Advances in Cancer Research Volume 52

ADVANCES IN CANCER RESEARCH VOLUME 52

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ADVANCES IN CANCER RESEARCH Edited by

GEORGE F. VANDE WOUDE NCI-Frederick Cancer Research Facility Frederick, Maryland

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

Volume 52

ACADEMIC PRESS, INC. Harcourl Brace Jovanovlch, Publishers

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COPYRIGHT 0 1989 BY ACADEMIC PRESS. INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER

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NUMBER:52-13360

CONTENTS

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

CONTRIBUTORS TO VOLUME5 2 . ..... PREFACE ...............................

ix xiii

Primary Chromosome Abnormalities in Human Neoplasia

HEIMAND FELIXMITELMAN Introduction ............. ............................ Cytogenetic Nomenclature . ........... SVERRE

I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII.

Data Base in Cancer C Acute Nonlymphocytic Leukemia . . ..................... Myelodysplastic Syndromes ............................. Chronic Myelopmlifera Acute Lymphoblastic Leukemia (ALL) ............................ Chronic Lymphoproliferative Disorders ... ................ Malignant Lymphoma .................................... .......................... Solid Tumors........ Oncogenes, Antioncogenes, an rrations ................... .................. Summary and Conclusions . .................. References ........................

2 4 9

18 22 24 27 30 37 38

T Cell Receptor and Immunoglobulin Gene Rearrangements in Lymphoproliferative Disorders M. D. REIS, H. GRIESSER,AND T. W. MAK I. 11. 111. IV. V. VI.

Introduction .......................................................... B Cell Antigen Receptor Structure, Function, and Gene Organization ......... T Cell Antigen Receptor Structure, Function, and Gene Organization. . . . . . . . . Clinical Applications of the Analysis of Immunoglobulin and T Cell Receptor Gene Rearrangements in Hematological Neoplasias . . . . . . . The Simultaneous Occurrence of the T Cell Receptor and Immunoglobulin Genes in Lymphoproliferative Disorders ................ Chrumowmal Translocations Involving the T Cell Receptor Genes . . . . . . . . . . . . References ............................................................ Note Addedin Proof .................................................... V

45 46 49 57 69 72 75 80

vi

CONTENTS

Structure. Function. and Genetics

of Human B Cell-Associated Surface Molecules

EDWARD A . CLARKAND JEFFREY A . LEDBETTER 1. Introduction . . . . . . . . . . . . . . ........................................ I1 . Major B Cell Differentiation gens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Other Biochemically Defined Surface Molecules ............................ on Pre-Band/or B Cells . . . . . . . . . . . . . . . . IV. Receptors on B Cells for Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Other Surface Molecules Expressed on Activated B Cells . . . . . . . . . . . . . . . . . . . . . VI . Surface Molecules Found on T Cells and Subsets of B Cells . . . . . . . . . . . . . . . . . . VII . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ........................ ............................

82 89 116 125

127 132 134 135

Adenovirus Proteins and MHC Expression SVANTE PAABO. LIV SEVERINSSON. MATS ANDERSON. INGRID MARTENS.TOMMY NILSSON. AND PER A . PETERSON I. I1 . I11 . IV.

V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ Adenoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adenovirus Gene Products Modulating MHC Cell Surface ression . . . . . . . . . . Functional Consequences of Adenovirus-Induced Modulation of MHC Class I Expression . . . . . . . . . . . . . . ........................ Summary and Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

151 152 154

157 160 161

Multidrug Resistance

ALEXANDER M . VAN DER BLIEKAND PIETBORST I. I1 . 111. IV. V.

VI . VII . VIII .

IX. X. XI . XI1 .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Drugs Affected by MDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . What Happens to the Drugs in MDR Cells? . . . . . . . . . . . Pharmacological Reversal of MDR . . . . . . . . . . . . . . . . . . . Alterations in MDR Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P-Glycoprotein Overproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amplified Genes in MDR Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Central Role of P-Glycoprotein Genes in MDR . . . . . . . . . . . . . . . . . . . . . . . . . P-Glycoprotein Structure Deduced from Sequence Comparisons . . . . . . . . . . . . . . . Diversity of P-Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutated P-Glycoprotein Genes with Altered Drug Transport Properties . . . . . . . . P-Glycoprotein Expression in Normal Tissue and Its Regulation . . . . . . . . . . . . . .

172 174 175

178 180 185 188 189

CONTENTS XIII. XIV. XV.

Coamplified Genes and Alterations Elsewhere in the Genome . . . . . . . . . . . . . . . . M D R i n t h e Clinic ..................................................... Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 192 194 195 197

Glutathione Transferases as Markers of Preneoplasia and Neoplasia

KIYOMISATO I. 11. 111.

IV. V. VI.

Introduct' .................................................... Marker E Preneoplasia ................................... Molecular Forms of Glutathione rases. . . . . . . . . . . . . . . . . . . . Glutathione Transferases as Preneoplastic Markers . . . . . . . . . . . . . . . Role(s) of Glutathione Transferases in the Mechanisms Underlying Multidrug Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . ................................................... .... ........... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205 207

241 242 243

Aberrant Glycosylation in Tumors and Tumor-Associated Carbohydrate Antigens

SEN-ITIROH HAKOMORI I. 11.

111.

IV.

V. VI. VII. VIII. IX. X.

XI. XII.

Introduction and Brief Historical Background (1929-1975) . . . ...... Tumor-Associated Glycolipid Antigens in Experimental Tumors . . . . . . . . . . . . . . . Tumor-Associated Carbohydrate Antigens in Human Cancers: Classification, Mosaicism of Expression, and New Procedures for Generation of Antibodies . . . . . . . .............................. Oncogenes and Aberrant Glycosylatio .............................. Antigens .......... Normal and Oncofetal Features of G1 Carbohydrate Glycoprotein Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . Alteration of Histo-Blood Group and Heterophile Antigens Expressed in Human Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aberrant Glycosylation in Preneoplastic Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . Requirements for Tumor-Associated Carbohydrate Antigens: Density of Antigens and Organizational Framework in Membranes . . . . . . . . . . . . Diagnostic Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor-Associated Carbohydrate Antigens as Targets for Therapeutic Applications ............................................ Summary and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

258 260

262 264

292 297 298 302 309 316

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

318 331

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

333

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CONTRIBUTORS TO VOLUME 52 Numbers in parentheses indicate the pages on which the authors' contributions begin.

MAT^ ANDERSON,Department of Cell Research, University of Uppsala, S-75124 Uppsala, Sweden (151) PIET BORST, Department of Molecular Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, T h e Netherlands (165) EDWARDA . CLARK, Department of Microbiology, University of Washington, Seattle, Washington 981 95 (81) H . GRIESSER,Ontario Cancer Institute, Toronto, Ontario, Canada M4X l K 9 (45) SEN-ITIROHHAKOMORI,T h e Biomembrane Institute, Seattle, Washington 98119 and Departments of Pathobiology, Immunology, and Microbiology, University of Washington, Seattle, Washington 98195 (257) SVERREHEIM,Department of Clinical Genetics, University Hospital, S-221-85 Lund, Sweden (1) JEFFREY A. LEDBETTER,Oncogen Corporation, Seattle, Washington 98121 (81) T. W. MAK, Ontario Cancer Institute, Toronto, Ontario, Canada M4X l K 9 and Departments of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada (45) INGRIDMARTENS,Department of Medical Virology, University of Uppsala, S-75124 Uppsala, Sweden (151) FELIXMITELMAN, Department of Clinical Genetics, University Hospital, S-221-85 Lund, Sweden (1) TOMMY NILSSON, Department of Immunology, Research Institute of Scra$@s Clinic, La Jolla, Cali&ornia 92037 (151) SVANTEPAWBO, Department of BiochemistTy, University of Calqornia, Berkeley, Calqornia 94720 (151) PERA. PETERSON, Department of Immunology, Research Institute of Scn3ps Clinic, La Jolla, California 92037 (151) M . D. REIS, Ontano Cancer Institute, Toronto, Ontano, Canada M4X 1K9 (45) ix

X

CONTRIBUTORS TO VOLUME 52

KIYOMISATO,Second Department of Biochemisty,Hirosaki University School of Medicine, Hirosaki 036, Japan (205) LIV SEVERINSSON, Ludwig Institute for Cancer Research, Uppsala Branch, BMC, S-75123 Uppsala, Sweden (151) ALEXANDER M. VAN DER BLIEK,Department of Molecular Biology, The Netherlands Cancer Institute, 1066 C X Amsterdam, The Netherlands (165)

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SIDNEYWEINHOUSE

PREFACE

This volume marks the retirement of Sidney Weinhouse from his dedicated work as Editor for Advances in Cancer Research. He assumed this post in 1961 when he edited Volume 6, together with Alexander Haddow. He has been responsible, first with Haddow and later with myself, for the subsequent 45 volumes. It is with much gratitude and true regret that I must accept Sidney’s decision to retire from the task that he has performed with so much circumspection and distinction. He always took the lion’s share of the work. Sidney Weinhouse can look back on a long and very distinguished career in biochemistry and cancer research. It started at the University of Chicago. where he received his Ph.D. and carried out wartime scientific activities. H is early research was on lipid metabolism. He was a pioneer in the use of isotopically labeled fatty acids and in the biosynthesis of amino acids in yeast. These studies led him to apply isotope methods to the study of tumors and to pursue his interest in the oxidative metabolism of glucose and other sugars. As this work progressed, Weinhouse obtained increasing evidence that conflicted with the views of Otto Warburg on the role of aerobic glycolysis in cancer cells. His publications in this area, including his G. H. A. Clowes Award Lecture of 1972 on “Glycolysis, Respiration and Anomalous Gene Expression in Experimental Hepatomas,” were largely responsible for the development of a more realistic view in a field that has suffered from both emotionalism and authoritarianism (see also Sidney Weinhouse: “The Warburg Hypothesis Fifty Years Later,” Guest Editorial, 2. Krebsforsch 87, 115-126, 1976). In the course of his work, Weinhousebecame interested in the minimal deviation hepatomas developed by H. P. Morris. He undertook an extensive series of investigationson these tumors, leading to important advances in carbohydrate metabolism as well as in the behavior of isozymes and their alterations in neoplastic cells (S. Weinhouse: “What Are Isozymes Telling Us about Gene Regulation in Cancer?” Guest Editorial,J. Natl. Cancer Znst. 68, 343-349, 1982). The results of these studies convinced Weinhouse of the great importance of aberrations in gene expression in the pathogenesis of cancer. His work forms a major part of the foundation of this now widely held view. In more recent years, Weinhouse has studied the metabolism of chemical carcinogens and started an investigation of inorganic pymphosphatase, believing that this key enzyme in cellular metabolism might play an important role in cancer. He has also turned his attention to a relatively new field for him, the possible role of nutrition in human cancer, on which he has prepared a special report for the American Cancer Society. xiii

XiV

PREFACE

The numerous contributions of Sidney Weinhouse have been recognized by many awards and distinctions of which only the most outstanding will be mentioned here. In biochemistry, he has been chairman of the Division of Biological Chemistry of the American Chemical Society and of the Committee on Biological Chemistry of the Division of Chemistry of the National Academy of Sciences-National Research Council. In cancer research, he has received the G. H. A. Clowes Award of the American Association for Cancer Research, the Papanicolau Award, and the National Achievement Award of the American Cancer Society. His election to the National Academy of Sciences confirms his stature as a truly outstanding scientist. He has assumed many editorial responsibilities, including the editorship of Cancer Research, the journal of the American Association for Cancer Research. He served on the editorial boards of numerous distinguished journals. He has also played a major role in the administration of scientific research in the United States. His positions include the Directorship of the Fels Research Institute at Temple University in Philadelphia, life membership on the Board of Directors of the American Cancer Society, and membership, often as chairman, on many national advisory bodies. I first met Sidney during my first 4 months in the United States as a predoctoral fellow at the Institute of Cancer Research, Fox Chase, Philadelphia, in 1950. Sidney headed the Department of MetabolicChemistry.The problemfocused scientific interest of Sidney and some others on the scientific staff, combined with an intense personal warmth, made a lasting impression that decisively influenced my own early scientific development and had similarly motivating effects on many other students and co-workers. It was a pleasure and a privilege to work with Sidney for more than a quarter of a century as co-editor of these Admances. His benevolent, soft-spoken personality will remain with us for many years to come. Sidney Weinhouse is succeeded by George Vande Woude as co-editor of Advances in Cancer Research. It is with great satisfaction that I see Sidney’s legacy deposited in such competent hands. I would like to extend my warmest welcome to George. GEORGE KLEIN

PRIMARY CHROMOSOME ABNORMALlTl ES IN HUMAN NEOPLASIA Sverre Heim and Felix Mitelman Department of Clinical Genetics, University Hospital, S-221-85 Lund. Sweden

I. Introduction 11. Cytogenetic Nomenclature 111. Data Base in Cancer Cytogenetics-An Overview IV. Acute Nonlymphocytic Leukemia A. inv(3)(q21q26) B. t(6;9)(p23;q34) C. t(8;21)(q22;q22) D. t(9;11)(~21-22;q23) E. t(15; 17)(q22;ql1-12) F. inv(16)(p13q22) G. +8 and Other Numerical Aberrations V. Myelodysplastic Syndromes A. Refractory Anemia without Excess of Blasts (RAWEB) B. Refractory Anemia with Ringed Sideroblasts (RARS) C. Chronic Myelomonocytic Leukemia (CMML) D. Refractory Anemia with Excess of Blasts (RAEB) and Refractory Anemia with Excess of Blasts in Transformation (RAEBT) VI. Chronic Myeloproliferative Disorders A. Chronic Myeloid Leukemia (CML) B. Polycythemia Vera (PV) C. Idiopathic Myelofibrosis/Agnogenic Myeloid Metaplasia D. Essential Thrombocythemia VII. Acute Lymphoblastic Leukemia (ALL) A. t( 1 ;19)(q23;p 13) B. t(4;11)(92 1 ;q23) C. del(6q) D. t(9;22)(q34;qll) E. Rearrangements of 14q32 and B Cell ALL F. Abnormalities Associated with T Cell ALL VIII. Chronic Lymphoproliferative Disorders A. Chronic Lymphocytic Leukemia (CLL) B. Prolymphocytic Leukemia (PLL) C. Hairy Cell Leukemia (HCL) D. Adult T Cell Leukemia (ATL) IX. Malignant Lymphoma A. Burkitt’s Lymphoma (BL) B. Non-Burkitt’s Non-Hodgkin’s Lymphoma (NHL) C. Hodgkin’s Disease (HD) X. Solid Tumors

1 ADVANCES IN CANCER RESEARCH, VOL. 52

Copyright 0 1989 h y Academic Press, Inc. All rights of reproduction in any form reserved.

2

SVERRE HEIM AND FELIX MITELMAN

A. Mixed Tumors of the Salivary Gland B. Small Cell Lung Cancer C. Renal Cell Carcinoma D. Bladder Carcinoma E. Uterine Leiomyoma F. Lipogenic Tumors G. Alveolar Rhabdomyosarcoma H. Synovial Sarcoma I. Meningioma J. Ewing's Sarcoma XI. Oncogenes, Antioncogenes, and Chromosome Aberrations A. Antioncogenes B. Oncogenes XII. Summary and Conclusions References

I. Introduction

The importance of karyotypic rearrangements in neoplasia has been the subject of heated debate ever since cellular pathologists, toward the end of the last century, described irregular cell divisions in tumors. These early observations were forged in 1914 into a systematic conceptual model by Theodor Boveri in what has become known as the somatic mutation theory of cancer. According to this hypothesis, nuclear changes, in particular chromosomal aberrations, are causative events in the transition from normal to neoplastic cell proliferation. Technical limitations prevented critical testing of the central idea in Boveri's reasoning until the 1950s. By that time, methodological improvements such as tissue culture techniques, hypotonic treatment of cells arrested in metaphase, and the air-drying method (excellent reviews of the historical background have been provided by Hsu, 1979, and Sandberg, 1980) opened up new possibilities for cytogenetic studies in oncology. The first spectacular breakthrough was not long in coming: Nowell and Hungerford in 1960 described the first characteristic neoplasia-associated karyotypic abnormality in man, the Philadelphia (Ph') chromosome, in patients with chronic myeloid leukemia (CML). This discovery seemed to perfectly epitomize the core concept of the somatic mutation theory: a distinctive chromosomal abnormality specifically associated with a particular malignant disorder. The decade following the Ph' discovery, however, did not see the expected steady increase in the reported number of tumor-specific abnormalities. The accumulated evidence instead indicated that CML might well be exceptional. Other malignancies were apparently not

CHROMOSOME ABNORMALITIES I N NEOPLASIA

3

characterized by consistent chromosomal changes; instead quite different aberrations were detected in what by all conventional criteria seemed to be indistinguishable neoplasms. Furthermore, the karyotypes were often very complex, containing numerous unidentifiable changes. As a consequence of these setbacks, the enthusiasm for a direct, causal role of primary chromosome abnormalities in human neoplasia waned, with many researchers taking the view that chromosome abnormalities in cancer and leukemia were probably randomly occurring epiphenomena of no direct pathogenetic importance. Such skepticism is seldom voiced today. Since the development of banding techniques around 1970, the discovery of which allowed unequivocal identification of individual normal and rearranged chromosomes, the evidence for an essential role of chromosomal changes in the pathogenesis of neoplastic lesions has been considerably strengthened. It is now established beyond doubt that most human tumors have karyotypic changes detectable with existing cytogenetic techniques. This conclusion is not restricted to malignant neoplasms; many benign tumors, too, are now known to contain characteristic karyotypic abnormalities. Furthermore, although the changes may vary from case to case and at times are quite complex, the overall aberration pattern is undoubtedly nonrandom, with some genomic sites involved in aberrations much more often than others. Of particular importance is the realization that many abnormalities are associated with distinctive disease variants, often revealing a cytogeneticmorphologic specificity that is fully comparable to the consistency seen between the Phl marker and CML. To these purely cytogenetic data implicating specific genetic changes in carcinogenesis may now be added the growing evidence of molecular specificity emerging from recombinant DNA studies. It appears that both currently known classes of directly cancer-relevant genes, the dominant oncogenes and the recessive antioncogenes, are located at just those genomic sites that are visibly involved in cancer-associated rearrangements. Hence, the last few years have witnessed a beginning understanding at the molecular level of the essential effects of cytogenetic changes in neoplasia. The sheer complexity of cytogenetic abnormalities in neoplastic cells has unquestionably added to the confusion regarding their importance in tumorigenesis. Greater clarity may be obtained if it is kept in mind that any chromosome aberration in a tumor cell can in principle be referred to one of the three following categories:

1. Primary abnormalities. These are essential in establishing the

4

SVERRE HEIM AND FELIX MITELMAN

neoplasm, and probably represent rate-limiting steps in tumorigenesis. They may occur as solitary cytogenetic changes, and are as a rule strongly correlated with tumor type. 2. Secondary abnormalities. The genomic instability of the tumor predisposes to further chromosomal mutations, leading to genetic and secondarily phenotypic variability within the tumor cell population. Darwinian selection invariably results, with the more fit subclones eventually outgrowing the others. Secondary abnormalities are thus important after the tumor has been established, in tumor progression, and reflect the clonal evolution during this disease phase. 3. Cytogenetic noise. Most chromosomal mutations confer no evolutionary edge on the cells, but may nevertheless be temporarily detectable as nonclonal aberrations. When the chromosomal instability in a tumor cell population is very pronounced, such noise abnormalities may obscure the pathogenetically important changes and completely dominate the karyotype. We shall in the present review concentrate on the primary abnormalities of human neoplasia. T h e types and importance of secondary aberrations have been the topic of several recent reviews, for example, Heim and Mitelman (1986a) and Nowell (1986).Before surveying the specific abnormalities known today, however, we shall recapitulate some of the basic conceps in cytogenetic nomenclature, and also briefly present an overview of the data on which all conclusions regarding chromosome changes in cancer are based. 11. Cytogenetic Nomenclature

A schematic illustration of the normal, male, G-banded human chromosome complement is presented in Fig. 1. The nomenclature of chromosome classification has been standardized at repeated international conferences, each of which has resulted in revised and improved recommendations to ensure a uniform cytogenetic terminology. The most recent and authoritative document in this regard is “An International System for Human Cytogenetic Nomenclature (1985),” or ISCN (1985), which incorporates all major decisions reached at previous conferences. The following descriptions are all based on the ISCN proposals. Transverse banding of chromosomes may be accomplished by any of numerous available methods. Each chromosome is seen as consisting of a continuous series of dark and light bands; thus no “interbands” exist. These bands define, together with regions, arms, and

5

NEOPLASIA

5

4

6

7

8

13

14

15

19

20

10

9

21

12

I1

22

X

Y

FIG. 1. Schematic illustration (idiogram) of the 24 different human chromosomes as they appear in G banding.

