) determinants (“Signal 1”) coded for largely by the K, D,and L regions (or, loci) of the mouse MHC, and the A , B, and, to a lesser extent, C regions of the human MHC. The noncytotoxic amplifier/ helper cells (proliferatinghelper cells, PHC, helper T lymphocytes,HTL), in

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contrast, are activated and prompted to extensiveproliferation by “lymphocyte-activating determinants” (LAD, or LD) encoded primarily by genes of the Zregion in the mouse and the D region in man. Amplification ofcytotoxic responses, through expansion and differentiation of CTL precursor clones to mature CTL, was ascribed to soluble factors (lymphokines) that are released by the activated amplifier cells (“Signal 2”),2 Development of alloantisera against T lymphocyte differentiation markers made for a more telling characterization of lymphoid responder populations. Employing sera against Lyt antigens of mouse T cells, a scheme of classification was proposed: Amplifier/helper cells were identified as Lyt 1+2-3-, cytotoxic precursor and effector cells directed toward alloantigens as Lyt 1-2+3+,and CTL reacting with modified self targets [and with (some) syngeneic tumor cells] as Lyt 1+2+3+(Cantor and Boyse, 1977). Probes with absorbed rabbit anti-human T-cell sera against a thymus-dependent differentiation antigen (TH,) permitted discrimination between human T lymphocyte subpopulations (Evans et al., 1978): killer cells as TH2+and amplifier cells as TH,-. More recently, monoclonal aniibodies to human T-cell differentiation antigens have come to hand. With aid of these reagents, noncytotoxic T cells that produce helper factors for the induction of alloreactive CTL have been classified as OKT4+ (Reinherz et al., 1980a) and CTL precursors and effectors (as well as suppressor cells) as OKT 4-, 5+, 8+ (Reinherz et al., 1980b;Zarling and Kung, 1980; Reinherz et al., 1981). A similar distinction has been put forward for the subsets involved in CTL generation against autochthonous cells infected with influenza virus (Biddison et al., 1981). Using another series of monoclonal antibodies, Ledbetter et al. (1981) recently classified helper/inducer T cells as Leu 1+2-3+ and CTL as Leu 1-2+3-. It now appears that there has been considerable oversimplification in the concepts of murine CTL genesis that took shape during the past years. The perspective of a sharp dichotomy between Lyt 1+2-3- noncytotoxic helper cells (reacting to Z region-coded determinants) and Lyt 1-2+3+ cytotoxic progenitor cells (responding to K and D determinants) has been revised. Alternate pathways of CTL induction, and the involvement of additional subpopulations of cells, have been discerned in experiments conducted with Description in the literature of “proliferative” responsiveness of T cells to MHC antigens is often interchangeablewith the assignment ofhelper-amplifier function. It must be pointed out, however, that whereas the initial riposte of CTL precursors to MHC epitopes is indeed marked by little if any cell multiplication, in contrast to the conspicuous division of helper (precursor) cells at the appropriate MHC antigenic signal, the specifically activated CTL-P clones, too, undergo considerable expansion eventually, upon further excitation by antigen-stimulated helper cells and/or their soluble lymphokine products.

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better defined, potent, monoclonal anti-Lyt antibodies and in mice that underwent mutations in different regions of the MHC (Swain, 1980, 1981a,b; Klein and Nagy, 1981). Collaboration between T-cell subsets in CTL genesis has been deemed mandatory largely on the basis of MLR assays in which the numbers of responding lymphocytes were limited, or the antigenic stimulus inadequate or inappropriate. Under optimum conditions of sensitization, however, cooperative interactions between recognizably distinct T-cell subsets may not be requisite to the same extent (Shortman ef al., 1978; Lutz, 1981; Weinberger et al., 198la). Moreover, recent studies with cloned, allosensitized splenocytes suggest that some T lymphocytes can both act cytotoxically against the relevant targets and produce T-cell proliferation-stimulating factor(s), i.e., to function as helper cell-independent CTL (Dennert et al., 1981a; Widmer and Bach, 1981). The role of MHC-encoded structures in cellular responsiveness of mice has come under searching scrutiny (J. Klein, 1979;J. Klein et al., I98 1 ;Klein and Nagy, I98 1). It has been found that the determinants that have been associated in the mouse primarily with excitation of cytotoxic alloreactivity (encoded in the K, D, and L loci; classically designated as CD; now termed Class I antigens; and characterized as 45,000-dalton glycoproteinsjoined to µglobulin) can provoke, under opportune culture conditions, both proliferative (presumably helper, to a large extent) and cytotoxic responses (J. Klein, 1976;Okada and Henney, 1980;Scott et a)., 1980;Lutz, I98 1); and that cytotoxic responsiveness can transpire without participation of the determinants that have been chiefly ascribed the regulatory function of inciting helper-amplifier cell division and the production of lymphokines (encoded in loci A and E of the I region; classically designated as LAD; now termed Class I1 antigens; the product of each consisting of two glycoproteins of 28,000 and 32,000 daltons) (Engers and MacDonald, 1976). It has also been shown that Class I1 alloantigens can excite, in appropriate sensitization and assay circumstances, cytotoxic responsiveness of the same order as can Class I antigens (Wagner et al., 1975;Juretit ef al., 1981). Similarly, human Class I1 antigens (encoded in the D/DR and SB regions) can provoke CTL reactivity and serve as its targets (Feighery and Stastny, 1979; S. Shaw ef al., 1980; Kavathas et al., 1981). It would thus appear that Class I and Class I1 determinants can perform the same roles-albeit perhaps not to an equal extent -when responder, stimulator, and target cell combinations and sensitization environment are suitable (Swain, I98 1b). Strictly speaking, designation of CD and LAD activity of MHC antigens should be in terms of the functions observed under given conditions, without hard and fast implications as to the mapping of the coding genes within the MHC chromosome area.

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Earlier thought on the association between certain T-cell differentiation characteristicsand T-cell functionality engendered in mouse MLR has also changed, many investigatorsno longer incliningto the view that helper/amplifier populations invariably express the Lyt 1 marker alone (i.e., Lyt 1+2-3-) and that all allocytotoxic effector populations are of Lyt 2 and 3 type (Le., Lyt 1-2+3+). Bach ef al. ( 1979a)suggesttwo alternativepathways of stimulation.Where responding and stimulating cells differ in both the K,D and I MHC regions, Lyt 1+ helper cells collaborate with Lyt 2+ cytotoxic precursors; where the difference is at K/D loci only, the cells involved are exclusively Lyt 1+2+,and can perhaps serve both in distinctive helper and effector capacities. An analogous outline has been proposed by Wettstein and Frelinger ( 1981) for CTL responses to minor histocompatibility antigens. Wagner and co-workers ( 1980c) distinguish between two classes of CTL precursors to H-2 alloantigens:the one dependent on T helper cells (Type I), consisting of Lyt 1+2+3+cells programmed to differentiateto Lyt 2+3+type; the other not dependent on T help (Type 11),and comprisingantigen-primed (or, memory) Lyt 2+3+cells. Simon ef al. (1 98 1) also find evidence for helper (Lyt 1+)-CTL precursor (Lyt 1+2+3+)collaboration in the maturation of CTL against H-2 as well as against other antigens. Klein and Nagy (1981) now advise the following scheme of interaction between MHC-coded determinants and T-cell subsets in the mouse: Class I alloantigens stimulate Lyt 1+2+and Lyt 2+, but probably not Lyt 1+,cells; they cannot drive differentiation of Lyt 1+2+to Lyt 1+ expression. Class I1 antigens,in contrast, stimulate Lyt 1+and also Lyt 1+2+(Vidoviket al., 1981) but not Lyt 2+cells, and are unable to propel differentiationofLyt 1+2+to Lyt 2+. All three T-cell subsets (Lyt I+, Lyt 2+,and Lyt 1+2+)have the potential for both proliferative and cytotoxic responsiveness. Thus, Lyt 1+ and Lyt 1+2+cells can proliferate and become cytotoxic upon stimulation by Class I1 alloantigens;Lyt 2+and Lyt 1+2+can proliferate (the former only to limited extent)and take on cytotoxicproperties when incited by Class I antigens.The pattern of cytotoxic responsivenessto “many MHC-restricted conventional antigens (hapten-modified cell-surface antigens, viral antigens, tumor-specific transplantation antigens), like that to mutant Class I alloantigens, is predominantly mediated by Ly-I+ Ly-2+cells” (Klein and Nagy, 1981). Other workers have postulated that CTL precursor and effector cells directed at allogeneic normal, syngeneictumor, and syngeneicbut modified normal mouse cells are all Lyt 1+2+3+(Shiku ef al., 1976; Al-Adra et al., 1980; Leclerc and Cantor, 1980a; Nakayama ef al., 1980; Teh and Teh, 1980a; Cerottini and MacDonald, 1981). Employing fluorescein-labeled monoclonal antibodiesand the sharply discriminatory cell sorter methodology, Ledbetter et al. ( 1980)found at least some Lyt 1 antigen to be present on

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all T lymphocytes. It appears possible, then, that precursor and mature CTL that have been stimulated by allogeneic MHC antigens, modified self-MHC antigens, and non-MHC antigens are predominantly of Lyt 1+2+3+type, but that those cells that express only low amounts of the Lyt 1 determinant escape detection by ordinary fluorescence microscopy techniques and by tests for susceptibilityto (weak)anti-Lyt 1 antisera and complement (Shaw et al., 1981). It has been reported, in contrast, that proliferating helper cells as well as CTL stimulated by Class I1 alloantigens in the mouse manifest only the Lyt 1 marker (Swain, 1980; Swain et al., 1981), and a significant proportion of proliferating noncytotoxic lymphocytes responding in secondary MLR have been characterized as Lyt 1-2-T cells (Alter and Bach, 1979). Conflicting observations have also been reported recently with regard to human cytotoxic T cells: Contrary to the commonly held view that CTL in man are OKT 4-8+, distinct clones of such cells have been identified variously as OKT 4-8-, OKT 4-8+, and OKT 4+8- (L. Moretta et al., 1981; Spits el af., 1982); Krensky et al. (1982) found CTL directed against DR antigens to be exclusively OKT 4+8-. It may be inferred from these observations that the Lyt and OKT markers correlate with the MHC subregion products that T lymphocytes recognize, but that they are not narrowly qualifying indicators of T-cell functional differentiation (Swain, 1980, 1981b;Swain et al., 1981;VidoviC etal., 1981; Krensky et al., I982), nor perhaps in themselves individually and specifically requisite to proliferative responsiveness or cytotoxic action [although inhibition of both proliferative and cytotoxic activity can be effected in mouse MLR by monoclonal anti-Lyt 2 marker antibodies (Hollander et al., 1980)l. Other than the Lyt and OKT families of molecules, helper T cells and CTL reveal characteristic surface properties that may distinguish between them. Marked differences have been reported between T-cell clones, mouse and human, that express helper and those that express cytotoxic effector activity in additional membrane antigens and membrane polypeptide patterns (Sarmientoetal., 1980;Bach etal., 1981;Pawelecetal., 1981).An"exclusive glycoprotein" (T 145) has been described in mature CTL of mice (Kimura and Wigzell, 1978). Another functional distinction between helper and cytotoxic cell precursors may lie in their different requirements for alloantigen binding; mouse CTL-P have been shown able to recognize and bind to alloantigens alone, whereas HTL-P in at least some circumstances bind to alloantigens only, or preferentially, where these are "seen" in conjunction with self-MHC products (Schnagl and Boyle, 1981; Weinberger el al., 1981a,b, 1982). Also, HTL-P have been found to be more sensitive than CTL-P to cyclophosphamide (CY) and y irradiation (Susskind and Faanes, 1981).

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In addition to the responding T lymphocytes, Ia+ macrophages (and perhaps other Ia+ adherent cells; antigen-presenting cells, APC) have been assigned an essential role in the primary in vitro generation of CTL against allogeneic normal and syngeneic modified and neoplastic cells, especially where sensitization conditions are suboptimal (Wagner et al., 1972; Davidson, 1977; Weinberger et af., 1980, 1981b; Gomard et af.,1981a). These accessory cells appear to serve in several capacities: presentation to HTL-P of (“processed”; subcellular?)antigenic moieties, in context of the APC Class I1 molecules, requisitely or preferably self, elaboration of soluble, nonspecific mediators, and the “conditioning” of culture media for the maintenance of lymphoid cell growth (Unanue, 1981). The events leading to the generation of allogeneic CTL in vitro transpire rapidly. Mouse responder cell proliferation reaches a peak after 72 - 96 hr incubation, that of human cells after 120- 144 hr; mature CTL are prominent in the cultures 24-48 hr thereafter. It is generally assumed that the actively proliferating T cells are (at first) largely those with helper function.2 Upon repeated stimulation with the same antigenic stimulators, proliferative and cytotoxic effector activitiesin the cultures peak in approximately halfthe time required at primary sensitization. Studies with metabolic inhibitors have led to the impression that multiplication of responder cells is generally a necessary step in the generation of CTL (Rollinghoffet af.,1973; Clark and Nedrud, 1974). However, CTL are sometimes produced even where responder cell division is minimal. This has been shown for primary cultures in which the stimulator cells were chemically fixed, UV irradiated, or heated and in which little if any responder proliferation takes place (Dennert, 1976; Meidav and Kedar, 1979). Similarly, extensive cytotoxic reactivity has been demonstrated to develop in cultured mouse lymphoid cells upon stimulation by allogeneic lymphoma T cell lines which do not provoke a significant proliferativeresponse (Collavoet af.,1976;Meidav and Kedar, 1979). And marked cytotoxic reactivity can be generated in secondary MLR cultures within 24 hr and in the presence of an inhibitor of DNA synthesis (MacDonald et al., 1975). Various means have been employed for delineation of the MHC antigens that incite and serve as targets of CTL activity, including analysis of CTL-mediated lysis in responder/eRector-target cell combinations of diverse genetic background, inhibition of cytotoxicity by specific antisera and unlabeled target cells, and selective adsorption of effector cells on target cell monolayers. It was shown already some time ago that the Kand D loci of the mouse are recognized by different CTL populations (Brondz, 1972). Subsequent studies revealed these loci to be highly polymorphic, animals of each genotype expressing several distinct antigens under allelic control at each locus (J.

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Klein, 1978;Klein and Figueroa, 1981) which are reacted against by distinct CTL subsets (Melief et al., 1977, 1981;Weyand et al., 1981). Polymorphism is also prominent for Class I1 antigens (J. Klein, 1978; Klein and Figueroa, 198l), and these too are recognized by CTL clonally (Fischer-Lindahl and Hausmann, 1980). Determinants encoded by other loci now considered as falling within the MHC and resembling Class I markers, e.g., Qa and Tla (J. Klein et al., 1981), can also elicit pronounced CTL responses under appropriate conditions (Wagner et al., 1980c; J. Klein et al., 1981). Specificity of the allocytotoxic response in mice is largely determined by “private” MHC antigens (Brondz et al., 1975). Where CTL crossreactivity occurs, it may be due either to cross-antigenicity of private determinants, cross-antigenicity between private and “public” determinants, or sensitization to shared public determinants (Peck et al., 1976). It is still an open question whether alloreactive CTL and allospecific antibodies evoked by the same murine stimulator cells discern the same or different MHC structures. Various Observationspoint to the possibilities that determinants distinguished serologically (“SD”) and those distinguished by means of reactive CTL represent distinct epitopes expressed on separate cell surface components, or distinct epitopes sited on different parts of the same entity, or identical epitopes associated with different surface structures (J. Klein, 1976; Bach et al., 1977). The suggestion has also been made that SD markers may represent the haptenic portion of an MHC molecule, and CD markers a camer structure (J. Klein et al., 1974). Segregation ofthe mouse MHC antigens that are revealed by serologic and by CTL assays is indicated by a number of observations. Target cells that had been stripped of determinants identified by cognate antibodies may remain subject to CTL recognition (Edidin and Henney, 1973) [although it must be noted that the stripping procedure of antibody-induced capping may fail to remove all such markers (Stutling et al., 1976)l. Gel chromatography separation of solubilizedcell membrane preparations has been found to yield fractions with separate activity in antibody and CTL tests (Sopori and Bach, 1977).Induction ofCTL responses in vitro and in vivo has been reported in a combination of mouse strains not differing one from the other in serologically defined determinants (Berke and Amos, 1973; Widmer et al., 1973). Cytotoxic reactivity has been seen by MLR-sensitized cells for target cells that share no serologically defined markers with the stimulator cells (Sonde1 and Bach, 1975). And loss or dilution of CTLdistinguished markers has been observed in certain tumor cell sublines which remain identical with the parental lines in expression of serologically distinguished entities (Russell et al., 1978; Portis and McAtee, 1979, 1981). It has also been noted that antigens differentially characterized on mouse splenocytes and leukemia cells by antisera and by CTL are differentially susceptibleto various manipu-

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lations. Meidav and Kedar (1 979) thus concluded that the latter, but not the former, are partially sensitive to heat (1 hr at 46°C or 30 min at 5 1”C), whereas the antibody-determined markers are altered to greater extent by TNBS modification; Schick and Berke (1978) observed that leukemic target cells preserve properties of antibody recognition after fixation with certain concentrations of formaldehyde while losing recognizability by corresponding CTL. Disassociationoflregion (i.e,, Class 11) determinants that stimulate HTL function and that are defined serologicallyhas also been noted (Peck et al., 1980). Human MHC loci, both Class I (A, B, and C ) and Class I1 (D/DR and SB), have also been shown to be highly polymorphic; individuals of different genetic constitution each express a number of determinants, private and public, under allelic control which stimulate and serve as targets for distinct CTL clones (Eijsvoogel et al., 1976; Dausset, 1981a; Kavathas et al., 1981; Kornbluth et al., 1981; Kristensen et al., 1981a,b; Reisfeld and Ferrone, 1981). CTL target antigens have been associated with the A, B, and C (Malissen et al., 1981) and DR (Ball et al., 1981; Dausset, 198lb) components that are also defined serologically. It has been suggested,however, that CTL may also be directed at epitopes other than those recognized by alloantibodies (Bradley et al., 1978; Biddison et al., 1980; Bach et al., 1981; Kristensen et al., 1981b; Kato et al., 1982). It must be emphasized that delineation of the cellular responses that are triggered by contact with foreign MHC (and other) antigens has come largely from analyses of IVS systems. The relevance of such findings to the nature of analogous immunologic responsiveness in vivo remains a still not fully answered question. b. Soluble NonspecciJicMediators. It was intimated some years ago that soluble mediators of lymphocyte origin are involved in T-T cell interactions, in a nonspecific stimulatory capacity (“Signal 2”) (Wagner et al., 1973; Wagner and Rollinghoff, 1978), and increasing importance is ascribed to such factors in CTL induction, activation, and propagation. Altman and Cohen (1975) reported that IVS of mouse T lymphocytes against allogeneic fibroblasts could be augmented by supernatants taken from short-term MLR cultures. A similar observation was recorded by Plate (1976) for the generation of CTL against allogeneic mouse lymphoid cells. Addition ofconcanavalin A (Con A) to cultures of mouse lymphoid cells that had been specifically primed but no longer exhibited marked killer activity was seen to awaken strong secondary cytotoxic responsiveness (Bonavida, 1977). Secondary CTL reactivity specific to the initially sensitizing alloantigens could be similarly induced in mouse and human lymphocyte cultures by introducing supernatants of corresponding secondary MLR systems (Ryser et al., 1978; Uotila et al., 1978). Primary cytotoxic responsivenessin

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the mouse to weakly immunogenic allogeneic stimuli (stimulating cells fixed with glutaraldehyde, or exposed to UV irradiation of heat; cell membrane fragments), and to syngeneic tumor cells, has been magnified by introduction to the sensitization mixtures of supernatants from MLR and from Con A-stimulated lymphocyte cultures (Talmage et al., 1977; Baker et al., 1978; Fyfe and Finke, 1979; Okada et al., 1979; Ryser et al., 1979; Rulon and Talmage, 1979; J. Shaw et al., 1980; Burton and Plate, 198I; Grimm et al., 1982b). Armerding and Katz ( 1974)designated as “allogeneiceffect factor” (AEF) a lymphokine capable of augmenting antibody responses, and of replacing T cells in such responses. Allogeneic effect factor is produced in secondary MLR in which the responder lymphoid cells are derived from irradiated mice preinjected with mixtures of syngeneic thymocytes and irradiated semiallogeneic (Fl) splenocytes. Recently, this factor has been shown to potentiate specific CTL formation in v i m , and to generate nonspecific cytotoxic reactivity in lymphoid cell cultures even in the absence of intentional antigenic stimulation (Altman and Katz, 1980). Large quantities of conditioned media rich in a variety of amplifying mediators have been obtained by exposing cultures of human mononuclear blood leukocytes to phytohemagglutinin (PHA) (Morgan et al., 1976), and cultures of mouse or rat splenocytes to Con A (Gillis and Smith, 1977). Connotation of the agents has been in terms of the properties they evince in defined test systems, viz. “costimulator,” “T cell replacing factor (TRF),” “secondary CTL inducing factor,” “T cell growth factor” (TCGF; interleukin-2, or IL-2) (Moller, 1980). It had been assumed that a single substance mediates the several effects; it now appears more probable, however, that distinct factors are involved in at least some of the activities. Thus, TRF has been shown to be distinct from TCGF, although they are similar in certain physicochemical properties (Schimpel el al., 1980; Watson and Mochizuki, 1980; Farrar et al., 1982); it is now evident that various stimulatory factors are elaborated under different stimulation circumstances and exert discrete influences on lymphoid, and nonlymphoid, cell populations (Moller 1980, 1981, 1982). The human T cell servingas a prime source of soluble helper factors during MLR responses has been identified as OKT 4+8- (Reinherz et al., 1980a; Palacios, 1982). In mouse T lymphocytes, polyclonal mitogenic stimulation and IVS to Class I1 alloantigens incite lymphokine production largely by Lyt I + (Wagner and Rollinghoff, 1978)or Lyt 1+5+7+(J. Shaw et al., 1980)cells, whereas at sensitization to Class I alloantigens it is preeminently Lyt 2+3+or Lyt 1+2+3+cells that yield the factors (Okada and Henney, 1980). Ability to induce helper factor production in MLR is largely lost in consequence of certain manipulations of the stimulatory cells (heating, UV irradiation,

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chemical fixation) and of disruption of their membrane (Okada et al., 1979; Scott et al., 1980; Wagner et al., 1980d). The agent that has attracted particular attention recently is IL-2, commonly produced by T lymphocytes under mitogenic (PHA and Con A) and alloantigenic stimulation (Gillis and Watson, 1981; Ruscetti and Gallo, 1981; Smith and Ruscetti, 1981; Farrar et al., 1982; Gillis et al., 1982). Enriched, high-titer preparations of IL-2 are now obtained from cultures of hybridomas and from polyclonal and cloned lymphoma cell lines of mouse and primate origin (as detailed in Section 111,A). The active principle in purified IL-2 preparations is a glycoprotein with a molecular weight of 13,000- 15,000 (human, rat) or 30,000- 35,000 (mouse). In both mouse and human cultures, macrophages have been ascribed an essential accessory function in T lymphocyte elaboration of the factor (Farrar et al., 1980b; Larsson et al., 1980; Teh and Teh, 1980b;Neefe et al., 1981). It is held that macrophages, upon appropriate stimulation, liberate a monokine (or macrokine) of 15,000 MW (lymphocyte-activating factor, LAF, now termed interleukin- 1, IL- 1)which incites IL-2-producingT cells that have been poised by contact with antigen or mitogen to make the lymphokine (Gery et al., 1972; Mizel, 1982; Oppenheim and Gery, 1982). Interleukin-2 has emerged as a key component in T-cell regulation and responsiveness (Moller, 1980, 1981, 1982; Paetkau, 1981; Smith and Ruscetti, 1981). Its stimulatory capacities, neither antigen-specific nor MHCrestricted, are manifold, in contradistinction to the more narrow range of influence exerted by some other antigen-specific and antigen-nonspecific helper factors. The documented activities of IL-2 in the mouse include enhancement in vitro ofT-cell proliferation in response to mitogenic stimuli, and of the generation of CTL to allogeneic normal, and syngeneic and autochthonous neoplastic targets; potentiation of allograft responsivenessof athymic and conventional mice in vitro and in vivo; augmentation of antibody responsiveness in lymphoid cell cultures; replacement of required macrophage and T helper functions in CTL sensitization cultures; and maintenance of continuous growth in culture of functional helper, cytotoxic, and suppressor T cell lines and clones. Recently, IL-2 has also been found capable of activating and sustaining the growth in vitro of mouse and human cytotoxic lymphoid cells having the characteristics of natural killer (NK) cells (see Section 111,C). In contrast to its prominent amplifying effects on T and NK cells, purified preparations of IL-2 appear to lack direct stimulatory action on macrophages and B lymphocytes. Immune interferon (IFN) has been identified as another component of MLR supernatants (Kirchner et al., 1979). It has been suggested (Farrar et al., 1981, 1982) that IL-2 controls the production of IFN in the mouse by a T-cell subset (probably of Lyt 2+3+type), and that IFN can facilitate the

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maturation and/or activation to cytotoxicity of CTL precursors, and of NK cells, reactive against allogeneic normal and syngeneic virus-infected and neoplastic cells(Lindah1et al., 1972;J. M. Zarling et al., 1978b, 1979;Djeu et al., 1979; Vgnky et al., 1980; Farrar et al., 1981). The association between IFN and NK cells will be discussed further below. T-cell- and macrophage-derived soluble helper factors other than IFN, IL- 1, and IL-2 have been reported to participate in CTL development (Finke et al., 1981; Okada et al., 1981; Raulet and Bevan, 1982; Reddehase et al., 1982;Zuberi and Altman, 1982).Thus, it has been suggested that production of IL-1 (Wagner et al., 1981) and probably also of IL-2 (Palacios, 1982) is controlled by one such additional lymphokine, “IL-3” (Hapel et al., 1981 ; Ihle et al., 1982), which is elaborated by Lyt I+ “inducer lymphocytes” (Wagner et al., 1981; Larsson, 1982). Production of IL-1 may also be facilitated by other mediators (e.g., colony-stimulating factor, CSF), produced by stimulated helper or inducer T cells (Farrar et al., 1982). It is thus seen from in vitro studies that soluble nonspecific mediators play a vital role in the generation of CTL, that they can substitute for that of certain participating immunocytes, and that they can magnify existingCTL reactivity when added to sensitization systems. The nature and kinetics of mediator elaboration in the intact animal and their importance to the organism’s cellular immunologic defenses await clarification. c. Summation. The recent studies on T-cell subsets, differential MHC alloantigen stimulation, and the role of nonspecific soluble mediators permit inference of a general pattern of in vitro CTL generation (Glasebrook and Fitch, 1980; Paetkau et al., 1980; Wagner et al., 1980b, 1981; Farrar et al., 1981, 1982; Kern et al., 1981; Klein and Nagy, 1981; Ruscetti and Gallo, 1981; Smith and Ruscetti, 1981; Watson, 1981; Weinberger et al., 1981a; Palacios, 1982).The caveat is in order, however, that inclusive validity ofthe scheme is still uncertain, that not all collaborative interactions commonly observed are in fact requisite in every instance, and that variant pathways may be followed (Paetkau, 1981). The core elements of the prototype model are the following: 1. Reaction specificity of mature CTL is imposed by interaction of Class I and/or Class I1 MHC antigens of the allogeneic stimulator cells with clonally distributed alloantigen-specificreceptors expressed on responder precursor cells (HTL-P and CTL-P). 2. The soluble nonspecific mediators identified as playing a prominent role in CTL generation -IL- 1, IL-2, and IFN -are neither antigen-specific nor MHC-restricted. 3. Activation of HTL-P requires both contact with the inciting alloantigen and the mediation (antigen presentation and/or macrokine release) of Ia+

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macrophages or macrophage-like cells; activation of CTGP demands contact with the inciting alloantigen only, and not, essentially, macrophage intermediacy. 4. Differentiation of CTL-P to mature, functional CTL involves two categories of signals. Signal 1 is effected by binding of the foreign MHC antigen to specific receptors on HTL-P and CTL-P. This signal serves to prepare (“poises”) antigen-selected T lymphocyte clones to respond to the inductive action of Signal 2, transmitted by the solublemediators(Lalandeet al., 1980; Robb et al., 1981; Herrmann et al., 1982).Thus, both HTGP and CTL-P possess two kinds of receptors: the former, receptors for MHC antigens (as these are presented by APC) with the stimulating properties of LAD markers (as per the classic definition) and nonclonally distributed receptors for IL- 1; the latter, receptors for MHC antigens behaving as CD epitopes and nonclonally distributed receptors for IL-2. Maturation of poised CTL-P to active CTL can proceed in the absence of macrophagesand HTL, provided exogeneous IL-2 is introduced to the IVS system. The generation of CTL to MHC alloantigensis thus presumed to transpire stepwise: 1. Attachment of mitogen to Ia+ macrophages, or the interaction of HTL-P bearing specific antigen receptors with the macrophage-borne antigen, leads to release of the macrokine IL- 1. 2. Interleukin-1 binds to correspondingreceptors on HTL-P. Where these cells have been readied by Signal I, the binding of IL-1 causes them to undergo division toward maturation as functional, IL-2-producing helper cells. 3. The other major T-cell subset, CTL-P, comes to express receptors for IG2 prominently upon poising by Signal 1. Clonal expansion of the poised CTL-P (or “IL-2 responder cells”)and their differentiationto mature CTL is conditioned on IL-2 stimulation, without further need for Signal 1. In the course of clonal CTL expansion and maturation, immune IFN is produced by the CTL or by other T cells, under IL-2 control, and may amplify the cytotoxic capacity of the fully mature CTL (Farrar et al., 1981).

The data available are suggestive of a similar sequence of events for CTL generation in syngeneic MLR (Lattime et al., 1982; Palacios, 1982). 2. Reactivity to MHC-Restricted Antigens Murine CTL induced by virus-infected (Zinkernageland Doherty, 1974) or chemically modified (Shearer, 1974)syngeneic cells, by minor histocompatibility antigens (Bevan, 1975),and by the male Y antigen (Gordon et nl., 1975) have been shown to react only, or preferentially, with corresponding

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targets of the same H-2 type as both stimulator and responder cells. Similar findings have also been reported for human CTL activated by Y antigen and by virally infected or chemically modified histocompatible lymphocytes (McMichael el al., 1977; Goulmy et al., 1979; Moss et al., 1981). This phenomenon of “MHC restriction” implies a dual specificity: Specific CTL-P and CTL express receptors reactive not only for the additional, non-MHC antigens but also for the self-MHC (predominantly Class I) molecules of the stimulating/target cells; HTL-P express receptors for both the additional antigens and for self MHC products (largely Class I1 determinants) on the APC which activate the lymphokine ~ascade.~ It is proposed that this dual recognition mechanism may be the only way for organisms to distinguish self from non-self (J. Klein et al., 1981; Benacerraf, 198I), and that the restriction imposed on T cells by the MHC is a major, if not the only, function of these cell components. Whereas HTL-P, CTL-P, and CTL must each “see” the foreign antigen in context of the appropriate MHC determinants, MHC restriction of HTL-CTL-P interaction has not been defined and may, in fact, not exist (Finberg and Benacerraf, 1981; Fink and Bevan, 1981). Two interpretations of MHC restriction have been proposed (Shearer et a]., 1977; Zinkernagel and Doherty, 1977, 1979; Blanden and Ada, 1978; Snell, 1978; Matzinger, 1981). According to the dual recognition model, the self-MHC structures and the foreign determinants (e.g., viral, haptenic, and other non-MHC antigens) on the stimulator/target cells are recognized separately by distinct receptors on the same reactive CTL. The altered-self model, in contrast, postulates a single CTL receptor that recognizes en bloc both the MHC and the other antigenic markers. Analysis of the specificities of cloned, diploid H-2k/H-2dCTL lines that recognize H-2k-plus-minorantigen 1 and cross-react with H-2d-plus-minor antigen 2 led Hunig and Bevan ( 1981, 1982)to conclude that recognition is of the integrated MHC-plus-foreign antigen complex rather than of either component independently. However, evidence has been brought forward for each of the receptor-MHC restriction models, and the data so far available are insufficient to permit definitive adjudication in favor of one or the other (Marx, 1981). The topographic arrangements of MHC-restricted target structures recognized by CTL remain unclear. A number ofpossibilities can be entertained in It is noted that even in the generation of CTL against alloantigens, requirements for macrophage cooperation are in some instances for APC of responder-cell type (perhaps the more so as the sensitization stimulus is limiting), i.e., for association between the sensitizing epitopes and self-MHCproducts (usually,Class 11) on the presentingcell. Although presentation of alloantgen can be effected at times by non-self APC (Weinberger et a!., 1982), CTL production may then be less efficient. Thus, a measure of “syngeneic preference” appears to apply as well, in APC- HTL-Pinteraction,t o the induction ofCTLdirected at allogeneic targets.

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keeping with both the dual recognition and the altered-self constructs of CTL receptor nature and MHC restriction. For the former, there can be envisaged the existence of MHC-coded and MHC-unrelated antigenic determinants as parts of the same polymolecular target entity, or placed on distinct target membrane entities that are closely associated by covalent or noncovalent bonds, or appearing on distinct, adjacent entities not linked chemically. For the latter, there can be considered similar eventualities of MHC and non-MHC molecular relationships, and also of changes in the antigenic nature of the MHC epitopes themselves, brought about by interaction with the “additional” antigens (for instance, by virus-associated proteins with enzymatic activity). Experiments in mice with virus-infected and chemically modified (normal and transformed) cells, employing techniques of cocapping, inhibition of CTL cytotoxicity by antisera against viral, haptenic, and H-2 antigens, and membrane protein separation (Germain et al., 1975; Schrader et al., 1975; Henning et al., 1976; Kvist et al., 1978; D. A. Zarling et al.,1978; Senik and Neauport-Sautes, 1979; Honeycutt and Gooding, 1980; Ciavarra and Forman, 1981) point to a close physical association between virus, or hapten, and MHC determinants on the membrane. On the other hand, Fox and Weissman (1979) demonstrated that H-2 and viral epitopes on lymphoma cells may be located on separate molecules. Lack of intimate association between extrinsic antigens and MHC products has also been suggestedby other investigators(Fenyo et al., 1977;Ciavarra et al., 198l;CiavarraandForman, 198l;D6mantetal., 1981). Thus,Hapelet al.(1 980a) and Ciavarra and associates (198 1) report observations pointing to the likelihood that viral determinants and H-2-containing membrane proteins associate noncovalently, but that although antiviral CTL recognize the formed complex the recognition is of native rather than of altered viral and H-2 entities. Moreover, the validity of some earlier studies which demonstrated cocapping of viral and H-2 components, and inhibition of CTL killing of virally infected target cells by anti-H-2 sera, must be questioned in light of the finding that many of the alloantisera employed were contaminated with antiviral gp70 antibodies (Milner et al., 1976), and on other grounds (Bourguignon et al., 1978). The generality of self-MHC restriction has been questioned, for both human and mouse systems. It has been found that supposedly restricted CTL populations (Dennert, 1976; Holden and Herberman, 1977; Stutman and Shen, 1978;Charmot and Mawas, 1979;Pfizenmaier et al., 1980; Siliciano et al.,1980; Seeley et al., 1981) and clones (Braciale et al,, 1981; von Boehmer and Haas, 1981) can also react with the non-MHC epitopes in the context of certain alloantigens, as well as with certain allogeneic target cells lacking the modifying determinant.

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Exceptionsand deviations from the rule of self-MHC restriction have been explained variously. It has been suggested that some altered- or modifiedself-MHC determinants are related structurally to some alloantigens (Burakoff et al., 1978;Finberg et al., 1980),or that they are in effect so “perceived” by responsive T cells (Perry and Greene, 1980). “Neoantigens” may be formed in consequence of some virus and chemical interaction with MHC components (McDevitt, 1980);whereas limited changes in MHC configuration may indeed result in self-restricted reactivity to the new epitopes, more pronounced alterations effected by the agents can lead to recognition of the self neostructure as a full-fledged alloantigen, and as cross-reactive with existing alloantigens (Dennert, 1980a; Siliciano et al., 1980). As T lymphocytes “learn” to recognize normal self-MHC determinants during differentiation in the thymus (Zinkernagel et al., 1978) or elsewhere (Lake et al., 1980; Marx, 1981; Wagner et al., 1981), some clones become capable of reacting, simultaneously, with modified self-MHC and with alloantigens of sufficient similarity (Benacerraf, 1981). It is argued that the repertoire (or complement) or peripheral CTL-P is “unlimited in that it includes self-MHC as well as allo-MHC restricted antigen-specific” clones, the latter, however, being represented with far lower frequency (Wagner et al., 1981); it is also possible that the affinity ofT-cell binding (by receptors on cells ofthe same or of different clones) to self-MHC and to allo-MHC in association with the extrinsic epitopes differs markedly. Evidence has been brought forward as well that presentation to HTL-P of both all0 and other antigens can be effected by allogeneic APC (Nagy and Klein, 1981; Ishii et al., 1981; Yamashita and Nakamura, 1981; Weinberger et al., 1982). The molecular nature, relative affinities, range of specificities, and clonal distribution of CTL and HTL receptors for self-MHC, allo-MHC, and other antigens clearly remain problematic, and the uncertainties are reflected in the still-ambiguous status of the rules of MHC product re~triction.~ Cytotoxic activity that appears to obey no constraints of MHC restriction, neither self nor allo, may also accrue from activation/expression of widely reactive NK or NK-like effector cells, especially as observed in the course of IVS and long-term in vitrucytotoxicity assay (E. Klein, 1981). Attachment of substances from fetal calf serum or other complex culture supplements to stimulator and target cells in culture can also give rise to the erroneous The designation “MHC restricted” is commonly thought to infer a demand for associative recognition of foreign, non-MHC antigen and a self-MHCproduct by reactiveT lymphocytes. It is evident from the previous discussion, however, that the requirement for associative recognition can often be met also by presentation of the non-MHC antigen in conjunction with do-MHC determinants. When refemng to an MHC-restricted reactivity without further specification,the eventualityof either or both these types of association must accordingly be left open.

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impression of nonspecific and/or non-self restricted CTL potency, due in fact to reactions directed at supplement antigens adsorbed to a variety of “target” cells (Forni and Green, 1976; Burton et al., 1978; Golstein et al., 1978) or to mitogen-like polyclonal activation of C T L P with diverse specificities. Self-restricted T cells have been shown to express surface markers similar to those possessed by alloreactivecells: HTL have been defined as Lyt 1+2-3(mouse) and OKT 4+8-(human) and CTL as Lyt 1+2+3+or Lyt 2+3+(mouse) and OKT 4-8+(human)(Ada et al., 1981; Biddison et al., 1981; Finberg and Benacerraf, 1981). 3. Reactivity to Tumor-AssociatedAntigens a. Tumor Antigenicity. Several distinct classes of TAA can come to expression on tumor cells, in various forms of molecular association with normally present constituents and with each other (Hellstrom and Brown, 1979;Weiss, 1980).The presence and activitiesof oncogenic(and/or passenger) viruses may give rise to several categories (Weil, 1978;Kurth et al., 1979; Law et al., 1980): components of the virion itself, cellular structures made under the genetic control of the viral genome, and host-coded structures, the virus acting to derepress repressed genetic information of the host cell. The antigenic and immunogenic (Sulitzeanu and Weiss, 1981) strength of virusassociatedtumor antigens varies from tumor to tumor, and for clones arising from the same neoplasm. Neoplasms induced by the same oncogenic virus commonly express some identical or related TAA, although individual-specific epitopes may also be borne by such growths (Vaage, 1968). The TAA potencies of neoplasms triggered by carcinogenic chemicals, irradiation, hormones, and certain physical imtants are also diverse, ranging from pronounced to marginal or nonexistent; and, where antigenicity is evident, variable degrees of uniqueness associated with individual tumors, histologic type, and carcinogenicstimulus have been noted. Some oncogenic agents may activate latent, or masked, viruses, and the TAA of tumors so initiated or superinfected may thus include the spectrum of virus-related determinants. Although some “spontaneous” neoplasms of animals appear to be immunogenically inert (Hewitt et al., 1976; Klein and Klein, 1977; Hewitt, 1978), there are growing indications that many spontaneous growths, in animals as well as in man, do manifest tumor-associated antigenic characteristics (Ioachim, 1976, 1980; Prehn, 1977; Baldwin et al., 1979; Hellstrom and Brown, 1979; Hellstrom et al., 1980; E. Klein et al., 1980; Old, 198l).s Serologic testing does not always reveal the presence of TAA, even of strongly immunogenic determinants. Thus, in describing their experience with chemically induced sarcomas of mice, long known to express individually distinct protective antigens, DeLeo et al. (1979)

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Two classes of antigens expressed by a variety of neoplasms are fetal (or embryonic) determinants (e.g., carcinoembryonic antigens, CEA) (Chism et al., 1978) and organ-specific markers (Weiss, 1980) that are not present, or present in only very small amounts or different topography, in corresponding normal, mature cells. These “displacement” antigens (displaced in ontogenic time or in tissue locality) may also serve as inducers of, and targets for, tumor-directed CMI responses (Weiss, 1980). It has also been proposed that the TAA displayed by some tumor cells may be closely related to normally occurring MHC-coded structures (Bortin and Truitt, 1980, 1981; Festenstein and Schmidt, 1981). By means of transplantation, serologic, and in vitro CMI tests, several workers have detected “alien” (or “inappropriate”) H-2 markers on murine tumors (Garrido ef al., 1977; Invernizzi el al., 1977; Martin et al., 1977; Rogers et al., 1979; Russell et al., 1979; Bonavida and Roman, 1981). Several human tumors have also been claimed to express such alien MHC determinants (McAlack, 1980; Festenstein and Schmidt, 198I). Compatible with these reports is the observation that cytotoxic cells (both T and NK) reactive against syngeneicmouse lymphoma and solid tumor cells can be generated in the course of culture with pooled normal allogeneic lymphocytes (Bach et al., 1980). Similarly, blood lymphocytes from cancer patients can develop strong cytotoxic reactivity toward autologous tumor cells following cultivation with allogeneic normal lymphocytes (J. M. Zarling ef al., 1978a; Strausser ef al., 1981; Vanky et al., 1981; Vanky and Klein, 1982a). It is conceivable that certain TAA are derepressed histocompatibility antigens, not come to expression on normal cells, or usually appearing self-determinants that have undergone a form of modification. Other findings indicate that TATA (of some neoplasms) and any deviant MHC components present are structurally distinct moieties (Chauvenet and Smith, 1978; Pliskin and Prehn, 1978; Parmiani et al., 1980; Rogers ef al., 1980). Analysis of the TATA in H-2 hemizygous isoantigenic variants of a somatic cell hybrid, derived from the fusion of a 3-methylcholanthrene (MCA)-induced sarcoma and a mammary carcinoma, led G. Klein ( 1977)to infer that the TATA of the MCA tumor does not represent modified H-2 determinants. Moreover, the suggestion has been advanced that certain observed that “despite prolonged immunization with strongly immunogenic tumors, resulting in a high level of transplantation resistance,the sera of such mice generallylack antibody having specificity for the immunizing tumor. In this regard, these tumor-specific antigens behave like antigensdeterminedby certain H-2 mutants;recognition of these mutant H-2 products appears to be entirely in the province of cellular immunity and does not result in the production of a humoral immune reaction.” Failure to demonstrate TAA in human neoplasms by the usually applied serologic probes must not be interpreted, accordingly,as firm proof for the absence of determinantscapable of evoking protective immunologic reactivities.

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supposedly new, alien components may already be present, cryptically, in the membrane of the normal parent cell (Robinson and Schirrmacher, 1979). Whatever the biologic natureofTAA, it is not improbable(G. Klein, 198 I ) that among the distinguishing antigenic markers of neoplastic cells, many if not all are products possible within the organism’s normal genetic potential but now appearing deviantly in time, locality, mode of expression, or quantity. Such determinants may be manifested by the variant cells in amount or form insufficient for the ready elicitation of immunologic responses,and perhaps especiallyof reactivitiesthat have defensive import, but adequate as targets of responses that have been brought about by specific andfor nonspecific hyperstimulation. Thus, for instance, the elicitation of cytotoxic responsiveness to autochthonous neoplasms by means of sensitization with pooled, normal allogeneic cells might be explainable on the basis of a cross-representation of certain epitopes on the autochthonous tumor and the allogeneic cells, pools of the latter constituting a more effective immunogenic stimulus (Weiss, 1980). A major obstacle to the definition of TAA in general, and especially of those that can induce rejection responses in vivo (i.e., TATA) and cytotoxic reactivity in vitro, lies with the circumstance that most studies so far have been with tumor lines established in tissue culture and with long-transplanted neoplasms. It must be assumed that the antigenic makeup of such cells is subject to considerable, frequent variation; fluctuations in the expression of MHC and TAA antigenicity have indeed been demonstrated for many tumors in the course of passage (Cikes et af.,1973; Finn et af.,1978; Celis et af.,1979;Hale et af.,1979; Rosenberg et af.,1980a;Greenberg et al., 1981a). Tumors maintained in culture, for even short periods oftime, may acquire new antigenicitiesin consequence of mycoplasma (van Diggelen et af.,1977) or C-type viral infections (or expression) (Lieber et af.,1974; Yefenof and Klein, 1974; Aoki et al., 1977), incorporate (heterologous) serum proteins from the tissue culture medium, as has been shown for cells cultured in FCS (Irie et af.,1974;Schlesinger, 1974;Forni and Green, 1976; Sulit et af.,1976; Golstein et af.,1978),express unusual amounts of oncofetal antigens (Gorczynski, 1976; Parker et af.,1977), and undergo a variety of other mutational and adaptive changes concomitant with the artificial growth circumstance. Any of these events can lead to the formation of new antigenic structures (not necessarily associated with the malignant state of the cells per se) able to generate CTL and to serve as target antigens, and thus erroneously identified as “TAA.” Conversely, tumor cell lines can lose, sometimes very rapidly, antigenic properties characteristic of the growth at isolation from its host.

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Some of these eventualities also apply, in varying degree, to neoplasms maintained by repeated passage in vivo. A prominent possibility is the loss or dilution of determinants capable of evoking protective immunologic responsivenessin the syngeneic organism (Fenyo et al., 1973;Celis et al., 1979; Greenberg et al., 198la). Moreover, alloantigenic differences between tumors and their strain or origin may arise in the course of prolonged breeding of “syngeneic animals” (Wainberg et al., 1977; Burton et al., 1978). This eventuality is supported by our recent findings with the Moloney virus-induced YAC lymphoma of A mice (H-28). Tumor cells tested in earlier passages (<50) could induce syngeneic humoral responses in vivo and cellular cytotoxic responses in vitro to an only limited extent, with no evidence of cross-cytotoxicity for H-2b normal and leukemia target cells (Kedar et al., 1976).After 200 passages in the syngeneicstrain, however, two sublines of this neoplasm have arisen in our colony: one, deficient in the original H-2” antigenicity and able to grow progressively in A/J (H-P), BALB/c (H-Zd),and C57BL/6 (H-Zb)mice, and a second, which exhibits (by serologic and cell-mediated cytotoxicity tests) reduced amounts of H-28, but displays antigens identical or cross-reactive with H-2b haplotype determinants (Kedar el al., 1982g). These findings imply that all inference drawn from studies with cultured or transplantable tumors as to antigenicity and immunogenicity must be cautious and reserved. Another problem pertaining to the characterization of tumor-associated antigenicity, and to the logistics of immunologic intervention in neoplastic diseases, lies with the likely differences between primary and metastatic foci. It has been proposed that distal neoplastic growths originate from variant clones, of different metastatic potential, that are represented in the original tumor cell population (Fidler and Kripke, 1977; Man, 1982). Cells in individual metastases may differ from those of the primary growth in diverse properties, including antigenicity and immunogenicity (Fidler et al., 1978; Nicolson et al., 1978; Schirrmacher et al., 1979; De Baetselier et al., 1980; Wiltrout and Frost, 1980; Fidler and Hart, 1981; Albino et al., 1981); immunoselective processes may play a role in determining, at outset, the dissemination capacity of distinct neoplastic subpopulations (Fidler et al., 1978). The varying behavior and fate in a given host of individual tumor niduses of the same origin may thus have, in some instances, an immunologic basis: Defensive immune responses may brake the development of one lesion without having an impact on another. Thus, for instance, Fogel and coworkers (1979) reported that C57BL/6 splenocytessensitized in vitro against primary Lewis lung carcinoma (3LL) implants, and highly cytotoxic for cells

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of the local tumor, were considerably less damaging for cells derived from subsequently developing pulmonary metastases. Conversely, splenocytes sensitized against metastatic cells proved significantly more cytotoxic for the corresponding targets than for cells from the primary growth. Only effector cells induced by metastatic variants could reduce pulmonary involvement following footpad injection to syngeneic animals of mixtures of the effector cells and 3LL cells of either primary or metastatic source. Bosslet and Schirrmacher (198 1) found that metastatic cells originating from a chemically induced lymphoma express distinct TAA, and are resistant to the action of CTL elicited by the primary neoplastic tissue. Brooks et al. (I98 1) showed that tumor cells taken from metastasesdevelopingin certain tissues of the rat frequently have alower sensitivity to NK cytotoxicity than test cells from the primary growth. If a similar diversity of antigenic properties between primary and satellite neoplasms applies as well to human cancer (Albino et al., 1981), the complement of CTL for systemic adoptive immunotherapy may have to include effector subsets directed at target determinants of both the local and the dispersed neoplastic cells that confront the patient. b. Antitumor Reactivityof CTL. CTL generated in vitroand in vivoagainst autochthonous and syngeneictumors display reactivity of varying degrees of specificity and MHC restriction (Burton et al., 1978; Burton and Warner, 1980; Wagner et al., 1980~). There has been considerable discussion regarding the nature of MHC restriction in the CTL response to TAA. In considering this question, a word of caution is in order, that applies as well to other antigens serving as CTL inducers and targets. Instances of a pronounced preference of reactivity for TAA- self-MHC target structures by CTL that have been correspondingly induced need not necessarily reflect an inability to respond to the antigens when these are presented in conjunction with allo-MHC products; unless shown otherwise, they could be explained as manifestations of a classic secondary response by effector cell populations to the specific sensitizing stimulus. Where CTL reactivity appears not to be conditioned on copresentation of TAA with self-MHC, the circumstance could be interpreted either as indicating the ability of CTL induced by TAA - self-MHC to cross-react well with that TAA in an allo-MHC constellation, or as freedom of the CTL directed at that antigen from any constraint of MHC assocation (as might be expected to be the case for TAA that in themselves resemble deviant MHC entities). Evidence in favor of what appears to be self-MHC restriction of CTL responsiveness to TAA comes from the frequent observation that CTL directed at experimental neoplasms preferentially recognize and destroy

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target cells sharing common MHC determinants with the sensitized lymphoid population (Wainberg et al., 1974; Plata et al., 1976;Trinchieri et al., 1976;BlankandLilly, 1977;Gomardetal., 1977;Duprezetal., 1978;Misko et al., 1980; Yefenof et al., 1980b; Lannin et al., 1981). Moreover, certain tumor cell sublines that are deficient in H-2 expression have been found unable to elicit and to be recognized by CTL (Tursz et al., 1977; Kaneko et al., 1978; Russell et al., 1978; Meruelo, 1979; Portis and McAtee, 1979; Dalianis et al., 1981; Plata et al., 1981). And, destruction of murine tumor cells by syngeneic CTL can sometimes be blocked specifically by relevant anti-H-2 sera (D. A. Zarling et al., 1978;Gooding, 1979;Green et al., 1980). Findings made in certain human tumor test systems are also compatible with a degree of self-MHC restriction in CTL reactivity. Lymphocytes sensitized in culture against autochthonous leukemia (Zarling et al., 1976; Lee and Oliver, 1978), Epstein - Barr virus (EBV)-transformed lymphoid (Moss et al., 198I), and some solid tumor (Vose et al., 1978a,b;Vanky et al., 1979,198 1) cells have been found capable of more effective attack against the autochthonous neoplasm than against allogeneic ones of the same histologic type. Conversely, patient lymphocytes sensitized against allogeneic growths sometimes prove ineffective in attacking corresponding autochthonous cancer cells (Taylor et al., 1979; Vinky et al., 1979). However, such observations can be interpreted instead as pointing to reactivity toward TAA specific to the individual neoplasm (Wainberg et al., 1974; Wainberg and Phillips, 1976; Vose et al., 1978b; Vinky et al., 1981). Other observations speak against the generalization of self-MHC restriction in CTL responses to neoplastic cells. Cytotoxic T lymphoid cell populations and clones sensitized against neoplastic cells of autochthonous or syngeneic origin, both mouse and human, not infrequently evince reactivity for similar allogeneic target cells, especially where long-term (1 8-40 hr) in vitro cytotoxicity assays are employed; allogeneic tumor sensitization sometimes leads to the generation of CTL potent toward syngeneic/autochthonous tumor targets; and antitumor CTL responsiveness across MHC bamers can sometimes be induced as well by normal stimulator cells (Holden and Herberman, 1977; Ting and Law, 1977; Burton et al., 1978; Collavo et al., 1978b; Stutman and Shen, 1978; Baker et al., 1979; Devens and Naor, 1979; Bach et al., 1980; Seeley et al., 1981; Strausser et al., 1981; Bach et al., 1982; Vanky et al., 1982). The suggestion has been made that cytotoxicity apparently not self-MHC restricted may be mediated by some CTL subsets within heterogeneous effector cell populations, and restricted reactivity by other subsets (Stutman and Shen, 1978). Some of the data claimed to support nonrestrictive CTL responsiveness may not be germane, however. The participation of NK and NK-like cells in

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the cytotoxic action of mixed, noncloned lymphoid effector populations must be excluded rigorously before there can be any imputation of non-self MHC-restricted CTL capacity. The question of the nature and extent of MHC restriction in CTL activity directed at TAA remains open. It would appear permissible to infer, tentatively, that CTL responsivenessdoes not invariably obey narrow constraints of self-MHC restriction, and that where such restriction is in effect, it may at times be circumvented by optimization of the conditions of induction. The relationship between various determinants on the surface of stimulator and target tumor cells has been the subject of numerous investigations.In at least some murine neoplasms, oncogenic virus proteins and other TAA appear to be physically associated with H-2 products (Blank and Lilly, 1977; Kvist et af., 1978; D. A. Zarling et af., 1978; Callahan et af., 1979). On the other hand, it has been shown that TAA can be separated readily from H-2 markers by detergent solubilization and fractionation techniques (Davies et af., 1974; Clemetson et af., 1976; Fox and Weissman, 1979; Honeycutt and Gooding, 1980) and that antisera against H-2 antigens sometimes fail to block the attack of antitumor CTL (Giorgi et af., 1982).investigations in our laboratories point to the induction by allogeneic mouse leukemia cells of distinct CTL populations in vivo and in vitro (Kedar and Bonavida, 1975; Devens et af., 1979a), one directed at TAA and the other at alloantigens. Indications that the inciting and target structures of antitumor CTL are not necessarily modified MHC products, nor polymolecular entities formed by intimate associationsofTAA with MHC, also come from the failure to detect marked changes in Class I and Class I1 determinants on several types of mouse leukemia cells (Meidav and Kedar, 1979; Watson et af., 1979; E. Kedar et af., unpublished observations, 1983). The responsiveness of CTL does not invariably fall within narrow limits of inducer and target cell specificity. Cross-reactivities between diverse normal and neoplastic cells have been observed frequently in both the generation and manifestation of CTL action; and CTL produced against a neoplasm of particular class often express a degree of cytotoxicity for unrelated ones. Such nonspecificity can arise, at times and in part, from the excitation of certain lymphoid cells to an undiscriminating, “promiscuous” cytotoxicity that extends beyond the boundaries usually set in defining immunologic reactions (E. Klein, 1982). Other explanations present themselves, however. Broadly manifested cytotoxic proclivity may be the consequence of recognition of discrete but widely distributed antigenic determinants. Prominent among such is the category of embryonic (“oncofetal”) antigens(BaldwinandRobins, 1977;Burtonetaf., 1977;Bartlettetaf., 1978; Chism et af., 1978);another is antigens associated with causative or common

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passenger viruses. Cross-reactivity may thus be due to specific, identical epitopes of prevalent occurrence that are shared by the diverse sensitizing and target cells, or to common structures characterized by some antigenic similarity. Putative nonspecificity may also be a gross artifact, resulting from the adsorption to cells in culture of antigenic substances from the medium. Evidence for the susceptibilityto specificCTL attack of a variety of human as well as of experimental animal tumors comes persuasively from in vitro investigations; in vivo observations have afforded corroborative information, if often only of a circumstantial nature. It would appear that diverse neoplastic cells are subject to CTL recognition and attack by virtue of their expression of certain tumor-associated antigens, regardless of whether any such are truly spec& to the neoplastic state, provided that the parameters of sensitization and effector/target interaction are favorable. The nature and topographic configurations of the target molecules on neoplastic cells that serve as sites for CTL attack remain largely undefined, however, for human cancers and for many of the tumors studied in laboratory animals. The extensive studies that have been conducted with tumor cells carrying C-type oncornaviruses exemplify the difficulties encountered in the precise definition of CTL target determinants. It has been proposed by many investigators that CTL induced by such cells in vitro and in vivo are directed predominantly at group-specific viral constituents, especially the envelope protein gp70 (Herberman et al., 1974;Plataet al., 1975, 1976;Shellam et af., 1976; Levy and Leclerc, 1977; D. A. Zarling et af., 1978; Hellstrom et af., 1979; Collins et al., 1980; Yefenof et al., 1980b). Moreover, CTL with reactivity for rodent leukemias and sarcomas can be induced in vitro by preparations of the intact or disrupted viral agents that infect the target cells (Shellam et al., 1976; Treves, 1978; Taniyama and Holden, 1979a); and specific CTL attack can sometimes be blocked by viral proteins (Bruce et af., 1976; Enjuanes et af., 1979).Other findings, however, suggest that gp70 and other virion constituents may not themselves be the specific CTL target epitopes (Watson and Bach, 1980). High-titer antisera directed at gp70 and other virion proteins have been found unable to block CTL interaction with infected murine leukemia cells (D. A. Zarling et af., 1978;Green et al., 1980). Working with solubilized, partially purified membrane components of RBL-5 leukemia cells, Alaba et af.( 1979)concluded that viral proteins gp70 and p30 cannot elicit tumor rejection responses irl vivonor induce antitumor CTL in vitro. Earlier studies in another mouse tumor system in which defined viral agents play a major etiologic role, mammary adenocarcinomas appearing in strains of mice that are pre- or neonatally infected with one of a family of mouse mammary tumor viruses (MMTV), have indicated that both virion

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and virus-coded host - cell constituents can elicit immunologic rejection responses in the intact animal, and that the spectrum of TATA also includes antigens unique to individual tumors (Weiss, 1969a,b). It would thus appear necessary to emphasize the likelihood that the cells of neoplasms can express, variously and competitively, immunity-evoking determinants specific to individual growths, even where these arise in the same organism, as well as determinants held in common by others of the same causation, tissue origin, stage of differentiation, and other mutual characteristics, and that the differential antigenic expression may diverge among clonal subpopulations and fluctuate as a function of tumor progression or passage. Attempts in the future at intelligent immunologic intervention in malignant diseases must be based on a delineation of the relevant antigens, on recognition of their constantly variable expression, and, consequently, on the very likely requirement for a corresponding individualization in the making and usage of immunologic weaponry. Information is still incomplete, especially for human tumor systems, but it appears that the nature and sequence of the events which lead to CTL generation against allo- and other antigens are on the whole analogous to those pertaining to anti-TAA responsiveness. Induction of CTL in culture to experimental animal tumors demands the participation of both functional T cells and APC (macrophages) (Mokyr and Dray, 1982), and, where the conditions of sensitization are suboptimal, collaboration between HTL-P and CTL-P and the activities of a variety of nonspecific soluble mediators are the more requisite (Glaser and Law, 1978; Fyfe and Finke, 1979; Glaser, 1979a,b; Burton and Plate, 1981; Gomard et af., 1981b; Hancock et af., 1981; Sinclair et af., 198la). Involvement of a seemingly antigen-specific soluble mediator, derived from lymphoid cells of tumor-bearing mice, has also been reported (Kilburn et af., 1981). The requirement for Ia+ macrophage cooperation, with a preference for APC histocompatible to the responder lymphocytes, has been reported by several investigators for CTL induction against neoplasms of mice (Igarashi et af., 1979; Mokyr et al., 1979;Taniyama and Holden, 1979b;Woodward et af.,1979;Glaser, 1980a; Gomard et al., 1981a). Specific antitumor CTL in the mouse have been characterized as being predominantly of Lyt 1+2+3+type, although Lyt 2+3+ cells have also been seen active (Shiku et af.,1976;Stutman and Shen, 1978; Leclerc and Cantor, 1980a);human CTL directed at EBV-transformed cells have been classified as OKT 8+(Zarling et af.,1981b) and as both OKT 4-8+ and OKT 4+8- (Spits et al., 1982). Although marked similaritiesare evident in the generation of CTL toward alloantigens,altered self-MHC and foreign antigens, and TAA, the eventuality must be taken into account that there exist differences as well in the

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induction of these reactivities, with regard to precise parameters and kinetics most favorable to the process. 4. Methodology of Production ofAntitumor CTL in Mixed Lymphoid

Cell - Tumor Cell Cultures a. General Considerations. Sensitization in vitro is effected by means of MLTC that bring together in various proportions living responder cells derived from lymphoid tissues and inactivated (by mitomycin C, irradiation, or otherwise) stimulator tumor cells in an enriched culture medium. For leukemias, lymphomas, mastocytomas, plasmacytomas, and other readily dispersed neoplasms the cultures are usually of cells in suspension (Wagner and Rollinghoff, 1973; Kedar et al., 1976); for solid tumors, responder lymphoid cells are usually seeded onto monolayers of inactivated tumor cells (Ilfeld et al., 1973; Golub and Morton, 1974; Kuperman et al., 1975) although mixed suspension systems have been employed (Sharma, 1976; Vose et al., 1978a). Under suitable conditions, reactive clones within the responder population undergo the proliferative and differentiationprocesses which bring them to cytotoxic effector reactivity within 5 - 8 days. The culture conditions satisfactory for induction of CTL against normal allogeneic cells in MLR also pertain, on the whole, to the generation of antitumor CTL (Wagner and Rollinghoff, 1973; Burton et al., 1975, 1978; Engers and MacDonald, 1976; Kedar et al., 1976; Sharma, 1976). For the latter, however, the parameters of optimal sensitization must be defined more stringently, and individually for each induction system, probably because the inciting TAA stimulus is commonly weak. Thus, for instance, the optimal responder/stimulator cell ratio has been found to vary over orders of magnitude (2: 1 - 10,000: 1) in different models. And changes in any one parameter (such as shape and capacity of the culture vessel) often set new optima for other variables (responder/stimulator cell ratio, total culture cell density, etc.) (Kedar et al., 1976, 1977). b. Responder Cells. In vitro sensitization against autochthonous and allogeneic tumor stimulator cells can be effected in MLTC with human responder cells obtained by density sedimentation on a Ficoll- Hypaque gradient (Boyum, 1974)of peripheral blood mononuclear leukocytes (PBL) from normal healthy donors (Svedmyr et al., 1974a;Sharma, 1976;Kedar et al., 1979b)and from cancer patients with active disease (Golub and Morton, 1974;Sharma and Terasaki, 1974b;Martin-Chandon et al., 1975;Vose et a/., 1978a; Vose and Bonnard, 1982) or in remission (Zarling et al., 1976; Lee and Oliver, 1978; Taylor et al., 1979). The lymphoid cells of some patients are capable of a degree of cytotoxic reactivity against their own tumors prior to any in vitro sensitization;the cells of others acquire such capacity only in

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the course ofspecific culture induction (Vose et af.,1978a).Patient PBL may show a lesser capacity than normal-donor PBL to undergo in vitro sensitization against allogeneic tumor cells (Sharma and Terasaki, 1974b). In murine systems, CTL production in culture has been successful with PBL, lymph node tissue, thoracic duct lymphocytes, and splenocytes from normal donors and with thymocytes from cortisone-treated animals (Wagner and Rollinghoff, 1973;Engers and MacDonald, 1976; Kedar et af., 1976);the spleen has been the most commonly employed and often the most reactive source of responder cells. We have obtained responder cell suspensions of high viability by gently squeezing the lymphoid tissues with forceps in coid culture medium supplemented with 2% FCS; gross aggregates are removed by gravity sedimentation for 5 min, and dead cells (as well as erythrocytes) by centrifugation for 10 min at 300g on a Ficoll-Hypaque layer. Efficacious CTL generation has also been reported for experimental animal responder cells from subjects with progressive neoplastic disease (Civin et af.,1979;Mokyr et af.,1979),from those in which a primary growth underwent spontaneous regression (Plata et af.,1975; Bruce et af.,1976; Glaser et af., 1976a; Cheever el af., 1977) or was surgically removed (Rollinghoff, 1974),and from animals immunized with inactivated neoplasticcells(Martin et af.,1973;Galili et af.,1978;Yefenofet al., 1980b). In some instances, lymphoid cells from animals with extensive tumor burden show a depressed responding capacity in MLTC (Treves et af.,1976; Mokyr et af., 1978); in others, strong cytotoxic responsiveness is evinced by such cells upon sensitization in vitro when adherent (suppressor?) cells are first removed (Mokyr ec al.,1979; Glaser, 1980b),and even without such deletion (Civin er af., 1979).Occasionally,cultivation of lymphoid cells derived from animals that bear progressive growths, underwent regression, or had been immunized with inactivated tumor cells leads to an activation of CTL activity even in the absence of further stimulation by the neoplastic tissue in culture (de Landazuri and Herberman, 1972;Galili et af.,1978;Wiltrout and Frost, 1980). Studies conducted in our laboratories with several different syngeneic tumors of mice (Kedar et af.,1976, 1978b,c; E. Kedar et af.,unpublished observations, 1983; Yefenof et af.,1980b)have led to the following findings on responder cell behavior. Synergistic responsiveness can be attained when the responder cell populations are mixtures composed of suboptimum numbers of splenic or lymph node cells, and of thymocytes which by themselves do not undergo more than negligible sensitization. Splenocytes from tumor-bearing animals often, but not always, respond only poorly to in vitro tumor stimulation, as do the cells of tumor hosts that had been treated with high doses (180-250 mg/kg) of cyclophosphamide (CY). In contrast, spleen cells from animals treated recently with smaller amounts of CY

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(40-100 mg/kg), or that survived Winn tests with the corresponding tumor challenge, or that were cured of established disease by chemoadoptive immunotherapy, are capable of a stronger responsivenessin MLTC than are naive splenocytes.A markedly elevated response capacity is also exhibited by splenocytes of normal mice that had been given, weeks or even months earlier, syngeneic or allogeneic sensitized lymphoid cells alone. With weakly immunogenic tumors, in vitro sensitization is appreciably effective only where the responder cells derive from primed donors. The magnitude of the CTL response that can be produced in culture is determined, over a certain range, by the numbers of specific HTL-P and CTL-P in the responder population (Le., the proportional representation of specifically reactive clones among the test cells). The frequency of syngeneic antitumor CTL-P in the lymphoid tissues of normal mice has been found by some investigators to be considerably lower ( 1 : 10,000; MacDonald et af., 1980) than that of CTL-P directed at allogeneic normal or neoplastic cells; other workers, however, have reported an equivalent representation of the precursor cells ( 1 :2000; Warren and Lafferty, 1979).In tumor-bearing hosts, splenocyte CTL-P frequency has been seen to increase appreciably (10- to 20-fold) with the onset of spontaneous regression (MacDonald et af.,1980). The frequency of CTL-P has been reported to be 5 - 10 times greater among lymphocytes infiltrating neoplastic masses in regressor mice than among PBL and spleen cells of the same animal (Brunner et af.,198 1). This observation is compatible with a role of CTL in the host’s defense against (some) tumors, and points to the tumor infiltrate as a possibly superior source of responder cells for IVS (Vose, 1982). Other investigators have found lymphoid cells accumulating at tumor niduses to be actively cytotoxic against the neoplasm, both in experimental animal (DeLustro and Haskill, 1978; Herberman et al., 1980) and in human (Vose et af.,1977; E. Klein ez al., 1980) tumor systems, although their cytotoxic potency, before and after further sensitization in culture, is often lower than that of lymphoid cells drawn from the blood or distal tissues, perhaps in consequence of an in situ contamination with suppressor elements (Vose and Moore, 1979). The absolute as well as relative numbers of CTL-P in lymphoid tissues may fall sharply following exposure of the animal to large doses of chemotherapeutic agents or irradiation (MacDonald et al., 1980; E. Kedar et al., unpublished observations, 1983). This circumstance must be considered in timing the harvest of responder cells from cancer patients under therapy. Macrophages play an important role in the in vitro generation of CTL against tumor cells. Their presence in responder cell preparations at a low frequency ( 1 - 5%) is requisite for successful sensitization of purified lymphocyte populations; at an elevated frequency (more than lo%),in contrast, they can be suppressive (Wing and Remington, 1977; Mokyr and Dray, 1982). The likelihood of macrophage suppressive activity is especially great

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when the cells are prominent in responder tissue derived from animals with progressive neoplastic involvement; partial depletion of macrophages can restore responsiveness (Mokyr et al., 1979; Glaser, 1980b). It has also been reported that neoplastic cells are capable of “switching on” macrophagemediated suppressor mechanisms, thereby aborting CTL induction in culture (Ting and Rodrigues, 1980a,b). In some instances, even extensive removal of macrophages from responder cells of tumor-bearing hosts, mouse and human, is conducive to CTL generation provided that IL-2 is added to the sensitization cultures (J. Shaw et al., 1980; E. Kedar et al., unpublished observations, 1983). The strength of the CTL response that can be induced in syngeneic mouse responder cells is usually lower than that attainable with allogeneic lymphocytes against similar tumors (Wagner and Rollinghoff, 1973; Plata et al., 1975; Kedar et al., 1976; Maki and Howe, 1976). Even the lower syngeneic reactivity generated in culture often exceeds, however, the response mounted by syngeneic animals in vivo (Plata et al., 1975; Wagner et al., 1980c). In human-cell MLTC systems, autochthonous and allogeneic responsivenessdeveloping against the same, freshly isolated stimulating tumor is, in many instances, equivalent (Vinky et al., 1982; Kedar et al., unpublished observations). Secondary in vitro sensitization of responder cells that had been primed either in vitro or in vivo usually leads to a more rapid and stronger CTL response than does MLTC sensitization of naive lymphocytes (Bernstein et al., 1976; Plata et al., 1976; Kedar et al., 1977; Yefenof et al., 1980b). Lymphoid cells ofboth murine and human origin can be cryopreserved(in liquid nitrogen) for at least several months without compromising their responder capability in allogeneic MLC (Du Bois et al., 1976) and in both allogeneic and syngeneic MLTC (Kedar et al., 1977, 1979b). Similarly, prolonged cryopreservation of CTL produced in culture does not reduce their cytotoxic potency upon thawing and testing (Kedar et al., 1979b; see also Table HI). It must be noted, however, that retention of activity by responder and effector cells after freezing, preservation, and thawing is contingent on effectuation of the procedures under well-defined, appropriate conditions. c. Stimulator Cells. Tumor cells of diverse origin and kind can be employed for IVS. The cells are usually first inactivated, by means of mitomycin C (30- 100 pg/ml, for 30-60 min at 37°C) or irradiation ( 5 - 10,000 R), thereby making for a unidirectional reaction and eliminating tumor cell overgrowth and neutralizing activities in the culture. Excessive concentrations of mitomycin C (>100 pg/ml) and excessive irradiation (>10,000 R) may, however, lower stimulator capacity. In some instances, untreated neoplastic cells have been found superior to inactivated ones in inducing

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CTL generation (Sharma, 1976). Where CTL production in vitro is camed out in the presence of metabolically active tumor stimulator cells, these must obviously be removed before the resultant effectors can be employed therapeutically. The eventuality must also be considered that small numbers of tumor stimulator cells that have been subjected to inactivation procedures retain viability; rigorous removal of all stimulator cells is in order, accordingly, before effector elements are readied for clinical utilization. In human systems,solid tumor cells freshly obtained from biopsy material (Vose et a!., 1978a; Kedar et al., 1982c),fresh and cryopreserved leukemia cells (Zarling et al., 1976; Lee and Oliver, 1978; Kedar et al., 1979b), and tumor cell lines maintained by passage in culture (Golub and Morton, 1974; Sharma and Terasaki, 1974b; Treves et al., 1977) have proven effective in IVS with autochthonous and allogeneic lymphoid cells. In experimental animal systems, tumor stimulation in IVS has been with spontaneously arising (Maki and Monaco, 1980)and induced primary neoplasms (Yefenof et al., 1980b),and with lines of passaged (in vivo and in vitro) solid (Treves et af., 1975; Kedar et al., 1982c) and lymphoid (leukemias, lymphomas, mastocytomas, and plasmacytomas) tumors (Wagner et al., 1973; Burton et al., 1975;Plataetal., 1975; Kedaretal., 1976;Cheeveretal., 1977;Mokyret al., 1978). It is usually difficult to obtain adequate numbers of tumor cells from fresh biopsy material. Mechanical and enzymatic disruptions of such tissues are not only tedious, but also result frequently in low yields of viable cells and in their reduced immunogenic potency (E. Klein et al., 1980; Russell et a/., 1980). It may be possible, however, to elevate the stimulator potency of tumor cells from enzymatically treated tissues by incubating them for a time sufficient to allow for regeneration of membrane components (Vanky et al., 1978).Another difficultythat must be overcome in resort to biopsy tissuesfor antitumor CTL induction is the presence of irrelevant and interfering elements (connective tissue, cells with suppressor activity, etc.); enrichment of the desired neoplastic stimulator cells in such preparations can be achieved by a variety of fractionation techniques (Vose et al., 1977; E. Klein et al., 1980).A rapid, simple fractionation method that ensures high yields of viable cells has recently been described from our laboratories (Kedar et al., 1982c). Human or murine solid tumor fragments are exposed for 5 - 10 min to a mixture ofcollagenase, DNase, and trypsin, and then passaged through a tissue sieve. Centrifugation on discontinuous Percoll gradients yields a fraction rich in large tumor cells (plus some macrophages, removable by adherence on plastic surfaces), and one in which the smaller, tumor-infiltrating lymphoid cells are concentrated. More than half the estimated number of tumor cells in the starting specimen are recovered by this procedure; the final cell suspension obtained is highly enriched in tumor cells

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(90- 9590),with a viability of 80 - 90%as assessed by vital dye exclusion. Such preparations have been employed successfully in MLTC systems. Newly isolated tumor cells are often coated by immunoglobulins,immune complexes, and other substances which may mask immunogenic determinants or otherwise interfere with stimulator capacity (Vhnky el a/., 1978; Ran el al., 1980; von Kleist et al., 1980). Mild proteolytic treatment, incubation for some hours, or merely repeated washing can effect removal of such material from the cells. In some cases of neoplastic disease,large numbers of neoplastic cells can be harvested from ascitic or pleural effusions. Such cells, too, may have to be subjected to appropriate manipulation before they can be used to stimulate CTL responses. Considerations of logistics and convenience have led many investigators to resort to cultured lines of tumor cells rather than to freshly harvested tissue. In some instances, tumor cells that have been maintained in culture, and even for short periods of time, are in fact more powerfully immunogenic in vitro and in vivo than cells taken directly from the parent neoplasm developing in host tissues (Bekesi and Holland, 1977; Jamasbi and Nettesheim, 1977,1979;Liu et d.,1977;Devens et d.,1979b, 1981;B. Klein el d., 1981). However, many primary autochthonous growths cannot be established as continuous lines in culture; and where this is possible, loss, gain, and modulation of antigenic determinants in the course of passage and the accretion of other artifacts, as indicated in a preceding section, often counterindicate employment of long-passaged tumor cells for attempts at CTL generation. Even where such cells can incite CTL responsiveness, it cannot be taken for granted that the effector cells obtained shall prove capable of recognizing and attacking neoplasms of the parent type growing in vivo. Resort could conceivably be had to human tumor cells grown in athymic mice. The relative homogeneity of neoplastic cell populations from this source and their availability in appreciable numbers over prolonged periods are clearly attractive features. On the other hand, the highly artifactual circumstances of human tumor growth in a distant species may result in markedly deviant expression of TAA characteristics; neither can there be excluded the risks of tumor cell infection by microbial or viral agents pathogenic for the tumor host to be treated with the effector products. Another possibility lies with the employment of subcellular and soluble tumor preparations for stimulation of CTL responses in vitro.6A number of

The disclaimer must be made that cytotoxic reactivity generated in respondercell populations, by any stimulator preparation, is often ascribed to specific CTL without provision of evidence that rules out participation of nonspecifically cytotoxic effector cells.

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advantages can be envisaged for this approach. Any likelihood that the yield of therapeutically intended effector elements will be contaminated with living neoplastic cells is sharply reduced. Antigenic determinants on the membrane of tumor stimulator cells that are not otherwise accessible to responder lymphocytes may be exposed, or acquire more effective immunogenicity, in consequence of the disruption procedures (Fish, 1978). The responsiveness of CTL can be directed at isolated, purified TAA entities, thereby lessening the risk of generating cytotoxic effectors capable of inflicting damage to normal host tissues (Tomonari, 1980). Suppressogenicdeterminants can be removed selectively from dissociated tumor cell preparations, leaving only the actively immunogenic moieties for optimal CTL induction (Brandchaft et al., 1976; B. Klein ef al., 1981; Kobrin et al., 1981; Pellis et al., 1981). However, subcellular preparations of tumor, and of allogeneic normal, cells are frequently ineffective in eliciting primary CTL responses in culture. They are more often potent in secondary stimulation. Solubilized H-2 moieties have been found nearly as satisfactory as the intact allogeneic cells for the evocation of CTL in secondary cultures (Engers et al., 1975; Wagner et al., 1976). Several groups have accomplished secondary in vifro CTL production in experimental animal systems by stimulation with tumor cell fractions and oncogenic virus preparations, employing responder lymphocytes from donors primed with the intact tumor cells (Bruce et al., 1976; Alaba and Law, 1978, 1980; Taniyama and Holden, 1979a); others have used disrupted material for in vivo priming and elicited secondary responsiveness with the intact neoplastic cells in vitro (B. Klein ef al., 1980, 1981; Kobrin et al., 1981). In human systems as well, subcellular moieties made from fresh or cultured neoplastic cells have been efficaciousin inducing CTL formation in autochthonous patient, and in healthy donor, responder lymphocytes (Morales et al., 1977; Sharma, 1979). It may be that the lesser sensitizing potency of subcellular antigens in primary than in secondary induction systems lies with a more pronounced dependence of the former on HTL function, incited by determinant structures that are destroyed in the process of cell disruption. In that event, addition of IL-2 (and perhaps other lymphokines) to primary sensitization cultures could be compensatory, and permit the usage there of subcellular stimulator preparations. Presentations of solubilized antigenic material by means of liposomes or artificial vesicles, or attached to the surface of inert particulate matrices, could conceivably also heighten the determinants’ immunogenic potencies (Burakoff and Mescher, 1982). Macrophages appear to play an especially important role in CTL induction when specific stimulation is with subcellular material. Thus, it has been shown that murine T cells with cytostatic (Treves, 1978;Treves et al., 1979b)

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and cytotoxic (Hellstrom and Hellstrom, 1976b)effector capacities could be induced in primary sensitization cultures with cell-free extracts of syngeneic and allogeneic tumor cells by first “pulsing” macrophage monolayers with the preparations and then adding the responder lymphoid cells. Similar exposure of macrophages to RadLV has been employed as a step in the generation of T cells with cytostatic action for mouse lymphomatous cells infected with the virus. Macrophage pulsing with solubilized tumor fractions has also proven effective for the generation of cytostatic T cells against some human neoplasms (Treves, 1978). The possibility deserves mention, in passing, that T cells may be capable, more often than is commonly taken into account, of cytostatic antitumor action even where they display little ifany cytotoxicefficacyin (short-term) in vitro assays. Such delimited reactivity against neoplastic cells may materialize in certain concatenations of circumstances- nature of responder and stimulator cells and of the sensitization milieu (Steinitz and Weiss, unpublished observations)-and it may play a significant role in the host’s defense against progressive neoplasia. As indicated in preceding sections, normal cells can also, on occasion, serve as stimulators of CTL with reactivity manifested against tumor targets. Thus, responder lymphocytes from normal donors and from tumor-bearing hosts, both mouse and human, have been shown to develop strong syngeneic/autochthonous antitumor cytotoxicity following sensitization in culture with pooled or single-donor lymphoid cells (J. M. Zarling et al., 1978a, 1981a; Bach et al., 1980; Strausser et al., 1981; VAnky et al., 1981). Monoclonal mouse CTL lines originating in lymphoid cell populations that had been stimulated in culture with allogeneic splenocytes frequently manifest cytotoxicity for tumors syngeneic to the responder/effector population (Bach et al., 1982). In some instances, induction of strong antitumor CTL activity in autochthonous human MLTC could be effected only by costimulation with normal allogeneic PBL (Zarling et al., 1976; Lee and Oliver, 1978; Gangel et al., 1980). Polyclonal activation of responder cells in the course of sensitization and the expression of shared or cross-reactive determinants between normal stimulator and neoplastic target cells have been proposed in explanation. Cryopreserved murine and human leukemia cells (Kedar et al., 1977, 1979b)as well as cells of human solid neoplasms (Kedar et al., unpublished observations) serve as efficiently to stimulate CTL generation in autochthonous, syngeneic, and allogeneic MLTC as do fresh stimulator cells. A potential logistic difficulty is thus resolved: large numbers of neoplastic cells can be drawn from patients in acute phases of malignant disease, stored at low temperatures, and employed for CTL induction as needed. d. Culture Conditions. The culture conditions that have been employed

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for the production of antitumor CTL are discussed in several recent articles (Burton et al., 1975, 1978; Engers and MacDonald, 1976; Sharma, 1976; Mokyr and Dray, 1982), and the subject is reviewed here only briefly. Culture vessels of various sizes and shapes have been used, including microtiter plates (Bruce ef a/., 1976), round-bottom culture tubes (Kedar et al., 1976), culture trays (Burton et al., 1975), Petri dishes (Golub and Morton, 1974), Marbrook flasks (Wagner and Rollinghoff, 1973),and other kinds of tissue culture flasks of up to 1 liter capacity and more (Plata et al., 1975; Kedar e? al., 1977, 1978b; Mokyr et al., 1978). Optimum culture volume depends on container size and shape. With large vessels, intermittent manual agitation can improve sensitization.Vessel suitability may vary with the type of tumor toward which CTL induction is essayed. Thus, we have found small-capacity flat-bottom vessels (e.g., 24-well Costar plates) superior to round-bottom tubes with some solid growths, whereas no appreciable difference was noted in the case of lymphoid neoplasms. Yields for CTL are usually higher per unit volume for flask than for tube cultures. For each type of culture vessel, optimum conditions of cell density, responder/stimulator cell (R/S) ratio, and culture volume should be ascertained empirically; the optimum parameters may vary for different responder- stimulator cell combinations. The media most commonly utilized are Dulbecco’s modified Eagle’s medium(DMEM)(Plata etal., 1975, 1976),RPMI 1640(Kedare?al., 1976), and EHAA (or, Click’s) medium (Click ef al., 1972; Peck and Bach, 1975) which contains nucleic acid precursors, Dulbecco’s modified Eagle’s medium and RPMI 1640 medium are usually supplemented with heat-inactivated FCS (for rodent MLTC) or human serum (for human MLTC), and EHAA with fresh mouse serum (for mouse MLTC). Addition to the media of larger than standard amounts of nonessential amino acids, folic acid, and sodium pyruvate tends to improve responder cell viability and CTL yields. HEPES buffer, 10-20 mM, is frequently used. 2-Mercaptoethanol (1 X 1 1 X 10-4 M ) has been shown to potentiate responder cell survival and sensitization efficacy in rodent systems (Cerottini et al., 1974; Burton e?al., 1975; Kedar et al., 1976);the effect may accrue from neutralization of toxic materials in the culture by the reducing agent, or from its mitogenic (Goodman and Weigle, 1979) and differentiation-promotingeffects (Harris et al., 1976) on T lymphocytes. In our laboratories, a beneficial action by 2-mercaptoethanol could not be detected in human MLTC (E. Kedar et al., unpublished observations. 1982). In short-term cultures (up to 7 days), fresh medium addition or replacement is not usually required except where mouse serum is a supplement, in which case introduction of fresh serum-containing medium every 2 - 3 days is recommended (Kedar et a!., unpublished observations).