6

SVERRE HEIM A N D FELIX MITELMAN

chromosome number, any position that may be discerned with the resolution currently obtainable in cytogenetics. To describe a given chromosomal position, the chromosome number (see Fig. 1)is stated first. Then the chromosome arm (“p” for the short arm, “q” for the long arm) is given. This is followed by the region in which the position is located. Regions, which are chromosomal areas delimited by distinctive landmarks, are numbered consecutively from the centromere outward. The band is provided last. There may be one or several bands within each region, and again numbering is consecutive from the centromere outward. When highresolution techniques (Yunis, 1981) are utilized, subbands may be obtained within the bands seen with standard methodology. These are described by punctuation followed by numbering after the standard band has first been identified. Thus four items of informationchromosome number, arm, region, and band-are needed to define a position on a chromosome. For example, 9q34 means chromosome 9, the long arm, region 3, band 4.At the high-resolution level, subband 3 within 9q34 is written 9q34.3. The usefulness of these nomenclature rules is demonstrated when structurally rearranged chromosomes are described. Structurally abnormal chromosomes are defined by their breakpoints, which are specified within parentheses immediately following the description of the type of rearrangement and the chromosome(s) involved. The following rearrangements will be encountered in this chapter. Translocation, abbreviated “t,” means that material is transferred between chromosomes. Deletion, abbreviated “del,” means the loss of chromosome material. Inversion, abbreviated “inv,” means that a segment has rotated 180” within a chromosome. For example, t(8;21)(q22;q22) means that a translocation has occurred between chromosomes 8 and 21. The breakpoint in chromosome 8 is in q22 and the breakpoint in chromosome 21 is in q22 of that chromosome. The chromosomal segments distal to the two breakpoints have been swapped. An additional example illustrates the description of deletions: de1(5)(q13q33) means that the segment between the breakpoints 5q13 and 5q33 has been lost. Plus (-t)and minus (-) signs are placed before the chromosome number to indicate gain or loss of whole chromosomes. They are placed after the symbol to indicate an increase or decrease in the length of a chromosome, a chromosome arm, or a region. A marker (mar) is a structurally abnormal chromosome. When the banding pattern is recognized, it may be adequately described using

CHROMOSOME ABNORMALITIES IN NEOPLASIA

7

the standard nomenclature; in other instances it remains as mar in the karyotvpe description. The possibility of detecting clonal karyotypic changes in any sample is naturally dependent on the size of the respective clones and on how many metaphases are analyzed. The minimum operational requirements for accepting an aberration as clonal are two cells with the same structural rearrangement or additional chromosome, or three cells with the same missing chromosome. Ill. Data Base in Cancer Cytogenetics-An

Overview

Descriptions of numerous new cases of cytogenetically abnormal neoplasms characterized with banding techniques are added each year to the scientific literature (Fig. 2). The aberrant karyotypes thus described have been compiled and published in catalog form (Mitelman, 1983, 1985). The rapid growth of information may perhaps best be illustrated by mentioning that, whereas the first two catalogs contained, respectively, 3844 and 5345 investigated cases, the third edition, in 1988, contains 9069 human neoplasms with chromosome aberrations (Mitelman, 1988). Impressive though these figures may seem, the picture they convey of the breadth of cytogenetic knowledge in neoplasia is to some extent misleading. The data are heavily biased toward hematological malignancies. Although these disorders account for only a small fraction of human oncological morbidity and mortality, as many as 86% of all tumors investigated by mid-1987 are bone marrow (75%) or lymph

1250 1000

750

500

250 n

1973 71 75 76 77 78 79 80 81 82 83 84 85 86

FIG.2. Annual increase, from 1973 to 1986, in the reported number of human neoplasms characterized with banding technique (cytogenetically abnormal tumors). Information on more than 9000 cases is currently available.

8

SVERRE HEIM AND FELIX MITELMAN

node (11%)neoplasms. The cancers, quantitatively by far the most important neoplasias in man, contribute only 14%.The shortcomings of existing data are even more apparent when the solid tumor group is subdivided: some of the clinically most important cancers, in particular many carcinoma types (squamous cell carcinomas of the lung and uterine cervix and adenocarcinomas of the breast and prostate being but four of the most prominent examples) have karyotypic profiles that are almost totally unknown. The main reason for this is technical: solid tumors, and especially carcinomas, have proved less amenable to chromosomal investigations than have myeloid and lymphatic neoplasms. Only in the very recent past have reports of solid tumor abnormalities begun to come forth in substantial numbers. Our knowledge of the karyology of cancers has also been hampered b y the frequently low technical quality of chromosome preparations. The chromosomes are, compared to blood or bone marrow chromosomes, often contracted and fuzzy, the spreading is poor, and banding is unsatisfactory. As a consequence, structural rearrangements frequently remain undefined in such preparations, thus reducing the value of the biological inferences to be drawn from karyotype data. Finally, many solid tumor studies were undertaken very late in the disease process, often of samples from effusion material rather than primary tumors. The karyotypic changes then found are mostly quite complex, with numerous numerical and structural abnormalities. Undoubtedly many of the changes represent cytogenetic noise (see above) or secondary changes acquired during tumor progression. The primary abnormalities may be exceedingly difficult to identify in this setting. All these difficulties notwithstanding, the gradual improvements of the data base have allowed significant conclusions about the pathogenetic role of chromosome changes in solid tumors. Here, as in the more extensively studied hematological neoplasms, the chromosomal changes are distributed throughout the genome in a strictly nonrandom manner. Several primary abnormalities have been identified, some of which are correlated with particular disease entities with a specificity quite comparable to that seen in leukemias and lymphomas. In the following sections we present a brief review of rearrangements for which a primary pathogenetic role in leukemogenesis and tumorigenesis is strongly suspected. The emphasis will be on the cytogenetic and pathogenetic features of the aberrations; clinical implications have largely been omitted. A recent and more extensive

CHROMOSOME ABNORMALITIES IN NEOPLASIA

9

discussion of the importance of chromosome aberrations in neoplasia may b e found in Heim and Mitelman (1987b), which may also be consulted for more extensive referencing. IV. Acute Nonlymphocytic Leukemia

Karyotypic abnormalities have been reported in roughly 2500 cases of acute nonlymphocytic leukemia (ANLL). The frequency with which clonal abnormalities are found in unselected series varies, but in state-of-the-art investigations may be conservatively estimated at about two-thirds of all cases. Some aberrations are found with remarkable consistency, indicating that their role in disease development is primary (Table I). Several of these abnormalities are associated with particular morphological ANLL subtypes, as defined, for example, by the French-American-British (FAB) classification, which denotes acute myeloid leukemias as MI-MG, based on the morphology of cells in Romanowsky-stained blood and marrow films and certain supplemental cytochemical reactions (see Bennett et al., 1976). By definition, these primary abnormalities are often found as the sole TABLE I PRIMARY CHROMOSOME ABNORMALITIESIN ACUTE NONLYMPHOCYTIC LEUKEMIA Rearrangement

inv(3)(q21q26) +4

-5 del(5q) t(6;W(p23;q34)

-7 del(7q) +8 t(8: 16)(pll;p13) t(8:21)i q22;q22) t(9;1l)(p21-p22;q23) t(9;22)(q34;qll) del/t(l l)(q13-q23) del It( 12p) t( 15;17)(q22;q11-12 12) inv(l6)(p13q22) del(2Oq)

Hematologic characteristic Dysmegakaryocytopoiesis Secondary ANLL, mostly &I4 Abnormal megakaryocytes and thrombocytosis Mz and M4 Secondary ANLL Secondary ANLL Mz and M4with basophilia Secondary ANLL Secondary ANLL

Ms with phagocytosis Mp with Auer rods and eosinophilia M;, mostly Msa MI and Mz M4 and Ms,mostly Msa Secondary ANLL, M 4or M Zwith eosinophilia M.3

and > 1 3 ~

M 4with eosinophilia Mfi

10

SVERRE HEIM AND FELIX MITELMAN

3

inv(3Mq21926)

FIG.3. The paracentric inversion inv(3)(q21q26) is associated with ANLL, with prominent megakaryocytic and platelet abnormalities.

detectable aberration; alternatively, they may be accompanied by secondary changes. A. inv(3)(q21q26) This paracentric inversion of the long arm of chromosome 3 (Fig. 3) is found primarily in ANLL patients with prominent megakaryocytic and platelet abnormalities. Similar hematological features are associated also with t(3;3)(q21;q26), with t(1;3)(p36;q21),and occasionally with other rearrangements affecting 3q21 or 3q26 (Bitter et al., 1985; Pinto et al., 1985; Bloomfield et al., 1985; Mertens et al., 1987a); it is possible that these latter aberrations may best be thought of as pathogenetically equivalent variants of inv(3). The molecular pathology of the rearrangement is unknown. Interference with the transferrin and transferrin receptor genes, located in 3q21 and 3q26, has been suggested as one pathogenetic possibility (Le Beau et al., 1986a). B . t(6;9)(p23;q34)

This translocation has been associated with bone marrow basophilia (Pearson et d.,1985), a feature not present in all t(6;9) leukemias (Heim et al., 1986). Most patients with t(6;Q)have been quite young, the leukemia has been Mz or Mq, and often a clinically manifest myelodysplastic syndrome has preceded full-blown ANLL. The pathogenetic mechanism is unknown. Although 9q34 is affected here as in CML (see below), at subband level the breakpoint in t(6;9) is distal to

CHROMOSOME ABNORMALITIES IN NEOPLASIA

8

21

11

t(8;21)(q22;q22)

FIG.4. The rearrangement t(8;21)(q22;q22) is associated with ANLL M2, with bone marrow eosinophilia and Auer rods.

the break in t(9;22), in 9q34.3, making it unlikely that similar molecular mechanisms are involved in the two disorders. C. t(8;21)(q22;q22) This, the single most common structural rearrangement (Fig. 4) in ANLL, was found in 15% of all ANLL patients reported at the Fourth International Workshop on Chromosomes in Leukemia 1982 (1984). Although occasional cases have cells with morphology corresponding to other subgroups, the vast majority of cases are classified as M2. Auer rods are frequently prominent, as is bone marrow eosinophilia. Several variants involving structural rearrangements of either 8q22 or 21q22 have been reported (Billstrom et al., 1987); hence, it is at present unclear which of the breaks is more important in pathogenesis. The essential molecular consequences of t(8;21) are unknown, but the cellular oncogene ets2 is moved from 21q to the derivative chromosome 8 (Sacchi et al., 1986).

D. t(9;11)(p21-22;q23) The nonrandom occurrence of structural rearrangements of l l q in patients with acute monoblastic leukemia (Ms), in particular the immature Msa subtype, was first pointed out by Berger et ul., who in 1982 reported l l q abnormalities in 12 of 34 MS patients. The rearrangement most commonly seen is a reciprocal translocation between chromosomes 9 and ll (Hagemeijer et d.,1982), i.e., t(9; 1l)(p21--22;q23); the other 1l q affecting changes may represent

12

SVERRE HEIM AND FELIX MITELMAN

variants of this abnormality. The changes are not always translocations: in several cases the only recognizable abnormality is a deletion of parts of Ilq. Diaz et al. (1986) have shown that in t(9;ll) the cellular oncogene c-etsl translocates from 11423 to 9p adjacent to the interferon genes, which are split by the 9p22 breakpoint. Whether this is pathogenetically important remains unknown.

E . t(15;17)(q22;q 11- 12) This is the highly specific translocation associated with acute promyelocytic leukemia (APL), or M3 and M ~ vas , these subtypes are known within the FAB classification. With increasing cytogenetic sophistication, the t( 15;17) is being found in steadily higher pro1984), portions of APL patients, and the Chicago group (Larson et d., which has played a leading part in describing this translocation, has suggested that practically all ANLL patients of this subtype will eventually b e shown to have rearrangements of these chromosomal sites. The molecular pathology of t(15;17) is unknown.

F. inv(16)(p13q22) The marrow morphology of ANLL patients carrying this abnormality is quite characteristic: the leukemia is of the myelomonocytic (M4) subtype, and disturbances of the eosinophilic lineage are particularly prominent, with both an excess of eosinophils and abnormal eosinophilic granulation (Arthur and Bloomfield, 1983; Berger et al., 1985; Larson et al., 1986). Variant rearrangements associated with the same hematologic features, mostly del(16)(q22), t( 16;16)(p13;q22), and translocation between 16q22 and other chromosomes, have also been reported. Le Beau e t al. (1985) have offered a hypothesis regarding the pathogenetic consequences of inv( 16). They found that the metallothionein (MT) multigene family was localized in 16q22, and that the 16q22 breakpoint split the MT gene cluster. Possibly this might interfere with intracellular zinc binding or storage, and hence affect granulocyte and monocyte differentiation. Alternatively, an as yet undefined oncogene might, as a result of the rearrangement, be recombined with sequences in the M T locus, leading to structural or regulatory abnormalities of oncogene function and ultimately to leukemia.

CHROMOSOME ABNORMALITIES I N NEOPLASIA

G. + 8

AND

13

OTHERNUMERICAL ABERRATIONS

Numerical,karyotypic abnormalities (Heim and Mitelman, 1986a) are common in ANLL. Trisomy 8 is the change most often seen and is apparently not restricted to any particular FAB subgroup. It occurs as the sole abnormality in 7% of all cytogenetically abnormal cases. If leukemias with multiple aberrations are taken into consideration, +8 is found at double that frequency. The other nonrandomly occurring numerical aberrations in ANLL, i.e., +4, -5, -7, +21, and -Y, together account for another 5%. The fact that each of these is often the only change merits their inclusion among the primary abnormalities. Monosomy 5 and monosomy 7, as well as the partial monosomies brought about by del(5q) and de1(7q), are associated with secondary ANLL. V. Myelodysplastic Syndromes

Several hematopoietic dysfunction states are covered by the umbrella diagnosis, myelodysplastic syndrome (MDS); terms such as preleukemia and dysmyelopoietic syndromes have also been used synonymously. MDS thus includes entities such as refractory anemia with or without blasts, nonregenerative anemia, sideroblastic anemia, hematopoietic dysplasia, and chronic myelosis. An attempt to reach an internationally acceptable, standardized nomenclature for the various MDS subgroups has been made by the French-American-British Study Group (Bennett et al., 1982). Their proposals recognize five MDS subtypes: refractory anemia without excess of blasts (RAWEB), refractory anemia with ringed sideroblasts (RARS), chronic myelomonocytic leukemia (CMML),refractory anemia with excess of blasts (RAEB), and refractory anemia with excess of blasts in transformation (RAEBT). Clonal chromosome abnormalities have now been reported in more than 700 MDS patients (reviewed in Heim and Mitelman, 1986b). The frequency of aberrations in unselected series varies (Second International Workshop on Chromosomes in Leukemia, 1979, 1980; Nowell, 1982; Knapp et al., 1985; Tricot et al., 1985; Jacobs et al., 1986; Yunis et al., 1986), but has mostly been below corresponding frequencies obtained in ANLL. The finding of acquired karyotypic abnormalities in myelodysplastic bone marrows confirms the presently held view that MDS is a neoplastic disorder. The aberration pattern varies among subtypes

14

SVERRE HEIM AND FELIX MITELMAN

TABLE I1 FREQUENCY OF PRINCIPAL PRIMARY KARYOTYPIC ABNORMALITIES IN MDS Abnormality (70)

Disorder

5q-

-5

-7

+8

deI/t(llq)

deUt(l2p)

RAWEB RARS CMML RAEB(T)

70 30 <5 30

(5 <5 <5

5 <5 20 30

15 25 20 10

<5 20

<5

<5 <5 15

10

10

10

(Table 11), although not as markedly as in ANLL. Overall, the karyotypic anomalies in MDS resemble ANLL changes, thus underlining the basic biological similarity between the two modes of disease presentation. This is particularly evident in the “gray zone” between ANLL and MDS, represented by the subgroups RAEB and FtAEBT. There is a definite tendency for MDS to gradually transform into ANLL; roughly one-third of all MDS patients ultimately become overtly leukemic. It is finally worthy of note that whereas some specific ANLL-associated abnormalities, e.g., t(8;21), t(15;17), and inv(16), are not found in MDS, the reverse situation is not seen. A. REFRACTORY ANEMIAWITHOUT EXCESS OF BLASTS (RAWEB) A deletion of the long arm of chromosome 5, del(5q), is the dominant aberration in RAWEB, where it is found in 70% of all abnormal cases. The MDS-del(5q) association was first pointed out by Van den Berghe and co-workers, and the same group has recently extensively reviewed cytogenetic and hematologic aspects of the “5q- syndrome” (Van den Berghe et al., 1985). The size of the 5q deletion (Fig. 5) varies considerably according to different reports. It has mostly been described as interstitial, with proximal breakpoint 5q12-14 and distal breakpoint 5q31-33. To what extent these differences reflect biological reality is unclear: one report (Mitelman et al., 1986) reexamined 15 cases of del(5q) associated diseases in which several different interpretations of the del(5q) had initially been offered, and found that the deletions in all cases actually appeared to be morphologically identical even at subband level, de1(5)(q13.3q33.1).The generality of this conclusion is debatable. It does emphasize, however, that cytogenetic heterogeneity may in some instances be less than is apparent from the karyotype descriptions.

CHROMOSOME ABNORMALITIES IN NEOPLASIA

5

15

del(SMq13q33)

FIG.5. A deletion of the long arm of chromosome 5, here illustrated as del (5)(q13q33),is often found in refractory anemia, where it gives rise to the characteristic “5s- syndrome.”

The exact breakpoint mapping in del(5q) is not only of academic interest; it has direct implications for the relative credibility of the different pathogenetic models that may be envisioned. If at least one of the breakpoints is constant in all cases, this would favor a position effect on gene(s) located in the breakpoint regions as the essential result of the deletion. On the other hand, if the breakpoints are widely divergent from case to case, this indicates that loss of a “minimal common segment” possessing antioncogenic activity (see Section XI) might be the essential event. At present, the data are insufficient to discard either hypothesis. The pathogenesis of the 5q- syndrome is unknown, as is the significance of the observation that the cellular oncogene c-fms (normally located in 5q31-33) is deleted (Nienhuis et al., 1985; Le Beau et al., 1986b). (RARS) B. REFRACTORY ANEMIAWITH RINGEDSIDEROBLASTS The three dominating cytogenetic abnormalities in this subgroup are del(5q), trisomy 8, and structural abnormalities involving 1l q . Each of these aberrations if found in roughly one-fifth to one-fourth of chromosomally abnormal RARS cases. C. CHRONICMYELOMONOCYTIC LEUKEMIA (CMML) Only three aberrations have been found in more than 10% of cytogenetically abnormal CMML cases. Monosomy 7 and trisomy 8 are both seen in about 20%, and structural changes involving 12p are seen in 15%. The 12p changes may be particularly common in

16

SVERRE HEIM AND FELIX MITELMAN

secondary disease, perhaps especially following Alkeran treatment (Wilmoth et al., 1985; Berger et al., 1986).Changes of chromosome 5 are no more common than can be accounted for by random involvement, in clear contrast to the situation in other MDS subtypes. D. REFRACTORY ANEMIAWITH EXCESS OF BLASTS (RAEB) AND REFRACTORY ANEMIAWITH EXCESS OF BLASTS IN TRANSFORMATION (RAEBT) These, the most leukemia-like MDS subtypes, have more chromosome aberrations than MDS in general. Thus, the greater the number of blasts, or, in other words, the closer the MDS is to ANLL, the greater is the chance of finding clonal changes in bone marrow samples. As in the other subtypes, the pattern of changes is nonrandom; del(5q) is found in 30%, monosomy 5 is found in 10%. Onefourth of all cytogenetically abnormal patients have monosomy 7. Finally, trisomy 8 is present in 10%. VI. Chronic Myeloproliferative Disorders

A. CHRONIC MYELOIDLEUKEMIA (CML) The dominating chromosomal change in CML is the Ph' marker, which originates through the reciprocal translocation t(9;22)(q34;qll) (Fig. 6). This rearrangement is detected in 85-90% of all CML patients, and the t(9;22) is also usually the only karyotypic abnormality during chronic-phase CML. With transition to accelerated phase and blast crisis, additional, secondary aberrations typically accrue, most commonly an extra Ph' marker, an extra chromosome 8, an

9

22

1(9;22)(qW;qll)

FIG.6. The rearrangement t(9;22)(q34;qll)leads to the formation of the Ph' marker, and is the characteristic abnormality in CML.

CHROMOSOME ABNORMALITIES IN NEOPLASIA

17

isochromosome for the long arm of chromosome 17 [i(17q)],and + 19 (Heim and Mitelman, 1987a). In a subgroup of Ph'-positive patients, the Ph' marker does not result from the usual 9;22 translocation. These variant translocations have traditionally been classified as either complex (meaning that, in addition to chromosomes 9 and 22, one or more other chromosomes are involved in the rearrangement) or simple (in which case the material from chromosome 22 is translocated onto a chromosome other than chromosome 9). More refined cytogenetic investigations, together with studies utilizing recombinant DNA technique, have now demonstrated that all variant translocations are in fact complex and involve interchanges between chromosomes 9 and 22 (Hagemeijer et al., 1984). The realization that both classic and variant Ph'-positive CMLs have in common recombination of sequences between 9q34 and 2 2 q l l strongly indicates that this constitutes a crucial step in CML development. The essential event at the molecular level is now thought to b e the movement of the cellular oncogene c-abl from 9q34 to 22ql1, where it fuses with another gene at what is called the breakpoint cluster region (bcr) to form a chimeric bcrlabl gene. The details of this rearrangement have helped explain many aspects of how cytogenetic changes may activate oncogenes, and will be discussed in Section XI. About 5% of CML patients have Ph'-negative CML. These patients may be cytogenetically normal, or may have other changes, mostly trisomy 8 and i(17q) (Heim and Mitelman, 1987a). Some Ph'-negative cases have been shown to contain submicroscopic changes involving 9q34 and 2 2 q l l that are pathogenetically equivalent to the visible translocation in Ph'-positive disease. On the other hand, many cases previously classified as Ph'-negative CML are now, on morphological grounds, being referred to other diagnostic categories, in particular the various forms of myelodysplasia (Pugh et al., 1985; Travis et al., 1986).It appears, therefore, that CML may be a cytogenetically even more homogeneous disease than has been appreciated in the past. Without doubt, t(9;22)(q34;qll) is the dominant cytogenetic change, and may, if pathogenetically equivalent molecular rearrangements are also included, turn out to be ubiquitous to all CML development.