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Serum is the most important supplement for MLTC systems, and heat-inactivated FCS the one most widely employed (at concentrations of 2 - 15%) with rodent cells. It appears desirable to test individual batches of FCS for satisfactory performance in syngeneic MLTC, even if they have proven adequate in allogeneic MLTC and MLR systems (E. Kedar et al., unpublished observations, 1982). Because FCS has been incriminated in the evocation of nonspecific cytotoxic, as well as of suppressor, reactivities, it has come to be replaced by mouse serum (0.5- 1.OYo) (Peck et al., 1977;Fogel et al., 1978; Dorfman and Wunderlich, 1980). For similar reasons, the serum supplement of choice in human MLTC is human serum (fresh or inactivated, either pooled AB or autochthonous, 10- 20%); however, autochthonous normal human serum has been found to elicit nonspecific cytotoxic activity in autochthonous MLR cultures (Tomonari and Aizawa, 1979),and autochthonous patient serum inhibitory effects on CTL generation in autochthonous MLTC (Vanky et al., 1978). The inimical influence of cancer patient sera may not be unexpected in light of the observations that neoplastic cells are capable of producing a variety of toxic and immune-neutralizing factors in the host organism (Israel and Edelstein, 1978). Responder cell density and R/S ratio are important variables, the optima falling within wide ranges. Usually favorable responder cell concentrations are 0.5 -2.0 X lo6cells/ml in microcultures, plate or tube, and up to 4 X lo6 cells/ml in macrocultures. Optimally efficacious R/S ratios must be determined for each system: they vary from 2: 1 to 500: 1 for leukemias, lymphomas, and plasmacytomas and from 2 : 1 to 10,000: 1 for solid tumors (Burton et al., 1978; E. Kedar et al., unpublished observations, 1982). Propitious R/S ratios are, on the whole, lower for secondary than for primary induction cultures (MacDonald, 1978), and they may differ markedly in autochthonous/syngeneic and allogeneic MLTC (Wagner and Rollinghoff, 1973) even where the same responder or stimulator cells are used. Sensitization cultures are incubated upright at 37°C in a humidified atmosphere of 5 3 % COz in air. Incubation is stationary (occasional, manual agitation for short periods is opportune in macrocultures) for up to 8 days in the primary sensitization system and for shorter periods in secondary cultures. The CTL response peaks on days 5 -8 in syngeneic murine MLTC and 1 - 2 days sooner where the responder cells are allogeneic (Wagner and Rollinghoff, 1973; Kedar et af., 1976). Maximum CTL responsiveness is reached at approximately similar times in autochthonous and allogeneic human MLTC (Sharma, 1976; Vose et al., 1978a; Kedar et al., 1979b, 1982~).When primary sensitization cultures are maintained for prolonged periods (in which case, replacement of part of the old medium with fresh is necessary every 5 - 7 days), CTL activity usually disappears after I2 - 25

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days; renewed stimulation with the sensitizing TAA preparation leads to a secondary response peak within 2 -4 days (Kedar et al., 1977). The recovery of viable responder lymphoid cells from primary murine MLTC on days 6-8 is 30- 50% of the initial number when the medium is supplemented with FCS and 15- 35% when supplemented with syngeneic mouse serum; in human PBL sensitization cultures supplemented with autochthonous or pooled AB sera, the recovery is often 50-200% (E. Kedar et al., unpublished observations, 1982). In general, viable lymphoid cell recovery is greater for secondary than for primary MLTC, in both murine and human systems. 5. Facilitation ofAntitumor CTL Induction

a. General Considerations. The failure of tumors to evoke protective immune responses does not of necessity lie with an absence of TAA. Immunogenically inert neoplasms may often express distinguishing antigens that can serve as targets for selective immunologic attack, but that are incapable of initiating the reactions (Greenberg et al., 1981a). Inability to provoke defensive reactivity-and this has been seen not only for growths arising spontaneously in experimental animals, but also for some intentionally induced (Baldwin, 1973; Baldwin and Price, 1976; Yefenof et al., 1980a,b)-may be for want of a particular property of TAA. For example, some tumors appear to be well capable of impelling specific CTL-P activity while lacking the faculty to energize HTL-P, presumably because of an inadequacy of determinants that function in a manner analogous to that of “LAD” alloantigens (Warren and Lafferty, 1979). Evidence is at hand that intrinsic immunogenic paucity of tumor cells can be overcome by modifying the relevant antigens of stimulator preparations employed in vitro, and by resorting to needed lymphokines; the aim of all such efforts is to achieve the generation of specific CTL with potency as well for the native, unmodified neoplastic variants. Even where tumor immunogenicity is as such sufficient, corresponding responsivenessmay be hampered by regulating mechanisms which function to restrict the extent ofimmunologic reactivity (Basten et al., 1980;Gershon, I980), and perhaps especially toward antigens related to normal-self epitopes. Thus, the responder and effector capability of lymphoid cells from donors with progressive malignant disease is often severely confined by suppressor cells, both T lymphocytes and macrophages, that may contribute to the progression of disease or come to the fore in its course (Martin et al., 1973;Gorczynski, 1974; Eggers and Wunderlich, 1975;Glaser et al., 1976b; Fujimotoet al., 1976;TrevesetaL 1976;Broderetal., 1978;Kirchner, 1978; J. G. Levy et al., 1979; Naor, 1979; Greene, 1980; Yefenof et al., 1980a;

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Herberman, 1981; Plater et a/., I98 1). Exclusion of suppressor elements from in vitro sensitization systems can improve markedly the production of CTL, and it is not inconceivable that the reactive cells then obtained can effect tumor inhibition when introduced in large numbers to the host even against a gradient of suppressor activity within the organism. In addition to the activities of different suppressor cells, a variety of other factors, of tumor or of host origin, specific and nonspecific, have been held responsible for inhibiting the generation or expression of antitumor immunity; these include antibody, TAA, and viral entities shed from the surface of living neoplastic cells or released at their death, antigen- antibody complexes, enzymes that inactivate cytotoxic immunologic elements, and stillundefined substances inimical to immunocyte viability or functionality (Hellstrom and Hellstrom, 1974; Bonnard et al., 1976; Davey et al., 1976; Kamo and Friedman, 1977;Price and Baldwin, 1977;Witz, 1977;Israel and Edelstein, 1978; Ting et al., 1979b; Doljansky, 1982). Attention must therefore be given to the exclusion of such elements from MLTC systems. Where serum from tumor-bearing hosts is to be employed as a medium supplement, contamination with inhibitory substances must be ruled out or their removal effected. Responder cells of similar source may have to be precultured so as to allow for depletion ofany such entities, and TAA-stimulating preparations must be examined for their presence. Where living neoplastic cells are used for stimulation, resort might be had to sublines that do not liberate interfering materials or do so to a lesser extent (Bonnard et al., 1976). Such measures may facilitate the production of CTL; the large question remaining is whether the effector cells now obtainable can function therapeutically in the environment ofhost tissues where the inhibitory agents may be in constant production (Dye and North, 1981). Inability to respond immunologically to TAA can be the manifestation of individual genetic dyscrasia. Thus, a deficiency in requisite MHC-linked “Ir gene” products, now thought to be the Ia (i.e., Class 11)-bearingmolecules that play a pivotal role in antigen presentation (J. Klein et al., 198 I), could explain states of idiosyncratic unresponsiveness to autochthonous/syngeneic neoplasms (Burton et al., 1978; Knowles et al., 1979; Greene, 1980; Wagner et al., 1980~).The genetic flaw could also represent situations “in which two H-2 molecules become involved in the response: one controls the generation ofhelper T cellsand the other ofsuppressor T cells. The latter then act on the former and suppress the response,” the interpretation of “Is” gene action now proposed by J. Klein et al. (198 I ). In such a circumstance, the evocation of specificsuppressor activity is unavoidable, set perforce from the outset of sensitization by nature of the associative antigenic stimulus. Another possibility is the occurrence in the T-cell receptor repertoire of “blind spots,” or “holes,” for certain antigen - self-MHC constellations

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(Nagy et al., 198 1). By modification-alteration of the relevant tumor cell constituents, it might prove feasible to create antigenic structures for which recognition molecules are extant in the T lymphoid complement, and which can incite active CTL generation in culture. Circumvention of defects in intrinsic response capability, elevation and modulation of responsivenessto poorly immunogenic TAA, and disembarrassment of the sensitization system from extrinsic interference are thus major tasks in the effectuation of CTL manufacture. Some of the approaches that have been taken to these ends are discussed in brief in the succeeding paragraphs; the experience gained in our laboratories is summarized in Table I. b. Tumor Cell Modijication. Various approaches to modification of tumor cells aimed at heightening their immunogenicity have been reviewed (Prager and Baechtel, 1973; Naor and Galili, 1977; Weiss, 1980). Studies conducted by us (Kedar and Lupu, 1978) revealed that CTL induced in vitro by lymphoid tumor cells that had been freshly obtained from a camer animal and appropriately modified generally manifest the same reaction specificities as do CTL incited by the corresponding unmodified stimulators. As seen from Table I, modification by certain chemicals and enzymes raises CTL responsivenessappreciably. The potentiation effect is, on the whole, similar when the cells are subjected to the procedures before, at the time of, or after inactivation with mitomycin C. Working with established tumor lines, Eggers et al. ( 1980) also reported a markedly strengthened immunogenicity of chemically modified stimulator cells, but found the cytotoxic effector cells generated to be reactive against a heterogeneous spectrum of target cells. The same modifying agent often has very different effects on different neoplastic cells, even where these are ofrelated type (Kedar and Lupu, 1978). The conditions of manipulation that are most efficacious also vary from tumor to tumor; under unfavorable conditions, modification can reduce stimulating potency. These observations support the proposition that the TAA of even cognate neoplasms possess distinguishing features. Tumor cells isolated from mice treated with tl?e chemotherapeutic agent DTIC have been shown to be more powerfully immunogenic, in vivo and in vitro, than those taken from untreated animals (Fioretti et al., 1980; Giampietri et al., 1981); this may be one case in point of a therapeutic impact of such drugs distinct from any straightforward toxicity for the target neoplasm. Chemical and enzymatic manipulation of neoplastic cells can be aniticipated to bring about direct changes in the composition and arrangement of existing cell surface antigenic determinants, an exposure of cryptic epitopes, or the creation of new antigenicities by haptenization. Such alterations may variously elevate the immunogenic strength of tumor cells for the auto-

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TABLE I METHODSEMPLOYED I N OURLABORATORIES FOR AUGMENTING THE INDUCTION AND EXPRESSION OF CTL GENERATED I N MLTC" MLTC system Method and optimal conditions Modification of stimulator tumor cells 2,4,6-Trinitrobenzenesulfonicacid (TNBS) M , 30 min, 37°C) M , 30 min, Iodoacetamide ( 37°C) Trypsin, papain (0.I - 1 mg/ml, 30 min, 37°C) Bromelain (0.I - I mg/ml, 30 min, 37" C) Edwn'chia co/i colicin (1 -4 units/ml, 30 min, 37°C)' Treatment of responder cell donor Cyclophosphamide (50- 100 mg/kg, 10 to 2 days before cell harvesting) Hydrocortisone succinate (2.5 mg, 10 to 2 days before cell harvesting) Manipulation of responder cells Depletion of nylon-adherent cells Depletion of Fc receptor-bearing cells Hydrocortisone succinate (

lo-* M )

Trypsin, papain (0.01-0.1 mg/ml, 30 min, 37°C) Neuraminidase (10-30 units/ml, 30 min, 37°C) Addition of nonspecific microbial immunomodulating agents to sensitization cultures MERd(0.5-2 pg/ml)

Muramyl dipeptidec (10- 100 pg/ml)

Tumor cells

Responder Amplification splenocytes index (range)*

RBL5; EL4

C57BL/6

1.8-6.7

RBLS; EL4

C57BL/6

1.5-4.2

RBL5; EL4 YAC EL4 YAC

C57BL/6 A C57BL/6 A

1.7-5.5 2.2-6.3 1.5-3.4

RBL5; EL4 YAC RBLS; EL4 YAC

C57BL/6 A C57BL/6 A

2.0-4.8

RBL5; EL4 YAC RBLS; EL4 YAC RBLS; EL4 YAC RBLS; EL4 YAC

C57BLJ6 A C57BL/6 A C57BL/6 A C57BL/6 A

I .8 - 3.9

EL4

C57BL/6

1.5-2.8

YAC RBLS: PXT Allogeneic leukemia (AML) Autochthonous lung carcinoma RBLS; EL4 YAC

A C57BL/6 Human PBL

2.2-6.4

Human PBL

2.0-6.2

C57BL/6

1.9-4.5

1.7-5.5

1.6- 3.5 2.3-6.6 1.7-2.9

1.8-3.7

A

(continued)

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TABLE I(continued) MLTC system Method and optimal conditions Soluble extracts ofBCG and Listerra monocytogenes (5-20 pg N/mly Manipulation of sensitized effector cells Papain, trypsin, bromelain (0.001-0.01 mg/ml. 30 min, 37°C) Separation of effector cell population on discontinuous BSA gradients and utilitzation of most active subpopulations (fractions 20-24%)

Tumor cells

Responder splenocytes

Amplification index (range)b

RBLS: EL4 YAC

C57BL/6 A

1.7-3.6

RBL5: EL4 YAC

C57BL/6

1.8-3.3

A

MLTC for 6 days, in tubes or small flasks, employing R/S ratios of 5 : 1-50: 1. Modification ofstimulator cells (10-20 X lo6cells/ml) performed prior to, during, or after mitomycin C inactivation. In the mouse systems, the responder cells were syngeneic splenocytes. The murine stimulator tumor cells were the following lymphomas: EL4, induced by DMBA in a C57BL/6 mouse, long passaged in vivo; RBL5, Rauscher virus induced in a C57BL/6 mouse, long passaged in vivo; PXT, radiation induced in a C57BL/6 mouse, long passaged in v i m ; YAC, Moloney virus induced in a Strain A mouse, long passaged In vivo. Cytotoxic activity of effector cells assessed by in vitro T r release (4 hr) assay. using effector/target ratios ranging from 1 : I to 30: 1. Cytotoxic activity expressed as lytic units (LU)/ lo6cells in effector population (see Section II,A,6,a for description of LU). Amplification index denotes ratio of LU obtained under amplification conditions/LU obtained in the same system under standard conditions. The values show the amplification index range based on three to six repeat experiments for each test system. ‘ Eschtrichia coli colicin obtained from medium of E. coli cultures (Farkas-Himsley and Cheung, 1976). MER, Methanol extraction residue of inactivated tubercle bacilli (Weiss, 1976). Muramyl dipeptide. a synthetic adjuvant with the structure of a mycobacterial cell wall component (Chedid et a/.. 1978). Kindly provided by Dr. C. Damais, Pasteur Institute, Pans. Soluble extracts of BCG and L. monocytogenes(Sharma et al., 1977). Kindly provided by Dr. P. Minden, National Jewish Hospital, Denver. rl

chthonous/syngeneic host, and the efficacyof immune defenses (Mitchison, 1970;Weiss, 1980):The stimulatory properties ofTAA, inherently low in the native neoplastic cell, may be potentiated in consequence of chemical or steric innovation, the forming of novel polymolecular associative-presentation structures, or the shaping of new camer-hapten effects; the new or bettered immune responses now engendered by the modified cells may have a recognition spillover for the native neoplasm in the host. The addition of new antigens to the stimulator cell profile might incite activation of corresponding HTLP, and the resultant lymphokine production facilitate responsiveness of CTL-P specifically directed at native TAA. Modification of the tumor cell could lead to a “breaking” of specificimmunologic unresponsiveness, including such as prevents reactivity against determinants closely

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related to, or identical with, normal self entities. In that event, CTL attack on a tumor might be viewed as a classic autoimmune phenomenon, but one hopefully of sufficient selectivity- by virtue of the variable expression of antigen on normal and on neoplastic cells, and/or an exaggerated susceptibility to attack by the latter-to enable tumor inhibition at an only tolerable cost to normal host tissues. Two other approaches to modification of tumor stimulator cells which have shown some promise in experimental systems are viral heterogenization and somatic cell hybridization. Infection of neoplastic cells with extrinsic viruses (Kobayashi et al., 1977, 1978)can add a variety of new antigenic determinants-virion, virus coded, virus derepressed-and cause changes in the cells’ existing antigenic topography; so can addition to the stimulator cells of allo- or organ-specific antigens by cell hybridization (G. Klein, 1977; Klein and Klein, 1979;Yefenof et al., 1982)or by liposome insertion into the cell membrane (Hapel et al., 1980b; Rutzky et al., 1982). c. Treatment of Responder Cell Donors. Suppressor cell functions in the intact organism can be reduced by different means (Broder et al., 1978;Naor, 1979), and such intervention can elevate the responder capacity in vitro of lymphocytes derived at appropriate times after donor treatment. We found that splenocytes and mesenteric lymph node cells of mice that had been given 2.5 mg hydrocortisone succinate or 50- 100 mg/kg CY develop a level of cytotoxic effector reactivity in syngeneic MLTC severalfold greater than that induced in responder populations from untreated subjects. The effect was evident for cells harvested 2- 10 days, but not 30 days, after donor treatment, and was treatment dose dependent. When the amount of CY was raised to 200 mg/kg, a marked reduction in splenocyte cytotoxic responsiveness took place; this was also the case when the donors were subjected to whole-body X irradiation ( 1 25 or 400 R) (Kedar et al., I979a). Magnification of CTL responsiveness in v i m against chemically modified normal and against syngeneic neoplastic cells has been noted by other workers for responder cells taken, respectively, from normal and tumor-bearing animals that had been treated with small quantities of CY (Rollinghoff et al., 1977; Glaser, 1979~;Hengst et al., 1980), adriamycin (Orsini et al., 1977; Ehrke et al., 1981), or hydrocortisone (Scheehter and Feldman, 1977).The usual interpretation of such findings is that suppressor cells, or their precursors, are more susceptible to limiting concentrations of certain immunosuppressive agents than are HTL and CTL and/or their precursors (Ferguson and Simmons, 1978; Bradley and Mishell, 1981). The observations also suggest another, “incidental” but perhaps important, modality of therapeutic action of drugs commonly employed in the management of cancer patients. Further appraisal would seem in order of the responder capability of

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lymphoid cells from tumor hosts that had been subjected to other manipulations of apparent resistance benefit. Thus, for instance, plasma exchange (Israel and Edelstein, 1978)and plasma perfusion over immobilized staphylococcal protein A (Terman et al., 1981) have been ascribed therapeutic value in patients with advanced malignant disease; the mode of action ofthe effects reported is unclear, but it is possible that it entails, in part, removal of subcellular inhibitory factors. Lymphoid cells of patients so relieved, temporarily, of their inimical influence might serve as more opportune responders for the generation of CTL. d. Manipulations of Responder and Eflector Cell Populations. In experiments conducted by us (Kedar et al., 1979a),splenocytes from normal mice were subjected to cell separation procedures or exposed to enzymes, before or after MLTC. Removal of nylon-adherent and Fc receptor-bearing cells prior to sensitization increased cytotoxic responsiveness of the remaining responder lymphocytes; no such effect was seen when the selective depletion was performed after 6 days of sensitization. Heightened CTL production following a similar processing of responder cells has also been reported by Treves et al. (1977, 1978, 1979a) in a human MLTC system; Mokyr ef al. ( 1979) recorded analogous observations for splenocytes of tumor-bearing mice upon removal, prior to sensitization, of glass-adherent cells. The augmentation so effected may result from a depopulation of suppressor cells or their precursors; suppressor cell activation occurs not uncommonly in the course oflymphoid cell culture and sensitization (Kedar et al., 1978d).There may also be other explanations for the effects on responder capacity that follow a reapportionment of cell types in the lymphoid population. That selective impoverishment of suppressor cells from responder mixtures can positively influence sensitization efficacy is indicated by other observations. We have found that addition to murine MLTC of hydrocortiM) can magnify syngeneic CTL responsiveness sone succinate ( two- to sixfold or more (Kedar et al., 1979a)(Table I). The stimulatory effect was evident for both spleen and lymph node responder cells; the effector cells produced had the same target specificities as those generated in ordinary MLTC. Increment in cytotoxic responsiveness was accompanied by a 60- 80%cell loss at term of sensitization culture (6 days), the surviving cells being predominantly large lymphoblasts. The potentiating action of the steroid hormone is thought to accrue from elimination of susceptible suppressor elements (especially of suppressor cell precursors) (Nachtigal ef al., 1975; Schechter and Feldman, 1977). It could also be accounted for, however, by the enrichment of specificantigen-reactive T cells in the culture, the steroid enhancing Signal 1-incited proliferation while damaging clones not reactive for the sensitizing antigens and therefore not poised to proliferative activity (Stavy et af., 1974). Removal of suppressor cell precursors

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and/or enrichment of relevant antigen-reactive cells have also been suggested as mechanisms of the stimulatory action of adriamycin in munne MLTC (Orsini et al., 1977; Ehrke et al., 1978, 1981) and of the cyclophosphamide derivative 4HP-CY in murine MLTC and human MLR(Cowens et al., 1981; Ozer et al., 1982; Smith et al., 1982). Other approaches could be taken to the exclusion of potential suppressor cells from responder cell mixtures. Among these are cell separation by density or velocity sedimentation (Small and Trainin, 1976), removal of subsets bearing receptors for histamine (Schechter et al., 1978),and elimination, by specific antisera and complement, of cells expressing determinants described as products of the “I-J” locus of the mouse MHC (Fujimoto et al., 1978; J. G . Levy et al., 1979; Greene, 1980). Exposure to neuraminidase or to low concentrations of proteolytic enzymes can potentiate sensitizability of responder cells, and the cytotoxic potency of effector populations after sensitization (Kedar et al., 1979a). Suggested explanations have been the exposure, topographic rearrangement, and facilitated mobility of receptor structures on the surface of the cells, and their enhanced metabolic activity or proliferation. Of the several efforts we have mounted to invigoration of the reactivity of (already sensitized)effector populations, the most effective has been concentration of the large-lymphoblast component by density sedimentation on discontinuous BSA gradients (Kedar et al., 1979a).These cells, collecting at the upper layers of the gradient (20-24% BSA), display a cytotoxic activity 5 - 19 times greater (per unit number of cells) than that evinced by the unseparated parent effector population (Table I). e. Nonspecific Biological Response Modifiers of Microbial Origin.It has been known for many years that a heterogeneous variety of microbial substances can modulate immunologic responsiveness in vivo, quantitatively and qualitatively, to seemingly unrelated antigens. Many such “nonspecific” microbial immunomodulators also exert appreciable influence on antibody and CMI responsiveness in in vitro systems. Their modalities of action may be diverse, even for the same agent (Weiss, 1979, 1980, 1983), including a dimension of specificity due to tumor cell modification by the BRM or to an (unrecognized) sharing of epitopes between it and the test antigenic entity. And the excitation of NK and NK-like cells effected by many BRM (Henney el al., 1978; Herberman et al., 1979)enforces caution in attempts to define their loci of impact. Therapeutic application of BRM to the patient entails the possible risks ensuant on any generalizeddigression from normal immunologic equilibria; it is also accompanied not infrequently by systemic or local toxicity. We turned our attention, accordingly, to the utilization of microbial BRM in sensitization cultures, with the aim of achieving maximum potentiation of

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CTL responsiveness at no hazard to the donor supplying the responder cells or to be treated immunologically. The substance most extensively examined by us is the methanol extraction residue (MER) fraction of nonliving tubercle bacilli (Weiss, 1976, 1983). Addition of MER to murine and human MLTC during the first 24 hr has appreciable consequence for the generation of cytotoxic responsiveness (Kedar el al., 1978a, 1979a,b). Small amounts of the agent (0.5-5.0pg/ml for murine MLTC; 0.25 - 2.0pg/ml for human MLTC) are strongly stimulatory, whereas quantities exceeding 10 pg/ml often proved markedly suppressive, even when the concentration was still below the threshold of gross toxicity for the responder cells. Incubation of responder cells with MER in the absence of tumor stimulator cells induces a level of cytotoxic responsiveness of the same order as that generated in syngeneic MLTC not exposed to MER; the cytotoxic reactivity resulting from incubation with MER alone is manifested against diverse tumor target cells, both syngeneic and allogeneic to the MER-stimulated lymphocytes, and against allogeneic, but not syngeneic, normal cells (mitogen-induced lymphoblasts). It appears that the stimulatory action of MER in MLTC, i.e., in the presence oftumor stimulator cells, represents the cumulative result of activation to seemingly nonspecific cytotoxicity and of amplification of the specific sensitization process (Kedar et al., 1978a).Murine splenocyteswere stimulated to a similar extent by MER in MLTC before and after removal of nylon wool-adherent cells, indicating a direct effect of the agent on lymphocytes. The synthetic adjuvant muramyl dipeptide and soluble extracts obtained from M . tuberculosis,BCG, and Listeria monocytogenesdisplay a potentiating effect in MLTC similar to that ascertained for MER (Table I). Levamisole, in contrast, proved only marginally stimulatory (at concentrations of 2- 10 pg/rnl) (Kedar et al., unpublished observations). Other investigators have also reported a potentiating influence in mouse and human MLTC for certain strains of BCG, other microbial entities, and poly(A-U) (Bernstein et al., 1979; Sharma and Odom, 1979; Alaba, 1980; Mokyr et al., 1980; Sharma, 1980). On the other hand, BRM can provoke suppressor function, in vilro as well as in vivo (Scott, 1972; Klimpel and Henney, 1978; Kendall and Sabbadini, 1981), and the same agent can accomplish immunologic activation in opposite directions under different experimental circumstances (Ben-Efraim el al., 1973). Microbial substances have so far occupied the center ofattention in efforts to potentiate cytotoxic responsiveness by means of BRM, but agents of other origin may also prove applicable. For instance, the hydrophilic polymer polyethylene glycol (PEG; MW 6000) has been shown to enhance lymphocyte proliferation in MLR and MLTC (Ben-Sasson and Henkart, 1977). We

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have found PEG capable of a significant amplification of both specific and nonspecificcytotoxic responsiveness in murine MLTC systems when added in concentrations of 1 -2Yo during the first 24 hr (Kedar et al., 1979a);PEG potentiation of cytotoxic reactivity in murine MLTC has also been recorded by others (Przepiorka et al., 1980). Availability in the future of microbial, and other, BRM with a defined bearing on the immunologic mechanism may bring this arm of cytotoxicity amplification to greater prominence (see also Sections II1,B and IV,B,4). J: Concluding Comments. Many of the methods employed for amplification of CTL (and other cytotoxic effector cell) responsiveness are most effective when MLTC is carried out at other than optimal R/S ratios, or otherwiseunder suboptimum circumstances. A limited availability oftumor stimulator and lymphoid responder cells of patient origin may often make for conditions far from ideal for the production of CTL to be used therapeutically, and it is in such situations that resort to various modalities of potentiation may be most telling. We have found, moreover, that recourse to several different techniques in the same system can lead to a cumulative magnification of reactivity, reaching levels of up to two orders of magnitude greater than those attainable under standard conditions (Kedar et al., 1979a). To what extent each form of manipulation serves to increase the numbers of cytotoxic effector cells, to elevate the cytotoxic potency and efficacy of each, and to free potentially reactive cells from suppressor and blocking influences requires further analysis.

6. Assay Systems for CTL Reactivity against Tumor Cells Correct assessment ofthe cytotoxic reactivity of effectorcell populations is possible only by careful regard to the methodology of testing, The various techniques in use may each reveal different aspects of the total cytotoxic capacity of an effector preparation. Reliance on a single assay is likely to furnish an only partial, and perhaps skewed, definition of potency; attainment of an accurate, inclusive view may necessitate examination by several means, under varied conditions of testing. Choice of the assays most appropriate must also be with reference to each type of target tissue. a. In Vitro Assays. The tests most frequently used are, with some modifications, the short-term (3 - 6 hr) s*Crrelease assay developed by Brunner et al. (1968, 1976; Bean et al., 1976a; Hirschberg et al., 1977) and the long-term’ ( 18 - 72 hr) microcytotoxicityassay of Takasugi and Klein ( 1970; Bean et al., 1976b; Oldham and Herberman, 1976). It is noted that the period oftime required for performanceof cytotoxicity assays, short and long term, is not necessarily indicative of the time that elapses between effector-target contact and initiation ofan irreversible chain ofpathologic changes in the target cell. The crucial interval

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Short-term assessment of isotope release from prelabeled target cells is the method particularly apt with nonadherent targets that are rapidly susceptible to immunologic injury. The test measures severe, assumedly irreversible, damage inflicted by attacking effector elements, i.e., cytotoxicity in the Sense of cytolysis. Commonly employed with cells of lymphoreticular origin, normal and neoplastic, the chromium release assay (sometimes also run long term) has been applied successfullyas well to certain solid tumor target cells that are adherent to the test vessel surface or are brought into suspension (Brunneret al., 1976;Brooks, 1978;Tameriusetal., 1978;Voseetal., 1978a; Dorfman et al., 1980). In some instances, neoplastic cells do not lend themselves as targets to chromium liberation assay, because of their limited uptake of the label or because of high rates of spontaneous label release; this is seen not infrequently for freshly obtained tumors. In such event, an estimation of effector cytolytic reactivity can be obtained by modifying the test to a competitive inhibition method, using antigenically related or cross-reactive cells that can be labeled as targets and those not suitably labeled as untagged blocking cells (Herberman et al., 1976; Chism et al., 1977; Yefenof et al., 1980b). We have recently effected changes in the Y r assay that have improved its Tumor cells (mouse and human, lymphoid and utility (Kedar et al., 1982~). carcinoma, freshly obtained and grown in culture) are chromated in a large volume of medium for 4 - 18 hr (instead of the standard I hr). After washing, the labeled cells are held for 2 - 4 hr at room temperature, and then washed again before employment in the test. All washings are with medium containing 5 - 10% serum. A considerably greater incorporation of the isotope is effected, especially by solid tumor cells, permitting assay with as few as 200 target cells. Spontaneous release of chromium by cells so labeled is relatively low (10-25%), even when incubation with test effector cells is long term (1 6 -24 hr). Isotopes other than W r (e.g., 1Z51UdR,[3H]proline, LIIIn)have been employed in tests based on target cell label release, and for both short- and long-term exposure to the effectorelements (Bean et al., 1976b;Oldham and Herberman, 1976; Brooks, 1978; Wiltrout et a!., 1978). The cytotoxic (cytolytic)potential of effector cell populations is estimated by determining the number of effector cells required for a given level of label release. The percentage values of specific, i.e., above background, release are may be very short (Martz, 1977; Berke, 1980), far briefer than the standard conduct of the test. The long test period may be in order to facilitate the incidence and intimacy ofcell interaction, and the progression to conspicuity of pathogenetic events triggered early, that enable a maximum disclosure of effector capacity. Even in long-term assays, however, presence of the effector elements may not be requisite after the initial phase in which injury is set.

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taken as the percentage values of specifically effected target cell destruction (lysis). By reference to a dose - response curve (percentage specific label release plotted against graded effector/target cell ratios on a logarithmic scale), the numbers of test effector cells needed to attain a given value of specificrelease from a given number of targets are ascertained (Cerottini and Brunner, 1974). The estimate of effector reactivity is expressed as lytic units (LU) per effector preparation or per unit number of cells in the preparation. For syngeneic tumor systems, we have defined as 1 LU the number of effector cells that cause 33% lysis of 2 X lo4 5*Cr-labeledtarget cells in 4 hr (Kedar et al., 1976). A more direct estimation of cytolytic effector cell numbers is afforded by the plaque assay of Bonavida et al. ( 1976)in which lytic plaques formed by effector cells seeded onto target monolayers are counted visually, and by the counting of single, killed target cells in effector- target cell conjugates that have been plated in a semisolid agar matrix (Grimm and Bonavida, 1979). This methodology enables evaluation of the cytolytic potency of single effector cells, i.e., the tempo at which target cells manifest the injury caused and the numbers of target cells destroyed per unit time. The Takasugi- Klein microcytotoxicity test, in which the numbers of monolayer target cells remaining viable (adherent) to the walls of microplate wells are counted under a microscope after incubation with effector cells, is appropriate for assessment of effector action against neoplastic cells that are adherent and that manifest inflicted damage only after some time. In common usage with solid tumortarget cells, the test providesan indication of cytostatic as well as of cytodestructive effector activities, cumulatively where both are exerted. Estimation of the numbers of cells surviving contact with the effectors has been simplified by terminal radioactive labeling of the adherent cell mass and resort to a standard calibration curve relating the radioactivity counts so obtained to known cell number (More et al., 1975; Schechter et al., 1976; Tamerius et al., 1978). Appraisement of effector reactivity by numeration of target cells surviving after prolonged exposure can be complicated by extraneous factors. Inimical culture conditions, unrelated to effector cell presence, may impinge on target cell survival; some target cells, on the other hand, may be able to proliferate during the test period. The cell number remaining at term may thus reflect the balance of effects elicited specifically by the effector cells examined -cell death, growth inhibition, and in some instances perhaps growth stimulation (Kall and Hellstrom, 1975; Prehn, 1977)-and by the culture environment as such, positive and deleterious, and conceivably both, to different target cell subpopulations. Secondary events of an immunologic nature may also transpire, including specific, de novo activation of additional CTL precursor and memory cells in the effector preparation and lymphokine excitation of

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other cells (NK, NK-like, macrophages) to nonspecific cytotoxicity. Some of these obstacles to a correct determination of the capacities of effector preparations also impede tests that measure target cell label release; where such assays involve long-term interaction between prelabeled targets and effectors, the additional eventualities of isotope toxicity and reutilization must also be taken into account. Other methods of effector cell evaluation are based on the detection of changes in target cell metabolism, occurring very rapidly after contact with the effectors and not necessarily leading to early cell death (Steinitz and Weiss, 1975; Steinitz et al., 1975). The nature and degree of effector cell sensitization can influence the character of their impact on target tissue. The gauging of specific CTL reactivity, by any test, is especially prone to error where the target cells have been maintained in culture. Attractive experimental models because of their homogeneity, such cells are subject not only to fluctuating changes in antigenic and other characteristics, but are often also very susceptible to nonspecific cytotoxic reactivity (Vanky et al., 1980). Tumors freshly harvested from the host, on the other hand, are more likely to yield cell populations that are heterogeneous in make-up, unstable in test culture, refractory to uptake and retention of isotopic labels, and contaminated with interfering factors of host origin (Perlmann and Cerottini, 1979). The choosing of an assay methodology and interpretation of the results must be, accordingly, with full account of the nature ofthe interacting cells, the diverse potencies of effector populations, and the limitations and artifacts inherent in each distinct test system. b. In Vivo Assays. The most relevant test ofeffector cell efficacy is, clearly, their ability to retard or abrogate already established progressive neoplastic disease in the living organism. Such assessment (discussed in Section IV), however, is cumbersome, imprecise, and time consuming. Resort is usually had, therefore, to a simpler methodology, the challenge protection, or neutralization, assays developed by Klein and Sjogren (1960) and Winn (196 I), which represent something of a compromise between in vitro appraisal and therapeutic evaluation. These tests are performed by mixing numbers of living tumor cells sufficient to initiate progressive disease with graded numbers of test effector cells, and injecting the mixtures, at once or after very brief incubation, into animals not previously exposed to the neoplasm. Injection may be by different routes- subcutaneous (sc), intramuscular (im), into the footpad (fp), intraperitoneal (ip), and intraveous (iv) (Rouse et al., 1972; Small and Trainin, 1975; Kedar et af., 1977, 1978~).The expression of effector cell protective capacity, judged by the occurrence and/or pace of tumor development from the inoculum, varies with its route of introduction, the ip and iv usually representing a much more acid test.

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The test animals may be normal or have been subjected to immunosuppressive pretreatment, usually sublethal X irradiation. In the latter event, it is possible to uti€ize animals that are allogeneic to the neoplastic and/or effector cells (Cohen et al., 1971b; Freedman et al., 1972; Rollinghoff and Wagner, 1973). Athymic nude mice are coming into increasing use as “hosts” for human tumor cells and for the assay of effector cells reactive against them (Kedar et al., 19820. In a variation of the Winn assay, 1251UdR-labeled tumor cells are inoculated into the footpad of mice, with or without effector cells, and the radioactivity emitted at the site followed for several days or weeks (Gorelik and Herberman, 1981; Gorelik et al., 1981). Where tumor growth takes place, radioactivity remains high for some time, whereas effective attack on the neoplastic cells is manifested by a sharp and rapid decrease. In another modification of the method, effector cells are injected systemically shortly before or after sc, im, or fp tumor challenge, and the fate of the local neoplastic inoculum followed visually over a period of time (Rouse et al., 1972, 1973;Small and Trainin, 1975;Burton and Warner, 1977;Treves, 1978). Local injection of mixed tumor-effector cell suspensions can have different consequence than separate introduction of each cell type by different route; thus, for example, Small and Trainin (1 975) reported that whereas inoculation of such a mixture led to enhanced tumor development in situ (the foot pad of mice), some retardation of growth of a fp tumor inoculum was effected when the same sensitized lymphoid cells were administered iv. Estimation of effector cell capacity in Winn-type assays is complicated by at least some participation of the test animal in responsiveness against the tumor inoculum. Effector cells often exhibit reduced capacities in Winn assays where the test animals have been irradiated (Zarling and Tevethia, 1973; Simes et al., 1975; Alaba and Bernstein, 1978). It must be assumed, nonetheless, that even experimentally immunosuppressed and athymic animals can offer some defense. The resistance functions mounted by the host in Winn test and similar protection assay situations are in part independent of the actions of the adoptively introduced effectorcells; they may also, however, reflect a collaborative endeavor, the test effector cells or subcellular entities acting to incite or recruit various host immunocytes (macrophages and/or T cells) to reactivity (Treves and Cohen, 1973;Zarling and Tevethia, 1973;Cohen and Livnat, 1976;Alaba and Bernstein, 1978;Fish et al., 1979; Ting et al., 1979a; Treves et al., 1979b; Scuderi and Rosse, 1981a,b). Effector cells displaying pronounced cytotoxic capacity in vitro not infrequently fail to afford protection in in vivo neutralization tests. It has been noted that effectorcells generated in primary MLTC are at times incapable of neutralizing a tumor inoculum decisively in vivo even though they display good cytotoxic reactivity in vitro, whereas lymphoid cells that had been

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derived from preimmunized or regressor donors and then undergone secondary stimulation in culture are potent in Winn-type tests (Bernstein, 1977; Cheever et al., 1977; Kedar et al., 1978c; Hellstrom ef al., 1979). In other instances, the reverse situation pertains, protective reactivity in vivo in the absence of discernible cytotoxicity in vitro. For that matter, correlation is often also lacking between Winn-type and true therapeutic assays, and between different techniques of in vitro estimation (Herberman, 1974; Howell et al., 1974; Engers and MacDonald, 1976; Glaser et al., 1976c; Matter and Askonas, 1976; Bernstein, 1977; Burton and Warner, 1977; Bartlett et al., 1978; Glaser, 1978; Fish et al., 1979; Hellstrom et al., 1979; Miller and Heppner, 1979;Robins et al., 1979;Cheever et al., 198la; Mokyr and Dray, 1982). The circumstance of host participation in in vivo test systems intended to reveal the capacities of extrinsic, presensitized effector cells is one likely explanation for the frequent faiIure at correlation between in vitroand in vivo assessmentsof CTL (and other effector) potency. A case in point is suggested by recent findings coming from adoptive transfer studies in mice and rats. Sensitized T cells of Lyt I+ (or Lyt 1+2+3+)(mouse) and W3/25+ (rat) type which manifest low, or no, cytolytic capacity in vitro can be effective in the rejection of syngeneic tumor (and allogeneic skin) grafts, and at times more so than Lyt 2+3+(mouse) and W3/25-, OX8+ (rat) CTL which are reactive in vitro (Fernandez-Cruz et al., 1980, 1982; Greene, 1980; Leclerc and Cantor, 1980b; Bhan ef al., 1981; Greenberg et al., 1981b; Loveland et al., 1981; Loveland and McKenzie, 1982a,b). It could be that amplifier activity effected by certain lymphocytes acting as HTL serves to bring into play indigenous host lymphocytes to more pronounced and/or durable CTL reactivity, or that interactions in vivo between the introduced cells and autochthonous immunocytes lead to the evolution ofrejection responsesof a different nature (perhaps of delayed hypersensitivity type) than those that may be transacted by direct CTL attack. The observation that preformed CTL are sometimes highly effective in Winn-type neutralization tests but only limitedly active, or not at all, when tested by adoptive transfer against established implants of the same tumor suggests that target cell-CTL contact is indeed not the only, and not necessarily the most efficacious, modality of the organism’s defense against alien tissue. Other explanations suggest themselves for the lack of test correlation. Cytotoxic reactivity in vitro may be directed at TAA that do not function effectively, if at all, as rejection antigens in vivo (e.g., embryonic antigens) (Baldwin and Robins, 1979). The demands on effector cell “viability” and normal behavior may be more stringent for the expression of antitumor efficacy in vivo, especially where disease is already in progress, than in vitro (Fefer et al., 1976; Greenberg et al., 1980). It has been shown that effector

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cells exposed to X irradiation, mitomycin C, and other agents that inhibit at least some physiologic functions can retain the facility for target destruction in vitro (Steinitz et af.,1975; Martz, 1977) while no longer being able to display it in the milieu of host tissues (Rouse et al., 1973; Zarling and Tevethia, 1973; Fish et al., 1979; Greenberg et al., 1979; Eberlein et af., 1982a). Altered “homing” patterns and a life span too short to permit expression of defensive potential in vivo bear likely responsibility for the therapeutic impotence (Rouse and Wagner, 1973; Burton and Warner, 1977; Kedar et af.,1978b; Lotze et af.,1980a). The eventuality must also be considered that effector cell preparations may include suppressor subsets that come to expression only at in vivo scrutiny (Small and Trainin, 1975; Treves et af.,1976; Greenberg et af., 1979; Schechter and Feldman, 1979). On the other hand, closely correlative results have been obtained by some investigatorsfrom parallel in vitro and in vivoevaluationsof the same effector preparation, against the same targets (Rollinghoffand Wagner, 1973; Rouse et al., 1973; Kedar et al., 1978b,c;Fernandez-Cruz et al., 1979). An interesting observation made repeatedly in our laboratories (Kedar et al., 1977, 1978b,c)is indicative of this test concurrence: Most mice that had survived Winn assays conducted some months previously in which MLTC-produced effector cells, either allogeneic or syngeneic, were employed to neutralize the neoplastic challenge, proved solidly resistant to rechallenge with massive implants of the same neoplasm. It was then found that splenocytes taken from long-term Winn assay survivors that had not been rechallenged, or from mice that had received the same allogeneic or syngeneic cytotoxic effector cells but that had not been subjected to any tumor challenge, could be sensitized in MLTC to a level of cytotoxicity categorically higher than usual, as assessed either in vitro(short-term W r release) or by Winn tests(i.e., complete protection of a high proportion of new test animals even against ip and iv injection of the test inoculum). It was also found that the therapeutic efficacy of effector cells was greater when these were generated in MLTC by splenocytes taken from donors that had survived a previous Winn assay, or that had been given an effector cell implant only. Correlations between in vitro and in vivo assessments of effector efficacy were noted by us as well in experiments in which lymphoid cells were cryopreserved before or after IVS (Kedar et al., unpublished, 1983). The attainment of such correspondence is of large importance in the strategy of immunologic intervention in patients. Concerted effort is in order toward establishment of germane tests that can yield information predictive of the therapeutic efficacy of reagents made in culture, even in the event not unlikely- that different test batteries may have to be developed for different clinical situations.

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B. OTHER EFFECTOR LYMPHOCYTES The central topics of this article are the induction and behavior of specifically reactive cytotoxic T lymphocytes. It is evident, however, that other lymphoid effector cells are produced as well in the course of intended CTL generation in vifro.Their coactivation complicates the interpretation of MLTC experiments and characterization of classic CTL. Some such other effector cells inhibit the expression of potential CTL responsiveness; others, of possible therapeutic interest in their own right, overshadow the activities of CTL or are mistakenly denominated as such. Some reference to these additional effectors is, accordingly, necessary to a discussion centered on CTL. 1. Nonspecifically Reactive Cyfotoxic Lymphocytes The designations “specific” and “nonspecific” are often employed imprecisely, without clear distinction between a speciJicifyof mechanism accruing from molecular analogies between interacting entities (molecular identity, similarity, or complementarity) and the scope of cause and expression of effects (Weiss, 1983).In an immunologic context, “specificity” should apply to reactions based on molecular (antigen - receptor) relationships, regardless of the breadth of distribution of the cognate structures, and “nonspecificity” to reactions not involving such analogies. Commonly, however, broadly manifested immunologic transactions are connoted as nonspecific even without ruling out the possibility that the phenomena are based on a molecular relationship of relevant but widely occumng determinants. In dissecting the modalities of effector cell action, the ready possibility of a misreading of specificityexpressed in diverse systems for nonspecificity must be kept in mind. Experiments conducted in many laboratories have shown that MLR and MLTC sensitization systems, animal and human, often lead to the generation not only of CTL specifically reactive to the inciting tissue, but also of lymphocytes cytotoxic for a wide array of normal and neoplastic cells, autochthonous, syngeneic, allogeneic, and occasionally xenogeneic to the effector population, and bearing no evident antigenic relationship to the stimulator cells (Kristensen ef al., 1974; Svedmyr ef GI., 1974b; Butterworth and Franks, 1975; Martin-Chandon ef al., 1975; Callewaert ef al., 1978; Jondal and Targan, 1978;Seeley and Golub, 1978;Treves el al., 1978;Karre and Seeley, 1979; Masucci ef al., 1980; Paciucci ef al., 1980; Bolhuis and Schellekens, 1981; Strausser el al., 1981; Vinky ef al., 1981; Zarling ef al., 1981b). Moreover, similarly “nonspecific” cytotoxic reactivity can be detected in lymphoid cell populations cultured alone, in the absence of any intended antigenic stimulation (Bevan ef al., 1974; Shustik et al., 1976;

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Zielske and Golub, 1976; Peck et al., 1977; Burton et al., 1978; Golstein et al., 1978; R. B. Levy et al., 1979; Ortaldo et al., 1979; Bartlett and Burton, 1982). Such “nonspecific” cytotoxicity has been designated variously “culture-induced cytotoxicity,” “promiscuous cytotoxicity,” “spontaneous killing,” “anomalous killing,” “activated lymphocyte killing,” and others. A number of distinct mechanisms may be envisaged for the broad reactivity. For one, CTL with specificityfor the inciting antigens can interact with any target cell that happens to share that antigenicity or to express related epitopes, no matter how distantly removed that cell may be phylogenetically. Second, specific antigenic excitation, giving rise to the production of IL-2, IFN,and other mediators, can result in the “transstimulation” (Augustin et al., 1979)of CTL-P clones that lack receptors for the stimulator antigens (Klein and Viinky, 1981). Third, foreign serum in the culture medium (e.g., FCS) can bring about a polyclonal activation of CTL subsets, each with its own specificity, even in the absence of any stimulator entities, for several reasons: the serum may contain antigens (embryonic, linked to stages of cell differentation, and other) that are also expressed by a heterogeneous variety of cells, and that are thus apt targets for the CTL generated in the culture; the serum may effect modifications in effector and/or stimulator cell membrane structures, and thereby cause new, specific sensitization circumstances; and it may act as a general mitogen (Zielske and Golub, 1976; Peck et al., 1977; R. B. Levy et al., 1979). And broadly manifested cytotoxicity appearing in sensitization cultures may be due not to the extended activities of classic, specific CTL, but rather to lymphocytes of NK or NK-like genre. “Natural killer” and “natural cytotoxic” (NK, NC) cells have indeed been ascribed a major role in culture-generated “nonspecific” CMI, the former reacting prominently against lymphoid neoplasms, the latter against anchorage-dependent solid tumor cells. It is beyond the scope of this article to discuss in detail this family of lymphoid effectors, to which much attention has been given in recent years. The reader is referred to a number of recent articles on the subject (Kiessling and Haller, 1978; Herberman et al., 1979; Herberman, l980,1982;HerbermanandOrtaldo,1981;Burtonetal., 1981; E. Klein, 1981, 1982; Roder et al., 1981; Stutman et al., 1980b, 1981). Consideration is here given, in brief, to only several aspects of NK- NC cells of particular relevance to the article’s central theme. There are several salient, distinguishing properties of NK-NC cells. Their cytotoxic action is expressed toward a heterogeneous variety of tumor cells, especially those passaged in culture, without evident specificity of antigenic recognition, and toward certain nonneoplastic cells (especially embryonic/ fetal tissue, virus-infected cells, lymphoblasts, some macrophages, and cer-

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tain cells of thymus and bone marrow). NK-NC activities do not appear to follow laws of MHC restriction, nor do they suggest the operation of classic immunologic memory. The cells are present in appreciable numbers in “normal,” unprimed organisms, and they differ, on the whole, from mature cells of well-defined T, B, and macrophage lineage. NK-NC cells are highly heterogeneous with regard to cell surface markers, range of cytotoxic reactivity, and susceptibility to activation by various agents. This variegation makes it difficult to trace their lines of descent. Some cells display markers that are associated with mature T lymphocytes (e.g., receptors for SRBC, and the OKT 10 determinant for human cells; Thy 1.2 and Lyt 2 and 5 for mouse) and/or with monocytes and polymorphonuclear leukocytes (e.g., OKM 1 for human cells, and asialoGM 1 for both human and mouse). Some express antigens not usually found on other immunocytes, e.g., NK 1 and 2 in the mouse and HNK- 1 (or, Leu 7) in man. The majority of NK-like cells, both human and rodent, show prominent azurophilic cytoplasmic granules, and have been categorized as large granular lymphocytes (LGL) (Timonen et al., 1981). NK-NC cells, human and mouse, can be propagated in culture for prolonged periods by means of IL-2; they can also produce the lymphokine, and they are activated by it (Dennert, 1980b; Dennert et af.,1981b; Herberman and Ortaldo, 1981; Minato et af.,1981; Nabel et al., 1981; Kuribayashi et af.,1981; Domzig and Stadler, 1982; Grimm et al., 1982a; Kedar and Herberman, 1982; Ortaldo et al., 1982; Timonen et af.,1982). These and other properties indicate that at least some of the subsets defined operationally as NK-NC should be considered as closely related to the classic T lymphocyte (Vanky and Klein, 1982a,b), perhaps as cells in the prethymic phase, or representing postthymic stages, preceding or succeeding that identified as the CTL. NK- NC-like cells tend to reach peak concentrations in sensitization cultures earlier than the typical CTL (Seeley and Golub, 1978; Karre and Seeley, 1979), more frequently express receptors for the Fc portion of IgG, and show greater “stickiness” for various surfaces(E. Klein, 1981; Bolhuis et af.,1982). Also in contradistinction to classic CTL, the appearance of NK-like cells in culture may not be dependent on cell division (Callewaert el al., 1978;Zarlinget al., 198la). Their generation in vitro can take place in the presence of the immunosuppressive agent cyclosporin A, which prevents that ofCTL (Landegren et al., 1981). A considerable proportion ofNK-NC cells and their precursors can be separated from CTL and CTL-P by Percoll density gradient centrifugation (Kedar et af.,1982f; Vose and Bonnard, 1982). NK-NC cells arising (activated) in culture also seem to differ from those