B. POLYCYTHEMIA VERA(PV) Roughly one-fifth of patients in this category have been reported to have recognizable chromosome aberrations. The dominating changes,

18

SVERRE HEIM AND FELIX MITELMAN

all of which may represent primary abnormalities, are del(20q), +8, +9, del(13q), and structural abnormalities of chromosome 1resulting in partial trisomy l q (Swolin et aE., 1986; Rege-Cambrin et al., 1987). C. IDIOPATHIC MYELOFIBROSI~AGNOGENIC MYELOIDMETAPLASIA In this category, chromosome aberrations have been described in somewhat more than 100 patients. The most common abnormalities have been numerical changes of chromosomes 7, 8, and 9, and structural changes of lq, 139, and 20q.

D. ESSENTIAL THROMBOCYTHEMIA No characteristic abnormality has so far been associated with this myeloproliferative disorder. Most patients have been karyotypically normal. VII. Acute Lymphoblastic Leukemia (ALL)

The relatively uniform morphology of leukemic bone marrows in acute lymphoblastic leukemia (ALL) is reflected in the FAB classification (Bennett et al., 1976),which recognizes only three morphologic ALL subgroups, i.e., L1, Lz, and Ls. More important than the morphologic subdivision is the immunophenotypic characterization of the proliferating blasts. Depending on where in development the maturation block in each respective leukemia occurs, the malignant cells exhibit a distinctive set of immunological characteristics corresponding to the immunocompetence of normal lymphocytic cells at that stage. The morphological and immunological characteristics have been combined with cytogenetic findings to form the MIC (morphologic, immunologic, and cytogenetic) classification (First MIC Cooperative Study Group, 1986). In the MIC system, B-lineage ALL comprises four maturation subgroups: early B precursor ALL, common ALL, pre-B ALL, and B cell ALL. The less common T-lineage ALL is subdivided into the immature early T precursor ALL and T cell ALL. In ALL, as in ANLL, roughly two-thirds of the patients may be found at diagnosis to have clonal chromosome abnormalities in their bone marrow cells. The most prominent aberrations, which also are the changes most likely to represent primary rearrangements, are summarized in Table 111.

CHROMOSOME ABNORMALITIES IN NEOPLASIA

19

TABLE 111 PRIMARY CHROMOSOME ABNORMALITIES IN ACUTE LYMPHOBLASTIC LEUKEMIA Rearrangement

Typical morphology

Typical immunophenotype

ti 1;11)(p32;q23) t(1;19)(q23;p13) t(2;8)(~12;q24) t(4;l l)(q21;q23)

LI L1 L3 L1, Lz

deMd t(8; 14)(q24;ql1) t(8; 14)(q24;q32) t(8;22)(q24;qll) dellt(9p)

L1, LZ

t(9;22)(q34;qll)

L1, Lz

t( 10;14)(q24;qll) t( 11;14)(p13;qll) t(l1;14)(q23;q32) t(l1;19)(~23;~13) del/t( 12p)

L1, Lo LI, Lz LI, Lz

Pre-B ALL Pre-B ALL B-ALL Early B-precursor ALL; mixed phenotype Common ALL T-ALL B-ALL B-ALL T-ALL or early T-precursor ALL Early B-precursor, common, or pre-B ALL T-ALL T-ALL

-

L3 L3 LI, Lz

-

Common ALL

A. t(1;19)(q23;p13) Although some uncertainty initially existed as to whether this translocation was to 19q or 19p, it has now been unequivocally demonstrated that the latter interpretation is correct, and that indeed the chromosome 19 breakpoint may be mapped to subband 19~13.3 (Michael et al., 1985). The aberration occurs in two forms: either the cells contain one normal chromosome 1 and one lq-, or, more commonly, they contain two normal chromosomes 1 and, in addition, the extra chromosome 1 material of the 19p+. The t(1;19) is strongly associated with the pre-B ALL phenotype, and the cells usually have L1 morphology (Shikano et ul., 1986). A cellular oncogene, c-ski, is located in lq23, and the insulin receptor gene is located in 1 9 ~ 1 3Whether . recombination or rearrangement of these loci occurs in t(1;19) is presently unknown. B . t(4;11)(92 1;q23)

This rearrangement has mostly been associated with very early leukemia debut, often as congenital leukemia. Opinions have been divided as to the precise nature of the leukemic cells: in conventional

20

SVERHE HEIM AND FELIX MITELMAN

light microscopy, they resemble lymphoblasts with L1 or L2 morphology. On the other hand, their sometimes unusual ultrastructural and cytochemical characteristics, which include a mixture of lymphoid and monocytic features, has lead to the alternative view that the immature blasts may instead belong to the myeloid lineage. These discrepancies have not yet been resolved, but more recent studies have detected beginning rearrangement of the Ig loci and an evolving immunophenotype indicative of early B cell differentiation (Crist et al., 1985; Stong et al., 1985). As a purely descriptive compromise, the leukemia has been called biphenotypic, without stating the precise nature of' the proliferating cells. The cellular oncogene c-etsl, which maps to 11q23-24, is translocated to the 4q- chromosome in t ( 4 ; l l ) (Sacchi et al., 1986). It may hence be speculated, although direct evidence to this effect is lacking, that altered c-etsl function may be of pathogenetic importance in t(4;ll)-associated disease.

C. del(6q) Deletions of the long arm of chromosome 6 are found in 5-10% of all cytogenetically abnormal ALL cases. The size of the deletion has varied considerably in different reports, but in most cases the breakpoints have been localized to 6q15 and 6q21. The cellular oncogene c-myb maps to 6q21-24, and has recently been shown to be retained in del(6q)-associated disease (Barletta et aZ., 1987). No rearrangement of the locus was detected, but c-myb messenger RNA levels were increased in del(6q)-carrying cells compared with malignant cells matched for lineage and stage of differentiation but lacking the 6q- marker. A pathogenetic role for c-myb in del(6q)-associated disease thus remains a distinct possibility. D. t(9;22)(q34;qll) This may be the single most frequent cytogenetic rearrangement in ALL, found in roughly 15% of all cases reviewed at the Third International Workshop on Chromosomes in Leukemia 1980 (1981).It is more often seen in adult patients than in children. The immunophenotype is in the majority of cases compatible with immature B cell differentiation, i.e., early B precursor ALL, common B cell ALL, or pre-B ALL. A crucial question is whether the t(9;22)(q34;qll)of CML and ALL

CHROMOSOME ABNORMALITIES IN NEOPLASIA

21

are identical at the molecular level. Is the molecular pathology the same in the two diseases? Some quite recent studies have at least partially answered this question (Chan et al., 1987; Clark et al., 1987; Kurzrock et al., 1987). Apparently the breakpoints in 2 2 q l l are not restricted to the bcr in ALL, but may sometimes be found proximal (5’) to this region. Thus, whereas a subset of ALL patients with t(9;22)(q34;qll)develop bcr-abl fusion genes that are indistinguishable from the molecular result in CML (see Section XI), another subgroup produces a unique abl-encoded tyrosine kinase of about 190 kDa. These cases therefore seem to represent an alternative mechanism of c-abl activation in leukemia, different from the one operating in CML. I n keeping with these findings, Kurzrock et al. (1987) suggested that Ph’-positive ALL might be viewed as a nosologic continuum. Those cases in which the essential molecular result is the formation of a bcr-abl chimeric gene coding for a BlOkDa protein product may represent the end stage of a previously unrecognized, clinically silent CML. Other patients, in whom c-abl is activated through a novel, different mechanism, develop ALL directly.

E.

REARRANGEMENTS OF

14q32 and B Cell ALL

Three translocations, t(8;14)(q24;q32) (Fig. 7), t(2;8)(p12;q24),and t(8;22)(q24;qll),are all associated with B cell neoplasia with remarkable specificity (Mitelman, 1981; Berger and Bernheim, 1985). They are found in both leukemias and lymphomas, in particular in Burkitt’s lymphoma, and are nearly always associated with the mature L3 morphology. The most common, t(8;14)(q24;q32), is found in 85% of cases, while the variants, t(2;8) and t(8;22), make up the remaining 15% of Burkitt-like leukemias. The details of oncogene deregulation in Burkitt-associated translocations will be discussed in Section XI. Let us here only briefly summarize that 8q24 contains the c-myc protooncogene, whereas 14q32, 2p12, and 2 2 q l l are the sites of the Ig heavy-chain locus and the K and A Ig light-chain loci. Through any of these three translocations, the current understanding is that c-myc comes under the influence of constitutively active transcription-stimulating sequences, whose normal function is to regulate the Ig loci, resulting in its transcriptional deregulation. The c-myc protooncogene is translocated to the Ig locus in t(8;14); in the variant translocations it remains on the derivative chromosome 8 but has parts of the Ig light-chain loci translocated into its immediate vicinity.

22

SVERRE HEIM AND FELIX MITELMAN

F. ABNORMALITIES ASSOCIATED WITH T CELLALL Structural aberrations involving 9p, i.e., t/del(9p),have been linked to a “lymphomatous” mode of presentation for T cell ALL with high tumor load, including bulky disease of lymph nodes, spleen, and mediastinum (Kowalczyk and Sandberg, 1983; Chilcote et al., 1985; Maseki et al., 1986a). The molecular pathology of the aberrations is unknown. Several translocations involving 14ql1, including t(8;14)(q24;qll), t(10;14)(q24;q11), and t( 11;14)(pl3;qll), have been associated with T cell neoplasia, often T cell ALL. Apart from the T-lineage association, no clinical stigmata have been consistently associated with these aberrations (Williams et al., 1984; Dube et al., 1986; Harbott et al., 1986; Shima et al., 1986). The common feature of these changes is obviously the involvement of 1 4 q l l . This band contains the T cell receptor (TCR) a-chain locus, which has been shown to be split by the translocations. It thus seems that marked similarities exist between B and T cell neoplasias: in B cell lymphomas and leukemias, recombinations between Ig loci and protooncogenes unleash the neoplastic process; in T cell neoplasms the corresponding recombinations occur between T cell receptor loci and protooncogenes. The oncogenes may in many cases be the same, for example, c-myc in 8924. So far, the a-chain TCR in 1 4 q l l has been the most frequently affected site in T cell neoplasms. It is to be expected that even the P-chain and y-chain receptor loci, which map to chromosome 7, will in some cases be found to be involved in T-cell-specific, tumor-associated aberrations. VIII. Chronic Lymphoproliferative Disorders

Several characteristic chromosome abnormalities have been associated with both B- and T-lineage chronic lymphoproliferative disorders (Juliusson et al., 1985; Han et al., 1986; Zech et ul., 1986), and a summary of the best candidates for a primary role is given in Table IV. We shall look at some of the diagnostic subgroups in more detail. A. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) Chromosome aberrations have been detected in more than 50% of patients with B cell CLL when the cultures have been stimulated with appropriate polyclonal B cell mitogens. The most common abnormality appears to be an extra chromosome 12, found in one-third

CHROMOSOME ABNORMALITIES IN NEOPLASIA

23

TABLE IV PRIMARY CHROMOSOME ABNORMALITIESIN CHRONIC LYMPHOPROLIFERATIVE DISORDERS Diagnosis

B lineage Chronic lymphocytic leukemia Prolymphocytic leukemia

Hairy cell leukemia WaIdenstrom’s macroglobulinemia Multiple myeloma

Rearrangement

+ 12 14q+ 14q+ t/del( 12p) deWp13) 14q+ 6q Unknown Chromosome 1 rearrangements 14q+

T lineage Chronic lymphocytic leukemia Adult T cell leukemia

Prolymphocytic leukemia Cutaneous T cell lymphoma/ SCzary’s syndrome/ mycosis fungoides

inv(14)(qllq32) t/del(l4)(qll) 14q+ 14ql1 rearrangements del(6q) 1 4 q l l rearrangements Chromosome 1 rearrangements t/del(6p)

of all cases. The second most common (25%) has been a 14q+ marker, originating through translocation of material from any of a number of different chromosomes onto 14q32. In some of these rearrangements, the Ig heavy chain locus in 14q32 has been shown to be split by the rearrangement, underscoring the principal similarity between these B cell abnormalities and the Burkitt-associated translocations. The much less frequently occurring T cell CLL is cytogenetically characterized by rearrangements affecting 14q11. The most consistent abnormality is inv(14)(qllq32), found in one-third of all cytogenetically abnormal T cell C L L s , but other rearrangements, most notably t(14;14)(qll;q32),may also b e seen.

B. PROLYMPHOCYTIC LEUKEMIA (PLL) The most common abnormality in B cell PLL is a 14q+ marker, found in one-half of all cytogenetically abnormal patients. The donor chromosome of the extra material translocated to 14q32 has not been

24

SVERRE HEIM AND FELIX MITELMAN

identified (Brito-Babapulle et al., 1987a). In T cell PLL, as in other T cell malignancies, the dominating changes involve 14ql1, often in the form of inv(14)(qllq32)(Brito-Babapulle et al., 1987b). C. HAIRYCELLLEUKEMIA (HCL) Very few cases have been investigated, but again a 14q+ marker is the most common change, encountered in one-third of all cases. The second most common aberration (20%)is 6q-.

D. ADULTT CELLLEUKEMIA (ATL) This leukemia is of particular interest because of the causal relationship to the HTLV-I retrovirus. Various rearrangements of 1 4 4 1 , probably the a-chain TCR locus, are the most frequent cytogenetic changes (Fujita et al., 1986), followed by 14q+ markers originating through translocations to 14q32. The 6q- markers are found in one-fifth of all cases. IX. Malignant Lymphoma

Based on clinical and histopathological criteria, the malignant lymphomas are classically subdivided into two main categories: Hodgkin’s disease (HD) and the non-Hodgkin’s lymphomas (NHL). Cytogenetic knowledge is largely restricted to the latter; the neoplastic cells of Hodgkin’s disease have hitherto proved quite resistant to chromosome analyses. More than 1000 NHL cases have been analyzed, and clonal chromosome abnormalities are found in about 90%(Yunis et al., 1984; Berger TABLE V PRIMARY CHROMOSOME ABNORMALITIES IN Burkitt’s lymphoma

Non-Burkitt’s non-Hodgkin’s lymphoma

MALIGNANT

LYMPHOMAS

t(2;8)(p12;q24) t(8;14)(q24;q32) t(8;22)(q24;qll) Structural changes of Chromosome 1 t(2;5)(p23;q35) +3 Structural changes of Chromosome 3 t/del(6p) del(6q) t(11;14)(q13;q32) t( 14;18)(q32;q21)

CHROMOSOME ABNORMALITIES IN NEOPLASIA

25

and Bernheim, 1985; Levine et al., 1985; Kristoffersson et ul., 1986). The most common changes are summarized in Table V. A. BURKITT’S LYMPHOMA (BL) A reciprocal translocation between chromosomes 8 and 14, t(8;14)(q24;q32) (Fig. 7), is found in four-fifths of all BL cases. In the remaining tumors one of the two variant translocations, t(2;8)(p12;q24) or t(8;22)(q24;qll), is present. As was pointed out briefly in the discussion of Burkitt-like ALL, the essential pathogenetic consequence of the rearrangement appears to be the juxtaposition of c-myc (normally located in 8q24) with transcriptionregulating elements whose normal function is to regulate the Ig heavy and light chain loci. The molecular details of these changes are discussed in Section XI.

B. NON-BURKITT’S NON-HODGKIN’S LYMPHOMA (NHL) Structural changes of chromosome 1are detected in about one-third of all NHLs. Breakpoints are found in both arms, and the type of aberration varies considerably. The changes of chromosome 1 occur mostly in addition to other, more specific abnormalities, and are hence probably secondary. The t(2;5)(p23;q35) translocation has been described in four cases of malignant histiocytosis (Kristoffersson et ul., 1985; Morgan et al., 1986). The effect of the rearrangement on the cellular oncogene c-fms in 5q is unknown. Numerical and structural changes of chromosome 3 have been described. Trisomy 3 appears to characterize a small subgroup of

14

t(8;14)(q24;q32)

FIG.7. The rearrangement t(8;14)(q24;q32) is specifically associated with Burkitt’s lymphoma and Burkitt-like leukemia.

26

SVERRE HEIM AND FELIX MITELMAN

chronic T-lineage neoplasms (Godde-Salz et al., 1986). Structural changes of chromosome 3 are found in one-fourth of all NHL cases, but the variability of breakpoints makes the evaluation of the specificity of these changes difficult. Translocations and deletions of the short arm of chromosome 6 have been reported in T cell lymphomas (Mecucci et al., 1985; Maseki et al., 1986b). In malignant lymphomas, as in other lymphatic neoplasms, a substantial subgroup of patients may be found with acquired deletions of the long arm of chromosome 6, del(6q). The possible importance of c-myb changes in this context has been mentioned. Rearrangements occur .that give rise to a 14q+ marker. Sometimes the donor chromosome supplying the material translocated to 14q32 is unknown. Among the fully identified translocations, two deserve separate mention. T h e first, t(11;14)(q13;q32), is found not only in B cell CLL but also in the corresponding lymphoma, namely, small cell lymphocytic lymphoma. The essential result is probably the split of the Ig heavy chain locus, leading to the juxtaposition of an oncogene from 1lq13, tentatively designated bcl-1, with regulatory elements from 14q32 (Erikson et al., 1984; Tsujimoto et al., 1985a). The second translocation, t( 14;18)(q32;q21), is the most common cause of a 14q+ marker in NHL, and is particularly associated with lymphomas with follicular growth pattern (Fukuhara et al., 1979; Yunis et al., 1984, 1987). The breakpoints have been examined with recombinant DNA methods (Tsujimoto et al., 1985b; Cleary et al., 1986).The 14q32 breakpoint occurs within the Ig heavy chain locus, near the 5’ end of the joining ( J ) segment, where diversity (D) segments are normally recombined with J segments to produce an active heavy chain gene. These findings are compatible with the hypothesis that the rearrangements splitting the Ig heavy chain do so as a result of mistakes in the normal V-D-J joining process. The 18q21 breakpoints cluster within a relatively small area. A putative oncogene, bcl-2, is as a consequence of the translocation moved to the derivative 14q+. In analogy with the prevailing hypothesis developed for the Burkitt-associated translocations, it is thought that the t( 14;18) recombines bcl-2 with transcription-stimulating elements from the Ig locus, ensuring oncogene deregulation and ultimately neoplastic growth.

C. HODGKIN’S DISEASE (HD) Only about 30 HD tumors have been successfully examined. The cytogenetic abnormalities found have largely resembled the aber-

CHROMOSOME ABNORMALITIES IN NEOPLASIA

27

rations of NHL (Kristoffersson et al., 1987), i.e., structural changes of chromosomes 1 and 11, 6q-, 14q+, and trisomy for chromosomes 3 and 7. X. Solid Tumors A summary of the most consistent aberrations presently known in solid tumor cytogenetics is provided in Table VI. We shall address only some of the most specific rearrangements, which hence are particularly likely to provide insights into the molecular mechanisms of solid tumor development.

A. MIXEDTUMORS OF

THE

SALIVARY GLAND

Three cytogenetic subgroups may be distinguished (Mark and Dahlenfors, 1986), namely, tumors with aberrations involving 3p21, 8q12, and 12q13-15. Often, but not always, the two first breakpoints are recombined in a t(3;8)(p21;q12).Possibly this may be considered a standard rearrangement, with the other 3p21 and 8q12 changes representing variant translocations. The salient molecular consequences of the translocations are unknown. This also goes for the third subgroup, the 12q-affecting aberrations, which occur independently of the 3p or 8q abnormalities.

B. SMALLCELLLUNGCANCER A deletion of the short arm of chromosome 3, mostly interpreted as de1(3)(p14p23), is a frequent finding in small cell lung carcinomas (Whang-Peng and Lee, 1985). Opinions are divided whether similar deletions may also be found in other lung tumors, such as adenocarcinomas and squamous cell carcinomas. It is unknown in what manner del(3p) contributes to carcinogenesis. The consistent loss of material, often with variable deletion breakpoints, is compatible with loss of antioncogenic activity as the essential mechanism. Positive evidence to this effect is presently lacking, however. C. RENAL CELLCARCINOMA

The most commonly reported cytogenetic abnormality in renal cell carcinomas is del(3p), or translocations between 3p and other chromosomes with loss of 3p material (Yoshida et al., 1986; Kovacs et al., 1987). These findings parallel those of small cell lung cancer, and

28

SVERRE HEIM AND FELIX MITELMAN

TABLE VI CONSISTENTLY OCCURRING CHROMOSOME ABNORMALITIESIN SOLIDTUMORS

TUMOR Alveolar rhabdomyosarcoma Bladder carcinoma

Breast carcinoma Ewing’s sarcoma Glioma Kidney carcinoma Large bowel carcinoma

Lipoma Malignant melanoma

Meningioma Myxoid liposarcoma Neuroblastoma Ovarian carcinoma Pleomorphic adenoma Prostatic carcinoma Retinoblastoma Small cell lung cancer Synovial sarcoma Testicular teratomaiseminoma Uterine carcinoma Uterine leiomyoma Wilms’ tumor

CHROMOSOME ABERRATION t(2;13)(q37;q14) Structural changes of Chromosome 1 i(5~) +7 -9 Structural changes of Chromosome 11 Structural changes of Chromosome 1 Structural changes of 16q t( 11;22)(q24;q12) dmin t/de1(3)(pll-21) t(5;14)(q13;q22) Structural changes of Chromosome 1 +7 + 12 Structural changes of Chromosome 17 t( 12)(q13-14) Ring Chromosomes t/del( l)(p12-22) t( 1;19)(q12;p13) t/del(6q)/i(6p) +7 - 22 t( 12;16)(q13-14;~11) del(l)(p31-32) HSR/dmin Structural changes of Chromosome 1 t(6; 14)(q21;q24) t(Np21) t/de1(8)(q12) t/del( 12)(q13-15) deUV(r122) del(10)(q24) Structural changes of Chromosome 1 i(6~) del(13)(q14)/-13 de1(3)(p14p23) t(X;18)(pll;qll) i(12-o) Structural and numerical changes of Chromosome 1 t(12;14)(~14-15;~23-24) Structural changes of Chromosome 1 t/del(ll)(pl3)

CHROMOSOME ABNORMALITIES IN NEOPLASIA

29

antioncogene loss has been a favored hypothesis as to the pathogenetic mechanism.

D. BLADDER CARCINOMA Among the more than SO bladder carcinomas so far successfully analyzed, five cytogenetic subgroups may be recognized (Atkin and Baker, 1985; Gibas et al., 1986). Structural changes of chromosomes 1 and 11 are present in one-third of all cases, but are highly variable, and may well represent secondary changes. Better candidates for a primary role are the aberrations i(Sp), trisomy 7, and monosomy 9, all of which have been repeatedly described as solitary changes. Nothing is known about how they might contribute to tumorigenesis.

E. UTERINELEIOMYOMA The reciprocal translocation t( 12;14)(q14-15;923-24) has been described as the sole aberration in four of five completely benign leiomyomas containing clonal abnormalities (Heim et al., 1988). The crucial genes involved in 12914-15 and 14q23-24 are unknown.

F. LIPOGENIC TUMORS Nonrandom chromosome abnormalities have been found in both benign and malignant lipogenic tumors. The most thoroughly investigated tumor type is the lipoma (Mandahl et al., 1987),where translocations involving 12q13-14 are found in half of all investigated tumors. The reason for this aberration pattern is unknown, but the int-1 protooncogene is located in this region and could possibly be of importance. A second cytogenetic subgroup of lipomas is characterized by the presence of unidentified ring chromosomes. Their role in pathogenesis is unknown. A specific translocation, t( 12;16)(q13;pll), characterizes myxoid liposarcomas (Turc-Care1 et al., 1986b; Mertens et al., 1987b). It is at present uncertain whether the 12q breakpoint in these cases is identical to the apparently similar breakpoint in lipomas. If so, why does the recombination of' 12q with 1 6 p l l result in the malignant tumor myxoid liposarcoma, whereas numerous other recombinations lead to benign neoplasms? The similarity between the 12q breakpoints in lipogenic tumors, uterine leiomyomas, and salivary gland tumors is also intriguing. Are the breakpoints identical? What

30

SVERRE HEIM AND FELIX MITELMAN

genes are involved? Neither question can at present be satisfactorily answered. G. ALVEOLAR RHABDOMYOSARCOMA

Although only few cases have so far been examined, it appears that the rearrangement t(2;13)(q37;q14) is specifically associated with this sarcoma (Turc-Care1 et at., 1986a).

H. SYNOVIAL SARCOMA A reciprocal translocation between chromosomes X and 18, t(X;18)(pll;qll), has been described in several cases of this tumor (Turc-Carel et al., 1987). I. MENINGIOMA This benign tumor has been extensively investigated (Mark, 1977; Zang, 1982; A1 Saadi et al., 1987), and it has been firmly established that the vast majority of cases are characterized by the loss of one chromosome 22. Occasionally, partial rnonosomy in the form of del(22q) has been found. Both observations are compatible with the hypothesis that antioncogene loss is the essential result of the cytogenetic aberration.

J. EWING’S SARCOMA This tumor is cytogenetically characterized by the reciprocal translocation t( 11;22)(q24;q12) (Turc-Care1 et al., 1984; Aurias et al., 1984). The same rearrangement is also found in the less common neuroepithelioma and Askin’s tumor, which has added to the suspicion that all these tumors may represent variants of basically the same neoplastic process. XI. Oncogenes, Antioncogenes, and Chromosome Aberrations

Overwhelming evidence indicates that genetic changes in somatic cells are the main causative events in turnorigenesis. This of course does not rule out that epigenetic phenomena may also be important in several neoplastic disorders: in all probability cancers go through several developmental stages before they become established, let

CHROMOSOME ABNORMALITIES IN NEOPLASIA

31

alone clinically recognizable. It is probable, therefore, that only some of these steps of multistage carcinogenesis involve genetic rearrangements. What are the essential genetic disturbances leading to neoplastic growth? How do cancer-associated chromosome abnormalities bring about the molecular rearrangements that unleash neoplasia? In principle, three classes of genes important in tumor development may presently b e distinguished. The least well-defined are the modulating genes, which are not directly involved in the neoplastic transformation per se but regulate the organism’s response to the neoplasm and hence the subsequent fate of the neoplastic cell population. No doubt hundreds of different loci contribute modulating influences in this sense, and a discussion of this class is outside the scope of the present review. The other two classes of cancer-relevant loci are presumably of direct importance in establishing at least some tumors. They are the recessive tumor suppressor genes or antioncogenes, and the dominantly acting oncogenes. A.

ANTIONCOGENES

Evidence that some cellular genes have the capacity to prevent neoplastic proliferation originally emanated from cell fusion studies (Stanbridge, 1984). When malignant and nonmalignant cells were experimentally fused, chromosomes supplied by the latter were found to ensure nonmalignant behavior by the resulting hybrids. When particular chromosomes were lost from the hybrid cells, the transformed phenotype reappeared. Independent evidence that loss of chromosomal material may lead to tumor development even in clinical contexts derives from studies of two childhood cancers that occur in both hereditary and spontaneous forms, retinoblastoma (RB) and Wilms’ tumor. Support for the concept that consecutive loss of recessive antioncogenes is an essential element in retinoblastoma development comes from three sources: studies of constitutional and tumor karyotypes, studies of a polymorphic enzyme linked to the RB locus, and examination of linked DNA restriction fragment length polymorphisms (RFLPs). The first firmly substantiated hypothesis portraying cancer development in general and retinoblastoma development in particular as multistaged emanated from an examination of age-specific tumor

32

SVERRE HEIM AND FELIX MITELMAN

incidences. Based on mathematical analyses of such data, Knudson (1971, 1983) proposed the following two-step model for tumorigenesis: he postulated that, in both hereditary and sporadic tumors, at least two independent steps are necessary. In the hereditary form, the first step, which corresponds to the inactivation of an antioncogene, has occurred in the germ line, and all somatic cells hence contain this initial change. T h e second step involves inactivation of the homologous antioncogene allele. This occurs as a stochastic event in a somatic cell. No phenotypic effect is detectable in the cells before both antioncogene alleles have been inactivated, which is why these genes at the cellular level behave as recessives (not to be confused with the autosomal dominant inheritance of the diseases they predispose to). I n sporadic tumors, basically the same two events must take place, but here they both occur in a somatic cell. Later evidence has completely borne out the scenario envisioned by Knudson (reviewed in Knudson, 1985, 1987; Cavenee et al., 1986; Benedict, 1987).Findings of constitutional deletions involving 13q14 in some retinoblastoma patients with bilateral tumors, and also the occasional finding in other cases of comparable changes restricted to tumor cells, indicated that the putative retinoblastoma antioncogene was located in 13q14. Later linkage studies on both protein level (using esterase D, which is closely linked to the RB locus) and at the level of DNA (using sets of linked RFLPs) have corroborated and further developed the conclusions surmised from cytogenetic data. These molecular studies have proved that both RB alleles are inactivated in the tumor cells even in such cases where chromosome 13 appears to be microscopically normal. Several mechanisms of inactivation of the wild-type antioncogene allele are operative (Fig. 8), and only some of them lead to visible cytogenetic changes. The other childhood tumor in which antioncogene inactivation is convincingly implicated is nephroblastoma, or Wilms’ tumor. Here, too, inactivation of both antioncogene alleles is necessary before neoplastic proliferation begins, and again the first step may at times have occurred in the germ line. The antioncogene locus of importance in Wilms’ tumor is located in llp13, and again both cytogenetic and RFLP linkage data support the two-step inactivation model. The genetic mechanisms whereby this inactivation may be achieved are identical to the pathways illustrated for retinoblastoma in Fig. 8. Do other tumors develop through antioncogene inactivation? The full answer to this question is unknown. It presently seems that inactivation of the antioncogene in 13q14 is important in retinoblasto-

CHROMOSOME ABNORMALITIES I N NEOPLASIA

rb

/

.

33

Nondisjunction

and reduplication

recombination Deletion including the Rb locus

Gene inactivation

the Rb locus

FIG.8. Mechanisms whereby the remaining wild-type antioncogene allele can be inactivated in the genesis of retinoblastoma. The same pathways are also available in other tumors developing through antioncogene loss, e.g., Wilms’ tumor.

mas and also osteosarcomas, but not in other neoplasms. The l l p 1 3 antioncogene locus is inactivated not only in Wilms’ tumor, but also in hepatoblastoma and rhabdomyosarcoma. Although these are the only two loci where it can be said with reasonable certainty that antioncogenes exist, suspicion is strong that other chromosomal regions may also harbor recessive tumor suppressor genes. Among the prime candidates in this respect are 22q (meningioma, acoustic neuroma), 3p (renal cell carcinoma, small cell lung cancer), and perhaps 5q and 6q (myeloid and lymphatic neoplasms, respectively). Whether loss of antioncogenes is also a major mechanism in some of the common cancers of adult life, for instance, colon and breast carcinomas, is an intriguing but as yet insufficiently explored possibility. What do antioncogenes do? How does their inactivation contribute to carcinogenesis? The term antioncogene is unfortunate, as is, for that matter, the term oncogene. In all likelihood antioncogenes do not operate through any inhibition of oncogenes; the two systems, of cancer-relevant genes are probably unrelated. The normal function of antioncogenes is unknown. This ignorance may soon be remedied, however, as at least the retinoblastoma-osteosarcoma gene now seems to be on the verge of complete chemical characterization (Friend et al., 1986; Lee et al., 1987). Unquestionably, greatly improved understanding of cell biology in general and of tumor

34

SVERRE HEIM AND FELIX MITELMAN

biology specifically will follow the precise description of this class of cancer genes.

B. ONCOGENES This group of evolutionary highly conserved housekeeping genes is, under physiological conditions, probably involved in the regulation of cellular proliferation and differentiation. Cellular protooncogenes have been picked up by retroviruses and slightly altered so that upon reinfection they may be oncogenic in a dominant fashion. Indeed, investigation of retroviral oncogenesis drew attention to this entire class of cancer genes. It now appears that changes affecting cellular protooncogenes, sometimes quite subtle changes, are essential steps in the development and progression of many human neoplasms (Weinberg, 1985; Temin, 1986; Klein and Klein, 1986; Bishop, 1987). The evidence implicating oncogenes stems from four principal sources :

1. Some experimental and human tumors contain activated oncogenes capable of transforming appropriately selected cells in vitro. 2. Some cellular oncogenes are activated when viruses are inserted in their vicinity. 3. Cellular oncogenes are often amplified in human tumors. 4. Cellular oncogenes are located at the same chromosomal sites that are involved in cancer-associated chromosome aberrations.

The last point is of particular relevance in the present survey. Although some protooncogenes, in particular those of the rus family, require point mutations for their activation, frequently chromosomal rearrangements seem to be the preferred pathway in oncogene activation. I n principle, oncogene activation through chromosomal recombination may be achieved through one of two mechanisms : either (1)the recombination leads to a qualitatively different oncogene, an oncogene whose protein product has cellular effects that are fundamentally different from those of the protooncogene’s, or (2) the rearrangement may lead to increased production, or untimely production, of a normal protooncogene product. We shall examine in detail two specific examples of how cancer chromosome abnormalities activate oncogenes. 1. myc Activation in Burkitt’s Lymphoma The cytogenetic abnormalities involved in this context are t(8; 14)(q24;q32)(Fig. 7) and the variant translocations t(2;8)(p12;q24)

35

CHROMOSOME ABNORMALITIES IN NEOPLASIA

Tdonmn

cCmu0m.n

Chromosome 8

5’1 Emn I

cCnmvnn

Chromosome 14 3’ C+ll8l-llI

13’

U

Emn I1 Exon 111 c-myc I ~ C U S

I

I

SHch

T.lomn

5‘

Jolnlng

Dhrm*

vwbh

19 Heevychaln locus

Tdonmn

1(8;14Hq24;q32) 3’

(3’ Elon I1

lg H

Exon 111

c-myc

FIG.9. Schematic illustration of the head-to-head recombination of c-myc and the Ig heavy chain locus in t(8; 14)(q24;q32). Vertical arrows indicate possible breakpoint positions. The functional result is deregulation of the oncogene.

and t(8;22)(q24;qll).The essential effect of the three translocations is presumably the deregulation of c-myc, a protooncogene normally located in 8q24, leading to increased or at least untimely production of a normal c-myc product (Croce and Nowell, 1985; Klein and Klein, 1985; Leder, 1985; Croce et al., 1987). The recombination between c-myc and the Ig heavy-chain locus in 14q32 is schematically illustrated in Fig. 9. The c-myc oncogene consists of three exons, the first (5‘) of which has stop codons in all reading frames. Thus, only exons I1 and I11 are translated into protein. The 8q24 breakpoint in t(8;14)(q24;q32) may vary by as much as 50 kb, but is always proximal, or 5’, of exon 11, which means that the entire coding region of c-myc moves to 14q32. The 14q32 breakpoint lies within the heavy chain locus, often in the joining ( J ) region. As a result of the translocation, c-myc, or at least the two coding exons, is recombined head-to-head (5’ to 5’) with sequences within the heavy chain locus which normaIly ensure constitutive transcription of immunoglobulin. This deregulation of c-myc presumably interferes with the regulation of normal cell proliferation in a neoplasia-developing manner, although the mechanism whereby this happens is largely unknown. It appears that the myc-encoded protein, a DNA-binding polypeptide, is normal or at least only insignificantly changed. The molecular results of the two variant translocations are identical to what happens in t(8;14). In t(2;8) and t(8;22), however, c-myc remains on 8q24, but is deregulated through the juxtaposition of Ig sequences from 2p12 and 2 2 q l l .

36

SVERRE HEIM AND FELIX MITELMAN

2. Constructon of a bcr-abl Fusion Gene in C M L The characteristic chromosome aberration associated with this disease is t(9;22)(q34;qll). Through this translocation a novel, chimeric gene, the bcr-abl fusion gene, is created, and the unique protein product encoded by bcr-abl is presumably a major step in the development of leukemia (Gale and Canaani, 1985; Groffen et al., 1987). Some of the details of the rearrangement are illustrated in Fig. 10. The breakpoints in 9q34 may be quite variable, spread out as they are over an area perhaps 100 kb or more upstream of the c-abl locus. In 22ql1, however, the breakpoints are grouped in a much smaller region, the breakpoint cluster region (bcr),spanning only 5.8 kb. The bcr region is part of a gene whose function is unknown but which extends both 5’ and 3’ of bcr. The bcr breakpoints occur after the second or third exons within the cluster region. Cells from different CML patients with widely variable 9q34 breakpoints have been shown to produce bcr-abl mRNA in which the abl segment begins at exactly the same nucleotide (Shtivelman et al., 1985). The uniformity at the mRNA level in spite of major size differences between different bcr-abl fusion genes is achieved in the following manner: the chimeric bcr-abl locus is transcribed into a giant nuclear precursor RNA, which varies in size according to the breakpoint position. During subsequent RNA splicing, however, the first abl exon, which lacks a “splice acceptor signal,” is excised together with intervening sequences. In the resulting mRNA, the second abl exon is joined to the last bcr exon before the fusion point.

cConhornere

....

T.lom.re

-

3’

Chromosome 22 S bcr cCon1rom*,r.

Tolomere

t (9;22Mq34;q11) 5’

-

m 3 bcr-abl fusion gene

FIG.10. The rearrangement t(9;22)(q34;qll)in CML leads to the formation of a chimeric abl-bcr gene which encodes a novel, presumably leukemogenic, polypeptide with tyrosine kinase activity. Vertical arrows indicate possible breakpoint positions.

CHROMOSOLMEABNORMALITIES IN NEOPLASIA

37

The small variation in breakpoint position within the bcr exon at the mRNA level may only result in the absence or presence of the small third exon, which codes for only 25 amino acid residues. The bcr-abl mRNA encodes a novel 210-kDa polypeptide present in CML cells instead of the normal 145-kDa ubl product. In the new polypeptide at least 25 N-terminal amino acid residues are replaced b y 600-700 residues encoded by bcr. This N-terminal alteration is presumably what renders the protein oncogenic. Why this is so remains uncertain. The protein’s tyrosine kinase activity is probably of importance, since the transforming ability appears to be tightly coupled to this ability. The target for phosphorylation within the cell is unknown. Receptors for several growth factors are tyrosine kinases, and it has been postulated that the bcr-ubl-encoded tyrosine kinase is an abnormal receptor molecule involved in proliferation regulation. The normal ligand for the receptor is unknown. The BL and CML situations have been mentioned specifically because they are the first two neoplasms in which the molecular effects of chromosome aberrations have been clarified in detail. In both cases oncogene activation was involved, in BL through a deregulation mechanism, in CML through a structural change of the protooncogene. Circumstantial evidence is gradually accumulating that these two mechanisms may also be operating in several other contexts where chromosome breakpoints and oncogene sites cluster to the same bands. This concordance is quite remarkable: no less than 22 of 30 oncogenes with known chromosome sites are located in the same bands as aberration breakpoints (Heim and Mitelman, 1987b). Continuing combined molecular genetic and cytogenetic investigations will undoubtedly further refine our understanding of the mechanisms that render cells neoplastic. Through such efforts, much will also be learned about how normal control of cellular differentiation and proliferation is achieved. XII. Summary and Conclusions

At the cellular level, cancer is a genetic disease; genetic changes in somatic cells are essential events in neoplasia. In a majority of cases these changes involve large enough blocks of genetic material to be visible in the microscope. The chromosome aberrations in neoplastic disorders are probably of three kinds: (I) primary abnormalities, which are essential steps in establishing the tumor; ( 2 ) secondary abnormalities, which develop only after the tumor has developed, but which nevertheless may be important in tumor progression; and

38

SVERRE HEIM AND FELIX MITELMAN

( 3 ) cytogenetic noise, which is the background level of nonconsequential aberrations. These latter changes are, in contrast to the primary and secondary changes, randomly distributed throughout the genome. The primary abnormalities, of which several dozens have now been identified, are mostly strictly correlated with particular diseases and even with histopathological subtypes within a given disease. This has been evident in the leukemias for some years already, and information now accumulating on solid tumor karyology indicates a similar situation. Clonal chromosome abnormalities are a feature of both benign and malignant neoplasms, although the changes are often less massive in the former. Apart from being clinically useful as a diagnostic technique and an aid in prognostication, tumor cytogenetics also plays a role in identifying those genomic sites which harbor genes essential in the pathogenesis of neoplastic lesions. So far, two functionally different classes of directly cancer-relevant genes have been detected, the oncogenes and the antioncogenes. There is every reason to believe that future investigations with cytogenetic and recombinant DNA methods will add to our knowledge of the biology of human neoplasia, in those tumor types where the characteristic genetic change is already partially known, and by identifying hitherto unknown karyotypic abnormalities.

ACKNOWLEDGMENTS Original work reported in this article was supported by the Swedish Cancer Society and the JAP Foundation for Medical Research.