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freshly isolated from tissues, with regard to membrane characteristics and cytotoxic reactivity; the former are more potently cytotoxic, and act on a broader spectrum of sensitive target cells (Bolhuis, 1980; E. Klein, 1981; Klein and Vinky, 1981; Bartlett and Burton, 1982; Bolhuis et al., 1982; Kedar et af.,1982b; V h k y and Klein, 1982a). It is uncertain to what extent the more pronounced activities of culture-induced NK- NC cells derive from a facilitated expansion and/or activation of subsets with such features normally active in the lymphoid population, or from a culture-favored differentiation of unique subsets that do not come to expression in viva Interferon, produced in the course of MLR and MLTC, is a potent activator of NK - NC cell activity developing in sensitization cultures (Kirchner ef al., 1979; Perussia et al., 1980;Kuribayashi el af.,1981). Tumor cells, especially if infected with viruses or mycoplasma, encourage IFN production (Trinchieri ef al., 1978; Beck et al., 1980; Cole ef af.,1981; Djeu et af.,1981, 1982), a circumstance that must be taken into account in the design and analysis of MLTC systems. The target structures at which NK -NC reactivity is directed have not been defined. Major histocompatibility complex and viral entities have been generally discounted, but recently reported findings do support the role of MHC antigens as recognition sites of at least some NK-like cells (Vinky et al., 1980;Viinky and Klein, 1982a,b).High-molecular-weight glycoproteins (Roder et al., 1979), and neutral sugars in glycoprotein or glycolipid complexes (Stutman et al., 1980a), have also been proposed as recognition determinants. Experiments in which effector cell populations are subjected to cold-target competition analyses, and to differential adsorption on target monolayers, have led some observers to posit a limited range of antigenic specificities for NK- NC action, widely distributed and variously expressed by diverse target cells, and of distinct subsets of the killer cells, each confined to the corresponding target (Phillips el a/., 1980; Ortaldo and Herberman, 1982).Other investigators, working with either noncloned or cloned effector populations, have not found evidence persuasive of an NK-NC receptor monoclonality, and tend rather to the view that the different cells susceptible to NK-NC action (and which may share discrete target antigens) are recognized by effector subsets each equipped with nonclonally distributed, multiple receptors (Kiessling and Wigzell, 1979; Dennert ef al., 1981b; Kedar et al., 1982e). Effector cells of NK - NC type have been attributed an important function in resistance against neoplastic growth and metastatic spread, perhaps indeed as a line of first defense (Hanna and Burton, 1981; Herberman and Ortaldo, 1981; Roder et al., 1981; Serrate et al., 1982). If this impression is confirmed, even in part, it might be possible to include, besides classic CTL, highly activated nonspecific killer lymphoid cells (preferentiallygenerated in

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culture under the appropriate conditions) in the armament for adoptive cellular immunotherapy (Kedar and Herberman, 1982). 2. Suppressor Cells (SPC) Cells with immunologic suppressor activities are activated in the course of murine and human MLR and MLTC and are capable of inhibiting the induction of proliferative and cytotoxic responses in culture (Rich and Rich, 1974; Fitch et al., 1976; Hirano and Nordin, 1976a,b; Sinclair et al., 1976; Hirschbergand Thorsby, 1977;Hodeseral., 1977;Schechteret al., 1978;Tse and Dutton, 1978;Treves et a].,1978, 1979a; Al-Adra et al., 1980; Sasportes et al., 1980).Cells with suppressor activity also arise in lymphoid cultures not undergoing (intentional) sensitization, especially where foreign serum is used as supplement; these suppressor cells affect antibody (Burns et al., 1975; Janeway et al., 1975; Parish, 1977) as well as CMI (Hodes and Hathcock, 1976; Ferguson et al., 1978;Kedar et al., 1978d)responsivenesswhen added to the relevant test systems. Culture-induced SPC have been characterized by most investigators as predominantly T lymphocytes, some adherent and others nonadherent, of Lyt 1+,Lyt 1+2+,or Lyt 2+ type in the mouse, and of Leu 2+, OKT 5+8+type in man. In murine sensitization cultures, the SPC that appear early (after 2 - 3 days) are relatively radioresistant and antigen specific; another set of SPC comes to the fore somewhat later, consisting of cells that are radiosensitive and antigen nonspecific. Some workers, however, consider the population of nonspecific SPC appearing in sensitization and in ordinary (i.e., not specifically stimulated) cultures to be largely non-T, macrophage-like cells (Treves et al., 1978, 1979a; Rollwagen and Stutman, 1981). The formation of suppressor cells in cultures intended to yield cytotoxic effector cells can jeopardize their therapeutic applicability. Even where cytotoxic capacity is clearly produced, a concurrent accretion of suppressor elements may make the preparation unsuitable. Thus, the accelerated tumor development that has been noted at times in experimental animals following joint implantation of in vitro-generated CTL and neoplastic challenge cells has been imputed in part to the presence in the effector preparation of SPC (Ilfeld et al., 1973; Small and Trainin, 1975;Schechter and Feldman, 1979). Experiments in our laboratories also indicate that mouse splenocytes cultured alone frequently promote tumor growth when injected to test animals together with the neoplastic challenge inoculum (Kedar et al., 1977). Greenberg et al. ( 1979)reported that nonstimulated, cultured spleen cells of mice can impede the therapeutic action of CTL when introduced together to tumor-bearing animals. Suppressor cells must be regarded, accordingly, not only as a likely source of failure at CTL generation and assessment, but as an

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important risk factor in the utilization of any lymphoid cells that had been maintained in vitro. The properties of murine SPC appearing in culture have come under extensive study; the reader is referred to several recent articles on the subject (Ferguson et al., 1978; Kedar et al., 1978d;Kedar and Schwartzbach, 1979; Rollwagen and Stutman, 1981). It remains to be determined whether SPC activity that becomes manifest in culture is primarily due to an expansion of already existing subsets of cells with such function, or to a differentiation/ maturation process of corresponding precursor cells. The mode(s) of SPC action also requires elucidation. Different mechanisms have been proposed, on the basis of in vitro studies. It may be that suppression is effected 'by virtue of SPC cytotoxocity for responder (Sinclair et al., 1976, 198lb) and/or stimulator (Fitch et ul., 1976; Sugarbaker and Matthews, 1981) cells, expressed specifically (i.e., SPC acting as CTL) or nonspecifically (SPC behaving as NK-NC cells). Alternatively, suppression may result from a cytostatic modality, SPC or certain of their products (for instance, prostaglandins) inhibiting the proliferation and/or differentiation of responder T lymphoid populations (Al-Adra and Pilarski, 1978; Rode et al., 1978; Webb and Nowowiejski, 1978; Novogrodsky et al,, 1979; Burton and Russell, 1981; h u n g and Mihich, 1981); it has been suggested that effects centered on IL-2 may be pivotal in genesis of the inhibition -reduced production of the lymphokine, its removal from the system, or unresponsiveness to it by relevant T lymphocytes (Maca et ul., 1979; Degiovanni and Schaaf-Lafontaine, 1981;Palacios and Moller, 1981; Kramer and Koszinowski, 1982). Suppression of CMI has been ascribed not only to intact SPC, but also to soluble factors, antigen specific and nonspecific, produced by them in culture (Engleman et al., 1978;Truitt et al., 1978; Kedar and Schwartzbach, 1979;Waksman, 1979;Beckwith and Rich, 1982;Kramer and Koszinowski, 1982). Several possibilities present themselves for reducing the dimension of nonspecific, and perhaps also of specific, suppressor activity in MLTC (Kedar and Schwartzbach, 1979):Depletion of macrophages from responder populations prior to sensitization lowers directly the numbers of one type of cell that can function as nonspecific SPC, and may also serve to interfere with any development of T suppressor cells that depends on macrophage help. Replacement of heterologous by homologous serum often constrains the emergence of suppressor reactivity in lymphoid cultures. Addition of small amounts of the MER tubercle bacillus fraction has similarly been shown effective. Some immunomodulators are capable both of facilitating and of restricting suppressor function in cultures and in vivo, depending on the concentration of the agent employed and on other experimental variables;

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under defined conditions, a highly selective impact on suppressor activity can be achieved (Zimber et al., unpublished observations, 1982). As discussed in Sections II,A,S,c and d, a variety ofdrugs, e.g., hydrocortisone and 4HP-CY, can also bring about a focused inactivation of SPC precursors. Suppressor cells that do appear in the course of MLTC may be removable by various means, e g , by specific antisera and complement (anti-I-J in the mouse) and by physical means of cell separation. Establishment of lymphoid clones with explicit HTL and CTL capacities can lead to availability of defined lymphocyte preparations devoid of any suppressor function. It must be emphasized, however, that failure to detect SPC sets within a lymphoid population by in vitro assays does not necessarily guarantee the absence of cells capable of causing immunologic suppression in vivo; the eventuality of their occurrence in effector preparations intended therapeutically is a ubiquitous concern. 111. T-cell Growth Factor: IL-2

Discovery of a lymphokine, IL-2, which enables the continued growth and numerical expansion in culture of functional T lymphocytes has afforded an important tool to immunology (Morgan et af., 1976; Gillis and Watson, 1981;Ruscetti and Gallo, 1981;Smith and Ruscetti, 1981;Rosenberg et al., 1982a,b).Some of the properties of IL-2 and its role in CTL generation have been discussed in preceding sections. Attention will be focused in the following paragraphs on certain aspects of IL-2 production, and on the usages of the agent in the potentiated induction, propagation, and in vivo employment of antitumor effector T cells. A. PRODUCTION OF IL-2

Crude preparations of 1L-2 were at first obtained by stimulating human PBL with PHA (Morgan et al., 1976; Ruscetti et al., 1977; Alvarez et a/., 1979;Bonnard et al., 1980)and mouse and rat splenocyteswith Con A (Gillis et af., 1978b; Rosenberg et al., 1978a). More expeditious procedures have been developed recently for producing human IL-2 in large amounts from tonsillar and splenic tissue (Kurnick et al., 1979; A. Moretta et af., 1981; Robb and Smith, 1981;Warren and Pembrey, 1981). Further improvement in production has come with the availability of T-cell leukemia and hybridoma lines (mouse, nonhuman primate, and human) which generate IL-2, constitutively or upon mitogen stimulation, to yields manyfold greater than those obtained from ordinary lymphoid cell suspensions, and with less of an admixture of other lymphokines (Farrar et af.,1980a; Gillis et af., 1980; Harwell et al., 1980; Shimizu et af.,1980;Gillis and Watson, 1981;Gooten-

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bergetal., 1981;Kappleretal., 1981;Okadaet al., 1981; Rabinetal., 1981; Schrader and Clark-Lewis, 1981;Stull and Gillis, 1981;Altman el al., 1982; Friedman et al., 1982). Efforts are now under way to apply techniques of genetic engineering toward a still more effective manufacture of the substance (Bleackley et al., 1981; Efrat et al., 1982; Gillis et al., 1982). Although initial T-cell responsiveness to IL-2 is conditioned on a prior stimulation by mitogen or specific antigen, subsequent propagation of T lymphoid populations deriving from progenitor cells that had undergone the primary excitation is effected by the lymphokine alone. [There is some indication of a direct, albeit low, mitogenic action of IL-2 itself on T cells taken from normal mice, not intentionally subjected to any other stimulus (Bodeker et al., 1980; Granelli-Piperno et al., 1981).] Interleukin-2 is assayed, accordingly, by quantifying its growth-stimulatory activity (viz. labeled thymidine uptake; direct estimation of cell numbers with time) for dependent T lymphocyte lines (Gillis et al., 1978b; Ruscetti and Gallo, 1981). The titers of IL-2 preparations obtained from cultures of normal human PBL can be raised by PHA stimulation of cells pooled from several donors, introduction of B lymphoid cell lines to the cultures, exposure ofthe cultures to limited X irradiation (1000-2000 R), and partial removal of adherent cells (Bonnard et al., 1980). The tumor promoter phorbol myristate acetate (PMA) has also been found an amplifier of IL-2 production (Efrat et al., 1982; Farrar et al., 1982). We have employed the following protocol in our laboratories for largescale production of IL-2 in high titers ( 10 -20 units/ml; stimulation index of 500 - 1500, as shown by thymidine incorporation of dependent T cell lines; cell doubling time of 20-24 hr): Rat IL-2 (for the propagation of murine T cells) is made by stimulating splenocytes (4 - 7 X 106/ml)pooled from 10 or more donor animals (ofthe W/Fu, Lewis, or Fischer strains, using cells ofany one strain, or 1 : 1 mixtures derived from two strains) with Con A ( 5 - 7 pg/ml where the medium concentration of FCS is 10Y0, I -2 pg/ml where FCS concentration is 1% or no FCS is used) for 24-48 hr. Human IL-2 (for propagating human T cells) is made by stimulating tonsillar lymphoid cells pooled from two to four donors(4 X 106/ml)with PHA-P (Difco),0.2 -0.5%, for 20-24 hr. The yields of IL-2 can be nearly doubled by harvesting the stimulation-culture supernatants after 24 hr and restimulating the cells with fresh mitogen, in fresh medium, for another 24 hr. Addition of PMA (10 ng/ml) increases the yield two- to threefold. In our hands, rat IL-2 proved superior to the mouse product for growing mouse T cells, and tonsillar IL-2 more potent than PBL-derived factor for human cells. Crude preparations of IL-2 are likely to contain appreciable amounts of the stimulating mitogen and of a variety of other mediators (e.g., IL-1;

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macrophage migration inhibitory factor, M I F macrophage-activating factor, MAF; colony-stimulating factor, CSF; immune IFN; B cell growth factor: IL-3; T cell replacing factor, T R F suppressor factors). Lectin contamination can affect adversely the survival and functionality of cultured T cells (Alvarez et al., 1981; Lotze and Rosenberg, 1981); the presence of other lymphokines and monokines may conflict with the action of IL-2 and/or becloud the interpretation of findings. Purification of IL-2 is, accordingly, desirable and often requisite. Mitogen-freepreparations of IL-2 can be obtained by limiting exposure of the producer cells to a short mitogen pulse (2-4 hr), followed by extensive washing, and by subjecting the IL-2-containing supernatants to differential (NH,),SO, precipitation andfor gel filtration. PHA can also be absorbed out by means of anti-PHA antibodies, thyroglobulin, and chicken or human erythrocytes (Kurnick et al., 1979; Fagnani and Braatz, 1980; Rosenberg et al., 1980b; Alvarez et al., 1981; Kahle ef al., 1981; Lotze and Rosenberg, 1981; Spiess and Rosenberg, 1981). Removal of other lymphokines and monokines from IL-2 is effected by a variety ofbiochemical techniques (Mier and Gallo, 1980, 1982;Mochizuki et al., 1980;Granelli-Piperno efal., 1981; Clark-Lewis and Schrader, 1982; Farrar et al., 1982; Gillis et al., 1982). OF ANTITUMOR CYTOTOXIC B. AMPLIFICATION RESPONSIVENESS IN MLTC

One possible cause for the incapacity of tumor cells to induce autochthonous/syngeneic CTL responsiveness can be their paucity in determinants required to stimulate HTL-P and lymphokine production. A low stimulator activity by murine and human tumor cells in vifrocan be overcome at times by adding to the sensitization cultures marginal amounts of mitogens (Warnatz and Scheiffarth, 1974) or the supernatants of lymphoid cultures that had undergone MLR or mitogen activation (Talmage et al., 1977;Baker et al., 1978; Ryser et al., 1979; Warren and Lafferty, 1979; Bertoglio ef al., 1980;Chapuis ef al., 1980;Mills and Paetkau, 1980;Burton and Plate, 1981 ; Gillis and Watson, 1981 ;Grimm efal., 198 I ;Scott and Finke, 1981;Kedar et al., 1982a,f). The augmented CTL responsiveness has been seen with autochthonous, syngeneic, and allogeneic responder cells, derived from normal donors and from donors with active neoplastic involvement or in remission. In these studies, the stimulating media preparations were either crude or partially purified, and identity of the responsible agents accordingly uncertain. Working with MLTC systems in which the responder cells were from either healthy or tumor-bearing donors, human and mouse, and the stimulator cells freshly explanted autochthonous or syngeneic leukemias and

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carcinomas, we observed a strong activating effect by partially purified, mitogen-depleted IL-2 preparations added to the cultures (2.5 - 10 units/ml) during the first 48 hr (Kedar and Herberman, 1982; Kedar et al., 1982a,f). Cytotoxic responsiveness in the IL-2-stimulated cultures was 3- to 1O-fold greater than that developingin absence of the agent, or when it was employed alone, without specific tumor stimulation; the consequence of joint tumor and IL-2 excitation was usually synergistic. Concomitant with thegeneration ofcytotoxic effectorcells directed at the stimulating tumor, there appeared in cultures supplemented with IL-2 noticeable cytotoxic activity for unrelated target cells, both such as are sensitive and as are resistant to the action of NK cells freshly derived from tissues; such nonspecific cytotoxicity also came to expression in IL-2-supplemented lymphocyte cultures not exposed to specific tumor stimulation. Other investigators have reported similar observations in mouse sensitization cultures (Mills and Paetkau, 1980; Burton and Plate, 1981). Working with an MLTC system of human melanoma cells and autochthonous patient PBL, Hersey et al. (198 1) found that the cytotoxic reactivity obtained in cultures containing both IL-2 and stimulator cells was no greater than that produced by incubating the responder cells with IL-2 alone; a broad cytotoxic capacity by effector cells developing in contact with the lymphokine was also noted. It thus appears that IL-2 can both potentiate specific CTL responsiveness against stimulating tumor cells, even where these display a limited immunogenicity, and impel1 nonspecific cytotoxic reactivity. The latter may be carried out by activated NK-NC-like cells, with a spectrum and/or intensity of cytotoxic capability greater than that of nonactivated effectors of this type (Grimm et al,, 1982a,b; Kedar et al., 1982e,f). OF SPECIFICALLY AND NONSPECIRCALLY CYTOTOXIC C. PROPAGATION LYMPHOID CELLS

Attempts at adoptive cellular immunotherapy in patients could founder on a shortage of autochthonous lymphoid cells available for the purpose. Interleukin-2 holds promise of overcoming the logistic obstacle, by facilitating not only the induction of effector cells but also by enabling their maintenance and considerable expansion. Preparations of IL-2 have been employed successfully in recent years for the long-term growth of HTL and CTL lines, cloned and uncloned, murine and human, deriving from effector cells produced in MLTC and MLR cultures of lymphocytes taken from the tissues of normal, sensitized, or tumor-bearing donors (Gillis and Smith, 1977; Collavo et al., 1978a; Fathman and Hengartner, 1978; Gillis et al., 1978a; Rosenberg et al., 1978b; Strausser and Rosenberg, 1978; Bach et al., 1979b; Baker et a/., 1979;

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Kurnicketal., 1979;ZarlingandBach, 1979;Csakoetal., 1980;Engersetal., 1980; Goulmy et al., 1980; Lotze et al., 1980b; MacDonald et al., 1980; Nabholz et al., 1980; Paetkau et al., 1980;Reisset al., 1980;Rosenberget al., 1980c; Schawaller et al., 1980; Schendel et al., 1980; Schreier et al., 1980; Wagner et al., 1980b Weiss et al., 1980; Cheever et al., 1981b; Fitch, 1981; Gillis and Watson, 1981;Giorgi and Warner, 1981;Glasebrook et al., 1981; Kornbluthetal., 1981;Bachetal., 1981;SmithandRuscetti, 1981;Eberlein et al., 1983b;Rosenberg et al., 1982a;V h k y et al., 1982;Vose and Bonnard, 1982). Increases in cell number of lo4- to 106-fold per month are made possible by the agent. Murine T cell lines can be so propagated for several years, by serial passage every 3 - 6 days; human lines can usually be maintained, similarly, for only several months. Supplementation of the cultures with feeder (“filler”) cells (irradiated leukocytes)encourages growth. It is not uncommon for cell lines to undergo a “crisis” period some weeks after culture initiation, during which a large proportion of the cells die; those surviving then adapt to the culture conditions and propagate for the extended times. A number of other important observations have been recorded in these studies on T lymphoid lines sustained with aid of IL-2 after sensitization. Continuous cultivation of antitumor CTL often elevates their ability to inflict injury to the corresponding target cells, including autochthonous neoplasms. Cells acclimated to grow in media supplemented with IL-2 lend themselves more readily to cloning. Many cloned lines deriving from specifically sensitized responder cells appear to retain their starting, specific helper or cytotoxic activities. Some lines, however, exhibit with time shifting patterns of reactivity, including the expression of cytotoxicity for allogeneic cells unrelated to the original stimulators and for normal autochthonousfsyngeneic cells; such deviation of reactivity is especially likely where cultures contain FCS and where the IL-2 preparation is crude. After prolonged passage, changes in cell surface antigenicity and karyotypic abnormalities may also appear; independence from an exogenous IL-2 supply and acquisition of neoplastic growth capability do not seem to develop. Parental karyotype and recognition specificity can be preserved by persisting restimulation during progressive culture with the original stimulators, and by ongoing feeder-cell supplementation. Fluctuations in the functionality of cloned cells can be reduced as well by repeated recloning. In uncloned sensitized T-cell populations, subpopulations with specific and nonspecific suppressor and with broadly nonspecific cytotoxic activity, as well as specific CTL, are prominently in evidence during extended cultivation. Although the properties of sensitized T lymphocytes during sustained

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growth in IL-2 are thus seen to be susceptibleto considerable diversification, and as not firmly predictable, these findings provide points of orientation in planning their usage. Cultures of lymphoid cells that had not been intentionally subjected to mitogenic or antigenic stimulation, in the donor or in vitro, can also be propagated for prolonged periods by means of IL-2 preparations, with the expression of broadly manifested, NK - NC-like cytotoxic reactivity. Even brief exposure of “normal” mouse and patient lymphoid cells ( 1 - 5 days) to mitogen-depleted, partially purified IL-2 can lead to an augmented cytotoxicity for a spectrum of both NK-sensitive and NK-resistant tumor cells (Kuribayashi et al., 1981; Minato et al., 1981; Grimm ei al., 1982a; Kedar and Herberman, 1982). In our hands, the cytotoxicity potentiation effected by IL-2 after 24-hr contact was consistently greater than that conferred on similar lymphoid populations by optimal amounts of fibroblast IFN (Kedar and Herberman, 1982). Kuribayashi and co-workers noted an additive stimulatory action by IL-2 and IFN;Minato and associatesreported that the mediators activate distinct effector subpopulations. Other studies have shown that continuing cultivation in crude IL-2 preparations of lymphoid cells that had not been stimulated with antigen or mitogen-murine and human, from normal and from tumor-bearing donors, cultured as derived from lymphoid tissue or separated into subsets -often gives rise to a strong cytotoxic reactivity that is broadly expressed toward autochthonous, syngeneic, allogeneic, and xenogeneic neoplastic cells, fresh and passaged, but seemingly to little if any injurious reactivity for normal, fresh lymphoid cells (as revealed by short-term assays)(Claesson and Olsson, 1980;Ortaldo et al., 1980, 1982; Price et al., 1980;Yron et al., 1980, 1982; Zaguri and Morgan, 1980; Lotze et al., 1981;Vose and Moore, 1981; Kedar et al., 1982d-f; Timonen et al., 1982). Several of the observations coming from our experiments deserve mention in brief. There were no appreciable differences in growth or nonspecific cytotoxicity patterns between propagated lymphoid cells taken from normal donors and those taken from donors with active neoplastic disease. The cells propagated in IL-2 were considerably more cytotoxic for fresh and passaged autochthonous/syngeneic neoplasms than were newly obtained human PBL and mouse splenocytes. Lymphoid cells drawn from mouse bone marrow and thymus, which lacked cytotoxic capacity against freshly explanted tumors when tested immediately after being obtained, developed strong cytotoxicity after growth in IL-2. After such growth for 2 - 3 weeks, human PBL that had been depleted at the outset of NK-NC activity (i.e., removal of LGL cells on Percoll gradients) nonetheless exhibited strong, nonspecific cytotoxicity for various neoplastic cells. Cytotoxicity for normal lymphoid cells-autochthonous, syngeneic, allogeneic; employed as such or after lectin stimulation -could

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often be demonstrated when the Y r release assay was extendedto 18 hr. The levels of nonspecific antitumor cytotoxicity developing in IL-2-propagated lymphoid cells was of the same, or even higher, order as that manifested by similar lymphoid cells specifically sensitized in MLTC, with no exposure to IL-2, for the corresponding targets. The finding of certain classic T-cell markers on IL-Zpropagated, nonspecifically cytotoxic lymphocytes has persuaded some investigatorsto consider these as polyclonally activated CTL. This proposition is not in keeping with their other properties: their broad cytotoxic capability, even across species lines and against both NK-sensitive and -resistant targets; their ability to function as effector cells in antibody-dependent cell-mediated cytotoxicity (ADCC);the augmentation of their reactivity by IFN;and their expression of other markers, including some associated with typical NK cells. It is suggested, accordingly, that activated NK- NC cells may represent a (functionally) distinct T subset, and may make a major contribution to the nonspecific cytotoxicity of lymphoid populations maintained in the presence of IL-2 (Grimm et al., 1982a; Kedar et al., 1982e,f;Kedar and Herberman, 1982).8 Ability of NK-NC cells to propagate in the presence of IL-2 without any (intentional) prior excitation by antigen or mitogen could be due to preexisting receptors for IL-2 on the cells, perhaps come to expression in consequence of contact with ubiquitous inciting stimuli in the host’s tissues or in the course of in vitro cultivation; IL-2 itself, servingas a weak mitogen, might effect the initial stimulus. Mouse and human lymphocytes with NK-like functionality have been cloned and cultured in IL-2 (Dennert et af., 1981b; Nabel et al., 1981 ;Brooks The overlap and lability of the characteristics of lymphoid killer cells have led to considerable confusion in their classification. They are commonly categorized on the basis of properties that fluctuate, even in cloned populations, with the conditions ofmaintenance and testing, that reflect only some of a range of possibilities for a particular cell type, and that may come to the fore not as firmly distinguishing traits but rather as variable consequences of (often unrecognized) impellents in the environment from which they were derived. It could well be that categorical distinctions between CTL, NK- NC, and activated NK-NCcellsare, at least in part, erroneous. In proposing an operational grouping of killer lymphocytes, E. Klein ( 1982) points to findings from studies with “cloned killer lines [which] suggest that at least a proportion oflytic cells act on different targets with different mechanisms. In one mechanism, lysis is based on the recognition of cell surface epitope, the other does not seem to involve antigen recognition but may be triggered by interaction between the cell surfaces, the nature of which is yet unknown. This effect ispolyclonal. . . . In a certain differentiation state Tcellsare capable to lyse targetsin either way. Other functional subsets may exert only one or the other action.” Thus, even a given effector cell may be capable, at the same moment, ofdifferent cytotoxic actions-and then be diversely classified on the basis of different assay systems. The caveat mufatismulandis must be introduced, accordingly, to the deliberations on killer cells in these pages: The discriminatory nomenclature and characterizations to which we resort may have a convenience of communicability in the field today, but are of doubtful biologic validity.

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et al., 1982; Kedar et al., 1982e,f;Ortaldo et al.. 1982; Pawelec et al., 1982; Riccardi et al., 1982; Sugamura el a!., 1982; Timonen et al., 1982). Our studies with 3 such cloned lines of human origin and with over 60 of murine origin have revealed a measure of heterogeneity among the clonal populations with regard to cytotoxic range, cell surface markers, and other properties. Other investigators have also found such heterogeneity among mouse NK-like clones (Dennert el al., 1981b; Brooks et al., 1982; Riccardi et al., 1982). The pronounced, albeit nonspecific, antitumor cytotoxicity of lymphokine-stimulated lymphoid subsets would suggest their employment therapeutically, especially where autochthonous neoplastic cells may not be available for the induction of specific CTL or where specific sensitization is not successful. On the other hand, the eventuality of their injurious action on normal tissues, their stringent dependence on IL-2, and certain of the other traits taken on by lymphoid cells at prolonged exposure to the lymphokine may severely constrain their utility. IV. Adoptive lmmunotherapy with Effector Lymphoid Cells Generated in Culture

A. GENERAL CONSIDERATIONS

Discussion in this article has been pointed throughout at the potential therapeutic employment of effector lymphocytes produced by IVS, and at the formidable problems in the way of realizing this potential. In the following pages, data that have accrued on adoptive immunotherapy in experimental systems with lymphoid effector cells made in vitro will be considered. Little information is available so far on clinical application of this therapeutic modality. The forseeable possibilities and difficulties of the approach to the treatment of human cancer have been surveyed by one of us elsewhere (Weiss, 1980),and only brief allusion to the clinical prospects is in order here.

I . Prerequisites The major prerequisites for successful adoptive immunotherapy with effector lymphocytes would appear to include the following. Adoptive immunotherapy should not be planned as a sole, independent method of intervention, but rather as a measure complementary to and intercalated with all others that can be brought into play in the course of disease (e.g., surgery, chemotherapy, radiotherapy, and possibly also other immunologic interventions, such as active specific immunotherapy and active nonspecific immunopotentiation). Neoplastic involvement of the host should be reduced as much as possible by conventional means before

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cycles of adoptive immunotherapy are initiated.9 The nature and timing of the cytoreductive and immunologic arms must be considered carefully with a view to preventing treatment antagonisms. Account must be taken of the likelihood that large numbers of neoplastic cells remain in the tissues even following reductive therapy, and that multimodal management of the patient may be an ongoing requisite. Ancillary procedures designed to diminish interference with the effectuation of immunologic treatment should be considered, such as repeated plasmapheresis (Israel and Edelstein, 1978; Terman et al., 1981) and curtailment of host suppressor function (North, 1982). It should be noted, nonetheless, that immunologic intervention in malignant disease may be of some efficacyon occasion even where tumor burden is extensive and conventional treatment has failed; this has been observed in some patients as well as in experimental subjects (Weiss, 1980, 1983). Adoptive immunotherapy should not be discounted out of hand, accordingly, even where massive debulking of the tumor host is not possible. The effector cells to be used must be devoid of appreciable cytotoxic reactivity against normal host tissues, and incapable of facilitating tumor growth directly or by suppressingimmune capability ofthe recipient or ofthe therapeutic implant itself. The recipient must tolerate the effector inoculum, which may have to be massive, and accept it at least for a time sufficient to allow expression of its cytotoxic/cytostatic antitumor potential. Where histoincompatible effector cells are utilized,judicious conditioning ofthe host with immunosuppressive agents might be necessary. The effector cells must be able to reach the host’s neoplastic niduses, and/or “home” to lymphoid centers from which they can subsequently translocate to the tumor foci or where they can act to recruit, by one or another mechanism, host immunocytes to defensive capability. Cells histocompatible with the recipient are the most likely able to fulfill these requirements (Degos et d.,1979; Greenberg et d , 1981c; Poupon et d.,1981). Both the theoretical considerations discussed in this article and experience already gained point to the probability that adoptive immunologic intervention must be mounted with attention to the individual circumstances pertaining in each instance, viz. the type of tumor, the total therapeutic strategy, the particular conditions of the host - tumor interaction at the time treatment is to be given. Clearly, such treatment individualization poses Multiple-modality treatment holds out potential advantages over and above an additive eradication of tumor cells differentially susceptible to each arm, and a constraining of the development of resistant cell populations. The rationale of combined therapeutic intervention has been discussed recently elsewhere (Weiss, 1980).

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trenchant difficulties. On the other hand, it may be a primary condition of efficacy, and the duration of many neoplastic processes, even once they have reached clinical dimensions, makes individualized adjustment and modulation of the treatment tactics not entirely phantasmagoric. The parameter of in vivo behavior of effector cells generated in culture has not been weighed in the preceding sections, and demands some deliberation here.

2. In Vivo Behavior of Effector Cells Generated in Vitro A major obstacle to the clinical application of effector lymphoid cells sensitized in culture may lie with possible impairments in their viability, deployment, and functioning within the tissues of the recipient. It has been reported (Rouse and Wagner, 1973; Burton and Warner, 1977; Kedar et af., 1978b;Lotze et al., 1980a)that large proportions of such (radiolabeled) cells are sequestered in the liver and lungs of experimental animals shortly after iv inoculation. It is questionable a priori, therefore, whether a sizable segment of implanted effector cells redistrihtes, intact, so as to gain target tumor deposits or lymphoid centers; forbiddinglylarge numbers may be required to act directly against the neoplasm or to mobilize host defenses. Experiments conducted in our laboratories on the problem of effector cell dispersal upon introduction to test animals (mice) have led to the following observations (Kedar et al., 1978b; E. Kedar et al., unpublished observations, 1983). Freshly explanted normal splenocytes and cultured splenocytesdistribute differently in the host's organs. Following systemicinoculation, cells that had been cultured for several days, with or without tumor stimulators, localize rapidly (within 2 hr) in lungs and liver (to equal extent), with only a small portion going to the spleen. At 24 hr, the lungs are nearly cleared of the introduced lymphocytes; a corresponding increase in cell number is evident in liver, with no appreciable increment in the spleen. Freshly explanted splenocytes, in contrast, are not sequestered in significant numbers within the lungs at either 2 or 24 hr, but rather settle, equally, in spleen and liver. Syngeneic and allogeneic sensitized spleen cells migrate similarly in all strains of mice tested (BALB/c, C57BL/6, A). The altered traffic in vivo of cultured cells could not be ascribed to the presence of heterologous serum (FCS) in the medium. Substitution of syngeneic mouse serum for FCS, and mild proteolytic treatment of effector cells grown in FCS prior to inoculation, did not affect the distribution patterns. On the other hand, the size of the implanted effector cells appears to affect their organ distribution. We separated splenic effector cell populations on BSA gradients after MLTC, and tested segregating subpopulations that were

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THERAPY WITH LYMPHOCYTES GENERATED I N VITRO

rich either in large lymphoblasts with potent cytotoxic reactivity in vitro, or in medium- and small-size lymphoid cells with low in vitro cytotoxicity (Table 11). At 2 hr, the small cells had deployed in a manner somewhat resembling that of fresh, unseparated splenocyte suspensions: relatively few in the lungs, an appreciable number in the spleen, although less than that of fresh cells, and approximately half of the inoculum in the liver. The large lymphoblasts were most deviant from the fresh splenocyte controls. Halfthe inoculum concentrated in the lungs at 2 hr and less than a tenth in spleen; at 24 hr, the lungs were largely clear, the spleen sector still small, and most of the cells resident in the liver. The behavior of the medium-size cells was intermediate, half seen in the liver at 2 hr and increasing to three-fourths by 24 hr, a third in the lungs early and then disappearing, and a small proportion in the spleen (but greater, at 2 hr, than that of the large cells). As will be described in Section IV,B,2, the therapeutic potency of the smaller lymphocytes was not substantially less than that of the large cells or ofan unseparated sensitized effector population. In most of the common experimental animal test systems, it must be stressed, the telescoped duration of malignant disease and the numerical relationships, absolute and relative, between neoplastic and immune effector TABLE I1 TISSUEDISTRIBUTION OF "Cr-LADELED, BSA GRADIENT-SEPARATEDA N D INTACT MOUSE SPLENOCYTE POPULATIONS ~~

Mean radioactivity (96 oftotal cpm recovered in tissues)a Blood (0.5 ml)

Liver

Spleen

Lungs

Cell population

2 hr

24 hr

2 hr

24 hr

2 hr

24 hr

2 hr

24 hr

Fresh normal, intact Sensitized, intact Sensitized, BSA fraction 24%(large lymphoblasts) Sensitized, BSA fraction 28% medium-size lymphoblasts) Sensitized. BSA fraction 35% (small-size lymphocytes)

3 2 2

2 1 1

45 20 9

47 25 17

41 42 40

47 69 75

11

36 49

4 5 7

3

2

17

20

50

74

30

4

4

ND*

29

ND

46

ND

21

ND

Values represent means of three replicate samples in one experiment. Tr-labeled viable, syngeneic splenocytes (2 X lo6)were injected iv into normal C57BL/6 mice (three animals per group). Radioactivity in the various tissues was determined at 2 and 24 hr; ca. 60 and 45%ofthe total counts injected were recovered, respectively. Similar results were observed in additional experiments performed in C57BL/6 mice and A mice, with both syngeneic and allogeneic splenocytes. Cell separation on BSA gradients was carried out as described (Kedar ef a/., 1979a). ND, not done.

248

ELI KEDAR AND DAVID W. WEISS

cells bear little semblance to the corresponding circumstances of neoplasia in nature (Weiss, 1978). These and other artifactualities make many experimental models doubtful testing grounds for the significance of effector cell vitality and tissue translocation tendencies. Therapeutic success could be attainable in such models, under highly selected conditions, despite serious defects in effector behavior. It must not be forgotten, however, that felicitous intervention in the natural history of malignant disease by effector cell implants is likely to pose far more rigorous demands. Thus, for instance, it may prove difficult to infuse sufficient numbers of effector lymphocytes where these are largely “end” cells, and incapable of response to further excitation in the recipient; even a dramatic antitumor capacity by such cells in vitro, in neutralization assays, and in “therapeutic” tests where the time dimension is grossly compressed may well be wholly irrelevant. Or an only temporary sequestration of inoculated effector cells in the capillary beds of certain organs may severely compromise their subsequent ability to act, directly or indirectly, against the tumor with the requisite perdurance. Even with elaboration of this question in experimental models of greater pertinence than those current today, it shall be necessary to test empirically, in the clinic, the therapeutic efficacies of cytotoxic and amplifier/helper cell subpopulations with different physical and biological properties and different migration behavior. Establishment of correlations between efficacy and such characteristics may then pave the way to a more reasoned employment of effector cell preparations. If clinicaltrial shall confirm the expectation that the therapeutic usefulness of effector cells generated in culture is conditioned on their “normal” deportment upon introduction to the patient, various possibilities of improvement, suggested by findings from animal experiments, could be explored. Thus, it has been reported (Mule et al., 1979) that lymphocytes freshly taken from immunized mice localize more effectivelyat tumor foci in syngeneic recipients if these are subjected to prior whole-body irradiation. Mathisen and Rosenberg ( 1980)noted that entrapment of fresh, xenogeneic lymphoid cells in the liver can be reduced, and their distribution to lymphoid organs expedited, by treating the recipient mice with reticuloendothelial blocking agents. Introduction of effector cells into blood or lymphatic vessels that are in communication with inoperable growths, and use of vasodilating drugs, may have a place in treatment tactics. Consideration of the pattern of neoplastic dissemination in the individual case could also guide the application of effector cell subpopulations; where metastasis is prominent to a particular organ, an effector cell preparation with preferential dispersal to that site could be most useful, even where the preparation is of little value in retarding tumor growth elsewhere.

THERAPY WITH LYMPHOCYTES GENERATED IN VITRO

249

B. ADOPTIVE IMMUNOTHERAPY IN EXPERIMENTAL SYSTEMS The ready demonstration of effector cell antitumor capacity in in vivo neutralization assays of the Winn type does not of necessity indicate their likely efficacy against established neoplastic disease. Some of the protective effects evinced in Winn assays may reflect damage inflicted to tumor cells before the cell mixture is injected to test animals, and introduction jointly (or in rapid sequence) of tumor and effector cells, the latter usually far outnumbering the former, is hardly a circumstance resembling the conditions of progressive neoplastic disease. Winn tests, and the various in vitro assays, can thus provide no more than a presumption of some degree of effector cell potency. Only assessment in animals already subject to a developing neoplastic process can afford evidence of therapeutic capability- and such evidence is, by nature of the common animal models, of only the most tenuous pertinence to the planning of immunologic intervention against spontaneous neoplasia in the primary host.

1. Therapeutic Employment of Eflector Cells Alone Attempts to intervene in neoplastic disease of experimental animals with effector cells only, i.e., without the effectuation of other therapeutic modalities, and employing lymphoid cells derived from immunized donors, have been under way for the past 20 years (Balme et al., 1962; Woodruff ef al., 1963b; Delorme and Alexander, 1964; Woodruff and Boak, 1965; Alexander, 1967, 1968; Fefer, 1969, 1974; Borberg ef al., 1972; Dullens el al., 1974; Rosenberg and Terry, 1977; Brendt and North, 1980;Dye and North, 1981;Shu et al., 1982).Success in these therapeutic attempts appeared to be contingent on introduction of large numbers of highly reactive cells (at least lo*immune lymphocytes are required to exert an appreciable effect against a 1-gm tumor in the mouse) and on their viability, metabolic functionality, and survival for some days in host tissues. Syngeneic effector cells were commonly more active than allogeneic ones. Depletion of suppressor elements in the treated animals generally elevated the efficacy of adoptive immunotherapy . The development of IVS technology, and the advantages held out by this approach to the procurement of effector cell preparations, prompted explorations of adoptive immunotherapy with lymphocytes activated in culture. Treves et al. (1975, 1976) reported that iv administration of syngeneic mouse splenocytes, obtained from normal donors and sensitized in v i m against the 3LL carcinoma of C57BL/6 animals, reduced appreciably the incidence of pulmonary metastases appearing after surgical removal of a primary tumor implant and led to an increased number ofcures. Splenocytes

250

ELI KEDAR A N D DAVID W. WEISS

that were not sensitized or that were cocultured with syngeneic fibroblasts were ineffective. Cells taken from mice carrying progressively growing tumors and then specifically sensitized in culture also exhibited little, if any, protective activity; however, spleen cells harvested after extirpation of the neoplasm proved efficaciousfollowing IVS. The presence of suppressor cells in the spleen of tumor-bearing hosts and reduction of these cells upon tumor removal are suggested explanations. Mixed findings have been recorded by other investigators. Some could detect, at best, a very limited therapeutic activity in mice with various disseminated neoplasms for syngeneic and allogeneic lymphoid cells taken from normal or immune donors and subjected to primary or secondary MLTC (Burton and Warner, 1977; Cheever et al., 1977, 1978; Kedar et al., 1978b,c). On the other hand, Fernandez-Cruz and associates (1979, 1980, 1982) demonstrated a high incidence of complete regression of large subcutaneously growing implants of several rat tumors following iv infusion of syngeneic lymphocytes that had been sensitized in vitro. The therapeutic effect appeared to be tumor specific;the active cells were splenocytesderived from animals with regressed tumors, or from animals immunized with inactivated tumors cells, and then (secondarily) sensitized in MLTC; the therapeutically reactive effector cells were not cytotoxic as assessed in vitro; and therapeutic results were improved when the test animals were subjected to X irradiation (400 R) before tumor challenge (presumably because of a depletion of suppressor cells). The impressions gathered, on the whole, from the efforts at adoptive immunotherapy as the sole treatment modality in laboratory rodents can be summarized as follows. Effector lymphocytes generated in vitro are usually efficacious only against relatively small numbers of tumor cells, and usually only where tumor challenge is effected shortly before introduction of the therapeutic implant. There is often no correlation between cytotoxic capability of effector cells as assayed in vitro and therapeutic efficacy. Cells that underwent secondary sensitization in culture (after primary sensitization in vivo) tend to be more efficaciousthan cells employed after primary sensitization in vivo or in vitro. The therapeutic action exerted is in most instances limited in extent, and it is closely contingent on selection ofapt experimental conditions. Changes in one or more of numerous variables-source of responder cells, parameters of sensitization, route of effector cell introduction, effector/tumor cell ratio, immunologic competence of the host, and others- frequently lead to very different results; under variant experimental concatenations, the consequences ofintervention can be either indifferent or beneficial, even in the hands ofthe same investigatorsworking with the same model system.

THERAPY WITH LYMPHOCYTES GENERATED IN VITRO

25 1

Whereas cure of an established neoplastic process, especially where dissemination has already occurred, is not commonly attained by adoptive immunotherapy alone, even a modest prolongation of survival need not necessarily be dismissed as insignificant. Thus, for instance, the YAC and EL4 leukemias of mice, models on which we have focused considerable attention in our laboratories (Kedar et al., 1978b,c),have a doubling time in viwo of ca. 14 hr; a prolongation in survival of only 2 - 3 days in consequence of immunotherapy may indicate a temporary reduction in tumor load of up to 909/0.Translated to the clinical situation of human neoplasia, an effect of this magnitude may have considerable therapeutic implication. Nonetheless, the validity of any such extrapolation is highly questionable, and efforts at more categorical therapeutic success are clearly mandatory in the experimental model systems. Such effort is represented by the employment of effector cells in conjunction with other treatment arms. 2. Therapeutic Employment of Efector Cells in Conjunction with Other

Therapeutic Modalities Lethal and sublethal whole-body X irradiation has been used for some time as a preparatory step to the application of hemopoietic, and of normal or immune lymphoid cells, in tumor immunotherapy; some therapeutic effects have been noted, but severe and even fatal GvH reactions occur frequently with histoincompatible grafts (Barnes et al., 1956; Math6 et al., 1960; Woodruff et al., 1963a; Boranic, 1968; Thompson and Mathk, 1972; Bortin et al., 1973, 1979; Fefer, 1974). It has been suggested that the GvH reactivity of allogeneic cells in immunosuppressed recipients may be responsible in part for the antitumor action. On the other hand, control of severe GvH disease (GvHD) poses a formidable task (Boranic and Tonkovic, 1970; Elkins, 1971; Fefer, 1974; Chester et al., 1975). Other groups have reported considerable success in the treatment of various murine tumors with freshly explanted lymphoid cells- syngeneic and allogeneic, normal and immune -in conjunction with sublethal chemotherapy, and with less intractable GvHD sequelae (Fefer, 1969, 1973, 1974; Mihich, 1969; Vadlamudi et al., 1971; Fassand Fefer, 1972; Kendeet al., 1975;FeferetaL 1976, 1982;Gotohdaetal., 1976;Putmanetal., 1978). Chemotherapy and adoptive immunotherapy often appear to act synergistically, and immune syngeneiclymphocytes were usually seen to be both safer and more effective than allogeneic effector cells. More recently, resort has been had to a therapeutic combination in mouse leukemias of high-dose CY, for a definitive reduction of tumor load, and fractionated total lymphoid irradiation (TLI) (Slavin et al., 1978) for facilitating the acceptance of histoincompatible bone marrow grafts (Slavin et al.,

252

ELI KEDAR A N D DAVID W. WEISS

1981).By this means, hemopoietic rescue and marked graft-versus-leukemia (GvL) effects have been achieved, and at little cost in GvHD. The chemotherapy arm of most of the successful chemoadoptive immunotherapy protocols in mice has been CY. This drug offers the advantages of a broad, direct antitumor action, rapid clearance from the circulation, and, at low doses, a preferential toxicity for suppressor lymphocytes and/or their precursors (Fefer, 1974;Mokyr et al., 1982;North, 1982).It may also be that CY modifies the membrane of neoplastic cells and renders them more susceptibleto immune attack (Borsoset al., 1976).CY has application as well in the temporary immunologic suppression of the recipient of allogeneic effector elements. Extensive investigations with in vitro-sensitized lymphoid cells and CY chemotherapy in several transplanted lymphomas of inbred mice have been conducted by Cheever et al. (1977,1978, 1980,1981a-c) and Greenberg et al. (1980, 198lb,c). Combined treatment with limited amounts of the drug and with lymphocytes that had been derived from syngeneic normal and immunized animals and were then sensitized (or resensitized) in MLTC was synergistically effective, often eliciting apparent cures. The therapeutic capacity of the cells did not correlate with cytotoxic capacity in vitro, and was tumor specific and MHC restricted. Their antitumor action was held to reflect two distinct mechanisms, an early, direct cytotoxicity for the target cells, and, subsequently, a recruitment of host immunocytes to defensive capability, presumably by a distinct subset of amplifier/helper cells in the sensitized T-cell population. Mokyr et al. (1981) and Mokyr and Dray (1982) successfully treated mouse plasmacytomas by a similar combined intervention, employing in vitro-sensitized effector cells that originated in animals with progressively growing tumors; suppressor cells were depleted from the donors by CY pretreatment before harvesting the cells. Experiments conducted in our laboratories with the EL4 and YAC mouse lymphomas revealed synergistic therapeutic effects for intervention with CY and with lymphoid cells from normal donors sensitized in primary MLTC (Kedar et al., 1978b). A large majority (80-95%) of animals were (apparently) cured, i.e., survived free of evident disease over the observation period of several months, when ip or iv challenge with lo3- los neoplastic cells was followed 1 - 3 days later by injection of moderate amounts of CY (80- 140 mg/kg) and, a day thereafter, by iv or ip introduction of 2 - 3 X 1O7syngeneic or allogeneic effector cells. Chemotherapy alone rarely effected cure of more than 50% of the subjects, and often of only a considerably smaller proportion, and the sensitized cells by themselves elicited only a moderate prolongation of survival time, with cures a rare occurrence (no more than 10%). More recently, we broadened the range of test parameters in chemoadoptive immunotherapeutic intervention against these tumors (Table 111). Mice

TABLE 111 CHEMOADOPTIVE IMMUNOTHERAPYOF ESTABLISHED LEUKEMIAS I N MICEWITH EFFECTOR LYMPHOIDCELLS(SPLENOCYTES) GENERATED in Vitro

Experiment number I

2

3

4

Tumop EL4 I X

los, ip

YAC I X los, ip

YAC I X los, iv

YAC 1 X lo6,sc

Percentage of mice surviving (day 100)

Mean survival time t SD of mice succumbing (days)

0 33

l6+2 29 t 5

85

37F4

80

36 F 5

+

0 29

16312 34 3

+

90

39 +- 4

+

87

3lF3

0 38

15+2 44 f 2

85

49t4

80

45 t 5

0 15 75

17t2 29F4 33+4

0 20 12

15t2 26 t 3 49 t 4

60

44 5 3

Treatmentb None Chemotherapy normal syngeneic cells Chemotherapy sensitized syngeneic cells Chemotherapy sensitized allogeneic cells

+ + +

None Chemotherapy normal syngeneic cells Chemotherapy sensitized syngeneic cells Chemotherapy sensitized allogeneic cells None Chemotherapy normal syngeneic cells Chemotherapy sensitized syngeneic cells Chemotherapy sensitized allogeneic cells

+ + +

None Chemotherapy Chemotherapy sensitized allogeneic cells

+

5

YAC I X los, ip

None Chemotherapy Chemotherapy fresh sensitized allogeneic cells Chemotherapy cryopreserved sensitized allogeneic cells

+ +

*

Tumor cells were inoculated on day 0 to syngeneic recipients ip, iv. or sc. Experimental groups consisted of 20- 30 mice each. '' Chemotherapy administered ip 5-6 days after tumor inoculation. In Exp. I , the animals received CCNU, 25 mg/kg; in Exp. 2- 5, they received CY. I50 mg/kg. Sensitized syngeneic or allogeneic splenocytes (20- 30 X lo6) were injected 24-48 hr after chemotherapy either ip (Exps. I . 2, and 5 ) . iv (Exp. 3), or into the tumor (Exp. 4). Similar results were obtained with sensitized lymph node cells. In Exp. 5, sensitized cells were cryopreserved after MLTC for 4 weeks before usage.

254

ELI KEDAR A N D DAVID W. WEISS

were challenged by various routes (ip, iv, sc) with lo5- lo6living tumor cells. They were treated ip on days 5 or 6 (after challenge)with CY ( 150mg/kg, for the YAC lymphoma) or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) (25 -30 mg/kg, for the EL4 lymphoma). Chemotherapy was given alone or was followed 24 - 48 hr later by introduction, as detailed in the table, of 2-3 X lo7 sensitized lymphoid cells, syngeneic or allogeneic. Normal lymphoid cells were employed as controls. At the time treatment was commenced, tumor load in the animals had probably reached, or exceeded, lo*cells. The different chemotherapy was chosen because it appeared from preliminary study that CY was ineffective against a large challengewith EL4, and CCNU even against a small inoculum of the YAC tumor. In none of these experiments (five representative ones are depicted in Table 111) were there any survivors at 100 days (term of the observation period) when no treatment was given. A maximum of 40% of the animals, and often fewer, survived on chemotherapy alone or on chemotherapy plus freshly obtained normal splenocytes. In contrast, 75 -90% commonly survived in the treatment groups receiving chemotherapy and sensitized spleen cells (60-7OYo in Exp. 5 , in which cryopreserved effector cells were used). Similar results were obtained when the effector cells were injected directly into an sc tumor mass (Exp. 4) and when they were introduced systemically. Cure rates consistently approaching 100% were achieved for both lymphomas when the effector cells were given twice, on days 7 and 12 after challenge(E. Kedar et al., unpublished observations, 1982).It was also noted in preliminary experiments that splenocytes taken from survivors of Winn and immunotherapy assays 45 - 90 days after their tumor challenge, and then restimulated in MLTC with the corresponding neoplastic cells, expressed considerably greater (two- to threefold) cytotoxic activity in vitro and therapeutic potency than effector cells harvested from the primary MLTC systems. In further experiments, we separated in vitro-produced effector cell populations on BSA density gradients and tested subpopulations of large-, small-, and intermediate-sized lymphocytes in chemoadoptive immunotherapy protocols against the two lymphomas. Despite marked differences in their cytolytic potency in vitro, the effector subpopulations exhibited similar therapeutic capacity (Table IV). Therapeutic action of allogeneic effector cells was not usually accompanied by discernible GvHD, even though the cells display strong cytotoxicity in vitro for normal lymphoid and for tumor cells of the same H-2 type as the stimulator cells used in MLTC (Kedar et al., 1977). The occasional symptoms of GvH reactivity seen tended to be transient and mild (Kedar et al., 1978b).It is not inconceivable that GvH and antitumor effects are mediated, in part, by different T lymphocyte subsets. In this event, it should prove

255

THERAPY WITH LYMPHOCYTES GENERATED I N VITRO

TABLE IV CHEMOADOPTIVE IMMUNOTHERAPYWITH EFFECTORCELLPOPULATIONS SEPARATED ON BSA GRADIENTS A N D in Vitro CYTOTOXICITY OF T H E CELLS" Survivors/total Treatment

EL4 systemh

YAC system'

None Chemotherapy alone Plus intact effector population Plus BSA fraction 24% effector cells Plus BSA fraction 28% effector cells Plus BSA fraction 35% effector cells

0/8 3/12 8/12 (36%) 9/ I2 (95%) 10/12 (21%) 6/12 (8%)

0/10 4/15 12/15 (27%) 10/15 (75%) 13/15 (18%) 8/15 (6%)

" The values in parentheses show percentage specific W r release from target cells effected in vitro by the same effector cell preparations employed for chemoadoptive immunotherapy; cytotoxicity values are means of four replicates. "C57BL/6 mice injected iv with I X 10' viable EL4 cells. Five days later. the animals received, ip, CCNU, 30 mg/kg. One day later, they received. where indicated, iv injection of 2.5 X 10' MLTC-sensitized syngeneic splenocytes, either the intact effector population or subpopulations obtained by fractionation on BSA gradients. Experiment terminated on day 100. In ritrocytotoxic activity of effector cells assessed in a 4-hr Y r release assay. effector/target (E/T) ratioof25/l. Strain A mice injectedipwith I X 105viableYACcells. Fivedayslater, theanimalsreceived, ip, CY, 140 mg/kg. One day later they received, where indicated, ip injection of 3 X lo7 MLTC-sensitized syngeneic splenocytes, asdescribed above. Experiment terminated on day YO. I n vifro cytotoxicity assay as described above, E/T ratio 30/1. L

possible to identify, isolate, and utilize effector subpopulations capable of a selective attack on the neoplasm. 3. Therapeutic Employment of Efector Cells Propagated with IL-2 Systemic adoptive immunotherapy in experimental rodents requires at least 10'- lo8effector cells, and it has been estimated that a minimum of 1 O 1 O cells would be needed for the treatment of patients (Rosenberg et al., 1982a,b). The recent availability of IL-2 as a means for expanding lymphocyte populations offers a solution to what might otherwise well be a defeating logistic obstacle. A number of reports have appeared recently on the therapeutic capacities of effector lymphocytes grown in media containing IL-2. T cells that were specifically sensitized in vitroand then cultured, cloned or noncloned, for prolonged periods with the lymphokine have been found by some investigators to be potently cytotoxic for mouse tumor cells in in vitro and local Winn-type assays, but in most instances only marginally therapeutic, if at all, when employed systemically without other forms ofintervention (Mills et al., 1980; Gillis and Watson, 1981; Giorgi and Warner, 1981). In

256

ELI KEDAR A N D DAVID W. WEISS

contrast, Eberlein et af. (1982b) achieved high cure rates against both localized and disseminated lymphomatous tumors (Friend virus induced) in mice by iv infusion of 5 X lo7effector cells (secondary MLTC) that had been propagated in IL-2. Administration of allosensitized cells grown in IL-2 has also been found to accelerate the rejection of corresponding skin allografts (Rosenstein et af., 1981). Mu16 and associates ( 1981) studied the tumor-neutralizing powers (in Winn assays) of tumor-infiltrating lymphocytes harvested from small and from large methylcholanthrene-induced mouse sarcomas and then cultured in IL-2. The interesting observation was made that cells taken from small growths, largely of Lyt 1+2+type, were specifically inhibitory in the test, whereas those derived from larger growths, predominantly Lyt 1-2+, exerted an enhancing effect. Sensitized effector cells propagated in IL-2, like those cultured in ordinary media, have proven therapeutically much more effective in mice when chemotherapy (CY) is added to the treatment regimen (Cheever et af., 198lb; Eberlein et al., 1982a,b; Kedar et af.,unpublished, 1983). The cells were found active even when tested after several months of growth in IL-2containing cultures, as were cloned CTL lines. Lymphocytes grown in the presence of IL-2 are strictly dependent on the agent-such cells have been seen to die within 48 hr after introduction to syngeneic animals (Gorelik et af.,1981)-and its sustained administration to animals treated with propagated effector cells can improve their therapeutic efficacy, presumably because of their facilitated survival (Cheever et af., 1982; Fernandez-Cruz et af.,1982; Palladino et af.,1982); the therapeutic potency of effector cells that had not been cultured in IL-2 was not elevated by treating the test hosts with the lymphokine (Cheever et af., 1982). An inhibitor of exogenous IL-2 has been identified in the serum of conventional, but not of nude, mice (Wagner et af.,1980b); serum concentration of this factor can be reduced markedly by eliminating a T-cell subset sensitiveto low doses of CY (Hardt et af.,1981). The method of IL-2 administration may be important. It was found (S. A. Rosenberg, personal communication, 1982) that the half-life of exogenous murine IL-2 in the circulation of mice is ca. 4 min, 2 hr, and 6 hr following, respectively, injection by iv, ip, and sc routes; application of the agent in a matrix of 15% gelatin, ip or sc, prolonged its half-life in the blood significantly. In contrast to investigators (Eberlein et af.,1982a) who could detect no therapeutic action by nonsensitized lymphoid cells that had been maintained in IL-2, we have found cells so grown after derivation from normal and from tumor-bearing mice to have antitumor reactivity. Populations of splenocytes expanded for 2 or more weeks in crude or in lectin-depleted, partially purified IL-2 exhibited strong and broad cytotoxicity for neoplastic

THERAPY WITH LYMPHOCYTES GENERATED IN VITRO

257

cells in vitro, and neutralizing capacity inWinn assays (Kedar et al., 1982d). Similar observations were made for IL-2-propagated, normal human PBL that were injected into nude mice together with human carcinoma cell lines (Kedar et al., 1982f). In preliminary chemoadoptive immunotherapy trials in mice bearing murine leukemia and carcinoma implants, clear therapeutic effects were noted upon repeated administration over several weeks of propagated normal-donor lymphoid cells and IL-2 (Table V). A temporary retardation of tumor growth in mice carrying sc and im neoplastic implants was also effected by repeated treatment with normal splenocytes grown in IL-2 and with preparations of the agent, even in the absence of cojoint chemotherapy. There was no evidence of severe GvHD in these experiments. The successful demonstration in our laboratories of antitumor reactivity by such nonsensitized lymphoid cells, in contradistinction to the negative experience of others, might be due to our usage of larger numbers ofthe cells, and of relatively crude preparations of IL-2 which perhaps provided a more potent, polyclonal stimulus. 4. Employment of Soluble NonspeciJic Mediators (Other Than IFN)

The possibility that soluble mediators of CMI can act therapeutically in neoplastic disease is arousing considerable attention, extending not only to known lymphokines and monokines but also to a heterogeneous variety of nonspecifically active BRM. The reawakened interest in the potentials of “nonspecific immunotherapy” is focused on defined reagents, and it promises to lead to new insights into immunologic function apart from any conceivable contribution to cancer treatment. Although essentially beyond the scope of this article, these approaches are of sufficient relevance and proximity to its central subject -the in vitro generation and utilization of effector lymphocytes -to warrant brief allusion here. Repeated administration of IL-2 preparations to immunosuppressed mice (CY, X irradiation) can appreciably augment the capacity of their spleen cells to respond to lectin stimulation, to undergo sensitization in MLR and MLTC, and to function as NK-like effectors (Kedar et al., 1982a). Potentiated lymphocyte functionality has also been seen to follow treatment of normal conventional and athymic mice with the agent (Wagner et al., 1980a; Conlon et al., 1982; Gillis et al., 1982; Kedar et al., 1982a; Kedar and Herberman, 1982). Development of pulmonary metastases in mice bearing fp implants of several different neoplasms could be prevented by sustained treatment with the lymphokine, although no effect was exerted against already-established metastatic foci (E. Gorelik, personal communication, 1982). Treatment of nude mice with IL-2 has been found to render the animals refractory to implantation of human tumor cells (Daudi) (Kedar and Herberman, 1982). Eradication of existing metastases in mice has been accomplished by

TABLE V CHEMOADOPTIVE IMMUNOTHERAPY OF LYMPHOMA A N D CARCINOMA IN MICEWITH IL-2 AND IL-2-hOPAGATED UNSENSITIZED SYNGENEIC SPLENOCYTESO Tumor-free survivors/ total (day 180)

Mean survival time fSD of mice succumbing (days)

Experiment number

Animals and tumor

1

BALB/c Carcinoma M 109, 5 X los ip, day 0

None CY, 200 mg/kg, ip, day 3 CY IL-2, 1 ml, ip, days 5, 7,9

018 2/10 4/10

37f2 48f4 67f7

C57BL/6 Thymoma 136.5, 1 X lo5ip, day 0

None CY, 200 mgjkg, ip, day 3 CY IL-2, I ml, ip, days 4,6,8 CY 15 X IWCLC, ip, days 4,8, 14

0/10 0/10

2/10 3/10

14f3 22f5 27f4 28 f 3

BALB/c M109, 1 X lo5 ip, day 0

None CY, 250 mg/kg, ip, day 7 CY IL-2, 1 ml, ip, days 8, 15,22 CY 15 X 106 CLC, ip, days 8, 15,22 CY CLC 1L-2, ip, days 8, 15,22

018 018 218 218 418

35 f 5 5725 6928 72 k 4 1 0 2 f 11

BALB/c ~ 1 0 9I ,x 105, sc,day 0

None CCNU, 35 mg/kg, ip, day 7 CCNU IL-2, sc and ip, days 8, 15, 22 CCNU + CLC, sc and ip, days 8, 15,22 CCNU CLC IL-2, sc and ip, days 8 15.22

018 118 218 218 518

61 2 6 78 2 10 8927 116212 1 4 8 f 19

2

3a

3b

Treatment

+

+ +

+ + +

+

+

+

+

* The tumors used were the M109 transplantable spontaneous lung carcinoma of BALB/c mice (Marks el a/..1977)and the 136.5 transplantable RadLV-induced thymoma of C57BL/6 mice (Yefenof ef al.. 1980b) in the syngeneic hosts. Rat IL-2 was depleted of lectin (Con A) and partially purified and concentrated by (NH,),SO,; each inoculation was with 40-50 units, ip (Exps. 1,2, and 3a) or ip (314 dose) andinto the tumor site (114 dose) (Exp. 3b). Cultured lymphoid cells (CLC) derived from the spleens of normal syngeneic mice were propagated in IL-2 for 2-6 weeks and injected ip (Exps. 1,2, and 3a) or ip (314 dose) and sc (114 dose) (Exp. 3b).

THERAPY WITH LYMPHOCYTES GENERATED IN VITRO

259

Fidler’s group (Fidler, 1980; Fidler et al., 1982)with injection of liposomes containing mixed lymphokine preparations, and of syngeneic macrophages that had been activated in vitro by liposome-camed MDP (the mycobacterial fraction previously described; see Table I). Inhibition of the growth of tumor implants, and oflung metastases developing from them, has been achieved in mice by repeated administration of AEF (Yen-Lieberman et al., 1982). A variety of soluble factors derived from the thymus [e.g., thymus humoral factor (THF), thymosin, thymopoietin] have been shown capable of correcting immunologic deficiencies, and of some therapeutic effects against progressive neoplastic disease (Friedman, 1979; Low and Goldstein, 1979;J. F. Bach, 1980;Gabizon and Trainin, 1981; Lau et al., 1982; Zatz et al., 1982). These recent reports are cited in illustration of a rapidly developing field of investigation. In what may be considered a parallel endeavor, already initiated some time ago, various attempts have been made to isolate from immune lymphoid cells soluble factors with specific reactivities, i.e., the ability to transfer to uncommitted immunocytes the information that would recruit, or transform, them to the corresponding, specific antitumor activities. The data, still tenuous, that have accrued so far on “immune RNA” (Pilch et al., 1978), “transfer factor” (Lawrence, 1978),and other aspects of this subject, are not for discussion here. The suggestion might be hazarded, however, that the approach of passive immunotherapy of neoplastic diseases could gain categorical impetus if it were to prove possible to replace effector cells by soluble, defined factors capable of similar, or more potent, specific and nonspecific immune excitation. OF CLINICAL APPLICATION C. THEPROSPECTS

Attempts at adoptive immunotherapy in cancer patients with lymphoid (sensitized and naive) and with bone marrow cells have been reported for the past 20 years (Rosenberg and Terry, 1977; Fefer et a/., 1982; Thomas et al., 1982). Limited, temporary responses were described in a few patients given normal-donor lymphoid cells, HLA matched or unmatched (Woodruff and Nolan, 1963; Symes et al., 1968; Yonemoto and Terasaki, 1972). More impressive results have been obtained in leukemic patients who received bone marrow transplantation following a regimen of otherwise lethal or supralethal radiochemotherapy. A considerable proportion of patients treated with marrow derived from identical twins or from HLA-matched first-degree relatives achieved long-term remission and apparent cure; however, severe and not rarely fatal GvHD frequently follows transplantation from donors other than identical twins (Santos, 1972; Math&et al., 1974; Fefer et al., 1974;Thomas et a]., 1975;Weiden et al., 198la). In keeping with observations in experimental animal models, the elicitations of GvH and of

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antitumor reactivity by allogeneic cells appear to go hand in hand in at least some instances; an inverse correlation has been reported between the occurrence and severity of GvHD and the incidence of patient relapse, and higher relapse rates have been noted in leukemia patients who were given identical-twin bone marrow than in those given allogeneic implants (Weiden et al., 1981b). Cross-leukocyte transfusion has been performed in solid tumor and leukemia patients immunized with each other's neoplastic cells, as has one-way implantation of PBL from patients cured of their disease and from patients immunized with the prospective recipient's neoplastic tissue. No persuasive information has come from these largely cursory, anecdotal studies (Nadler and Moore, 1969; Humphrey et al., 1971; McPeak, 197 1; Krementz et al., 1974; Neff and Henneking, 1975; Rosenberg and Terry, 1977). The logistic curtailments on availability of human lymphoid cells sensitized in vivo against tumors, and the risks of human immunization with neoplastic tissue preparations (Scanlon et al., 1969, have led investigatorsto turn to antitumor effector cells generated in culture. The clinical essays reported to the present with allogeneic or autochthonous lymphoid cells stimulated in vitro by tumor cells (Trouillas and Lapras, 1969; Aust et al., 1970; Moore and Gerner, 1970; Seigler et al., 1972, 1976; Thomas et al., 1980), IL-2 (Lotze et al., 1980a; Slankard-Chahinian et al., 1980; Rosenberg et al., 1982b), or mitogens (Cheema and Hersh, 1972; Frenster and Rogoway, 1970;Frenster, 1976;Mazumder et a/., 1982a,b)have been exploratory and tentative. In most instances, the patients treated had far-advanced disease, the potency of the effector cells was uncertain, and their numbers may have been far below required thresholds, HvG and GvH reactivities were not always controlled, and wide testing of the parameters of direct and ancillary treatment has not yet been brought to the clinic. That these very preliminary attempts did not produce striking therapeutic results is of little meaning, therefore. It does appear so far that even large numbers ofvariously activated effector cells (up to 2 X lo", over a series of injections) can be administered safely to patients, as to chimpanzees (Slease et al., 1981), but clearly, adoptive immunotherapy is still to be put to careful, systematic clinical trial. Allusion has been made repeatedly throughout this article to the design that is envisaged for this approach, and the scheme need only be summarized in outline here: Tumor cells are obtained from the patient (from blood, effusions, or tissue removed surgically) and cryopreserved ready for employment in MLTC stimulation. The patient is treated conventionally to effect maximal reduction oftumor load, and remission ofactive disease. For use as responder cells,

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PBL are drawn during disease-free interval, where possible; they may be taken as well from solid tumor patients, during active disease, but in all instances their harvesting should be timed so as to reduce any inimical influences on cell number and functionality that may accrue from the treatment regimen. Usage of allogeneic responder cells, from normal donors or from long-term survivors of similar disease, could be considered. The responder cells can be used at once, or after cryopreservation. Mixed lymphocyte- tumor cell culture is performed with intact lymphoid cell populations or with responder cell subsets, utilizing the patient’s autochthonous neoplastic tissue for stimulation, or allogeneic tissue where autochthonous is not available, and resorting to the various possibilities of amplified sensitization that have been described. The effector cells harvested after primary (6 - 8 days) or secondary (3- 4 days) MLTC are utilized therapeutically at once or after cryopreservation, as such or as selected, reactive subpopulations. Their numbers can be expanded, as needed, with IL-2; all effector preparations must be screened for contamination with microorganisms and viruses. Treatment is initiated at “appropriate” times in the course of the disease, with large numbers of effector cells (1O’O or more, by repeated administration) infused systemically or into vessels connecting with tumor masses. Treatment might be deemed most apt during initial remission or at the sign of incipient relapse or disease progression, and when the patient’s immunologic functioning is as nearly unimpaired as possible, but only experience shall provide reliable indication of the optimal timing of intervention in different disease circumstances. Adoptive immunotherapy with IL-2-propagated effector cells is likely to demand repeated administration of the free lymphokine. The immunologic regimen must be intercalated appropriately with all other arms of continuing patient management. The arguments that can be advanced, theoretically, for and against the use of autochthonous and of allogeneic stimulator and responder cells have been presented in preceding sections of this paper, and elsewhere (Weiss, 1980). “Ancillary” measures may prove of crucial importance in facilitating expression of the potentials of adoptive immunotherapy. Rejection of allogeneic effector cells could be slowed by cautious immunosuppression of the patient. Both GvH and HvG reactivities may be reduceable by the recently developed technique of fractionated total lymphoid tissue irradiation (Slavin et al., 1978, 1979, 1983; Strober et al., 1979; Najarian et al., 1981). Bone marrow transplantation might be of value as an adjunct to the introduction of effector lymphocytes in patients that had been subjected to intensive chemotherapy or irradiation, in aiding hemopoietic restoration and perhaps also as direct contribution to immunologic antitumor defenses; beneficial effects have been reported in experimental animals and cancer patients for autochthonous marrow implantation following cytoreductive

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treatment (Tobias and Tattersall, 1976;Graze and Gale, 1978; Dicke et af., 1981; Gorin et af., 1981; Netzel et af., 1981). The risk of reintroducing neoplastic cells cryptic in autochthonous marrow could be lowered by resorting to techniques for the removal of any such cells (Dicke et af.,1978; Krolick et af., 1981, 1982; Raso et af.,1982), and the risks of GvHD in consequence of introducing allogeneic, normal-donor marrow by depleting the implant of mature T lymphocytes (Reisner et af.,1981;Wulff et af.,1981; Sharp et af.,1982; Vallera et af.,1982). Substances that interfere with the mounting and effectuation of immune attack on neoplasms may have to be removed from the tissues of the patient, or diluted, by plasmapheresisand other means; and active potentiation of the patient’s innate immunologic capabilitiesmay be requisite to assure the most efficacious collaboration between autochthonous and implanted immunocytes. The successes achieved in the past several years with adoptive immunotherapy in murine experimental systems are encouraging, but the attitude to clinical application must be, at this point, highly cautious and skeptical. Some of the formidable difficulties that will confront translation of this therapeutic modality from laboratory to clinic have been described in this article. Others, still unanticipated, will undoubtedly present themselves as further information develops. Much more work is required, in animal models and in Phase I and Phase I1 patient trials, before the chances of clinical success can even be approximated. What is evident today is that the endeavor is likely to contribute important new insights into immunologic function and antitumor defenses. For this reason alone, it is eminently worth the pursuit, and it would be unfortunate if premature enthusiasms and forecasts were, once again, to compromise advancement of an interesting line of investigation in tumor immunology. ACKNOWLEDGMENTS The experimental work here cited from our laboratories was supported by research contracts and grants from the following: NIH Contract NOI-CB-64050; CONCERN Foundation, Inc. of Los Angeles; Ahmanson Foundation ofLos Angeles; Shaklee FoundationofCalifornia; Mr. and Mrs. Dan Belin, Mr. Alan Liker, Mr. and Mrs. Stewart Resnick, and Dr. and Mrs. Wilbur Schwartz of Los Angeles; Lautenberg Endowment Fund of the American Friends of Hebrew University; Society of Research Associates of the Lautenberg Center; Dr. “I” Fund Foundation of New York Aaron Shapiro Memorial Fund; Densen Family Fund; Harold 9. Abramson Memorial Fund; Mr. Stanley Stem, New York; Seymour B. Feldman Leukemia Fund of New Jersey; Leukemia Research Foundation, Inc. of Chicago; Carl Howard Litvin Fellowship for Leukemia Research of Chicago; Mr. and Mrs. J. Ira Hams of Chicago; Mr. Carl H. Lindner of Cincinnati, Ohio; and the Jerome D. Mack Family Trust of Las Vegas. The authors acknowledge with much appreciation the support that made possible our research program, and they expresstheir warm thanks to Drs. Eva Klein and Steven J. Burakoff

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for valuable advice and suggestions with regard to sections of this article, and to Mrs. Roberta Shaked for the preparation of this manuscript.

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CELL SURFACE GLYCOLIPIDS AND GLYCOPROTEINS IN MALIGNANT TRANSFORMATION' G. Yogeeswaran Depanment of Microbiology and Hubert H. Humphrey Cancer Research Center Boston University School of Medone. Boston. Massachusetts

289 29 1 29 1 293 296 296 299 299 304 309 310 315 315 318 320 32 I 322 321 327 330 33 I

I. Introduction. 11. Structure and

A. Cultured Cells .........

B. In Human LymphoproliferativeDiseases C. Epiglycanin................................................. V. Sialoglycoconjugatesand M A. Sialic Acid Content ........

D. Sialyltransferases.................................................................................... E. Sialic Acid and Immunogenicity ............................................................................ V1. Serum Glycoconjugates and Glycosyltransferases- Diagnostic Value and Pathophysiological Significance.............................................................. ...... ............... 332 ............ ........ 336 VII. Summary and Prospects References ......................................................................... ........ 338

I. Introduction

In the last two decades, considerable research effort has been focused on defining changes in cell surface membrane molecules in neoplastic transformation. The emphasis on acquiring new insights into the transformationdependent membrane changes stems from the observations that the cell surface membrane may determine several properties of malignant cells such as increased growth rate, prolonged survival, decreased adhesion, loss of Publication Number 105 of Hubert Humphrey Cancer Research Center. 289 ADVANCES IN CANCER RESEARCH, VOL. 38

Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-006638-6

290

G . YOGEESWARAN

contact inhibition, increased invasiveness and motility, expression of repressed antigens, and escape from immune destruction (reviewed in Wallach, 1968). Particular attention has been paid to defining biochemical changes in cell surface glycosphingolipids (GSL)and glycoproteins (GP) that take place in various types of malignant transformation. Glycosphingolipids and most GPs are integral and intrinsic components of the plasma membrane; that their carbohydrate components are accessible on the exterior of cells is demonstrated by their reactivity to antibodies and lectins and by their removal by various glycosidasesand proteases. Antibodies raised against GSLs often recognize the terminal carbohydrate moieties of lipid (Hakomori and Young, 1978). Hakomori and co-workers showed that cell surface ceramide pentasaccharide (Forssman antigen - GSL) is highly reactive to anti-Forssman antibodies in transformed hamster cells (Kijimoto and Hakomori, 1969). Various blood group structures on the surface of erythrocytes and tissues, whose antigenic determinants are carbohydrates, react with various specific lectins (reviewed in Kabat, 1970). Certain lectins that are shown to induce mitogenesis in lymphocytes exert their action by binding to cell surface carbohydrate determinants (Nowell, 1960). Data derived from other approaches, such as cell surface labeling using galactose oxidase (Gahmberg and Hakomori, 1973a; Steck and Dawson, 1974) and the lectin binding studies of Oseroff et al. (1973), lend additional support to the idea of exterior localization of oligosaccharide moieties of GP. Twothirds ofcell-bound sialic acid (in GSLs and GPs) can be removed from intact cells by neuraminidase (NANase) in CHO, L, HeLa, and BHK cell lines (Kraemer, 1967), and the treatment does not affect viability. Indeed, NANase treatment and/or trypsin treatment of quiescent cells stimulate them to divide (Vaheri et al., 1972). A very high proportion of cell surface sugars is released by mild trypsinization without loss of cell integrity (Shen and Ginsberg, 1968; Onodera and Sheinin, 1970). These studies clearly demonstrate that the carbohydrate residues of GSL and GP are exposed to the cell exterior. As a result of increased growth and understanding in the field ofcell surface glycoconjugate biochemistry, several reviews (Hakomori, 1975a, 1981 ; Morre et al., 1978; Fishman and Brady, 1976;Emmelot, 1973; Hynes, 1976; Warren et al., 1978; Atkinson and Hakimi, 1980;Pearlstein et al., 1980a),a monograph (Vaheri et al., 1978),and a recent book on the surfacesof normal and malignant cells (Hynes, 1979) have been written on this subject. This article will be concerned with reviewing past and recent literature on the structure and function of glycoconjugates, transformation-associated GSL and GP changes, the relationship of these membrane glycoconjugatechanges in altered growth properties, tumorigenesis, and antigenic changes. The last section will deal with glycoconjugatesthat are released from the tumor cells

GLYCOLIPIDS AND GLYCOPROTEINS IN CANCER

29 1

through turnover, secretion, and shedding. The appearance of these glycoconjugates and glycosyltransferases in sera of tumor-bearing animals and humans may have diagnostic and prognostic value, and may also be important determinants in the pathophysiology of cancer. 11. Structure and Function of Glycosphingolipids and Glycoproteins

A. STRUCTURE OF GSL Research work done in the past 20 years or so in the field of complex carbohydrates has resulted in the discovery of a great degree of structural diversity among GSLs and GPs of eukaryotic cells. Many structurally different GSLs and GPs are encountered on the surfaces of cells by virtue of the occurrence of different monosaccharide content, monosaccharide sequence, and the nature of linkage and anomeric configuration between the constituent monosaccharides. Although over 100different monosaccharides are found in various biological systems, only about seven monosaccharides are seen in the oligosaccharidesof mammalian cell surface GPs and GSLs. These are sialic acid (acylneuraminic acid, NAN), N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), D-galactose (Gal), D-mannose (Man), D-glucose (Glu), and L-fucose (Fuc). Glycosphingolipid from mammalian cells lacks mannose, but often contain glucose as the innermost sugar attached to the lipid moiety of this molecule. Glycosphingolipids are composed ofthree basic structural units: a base, fatty acid, and carbohydrate. The lipid moiety of GSL contains a long-chain amino-alcohol base, the most common unit being 4-sphingenine (sphingosine), to which one of a variety of fatty acids of varying chain length (Cl.,-C26)is linked via an amide bond. This structure is called ceramide (C). The carbohydrate units, comprising the hydrophilic component, are linked to ceramide via a glycosidic bond between the sugar hydroxyl group and the primary hydroxyl group of the sphingosine unit. The individual monosaccharides of GSLs and GPs are linked together by a glycosidic bond between the anomeric carbon of a monosaccharide (carbon 1 of hexoses, and carbon 2 of NAN) and one of several hydroxyl groups ofthe other monosaccharides ( 1 1 2 , l 1 3,l 1 4 , l 1 6, 2 1 3, 2 1 8) resulting in a (perpendicular) or p (planar) configuration. More complex oligosaccharide-containingGSLs are formed from simple ones, by the sequential addition of monosaccharides from nucleotide sugars by membrane-bound glycosyltransferases as shown in Fig. 1 (reviewed in Schachter and Roseman, 1980). Glycosphingolipids can be subdivided into three major families: neutral GSL, gangliosides, and sulfato GSL. Gangliosides are characterized by the presence of sialic acid, and sulfato GSLs contain carbohydrates substituted

Ceramide I

i

C-Glu

(c) (CM)

(Ceramide Monoherosidel

C-G~U-G~~-G~~NAC-G~~-FU= (A-Glycolipid)

I

GalNAc

C-Glu-Gal-GalNAc-Gal-

I

NAN

-blood

Group GSL-

--Neutral

GSL-

-Asialogangliasides-

C-Glu-Gal-GalNAc-Gal

(GTla)Nk-NAGQ) NAN-NAN I

-Pathway-I-

- Pathway-I1

I

NAN-NAN

~

Gang1 iosides FIG. 1. Probable biosynthetic pathway of major GSL classes of cell membrane, indicating various known examples of blocks in the biosynthesis ofGSL homologs. Arrows indicatesteps inhibited in the biosyntheticpathwayand numbersindicatea random ordenngofknown blocks(seedetai1s in text). The system of nomenclature of gangliosides is that of Svennerholm ( 1964).Terms given in parentheses refer to certain trivial names for GSLs. For details of glycosidic linkage and anomeric configurations, see a general description in Fig. 2.

293

GLYCOLIPIDS A N D GLYCOPROTEINS IN CANCER Classes of GSL Neutral GSL

Structure

Series Prefix

C-Glc(4.1 H)Gal(4,1tx)Gal (3,Ifi)GalNAc

Globo

C-Glc(4.1 B)Gal13,1~1Gal [ 3.1 LOGalNAc

Globoiso

Gangliosides

C-Glc (4,IY)Gal(4,1il)GalNAc ( 3 , I R ) Gal

Ganglio

Blood Group GSL

C-Glc (4,18IGal(3,1 B l GlcNAc (3.1 8)Gal

Lacto

C-Glc (4,1B)Gal(3,1n l GlcNAc (4,1B ) Gal

Lactoneo

FIG. 2. Major core structure of five GSL series described under the systematic nomenclature (Sweeley and Siddiqui, 1977). See text for details.

with sulfate ester groups. For the purpose of a recently proposed systematic nomenclature, the GSL can be divided into many series as shown in Fig. 2. This system of nomenclature (reviewed in Sweeley and Siddiqui, 1977) uses certain prefixes which imply the core structure for these series of GSL as shown in Fig. 2. However, the majority of the papers reviewed in this article use a system of trivial nomenclature and abbreviations to describe GSL; these symbols and abbreviations are preferred in this article. The class of complex neutral GSLs, globoside and Forssman GSL contain a globo-core structure (Fig. 2). Complex gangliosides and asialogangliosides contain a ganglio-corestructure and these two classes of molecules are derived from a common precursor, ceramide lactoside (CL) (see Fig. 1). The blood group GSL, belonging to the category of neutral GSLs (Figs. 1 and 2), contains a lacto- or lactoneo-core structure. Although over 7 1 structurally different GSLs have been discovered in mammalian cells (reviewed in Macher and Sweeley, 1980), only the major GSLs shown in Fig. 1 and Table V will be discussed in this article with reference to malignant transformation. It is a generally held view that GSLs enrich and are present in the outer leaflet ofthe plasma membrane of the lipid bilayer, with its carbohydrate moiety oriented toward the cell exterior. The evidence for this concept comes from the studies of the erythrocyte membrane (Gahmberg and Hakomori, 1973a; Steck and Dawson, 1974). The generality of this concept to other cell types awaits further work. B. FUNCTION OF GSL A high concentration of GSLs found in the outer leaflet of the plasma membrane suggests that these lipids may confer structural rigidity to the membrane because of the presence of the ceramide structure, which contains both hydrogen acceptor moiety (amide carbonyl) and a hydrogen donor (secondary hydroxyl of sphingosine), and sugar residues (Abrahmsson et al., 1977). A higher rigidity of phospholipid liposomes containing GSLs has been

294

G. YOGEESWARAN

noticed in comparison to phospholipid liposomes without GSL (Sharom and Grant, 1977). Other functions attributed to GSLs, such as cell surface receptor for various substances, cell surface markers, and antigens, are conferred by the carbohydrate moieties (Table I). The role of gangliosidebound carbohydrates as receptor for toxins, hormones, viruses, interferon, fibronectin, lymphokines, and serotonin may not be exclusive for gangliosides, because a number of proteins bearing ganglioside-oligosaccharides are demonstrated by two recent studies (Tonegawa and Hakomori, 1977; Fukuda et al., 1979). Although it has been suggestedthat the GM, ganglioside is the cell surface receptor for cholera toxin (Van Heyningen et al., 1971; Holmgren et a!., 1973),the interaction per se does not indicate that GMlis the real cell surface receptor for cholera toxin in all cell types, because toxin-sensitive adipocytes and adrenal cells lack GMl (Kanfer et al., 1976), and in mouse LY and human KB cells, the toxin receptor is trypsin sensitive (Grollman et al., 1978). This indicates that certain GPs bearing GM1 determinants may play a receptor role as suggested by Tonegawa and Hakomori (1977). Van Heyningen (1967) and Ledley et al. (1977) demonstrated that GTlaand G, could inhibit the action of tetanus toxin on cells, suggestingthat these gangliosides could be involved in the receptor for the hormone. It has been suggested that gangliosides are the receptor for botulinum toxin (Simpson and Rapport, 1971) and gonococcus pilli-protein (Buchanan et al., 1978). GD1b ganglioside has been shown to inhibit the action of thyrotropin (Mullin et al., 1976) and G, ganglioside inhibits the action of chorionic gonadotropin and leuteinizing hormones (Lee et al., 1976, 1977). The proposed ganglioside receptor for thyrotropin, G D l b , is shown to be enriched in target tissue thyroid plasma membrane (Mullin et al., 1976). Besancon and Ankel(l974) showed that G M z ganglioside inhibits the antiviral effect of mouse p-interferon. Vengris et al. (1976) supported the observations of Besancon and Ankel (1974) by enrichment of the putative p-interferon receptor ganglioside in GMz-deficientcells, which resulted in interferon responsiveness in these cells. However, a very recent study by Belardelli et al. (1982) showed that the gangliosides may be either nonspecifically bound to the interferon by the hydrophobic interaction or may serve as low-affinity receptors on cell surfaces (Grollman et al., 1978). The real high-affinity receptor for interferon may be a protein conferring species specificity for interferon action (Belardelli eta!., 1982).Gangliosidessuch as G,,, and GTlb inhibited cell attachment on a fibronectin - collagen layer suggesting that these gangliosides on cell surfaces may serve as interactants in the intercellular matrix. A fucosylated gangliopentaosyl ceramide GSL abundant in macrophages and granulomas is considered to be a lymphokine receptor,

GLYCOLIPIDS AND GLYCOPROTEINS IN CANCER

295

TABLE I FUNCTIONS OF MAMMALIAN GLYCOSPHINGOLIPIDS Postulated functions Structural component of PM conferring structural rigidity Cell surface receptor Bacterial toxins Cholera toxin-G,, Tetanus toxin-GT,,, G, Botuliiium toxin-G,,, Gonococcus piIli-G, I Glycoprotein hormones Th yrotropin-G,, , Chorionic gonadotropin and leutinizing hormones-G, Virus Sendai virus-GTla,G,

Interferon Interferon only-G,, Fi bronectin GDI,,G T I ~ Lymphokines Macrophage migration inhibitorfucosylgangliopentaosylceramide Serotonin Gm Cell surface antigens and markers A, B, H, Lewis, Ii blood groups Forssman determinant Teratocarcinoma differentiation antigen Tumor-associated antigens (see Table V) T cell marker NK cell marker Transport Cholera toxin effect on Na+-K+ transport gangliosides Na+ transport, K+ effux

Reference Sharom and Grant (1977)

Van Heyningen efal. ( I 97 1); Holmgren ef al. ( 1973) Van Heyningen ( I 967); Ledley ef al. (1977) Simpson and Rapport (197 I ); Kitamura et a/. (1 980) Buchanan ef a/. (1978) Mullin ef a/. (1976) Lee ef al. (1976, 1977) Haywood (1974); Markwell ef a/. (1981); Holmgren ef 01. ( 1980) Besancon and Ankel (1974); Vengris ef a/. ( 1976): Ankel et al. ( 1980) Kleinman ef a/. (1979) Higgins ef a/. ( 1978); Miura ef a/. ( 1 979) Woolley and Gommi (1 963) Hakomori ef a/. (1977a); Koschielak el a/. ( 1979) Siddiqui and Hakomori ( 1 97 1) Stern ef al. (1978): Muramatsu ef a/. (1979): Nudelman et a/. (1980); Kapadia ef a/. (1981) Stein ef a/. (1978) Kesai ef al. ( I 980); Young ef al. (1980) Van Heyningen et al. (1971) Karlsson (197 1)

296

G. YOGEESWARAN

because it strongly inhibits the action of macrophage migration inhibition factor (Higgins et al., 1978; Miura et al., 1979). The carbohydrate moieties of various GSLs serve as antigenic determinants and markers on cell surfaces. Several such antigens are described in Table I. GSLs are also considered as tumor-associated cell surface antigens and these will be discussed in Section III,D. Glycosphingolipids are also involved in ion transport functions of the membrane. Karlson et al. (197 1) have shown that sulfatides may be involved in the K+-selectivecofactor site of Na+- K+-dependant adenosine triphosphatase. Other indirect evidence from cholera toxin studies suggests that gangliosides may also be involved in Na+- K+ transport (Van Heyningen et al., 197 1). Gangliosides also regulate cell surface levels of Ca2+by direct binding via sialic acid residues. For example, gangliosides are shown to be a Ca2+-bindingcofactor in synaptic transmission (Svennerholm, 1980). OF GLYCOPROTEIN OLIGOSACCHARIDES C. STRUCTURE

The oligosaccharides of GPs also comprise the same monosaccharides as GSLs (see above) with the exception of glucose and with the inclusion of mannose. The oligosaccharides of GPs are of two types: (1) simple alkalilabile (0-linked) oligosaccharide chains bound to serine (Ser)/or threonine (Thr), containing GalNAc, Gal, GlcNAc, and NAN; and (2) complex alkali-stable (N-linked) oligosaccharides bound to asparagine (Asp) residues of the polypeptide chain, containing Man, GlcNAc, Gal, NAN, and Fuc. A few examples of complete oligosaccharide chains of membrane GPs are shown in Fig. 3. The number and type ofN-linked and/or 0-linked oligosaccharides attached to the polypeptide chain of proteins vary among different GPs. The branching of oligosaccharidechains of asparagine-linkedcomplex oligosaccharides also varies from two to four branches per oligosaccharide (see Fig. 3, structures 1-4).