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T CELL RECEPTOR AND IMMUNOGLOBULIN GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS M. D. Reis,* H. Griesser,* and T. W. Mak*,t *

Ontario Cancer Institute, Toronto, Ontarlo, Canada M4X 1K9

t Departments of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada

I. Introduction 11. B Cell Antigen Receptor Structure, Function, and Gene Organization A. Creation of a Functional Immunoglobulin Gene and Generation of Antibody Diversity B. Sequence of Activation and Rearrangements of Immunoglobulin Genes 111. T Cell Antigen Receptor Structure, Function, and Gene Organization A. The Role of T Cells in Immune Responses B. The Structure of the T Cell Antigen Receptors and the Cloning of the Genes Encoding Their Polypeptides C. Genomic Organization of T Cell Receptor Genes D. Somatic Rearrangement of Gene Elements and Sequence of T Cell Receptor Gene Rearrangements IV. Clinical Applications o f t h e Analysis of Immunoglobulin and T Cell Receptor Gene Rearrangements in Henlatological Neoplasias A. Chronic T Cell Malignancies B. T Cell Lymphomas C. T Cell Acute Lymphoblastic Leukemia (T-ALL) D. Ty Lymphoproliferative Disorder E. Lymphomatoid Papulosis F. B Cell and Pre-B Cell Malignancies G. Other Hematopoietic Malignancies H. Acute Myeloblastic Leukemia (AML) V. The Simultaneous Occurrence of the T Cell Receptor and Immunoglobulin Genes in Lymphoproliferative Disorders VI. Chromosomal Translocations Involving the T Cell Receptor Genes References Note Added in Proof

I. Introduction The development and applications of recombinant DNA technology have led to remarkable advances in several areas of biology. Two fundamental achievements in immunology in the last 10 years have 45 ADVAhCES Ih CANCER RESEARCH, VOL 52

Copyright 0 1989 hy Academic Press, Inc All righta of reproduction in any forin resewed

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been the solution to two critical issues: the mechanisms operating in the generation of antibody diversity, and the nature of the T cell antigen receptor. Because of the cloning of immunoglobulin (Ig) and T cell receptor (TcR) genes, genetic probes have become available; these are used to study the activation of these genes in B and T cell ontogeny, to study the fine details of antibody generation, and to study the uniquely specific TcRs. They have also been used as powerful diagnostic tools for the detection of clonal lymphoid expansions, for determination of cell lineage in the great majority of lymphoproliferative disorders, and for monitoring therapy and detecting residual clonal populations in these lymphoid disorders. Several examples of recurrent specific translocations involving Ig and TcR genes in lymphoid malignancies are known and the role of these events in the pathogenesis of these group of neoplasias is being pursued. The molecular aspects of the recognition of self and foreign antigens and the possible links between histocompatibility antigens and the TcR repertoire are the subject of intense studies. A potential exists, for therapeutic purposes, for the use of the available knowledge regarding the structure of these receptors. I n this review, we will pursue some of these issues.

I I . B Cell Antigen Receptor Structure, Function, and Gene Organization

B cells, as precursors of antibody-producing cells, are the effector arm of the humoral immune response system. In this system, the immunoglobulin (Ig) molecules serve as cell membrane antigen receptors and have the ability to react with soluble antigens. Studies on the B cell antigen receptors have preceded those of T cell antigen receptors because of the large amounts of Ig that are available from Ig-producing myelomas. The basic units of these polypeptides form a heterodimer, consisting of a pair of two identical light (L) and two identical heavy (H) chains. These molecules are encoded by three unlinked gene families, K and h light chain genes, and heavy chain genes. Amino acid sequences of these chains reveal a remarkable pattern, with marked variation in the sequences of the N-terminal portion of the molecule when compared to each other. Contrasting with this, the sequences of the C-terminal half of the molecules of a given class are practically identical. Antigen recognition has been ascribed to the variable regions of the light and heavy chains, which

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contain hypervariable segments that have been implicated in antigen contact. It is clear from these studies that heavy and light chains consist of a series of homology units or domains.

A. CREATION OF

A

FUNCTIONAL IMMUNOGLOBULIN GENEAND

GENERATION OF ANTIBODY DIVERSITY The important question of how a limited number of genes can generate a vast number of antibodies with unique specificities was elucidated by Tonegawa (1983). It was found that the genes specifying the structure of each Ig molecule are organized as discontinued DNA segments in their germ line configuration, the form present in essentially all non-B lymphoid cells. As a mandatory step toward B cell differentiation, an uncommitted pre-B cell undergoes an orderly sequence of events, activating Ig genes and resulting in somatic recombination; that is, these genes become rearranged. The genes for K and h chains contain variable (V) and joining ( J ) gene segments, and constant (C) region genes. The heavy chain genes contain additional diversity (D) segments. The rearrangement and joining of one of each V-(D)-J segments lead to the assembly of a V region gene whose product is an Ig variable region polypeptide. Several genetic mechanisms are thought to contribute to the generation of antibody specificities, among them the multiple germ line variable region gene segments and the random combinatorial joining of the numerous V, D, and J segments. Junctional site diversity is another mechanism, with the flexibility occurring at the junction of the variable region gene segments during recombination. Additional junctional diversity may occur in the heavy chain junctions due to the insertion of one to several nucleotides at the ends in a templateindependent fashion (N insertion), perhaps by the enzyme terminal deoxynucleotidyltransferase (TdT) (Alt et al., 1982). Finally, the repertoire of antibodies can be further expanded as a result of somatic hypermutation, leading to changes in the amino acid sequences of the variable region of the immunoglobulin molecule, as in a fine-tuning modification resulting in increased antibody affinity for a given antigenic epitope. For additional details on the generation of antibody diversity, we suggest a review by Tonegawa (1983). Each B cell produces antibodies with a single type of variable region sequence. With the exception of the variability resulting from somatic mutation, a mature B cell and its progeny will have the same

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ET AL.

variable region sequence and will synthesize either chains, but not both.

K

or A light

B. SEQUENCE OF ACTIVATION AND REARRANGEMENTS OF IMMUNOGLOBULIN GENES A hierarchy of immunoglobulin gene recombinations has been demonstrated, starting first with rearrangement of heavy chain genes, followed by K and A light chain genes. The initial event brings about the joining of a D H with a JH segment of the heavy chain gene (Ah et al., 1982). This is followed by a VH segment combining with this DJ junction. The initial attempt to form a VDJ joining may be successful, in which case a p cytoplasmic heavy chain may be synthesized, or may be aberrant, with an incomplete heavy chain being produced. It has been postulated that a productive VH-DH-JH rearrangement resulting in the synthesis of a heavy chain polypeptide inhibits further heavy chain rearrangements. Upon complete heavy chain rearrangement, attempts at light chain recombination generally follow, involving the K genes first. If VK and J K are successfully joined in a cell already possessing a complete VHDHJHrecombination, a p, K surface Ig will result. If both K alleles rearrange aberrantly or are deleted, which frequently occurs, the cell next attempts to rearrange the A genes; if effective, this results in a p, A surface Ig; if ineffective or aberrant, there is no synthesis of light chain and the cell remains at the pre-B stage. It should be mentioned that the flexibility of joining of VH, DH,and JH may lead to nonproductive rearrangements relatively frequently, and the sequence of rearrangements of heavy and light chain genes is also prone to errors, which may result in a significant portion of B cell precursors incapable of further expansion. A detailed description of the genomic organizaton of the immunoglobulin genes will not be developed here, since many reviews are available on this topic (Tonegawa, 1983; Waldmann, 1987b). The utilization of molecular biological techniques has resulted in enormous progress in the understanding of important biological events in B cell development, and in procedural applications of' practical medical importance. Korsmeyer and co-workers (1981) first proposed the analysis of patterns of Ig gene rearrangements as a powerful and sensitive tool to assess clonality in B cell proliferative disorders. This is now used to determine cell lineage and clonality and to diagnose B cell malignancies and monitor their therapy, and has also provided insights into mechanisms of malignant transfor-

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mation in some B cell diseases. An example of the latter is the observation that chromosomal translocations in Burkitt’s lymphoma involve the locus containing the c-myc oncogene on chromosome 8 and the genes for either immunoglobulin heavy, K light, or X light chains, on chromosomes 14, 2, and 22, respectively (Klein, 1983; Croce and Nowell, 1985). Ill. T Cell Antigen Receptor Structure, Function, and Gene Organization

A. THE ROLEOF T CELLSIN IMMUNE RESPONSES T cells mature in the thymic environment and their precursors derive from hemopoietic stem cells. There are several subtypes of T cells, including those that mediate regulatory function, such as help, as well as those involved in effector functions, such as the lysis of cells bearing antigens on their surface, and the production of lymphokines. The existence of a distinct subclass of T cells involved in suppression alone is not clear. Collectively, T cells are responsible for the cellular immune responses. To carry out their functions in immune response, T cells must be able to recognize a wide array of foreign antigens. Unlike immunoglobulins, which are able to recognize free (soluble) antigens, T cells recognize foreign antigens only in the context of their own cells’ major histocompatibility complex (MHC) gene productsthe HLA system in humans, the H2 system in mice-in what is termed MHC restriction (Zinkernagel and Doherty, 1975). CD4 (T4) helper and a minority of CD4 (T4) cytotoxic cells corecognize an antigen and structures of class I1 MHC products, whereas CD8 (T8) cytotoxic T cells corecognize an antigen in conjunction with class I MHC molecules. B. THE STRUCTURE OF THE T CELLANTIGENRECEPTORS AND THE CLONING OF THE GENESENCODING THEIRPOLYPEPTIDES

Before the T cell receptor genes were cloned, the TcR was first identified as a distinct structure present only on T cells (Haskins et al., 1983; Kappler et al., 1983; Acuto and Reinherz, 1985). Anticlonotypic antibodies were raised against lines of cloned T cells and reacted only with the clones of cells used to elicit antibody production. The antibodies derived from functional cell lines were capable of inhibiting the functional activity of the respective originator clone. Under nonreducing conditions, these antibodies were used to

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immunoprecipitate structures from human T cells of approximately 90 kDa, separated under reducing conditions into two components of approximately 50 and 40 kDa. The larger and more acidic glycoprotein was called a chain, while the smaller and more basic polypeptide was termed p chain. Therefore, the intact TcR is a disulfidelinked heterodimer. This heterodimer is part of the macromolecular complex that includes the invariant peptides of the CD3 (T3) complex. Upon recognition of an antigen by the TcR, the CD3 (T3) complex is apparently responsible for transducing the signal to the interior of the cell, thus initiating the cellular response. This causes an increase in cytosolic free calcium and the activation of the protein kinase system (Weiss and Stobo, 1984). These events lead to a series of steps in which previously silent genes become expressed, such as the ones encoding the interleukin 2 (IL-2) molecule and IL-2 receptors. The production of IL-2 and the expression of the IL-2 receptors are critical in terms of determining the magnitude and duration of immune response (Smith, 1984). Transfection experiments on mutant T cell lines have shown that a complete T3-TcR complex must be on the cell surface for responses to antigens or anticlonotypic antibodies to occur, and that the components of the T3 complex and the TcR heterodimeric polypeptides comodulate each other (Ohashi et al., 1985). Employing molecular techniques, two groups independently isolated cDNA clones from human (Yanagi et al., 1984) and murine (Hedrick et al., 1984a) sources that coded for the p chain of the T cell receptor. They first used subtractive hybridization and differentiatial screening to isolate a number of cDNA clones that appeared only in T cells. Some of these cDNA clones were shown to undergo somatic rearrangements in clones of functional T cells and leukemic cells of thymic origin (Hedrick et al., 1984a; Toyanaga et al., 1984). Upon comparison of the partial protein sequence of the TcR p chain with the deduced amino acid sequence of the’se cDNAs, it became clear that the cDNAs contained the genes coding for the /3 chain. Furthermore, the primary structure of the proteins deduced from the cDNAs sequence revealed significant homology with the products of the Ig and MHC genes (Yanagi et al., 1984; Hedrick et al., 198413; Hannum et aZ., 1984). The isolation of the cDNAs encoding the a chain followed the application of similar techniques and the use of oligonucleotide probes deduced from the partial protein sequence of the TcR a chain (see Kronenberg et ul., 1986; Toyonaga and Mak, 1987). The deduced primary structure of the cr and p chain polypeptides of the TcR showed them to be composed of a variable and a constant

GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS

51

domain connected by a diversity segment (at least for the p chain) and a joining segment. Each chain can be construed as having seven regions: a hydrophobic leader region of 18-29 amino acids, a variable segment of 88-98 amino acids, a joining segment of 14-21 amino acids, a constant region of 87-113 amino acids, a connecting peptide, a transmembrane region of 22-24 amino acids, and a cytoplasmic tail of 5-12 amino acids (Kronenberg et al., 1986). There is a similarity in the relative positions of the cystine residues responsible for the intrachain disulfide bonds in a TcR chain and murine and human Ig proteins. The overall structure of each chain is very similar to that of a light chain Ig molecule, except that they include an extended transmembrane and a cytoplasmic portion. There is also a 15-50% amino acid sequence homology between the Ig and TcR chains. The concept of an Ig gene superfamily was created, defined as a group of multigene families in single gene copies related by sequence, suggesting that they have evolved from a common primordial precursor, but are not necessarily related in function (Hood et al., 1985). Other members of the superfamily include Thy-1, CD8 (TS), CD4 (T4), MHC class I, MHC class 11, and the poly(1g) receptor for polymeric IgA and IgM molecules. A third T-cell-specific gene has been cloned and designated y chain gene (Saito et al., 1984). These genes undergo somatic rearrangement during early stages of T cell development and had no known function until recently. The fact that most reports of y chain genes cDNA sequences indicate that they were derived from nonfunctional messages added to the puzzle. However, utilizing antibodies directed against the constant region of the human y chain, y chain protein could be shown by immunoprecipitation to be present on CD4negative, CD8-negative thymocytes from patients with primary immunodeficiency states (Brenner et al., 1986)and on approximately 3% of peripheral blood CD4-negative, CD8-positive T cells from normal individuals (Bank et al., 1986). These subtypes of thymocytes and peripheral T cells have TcR y chain associated with the T3 peptide but lack mature TcR /3 or a chain messages. Soon thereafter, there was a report of functional y cDNAs from athymic nude mice (Yoshikai et al., 1986), and this observation raised a possible role for the y chain as a component of a hypothetical second T cell receptor, an alternative receptor functioning in some circumstances. Furthermore, to add more evidence in favor of a second T cell receptor, a polypeptide of about 55-62 kDa could be coprecipitated with y chains, and it was tentatively called 6 chain (Brenner et al., 1986; Bank et al., 1986; Weiss et al., 1986). Very recently, there were reports of a

52

M.D. REIS ET AL.

“constantlike” gene located 3‘ to the V, genes and 5’ to the J a genes, whose deduced amino acid sequence is consistent with a 6 polypeptide chain (Chien e t al., 1987; Takihara e t al., 1988). The y-6 heterodimer also forms complexes, with the CD3 (T3) molecules. C. GENOMIC ORGANIZATION OF T CELLRECEPTOR GENES

Multiple bands were observed when full length a or p chain cDNAs were used to probe germ line genomic DNA from fibroblasts (Yanagi et al., 1984; Toyonaga et al., 1984; Minderi et al., 1985). DNA from cloned T cells or leukemic T cell lines also revealed several bands, although the band patterns were different from those representing germ line configuration and from each other. Comparison of the DNA from fibroblasts and from leukemic cells from the same patients showed that the differences in the band patterns were not due to an inherited restriction fragment-linked polymorphism. Taken together, these findings indicated that distinct somatic rearrangement of TcR genes had occurred in different T cell clones. 1. Organization o f t h e T Cell Receptor p Chain Genes The TcR p genes are located on chromosome 7 in man (7q34) and chromosome 6 in mouse (Caccia e t al., 1984; Morton et al., 1985). In their germ line configuration, these genes are composed of discontinuous genetic segments which bear resemblance to variable, diversity, joining, and constant immunoglobulin gene elements, and as such are referred to in the same manner (Fig. 1).Approximately 100 Vp gene segments are estimated to exist in man, in over a dozen Vp families (Toyonaga and Mak, 1987). The total number of Vp gene segments is considerably smaller than the number of K and heavy chain immunoglobulin V genes. A leader sequence is at the 5‘ end of each V Val

Van

v.2

Alpha

I

Beta

I I I . 1 - 1

-

VYl

Vpz

vr2

“pa

Vpn

Dgi

::I I

v711

Jpi.1-6 :I-

JyPl J 7 P J 7 l C y l

C,

Jai-y

-

--Vpi

Gamma

Van DgJa Ca

I

; Cpi

-

Dp2 Jpz.1.7 ;

JyP2 J y 2

L

Cgz

Cy2

FIG.1. Schematic representation of the germ line genomic organization of the TcR a, 7 , and 6 genes. Note the location of the TcR S genes within the TcR a locus. Reproduced with permission from M. D. Reis, H. Griesser, and T. W. Mak. In “Recent Advances in Haematology 5” (V. Hoffbrand, ed.). Churchill-Livingston, Edinburgh, 1988.

P,

GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDER!

53

segment, separated from the rest of the coding sequences by an intron. Immediately 3’ of the coding sequences are highly conserved heptamer and nonamer sequences, separated by a nonconserved spacer (Siu et al., 1984). A region of about 20 kb contains the diversity and joining segments and constant region genes. There are two highly conserved C region genes approximately 10 kb apart, the 5’ C gene designated Cpl and the 3’ gene designated Cpz (Toyonaga et al., 1985). They are also similar in their general organization, with their first two exons coding for most of the extracellular Ig-like globular domain and a shorter domain containing an extra cystine, presumably important in heterodimer formation; the third exon contains sequences corresponding to part of the transmembrane region, and the last exon contains the sequence coding for a short cytoplasmic domain, as well as a 3‘ nontranslated region of the p chain message. The introns and the 3’noncoding region exhibit little similarity to each other, whereas the coding sequences of the two C regions are highly homologous, having only a four-amino acid difference between them. The lack of homology between the 3’ nontranslated sequences has been used to distinguish the transcripts from the two C regions. About 4 kb 5’of each C region is a cluster ofjoining segments. In the human, the Jpl cluster and the Jpz cluster (each with six functional segments) are upstream of C,1 and Cpz, respectively. There are more functional Jp segments than there are JH or J K segments in humans. The highly conserved heptamer and nonamer structures, separated by a spacer, are located adjacent to the 5’ end of each J segment. A single D segment is found 650 base pairs (bp) 5’ of each J cluster. Each D segment is flanked on its 5’ side by a heptamer and a nonamer with a 12-base spacer and on its 3‘ side by the heptamer and nonamer with a 23-base spacer. The presence of the heptamers and the nonamers with their varying spacer sequences satisfies the 12/23 bp rule, whereby recombination occurs only between a pair of Ig or TcR gene segments, one with a 12-nucleotide spacer and the other with a 23-nucleotide spacer, each 12 nucleotides representing a full turn in the DNA helical structure (Fig. 2). Since each DO is flanked on its 5’ by 12-base spacer and on its 3’ side by 22-base spacer, both VJ and D D joinings are theoretically possible, perhaps expanding the diversity of the Jp gene products (Chien et aZ., 1984; Clark et al., 1984; Kavaler et al., 1984). Also, a DO gene segment can be linked to a V, gene in all three translational reading frames, which does not occur with Ig heavy D segments. Somatic hypermutation, resulting in further V region diversification, is seen in Ig genes but has not yet been shown for TcR V region genes. However, the possible linking of the Dp gene to a V,

54

M.D. REIS ET AL.

I-(-

Dy?

-1

FIG.2. Schematic representation of the heptamer, nonamer, and spacer sequences flanking the gene segments of the Ig and TcR genes. Modified from M . D. Reis, H. Griesser, and T. W. Mak. In “Recent Advances in Haematology 5” (V. Hoffbrand, ed.). Churchill-Livingston, Edinburgh, 1988.

gene in all three translational reading frames is a form of somatic diversification mechanism occurring only in T lymphocytes. The other strategies employed to generate diversity are commonly used in the production of Ig and TcR molecules. They include multiple germ line segments, random combinatorial joining of the segments, the combinatorial association of the polypeptide subunits, and junctional diversity either from variability at the site at which gene segments may be joined or from random addition of nucleotides thought to be mediated by the enzyme TdT (N insertion). In view of the similarity of the overall organization of the two C region genes and the associated D and J gene segments, it has been proposed that these tandem structures arose through gene duplication (Toyonaga et ul., 1985; Kronenberg et al., 1986). A comparison of the human and murine TcR p genes shows remarkable conservaton of the coding region when one considers that the two species diverged some 70 million years ago.

2. Organization of the T Cell Receptor CY Chain Genes These genes are on human chromosome 14, in the 14qll-12 region, with the variable region being proximal and the constant region distal,

GENE REARRANGEMENTS I N LYMPHOPHOLIFERATIVE DISORDERS

55

in relation to the centromere (Caccia et al., 1985; Croce et al., 198513). The genomic organization of the CY chain V gene segments is very similar to that of the p chain and Ig genes. It is estimated that there are approximately 16 V, families in the human, containing a total of 70-80 different V region segments. By using pulse field gel electrophoresis, it has been possible to obtain a physical macrorestriction map of the entire human CY locus, which spans close to 1,000,000 bp and includes the V,, J,, and C, genes. The V, segments are spread out over 750,000 bp (Griesser et al., 1988b). There is only one a constant region in human and mouse, divided into four exons, with the first two exons encoding the extracellular domain and the third encoding the transmembrane and cytoplasmic portion of the protein. The 3' untranslated sequence occupies its own exon.The J gene segments are most unusual, in that they occupy a region of approximately 80 kb 5' of the C, region, and there appears to be more than 50 individual segments in number. An a diversity segment has not been identified yet. The TcR a genes have also been shown to undergo somatic rearrangement in T cell neoplasms and T cell lines as well as in some precursor B cell ALLs (Sangster et al., 1986; Hara et al., 1988).