D. FUNCTION OF GP Various known functions of GPs are listed in Table 11. Many of these functions are attributed to carbohydrate structures of GP, while in certain other GPs the sugars have no function. For example, sialic acid residues of various GPs determine their circulating half-life. Upon removal of sialic acid, many GPs are cleared from circulation, through binding ofpenultimate galactosyl or galactosaminyl groups by hepatocytes (Ashwell and Morrell, 1974).The carbohydrate moieties ofGP play a receptor role for cholera toxin (Tonegawa and Hakomori, 1977; Fukuda et al., 1979), Sendai virus (Markwell and Paulson, 1980), and serotonin (Carrol and Sereda, 1968). In other

N- L I NKED

0-L I NKED G

“4;v

I

t6,p?.72

A S W C ~ C N A C GL l c N A c a M a n

~

c

N

A Gc a l ~-

NAN

1

‘klcNAcL4.1

Gal-13‘2

NAN

I

0 ASn-GlcNAc

I

T

ClCNAc

1

-

ClcNAc

Man

t_

1 \

Man

‘G~cNAc ,GlCNAc

‘GlcNAc

NAN

-

G a l -NAN

-

G a l -NAN G a l c _ NAN

-Gal-NAN

FIG. 3. Some examples of oligosaccharide structures of major cell surface GPs. ( I ) Vesicular stomatitis virus envelope GPoligosacchande (Readinger a/.. 1978). (2)MajoroligosaccharidestructureofcalfthymocyteplasmamembraneGP(Kornfeld, 1978). (3)Rabbit liverplasma membrane GP oligosaccharide structure (Kawasaki and Ashwell, 1976). (4) BHK polyomavirus-transformed cell membrane glycopeptide (Ogata er a/.. 1976). (5). (6),and (7) are alkali-labile oligosaccharide structures from epiglycanin (Codington ef a/.. 1975b Van den Eijnden, 1979). Structures ( 6 )and (7) are also present in B16 mouse melanoma cell surface GP (Bhavanandan er a/.. 1977).

298

G. YOGEESWARAN

TABLE I1 FUNCTIONS OF MAMMALIAN GLYCOPROTEINS Postulated functions Homeostasis Sialic acid on GP determines the survival of serum GP and tumor-associated GP Cell surface receptor Toxin Cholera toxin-(;,, like oligosaccharideganglioprotein Hormone Insulin receptor-GP Virus Sendai virus-GP Serotonin Sialic acid of GP Cell surface antigens and markers A, B, H, MN blood group structures Ii determinant-polylactosamine-GP Carcinoembryonic antigen a-Fetoprotein Histocompatibilityantigen Ly and OKT antigens of lymphocytes Surface GP on subsets of T cells, B cells, and other blood cells

Reference Ashwell and Morel1 ( 1974)

Tonegawa and Hakomori (1977) Cuatrecassasand Illiano (197 I ) Markwell et al. (1981) Carrol and Sereda (1968) Takasake and Kobata (1976) Fukuda et a/. (1979) Gold and Freedman ( 1965) Abelev et a/. (1963) Nathenson and Cullen (1974) Gahmbergand Andemon( 198I); Andersson and Gahmberg ( I 979)

Immune system Immunoglobulin-complemenrmediated cytotoxicity

Koide ef a/. (1977)

Adhesion and aggregation Fibronectin binding to collagen Neural cells Terratoma

Yamada ef a/.( 1 978) Hausman and Moscona (1975) Oppenheimer (1975)

Transport RBC-anion transport-GP

Ho and Guidotti (1975)

Survival of hormones and enzymes, and activity of enzymes GP hormones and glycoenzymes

Reviewed in Warren et a/. ( 1 978)

glycoprotein receptor molecules such as insulin (Cuatrecassas and Illiano, 1971), the carbohydrate moieties play no functional role. Among cell surface markers and antigenic glycoproteins,the carbohydrate moieties play a role as antigenic determinant. For example, in A, B, H, MN, and Ii blood group substances, the carbohydrate residues act as the antigenic structures. In

GLYCOLIPIDS A N D GLYCOPROTEINS IN CANCER

299

contrast, in other cell surface markers and antigens such as histocompatibility antigen (Nathenson and Cullen, 1974),carcinoembryonic antigen (Gold and Freedman, 1969, a-fetoprotein (Abelev et al., 1963), and lymphocyte markers, the carbohydrate moieties are not responsible for their function or antigenicity. Although fibronectin is a glycoprotein, the adhesive properties responsible for binding to collagen and cell spreading are not determined by the carbohydrate residues (Olden et a]., 1979). Anion transport protein of erythrocyte is a glycoprotein, but the carbohydrate residues are not involved in the transport function (Ho and Guidotti, 1975). 111. Glycosphingolipids and Malignancy

A. CULTURED CELLS 1. Transformation-Associated GSL Alterations in Fibroblast and

Undiferentiated Cells Historically, transformation-associated GSL alterations were first discovered in cultured cells by Hakomori and Murakami (1968). Since this first report, there has been tremendous growth in this field, and Table 111lists the observations made in GSL changes associated with cell transformation. Changes in GSL composition have been observed as one ofthe characteristic phenotypic alterations found in various transformed fibroblasts of hamster, mouse, rat, chicken, and human irrespective of the agent of transformation, namely, DNA viruses, RNA viruses, DNA- and RNA-thermosensitive viruses, and chemical carcinogens (Table 111). Reduction in the oligosaccharide chains (Type 1 change) of neutral GSL (Hakomori et al., 1971a; Robbins and MacPherson, 1971;Gahmberg and Hakomori, 1975),gangliosides (Hakomori and Murakami, 1968; Mora et al., 1969; Brady and Mora, 1970; Siddiqui et al., 1970; Yogeeswaran et al., 1972; Diringer et al., 1972; Brady and Fishman, 1974; Langenbach, 1979, and fucolipids (Steiner and Steiner, 1976;Skelley et al., 1976;Itaya et al., 1976)was the common change noticed in transformants. Occasionally, precursor GSL accumulates as a consequence of the loss of higher GSL homologs (Hakomori and Murakami, 1968; Mora et al., 1969; Brady and Fishman, 1974; Yogeeswaran et a]., 1972).However, in other studies, an elongation ofthe oligosaccharidechains of GSLs (Type 2 change) was noticed following viral transformation (Yogeeswaran et al., 1972; Diringer et al., 1972). A third type of GSL alteration associated with cell transformation has to do with the appearance of new GSLs which are present in trace amounts in progenitor cells. For example, Gahmberg and Hakomori ( 1975)observed a high concentration of paragloboside (an isomer of globoside) in hamster cells transformed by polyoma-

TABLE 111 ALTEREDGSL PATTERN IN TRANSFORMED CELLS IN CU LTU RE Species, tissue of origin, and cells

W

a a

Hamster Fibroblast BHK-C/ 13 BHK-C/ 13 Nil-2E Nil-2

Agent of transformation

Polyomavirus Rous sarcoma virus Polyomavirus Simian virus 40

Nil-ICl, 2C1 Nil-2K Mouse Fibroblast 3T3 3T3

Polyomavirus

3T3

Polyomavirus Polyomavirus Simian virus 40 Simian virus 40 Simian virus 40

AL/N 3T3

Friend virus Simian virus Murine sarcoma virus

Glycosphingolipid change

CDf GMd, CDt CTJ, Forssman GSLJ CT, globoside Forssman GSLJ GM,J,globoside, CT, Forssman GSLJ Globoside, Forssman GSLJ, paragloboside G ~ 3 i r

GM,, GM,,

GMl,

GD,, absent, GM3t Go,, absent

GMi, GDh absent, GM3t GM31,

Gist

GM3J,G,,,1 after a few generations GM3?

GMl1, GDIaJ,

GM3?

Reference

Hakomori and Murakami (1968) Hakomori et al. (1968) Hakomori ef al. ( 197 I a) Hakomori ef al. ( I97 I a) Robbins and MacPhenon (1971) Sakiyama and Robbins (1973) Gahmberg and Hakomori (1975)

Brady and Mora ( 1970) Yogeeswaran ef al. (1972) Yogeeswaran ef al. (1972) Yogeeswaran et al. ( 1 972) Diringer ef al. (1972) Diringer ef al. (1972) Mora et al. (1969)

3T3 Embryonic

3T3 L cell

Murine leukosis virus Murine sarcoma virus (Kirsten strain) Simian virus 40 Herpes simplex virus Dimethylbenzanthracene Methylcholanthrene Kirjtenmurine sarcoma Low and spontaneous hybrids

GM3t,

GhlZir G M I i ?G D I s i

GM3t3 GMZt, G M l l ? G D I a i

Complex fucolipidl Complex fucolipidi GM3t GMZ1. G D l b t

GD,& new GSL. Asialo-GM,t Complex long chain J, GSLJ,

Brady and Fishman (1974) Brady and Fishman ( 1974) Steiner and Steiner (1975) Steiner and Steiner (1975) Langenbach pf al. (1976) Langenbach ef al. ( 1976) Rosenfelder ef al. (1977) Itaya ef al. ( 1976)

GM3f

W

Rat Embryonic Kidney fibroblast Hepatocytes Chicken, embryo Fibroblast

0

Human Fibroblast Lymphblastoid Chicken, embryo Hamster Fibroblast BHK BHK-DMN-B

Meth ylcholanthrene Munne sarcoma virus N-Fluoren ylphthalamide

GM3ir G M 2 i ~G D l b i

Long chain fucolipid 1 GDla17

GM3t

Langenbach (1975) Skelley ef al. ( 1 976) Brady ef al. ( 1969)

Rous sarcoma virus Rous sarcoma virus

GDabsent GM3C, GD3J

Siddiqui e/ a/. (1970) Hakomori ef al. ( I97 I a)

Simian virus 40 Simian virus 40 fs mutant ASV

CMt, CDl, G ~ 3 1 G Mabsent. ~ GMZt CMJ, CDt, CTf, globosidet No change in GSL GM~J

Hakomori ef al. (1968) Hakomori el a/. (1971a) Levis el a/. (1976) Warren el 01. ( 1972) Hakomori ef al. (1977b)

fs mutant polyoma Dimethylnitrosamine

CMt, CDt, CTJ

fx mutant RSV

GM34,

cDt

Gahmberg er al. (1974) Bueler and Moolten ( I 975)

302

G. YOGEESWARAN

virus. Another example of this type of change is the appearance ofasialo-GM, in Kirsten murine sarcoma virus (Ki-MuSV) transformed BALB/3T3 cells (Rosenfelderet al., 1977;Yogeeswaran and Stein, 1980).These GSL changes seen in different types of transformation are not incidental “late” phenotypic changes associated with an outgrowth of a clonal population of transformant, because in RNA virus transformation, where all normal cells are transformed in a relatively short time, several investigators have observed a consistent GSL change associated with transformation (Hakomori et al., 1968; Siddiqui et al., 1970; Hakomori, 1970). In other studies in which temperature-sensitive (ts)mutant DNA and RNA viruses have been used as the transforming agent, GSL changes were seen as very early membrane changes associated with the activation of transforming (src)gene (Hammarstrom and Bjursell, 1973; Gahmberg et al., 1974; Hakomori et al., 1977b3. Buehler and Moolten ( 1975) also concluded that the transformation event can result from a mutation affectingGSL metabolism by using a ts mutant of the dimethylnitrosamine-transformed BHK cell line. However, in two other reports, gross biochemical change was not observed with cell transformation by ts mutant viruses (Warren et al., 1972; Itaya and Hakomori, 1976). Many of the transformation-associated GSL alterations described above indicated a gross change in chemical composition as evidenced by the use of chemical and radiochemical techniques. With the advent of cell surface labeling techniques and production of antibodies to GSLs, alterations in the organization and cell surface exposure of GSL have been noticed in several transformants. For example, Hakomori and Kijimoto (1972)and Gahmberg and Hakomori (1975) reported an increase in the cell surface exposure of globoside and Forssman GSL in transformed hamster fibroblasts although the chemiquantity is decreased relative to control cells. In another study, Itaya and Hakomori (1976) reported an increase in cell surface exposure of asialo-GM,in the transformed state of ts SV40-transformed 3T3 cells although these cells had no gross change in chemical composition of GSL. The study of cell surface GSL of transformants using these techniques may provide additional evidence of other subtle changes associated with malignancy. 2. Alterations in GSL Metabolism in Transformed Cells Seeking the metabolic basis of GSL alterations, a number of investigators measured the activities of glycosyltransferases in transformed cells (Fig. 1). Cumar et al. (1 970) and Fishman et al. ( 1972) observed that G,,:UDPGalNAc-galactosaminyltransferasewas drastically reduced (Fig. 1, Block 3) in SV40 and polyomavirus-transformed mouse 3T3 cells as compared to nontransformed 3T3 cells. These transformants did not show altered levels of sialidase,indicating that a decrease in GM,and higher gangliosidesis due to

GLYCOLlPlDS A N D GLYCOPROTEINS IN CANCER

303

an impairment in synthesis rather than to an increased degradation of gangliosides. Impairment ofGSL synthesiswas also observed in other studies of transformed hamster fibroblast (Kijimoto and Hakomori, 197 I ) which showed a 10- 50%reduction of CD:UDPGal-a-galactosyltransferase(Fig. 1, Block 1).Den et al. ( I97 1) observed that the enzyme synthesizing GM3 from CD was reduced about 15%from the normal cell level (Fig. 1, Block 2). In another report, Den et al. (1974) reported that “flat” revertants of polyomavirus-transformed hamster cells showed a two- to fourfold increase in “blocked” enzyme activity as compared to the transformed cells. However, this gain in the enzyme activity in nontumorigenic “flat” revertants was only 10% above control cells, indicating a lack of perfect correlation between altered glycosyltransferaseand the transformed state. These authors did not study the levels of ganglioside catabolic enzyme to explain their negative result. A decrease in G,,:UDPGalNAc-galactosaminyltransferase was also observed in RNA virus-transformed 3T3 cells as well (Mora et al., 1973). Deletion of GM,and increase of GM2,due to a loss of specific galactosyltransferase (Fig. 1, Block 3), was observed in Ki-MuSV-transformed 3T3 cells (Fishman et al., 1974).In contrast to the earlier claims of Cumar et al. (1970) and Yogeeswaran and Hakomon (1975) who showed that the levels of sialidase were unaltered in transformants, Schengrund et al. (1973, 1976) observed a significantincrease in sialidaseactivity in transformed cells. Thus, it appears that altered neutral GSL and ganglioside composition in the transformants may be due to a decrease in synthesis and possibly to an increase in the catabolism of GSL. Several nontransformed cultured cells show an increase in the quantities of certain GSL during cell contact, and this response has been termed “contact-extension response” (Hakomori, 1970). This cell density-dependent GSL augmentation was observed in CT glycolipid in BHK cells (Hakomon, 1970), Forssman GSL and G M , in Nil cells (Sakiyama et al., 1972; Critchley and MacPherson, 1973), GD,, in 3T3 (Yogeeswaran and Hakomori, 1975) and C3H mouse fibroblast (Langenbach and Kennedy (1978), and G,, and GMlin human fibroblast (Hakomori, 1970). The enzymatic basis of this GSL augmentation in cell density-inhibited nontransformed cells was shown to be due to an enhancement of the activity of glycosyltransferase (Kijimoto and Hakomori, 1971; Chandrabose et al., 1976)and/or suppression of sialidaseactivity (Yogeeswaran and Hakomori, 1975). The transformed cells generally lacked this “contact-extension response” (Hakomori, 1970; Sakiyama et al., 1972; Critcheley and MacPherson, 1973; Yogeeswaran and Hakomori, 1975). Interestingly, chemically transformed mouse embryo cells showed a pattern of GSL similar to nontransformed cells, but lacked the cell density-dependent GDl, enhancement as compared to control cells (Langenbach and Kennedy, 1978).

304

G. YOGEESWARAN

However, the lack of contact-extension response of GSL metabolism does not always correlate with increased tumorigenicity (Sakiyama and Robbins, 1973), and highly malignant Ehrlich ascites carcinoma shows this response (Prokazova et af., 1978). B. TUMORS 1. Animal Model Systems

Extensive work has been done to define GSL changes associated with malignant transformation using in vivo tumor tissue (see Table IV). In these studies, the investigators have compared the GSL pattern ofthe tumor tissue with surrounding normal tissue, or a transplantable tumor has been compared with a corresponding normal tissue of origin for the tumor. The majority of earlier workers in this field compared the GSLs ofpopular Morris minimal deviation rat hepatomas with control liver. The pioneering work done in this field is by Siddiqui and Hakomori (1970) and Cheema et af. ( 1970), who showed a decrease in G M 3 , GDlb, and GTlb and an increase in G D l a in hepatoma relative to control liver tissue (Fig. 1 , Block 7). These investigators also noticed an overall increase in theganglioside content in tumor tissue although there was a simplification of ganglioside pattern relative to control liver tissue. Subsequent work done by Dnistrian et af.( 1977, 1979) extended these studies on Morris hepatoma and showed that the altered ganglioside pattern seen in tumor tissue is demonstrable in isolated plasma membrane fractions as well as other inner membrane fractions. In addition, Dnistrian et al. (1977) showed an increase in ceramide monohexoside (CM), ceramide dihexoside (CD), and an overall increase in ganglioside sialic acid. Keenan and Morre (1973) reported an accumulation of GMland a loss of GDlaand G,,b in the cell membranes, a striking decrease in G,,:CMPNAN-sialyltransferase, and an overall increase in ganglioside content (Fig. 1, Block 5 ) in dimethylbenzanthracene-induced mammary carcinoma compared to mammary tissue in female rats. In order to establish the general significance of ganglioside changes in tumorigensis, Merritt et al. (1978a) used an in vivo hepatocarcinogenesis model. They observed that the development of tumors was slow with the carcinogen (N-fluorenylacetamide) so that temporal changes in the biochemical and morphological development of carcinogenesis could be easily identified with this model system compared to previous models. They found that the total ganglioside levels were elevated in all but poorly differentiated tumors. Their study also documented that the ganglioside pattern showed a progressive simplification from hyperplasia to malignant hepatoma. The enzyme G,,:CMPNAN-sialyltransferase responsible for synthesis of G,,

GLYCOLlPlDS AND GLYCOPROTEINS IN CANCER

305

leading to disialoganglioside pathway was decreased (Fig. 1, Block 6), contributing to a decrease in GD, and G,,,. The enzyme GD,,:CMPNANsialyl-transferase responsible for synthesis of GTlbwas also decreased and enzymes responsible for the monosialogangliosidepathway increased (GM3, GM,, and G,,, synthetases) in the hepatoma tissue (Memtt et af., 1978b). Thus, their study confirmed that ganglioside alterations may be an early event in tumorigenesis. The appearance of high levels of a novel fucosylasialo-G,, ganglioside was described in another type of transplantable hepatoma induced by N-2,7fluorenyl bis-trifluoroacetamide, whereas fucosylasialo-G,, was a minor component in the control liver tissue (Baumann et al., 1979).In addition to these qualitative fucolipid changes, the quantity of total fucolipid and cellular fucose content increased in hepatoma tissue. Simplification of complex gangliosides was also demonstrated in several transplantable avian lymphomas and was shown to be due to a block in synthesis (Keenan and Doak, 1973).

2. Human Gastrointestinal and Respiratory Tract Neoplasms In contrast to transformed cultured cells and malignant animal tumors that showed a qualitative change in GSL pattern associated with transformation, a few earlier studies of human tumors showed only a quantitative change in GSL (Table IV). For example, the studies of GSL composition of lung adenocarcinomas and squamous cell carcinomas showed an increase of CM, CD, ceramide trihexoside (CT), globoside, and all the ganglioside homologs with no change in GSL pattern (Narasimhan and Murray, 1979). Similar findings were made in a study of squamous and small cell lung carcinomas which showed an increase in CM and CD. However, in other lung adenocarcinomas, sulfatide increased but its precursor GSLs such as CM and CD decreased relative to control lung tissue (Yoda el al., 1979). Several reports of gastric and colon tumors indicate an increase in various classes of GSL, but an absence of qualitative change. Twenty-five human gastric and 1 1 colon adenocarcinomas were analyzed for total content and ganglioside pattern by Keranen et af.(1976), and they found an increase in the content of all ganglioside homologs. Siddiqui et al. (1978) also found an increase in CD, galactosylceramide,Leb-GSL,and sulfatide in human colon tumors compared to normal tissues. In other studies, Pacuszka et al. ( 1980) also observed an increase in C-Glu, C-Gal, CD, and certain fucolipids in gastric and colon tumors. Interestingly, studies by Hakomori et af.(1977~) interpreted these results as an example of anomalous GSL expression in tumors, and these GSL changes could be related to a previously described increase in “A-like antigen.” Hattori et af. (1982) showed that in several human gastric cancers, although neutral GSL, sulfatide, and GM3ganglio-

TABLE IV ALTERED GSL PATTERN IN TUMOR TISSUES Species, tissue of origin, and cells Aniamal models Rat, hepatoma Moms, 5 123,7800 Moms minimal deviation Mammary Carcinoma Moms hepatoma 5 123TC Moms hepatoma 5 123TC

Agent of transformation

Glycosphingolipid change

Reference

N-2-Fluorenylphthalamic acid N-2-Fluorenylphthalamic acid 7,12-Dimethylbenzanthracene N-2-Fluorenylphthalamic acid N-2-Fluorenylphthalamic acid

Siddiqui and Hakomori (1970) Cheema et af. ( 1970) Keenan and Morre (1973) Dnistrian ef al. (1 977) Dnistrian ef al. ( 1979)

Hepatoma

N-2-Fluoren ylacetamide

Memtt et al. (1978a)

Hepatoma HTC, H35

N-2,7-Fluorenylbistrifluoroacetamide Methylcholanthrene

Baumann ef a/. (1979)

RPL 12 virus ? Spontaneous

Keenan and Doak (1973) Yogeeswaran et al. ( 1 978)

Mammary carcinoma Metastasizing and nonmetastatic Avian, lymphoma Mouse, melanoma Human tumors Brain tumon

Skipski et uf.(1981)

Oligodendroglioma, astrocytoma, glioma Neuroblastoma Astrocytoma, glioma Glioma, astrocytoma Gastrointestinal and respiratory tract Adenocarcinoma

Spontaneous Spontaneous Spontaneous Spontaneous

Keranen et al. ( 1976)

Spontaneous

No change in ganglioside profile, increase in all gangliosides Globosidef ,Forssmanf All ganglioside 7, CDt, Leb-GSLt, sulfatide 7 No change in profile, all gangliosides 7, CMt, CDt, CTt, globoside f CMt, CDt

Spontaneous Spontaneous

CMJ, CD1, sulfatidet CDf, fucoglycolipids t

Yoda et af. ( 1979) Pacuszka ef al. (1980)

Spontaneous

GM3f,sulfatide 7, fucoglycolipids f, blood group A-GSLf

Hatton ef ul. ( 1982)

Spontaneous Spontaneous

CMf, CDf G M I f, GD,,f

Hildebrand ef al. (197 1) Hildebrand ef al. (1972)

Spontaneous

Gastric carcinoma Colon adenocarcinoma

Spontaneous Spontaneous

Lung adeno, squamous Adeno, squamous carcinoma Lung, squamous and small cell carcinoma Lung adenocarcinoma Gastric and colon carcinoma Gastric carcinoma

Spontaneous

Reticuloendothelial CML, CLL CML, CLL

Kanazawa and Yamakawa ( 1974) Shochat ef al. (1977) Yates et a/. (1979) Traylor and Hogan ( 1980)

Hakomori et al. (1977~) Siddiqui el nl. (1978) Narasimhan and Murray (1979) Yoda et al. (1979)

308

G. YOGEESWARAN

sides increased only quantitatively, fucolipids increased and blood group A-like GSL increased in 0 blood group patients. Thus, the observations of Hatton et al. (1982) and Hakomori et al. (1977a) may be similar and emphasize that qualitative changes in GSL do occur in some human cancers, althoughthese changesare restricted to a certain class ofGSLs in carcinomas, and more common neutral GSL and gangliosides do not show the transformation-associated changes observed in cultured cells (see Section III,A, 1). 3. Human Brain Neoplasms Earlier studies by Kanazawa and Yamakawa (1974) on the neutral GSL composition of human oligodendrogliomas, astrocytomas, and gliomas showed an increase in CM and CD, positively correlatingwith the increase in malignancy (see Table IV). Schochat et al. (1977) observed that total polysialoganglioside content increased in several human neuroblastoma tumors and reflected the clinical status of the disease. Yates et al. (1 979) also observed an increasein polysialogangliosidesin severalhuman astrocytomas and glioma samples reflecting the degree of malignancy. In a more recent study, Trayler and Hogan ( 1980)observed that G D l a , G,,, ,and G, decreased in human gliomas and astrocytomas, paralleled by an increase in lower ganglioside homologs such as GM, ,GM,, and GDz, These observations were similar to the simplification of ganglioside profile seen in many transformed cultured cell systems (see Section III,A, 1). 4. Human Reticuloendothelial Neoplasms Leukocytes isolated from chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL) showed an increase in CD and CM relative to control leukocytes from normal healthy individuals (Hildebrand et al., 1971). The same group of investigators observed an increase in total ganglioside content reflected by the major gangliosides such as G M i and GDla in CLL and CML patients relative to control lymphocytes (Hildebrand et al., 1972). Recent studies by Macher and collaborators have shown that there were no malignancy-associated neutral GSL changes in human acute and chronic myeloid leukemia (AML, CML) in comparison to normal neutrophils (Mock et al., 1981). Their studiesalso showed that neutral GSL will aid in distinguishing the types of leukemias. The CLL cells are characterized by the presence of CM, CD, CT, and globoside, whereas CML and AML cells contain lactoneo- and polygalactosyl-GSL (Lee et al., 198la,b). However, a minor quantitative difference was seen between normal lymphocyte and CLL cells. Peripheral blood lymphocytes contained more CD than CM, whereas the reverse was true in CLL (Lee et al., 1981b).

GLYCOLIPIDS AND GLYCOPROTEINS IN CANCER

309

CONTROL,TUMORIGENESIS, C. ROLEOF GSL IN GROWTH AND METASTASIS Cell surface GSL can be considered to play an important role in growth control as indicated by the following lines of evidence. ( 1) The loss ofgrowth control seen in all transformed cells in culture and in in vivo transformation often results in an alteration of GSL composition (see Sections III,A and B and Tables I11 and IV). The most compelling evidence for the causal nature ofGSL in growth control comes from observations of GSL changes seen in ts transformation (Table 111). (2) The second line of evidence for the potential role ofGSL as a candidate for receiving growth regulatory signals comes from the observation of a striking metabolic change in GSL (contact-extension response) seen in cell density-inhibited cultures which may be regarded as one of the molecular bases of the phenomen of “contact inhibition of growth,” although this phenomenon is seen in certain transformed and tumorigenic cells (see Section III,A,2). (3) Several studies demonstrated that an increase in GSL synthesis and exposure occurs at the G, and S phases of the cell cycle (Gahmberg and Hakomori, 1974, 1975;Chatterjee et al., 1973, 1975). In addition, GSL synthesis increases in mitogen-activated lymphocytes (Narasimhan et al., 1976; Rosenfelder et al., 1978, 1979). (4) Exogenous addition of neutral-GSL (globoside, asialo-GM2)and gangliosides to growing normal and transformed cultures resulted in a decreasein saturation density, inhibition of growth, increase in adhesion to the substratum (Laine and Hakomori, 1973; Brailovsky ez al., 1973, 1974; Keenan et al., 1975; Yogeeswaran, 1981), and particularly an extension of the prereplicative phases (G and S) ofthe cell cycle (Laine and Hakomori, 1973).(5) Lingwood and Hakomori ( 1977)observed that the addition of Fab fragments of certain cell contact-sensitive GSLs (GMS, globoside) to nontransformed cells resulted in a reduction in saturation density, similar to cell density-dependent inhibition and this treatment also resulted in a contact extension response of membrane GSL as described in Section III,A,2 (Lingwood and Hakomori, 1977). (6) Young et al. (1978) observed that GSL covalently attached to Sepharose absorbed certain proteins from fetal calf serum which, when applied to cells, stimulated cell growth, thereby suggestingthat GSL could be a receptor for serum growth factor. The speculation that GSL could serve as a cell surface receptor for serum growth factor has to be demonstrated at the cellular level, and the possibility that the absorption of serum growth factor on an affinity column may be due to nonspecific hydrophobic interaction of the acyl chain of GSL attached to Sepharose has to be ruled out. The role of GSL in tumorigenesis has been addressed in a number of studies. Although many alterations in GSL composition have been noticed (see Tables 111 and IV), only certain GSL changes correlated with tumori-

3 10

G. YOGEESWARAN

genesis. For example, Sakiyama and Robbins (1973) reported that the increase in the chemical quantity of G,, and long-chain neutral GSL correlated positively with tumorigenicity when a large number of low and high malignant clone hybrid cells, and segregants of hybrid cells have been compared (Itaya et al., 1976).Menitt and co-workers observed an increase of ( 3 ~ 3GM1, , and GD,, and an accompanying decrease of G,, ,GDIb,and GTlb with tumorigenic progression in their rat liver carcinogenesismodel (Memtt et al., 1978a). A number of investigators have studied the GSL composition of metastatic tumors compared to nonmetastatic control. The role of GSL in tumor cell metastases is a poorly understood subject as is any other function of GSLs. However, several interesting observations have been made regarding GSL in metastatic tumor lines in comparison to nonmetastatic lines. For example, several rat ascites hepatoma cell lines with low metastatic potential contained CT, globoside, and G,, , whereas their metastatic derivatives lacked CT, globoside, and G,, but contained asialo-G,,, asialo-G,, ,and fucosylasialo-G,, (see Fig. 1, asialoganglioside pathway) (Matsumoto and Taki, 1975;Taki et al., 1979a,b;Hirahayashi et al., 1978).Yogeeswaran and Stein ( 1980) also observed a decrease in G,, and an increase in asialo-G,, and GMZ and a positive correlation between cell surface-exposed asialo-G,, and metastatic potential by comparing a family of MuSV-transformed metastatic variants of BALB/3T3 cells. These studies on metastatic variant rat hepatomas by Taki and collaborators and mouse reticulum cell sarcoma by us strongly suggest that ganglioside-G,, is made from asialo-G,, rather than from G,, (see Fig. 1, alternate pathway 111) in highly metastatic cell lines (Yogeeswaran and Stein, 1980). These observations suggest that the switching of the ganglioside pathway could result in the accumulation of novel GSL antigens, such as asialo-G,,, asialo-G,, , and fucosylasialo-G,, on the surfaces ofthese metastatic cells (Rosenfelder et al., 1977;Taki et al., 1981). In a recent report, Skipski et al. ( 1981) showed that metastatic transplantable rat mammary carcinoma tumors have higher levels of gangliosides and an enrichment of G,, , GDla,GDIb, GTla,GTLb, and G, compared to nonmetastatic tumors. Their results also favor the hypothesis that the disialoganglioside pathway is the preferred pathway in metastatic cells, and gangliosides may be formed predominantly via the monosialoganglioside pathway in nonmetastatic cells (Skipski et al., 1981).

D. GLYCOSPHINGOLIPID ANTIGENS I . Human and Experimental Tumors The carbohydrate moieties of GSLs are the immunodominant portion of the molecule. Antigenic activity of GSLs of human tumor tissues was

GLYCOLIPIDS AND GLYCOPROTEINS IN CANCER

31 1

discovered by immunization of tumor tissues in rabbits over 50 years ago (Witebsky, 1929). A few examples of tumor-associated surface membrane GSL antigens are found in human and experimental tumor model systems, as shown in Table V. Rapport and Graf( 1961) isolated a GSL called cytolipin H in human epidermoid carcinoma, which inhibited xenogeneic antisera raised against the tumor. The complement fixation reaction displayed by the lipid was strongly inhibited by lactose, indicating that carbohydrate moieties were involved in the antigenicity. Another tumor GSL antigen, cytolipin R, was discovered in Murphy-Strum rat lymphosarcoma (Rapport and Graf, 1967).The antigen was not present in significantamount in organs ofcontrol rats. Makita and Seyama (1971) showed that Forssman reactivity could be detected in in vivo-grown polyoma-induced hamster sarcoma (BHK-Py), whereas nontransformed cells did not show such activity. Hakomori and co-workers have provided three examples of specific glycolipid haptens in three experimental tumor model systems. The first example concerns the presence of paragloboside,a ceramide tetrasaccharide isomer of globoside in Nil-Py hamster tumor cells. Rabbit antisera directed against Nil-Py tumor contained antibodies reacting with a tumor GSL paragloboside as demonstrated by complement fixation reaction. Antisera prepared against nontransformed Nil hamster cells did not react with paragloboside (Sundsmo and Hakomori, 1976). Another example of tumor-specific GSL is asialoG,, ,a ceramide trisaccharide seen in Ki-MuSV-transformed nonproducer BALB/3T3 cells. This line contains large quantities of asialo-G,, (20-fold higher) compared to nontransformed 3T3 cells. Rabbit antiserum against asialo-GM2showed a strong direct immunofluorescence against tumor cells, whereas 3T3 control cells did not show the presence of this surface GSL antigen (Rosenfelder et al., 1977). The ability of tumor-associated GSL antigens to elicit a positive immune response has not been established during a progressive tumor growth. However, Young and Hakomori (1981) have shown that by passive immunization against anti-asialo-GM2antibodies in DBA mice injected with a lymphoma tumor cell line, L5178Y, tumor rejection was possible. Interestingly, various tissues and organs of DBA mice do not contain detectable levels of this GSL (Young et al., 1981). Another excellent example of GSL serving as tumor-associated antigen comes from the work of Huet and Ansel (1977) who showed that vesicular stomatitis virus (VSV) grown in SV40-transformed hamster cells resulted in the isolation of a polar GSL (Ansel and Huet, 1980), which, when injected in hamster as a liposome, resulted in the suppression of growth (Huet and Ansel, 1977).

2. Modijication of Blood Group GSL Antigens in Human Tumors The expression of blood group GP and GSL antigens on normal and tumor cells has been extensively reviewed by Hakomori ( 1975b).Therefore,

TABLE V GLYCOSPHINGOLIPID ANTIGENS OF

W

N

Species, tumor Animal models Rat, lymphosarcoma Hamster, BHK-Py sarcoma Hamster, Nil-Py sarcoma MOUX,B-MUSV 3T3 sarcoma Human studies Epidermoid carcinoma Gastric carcinoma

TUMORTISSUES

Tumor antigen, structure

Changes Seen in tumors

Cytolipin R C-Glu-j?GalaGal-f#iaiNAc Forssman

New expression of GSL antigen Increased expression of a

Rapport and Graf ( I 96 1); Laine er af. (1972) Makita and Seyama (197 1)

Paraglobside C-Glu-fial-fialNAc-fial Asialo-G,, C-Glu-/it3al-palNAc

New expression of GSL antigen

Sundsmo and Hakomori (1976)

Increased expression of a normal antigen

Rosenfelder er al. (1977); Yogeeswaran and Stein ( 1980)

Increased expression of a normal antigen Deletion of antigen in GP fraction

Rapport and Graf ( I 96 1)

C-Glu-BGal-aGal-PGalNAc~alNAc normal antigen

Cytolipin H CGlu-/iGal A antigen P-Glu-fial-~lcNAc-PG$1-cuGalNAc aFuc

Reference

Masamune ef al. (1958)

Gastric carcinoma

B-antigen

P-Glu-jPGal-@lcNAc-jK3~l-&al

Deletion of antigen in G P fraction

Kawasaki (1958)

A, B, H antigens reduced, absent

Davidsohn et al. (1969)

aFuc Adenocarcinoma of GI tract, ovary, squamous cell carcinoma of skin, larynx. bladder Gastric and colon adenocarcinoma Breast, gastric. colon carcinoma GI tract adenocarcinoma Several adenocarcinomas Gastric carcinoma

A antigen, B antigen, and H antigen

in GSL and/or G P -Glu-fial-jPGlcNAc-@yl ~FUC Blood group trisaccharide C-Glu-flal-fllcN Ac T antigen

k hapten C-Glu-/K3al-@FNAc-fial aFuc X Hapten C-Glu-/Gal-jPGlc,NAc-jPGal 3aFuc Forssman C-Glu-@alirGal-jPGaI"c-&alNAc

Increase in this trisaccharide

Watanabe and Hakomori ( 1976)

Precursor to M N antigen (7 antigen) elevated in G P fractions New antigen foreign to Leb individual

Springer and Desai (1977)

An isomer of L e , seen in Leb individual. foreign to the host

Yangand Hakomori (1971)

Foreign antigen expressed B, 0 individuals

Hakomori ef al. ( I977c)

Hakomori and Andrews ( 1970)

3 14

G. YOGEESWARAN

this topic will be discussed only briefly here. Blood group antigens are gene-dependent carbohydrate structural determinants expressed in GP and GSL of body fluids, secretions, and membranes. Blood group antigens are believed to confer “individuality” to cells just as do histocompatibility antigens. A widespread distribution of blood group ABH and Lewis antigens has been demonstrated in blood cells, blood serum, endothelial cells, mucus-secreting cells, nonmucous glands, and epithelial cells using immunofluorescence techniques (Szulman, 1966). The modification of blood group structures in human cancers is given in Table V. Alterations of blood group antigens ha.ve been recognized in several human tumors. These changes include a deletion of host blood group antigen, accumulation of precursors of blood group structures, and appearance of new blood group determinants foreign to the host. These changes are summarized in Table V. Deletion of blood group determinants in certain tumors was first recognized over 30 years ago (Oh-uti and Tohokj, 1949). About 10years later the nature of such biochemical changes in tumor tissues was demonstratedin GP fractions (Masamuneet al., 1958;Kawasaki, 1958). Further extensive studies using imrnunofluorescencetechniques and agglutination reactions by Davidsohn et al. (1 969) revealed a reduction or absence of ABH isoantigenic activity in carcinoma of the intestine and ovary and in several squamous cell carcinomas. The loss of A and B blood group determinants in these epithelial tumors has been shown to result from a deficency of respective glycosyltransferase (Fig. 1, Block 8) and not from a loss in glycosylhydrolase (Stellner et al., 1973). Deletion of ABH blood group activity has been recognized in several oral epithelial tumors (Prendergast et al., 1968). The second class of change involving the accumulation of precursor blood group substances involves Lewis antigen, precursor blood group core structure, and precursor T antigen of the MN blood group. Lewis antigenic activity of blood group GP of gastric cancer has been variously reported, some cases showingan enhancement of Le”activitywith accompanying decrease of Leb, and some cases showing an enhancement of H-Leb activity (Iseki et al., 1962). Lea blood group active GSL has been shown to accumulate in human adenocarcinoma of Lebblood type, which is understood to be the result of blocked synthesis of Leb resulting in precursor accumulation (Hakomori and Andrews, 1970).A GSL with the structure of ceramide-Glua(4-1)Gda(3-l)GlcNAc, a precursor for various blood group structures was found to accumulate in many gastric and colon adenocarcinomas (Watanabeand Hakomori, 1976).The T antigen [GalNAc(4-l)Gal], a precursor for MN blood group structures,has been shown in many cases of breast, colon, and gastric carcinomas by Springer and Desai (1977). The third class of change in blood group determinants involves the appearance of

GLYCOLIPIDS AND GLYCOPROTEINS IN CANCER

315

incompatible blood group substances in various human tumors. These include the appearance of A-like antigen in tumors of 0 and B blood type individuals (Hakomori et al., 1977c; Hakkinen, 1970) and X hapten (an isomer of Le") seen in Lebgroup individuals (Yang and Hakomori, 1971). Hakomori and co-workers have recently shown that the appearance of an incompatible blood group substance with A-like activity is a Forssman GSL antigen (Hakomori et al., 1977c), and a novel Forssman GSL with a ceramide trisaccharide structure (Yokota et al., 1981) occurs in several gastric carcinomas and hepatomas, respectively. These observations are highly significant because 70-80%of humans are Forssman negative (Hakomori et ul., 1977c).Levine and co-workers observed that cancer patients with blood group P contained an incompatible p, antigen with a ceramide pentasaccharidestructure (Levine, 1976). Recently, Gupta et al. (1979) have identified a GSL antigen shed by human malignant melanoma in cell culture and this GSL strongly reacted with a monoclonal antibody against this melanoma cell line. Similarly, Dippold et al. ( 1980)showed that monoclonal antibodies against another human melanoma react with a GSL fraction isolated from this cell. Nakahara et al. ( 1980)indicated that several human lymphoblastic leukemia cells contain an antigen asialo-G,, which is a cell surface marker for natural killer (NK) lymphocytes and other leukocytes (reviewed in Herberman and Ortaldo, 1981). IV. Glycoproteins and Malignancy

Alterations in cell surface membrane-bound GPs have been demonstrated in many transformed cells and tumors. These changes can be classified into three types: (1) a loss of high-molecular-weight GP (fibronectin); (2) an increase in certain cell surface GPs in lymphoproliferative diseases which serve as cell surface markers for these neoplasms; and (3) an increase in novel GP such as epiglycanin. In addition, many cell surface GPs may undergo additional changes in carbohydrate components, particularly an increase in fucose and sialic acid and an increase in branching of asparagine-linked oligosaccharides. Because of the very general nature of these carbohydrate moieties, these changes will be discussed in a separate section. A. FIBRONECTIN

A number of reviews have been written on the subject of fibronectin changes in oncogenic transformation (Hynes, 1976; Vaheri and Mosher, 1978; Yamada, 1978; Vaheri, 1978; Pearlstein et al., 1980a);therefore, this subject will be covered only briefly here. Deletion of a high-molecular-weight

316

G. YOGEESWARAN

major cell surface GP (1 - 3%of total protein) was demonstrated simultaneously by several groups in the early 1970s in various transformed cells. This protein was variously termed by the investigators who discovered the molecule galactoprotein a (Ga-P a), large external transformation-sensitive protein (LETS), cell surface protein (CSP),zeta protein, and soluble fibroblast (SF) antigen, and is now called by a formalized name, fibronectin.This major cell surface GP is seen in fibroblasts, myoblasts, and certain epithelial cells. Fibronectin is found to be extrinsically associated with plasma membrane, and is also localized in pericellular regions and shed to the exterior. Immunofluorescencestaining of fibronectin detected this molecule on the cell surface as a fibrillar network concentrated in areas of cell contact (Birdwell and Gospabanowicz, 1978). Various groups of investigators have detected this protein on nontransformed cells using lactoperoxidase-catalyzed iodination (Hynes, 1973; Hogg, 1974), galactose oxidase-NaB3H, reactions (Gahmbergand Hakomori, 1973b),metaboliclabeling(Robbins et al., 1975), and immunological methods (Vaheri and Ruoslahti, 1974). The work on the isolation, characterization, and function of fibronectin was accelerated by the ease of isolation of this molecule using urea treatment of cultured cells (Yamada and Weston, 1974) and purification by gelatin affinity chromatography (Engvall and Ruoslahti, 1977). Fibronectin is present on the cell surface as a dimer of 440,000 daltons (440K) (McConnell et al., 1978) and consists of two polypeptide chains of 220K daltons. The polypeptide chains are held together by many disulfide bonds (Ali and Hynes, 1978).Fibronectin containstwo monosialodecasaccharideand three asialodecasaccharidechains per polypeptide chain, all of which are glucosamine-containing complex oligosaccharides(Fukuda and Hakomori, 1979). Although the loss of fibronectin in transformed cells is mediated by a transforming event (Rieber and Irwin, 1974; Marciani and Bader, 1975; Weber and Fries, 1979), the loss of fibronectin may not relate to increased tumorigenicity (Der and Stanbridge, 1978; Kahn, 1979). Loss of fibronectin has been observed in various in vivo tumors as well. For example, Vaheri et al. ( 1976) and Lubitz et al. (1 980) observed a loss of fibronectin in malignant gliomas and breast carcinomas (Lobart-Robertet al., 1980). However, Lloyd et al. ( 1979) did not observe a correlation between malignancy and loss of fibronectin in their studies of cultured human melanoma, astrocytoma, and carcinoma of the ovary, bladder, stomach, and cervix. Studies by TaylorPapadimitriou et al. ( 198 1 ) showed that both normal and malignant mammary epithelial cells synthesize fibronectin, but malignant cells lose more than 90% of the molecule. Stenman and Vaheri (1 98 1) looked for the presence of fibronectin on the cell surface of a large number of human carcinomas, sarcomas, and melanomas and found there was no correlation

GLYCOLIPIDS AND GLYCOPROTEINS IN CANCER

317

between the presence of fibronectin and malignancy; however, sarcoma tissues contained more fibronectin than carcinomas, and the investigators proposed that the screening for fibronectin in human tumors may be useful for the distinction of carcinomas and sarcomas. Loss of fibronectin by transformed cells often accompanies the appearance of fibronectin in culture medium or in plasma of tumor-bearing hosts. An immunological identity has been establishedbetween cell surface fibronectin and plasma cold-insoluble globulin (Ruoslahti et af., 1973; Vaheri and Ruoslahti, 1974). Pearson et al. ( 1979) demonstrated fibronectin-like proteins in peritoneal and pleural fluids from patients with malignant disease. Yamada and Kennedy (1 979) demonstrated that plasma fibronectin-like proteins are similar but not identical t>>cell surface fibronectin, by their observations of similar collagen binding and dissimilar hemagglutination reaction and ability to restore normal morphology in transformed cells. The mechanism of fibronectin loss from transformed cells was first thought to be due to increased proteolysis because of the extreme sensitivity of the molecule to proteases (Hynes, 1973). This view was further supported by the observation of increased proteases seen in transformation (Unkless et af.,1973;Ossowski et al., 1973).However, subsequent studies ruled out this possibility (Hynes and Pearlstein, 1976; Yang et al., 1980). Olden and Yamada (1977) found the decrease in fibronectin in transformed cells may be due to a number of factors, such as a reduction in synthesis, increased degradation, and loss from surface by shedding. Recent studies suggest that other chemical modification of the fibronectin molecule itself, such as increases in sulfate groups seen in shed fibronectin in melanoma cells (Wilson et af.,1981) and increased sialylation (Critchley et af.,1976;Wagner et al., 1982), might result in its loss. These changes would result in the increase in net negative charge on the molecule, which might alter the adhesive and binding properties of fibronectin to other cell surface constituents. The significance of the loss of fibronectin in cell transformation may be best judged by several properties associated with the fibronectin molecule. Addition of fibronectin to transformed cells in culture is shown to promote adhesion and a morphological reversion to a nontransformed state (Yamada et al., 1978).Both cell surface fibronectin (Engvall and Ruoslahti, 1977)and plasma fibronectin (Pearlstein et al., 1976) have binding sites for collagen, indicating that an important function of the molecule is as an adhesion material between cells in organizing tissue structure. Fibronectin also binds to other cell surface molecules such as proteoglycan (Culp et af., 1979), sulfated proteoglycan (Perkins et al., 1979),and gangliosides or gangliosidelike oligosaccharides(Kleinman et af.,1979). Bensusan et al. (1978) showed

318

G. YOCEESWARAN

that fibronectin is the collagen receptor on platelet membranes, which explains the ability of platelet aggregates to interact with vascular endothelium which is rich in collagen.

B. INHUMAN LYMPHOPROLIFERATIVE DISEASES Cell surface marker analysis and glycoprotein patterns of neoplasms involving the human hematopoietic system have been extensively studied to help in the diagnosis of these cancers. Studies of the GP profile of these neoplasms using surfacelabeling by the galactoseoxidase sodium borotritide reaction followed by polyacrylamide gel electrophoresis and fluorography have been described to characterize the phenotype of these cells by Anderson, Gahmberg, Nilsson, and their collaborators (Table VI). These neoplasms may belong to various hematopoietic lineages: T cell, B cell, granulocyte, monocyte, and erythroid. In cancers of the hematopoietic system, the differentiationcapacity is not completely lost due to malignant transformation, and, therefore, several GP and functional characteristics are seen in these cells. It should be emphasized that GP markers described in these neoplasms are not tumor-specific structures, but instead express differentiation markers typical for these cells at a particular stage of maturation. The function of some of these tumor-associated GPs is beginning to be understood. For example, B cell GP210 contains C3b complement receptor (Fearon, 1980) and transferrin receptor (T9) is a GP (Terhost et al., 1981; Trowbridge and Omary, 1981). Normal human thymocytes and T cells contain GPs in the range of 160,000- 200,000 MW and another 120,000-MWGP component ( A n d e s son et al., 1980; Judd et al., 1980;Andersson and Gahmberg, 1978). A study of the T cell leukemia cell lines (Andersson et al., 1977)and T leukemia cells from cancer patients (Andersson and Gahmberg, 1978) showed four highmolecular-weight GPs (GP160, 165, 180, 200), resembling normal T cells. The main difference in GP in T cell leukemias is the strong labeling of GP 120- 130 due to an increase in glycosylation (Andersson and Gahmberg, 1978). B lymphocytesand B cell neoplasms have certain GPs distinct from T cells (Andersson et al., 1976).Glycoproteinsof 2 10,000,31,000,and 24,000 MW were seen in B cells (Andersson et al., 1976) and various Burkitt and lympocytic lymphoma lines (Trowbridge et al., 1977; Nilsson et al., 1977), with a typical intensification of GPs of 3 1,000and 24,000 MW in tumor cell lines compared with normal B cells. Recently, Andersson et al. (1981) have characterized a leukemia called “hairy cell leukemia,” based on butyrateinduced ruffling of the cell surface, which has many B cell characteristicGPs and a specific GPl 10. Various cases of chronic lymphocytic leukemia cells

TABLE VI ALTERATIONSOF CELL SURFACE GPS OF RETICULOENDOTHELIAL NEOPLASMS Normal and neoplastic cells Thymocytes and T cells

T cell leukemia lines MOLT-4, JM Non-T, Non-B cell lines MALM-I, NALM-I6 T cell leukemic cells Non-T. Non-B leukemic cells B lymphocytes Burkitt and lymphocytic lymphoma lines Ramos, P3H, U698, U714, Daudi, myeloma Hairy cell leukemia line: Jok. 1 Chronic lymphocytic leukemia (CLL) Myeloid leukemia, acute lymphocytic leukemia Normal granulocytes Normal monocytes Monocyte-likecell lines U937, DH-2, DHL-4 Promyelocytic cell line HL60 Erythrocytes Erythroleukemia cell line K562

Characteristic surface glycoproteins (MW X I&*)

Reference

GP 160-200 GP120 GP160, 165, 180.200 GP120- 130 GP100- I30 GP200 Resembles T cell G P GP210,GPI20-130.GPlOO GP2 10, HLA-A, B. C. D (la antigen). GP3 I , 24 GP2 10. GP3 1,24. GP87/85. GP7 I /69

Andersson et a/. (1980) Andersson and Gahmberg ( 1979) Anderson et al. (1977)

GP210. HLA-D (GP31,24), GPI 10 GP2 10. GP3 I, 24 GP2 10, GP3 1,24, GPl30, glycophorin A

Anderson ef a/. (1981) Andersson ef al. (1979b) Andersson et al. ( 1979a.b)

GP 130 GPI 30, GP200 GP I20 GPI 30, GP95 GP I60 Glycophorin A. B, Band 3 G P Glycophorin A, GP104, Band 3 GP

Gahmberg and Anderson (unpublished) Breard et al. ( 1980) Nilsson el al. ( I9GO)

Gahmberg el a/. (1980) Anderson and Gahmberg ( 1979) Anderson and Gahmberg (1979) Anderson et al. (1976) Trowbndge et al. ( 1 977) Nilsson et al. (1977)

Gahmberg et al. ( 1979) Marchesi et al. (1976) Karhi and Gahmberg ( I 980); Gahmberg et al. ( 1978); Gahmberg ef al. ( 1979)

320

G. YOGEESWARAN

contain B cell-specific GP2 10, GP3 1, 24, surface immunoglobulins (Andersson et al., 1979a). Surface G P patterns of acute myeloid leukemia resemble those of acute lymphocytic leukemias, containing GP2 10, GP3 1, 24 (Andersson et al., 1979b).In the same study, Anderson et al. (1979b) also reported that granulocytic leukemia cells contain a GP profile similar to granulocytes with a characteristic GPl30. Normal granulocytes contain a GP of 130,000 MW with a large amount of 0-glycosidic linkages in the molecule (Gahmberg and Anderson, unpublished results). These proteins may be related to chemotactic properties as evidenced from the studies of a promyelocytic cell line which lacks this GP, and, upon induction ofdifferentiation, gains this protein and acquires chemotaxis (Gahmberg et al., 1979a). Monocytes contain GP130 and a monocyte-specific GP200 (Breard et al., 1980). Various human histiocytic lymphoma cell lines exhibit a monocytic characteristic GP120, 130, and a unique GP with a molecular weight of 95,000. A human erythroleukemic cell line exhibits a surface GP characteristic of erythrocytes, such as glycophorin A, Band-3 GP, and a unique GP104 (Gahmberg et al., 1979a; Karhi and Gahmberg, 1980). In addition, certain acute leukamia cells exhibit glycophorin A (Anderson et al., 1979b). From the above-mentioned discussion, it is apparent that the majority of surface GPs expressed on the surface of hematopoietic neoplasms are not unique, although, occasionally, certain GPs intensify and others decrease. These quantitative inverse changes in GP profile may reflect acquisition or suppression of a lymphocytic or myelocytic phenotype. Despite the lack of tumor-specific GP changes in these lymphoproliferative diseases, small increases in the apparent molecular weight of certain proteins are noticed, and this may be due to an increase in glycosylation of the polypeptide chain (Anderson and Gahmberg, 1979). C. EPIGLYCANIN Codington and co-workers described the presence of a unique large intrinsic plasma membrane GP called epiglycanin in certain mouse TA3-Ha mammary adenocarcinoma tumors (Codington et al., 1975a). This tumor isolate, TA3-Ha, does not exhibit strain specificity when compared with its parental tumor, TA3-St (Friberg, 1972).Codington et al. (1 975a) correlated the appearance of epiglycanin on the surface of TA3-Ha with the allotransplantable characteristics of this line. The allotransplantable TA3-Ha cell line absorbed H-2 antibodies poorly, but after sonication or cell disruption absorbed more antibodies, whereas the parental TA3-St absorbed H-2 antibodies to the same degree after these treatments (Sanford et al., 1973). Codington and co-workers postulated that the presence of epiglycanin on the

GLYCOLIPIDS ANDGLYCOPROTEINS IN CANCER

32 1

surfaces of TA3-Ha cells results in decreased H-2 expression and confers allotransplantable properties (Codington et al., 1975a). Their hypothesis was further supported by studies of several hybrid cell lines between TA3-Ha and an embryonic fibroblast of congenic mice, which showed a good inverse correlation between the presence of cell surface epiglycanin and absorption of H-2 antibodies, and an extent of allotransplantability (Codington et al., 1978). Biochemical studies showed that epiglycanin is a large rod-like GP of 500,000 MW which has over 500 O-glycosylatedoligosaccharideside chains (Codington et af.,1975b; Van den Eijnden et al., 1979) (see Fig. 3, structure for epiglycanin oligosaccharides). The long rod-like structure and high degree ofglycosylation in the epiglycanin molecule confer the ability to cover the H-2 structure, presumably by steric hindrance. The oligosaccharide structures of epiglycanin bind to Viceu gruninea lectin (Codington et ul., 1975a),and this unique property may aid in identifying this type of oligosaccharide in GP on cell surfaces. Another example of an abnormal glycoprotein on the surface of tumor cells comes from the studies of Carraway and collaborators (Buck et al., 1979).A rat ascites mammary carcinoma line MAT-B 1 was restricted in its transplantation to rat strains and same species. A derivative of this cell, MAT-Cl, is transplantable across the species barrier into mice and is characterized by a distinctive change in carbohydrate moieties ofa GP(Buck et al., 1979). The xenotransplantable cell line, MAT-C I , contained several times more sialic acid and highly branched oligosaccharides attached to serine and threonine residues on the polypeptide. Ohno et al. (1977) also observed that an IgA-synthesizing plasmacytoma cell showed decreasing levels of anti-H-2 antibody absorption during animal passages. Subsequent studies by Tokuyama and Migita ( 1978) showed that the animal passaged tumors that had poor anti-H-2 antibody absorption also possessed a highly sialylated G P which was thought to mask the H-2 structure. Epiglycanin and other highly sialylated GPs that cover H-2 structures are also shown to be responsible for increasing the metastatic potential of the tumor cells. For example, Cooper et ul. (1977) have reported a correlation between cell surface epiglycanin and metastatic potential in TA3-Ha variant cell lines. In the following section we will consider in detail the role of sialic acid in malignancy. V. Sialoglycoconjugatesand Malignancy

Sialic acid is present both in free form or bound to aligosaccharidechains of membrane-bound GP and gangliosides in higher organisms. It occupies predominently a terminal position, a(2 -, 6) in oligosaccharidesof GPs, but

322

G. YOGEESWARAN

in GSLs it can be attached internally a(2 -, 3) in the carbohydrate backbone (see Figs. 1 and 3). Sialic acid is attached to galactose or N-acetylgalactosamine residues of G P and GSL (Figs. 1 and 3). Considerable literature has accumulated on the biological roles of sialic acid in which it is said to be important in the physiocochemical properties of GPs and transport of GPs inside the cell, in the survival of GPs and circulating cells (RBC, WBC, platelets, and tumor cells), and as the receptor for various substances and infectious agents (reviewed in Yogeeswaran, 1980; Codington and Jeanloz, 1976). Several studies comparing the properties of transformed cells and sialic acid suggested that cell surface sialic acid might play a role in determining some properties that contribute to metastatic potential, including an increased capacity to aggregate platelets (Pearlstein et al., 1980b), adhesion (Weiss, 1963; Berwick and Coman, 1962;Kemp, 1970),ability to implant in various organs (Weiss et al., 1974;Sinha and Goldenberg, 1973;Bosmann et al., 1973;Yogeeswaren et al., 1978),cellularmotility(SalkandLanza, 1980), deformability (Weiss, 1969, invasiveness(Yarnell and Ambrose, 1969),and immunogenicity of tumor cells (Salk and Yogeeswaran, 1979;Yogeeswaran and Salk, 1978). A. SIALIC ACIDCONTENT Ambrose and co-workers and later Fuhrman (reviewed in Warren et al., 1978) indicated that the electrophoretic mobility of malignant cells toward the anode was higher than for normal cells and was reversible by NANase treatment of malignant cells. Prompted by this observation, several investigators studied the composition of sialic acid (total cellular sialic acid in GPs and GSLs) in normal and transformed cells (Table VII). No consistent pattern of change in sialic acid was observed in these earlier studies using cultured transformed cells. An increase in total sialic acid in cultured transformed cells was observed by Warren and co-workers (Hartmann et al., 1972),Yogeeswaran and collaborators (Yogeeswaran, 1972;Yogeeswaran et al., 1979; Yogeeswaran and Salk, 1981), Perdue et al. (1971), Meager et al. (1979, Nigam and Canter0 (1973), and Shen and Ginsberg (1976) (Table VII). But in other transformed cells, no change or a decrease in sialic acid content was observed (Ohta et al., 1968; Meezan et al., 1969; Culp et al., 1971; Grimes, 1971; Hartman et al., 1972; Yogeeswaran, 1972; Grimes, 1973)(see Table VII). One possible explanation for these conflictingfindings was made by Perdue et al. (1972). The quantity of sialic acid bound to sialyl components (gangliosides and sialyl-GP-S-GP) in plasma membranes in uninfected and virus-transformed cells showed a correlation with cell shape. In general, cells that were more fusiform in shape had more sialyl components. Cells that were more rounded and less well anchored had less sialic

TABLE VII ACIDCONTENT OF NORMAL AND TRANSFORMED CELLSINCULTURE AND TUMORS TOTALSIALIC

Normal and transformed cells" Cultured cells BHK BHKn Py-BHK 3T3 Py-3T3 3T3 SV-3T3 3T3 SV-3T3 3T3 SV-3T3 ST3T3 Chinese hamster ChHE-SV ChHE-Py Chick embryo fibroblast CEF-RSV BHK RSV-BHK (Bw) RSV-BHK (SR) CEF ALV-CEF RSV-CEF (flat)

Sialic acid content (nmoles/mg protein) Normal

Transformed

Relative change

8.1 8.8

Reference

6.7

1

5.5

1

Less

-L

Ohta et al. ( 1 968) Ohta et al. ( 1968) Ohta ef al. ( 1968) Ohta et al. (1968) Ohta ez al. ( 1968) Meezan et al. ( 1969) Meezan ef al. ( 1969)

10.6

1

Grimes ( 1970)

9.7 11.0

1

1

Culperal.(1971) Culp el al. (1971)

12.1 21.0

1 t

Hartmann ef al. ( 1 972) Hartmann et al. (1 972)

32.6

t

Hartmann et al. (1972)

6.8 6.9

No change

No change

Hartmann ef a/. (1972) Hartmann ef al. ( 1972)

14.0 More 13.2 16.2 19.9 24.4 6.7 106.0 1 14.0

t

77.0

1

Perdue er al. ( 1971 ) Perdue ef al. ( 1971 )

(continued)

TABLE VII (conlinued)

Sialic acid content (nmoles/mg protein) Normal and transformed cellsu

W

N

P

RSV-CEF (round) ASV-CEF 3T3 SV-3T3-479 SV-3T3-A26 SV-3T3-CE56 Py-3T3 BHK CIZTSVS-S CIZTSVS-R BHK Py-BHK BHKJC I3 BHKJCI 3-AL SV-BHKZJC13 Py-BHKJCIZ 3T3-A3I 3T3-MSV85C13(LM) 3T3--52 1 (IM) 3T3-KA31 (HM) 3T3-K234 (HM) PWZO-MH) (LM) PW20-WRL (LM) PW20-RI (HM) PW20-R2 (HM)

Normal

Transformed

Relative change

67.0 79.0

Reference Perdue et a/. ( 1971) Perdue er al. ( 1971)

8.8

10.8

t

8.9 14.7 9.9

No change

41.1 65.8

t

5.8

1

35.4

t t t

8. I

Yogeeswaran (1972) Yogeeswaran (1972) Yogeeswaran ( 1972) Y ogeeswaran ( 1972) Nigam and Cantero (1973) Nigam and Cantero ( 173) Grimes (1973) Grimes(1973)

10.0 9.4 15.9

Meager et al. ( 1975) Meager et al. (1975) Meager et al. (1975)

10.5

Yogeeswaxan et al. (1 979) Yogeeswaran et al. (I 979) Yogeesman et al. ( 1979) Yogeeswaran et al. ( 1979)

8.9 10.1

9.6 9.7 6.9 6.4 14.6 13.9

t t

Yogeeswaran and Salk (1981) Yogeeswaran and Salk (1 98 1)

More

t

Barker et a/. (1959)

In vivo Normal human tissues Colon adenocarcinomas

Less

u N v,

Duodenum carcinoma Breast carcinoma Rat liver PM Hepatoma-484 PM Rat liver PM Hepatoma-484 PM Hepatoma-48 I A PM Mouse liver PM Hepatoma- I47042 PM Hepatoma-4 I89 PM Hepatoma- 143066 PM Human normal tissue Pancreas adenocarcinoma (P) Pancreas adenocarcinoma (M) Skin carcinoma (P) Skin carcinoma (M) Rat mammary tissue Mammary carcinoma Normal human lung tissue Lung carcinoma Rat liver Hepatoma PW20-R,, (H) primary PW20-R2RI primary PW20-R 1 primary PW20-R2 primary Rat liver Hepatoma-RTL, (LM) Hepatoma-RTL, (M) Hepatoma-RTL, (M) Hepatoma-RTL, (LM) Hepatoma-RTL, (M) Hepatoma-H7 (LM)

More More

t t

44.0

?

Barker el a/. ( 1959) Barker ei a/. (1959) Benedetti and Emmelot ( 1967) Benedetti and Emmelot ( I 967)

45.0 61.0

t t

Emelot and Bos (1972) Emmelot and Bos ( 1972)

33.0 33.0 30.0 36 1 .0 27.0 28.0

Emmelot and Bos (1972) Emmelot and Bos (1972) Emmelot and Bos ( 1972)

More More More More

Mabry and Carabelli (1972) Mabry and Carabelli (1972) Mabry and Carabelli (1972) Mabry and Carabelli (1972) Keenan and Morre (1973) Keenan and Morre ( 1973) Bryant et a/. ( 1974) Bryant er al. ( 1974) Menitt ef al. (1978a) Menitt et a/.( 1 978a) Yogeeswaran and Salk (unpublished) Yogeeswaran and Salk (unpublished) Yogeeswaran and Salk (unpublished) Yogeeswaran and Salk (unpublished)

Less

19.6 48.6

t

55.6

t

41.0 7.3 7.8 9.6 8.4

t

22.5 21.0

t

4.5 16.0 15.6 8.3 9.2 6.2 25.3

Kloppel and Morre ( 1980) Kloppel and Morre ( 1980) Kloppel and Morre ( 1980) Kloppel and Morre (1980) Kloppel and Morre ( 1980) Kloppel and Morre (1980)

LM, Low metastatic; IM, intermediately metastatic: HM. high metastatic; PM, plasma membrane: P, primary tumor.

326

G. YOGEESWARAN

acid. Thus, anchorage to a plastic substratum, adhesiveness, and cell shape may regulate the content of sialic acid per cell (Perdue et al., 1972). These unresolved variables in the use of cultured transformed cells (Ohta et al., 1969;Meezan et al., 1969;Culp et al., 197 1;Hartmann et al., 1972;Grimes, 1971, 1973; Yogeeswaran, 1972), make it difficult to draw definite conclusions about sialic acid change and cell transformation. In contrast to the lack of consistent changes in sialic acid composition of cultured transformed cells, studies with malignant tumors in vivo by a number of investigators have consistently revealed an elevation of both total and plasma membrane-bound sialic acid relative to normal surrounding tissues (see Table VII) (Barker et al., 1959; Benedetti and Emmelot, 1967; Emmelot and Bos, 1972; Mabry and Carubelli, 1972; Keenan and Morre, 1973; Bryant et al., 1974; Merritt et al., 1978a; Kloppel and Morre, 1980). Changes in sialic acid content with reference to tumor cell metastases is a subject of renewed interest because of many postulated functions of sialic acid in metastatic cells. The levels of total cell sialic acid in human metastatic lesions (liver and lymph node metastases) were higher than in the respective primary pancreatic adenocarcinoma and skin melanoma tumors (Mabry and Carubelli, 1972). Subsequent studies from several laboratories revealed no consistent increase in sialic acid in metastatic tumor systems as compared to nonmetastatic controls. For example, Yogeeswaran et al. (1979) and Kloppel and Morre ( 1980)did not observe a consistent increase in total sialic acid content in cultured metastatic variant mouse sarcoma lines and transplantable rat hepatoma tissues, respectively. However, in other studies Skipski et al. (198 1) and Yogeeswaran and Salk ( 1981) observed an increase in total sialic acid content in metastatic mammary carcinomas and PW20 sarcoma lines compared with nonmetastatic controls. Morre and collaborators observed interesting changes in sialic acid content in liver tissue from fetal to adult stagesand during carcinogenesis(Morre et al., 1978). Sialic acid levels were elevated in fetal and newborn rat livers. The sialic acid levels dropped sharply a few weeks after birth and remained low for all age groups. Following administration of carcinogen, sialic acid levels began to rise and maximum levels were attained in poorly differentiated and well-circumscribed hepatoma. They found a lowering of sialic acid in certain poorly circumscribed, rapidly growing hepatomas as compared with slow-growing, well-circumscribed hepatomas (Morre et al., 1978). Thus, their observation made the important point that elevation of sialic acid seen in malignancy is not due to an increase in growth rate, but the contrary is seen in malignant tumors. The authors suggested that a decrease in sialic acid seen in certain transformed cells (see Section V,A) in culture may be related to rapid growth, and implied the need to characterize the cultured cells for their tumorigenic and metastatic properties in vivo (Morre et al., 1978).

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B. GANGLIOSIDE CHANGES In Section 111, ganglioside changes associated with transformation have been reviewed (Tables 111 and IV). Several studies described in this section showed an increase in lipid-bound sialic acid in cultured transformed cells (Brady and Mora, 1970;Mora et al., 1969;Brady et al., 1969;Yogeeswaran et al., 1972; Brady and Fishman, 1974), animal tumors (Cheema et af.,1970; Keenan and Morre, 1973; Dnistrian et al., 1977, 1979; Memtt et al.. 1978a; Skipski et af.,198l), and human tumors (Traylor and Hogan, 1980;Keranen et af.,1976; Siddiqui et af.,1978; Narasimhan and Murray, 1979; Hattori et af.,1982). The increase in ganglioside content and lipid-bound sialic acid as seen in transformed cells is related to tumorigenesis (Sakiyama and Robbins, 1973; Itaya et al., 1976; Memtt et al., 1978a) and may be a reflection of altered ganglioside metabolism (see Section 111,A,2) which results in the accumulation ofcertain precursor ganglioside homologs due to a block in the biosynthetic pathway leading to complex gangliosides. C. SIALOGLYCOPEPTIDES OF CELLSURFACE PROTEINS Sialoglycopeptidesare the oligosaccharide remains of extensive proteolytic digestion by Pronase (a mixture of broad specificity proteases) of trypsinates (mild trypsinization) of cell surface glycoproteins from intact cells. Warren and collaborators analyzed the glycopeptides from normal and transformed cells labeled with radioactive fucose or glucosamine by Sephadex-50 chromatography columns and found an increase in molecular weight of glycopeptides A and B in malignant transformed cells compared with controls (Buck et al., 1970, 197 la,b). Glycopeptides ofgroup A (MW 4600) and group B (MW 3600-3800) contained fucose, mannose, galactose, N-acetylgalactosamine,N-acetylglucosamine,and sialic acid (Warren et al., 1973). These glycopeptides contain the asparagine-linked complex type oligosaccharides (Levy-Genshimol, 1977). Changes in high-molecularweight glycopeptides A and B were observed in several subcellular fractions such as nuclei (Keshgegian and Glick, 1973; Muramatsu et af., 1976), mitochondria, lysosomes, and endoplasmic reticulum (Muramatsu et af., 1976). Warren and co-workers observed that although the increase in high-molecular-weightglycopeptide A is somewhat related to growth rate of cells, the surfacesoftransformed cells had a lot more glycopeptidethan could be accounted for by an increase in growth rate (Buck et al., 1971b). Van Beek et af.(1975) also showed that the appearance of glycopeptide A is correlated with malignancy, because lectin stimulation and activation of lymphocytes did not result in the appearance of this structure in comparison with malignant leukemic cells. The appearance of high-molecular-weight glyco-

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peptide A is seen in several RNA virus-transformed cells (Buck et af.,1970; Fishman et af., 1974; Grimes et al., 1977), DNA virus-transformed cells (Buck et al., 1971a), and temperature-sensitive RNA virus-transformed cells (Warren et af., 1972; Pietropaolo et af., 1977). Thus, the presence of high-molecular-weight sialoglycopeptidesin transformed cells is found in several types oftransformed cells and solid tumors (Warren et af.,1975).This is found to be true in transformants of several species (reviewed in Warren et al., 1978). The appearance of group A glycopeptides is also seen in rat hepatoma (Van Beek et al., 1973, 1977), human leukemic cells, B cell lymphoma lines and Epstein-Ban virus-infected cells (Van Beek et al., 1975, 1978, 1979), mouse mammary carcinomas (Smets et af., 1977), neuroblastoma (Hudson and Johnson, 1977; Glick et af.,1976), chemically transformed rat epithelial cells (Whyte and Loke, 1978),and human gliomas and osteosarcoma (Van Beek et af.,1978).The chemical basis of the increase in molecular weight ofglycopeptide A was analyzed. Severalgroups in earlier reports showed that neuraminidase (NANase) treatment of glycopeptide A resulted in a reduction of molecular weight and the material was similar in chromatographic behavior to normal-cell glycopeptide A. This observation led to the proposal that the increase in molecular weight of glycopeptideA is simply due to an increase in sialic acid residue (Buck et al., 1970, 1971a,b; Warren et af.,1973;Van Beek et af.,1973).However, this simple explanation was questioned by Ogata et al. (1 976) who observed that 70’%0of the group B glycopeptide of transformed cells, bound to concanavalin A-Sepharose, whereas group A glycopeptide from the same cell bound only 20% and failed to bind even after NANase treatment (Ogata et af.,1976). However, following digestion with NANase, /I-galactosidase,and /I-N-acetylglucosaminidase, the remaining core-glycopeptides of both group A and B glycopeptides bound to concanavalin A-Sepharose. The core structure of group A and B gl ycopeptideswas cleaved by endo-P-N-acetylglucosaminidase resulting in a fucosyl-N-acetylglucosaminepeptide. On the basis of these studies, Ogata et af. (1976) proposed that the oligosaccharide of group A glycopeptide contains three to four branches and the following structure: (NAN-GalGlcNAc),-Man,-(GlcNAc),-(Fuc)-Asn, whereas the group B glycopeptide might contain two branches and the following structure: (NAN-GalGlcNAc),-Man,-(GlcNAc),-(Fuc)-Asn(see Fig. 3, structure 4 for group A glycopeptide). Glick et al. ( 1973) observed that several dimethylnitrosamine-transformed hamster fibroblastsin culture did not possess high-molecular-weight glycopeptide, although these cells lost growth control, changed in morphology, and grew on soft agar. However, tumors that arose as a result of injection of these transformants into animals gained glycopeptide A structures in their GP. In another study, Glick et al. ( I 974) obsem? that the concentration of

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glycopeptide A obtained from trypsinates of several polyomavirus-transformed clones of hamster fibroblastscorrelated with the dose of cells required to produce a positive incidence of tumors in viva Similarly, Smets et al. ( 1976) have found that SV40-transformed 3T3 cells that successfully formed tumors contained group A glycopeptide in tumor masses, whereas these glycopeptides were absent in the corresponding cultured cells. This study also shows a positive correlation between the presence of glycopeptideA and tumorigenicity. A recent study by Richards et al. (198 1) also showed a positive correlation between the presence of sialylfucoglycopeptide A and tumorigenicity in several tumorigenic and nontumorigenic mammary epithelial cells. Recently, the relationship between cell surface sialic acid and metastatic potential has been investigated by us and others. Sialoglycoconjugatesand metastatic potential of a family of tumorigenic MSV-transformed variants of BALB/3T3 lines were characterized (Yogeeswaran el al., 1979, 1980). We observed that although total cellular sialic acid was not appreciably increased in high metastatic cells, NANase-releasable sialic acid and degree of sialylation of Gal and GalNAc groups measured by galactose oxidase- sodium borotritide labeling of NANase-treated versus untreated cells showed a highly significant positive correlation with the incidence of metastases (Yogeeswaranet al., 1979).Cell surface GP detected by surface labeling with galactose oxidase- sodium borotritide showed an enrichment of several sialo-GPs in metastastic cells, whereas these GPs are poorly sialylated in nonmetastatic cells, nontumorigenic 3T3, and flat revertants of transformed cells (Yogeeswaran et al., 1979).In a subsequent study, we have extended the correlation between sialic acid and tumor cell metastases in a variety of murine sarcomas, carcinomas, and melanomas. Our study revealed a significant positive correlation between degree of sialylation ( r = 0.9, p < 10-12), NANase-releasable sialicacid ( r =0.74,p
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glycopeptidesisolated from the WGARcell lines had a reduction in molecular weight compared with control high metastatic melanoma cells. No difference was observed in other 0-glycosidic glycopeptidesand mannoserich glycopeptides. However, complex N-linked glycopeptides of WGAR lines had a reduction in sialic acid residues by a half per glycopeptide chain. Thus, the findings of Jumblatt et al. (1980) and Finne et al. (1980) are consistent with the proposed role for sialic acid and tumor cell metastases discussed here. Another class of glycopeptideisolated from tumor cell surfaceGP contains highly sialylated oligosaccharides bound to serine or threonine residues of the polypeptide chain via an 0-glycoside bond. An example of this type of 0-linked sialylated glycopeptide is seen in the epiglycanin molecule (Van den Eijnden et al., 1979). Bhavandan et al. (1977,198 1) also describedhighly sialylated 0-glycosidically linked oligosaccharides in greater abundance in human melanomas compared with melanocytes. The glycoprotein fraction with a MW of 100,000 from the plasma membrane of human melanoma cells alsocontainssialylated 0-glycosidically linked oligosaccharidesrelative to control melanocytes (Umemoto et al., 1981).

D. SIALYLTRANSFERASES The mechanism by which cell surface sialic acid increasesamong the sialyl componentsof malignant transformed cells is not clearly understood. Only a few studies have been done to elucidate the mechanism of elevated sialylation in malignant cells. Warren and co-workers observed that transformed cells contain 3- to 10-fold more desialyzed glycopeptideA:CMPNAN-sialyltransferase than control cells. Interestingly, when “nonspecific” asialoserum glycoproteins were used as acceptors for sialyltransferase, the transformed cells had levels of activity similar to normal cells, indicating that these sialyltransferaseshave a high degree of substrate specificity (Warren et al., 1973). Grimes (1973) and co-workers observed a lack of correlation between increased sialyltransferaseand the transformed state (Grimes, 1973; Patt et al., 1975) which may be due to the use of inappropriate substrates. Onodera et al. ( 1976) observed that the sialyltransferase activity and sialoglycopeptide increase were correlated with the transformed state using temperature-sensitive mutant virus-transformed cells. Bosmann and Hall (1 974) reported an elevation of tumor cell sialyltransferase in an extensive study of several patients with carcinomas as compared to normal tissues. Sialyltransferaseis also believed to exist on the plasma membrane (ectoenzyme), a subject much debatedamongscientists.The detection ofcell surface sialyltransferaseinvolved the incubation of intact cells with CMPNAN, and the transfer of radioactive NAN was considered to indicate the activity of the

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enzyme. Using this approach Bosmann and co-workersshowed an elevation of ectosialyltransferase in transformants (Bosmann et al., 1975; Bosmann, 1972,1973). In contrast, studies by Patt and Grimes( 1974)and Lamont et al. ( 1974) showed no change in ectosialyltransferase activity in transformants relative to controls. Subsequent studies by Deppert and Walter (1978) showed that the assay conditions were harsh and that cells were damaged under these conditions. The possibility of the breakdown of CMPNAN to free NAN and internalization and intracellular incorporation was raised. Bernacki and collaborators (Bernacki and Porter, 1978;Porter and Bernacki, 1975), using gentle techniques, showed that 10- 15% of L1210 mouse leukemic cell-associated sialyltransferase may be on cell surfaces. In their studies, breakdown of CMPNAN was ruled out (Bernacki and Porter, 1978).

E. SIALIC ACIDAND IMMUNOGENICITY The role of sialic acid in regulating antigenic expression in tumor cells in the host is indicated in many reports. Prat et al. (1975) have reported NANase-sensitive augmentation of certain antigenic determinants on G P isolated from tumor cells. Sialic acid is also seen to play a role in “masking” indirectly by negative charge repulsion in normal (Springer and Ansell, 1958) and tumor cells (Currie and Bagshawe, 1968a; Prat el al., 1975; Shearer et af.,1977). Neuraminidase treatment of normal cells- normal human lymphocytes (Lundgren and Simmons, 1971; Rogentine and Plocinik, 1974), bone marrow cells (Im and Simmons, 1971), and fetal tissues (Simmons et al., 197la)-also increases their immunogenicity. Shearer et al. (1977) demonstrated that variant L cell lines, isolated by immunoselection with antiserum raised against the parent cell line, showed an increase in cell surface sialic acid and resisted killing by antibody and complement. Neuraminidase treatment of such variant cell line restored susceptibility to killing by antisera to the parental cell with the addition of complement. Reduced transplantability of NANase-treated tumor cell has been reported in allogeneic tumor systems (Cunie and Bagshawe, 1968a,b) and syngeneic tumor systems (Simmons er al., 197lb,c; Bekesi er al., 1971; Rios and Simmons, 1973). In an attempt to determine if the effect of NANase could be applied to the clinical management of human cancers, Simmons and his colleagues first tested this approach in experimental animal tumor models (Rios and Simmons, 1974),and later Bekesi et af.(1 976) applied this approach in clinical trials with patients suffering with acute myelocytic leukemia. This “NANase therapy” resulted in partial success (Bekesi er al., 1977). Tumor cell lines of B 16 mouse melanoma, MCA-induced fibrosarcoma, and mammary carcinoma were shown to regress in syngeneic mice

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when the animals were immunized with live tumor cells pretreated in vitro with NANase and mitomycin C (Simmons and Rios, 1972, 1974; Rios and Simmons, 1973). Intratumoral injection of NANase in mice bearing transplantable methylcholanthrene-induced pulmonary squamous and Lewis lung carcinomas resulted in inhibition of tumor growth and total regression of tumors in some mice (Alley and Snodgrass, 1977). Several mechanisms have been proposed to explain the “NANase effects” on tumor growth seen in the above-mentioned studies: (1) removal of cell surface sialic acid increases antigenicity of tumor cells by exposing new immunogenic sites and cryptantigens; (2) removal of cell surface negative charges contributed by sialic acid residues of cell surface gangliosides and S-GPs can increase accessibility to binding and killing by lymphocytes and macrophages; (3) removal of sialic acid unmasks receptor sites for naturally occurring IGM antibodies, which bind to cells and kill them by a complement-mediated reaction; and (4) regulation of the activation of the alternate complement pathway via a low density of cell surface sialic acid (Fearon, 1978; Nydegger et al., 1978). Sufficient data are available to support each of these proposed mechanisms. Mechanisms (3) and (4) may be interdependent. For example, Codington and co-workers showed that S-GP such as epiglycanin can mask H-2 antigen on the cell surface (Fribergand Lilliehook, 1973; Sanford et al., 1973) thereby affecting H-Zdependent T cell killing of tumor cells. Neuraminidase treatment of cultured L 12 10 leukemic cells has resulted in increased susceptibility to killing by peritoneal macrophages taken from normal animals (Sethi and Brandies, 1973), and an inverse correlation between cell surface sialic acid and susceptibility to lysis by NK cells (Yogeeswaran et a/., 198 1). Studies by Sanford and Codington (1971) and Rosenberg and Schwartz (1974) showed that NANase-treated tumor cells are particularly sensitiveto lysis by naturally occurring IGM antibodies and complement. The work of Shearer et al. (1 977), described above, may be explained by the activation of alternate complement pathway leading to the lysis of an NANase-treated L cell variant line. VI. Serum Glycoconjugates and Glycosyltransferases-Diagnostic Value and PathophysiologicalSignificance

Elevated levels of serum glycoconjugatessuch as normal serum GPs and tissue markers, and also elevated levelsof carbohydrate transferring enzymes (glycosyltransferases), have been noticed in various pathological states (reviewed in Bacchus, 1975; Rennert, 1978). Tumor cells seem to shed their surface components (GPs, GSLs, and various enzymes) more than normal cells. The phenomenon of cell surface shedding of molecules, its role in normal

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membrane protein turnover, and its importance in certain disease processes have been extensively reviewed recently by Black (1980), and therefore the subject will be only briefly discussed here. Spontaneous shedding of cell surface components increases with cell activation (during cell growth mitogen activation in normal cells), transformation, and malignant growth. The rate ofprotein synthesis in growing diploid human fibroblast was three times higher than in resting cultures (Meedal and Levine, 1978). Similarly, the rate of carbohydrate synthesis was four times higher in growing cells than in nongrowing control cells (Kaplan and Moskowitz, 1975). These studies imply that the process of shedding and increased turnover of surface components in normal cells is a natural physiological phenomenon accompanying growth and cell activation. However, shedding occurs continuously in cancer cells and may be responsible for a part of the pathophysiologicalstate of cancer. The phenomenon of shedding may be mediated by an increase in cell surface proteases, a decrease in cross-linking of membrane components by transglutaminases, a disruption ofcytoskeletal elements, and a decrease in membrane-bound calcium and other membrane protein modifications (reviewed in Black, 1980). A number of studies indicate that metastatic cells are less immunogenic and shed more of their surface components very rapidly (Alexander, 1974; Davey et a/., 1976; Jamasbi et al., 1978). For example, Kim et al. (1975) have shown that metastatic rat mammary carcinomas have less membrane-associated glycocalyx and higher levels of circulating tumor antigens in serum compared with nonmetastatic controls. In other studies, Bystryn (1977) and Rahman et af. (1977) showed that shedding of labeled human melanoma antigens is very rapid; in 3 hr, about 50%of melanoma surface antigen could be released. Large amounts of free tumor antigens have been found in sera(Rao and Bonavida, 1977;Lopez and Thompson, 1977; Murray et af.,1978; Taub et af.,1978).While many of the studies discussed above have focused on the shedding of tumor antigens, several other reports have shown increased levels of serum gangliosides (Skipski et al., 1975; Kloppel et af.,1977, 1978; Langle et af.,1979; Kloppel and Morre, 1980)and total sialoglycoconjugates(Bernacki and Kim, 1977) in tumor-bearing animals and humans, although the antigenic nature of the material was not defined. The effect of the growth of malignant hepatoma on the serum ganglioside (sialyl-GSL)profile was studied by Skipski et af.( 1979, and they noticed that the tumor serum ganglioside profile closely resembled that of tumor tissue and differed from that of normal serum. This study suggests that tumor GSLs could be detected in serum, and, therefore, may be a useful indicator of malignant hepatomagrowth. In another study, Lo et al. ( 1980)found that all cases of human astrocytoma showed an increase in serum gangliosides, whereas a screening of other types of human brain tumors including pitutary

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adenoma, ependymoma, teratoma, and other intracranial tumors did not alter serum ganglioside levels. A recent report by Morre and collaborators showed that serum-bound neutral-GSLs in hepatoma-bearing animals were five to nine times higher than in control animals, and, particularly, glucosylceramide increased 10- to 20-fold in serum and may be used in cancer detection (Walter et al., 1980). Elevation of serum sialic acid was unique to tumor growth; other diseases and inflammation did not elevate serum sialic acid levels (Kloppel el al., 1978). The levels of ganglioside-bound sialic acid correlated strictly with tumor growth, and excision of tumor often resulted in a drop in serum sialic acid to near normal levels (Kloppel et al., 1977). Bernacki and Kim (1977) and Harvey et al. ( 1981) observed a good correlation between the levels of serum sialoglycoconjugatesand the size of rat mammary carcinoma and other human malignant tumors. Interestingly, in the rat study, the metastasizing tumors seemed to shed more sialoglycoconjugatesthat did nonmetastasizing tumors (Bernacki and Kim, 1977). Tatsumura et al. (1 977) observed that serum GP-bound fucose increased in patients with malignant tumors and benign tumor-bearing cases did not show these changes. Waalkes et al. (1978) reported that sialic acid, fucose bound to glycoconjugates, and the levels of carcinoembryonic antigens are the most useful markers for following the course of human metastatic breast carcinoma. Other carbohydrates, such as mannose and galactose, bound to glycoconjugates showed increases in sera from certain tumor-bearing patients and not in other cases (Waalkes et af.,1978).Silver et al. (1980)also found that sialic acid bound to serum-GP increased consistently in a study of 59 melanoma patients. Serum glycosyl transferases such as fucosyl-, galactosyl-, and sialyltransferases (terminal transferases) involved in the transfer of these sugars to GPs and GSLs have been shown to be elevated in humans suffering from several types of cancer. Increases of serum galactosyltransferaseactivity have been seen in humans with liver cancer (Kim et al., 1972)and rats with carcinomas (Chatterjee and Kim, 1977).Elevation in the levels of serum galactosyltransferase, particularly isoenzyme 11, was found in several carcinomas of the lung, breast, stomach, colon, and pancreas and in chronic lymphocytic leukemia (Weiser et al., 1976; reviewed in Weiser and Wilson, 1981). Interestingly,the level ofthis isoenzyme I1 ofgalactosyltransferasewas found to be higher in patients with disseminated tumors, compared with subjects with local tumors (Podolsky et af., 1978). The activity of sialyltransferasewas found to be increased in patients with breast cancer when compared with patients having benign diseases and healthy control subjects (Kessel and Allen, 1975; Henderson and Kessel, 1977; Ip and Dao, 1978). Elevation of sialyltransferasewas found in metastatic mammary carcinoma-bearing rats and correlated with tumor size, but