3. Organization of the T Cell Receptor y Chain Genes The human TcR y chain genes are located on the short arm of chromosome 7, in the 7p15 region (Murre et al., 1985). In the human, the total number of y variable gene segments has been determined to be 11 (Kimura e t al., 1987; Forster et al., 1987). Five joining gene segments and two constant region genes have been found, arranged as Jal-Col and J,z-C,z (Murre et al., 1985).Each y gene is flanked by a heptamer separated from a nonamer by a spacer. The deduced amino acid sequence of the y chain has a low but distinct homology to immunoglobulin V, J, and C regions; portions similar to the transmembrane and intracytoplasmic region of integral plasma membrane proteins; and a cystine residue at the position expected for a disulfide bond Iinking two subunits of a dimeric membrane protein molecule (Hayday et al., 1985). 4 . Organization of the 6 Chain T Cell Receptor Genes

The rather recent description of a constant region gene situated within the murine TcR a locus and approximately 85 kb 5' of the CY constant region (Chien et al., 1987) was followed by the description of its human counterpart (Takihara et al., 1988);the two regions are 80% homologous to each other. The human Cs does not cross-hybridize to C,. The Cs exons span a region of 5 kb. Three J segments have been found upstream of Cg, with flanking heptamer and nonamer

56

M.D. REIS

ET AL.

recombination sequences. A few Vs segments have been sequenced, and two Ds genes also have been found (Hata et al., 1987; Takihara et al., 1988). D. SOMATIC REARRANGEMENT OF GENEELEMENTS AND SEQUENCE OF T CELLRECEPTOR GENEREARRANGEMENTS As w e have seen, the recombination of segments flanking the V, D, and J segments of the TcR genes are very similar for immunoglobulin and T cell receptor genes. In fact, it has been recently shown that the same recombinase may be operative in the joining of these gene segments (Yancopoulos et al., 1986), and this may explain why in some T cell lines the Ig heavy chain genes are rearranged and in some B cell lines the TcR genes are rearranged. The first event in TcR p chain rearrangement is a DJ joining, similar to what occurs with Ig genes. This rearrangement can sometimes result in truncated transcripts being expressed, of approximately 1.0 kb for the p chain (containing only D, J, C and sequences 5' of the D segment) and 1.3kb for the a chain (consisting of J, C, and 5' J germ line sequences). These incomplete or nonproductive transcripts can be occasionally found in B cells. A final recombination event brings a V region gene adjacent to the D or J segments, resulting in the expression of a complete 1.3- or 1.6-kb message derived from the p and a chain genes, respectively. The y and 6 genes also undergo somatic rearrangement. The complete y chain message is 1.7 kb long. With respect to the 6 genes, analysis of several T cells lines have shown no transcripts or the presence of transcripts of 2.2, 1.8, 1.5, and 1.2 kb, with the 1.8- and 1.2-kb messages being expressed mainly in thymocytes (Takihara et al., 1988). The mechanisms that can produce these four transcripts are not known at this time, and possible explanations include alternate splicing, production of transcripts from rearranged and nonrearranged genes, or messages with alternate polyadenylation sites. In a fashion similar to what occurs with Ig genes, there appears to be a hierarchy in the rearrangement and expression of the TcR genes. Analysis of transcripts of murine fetal thymic cells has shown that the TcR y and /3 genes are the first to be arranged (Royer et al., 1984; Raulet et al., 1985; Samelson et al., 1985). According to these studies, TcR a genes were the last to be rearranged and expressed, leading then to the production and cell surface expression of the TcR a i p heterodimer. Studying human lymphoid leukemias, Davey et al. (1986) have shown that expression of human TcR genes parallels that

GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS

57

seen in murine cells, and supports the view of a hierarchy of TcR gene rearrangements in T cell ontogeny. TcR y genes are rearranged first, followed by TcR p and then by TcR a activation. At this time, the order of appearance of rearrangement of the TcR 6 gene has not been unequivocally demonstrated. In one study looking at rearrangements of the TcR 6 gene in lymphoid processes, the 6 gene has been shown to be rearranged only in T cell malignancies with TcR y and p gene rearrangements, and no change of the TcR 6 gene was seen in tumors clearly containing TcR y and p rearrangements (Tkachuk et al., 1988). Thus, it is tempting to speculate that the 6 chain genes may rearrange after the a and p chain genes. The authors caution that more samples of T cell malignancies and nonmalignant T cells should be studied before any definitive conclusion is made. Furthermore, the TcR 6 gene has also been found to be rearranged in a large number of non-B, non-T acute lymphoblastic leukemia ( J . Hara, personal communication), and even occasionally in acute myelogenous leukemia (M. Minden, unpublished data).

IV. Clinical Applications of the Analysis of Immunoglobulin and T Cell Receptor Gene Rearrangements in Hematological Neoplasias Rearrangement of the immunoglobulin or of the T cell receptor genes is a mandatory step within lymphoid cells for commitment to the B or T lymphoid lineage, respectively. This DNA rearrangement is expected to result in the assembly of functional genes and is associated with changes in their structure that will alter the original germ line configuration. If appropriate restriction endonuclease enzymes and DNA probes are used to analyze a clonal cell population, these changes can be detected on a Southern blot. The analysis of Ig and TcR genes has had an impact in determining the monoclonal, oligoclonal, or polyclonal status of abnormal lymphocytic proliferations; in defining the origin of lymphoid neoplasias of unclear lineage with the methods then available; in the diagnosis, definition of disease extension and monitoring of therapy in lymphoid malignancies; in defining the genetic events taking place at different stages of B or T cell ontogeny and the defects resulting in failure of maturation of B and T cell precursors; and in searching for the possible role in malignant transformation of specific translocations seen in lymphoproliferative disorders, involving Ig or TcR genes and transforming oncogenes. For Southern blot analysis, high-molecular-weight DNA is extracted from the cell population targeted for study, such as

58

M.D. REIS ET AL.

mononuclear cells circulating in the peripheral blood, lymph node, or bone marrow samples, or a tumoral growth in the skin. Nonlymphoid DNA should also be extracted as a source of DNA in the germ line configuration for both Ig and TcR genes. The DNA is digested with restriction endonuclease(s) carefully chosen based on information available on the restriction maps and on the DNA probe(s) one intends to use. The rationale for this is to use restriction endonuclease/probe combinations that allow one to distinguish not only the rearrangements from the germ line Ig or TcR gene patterns, but also how many alleles underwent rearrangements, where in the gene the rearrangement occurred, and the gene segments deleted or involved in this recombination process. The DNA samples are then sizefractionated by agarose gel electrophoresis, transferred onto a filter, and hybridized to 32P-labeled DNA probe(s) for Ig or TcR genes, followed by proper washings and autoradiography. A polyclonal B or T cell population is expected to contain many different rearrangements of the Ig or TcR genes, respectively, each yielding restriction fragments of different sizes. Collectively, none of these rearrangements, in particular, can be seen as a new band on Southern analysis, as a result of a lack of sensitivity of the methods currently used to detect them. However, if a monoclonal expansion is present, representing a single cell’s progeny, it will have a unique identifying DNA rearrangement pattern, specific for that population, identifiable as a new single-sized fragment(s) containing DNA sequences that hybridize to the probe used. The multiple copies of the unique fragment(s) in the DNA from a clonal process allow the rearrangement to be detected and distinguished from the bands representing the unrearranged (germ line) form of the gene. In terms of sensitivity, the detection of clonally rearranged Ig or TcR genes by Southern blot analysis can be seen when the clonal population comprises as little as 1% of the total cell population, using current hybridization techniques (Arnold et al., 1983; Minden and Mak, 1986). The question of whether one is dealing with a clonal versus polyclonal lymphoid expansion has obvious clinical importance. Several markers have been used, each with its own intrinsic limitations. Chromosomal markers can certainly indicate clonal origin, but are not present in every single lymphoproliferative disorder. Glucose 6phosphate dehydrogenase (G,6PD) analysis may also reveal clonality of a lymphoid proliferation, but is of value only in female patients heterozygous for this enzyme. Another approach is to use antibodies directed against cytoplasmic or surface markers; the antibodies bind to antigenic determinants on the molecules of differentiation antigens

GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS

59

present in different subsets of lymphoid cells or to Ig heavy or light chains in the cytoplasm or on the surface of B cells. A problem with the latter approach is that no marker exists for T lymphocytes that is universally accepted as an indicator of monoclonality. The advent of the analysis of Ig or TcR genes in determining clonality and lineage has greatly aided in the diagnostic approach to lymphoid expansions where phenotyping is uncertain or difficult. Defining clonality for cells of the B or T lymphoid series may be of value in the differential diagnosis of the malignancies from atypical lymphocytic proliferations. A diagnosis of lymphoma is usually unambiguous on histologic examinations by experienced pathologists, but in a significant minority of cases this is rather difficult since antigenically stimulated polyclonal lymphocytes may resemble neoplastic lymphocytes. Two notes of caution are advisable. First, it is important to bear in mind that monoclonality does not necessarily equate to malignant phenotype for the cell population being studied, since benign monoclonal proliferations are known to exist for either B or T lymphocytes (Davey and Waldmann, 1986; Reis and Mak, 1987). Second, the presence of inherited polymorphism in Ig or TcR gene DNA may be mistaken as rearrangements in lymphoid proliferations, creating possible difficulties in interpreting the analysis of rearrangements of those genes. In practical terms, such polymorphisms appear to be rare for Ig heavy and K light chain genes, and for TcR genes. An internal control for such inherited polymorphism is the inclusion in the samples to be analyzed of nonlymphoid DNA, for example, DNA obtained from granulocytic cells in the buffy coat of a peripheral blood sample or from skin fibroblasts from the same patient. Once a particular Ig or TcR gene rearrangement pattern has been established for a patient’s lymphoid malignancy, it can serve as a highly sensitive tumor-specific marker, improving the ability to identify tumor cells that persist after therapy and helping in the early detection of a recurrence (Waldmann et al., 1985a; Minden and Mak, 1986). For B lymphoid neoplasms, the question of in vivo clonal progression, biclonality, and the emergence of a malignant clone from polyclonal populations of virally stimulated cells has been reviewed in detail (Waldmann, 198713). A. CHRONICT CELLMALIGNANCIES The mature T cell leukemias and lymphomas constitute a group of heterogeneous disorders and include T cell chronic lymphocytic leukemia (T-CLL), T cell prolymphocytic leukemia (T-PLL), adult T cell leukemia (ATL), and cutaneous T cell lymphomas (mycosis

60

M.D. REIS ET AL.

TABLE I REARRANGEMENT OF TcR p CHAIN GENESIN LEUKEMIA OR LYMPHOMA ~

~~

No. of cases

Rearranged TcR p

Rearranged Ig H C

127 50 56 20 56 33 17 6

126 50 49 20 55 33 13 5

18 (13 not done) 1 (6 not done) 0 (11 not done) 0 (1 not done) 1 (39 not done) 0 (8 not done) 0 (4 not done) Not done

77 59 45 2

19 6 5 0

76 59 45 2

6 37 12 68

4 9 9

(1)T cell malignancies

T-ALL ATL T cell lymphoma T cell prolymphocytic leukemia SCzary syndrome/mycosis fungoides T-CLL T8 lymphocytosis Lymphomatoid papulosis (2) B cell and pre-B cell malignancies ALL Lymphoma B-CLL Myeloma (3) Other hematopoietic malignancies Lennert’s lymphoma Hodgkin’s disease AIL AML ~~

6

0 2 (1 not done) 4 (also TcR p) 6 (also TcR p in 2 cases)

~

Abbreviations: T-ALL, T cell precursor acute lymphoblastic leukemia; ATL, adult T cell leukemia; T-CLL, T cell chronic lymphocytic leukemia; ALL, common non-B, non-T cell ALL, cALLa+;B-CLL, B cell chronic lymphocytic leukemia; AIL, angioimmunoblastic lymphadenopathy; AML, acute myeoblastic leukemia. See text for references. Reproduced from Reis et al. (1988).

fungoides and Sezary syndrome). Virtually all of the cases reported have shown rearrangement of the TcR /3 genes (see Table I). T-CLL represents approximately only 2% of all cases of chronic lymphocytic leukemia. In most cases of T-CLL, the immunophenotypes are identical to those of the cutaneous T cell lymphomas, that is, CD3-positive, CD4-positive, and CD8-negative. TcR /3 gene rearrangements were seen in 33 of 33 cases studied. T-PLL is also uncommon; it represents approximately 2% of the prolymphocytic leukemias and is a rather aggressive variant of CLL, with marked lymphocytosis, massive splenomegaly, moderate splenomegaly, and minimal peripheral lymphadenopathy. T-PLL cells are also usually CD3-positive, CD4-positive, and CD8-negative. TcR /3 gene rearrangements have been shown in 20 out of 20 cases reported. Mycosis fungoides (MF) and Skzary syndrome are cutaneous lymphomas of mature T cells with cerebriform nuclei. Patients with

GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS

61

S6zary syndrome have exfoliative erythroderma, generalized lymphadenopathy, and circulating malignant T cells with a CD2-, CD3-, CD4-, and CD5-positive phenotype. TcR /3 genes were found to be rearranged in 55 of 56 cases of S6zary syndrome and mycosis fungoides (one case was also shown to have Ig heavy chain gene rearrangement). In cases of mycosis fungoides, the draining lymph nodes were demonstrated to harbor tumor cells. It may be difficult to histologically differentiate inflammatory from malignant T cell infiltrates in lymph nodes accompanying chronic skin disease. Weiss and co-workers (1985) demonstrated clonal rearrangement of the TcR p receptor genes not only in lymph nodes where M F cells were unambiguous, but also in lymph nodes of patients with M F considered to contain only benign reactive cells as determined by histological analysis. In a recent report, TcR /3 and y genes were rearranged in three cases of cutaneous T cell lymphomas; in four cases of T-CLL, the p gene was rearranged in all cases, whereas the y gene was rearranged in three samples (Aisenberg et al., 1987). ATL was initially described in Japan and is a rapidly fatal T cell lymphoproliferative disorder, whose malignant cells also have a tendency to infiltrate the skin. This entity runs a rather aggressive course in most instances, and is associated with hypercalcemia and pulmonary infiltrates. Cases have also been found clustered within families and in certain geographic areas, such as southwest Japan, the Caribbean basin, the southeastern United States, and certain parts of Africa. It has been shown that a retrovirus, called HTLV-I, is the etiological agent of ATL, and the virus has been found integrated into the genome of ATL cells. ATL cells have a phenotype identical to that of S6zary cells. TcR /3 genes were found to be rearranged in 20 of 20 cases of ATL. T h e references for the studies of TcR /3 genes in mature T cell malignancies are Aisenberg et al. (1985), Baer et al. (1985), Bertness et al. (1985), Flug et al. (1985), Isaackson et al. (1985), Minden et al. (1985), O’Connor et al. (1985), Pelicci et al. (1985), Rabbitts et al. (1985b), Waldmann et al. (1985b) and Weiss et al. (1985). In more than 25 cases of mature T cell neoplasia analyzed, the TcR y genes have been found to rearrange in the vast majority of cases (LeFranc and Rabbitts, 1985; Davey et al., 1986; Quertermous et al., 1986; Aisenberg et al., 1987).

B. T CELLLYMPHOMAS The TcR /3 genes were rearranged in most of the non-Hodgkin’s T cell lymphomas analyzed (49 out of 56). In one study, two cases of T cell lymphoma showed rearrangement of TcR y gene but the TcR /3

62

M.D. REIS

ET AL.

gene was in germ line configuration. Of four samples of T cell lymphoblastic lymphoma examined, two showed rearrangements of the TcR p genes, but in the other two the /3 genes were in the germ line configuration. None of these cases had rearranged Ig heavy or K light chain genes (Bertness et al., 1985; Flug et al., 1985; Isaackson et al., 1985; O’Connor et al., 1985; Pelicci et al., 1985; Griesser et al., 1986a; Williams et al., 1987; Matsuoka et al., 1987). Tkachuk et al. (1988), using two JS genomic probes, analyzed 14 samples of T cell lymphoma and found TcR 6 gene rearrangement or deletion in 6 cases. In all cases with TcR 6 rearrangement, the TcR /3 and y had also rearranged. C. T CELLACUTE LYMPHOBLASTIC LEUKEMIA (T-ALL) Of the approximately 130 cases reported, one or both alleles of the TcR p chain genes have been found to rearrange in a11 except one of the cases analyzed (Aisenberg et al., 1985,1987; Bertness et al., 1985; Davey et al., 1986; Flug et al., 1985; Hara et al., 1987; Kitchingman et al., 1985; Minden and Mak, 1986; Minden et al., 1985; O’Connor et al., 1985; Pelicci et al., 1985; Rabbitts et al., 198513; Tawa et al., 1985, 1987; Waldmann et al., 1985a; Williams et al., 1987). When performed, cell surface phenotypic analysis of T-ALL showed a heterogeneous pattern, with no single marker present in all cases. Some cells were found with rearranged TcR p genes but no expression of the CD3 (T3) cell surface antigen. Rearrangements of the TcR y chain genes have been detected in 38 of 38 cases examined (Davey et al., 1986; Aisenberg et al., 1987; Yara et al., 1987; Tawa et al., 1987). Davey and co-workers found rezirrangements of the TcR y genes in all patients whose leukemia expressed the CD2 (T11) antigen. In 3 cases of CD7-positive, CD2-negative T-ALL, considered as stem cell or very early pre-T leukemias, they also showed the TcR /3 genes to be in germ line configuration in all instances, whereas the TcR y genes were rearranged in two cases and unrearranged in one. As for the TcR 6 chain gene, Hara and co-workers (1988) have examined 19 cases of T-ALL. In 5 out of 7 CD3- cases and in 4 out of 12 CD3+ cases, there was biallelic rearrangement of the TcR 6 genes. In 8 other CD3+ cases, 3 had a single allelic TcR 6 rearranged, with TcR a rearrangement on the other allele; the other 5 cases of CD3+ T-ALL showed deletion of the 6 loci, with biallelic rearrangement of the TcR a genes. In the same series, 6 cases of T-ALL were analyzed for transcription of TcR genes, and 5 were found to express the TcR 6 mRNA; the one case that did not have TcR S mRNA did express TcR y and a truncated (1.0 kb) TcR p mRNA, and was presumed to derived

GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS

63

from the most immature T lineage cells. These authors suggest that TcR a gene rearrangement may take place after rearrangements of the TcR 6 genes with concurrent deletion of the rearranged 6 genes in T cell differentiation. Immunoglobulin probes were used in 114 of the cases mentioned above and were studied for the structure of the TcR p gene; 18 showed rearrangements of the Ig heavy but not of Ig light chain genes. T h e incidence of Ig heavy chain gene rearrangements in T-ALL is substantially higher (17 of 107) than that observed for mature T cell malignancies.

D. Ty LYMPHOPROLIFERATIVEDISORDER Clonal rearrangement of the TcR p genes has been observed in peripheral blood of 13 of 17 patients with CD8 (T8) y lymphocytosis or cytotoxic-suppressor lymphocytosis and neutropenia (Aisenberg et al., 1985; Bertness et al., 1985; Rambaldi et al., 1987; Waldmann et al., 1985a). Ty lymphoproliferative disorder appears to be a heterogeneous process, with lymphocytosis of large granular lymphocytes bearing receptors for IgG. The clinical course is benign, with increased incidence of infection, splenomegaly, and long-standing arthropathy in one-third of the patients, serologically identical to rheumatoid arthritis (Aisenberg et al., 1981; Reynolds and Foon, 1984). The majority of expanded lymphocytes in this entity express the CD2 (T1l)-positive, CD8 (T8)-positive, HNK1-positive phenotype and are usually C D 3 (T3)-positive also. This disorder has been viewed as a reactive rather than a neoplastic process by some, or a form of chronic leukemia by others. In view of the demonstration of clonality by analysis of the TcR p gene structure, one group has equated T8 y lymphocytosis to a benign monoclonal gammopathy (Aisenberg et al., 1985). Further analysis has shown that patients with a proliferation of large granular lymphocytes expressing CD2 and HNKl antigens, but not the CD3 antigen, present a different pattern of rearrangement and expression of the TcR genes, with retention of germ line configuration by the TcR p and y genes, lack of TcR y and a mRNA in cultured cells from such patients, but production of a truncated 1.0-kb TcR p gene message. The existence of two forms of Ty lymphocytosis parallels the observations that CD2-positive, CD3positive NK cell clones expressed full-length TcR a and p mRNA, whereas CD2-positive, CD3-negative NK clones expressed only a 1.0-kb truncated TcR p but no TcR a transcripts. A predominant T cell clonal population, shown by analysis of TcR p gene structure, has been seen in one patient with CD4 (T4) lymphocytosis (M. Minden, unpublished observation).

64

M.D. REIS E T AL.

E. LYMPHOMATOID PAPULOSIS Lymphomatoid papulomatosis is a chronic disease with a relatively benign course, characterized by repetitive episodes of self-healing papular lesions, which eventually ulcerate and then resolve as scars. However, the histological appearance of the lesions is consistent with that of a malignant lymphoma, in that they contain mononuclear cells along with a prominent population of large atypical cells that either have cerebriform nuclei or resemble Reed-Sternberg cells. Weiss and co-workers (1986) studied tumor tissue from six patients with this entity and found evidence of clonality in five of them using a TcR p gene probe, a result confirmed in four samples probed with TcR y DNA. The patient whose specimen did not show clonality with either the TcR p or y probes had a scant subepidermal infiltrate, with a comparatively low number of atypical cells. The finding of three rearranged bands in the sample from a patient was suggestive of the presence of at least two clonal populations, since rearrangements of both TcR p gene alleles in a monoclonal proliferation would result in only two rearranged bands. In another patient, varying patterns of TcR /3 gene rearrangement were seen in three separate specimens, suggesting that different lesions may contain different T cell clones.