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not in nonmetastatic mammary tumor-bearing animals (Bernacki and Kim, 1977). In contrast, a recent study by Evans et af.(1980) showed that Fisher rats bearing R3230AC transplantable mammary adenocarcinoma did not show elevation of serum sialyltransferase until the tumors reached 20 g or until tumors had been present in the animals for 2 1 days or longer. Serum sialyltransferaseincreased in serum from 2 1 days to 25 days, concurrent with the increase in sialyltransferaseactivity in liver tissue. When the tumors were excised, the serum enzyme levels returned to control values in 4 days, suggesting a half-life of 2 days for the enzyme in serum. The authors concluded that liver may be the origin of serum sialyltransferase which increases in response to tumor growth and elaborates the enzyme in serum (Evans et af.,1980). But the author’s conclusions may be misleading because liver is the site for clearance of several circulating proteins and sialyltransferases which may be picked up by hepatocytes (Ashwell and Morrell, 1974)or by Kupffer cells (Toth et af.,1982);thus the increase in liver enzyme activity may be a reflection of a normal physiological mechanism for clearance of glycoproteins and glycoenzymes. Levels of both fucosyltransferaseA, which transfers fucose from GDPfucose to galactose residues of acceptor proteins, and galactosetransferase in cancer patients with colon carcinoma overlap considerably with levels in benign disease and healthy individuals. In contrast, the levels of fucosyltransferase B, which transfers fucose to GalNAc residues of acceptor-GP, have been shown to be useful indicators of growing colon carcinoma (Chatterjee and Kim, 1977;Bauer et a!., 1978). Bauer et af.(1978) observed a striking decrease in fucosyltransferaseB levels in the serum of patients after successful therapy (surgery, chemotherapy, and radiation). A number of mechanisms exist by which shed cell surface components (GPs and GSLs) in plasma can facilitate tumor escape, and the subject has been extensively reviewed by Black ( 1980). Shed tumor antigen or antigen antibody complexes can mediate blocking of cell-mediated immune reactions (Bonavida, 1974; reviewed in Pierce and Baldwin, 1977). Immune complexes in sera of tumor-bearing host contain tumor antigens of varying specificities(Loughbridgeand Lewis, 1971) or fetal antigens (Costanza et af., 1973) or viral antigens (Oldstone et al., 1974). Having evoked a humoral immune response, such antigens are likely to combine with shed antigens from the tumor or, by directly stripping surface antigens from cell surfaces, bind to the surface of tumor cells. In a high percentage of human sera (50-609/0), the presence of immune complexes (Ag-Ab) can be demonstrated (Rossen ef af., 1977; Theofilopoulos et al., 1977). Although the immune complexes are shown to act as blocking factors in immune reactions, the exact mechanism is not clear; presumably, the immune complexes bind to Fc receptor on K cells that mediate antibody-dependent cell-

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mediated cytotoxicity (Prather and Lausch, 1976) and suppressor cells (Friedman et al., 1977). Shed cell surface antigen or Ag- Ab complexes can also substitute for suppressor cells. For example, several investigators showed that the injection of soluble tumor antigens, tumor extracts, or X-irradiated tumor cells in rodents prior to tumor challenge can enhance tumor growth (Rao and Bonavida, 1976; Embleton, 1976; Hellstrom and Hellstrom, 1978). Tumor enhancement by these shed surface molecules or the Ag- Ab complexes thereof was due to the induction of a radiosensitive population of T cells (T-suppressor cells) (Hellstrom and Hellstrom, 1978). Recently studies by Langle et al. (1979) and Whisler and Yates (1980) have indicated that membrane gangliosides from human brain tissue in micellar or liposomal forms can inhibit mitogen-induced proliferative response. These model studies with brain gangliosides(Langleet al., 1979;Whisler and Yates, 1980) suggest that gangliosides shed into Serum of the tumor-bearing host can be immunosuppressive by yet another mechanism, different from those considered earlier. Studies by O’Kennedy et al. ( I 980) showed that total, spontaneously released, and NANase-releasable sialic acid from lymphocyte cell surfaces of patients with metastatic cancers was higher than in the case of nonmetastatic patient lymphocytes, and these lymphocytes had an impaired T cell function. This study indicates the potential role of elevated serum sialoglycoconjugateswhich may be taken up by lymphocytes; these components may contribute to an immunosuppressive state in cancer. VII. Summary and Prospects

Although the functions of membrane GSLs and GPs are not clearly understood, many important functions have been postulated for the carbohydrate moieties ofthe molecule. Several lines of evidence suggest that GSLs are involved in growth control. In accordance with this hypothesis, cell transformation always results in a variety of alterations in different classes of GSL. Only certain of these transformation-associated GSL changes correlate with tumorigenesis and metastasis. GSL changesassociated with malignancy do not stop at the first stage of a transforming event, but continue on as a cascade of changes with progression of the malignancy. Changes in GSL composition of transformed cells seem to occur not only at the surface membrane, but also at other inner membranes where GSLs are localized. Altered GSL composition seen in transformed cell membrane is due to a repression of glycosyltransferasesand probably is mediated by an increase in catabolism as well. These transformation-associated changes in membrane GSL often result in an antigenic change and Several tumor-associated GSL antigens are described in various tumor tissues. A few unique CSL antigens are known in experimental and human tumors, and these findings invite the approach of using either toxin-conjugated antibodies (Urdal and Hakomori,

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1980) or antibodies alone directed against these GSL in immunotherapy (Young and Hakomori, 1981). Malignant transformation also results in various GP changes. These include a loss of fibronectin which binds to collagen, proteoglycans, and gangliosides and acts as an intercellular matrix GP. Therefore, the loss of fibronectin might result in a release of tumor cells from the site of origin. However, the loss of fibronectin seen in transformed cells does not correlate with tumorigenic potential. Several GP markers that appear in various human lymphoproliferative malignancies are not cancer specific, but may reflect certain stages of differentiation in these cells. Whole-cell sialic acid, total ganglioside content, and sialic acid bound to GP oligosaccharides are increased in various types of transformed cells and may reflect a general increase in cell surface negative charge. More recent studies suggest that the cell surface sialic acid and degree of sialylation correlate with metastatic potential in different types of cancers. Elevated cell surface sialoglycoconjugatesseen in malignant tumor cells may influence several properties that facilitate tumor cell metastasis. Malignant tumor cells also release cell surface glycoconjugates and glycosyltransferases in plasma during tumor progression and offer the potential for use in cancer diagnosis. These released tumor products may be responsible for blocking humoral and cell-mediated immune responses and also cause a general immunosuppressive state by inhibiting lymphocyte proliferation. So far, many of the research efforts in the area of glycoconjugates have focused on defining carbohydrate changesassociated with malignancy. Here, I will consider five potential areas in research that may be directed toward altering glycoconjugates on tumor cell surfaces to cause a reversion of the transformed phenotype. The ultimate goal, of course, is a complete tumor regression. 1. An approach to decrease the concentration of cell surface sialoglycoconjugates by the use of CMP-NAN analog which will inhibit sialyltransferase activity has been made (Klohs et al., 1979;reviewed in Bernacki et al., 1978). 2. Other approaches that affect glycosylation of glycoconjugates, using excesses of glucosamine, glycosamine analog, and a GP-N-glycosylation inhibitor such as tunicamycin (Bernacki et a!., 1977; Milenkovic and Johnson, 1980) are shown to be useful in modifying tumor cell surface carbohydrates and inhibiting tumor growth. 3. Certain corticosteroid hormones or their analogs can restore normal morphology and enhance tumor cell adhesion (Armelin and Armelin, 1978). Corticosteroids also prevent shedding of cell surface molecules such as plasminogen activator (Viaje et al., 1977;Vassalli and Reich, 1977),increase activity of glycolipid glycosyltransferase (Dawson and Kernes, 1978), and

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decrease net cell surface charge and sialoglycopeptidesin GP (Behrens, 1976) in various tumor cells. Therefore, the use of corticosteroidsin altering cell surface carbohydrates could be of value. 4. Studies by Sporn et al. (1976) and Colburn et al. (198 1) showed that retinoic acid can exert a pronounced antipromoter effect on phorbol estertreated cells in culture and in tissues in viva Recent studies by Srinivas and Colburn ( 1982) showed that retinoic acid can reverse gangliosidepatterns of phorbol ester-treated cells so they more closely resemble control untreated cells. These studies indicate the potential usefulness of retinoids in normalizing the altered ganglioside metabolism seen in the transformed state. Recent work by Morre and collaborators also demonstrates that retinoids can prevent tumor cell metastases in a transplantable hepatoma model (Morre el al., 1980). These investigators also showed that retinoids alter ganglioside metabolism in metastatic hepatoma so that it resembles more closely the metabolism in nonmetastatic tumors. Studies by Morre et al. (198 1) and Srinivas and Colburn ( 1982) show that retinoids can normalize the ganglioside pattern of an aberrant cell. The changes in gangliosides that occur are a decrease in the class of di- and trisialogangliosides which are suggested to be the fibronectin receptor on cell surfaces (Kleinman et al., 1979). Retinoids also restore fibronectin in transformed cells (Patt et al., 1978) and increase mannose-rich oligosaccharides on GP of spontanously transformed 3- 12 cells (Sasak et al., 1980). Thus retinoids can normalize the composition of fibronectin and gangliosides and increase GP-glycosylation on tumor cell surfaces, thereby promoting intercellular linkages that might prevent tumor cells from breaking away in metastatic spread. More work in this area may prove to be of vital importance. 5. Generation of large amounts of monoclonal antibodies against tumor cell surface carbohydrate structureswill aid in tumor diagnosis as well as in immunotherapeutic approaches. Thus carbohydrate-directed research on potential cancer therapy may offer useful results in the future. ACKNOWLEDGMENTS The author wishes to expresshis gratitudeto his previous mentors, Dr.R. K. Murray and Dr. S. Hakomori, for their encouragement during the earlier phases of the work cited in this article; his thanks are also due to Dr. P. Black for his ideas and helpfuldiscussion on the phenomenon of cell surface shedding presented here. He is also grateful to Dr.I. Taylor and Ms. C. Peirce for correctingthis manuscript and to Mrs. Anne Brennan and Mrs. Beverly Wyche for typing it.

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INDEX A N-Acetyl-2-aminofluorene. repair of DNA modificationscaused by, 43 -48 Actinomycin B as inhibitor of leukemia cell differentiation, 142 Adrenarche, cervical cancer and, 107- I 10 Agglutinins, stimulation of leukemia cell differentiation by, I32 Alkyllysophospholipids,stimulation of leukemia cell differentiation by. 128 Antioxidants as inhibitors of leukemia cell differentiation, 144- 145 Arginase, stimulation of leukemia cell differentiaiim by, 127

B Bacillus Calmeffe-GuPrin (BCG), stimulation of leukemia cell differentiation by, 131-132 Bacterial cell wall skeletons, stimulation of leukemia cell differentiation by. 131 - 132 Brain tumors, glycosphingolipidchanges in, 308 Breast cancer epidemiological aspects of, 78-66 genesis of, 104- 107 mammocarcingenic hydrocarbon role in 105-106 steroid hormone receptors in, 61 -75 steroid metabolism role in, 77 - I 19

Cell surface glycolipids, role in malignant transformation, 289-350 Cervical cancer epidemiological aspects of, 78-86 genesis of, 107hormonal aspects of, 9 I - 92 steroid metabolism in, 77- I 19 Chemotherapy of myeloid leukemia, 12 I - I69 Chick oviduct, estrogen response of, 96 Chloroquine, stimulation of leukemia cell differentiation by, 131 - 132 Chromatin, structure of, role in DNA repair, 49-50 Colony-stimulatingfactor. stimulation of leukemia cell differentiation by, 124- I27 Cytochalasin B as inhibitor of leukemia cell differentiation, I42 Cytochrorne oxidase, induction in leukemia cell differentiation, 137 Cytoplasmic proteins, induction in leukemia cell differentiation, 137 - 138 Cytotoxic lymphocytes (CTL) antitumor reactivity of, 200-205 assay systems for, 224-230 induction of, 215-224 nonspecifically reactive, 23 I -235 production, 240-244 production of, 205 2 I 5

-

D D-factor, stimulation of leukemia cell differentiation by, 124- 127, 132 - 134 DMSO,stimulation of leukemia cell differentiation by, 132 DNA, OV-induced pyrimidine dimers of, repair of, 36 - 38 DNA excision-repair, 23-59 06-alkyl acceptor protein in, 27-36

C CAMP,stimulation of leukemia cell differentiation by, 129- 130 Carcinogenesis epidemiology and endocrinology of, 101- 104 host steroid metabolism in, 77- I I9 3.5 I

352

INDEX

DNA excision-repair (contd.) chromatin structure and, 49-50 N-glycosylases in, 27 -36 inducibility of, 33-36 inhibition of, 50- 52 initiation of, 23-59 of modificationscaused by N-acetyl-2-aminofluorene, 43 -48 pol ycyclic aromatic hydrocarbons, 38-43

E Effectorlymphocytes, culture and generation Of, 181-237 Endometrial cancer epidemiological aspects of, 78 - 86 genesis of, 1 10- I 13 hormonal aspects of, 92 -95 steroid metabolism in, 77- I19 Enzymes, induction in leukemia cell differentiation, 135- I37 Epiglycanin, malignancy and, 320-32 I Estrogen receptors, role in human breast cancer, 61 -75 Excision-repair of DNA, 23-59

F Fibronectin, malignancy and, 3 I5 -3 1 8 Formamidopyrimidine N-gl ycosylases, role in DNA excision-repair, 29- 3 I

G Gastrointestinal tumors, glywsphingolipid changes in, 305 -308 Glucocorticoid hormones, stimulation of leukemia cell differentiation by, 129-130 Glucose-6-phosphatase,induction in leukemia cell differentiation, 137 Glycolipidsof cell surface, role in malignant transformation, 289- 350 Glycoproteins (GP) functions of, 296-299 table, 298 malignancy and, 3 I5 - 32 1 Glycosphingolipids (GSL) antigens of, in tumors, 310-315 function of, 293-296 table, 295

Glycosphingolipids(GSL) (contd.) malignancy and, 299 - 3 15 in cultured cells, 299-304 in growth control and metastasis, 309-310 in tumors, 304-308 Structure Of, 291 -293 N-GI ywsylases role in DNA excision- repair, 27 - 36 inducibility of, 33-36 Glycosyltransferasesin serum, diagnostic value for malignancy, 332-336

H H-2 antigens of SJL reticulum cell sarcomas, 13-14 Histocompatibility complex antigens, lymphocyte reactivity to, 181- 192 Histones, stimulation of leukemia cell differentiation by, 127 Histone H3 as inhibitor of leukemia cell differentiation, 145 HL-60 leukemia cell line, 123 differentiation induction in, 153- 159 Hodgkin’s disease, SIL reticulum cell sarcoma relationship to, 2,7 Humans breast cancer in, role of steroid hormone receptors in, 61 -75 glycosphingolipid changes in tumors of, 304 - 308 Hypoxanthine N-glycosylase, role in DNA excision -repair, 27 -29

I IL-2 growth factor, 237-244 effector cells propagated by, 255 -257 production of, 237-239 Immunogenicity, sialic acid and, 331 -332 Immunological surveillance concept, 1 -2 Immunotherapy of tumors adoptive, 179- 180,244-262 in experimental systems, 249-259 possible clinical use, 259 - 262 theoretical aspects of, I7 1 - 177 Insect growth, steroid hormone role in, 96-97 Interferons, stimulation of leukemia cell differentiation by, 130- 13 1

353

INDEX

K K562 leukemia cell line, 123 differentiation induction in, 159- 160 KG-1 leukemia cell line, 123 differentiation induction in. 160- 161

L Lectins, stimulation of leukemia cell differentiation by, 132 Leukemia cell, differentiation induction of, 121- 169 Lipids, stimulation of leukemia cell differentiation by, I27 - 129 Lipogenic enzymes, induction in leukemia cell differentiation, 137 Lymphoproliferativediseases, glycoprotein role in, 3 18- 320 Lysosomal enzymes, induction in leukemia cell differentiation, I35 - 136

M MI leukemia cell line, 123 sensitization of, 147- 148 therapy of mice by, 149- I52 ML- 1 and ML-3 leukemia cell lines, 123 differentiation induction in, 161 Malignancy glycoproteinsand, 3 I5 - 32 I glycosphingolipidsand, 299 - 3 15 sialoglycoconjugatesand, 32 1 -332 Malignant transformation, cell su&ce glycolipids and, 289-350 Membrane components in myeloid leukemia cell differentiation, 134- 135 3-MethyladenineN-glycosylase, role in DNA excision-repair, 29-31 7-MethylguanineN-glycosylase, role in DNA excision-repair, 29-31 Myeloid leukemia chemotherapy of, I 2 1 - 169 Myeloid leukemia cell lines description of, 122- 123 induction ofdifferentiation in, 123- 148 by antibacterial agents, I3 I - 132 cytoplasmic proteins, 137- 138 enzyme role, 135- I37 feedback control mechanisms, 141 - 142 by glucocorticoid hormones, prmtaglandins, and CAMP, 129- 130

Myeloid leukemia cell lines (contd.) in human leukemia cells, 152- 162 inhibitors of, 142- 145 by lipids, I27 - 129 proliferation-change potential in, 138- 141 by proteins, 124- 127 sensitizers of, 147- 148 by synthetic polyribonucleotidesand interferons, 130- 13I by vitamins, 130 therapy ofanimals with, 148- 152

0 Oral contraceptives,endometrial cancer and, 112- 1 I3

P Phorbol esters as inhibitors of leukemia cell differentiation, 143- 144 Poly-L-arginine as inhibitor of leukemia cell differentiation, 145 Polycyclic aromatic hydrocarbons, DNA modifications caused by, repair of, 38-43 Poly-L-lysine as inhibitor of leukemia cell differentiation, 145 Polyribonucleotides,synthetic, stimulation of leukemia cell differentiation by, 130-131 Prostaglandin(s) as inhibitors of leukemia cell differentiation, 142-143 stimulation of leukemia cell differentiation by, 129- I30 Prostaglandin synthetases, induction in leukemia cell differentiation, I36 - I37 Proteins, stimulation of leukemia cell differentiation by, 124- 127,153- 154 Puromycin as inhibitor of leukemia cell differentiation, 142 Pyrimidine dimer N-glycosylase, role in DNA excision- repair, 3 1 - 33

R R453 leukemia cell line, I23 sensitization of, 148 Respiratory tract tumors, glycosphingolipid changes in, 305- 308

354

INDEX

Reticuloendothelialtumors glycoprotein changes in, 3 19 glycosphingolipidchanges in, 308 Retinoids as inhibitors of leukemia cell differentiation, 143- 144 Reverse transcriptase, induction in leukemia cell differentiation, 136- 137

S Sialic acid immunogenicity and, 33 1-332 of malignant cells, 322 -326 Sialoglycoconjugates malignancy and, 32 1 -332 gangliosidechanges, 327 sialic acid content, 322-326 in serum, diagnostic value of, 332-336 Sialoglycopeptidesof transformed cell surface proteins, 327 -330 Sialyltransferasesof transformed cells, 330-331 SJL reticulum cell sarcomas,1-22 cellular interactions with host cells, 18- 20 development in vim, 7-9 etiology of, 5 histopathology and characteristicsof, 3- 7 in culture, 6 Hodgkin's disease, relationship to, 2, 7 host cell infiltration by, 7-9 host immune response against tumors of, 10-13

immune competence of normal and mice-bearing tumors of, 8-9 in vivo regulation of growth of, 17-210 antigen-reactivecell opsonization, 17-18 dependence on host response, 18 - 19 maintenance of tumor lines, 5-7 origin of, 3 - 5 pathogenesis of, 3 transplantation resistance of, 9- 10 tumor-associatedantigens of, 13- 17 H-2 antigens of, I3 - 14 hybrid IE/c antigens on, 14- 17 Steroid hormone receptors cell kinetics of, 68 cell morphology of, 67-68 in human breast cancer, 61 -75 physiology, 63-66 prognosis, 68-69

Steroid hormone receptors (conrd.) role in therapy, 69 - 72 measurement of, 62-63 pathology Of, 66-68 Steroid hormones biology and molecular biology of, 95 - 10 1 endogenous and urinary, metabolic relation among, 94 Steroid metabolism, role in various cancers, 77- 119 Suppressorcells (SK), formation and properties of, 235-237

T Thymidine analogs as inhibitor of leukemia cell differentiation, 142 Thymine glycol N-glycosylase, role in DNA excision-repair, 31 -33 Tumors immunotherapy of, 17 1 - 180 adoptive, 179- 180,244-262 Tumor-associated antigens (TAA) reactivity to, 196-205 of SJL reticulum cell sarcomas, 13 - 17 Tumor neoantigens, 2 Tumor promoters, leukemia cell differentiation by, 155-156 Tumor virus, steroid hormone role in effects of, 97- 100 Tunicamycin, stimulation of leukemia cell differentiation by, 13 1 - 132

U Uracil N-glycosylase, role in DNA excisionrepair, 27-29 Urea N-glycosylase, role in DNA excisionrepair, 3 1 - 33 Urine, neutral steroids in, 88

v Vaginal epithelium, steroid hormone role in effectsof, 100-101 Vitamins, stimulation of leukemia cell differentiation by, 130

W WEHI-3B leukemia cell line, 123

CONTENTS OF PREVIOUS VOLUMES

Volume 1 Electronic Configuration and Carcinogenesis C. A . Coulson Epidermal Carcinogenesis E. V. Cowdry The Milk Agent in the Origin of Mammary Tumors in Mice L. Dmochowski 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 Charles Heidelberger The Carcinogenic Aminoazo Dyes James A . Miller and Elizabeth C. Miller The Chemistry o f Cytotoxic Alkylating Agents M . C. J . Ross Nutrition in Relation to Cancer Albert Tannenbaum and Herbert Silverstone Plasma Proteins in Cancer Richard J . Winzler

Carcinogenesis and Tumor Pathogenesis I . Berenblum Ionizing Radiations and Cancer Austin M. Brues Survival and Preservation of Tumors in the Frozen State James Craigie Energy and Nitrogen Metabolism in Cancer Leonard D. Fenninger and G. Burroughs Mider Some Aspects of the Clinical Use of Nitrogen Mustards Calvin Z Klopp and Jeanne C. Batemnn Genetic Studies in Experimental Cancer L. w. Law The Role of Viruses in the Production of Cancer C. Oberling and M. Guerin Experimental Cancer Chemotherapy C. Chester Stock

AUTHOR INDEX-SUBJECT INDEX

Volume 2 The Reactions of Carcinogens with Macromolecules Peter Alexander Chemical Constitution and Carcinogenic Activity G. M. Badger 355

AUTHOR INDEX-SUBJECT INDEX

Volume 3 Etiology of Lung Cancer Richard Doll The Experimental Development and Metabolism of Thyroid Gland Tumors Harold P. Morris Electronic Structure and Carcinogenic Activity and Aromatic Molecules: New Developments A . Pullman and B . Pullman Some Aspects of Carcinogenesis I? Rondoni Pulmonary Tumors in Experimental Animals Michael B. Shimkin

356

CONTENTS OF PREVIOUS VOLUMES

Oxidative Metabolism of Neoplastic 'ITssues Sidney Weinhouse AUTHOR INDEX-SUBJECT INDEX

Volume 4 Advances in Chemotherapy of Cancer in Man Sidney Farber, Rudolf Toch, Edward Manning Sears, and Donald Pinkel The Use of Myleran and Similar Agents in Chronic Leukemias D. A . G.Calton The Employment of Methods of Inhibition Analysis in the Normal and TumorBearing Mammalian Organism Abraham Godin Some Recent Work on Tumor Immunity I! A. Corer Inductive Tissue Interaction in Development Clifford Grobstein Lipids in Cancer Frances L. Haven and W R. Bloor The Relation between Carcinogenic Activity and the Physical and Chemical Properties of Angular Benzacridines A . L a c a s s a g n e , N. P. Buu HOT, R . Daudel, and E Zajdela The Hormonal Genesis of Mammary Cancer 0. Miihlbock AUTHOR INDEX-SUBJECT INDEX

Volume 5 Tumor-Host Relations R. W.Begg Primary Carcinoma of the Liver Charles Berman Protein Synthesis with Special Reference to Growth Processes both Normal and Abnormal l? N. Campbell The Newer Concept of Cancer Toxin War0 Nakahara and Fumiko Fukuoka Chemically Induced Tumors of Fowls l? R. Peacock

Anemia in Cancer Vincent E. Price and Robert E. Greenfield Specific Tumor Antigens L. A . Zilber Chemistry, Carcinogenicity, and Metabolism of 2-Fluorenamine and Related Compounds Elizabeth K. Weisburger and John H. Weisburger 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. Krementz Viral Etiology of Mouse Leukemia Ludwick Gross Radiation Chimeras l? C. Koller, A. J . S. Davies, and Sheila M.A. Doak Etiology and Pathogenesis of Mouse Leukemia J. E A . P. Miller Antagonists of Purine and Pyrimidine Metabolites and of Folk Acid G. M . Timmis Behavior of Liver Enzymes in Hepatocarcinogenesis George Weber AUTHOR INDEX-SUBJECT INDEX

Volume 7 Avian Virus Growths and Their Etiologic Agents J. W Beard Mechanisms of Resistance to Anticancer Agents R. W. Brockman

CONTENTS OF PREVIOUS VOLUMES Cross Resistance and Collateral Sensitivity Studies in Cancer Chemotherapy Dorris J . Hutchison Cytogenic Studies in Chronic Myeloid Leukemia W. M . Court Brown and Ishhel M. Tough Ethionine Carcinogenesis Emmanuel Farber Atmospheric Factors in Pathogenesis of Lung Cancer Paul Kotin and Hans L. Falk Progress with Some Tumor Viruses of Chickens and Mammals: The Problem of Passenger Viruses G.Negroni AUTHOR INDEX-SUBJECT INDEX

Volume 8 The Structure of Tumor Viruses and Its Bearing on Their Relation to Viruses in General A . F: Howatson Nuclear Proteins of Neoplastic Cells Harris Busch and William J . Steele Nucleolar Chromosomes: Structures, Interactions, and Perspectives M. J . Kopac and Gladys M. Mazeyko Carcinogenesis Related to Foods Contaminated by Processing and Fungal Metabolites H.E Kraybill and M. B. Shimkin Experimental Tobacco Carcinogenesis Ernest L. Wynder und Dierrich Hoffman AUTHOR INDEX-SUBJECT INDEX

Volume 9 Urinary Enzymes and Their Diagnostic Value in Human Cancer Richard StambauRh and Sidney Weinhouse The Relation of the Immune Reaction to Cancer Louis V. Caso Amino Acid Transport in Tumor Cells R . M . Johnstone and I? G. Scholefield

357

Studies on the Development, Biochemistry, and Biology of Experimental Hepatomas Harold P Morris Biochemistry of Normal and Leukemic Leucocytes, Thrombocytes, and Bone Marrow Cells I . E Seitz AUTHOR INDEX-SUBJECT INDEX

Volume 10 Carcinogens, Enzyme Induction, and Gene Action H. V. Gelboin In Vitro Studies on Protein Synthesis by Malignant Cells A. Clark Griffin The Enzymatic Pattern of Neoplastic Tissue W. Eugene Knox Carcinogenic Nitroso Compounds P N. Magee and J . M. Barnes The Sulfhydryl Group and Carcinogenesis J. S. Harrington The Treatment of Plasma Cell Myeloma Daniel E. Bergsagel, K. M. Griffith, A. Haut, and W. J . Stuckley, Jr. AUTHOR INDEX-SUBJECT INDEX

Volume 11 The Carcinogenic Action and Metabolism of Urethran and N-Hydroxyurethran Sidney S. Mirvish Runting Syndromes, Autoimmunity, and Neoplasia D. Keast Viral-Induced Enzymes and the Problem of Viral Oncogenesis Saul Kit The Growth-Regulating Activity of Polyanions: A Theoretical Discussion of Their Place in the Intercellular Environment and Their Role in Cell Physiology William Regelson

358

CONTENTS OF PREVIOUS VOLUMES

Molecular Geometry and Carcinogenic Activity of Aromatic Compounds. New Perspectives Joseph C . Arcos and Mary E Argus CUMULATIVE INDEX

Role of Cell Association in Virus Infection and Virus Rescue J . Svoboda and 1. Hlotanek Cancer of the Urinary Tract D . B. Clayson and E. H . Cooper Aspects of the EB Virus M . A . Epstein AUTHOR INDEX-SUBJECT INDEX

Volume 12 Antigens Induced by the Mouse Leukemia Viruses G. Pasternak Immunological Aspects of Carcinogenesis by Deoxyribonucleic Acid Tumor Viruses G . 1. Deichmun Replication of Oncogenic Viruses in VirusInduced Tumor Cells-Their Persistence and Interaction with Other Viruses H . Hanqfusa Cellular Immunity against Tumor Antigens Kurl Erik Hellstrom and Ingegerd Hellstrdm Perspectives in the Epidemiology of Leukemia Irving L . Kessler and Abraham M . Lilienfeld AUTHOR INDEX-SUBJEm INDEX

Volume 13 The Role of Immunoblasts in Host Resistance and Immunotherapy of P r h a r y Sarcomata F! Alexander and J . G . Hull Evidence for the Viral Etiology of Leukemia in the Domestic Mammals Oswald Jarrett The Function of the Delayed Sensitivity Reaction as Revealed in the Graft Reaction Culture Haim Ginsburg Epigenetic Processes and Their Relevance to the Study of Neoplasia Gajanan V. Sherbet The Characteristics of Animal Cells 'Transformed in Vitro Ian Macpherson

Volume 14 Active Immunotherapy Georges Mathe' 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 hogression of Leukemia Donald Metca(f Complement and Tumor Immunology Kusuya Nishioka Alpha-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors G . I . Abelev Low Dose Radiation Cancers in Man Alice Stewart AUTHOR INDEX-SUBJECT INDEX

Volume 15 Oncogenicity and Cell Transformation by Papovavirus SV40: The Role of the Viral Genome J. S. Butel, S . S . Teverhia, and J . L . Melnick Nasopharyngeal Carcinoma (NPC) J . H . C. Ho Transcriptional Regulation in Eukaryotic Cells A . J . MacGillivray, J . P a u l , and G . Threlfall

CONTENTS OF PREVIOUS VOLUMES

359

Atypical Transfer R N A s and Their Origin in Neoplastic Cells Ernest Borek and Sylvia J. Kerr Use of Genetic Markers to Study Cellular Origin and Development of Tumors in Human Females Philip J . Fialkow Electron Spin Resonance Studies of Carcinogenesis Harold M. Swartz Some Biochemical Aspects of the Relationship between the Tumor and the Host V. S . Shapot Nuclear Proteins and the Cell Cycle Gary Stein and Renato Baserga

Some Aspects of the Epidemiology and Etiology of Esophageal Cancer with Particular Emphasis on the Transkei, South Africa Gerald I? Warwick and John S . Harington Genetic Control of Murine Viral Leukemogenesis Frank 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, Jr. Mammary Neoplasia in Mice S. Nandi and Charles M . McGruth

AUTHOR INDEX-SUBJECT INDEX

AUTHOR INDEX-SUBJECT INDEX

Volume 16

Volume 18

Polysaccharides in Cancer Vijai N . Nigam and Antonio Cantero Antitumor Effects of Interferon Ion Gresser Transformation by Polyoma Virus and Simian Virus 40 Joe Samhrook Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing? Sir Alexander Haddow The Expression of Normal Histocompatibility Antigens in Tumor Cells Alena Lengerova

Immunological Aspects of Chemical Carcinogenesis R . W. Baldwin lsozymes and Cancer Funny 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. Specfor, and Robert A . Good Recent Observations Related to the Chemotherapy and Immunology of Gestational 1,3-Bis(2-Chloroethyl)-l-Nitrosourea C horiocarcinoma (BCNU) and Other Nitrosoureas in K. D. Bagshave Cancer Treatment: A Review Glycolipids of Tumor Cell Membrane Stephen K. Carter, Frank M . Schohel, Jr., Sen-itiroh Hakomori Lawrence E. Broder, and Thomas P. Chemical Oncogenesis in Culture Johnston Charles Heidelherger

AUTHOR INDEX-SUBJECT INDEX

AUTHOR INDEX-SUBJECT INDEX

Volume 17 Volume 19 Polysaccharides in Cancer: Glycoproteins and Glycolipids Vijui N . Nigam and Antonio Cantero

Comparative Aspects of Mammary Tumors J . M . Hamilton

360

CONTENTS OF PREVIOUS VOLUMES

The Cellular and Molecular Biology of RNA Tumor Viruses, Especially Avian Leukosis-Sarcoma Viruses, and Their Relatives Howard M. Temin Cancer, Differentiation, and Embryonic Antigens: Some Central Problems J . H. Coggin, Jr. and N . G. Anderson Simian Herpesviruses and Neoplasia Fredrich W.Deinhardt, Lawrence A . Falk, and Lauren G . Wove Cell-Mediated Immunity to Tumor Cells Ronald B. Herberman Herpesviruses and Cancer Fred Rapp Cyclic AMP and the Transformation of Fibroblasts Ira Pastan and George S. Johnson Tumor Angiogenesis Judah Folkman SUBJECT INDEX

Volume 21 Lung Tumors in Mice: Application to Carcinogenesis Bioassay Michael 8.Shimkin and Gary D.Stoner Cell Death in Normal and Malignant Tissues E. H. Cooper, A. J. Bedford, and T. E. Kenny The Histocompatibility-Linked Immune Response Genes Baruj Benacerraf and David H . Katz Horizontally and Vertically Transmitted Oncornaviruses of Cats M. Essex Epithelial Cells: Growth in Culture of Normal and Neoplastic Forms Keef A. Rafferty, Jr. Selection of Biochemically Variant, in Some Cases Mutant, Mammalian Cells in Culture G. B. elements The Role of DNA Repair and Somatic Mutation in Carcinogenesis James E. Trosko and Ernest H. Y. Chu SUBJECT INDEX

Volume 20 Tumor Cell Surfaces: General Alterations Detected by Agglutinins Annette M . C. Rapin and Max M . Burger Principles of Immunological Tolerance and Immunocyte Receptor Blockade G. J . V. Nossal The Role of Macrophages in Defense against Neoplastic Disease Michael H. Levy and E. Frederick Wheelock Epoxides in Polycyclic Aromatic Hydrocarbon Metabolism and Carcinogenesis I! Sims and I? L. Grover Virion and Tumor Cell Antigens of C-Type RNA lbmor Viruses Heinz Bauer Addendum to “Molecular Repair, Wound Healing, and Carcinogenesis: Tumor Production a Possible Overhealing?” Sir Alexander Haddow SUBJECT INDEX

Volume 22 Renal Carcinogenesis J. M. Hamilton Toxicity of Antineoplastic Agents in Man: Chromosomal Aberrations, Antifertility Effects, Congenital Malformations, and Carcinogenic Potential Susan M. Sieber and Richard H. Adamson

Interrelationships among RNA Tumor Viruses and Host Cells Raymond V. Gilden Proteolytic Enzymes, Cell Surface Changes, and Viral Transformation Richard Roblin, lih-Nan Chou, and Paul H . Black Immunodepression and Malignancy Osias Stutman SUBJECT INDEX

CONTENTS O F PREVIOUS VOLUMES

Volume 23 The Genetic Aspects of Human Cancer W. E. Heston The Structure and Function of Intercellular Junctions in Cancer Ronald S. Weinstein, Frederick B. Merk, and Joseph Ahoy Genetics of Adenoviruses Harold S. Ginsherg and C. S . H. Young Molecular Biology of the Carcinogen, 4-Nitroquinoline I-Oxide Minako Nagao and Takashi Sugimura 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 and Jana Hillova SUBJECT INDEX

Volume 24 The Murine Sarcoma Virus-Induced Tumor: Exception or General Model in Tumor Immunology? J . I? Levy and J . C. Leclerc Organization of the Genomes of Polyoma Virus and SV40 Mike Fried and Beverly E. Griflin P,-Microglobulin and the Major Histocompatibility Complex Per A. Peterson, Lars Rask, and Lars Ostberg Chromosomal Abnormalities and Their Specificity in Human Neoplasms: An Assessment of Recent Observations by Banding Techniques Joachim Mark Temperature-Sensitive Mutations in Animal Cells Clairdio Basilico

36 1

Current Concepts of the Biology of Human Cutaneous Malignant Melanoma Wallace H . Clark, Jr., Michael J . Mastrangelo, Ann M. Ainsworth, David Berd, Robert E. Bellet, and Evelina A . Bernardino SUBJECT INDEX

Volume 25 Biological Activity of Tumor Virus DNA F: L. Graham Malignancy and Transformation: Expression in Somatic Cell Hybrids and Variants Harvey L. Ozer and Krishna K.Jha Tumor-Bound Immunoglobulins: I n Situ Expressions of Humoral Immunity Isaac P. Witz The A h Locus and the Metabolism of Chemical Carcinogens and Other Foreign Compounds Snorri S . Thorgeirsson and Daniel W. Nebert Formation and Metabolism of Alkylated Nucleosides: Possible Role in Carcinogenesis by Nitroso Compounds and Alkylating Agents Anthony E. Pegg Immunosuppression and the Role of suppressive Factors in Cancer Isao Kamo and Herman Friedman Passive Immunotherapy of Cancer in Animals and Man Steven A . Rosenherg and William D. Terry SUBJECT INDEX

Volume 26 The Epidemiology of Large-Bowel Cancer Pelayo Correa and William Haenszel Interaction between Viral and Genetic Factors in Murine Mammary Cancer J . Hilgers and F! Benrvelzen Inhibitors of Chemical Carcinogenesis Lee W. Wattenberg

362

CONTENTS OF PREVIOUS VOLUMES

Latent Characteristics of Selected Herpesviruses Jack G. Stevens Antitumor Activity of Corynebacterium parvum Luka Milas and Martin T. Scott SUBJECT INDEX

Volume 27 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 Disease: Origin of Choriocarcinoma, Invasive Mole and Choriocarcinoma Associated with Hydatidiform Mole, and Some Immunologic Aspects J . I. Brewer, E. E. Torok, B. D. Kahan, C. R . Stanhope, 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 Treatment of Acute Leukemia E. Donnall Thomas, C. Dean Bucknrr, Alexunder Fefer, Paul E. Neiman, and Rainer Storb Susceptibility of Human Population Groups to Colon Cancer Martin Lipkin Natural Cell-Mediated Immunity Ronald B . Herberman and Howclrd T. Holden SUBJECT INDEX

Volume 28 Cancer: Somatic-Genetic Consideraticins E M. Burnet Tumors Arising in Organ Transplant Recipients Israel Penn

Structure and Morphogenesis of Type-C Retroviruses Ronald C . Montelaro and Dani P. Bolognesi BCG in I’umor Immunotherapy Robert W. Baldwin and Malcolm V. Pimm The Biology of Cancer Invasion and Metastasis Isaiah J . Fidler, Douglas M.Gersten, and Ian R. Hart Bovine Leukemia Virus Involvement in Enzootic Bovine Leukosis A. Burny, E Bex, H . Chantrenne, Y. Cleuter, D. Dekegel, J . Ghysdael, R. Kettmann, M. Leclercq, J . Leunen, M. Mammerickx, and D. Portetelle Molecular Mechanisms of Steroid Hormone Action Stephen J . Higgins and Ulrich Gehring SUBJECT INDEX

Volume 29 Influence of the Major Histocompatibility Complex on T-cell Activation J . E A . P. Miller Suppressor Cells: Permitters and Promoters of Malignancy? Duvid Naor Retrodifferentiation and the Fetal Patterns of Gene Expression in Cancer Jose Uriel The Role of Glutathione and Glutathione STransferases in the Metabolism of Chemical Carcinogens and Other Electrophilic Agents L . E Chasseaud a-Fetoprotein in Cancer and Fetal Development Erkki Ruoslahti and Markku Seppala Mammary Tumor Viruses Dan H. Moore, Carole A. Long, Akhil B. Vaidya, Joel B. Sheffield, Arnold S. Dion, and Etienne Y. Lasfargues Role of Selenium in the Chemoprevention of Cancer A . Clark Grqfin SUBJECT INDEX

CONTENTS OF PREVIOUS VOLUMES

363

Volume 30

Volume 32

Acute Phase Reactant Proteins in Cancer E. H. Cooper and Joan Stone Induction of Leukemia in Mice by Irradiation and Radiation Leukemia Virus Variants Nechama Haran-Ghera and Alpha Peled On the Multiform Relationships between the Tumor and the Host V. S . Shapot Role of Hydrazine in Carcinogenesis Joseph Bal6 Experimental Intestinal Cancer Research with Special Reference to Human Pathology Kuzymir M . Pozharisski, A l e x e i J . Likhavchev, Vakri E Klimashevski, and Jacob D. Shaposhnikov The Molecular Biology of Lymphotropic Herpesviruses Bill Sugden. Christopher R. Kintner, and Willie Mark Viral Xenogenization of Intact Tumor Cells Hiroshi Kobuyashi Virus Augmentation of the Antigenicity of Tumor Cell Extracts Faye C. Austin and Charles W Boune

Tumor Promoters and the Mechanism of Tumor Promotion Leilu Diamond, Thomas G. O’Brien, and William M. Baird Shedding from the Cell Surface of Normal and Cancer Cells Paul H. Black Tumor Antigens on Neoplasms Induced by Chemical Carcinogens and by DNAand RNA-Containing Viruses: Properties of the Solubilized Antigens Lloyd W. Law, Michael J . Rogers, und Ettore Appella Nutrition and Its Relationship to Cancer Bandaru S . Reddy, Leonard A . Cohen, G . David McCoy. Peter Hill, John H. Weisburger, and Ernst L . Wynder

INDEX

The Epidemiology of Leukemia Michael Alderson The Role of the Major Histocompatibility Gene Complex in Munne Cytotoxic T Cell Responses Hermann Wagner, Klaus Pfizenmaier, and Martin Rollinghoff The Sequential Analysis of Cancer Development Emmanuel Furber and Ross Cameron Genetic Control of Natural Cytotoxicity and Hybrid Resistance Edwwrd A . Clark and Richard C . Harmon Development of Human Breast Cancer Sefton R. Wellings

The Cultivation of Animal Cells in the Chemostat: Application to the Study of Tumor Cell Multiplication Michuel G. Twey Ectopic Hormone Production Viewed as an Abnormality in Regulation of Gene Expression Hiroo Iniuru The Role of Viruses in Human Tumors Harald zur Hausen The Oncogenic Function of Mammalian Sarcoma Viruses Poul Andersson Recent Progress in Research on Esophageal Cancer in China Li Mingxin (Li Min-Hsin), Li Ping, and LI Buorong (Li Pao-JimgJ Mass Transport in Tumors: Characterization and Applications to Chemotherapy Rakesh K. Jain, Jonas M. Weissbrod, clnd James Wei

INDEX

INDEX

Volume 31

INDEX

Volume 33

364

CONTENTS OF PREVIOUS VOLUMES

Volume 34 The Transformation of Cell Growi:h and Transmogrification of DNA Synthesis by Simian Virus 40 Robert G . Martin Immunologic Mechanisms in UV Radiation Carcinogenesis Margaret L. Kripke The n m o r Dormant State E. Federick Wheelock, Kent J . Weinhold, and Judith Levich Marker Chromosome 1 4 6 in Human Cancer and Leukemia Felix Mitelman Structural Diversity among Retrovirall Gene Products: A Molecular Approach to the Study of Biological Function through Structural Variability James W. Gautsch, John H . Elder, Fred C . Jensen, and Richard A. Lerner Teratocarcinomas and Other Neoplasms as Developmental Defects in Gene Expression Beatrice Mintz and Roger A. Fleisthman Immune Deficiency Predisposing to Epstein-Barr Virus-Induced Lymphoproliferative Diseases: The X-Linked Lymphoproliferative Syndrome as a Model David T. Purtilo INDEX

Volume 35 Polyoma T Antigens Walter Eckharr Transformation Induced by Herpes Simplex Virus: A Potentially Novel Type of Virus-Cell Interaction Berge Hampar Arachidonic Acid Transformation and Tumor Production Lawrence Levine The Shope Papilloma-Carcinoma Complex of Rabbits: A Model System of Neoplastic Progression and Spontaneous Regression John W Kreider and Gerald L. Bartlett

Regulation of SV40 Gene Expression Adolf Graessman, Monika Graessmann, and Christian Mueller Polyamines in Mammalian Tumors, Part I Giuseppe Scalabrino and Maria E. Feriolo Criteria for Analyzing Interactions between Biologically Active Agents Morris C.Berenbaum INDEX

Volume 36 Polyamines in Mammalian Tumors, Part I1 Giuseppe Scalabrino and Maria E. Ferioli Chromosome Abnormalities in Malignant Hematologic Diseases Janet D. Rowley and Joseph R. Tesra Oncogenes of Spontaneous and Chemically Induced Tumors Robert A. Weinberg Relationship of DNA Tertiary and Quaternary Structure to Carcinogenic Processes Philip D. Lipetz, Alan G. Galsky. and Ralph E.Stephens Human B-Cell Neoplasms in Relation to Normal B-Cell Differentiation and Maturation Processes Tore Godal and Steinar Funderud Evolution in the Treatment Strategy of Hodgkin’s Disease Gianni Bonadonna and Armando Santoro Epstein-Barr Virus Antigens-A Challenge to Modern Biochemistry David A. Thorley-Lawson, Clark M. Edson, and Kathi Geilinger INDEX

Volume 37 Retroviruses and Cancer Genes J . Michael Bishop Cancer. Genes, and Development: The Drosophila Case Elisabeth GateSf Transformation-Associated Tumor Antigens Arnold J . Levine

CONTENTS OF PREVIOUS VOLUMES Pericellular Matrix in Malignant Transformation Kirri Alirulo trnil Antti Vuhi,ri

Radiation Oncogenesis in Cell Culture Curniiu Borek

Mhc Restriction and /r Genes Jon Kleiti und Zdtun A . Ntrgy Phenotypic and Cytogenetic Characteristics of Human B-Lymphoid Cell Lines and

365

Their Relevance for the Etiol6gy of Burkitt's Lymphoma Kentietli Nilsson und Georgr. Klt~in Translocations Involving /g Locus-Carrying Chromosomes: A Model for Genetic Transposition in Carcinogenesis George> Klein arid Gilbert Lenoir INDEX

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