F. B CELLAND PRE-B CELLMALIGNANCIES 1. Mature B Cell Malignancies There have been numerous reports on the analysis of rearrangements of immunoglobulin genes in lymphoid neoplasias of immature and mature B cell phenotypes. Rearrangements of at least one Ig heavy chain plus one light chain gene were found in all malignant cell populations from patients with a mature B cell or plasma cell malignancy, such as chronic lymphocytic leukemia, prolymphocytic leukemia, multiple myeloma, Waldenstrom’s macroglobulinemia, B cell follicular and diffuse lymphoma, or Burkitt’s lymphoma. In the majority of cases, the use of a J H probe produced two bands, indicating an effective V-D-J rearrangement on one chromosome and a presumably nonproductive rearrangement of the Ig heavy chain genes on the alternate chromosome. The use of only one light chain gene allele of the selected light chain type was also observed. The excluded allele may remain in the germ line configuration, may be deleted, or may be nonproductively rearranged. Human B cells express the K light chain type in about 60% of the cases; the cells expressing A light chains

GENE REARRANGEMENTS IN LYMPHOPHOLIFERATIVE DISORDERS

65

undergo the obligatory A gene rearrangement. The B cells producing K light chains retain their h genes in the germ line configuration, whereas the K genes in A-producing B cells do not remain in the germ line form but are either rearranged or deleted [for review of Ig gene rearrangements in B cell disorders, see Waldmann (1987a,b)]. In recent years, several studies have addressed the question of whether the TcR genes retain the germ line configuration in mature B cell disorders. Rearrangements of the TcR p genes have been detected in 5 of 45 cases of B-CLL studied (Waldmann et al., 1985a; Pelicci et al., 1985; Aisenberg et al., 1987). In Aisenberg’s study, no rearrangements of TcR y genes were found. In a few cases, TcR p gene rearrangement was observed with,one restriction enzyme but was not confirmed in DNA digested with other enzymes, thus favoring TcR p gene polymorphism instead of the conventional rearrangements seen in T cell populations. TcR y chain gene rearrangements have not been observed in B-CLL. Of 59 examples of B cell non-Hodgkin’s lymphoma studied, 6 have shown TcR /3 gene rearrangement (Pelicci et al., 1985; Griesser et al., 1986 a,b; Williams et al., 1987). All 14 samples of B cell lymphomas and B cell lines studied with TcR 6 genes have shown these genes to be in germ line configuration. In all these cases, at least one Ig H allele was rearranged, with or without concurrent TcR p or y rearrangements (Tkachuk et al., 1988). Also, two samples of Iymphoid hyperimmune reaction had their TcR loci in the germ line configuration. Thus, in about 10% of the chronic B cell neoplasias, the TcR genes are rearranged, an incidence higher than the occurrence of Ig gene rearrangement in neoplasias of mature T cell origin.

2. Precursor B Cell Acute Lynzphoblustic Leukemia (ALL) Virtually all cases of non-B, non-T ALL have demonstrated rearrangements when analyzed with a J H immunoglobulin probe, and are thus considered as precursor B cell ALL, together with pre-BALL. Of 77 samples of precursor B-ALL studied with a TcR p probe, rearrangements were detected in 19 (Aisenberg et al., 1985; Minden et al., 1985; Pelicci et al., 1985; Davey et al., 1986). More recently 77 cases of precursor B-ALL have been analyzed with J H and TcR /3 and y probes. Ig heavy genes were rearranged in 76 of 77 samples, in combination with TcR y rearrangement in 20 cases, and with TcR y and /3 genes rearrangements in an additional 18 cases. Therefore, the TcR y genes do rearrange in approximately 50% of the cases of

66

M.D. REIS ET AL.

precursor B cell ALL, which confirms that TcR y gene rearrangement is not a reliable indicator of cell lineage. In contrast to rearrangements of the two TcR y genes seen in T cell ALL, one TcR y gene remains in the germ line configuration in the majority of cases of precursor B-ALL in which y rearrangements occur (Asou et al., 1987; Tawa et al., 1987; Williams et al., 1987). A recent study has examined 18 cases of T-ALL and 46 cases of precursor B-ALL with J H , TcR p, y , and three TcR J a probes (Hara et al., 1987). TcR a chain gene rearrangements were observed in only 2 of 18 T-ALL cases, indicating that perhaps the majority of T-ALLs would have rearrangements involving J n segments located upstream of the probes used. In contrast, rearrangements of the TcR a genes were demonstrated in 15 out of 46 precursor B-ALLs. Of these cases, 9 also had rearrangements of both TcR p and y genes, whereas the remaining 6 had rearrangement of neither of these genes. The same authors (Hara et al., 1988) examined 29 samples of precursor B cell ALL with all TcR genes, and found TcR 6 gene rearrangement in 20 cases (69%), a frequency higher than those for the TcR CY (59%), y (52%)or p (31%) genes, and made the observation that the TcR 6 gene seems to be the earliest rearranging TcR gene, followed by TcR y and p. Thus, it appears that TcR a and 6 chain gene rearrangements are not specific for cells of T lymphoid lineage and that TcR y and/or p gene rearrangements do not seem essential for TcR a and 6 gene rearrangements, at least in the case of precursor B cell ALL.

G. OTHERHEMATOPOIETIC MALIGNANCIES Other Types of Lymphomas

Other lymphomas include Lennert’s lymphoma, angioimmunoblastic lymphadenopathy, and Hodgkin’s disease, disorders in which the presence and lineage of a clonal population had not been clearly demonstrated previously. a. Lennert’s Lymphoma. The lesion of Lennert’s lymphoma is characterized by a high content of epithelioid cells and some large blast cells dispersed among atypical lymphocytes. Some of the large blast cells have the appearance of Reed-Sternberg cells, and at one time this lymphoma was felt to be a special variant of Hodgkin’s disease. TcR /3 gene rearrangement has been seen in six of six cases examined, with Ig genes remaining in the g e m line configuration (Griesser et al., 198615; O’Connor et al., 1985). The TcR y genes have

GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS

67

also been found to-be rearranged in the cases examined (Griesser et

al., 1986a). As for the TcR 6 genes, they were found completely or partially rearranged in three out of five samples of Lennert’s lymphoma. The band pattern in two cases suggested that only one allele was rearranged. All five cases had the TcR y and p genes rearranged (Tkachuk e t al., 1988). b. K i 1’ Lymphomas. The monoclonal antibody Ki 1+detects a cell surface antigen present on Reed-Sternberg cells in Hodgkin’s disease, on activated normal T lymphocytes, and on HLA-DR+ lymphoma cells in some large cell anaplastic lymphomas (Stein et al., 1985). The latter type of lymphoma is designated as Ki 1+lymphoma. Of 10 cases examined, 4 revealed rearrangements of the TcR p genes alone, 3 showed rearranged TcR p and Ig heavy genes (but not Ig light chain genes), and 3 were in germ line configuration for both TcR p and Ig heavy chain genes (Griesser et al., 198613).The TcR 6 genes were rearranged in 5 out of 11 samples examined, all showing TcR p and y rearrangements and germ line configuration for Ig H genes (Tkachuk et al., 1988). They observed that this heterogeneous rearrangement pattern suggests that these tumors represent poorly differentiated lymphomas that do not all share similar origins. c . Angioimmunoblastic Lymphadenopathy (AIL).The TcR /3 genes were rearranged in 9 of 12 cases analyzed (Bertness et al., 1985; Griesser et al., 1986b). Ig heavy chain genes were rearranged in 4 of those 9 cases and one of these also revealed rearrangement of the Ig K light chain gene. d . Hodgkin’s Disease. Reports of analysis of TcR and Ig genes in Hodgkin’s disease have been inconclusive and at times contradictory. A faint but noticeable rearranged band was detected in 4 of 11 cases analyzed with TcR p gene probes (Bertness et al., 1985; Griesser et al., 1986b). It is interesting that one of the cases studied had approximately 50% Reed-Sternberg (RS) cells, and no rearrangement was seen in that sample. The immunoglobulin genes were in germ line configuration in all of these 11 cases. Later, Griesser et al. (1987) reported the analysis of 22 cases of Hodgkin’s disease (HD).Quantitative immunophenotypic analysis showed a predominance of CD3positive T cells in all subtypes of HD except for the lymphocytic depletion (HDLD) subtype. Of the five cases of HDLD, three had rearrangements of TcR y genes alone, and two of Ig heavy chain gene alone. Only five samples [two of lymphocytic predominance (HDLP), two of mixed cellularity (HDMC), and one of nodular sclerosis (HDNttS)] retained both their TcR and Ig genes in the germ line

68

M.D.

KEIS ET AL.

configuration. The remaining 12 patients exhibited rearrangements of TcR y genes only or both TcR y and /3 genes. They concluded that Hodgkin’s lymphomas contain clonal lymphoid populations and that different rearrangement patterns may be associated with different subclasses of HD. Knowles and co-workers (1986) detected minor clonal populations in 3 of 18 cases of HD. Clonal Ig or TcR rearrangement was not seen in cases of H D containing more than 25% RS cells. Their conclusion was that H D is predominantly composed of polyclonal B and T cell populations, that minor B or T cell populations unrelated to RS cells can be occasionally found in HD, and that RS cells do not represent clonal B or T cell expansion. There seems to be a consensus that the question of whether the clonal populations found represent the neoplastic cells or a clonal proliferation in response to some antigen is still unanswered. Recently, probing of six cases of H D revealed TcR S rearrangement in only one sample; all six samples had TcR p and y rearrangement (Tkachuk et al., 1988).

H. ACUTE MYELOBLASTIC LEUKEMIA (AML) The TcR p chain genes were shown to be rearranged in 3 out of 24 patients with AML (Cheng e t al., 1986). All of these patients expressed myeloid cell surface markers. Two of the cases showing rearrangement were positive for TdT and one of these patients was positive for the T cell surface marker T6 (CD1). In another study, Seremetis and co-workers (1987) found a high frequency of Ig and/or TcR p gene rearrangement (60%) in cases of AML expressing TdT. Of 13 TdT-positive AML, rearrangements were seen for the TcR p genes only in 4 cases, for the IgH genes only in 3 cases, and for both TcR p and IgH genes in 1 case. In contrast, of 25 TdT-negative AML, 1 case showed rearrangement of the TcR p genes only, and another showed rearrangement of the IgH genes (frequency lower than 8%). Their conclusion was that the close association between TdT expression and Ig/TcR p gene rearrangements detectable in TdT-positive AML further suggests a role for TdT in the mechanisms leading to the assembly of diverse Ig or TcR p genes. Of note is that approximately 10% of the cases of AML do express TdT, as evidenced by the use of sensitive techniques for detection of this enzyme, initially considered a lymphoid lineage-associated marker. It appears, from the cases reported thus far, that AML patients whose blasts express TdT have a poor prognosis. Preliminary observations indicate that the TcR 6 genes may be occasionally rearranged in cases of AML (M. Minden, unpublished observations).

69

GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS

V. The Simultaneous Occurrence of the T Cell Receptor and Immunoglobulin Genes in Lymphoproliferative Disorders

The utilization of monoclonal antibodies directed against the socalled lymphoid cell developmental antigens has greatly helped to define the cell lineage and stage of differentiation of the cells of most lymphoproliferative disorders. In some of these disorders, however, immunophenotyping procedures still fail to detect either clonality or the lineage of the abnormal cells in some of these lymphoid expansions. It is difficult to detect clonality in T cell disorders by phenotypic analysis, especially in T-cell-rich processes such as Lennert's TABLE I1 T CELLRECEPTORSAND IMMUNOGLOBULIN GENEREARRANGEMENTS IN LYMPHOPROLIFERATI\7EDISEASES" ~

+ + +' + + - + -

IgH IgL TcR y TcR p TcR 6" TcR a Leukemias T-ALL B-ALL Non-T, non-B ATL AML Lymphomas

T B Lennert Ki l'(CD30) Hodgkin's AIL Hyperreactive Possible lineage ~

~~

-

f C

-

-

-

-

NT

-

-

0 0 0 0 70

0 70

0 30 0 0 0

0 0 10

0 0 0 0 50 0 100

0 60 0 0 0 0

0 0 0

0 40 0 0 0

0

0 0

B

B

0 0

0

0

0 0 10 0 0

+c

+<

+'

-

-

-

+

-

+

c

-

+

-

N

T

-

+ -

-

+

-

+ -

+ -

+'

+ L

-

t

-

+ +

+ +

+ + -/+

0 0 0 0 0

0 0 10 0 0

0 0 20 0 0

0 0 10 0 0

0 0 20 0 0

10 0 30 0

0 0 0 30 0 20 0 Pre-B

0 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 30 0 25 0

_

0 0

-

-

+ -

-/+

5

Pre-T

0 0 0 20 10 0 0

-

-

+ +

+ + +

-

-I+

-I+

45 0 0 0 5

45 0 0 100 15

60 0 40 0 30 25 0 T

40 0 60 20 0 30

0 T

~

The configurations of the T cell receptor (TcR) and immunoglobulin (Ig) genes in cells from a variety of lymphoproliferative diseases are summarized. Germ line (-) and rearranged (+) structures of these genes are denoted. The distributions of the individual types of disorders with a specific pattern of rearrangement are expressed as percentage (approximate) of the total number of cases examined. T-ALL, T cell acute lymphoblastic leukemia; B-ALL, B cell acute lymphoblastic leukemia; non-T, non-B, common acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; ATL, adult T cell leukemia; Ki 1- (CD30), anaplastic large lymphoma positive for the surface antigen Ki 1 (CD30); AIL, angioiminunoblastic lymphodenopathy. NT, not tested. Analyses included both rearrangement and deletion of the S chain genes. Patterns most likely representing only DJ rearrangements. a

70

M.D. REIS ET AL.

and large cell anaplastic (Ki 1+)lymphomas. As we have seen, genotypic analysis with the use of Ig and TcR genes has confirmed the B or T cell origin of most cases of lymphoproliferative disorders, but has also revealed complex gene rearrangement patterns in a few cases, with both Ig and TcR genes being rearranged in the same tissues (Table 11), thus raising questions as to the meaning of immunogenotypic studies for definition of lymphoid lineage in these few cases. The occurrence of TcR and Ig gene rearrangements in AML makes these issues even more complex, forcing one to consider the question of lineage promiscuity (Cheng et al., 1986; Seremetis et al., 1987). Upon analyzing the available literature, one observes that the frequency of bigenotype is higher in B cell than in T cell lymphoid proliferatons, and this applies to neoplasms of immature and of mature lymphoid phenotypes. There are quantitative differences in the patterns of rearrangements, however, with usually only one Ig heavy chain allele rearranging in T cell disorders, with Ig light chain genes remaining in the germ line configuration. As for TcR /3 and y genes, the overwhelming tendency is for only one allele of those genes to rearrange in B cell disorders, as opposed to both alleles, as seen in T cell neoplasms. One question that naturally arose when these dual genotypic patterns started to emerge was whether both B and T cell clonal expansions were not actually present in the tumors analyzed, thus explaining the cross-lineage rearrangements. This is unlikely, however, since the malignant cells in the cases of B cell leukemias with double genotype were in fact immunocytochemically of pure B cell phenotype. Moreover, the equal intensity of the rearranged TcR and Ig gene bands on Southern blot autoradiography suggested that these rearrangements occur in cell populations of equal size. The concept of lineage promiscuity (Greaves et al., 1986),with respect to Ig and TcR gene rearrangements in the same lymphocyte, then comes to the fore. The most likely explanation for the phenomenon of lineage promiscuity at the gene level is the accessibility of the Ig and TcR gene loci to the recombinase enzyme, which facilitates the recombination of discontinuous subunits of these genes (Yancopoulos et al., 1986). The inappropriate rearrangements only involve recombination of a D with a J segment (the first event in recombination), but not a V segment with an assembled DJ recombinant (the second step in a complete recombination process). Complete (VDJ) rearrangement of the Ig heavy chain genes in T cell neoplasms, or of TcR /3 chain genes in B cell disorders, has not been reported so far. Moreover no full-length RNA transcripts corresponding to the gene rearrangements described above have been detected. Therefore, it seems that neither

GENE REARRANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS

71

TcR y and a gene rearrangement nor incomplete TcR /3 and Ig H DJ joining should be accepted as indicative of lineage commitment. Rearrangements of both TcR and Ig genes are substantially more frequent in lymphoproliferative disorders whose lineage is difficult to ascertain on the basis of histological or immunological analysis. Such entities would include Ki 1+lymphomas, AIL/AIL-like lymphomas, and non-T cell ALL (Table 11). Griesser and co-workers (198613) detected a high proportion of tumor cells with rearrangement of both Ig and TcR genes in cases of Ki 1+lymphomas and AIL. The TcR y genes were most often rearranged, with no apparent bias toward rearrangements of either their Ig H chain or their TcR p chain genes in these cells. In these cases, it is possible that the malignant transformation occurred in a lymphoid precursor cell that became frozen at a stage of differentiation prior to commitment to either B or T cell lineage. In some cases of AIL and Ki 1' lymphomas, there were rearrangements of the TcR p, y , and 6 chain genes, while the Ig heavy and light chain genes remained in their germ line configuration, indicating T cell origin. When this occurred, the T cell phenotype was confirmed by immunophenotypic analysis in most cases. The non-T cell acute lymphoblastic leukemias form a most heterogeneous group of lymphoid disorders in terms of their immunogenotype. A large proportion of non-T cell ALL samples have shown rearrangements not only of the Ig H chain genes but also of the TcR y and/or a and p chain genes (Tawa et al., 1985, 1987; Asou et al., 1987; Hara et al., 1987). In some of these precursor B cell ALLs, Ig H chain and any combination of TcR 6, a , and y chain gene rearrangements occur, with or without TcR p chain gene rearrangement, even if the leukemic population expresses only cytoplasmic Ig p chains (Tawa et al., 1987; Hara et al., 1987, 1988; Norton et al., 1988). Several possible considerations have been put forward to explain the matter of dual genotype of some lymphoid tumors, with the simultaneous occurrence of Ig and TcR gene rearrangements being either a normal or an abnormal feature of lymphoid differentiation pathways (Pelicci et al., 1985; Davey et al., 1986; Griesser et al., 1987). Several studies have reported the occurrence of Ig H chain gene rearrangements and expression in mouse thymocytes and T lymphocytes, thus strongly suggesting that dual genotypes may also occur in normal cells (Kemp et al., 1980; Kurosawa et al., 1981). This phenomenon may have some functional significance and may identify a subpopulation of bipotent lymphoid cells, with properties of both B and T cells. This is unlikely, however, since the bigenotypic tumors

72

M.D. REIS ET AL.

examined have maintained a pure B or T cell phenotype. Griesser and co-workers (1988a)have proposed that the complex Ig and TcR gene rearrangement patterns are not necessarily arbitrary, but may reflect a clonal population arising from an undifferentiated hematopoietic cell that underwent Ig and TcR rearrangements prior to lineage commitment. Alternatively, the dual genotype may mirror a stage in lymphoid differentiation in which a committed B or T cell maintains the potential of lineage switch, with the fidelity of the immunophenotyping then reflecting the final lineage commitment of the cell, and the dual genotypic markers remaining as an irreversible footprint of the previous commitment (Pelicci et al., 1985). Another possibility is that the dual genotype may reflect the simultaneous activation in T and B cells of common molecular mechanisms, regulating the rearrangement of the Ig and TcR loci. Alternatively, the phenomenon of Ig and TcR gene rearrangements in immature B leukemias may represent events that are not on the normal B cell maturation pathway, and the high incidence of dual genotype in precursor B cell leukemias may be due to the lack of signals present on more mature cells that terminate gene rearrangements (Davey et al., 1986). Recombinases responsible for Ig and TcR gene rearrangements may remain active until an effective stop signal (mature Ig or TcR molecules) is produced, with suppression of the recombinase(s) or closing of open chromatin sites. If a productive mRNA leading to the expression of immunoglobulin products is not present in a B cell precursor, the recombinase may remain active, with resulting activation of both Ig and TcR genes; if a transforming event occurs at this stage, this could lead to the clonal expression of a population of cells with dual rearrangements. On the other hand, the dual genotyping could be present in cells that are destined to a dead end, such as is the case with numerous thymocytes whose fate is death, perhaps as a result of defective gene rearrangements. If such cells are the target of a transforming event, they could give rise to a bigenotypic clone of neoplastic cells and escape cell death. Another possibility is that the dual genotype results from a transformation-related event that reflects the derangement of the genetic machinery or program regulating differentiation in the neoplastic cells. VI. Chromosomal Translocations Involving the T Cell Receptor Genes

A great deal of information has been obtained in recent years, at the molecular level, regarding the involvement of certain genes in recur-

GENE REARKANGEMENTS IN LYMPHOPROLIFERATIVE DISORDERS

73

rent chromosomal translocations in a number of tumors. Examples of these are the t(9;22) in chronic myeloid leukemia (Gale and Cannani, 1985), and the translocations seen in Burkitt’s lymphoma, involving chromosome 8 (where the c-myc oncogene locus is situated) and either chromosome 2, 14, or 22) containing the loci for the Ig light K , heavy, and light chain genes, respectively) (Klein, 1983; Croce and Nowell, 1985).There have been reports of recurrent translocations in T cell disorders, involving the 7q33 and 7p13 regions of the TcR p chain genes (Morton et al., 1985; Hecht et al., 1985; Isobe et al., 1985) and TcR y chain genes (Rabbitts et al., 1985a), respectively. As depicted in Table 111, however, the locus containing the TcR a chain genes (14qll-13) is more often involved in translocations in T cell disorders. Three examples of reciprocal translocations involving the TcR a locus are t(8;14)(q24;q12) (Shima et al., 1986; Erikson e t a]., 1986), t(11;14)(p13;q12)(Williams et al., 1984; Lewis et al., 1985), and t(10;14)(q24;qll))Dub6 et al., 1986; Hecht et al., 1985). An inversion of chromosome 14 has also been described (Williams et al., 1984; Hecht et al., 1984; Zech et al., 1984; Croce et al., 1985; Baer et al., 1985; Denny et al., 1986b). A full discussion of these translocations is beyond the scope of this review. However, given the potential significance in terms of being important in the genesis of malignant transforming events, we will briefly discuss some of these translocations. In the studies of t(8;14)(q24;q12), the breakpoint has occurred between V, and C,, with the translocation involving the J, segments and resulting in the C, gene being translocated to a region immediately 3‘ of the c-myc oncogene. It is possible that activation of the c-myc gene by the TcR a chain gene elements occurs in a manner similar to the activation of this oncogene seen in Burkitt’s lymphoma. With regard to the t( 11;14)(pl3;qll), the exact nature of the gene involved in the p13 region is not known. This locus has been implicated in the development of Wilm’s tumor and it is possible that either this Wilm’s tumor-associated gene or another as yet unidentified gene, which may represent a new oncogene, is involved in the malignant transformation as a result of its altered expression or structure, secondary to the translocation into the TcR a chain locus. The t( 10;14)(q24;qll)translocation has been reported in some cases of T-ALL and high-grade T cell lymphoma. Analyzing somatic cell hybrids between mouse leukemic T cells and human leukemic T cells carrying the t(10;14) translocation, Kagan et al. (1987) have found that the breakpoint on chromosome 10 is distal to the TdT gene, and that the C, gene was translocated to chromosome 10, with the V, gene remaining on the 149 chromosome. They suggested that C, was

74

M.D. REIS ET AL.

TABLE 111 T~ANSLOCATIONS INVOLVING T CELLRECEP~OR GENES’ Disease Pre-T (T cell)

Translocation

Loci involved

t(1;14)(~32;qll) L-myc?-TcR N-ras?-TcR a src-2?-TcR (Y t(8;14)(q24;qll) c-~Yc-TcRa

t(10;14)(q23;qll) TdT?-TcR o~Lc?-TcR t(ll;l4)(pl3;qll) Wi1ms’-TcR a om?-TcR a t(12;14)(q24;qll) o~c?-TcR inv(l4)(qllq32) Ig H-TcR a t(14;14)(qIl;q32) Ig H?-TcR a akt?-TcR a o~c?-TcR t(7;9)(q34;q33) TcR p-abl? Pre-B (B cell) leukemia/ lymphoma Ataxia-telangectasia

inv(14)(qllq32)

Ig H-TcR

inv(7)(p15q33) t(7;14)(p13;qll) t(7;14)(q34;qll) inv(14)(qllq32) t(14;14)(qll;q32)

TcR y?-TcR p? TcR y?-TcR a? TcR P?-TcR a? TcR a?-Ig H? TcR a?-Ig H?

(Y

Reference Kurtzberg et al. (1985) Williams et al. (1984); Mathiew-Mahul e t al. (1985); Erikson et al. (1986); Shima et al. (1986); Dub6 et al. (1986); Smith et al. (1986) Kagan et al. (1987) Williams et al. (1984); Sadamori et al. (1985); Erikson et al. (1985); Lewis et al. (1985) Sadamori et al. (1985) Zech et al. (1984); Baer et al. (1985); Denny et al. (1986b) Shah-Reddy et al. (1982); Sadamori et al. (1985) Hecht et al. (1984); Smith et al. (1986b) Denny et al. (1986a) Hecht et al. (1975); Welch and Lee (1975); Kaiser-McCaw et al. (1975); Aurias et al. (1980); Scheres et al. (1980); Taylor (1982); Kohn et al. (1982); Fukuhara et al. (1983); Battney et al. (1983);

Reproduced from Reis et aE. (1988).

translocated to a putative protooncogene located proximal to the breakpoint at 10q24, and proposed calling the protooncogene TCL3, the deregulation of which would lead T cell leukemia. The inv(14)(qllq32) has been observed in lymphomas and chronic T cell leukemias, in virus-associated T cell leukemias, and in T cell

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75

monoclonal expansions in patients with ataxia-telangiectasia. Two breakpoints occur on the long arm of the inv(14) chromosome. Denny et al. (1986a) determined that the telomeric breakpoint creates a fused structure in which an Ig V H gene segment joins a TcR segment. This VH-JolCajoining is productive, resulting in the formation of a hybrid gene, part Ig and part TcR, that is transcribed into mRNA with a completely open reading frame. This telomeric joining has been seen in T cell disorders and in at least one case of B-ALL (Denny et al., 1986a). Baer and co-workers (1987), studying the centromeric breakpoint of inv(l4), showed a reciprocal VH-Ja joining involving gene segments different from those that constitute the join at the telomeric breakpoint. Since a protooncogene is not known to be involved in this aberration, it is tempting to speculate that the fusion products of this inversion may play a role in oncogenesis. However, it is interesting to note that this inversion is occasionally seen in stimulated normal T cells and in T cells from patients with ataxiatelangiectasia (Kirsch et al., 1985). ACKNOWLEDGMENT The authors are grateful to Diana Quon for her expertise and dedication in preparing this manuscript. MDR is now at Sunnybrook Medical Centre, Department of Laboratory Hematology, University of Toronto, Toronto, Canada. HG is now at the Institute of Pathology, Christina1 Albrecht University, Kiel, Federal Republic of Germany.

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NOTEADDEDIN PROOF. Since the preparation of this manuscript, a few investigators have documented the involvement of the TcR 6 locus in translocations seen in T-ALL, t ( l l ; l 4 ) ( p l 3 ; q l l )(Boehm et al., 198813; Champagne et al., in press), and in the T cell line R P M l 8002, t(11;14) (p15;qll) (Boehm et al., 1988a)

STRUCTURE, FUNCTION, AND GENETICS OF HUMAN B CELL-ASSOCIATED SURFACE MOLECULES Edward A. Clark Department of Microbiology, Universlty of Washington. Seattle, Washington 98195

Jeffrey A. Ledbetter Oncogen Corporation, Seattle, Washington 98121

I. Introduction A. Scope of Review B. B Cell Activation and Differentiation Pathway C. Brief Historical Overview 11. Major B Cell Differentiation Antigens A. Overview of Expression and Function B. CD20: Regulator of B Cell Activation C. CD19: Regulator of Pre-B and Resting B Cell Function D. CD21: Receptor for C3d and Epstein-Barr Virus E. CD22: A Bridge for Surface Ig? F. CD23: Both a Receptor for IgE and a Soluble Factor G. CDw40/BLCa: Receptors for B Cell Progression Signals H. Bgp95: A Unique 95-kDa Glycoprotein Antigen Involved in B Cell Activation 111. Other Biochemically Defined Surface Molecules on Pre-B and/or B Cells A. CD10: The Common Acute Lymphocytic Leukemia Antigen B. CD24 C. CD37 D. CD39 E. BLA: AGIycolipid Globotriaosylceramide F. Other Molecules Expressed on Resting B Cells IV. Receptors on B Cells for Cytokines A. IL-2 Receptors B. IL-4 Receptors C. IL-6 Receptors D. Receptors for Other Factors V. Other Surface Molecules Expressed on Activated B Cells A. Markers Expressed Early after Activation B. Markers Expressed Late after Activation VI. Surface Molecules Found on T Cells and Subsets of B Cells A. CD5 B. CD2, CDlc, and CD28 VII. Concluding Remarks References 81 ADVANCES IN CAKCER RESEARCH, VOL. 52

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reselved.

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I. Introduction

A. SCOPEOF REVIEW

B lymphocytes are exceptionally motile and social cells that are directed and regulated through their cell surface molecules by a number of different kinds of cell-cell interactions and soluble signals. From the moment a B lymphocyte progenitor (pro-B) cell becomes committed to become a pre-B cell, to the last step in the terminal differentiation of a plasma cell, B lineage cells are interacting with other lymphoid cells and/or various kinds of accessory cells (Kincade, 1987). In hematopoietic tissues B lymphoid progenitors are receiving signals, e.g., from accessory stromal cells (Dorshkind, 1986; Kincade, 1987); they then migrate to specific regions in the central lymphoid tissues, presumably through interactions with specific homing receptors (Jalkanen et al., 1986; Heinen and Tsunoda, 1987).In the central lymphoid tissues, the cycle of B lineage cell-accessory cell interaction, followed by activation and homing to defined compartments, is repeated. The migration of mature B cells to B-cell-specific regions in peripheral lymphoid tissues also requires specific ceII-cell adhesion interactions (Jalkanen et al., 1986; Woodruff et al., 1987; Springer et ul., 1987). Finally, the activation of B lymphocytes in peripheral lymphoid tissues may involve cognate interactions with T cells and accessory cells (such as follicular dendritic cells), followed by a characteristic subsequent migration into germinal centers and eventually the medullar cords (Klaus and Humphrey, 1986; Steinman et al., 1986; Heinen and Tsunoda, 1987). Each distinct interaction that B lymphocytes have with other cells during differentiation presumably is mediated through specific cell surface molecules expressed on B cells or subsets of B cells. B cells must also express specific receptors for the various soluble factors that regulate B cell growth differentiation (Kerhl et al., 1984; Kishimoto, 1985; Hamaoka and Ono, 1986; Paul and Ohara, 1987; Kishimoto and Hirano, 1988). Thus, it is not surprising that about 20 biochemically distinct surface polypeptides have already been identified that are only expressed or principally expressed on human B lineage cells (Nadler, 1986; Ling et ul., 1987). The purpose of this review is to describe the structures and possible functions of this intriguing group of molecules. Surface molecules primarily associated with B lineage cells most likely function as receptors for signals required uniquely by these cells during their maturation or activation. Thus, a better understanding of this group of lymphocyte molecules should help

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elucidate the mechanisms that regulate normal or malignant B lineage cells. B. B CELLACTIVATION AND DIFFERENTIATION PATHWAY Detailed descriptions of the development and activation of B lymphocytes can be found in several excellent reviews (Kishimoto, 1985; Hamaoka and Ono, 1986; Melchers and Anderson, 1986; Paul and Ohara, 1987; Kincade, 1987; Jelinek and Lipsky, 1987; Cooper, 1987; Kishimoto and Hirano, 1988). The major steps in B cell differentiation are shown in Fig. 1. Initially, a lymphoid progenitor cell commits to differentiate along the B cell lineage (pro-B stage). Sterile transcripts of unrearranged heavy (H) /.L chain gene segments are expressed and then H chain variable (V), diversity (D),joining (J), and constant (C) gene segments are rearranged and cytoplasmic /.L chains are expressed (pre-B stage) (Cooper, 1981; Alt et al., 1987). The pre-B pool is expanded and then some pre-B cells rearrange and express light (L) chain genes. Cells with both functional /.L and L chains then express membrane surface immunoglobulin M (sIgM) (immature B stage), and upon further maturation also express surface IgD (sIgD) (mature B, or resting B, stage). All of the steps up to this point occur in the absence of foreign antigen (Alt et al., 1987). The subsequent steps which normally require antigen or cross-linking of sIg, are sometimes referred to as the B cell “activation” pathway. Human B cell immunobiologists currently investigate these steps most extensively, because mature and activated B cells are readily obtainable, for example, blood or tonsillar lymphocytes. After resting B cells are triggered, they enlarge and express elevated levels, for example, of class I1 major histocompatibility complex (MHC) antigens Pro-B

Pre-B

Immature B

Mature 0

Activated B

Blast 0

Plasma

Cell

FIG.1. The human B cell differentiation pathway. It begins when a pro-B cell commits to the B cell lineage; next, pre-B cells express cytoplasmic p chains, then light chains are expressed as immature B cells express surface IgM. Mature B cells express both surface IgM and IgD and, after activation, enlarge and subsequently divide as B cell blasts. Some blasts subsequently mature into IgM-secreting plasma cells or become memory B cells.

84

EDWARD A. CLARK AND JEFFREY A. LEDBETTER Cell Stage Pre-B

Cell Induction C y c l e Soluble Cell F a c t o r Surface Go G1/S

Resting B

Blast B Antibody Producing

BCGF

CD19

GO

IL-4

slg CD20

G1

BCGF LT IL-2 IL-5 IL-6 IL-2 IFN-X

CDw40 CD23 BLCa

S

Inhibition Soluble Cell Factor Surface

IFN-g

TGF-D IFN- s(

CD19 Fcr

CD19

FIG.2. The different stages of pre-B and B cell activation and differentiation are regulated by soluble factors and signals via cell surface differentiation markers. These signals can be grouped as either inducing or inhibiting further activation or differentiation. The ligands for the cell surface structures listed could be either on cell surfaces or soluble. Competence signals induce cells to leave Go and enter the cell cycle, while progression signals induce activated cells to enter S phase.

(activated B stage). With further stimulation, activated B cells enter the cell cycle and begin to divide (B blast stage) and eventually may differentiate into antibody-producing cells (APC, plasma cell stage) or memory B cells. The soluble and cell surface molecules that either promote or inhibit steps in the B cell activation pathway are shown in Fig. 2. According to this scheme, some signals, exemplified by the soluble factor interleukin 4 (IL-4), also known as B cell-stimulating factor 1 (BSF-1; Paul and Ohara, 1987), act as “competence” signals or cocompetence” signals with antigen to induce resting B cells to leave Go and enter an early point in the cell cycle, sometimes referred to as Go. or GI, (O’Keefe and Pledger, 1983). “Progression” signals, such as IL-2, IL-5, or low-molecular-weight (12-kDa) or highmolecular-weight (60-kDa) B cell growth factors (BCGF), by contrast, on their own cannot stimulate resting B cells to enter the cell cycle, but rather stimulate certain activated B cells to traverse the cell cycle, to enter S phase, and to divide (Kehrl et al., 1985; Ambrus et al., 1985; Mehta et al., 1985; Kishimoto, 1985; Kishimoto and Hirano, 1988). Recently, recombinant lymphotoxin (LT) and tumor necrosis factor (TNF) were shown to have potent B cell growth factor (BCGF)activity (Kehrl et al., 1987a,b). Furthermore, certain low-molecular-weight BCGF preparations were found to contain very high levels of active LT (e.g., 100 ng/ml), and much of their BCGF activity was blocked by “

B CELL-ASSOCIATED SURFACE: MOLECULES

85

anti-LT sera. Thus, most or all of the activities attributed to BCGF (low) may be due to LT. Another set of overlapping signals, such as IL-2 and IL-6 (BSF-2, IFN-BB), sometimes referred to as “differentiation” or “maturation” factors, has the ability to promote activated and/or dividing B cells to differentiate into antibody-producing cells. Finally, some signals, such as the transforming growth factors (TGF) ,Bl and p2, inhibit B cell activation, but much less is known about this class of molecules and about the stages in the B cell activation cycle they affect (Kehrl et al., 1986; Smeland et al., 1987b; Clark et al., 198813). It should be noted that this linear scheme of activation and differentiation is oversimplistic and does not consider possible heterogeneity in B lineages. It is used here for the sake of discussion (Kishimoto, 1985). Although a great deal already is known and has been written about the soluble signals that either promote or inhibit B cell activation (e.g., Melchers and Anderson, 1986; Paul and Ohara, 1987; Kishimoto and Hirano, 1988),much less is known or has been discussed about signals regulating B cells that are mediated via cell-to-cell surface interactions. In this review we describe a number of B cell-associated surface molecules that may function to regulate B cell activation. The possible sites of action of some of these surface molecules are also summarized in Fig. 2. Agonistic or antagonistic monoclonal antibodies (MAbs) have been used to determine the possible function of most of these molecules, and with the exception of CD21 and CD23, the natural ligands for these molecules have not yet been identified. As described in detail below, it is unlikely that these surface molecules are all receptors for soluble factors with signaling activity similar to the MAb. Cell surface-associated ligands for several T cellassociated surface molecules have been identified, including CD2 (LFA-3), CD4 (class I1 MHC), CD8 (class I MHC), and CD18ILFA-1 (I-CAM-l/gp80) (see Cobbold et al., 1987; Springer et al., 1987; Ledbetter and Clark, 1988). Thus, it is likely that many of the cell surface molecules we discuss in this review also have ligands found on the surface of hematopoietic cells.

OVERVIEW C. BRIEFHISTORICAL 1. Heteroantisera Used to Define Lymphocyte Lineages The field of cell surface immunology is greatly indebted to the pioneers E.A. Boyse and L.D. Old (see Boyse and Old, 1969), who were among the first to characterize “lymphocyte differentiation

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EDWARD A. CLARK AND JEFFREY A. LEDBETTER

antigens,” a term they coined, and who helped set the standards for serologic, biochemical, and genetic characterization of lymphocyte surface molecules. Their early work defining such murine antigens as Lytl, Lyt2, and TL, as well as other early studies of murine markers, has been extensively reviewed (Snell et al., 1975; Katz, 1977; McKenzie and Potter, 1979). Because the major theme of this review is the structure and function of human B cell surface molecules, we refer the reader elsewhere for detailed reviews of murine B lymphocyteassociated markers of B cell markers (Katz, 1977; McKenzie and Potter, 1979; McKenzie and Zola, 1983; Moller, 1983; Morse et al., 1987) By 1970 it was shown that the presence or absence of differentiation antigens on lymphocytes could be used to identify functionally distinct subsets. Raff (1969) and Schlesinger and co-workers (see Schlesinger, 1972) showed that the differentiation antigen, Thy-1, is selectively expressed on a subpopulation of lymphocytes, T cells. Soon thereafter, Takahashi and co-workers (1970) identified an alloantigen, PC1, that was restricted to antibody-forming cells and plasma cell tumors, and was not found on thymocytes. This finding added further support to the notion that immunoglobulin-producing cells and T cells are derived from distinct lineages. About the same time, Raff and co-workers (1970) clearly demonstrated in the mouse that B cells express surface Ig, confirming earlier indirect studies in the rabbit (Sell and Gel], 1965). Human B cells were found to express surface IgM or IgG, and the Ig+ cells were absent in patients with Burton’s agammaglobulinemia (Froland et al., 1971; Grey et al., 1971). Once B cells could be identified by the expression of surface Ig, it was possible to measure whether other markers were on Ig+ cells. Fc receptors for IgG (Dickler and Kunkel, 1972; Moller, 1973) and receptors for C3 components of complement (Bianco et al., 1970; Bianco and Nussenszweig, 1971; Jondal et al., 1972; Ross et al., 1973) were found on Ig+ B cells. Soon after the discovery that heteroantisera to Thy-1 could be used to identify murine T cells, Raff and co-workers (1971) used an analogous approach to develop rabbit heteroantisera specific for murine B cells. The antisera was B cell specific yet did not react with immunoglobulin, implying that mature B cells, like T cells, express characteristic differentiation antigens. Later, other B cell-associated differentiation antigens were defined in mice with alloantisera such as Lyb-2, Lyb-3, Lyb-4, and Lyb-5 (see Moller, 1983). Following the general approach of Raff et al. (1971), Greaves and Brown (1973)

B CELL-ASSOCIATED SURFACE MOLECULES

87

developed a rabbit heteroantiserum to human B chronic lymphocytic leukemia (CLL) cells. The absorbed antisera reacted with Ig+ cells, B lymphoblastoid cell lines, and B cell malignancies, but not with T cells or T cell or myeloid leukemias. This work set the stage for later attempts to make specific antibodies to human B cell differentiation antigens. Greaves and co-workers (1975, 1980) also went on to define the B cell-associated common acute lymphoblastic leukemia antigen (CALLA, CD10). About the same time, Thomas and Phillips (1973) described a heteroantiserum raised against a Burkitt’s lymphoma (BL) cell line that was relatively specific for B cell blasts and did not react with resting T cells or B cells. This approach was later used by others to develop monoclonal antibodies to B cell-associated activation markers (see Section V). In the mid-1970s a number of heteroantisera and alloantisera were developed that reacted with human B cells but not T cells (see, e.g., Winchester et al., 1975; Chess and Schlossman, 1977; Ferrone et al., 1978).Many of these antisera turned out to be specific for HLA class I1 antigens, the discussion of which is beyond the scope of this review. In notable exceptions, Balch and co-workers (1978; Ades et al., 1980) identified a 68,000-Da marker on human B cells with a rabbit antimacaque B cell xenoantiserum, and Wang et al. (1979) characterized a 54,000-Da antigen on human B cells with a rabbit antihuman xenoantiserum. The antiserum developed by Wang et aZ.stimulated the proliferation of human tonsillar B cells, the first such report of an agonistic antisera to a human B cell differentiation marker. 2. Monoclonal Antibodies to Leukocyte Surface Markers In their review of murine lymphocyte antigens in 1979, McKenzie and Potter describe in detail methods of xenoantisera alloantisera production, but the monoclonal antibody technology developed by Kohler and Milstein (1975) is not even mentioned. Soon after that report, MAb revolutionized cell surface immunology. After the first MAb to cell surface antigen was reported (Galfare et al., 1977), MAbs to lymphocyte differentiation antigens were produced (Williams et al., 1977). Within a short time the first MAbs to human cell surface antigens had been made (Barnstable et al., 1978; Parham and Bodmer, 1978). Once McMichael et al. (1979) described the first MAb to a human T lymphocyte differentiation antigen (now appropriately called C D l ) , the floodgate was opened and a number of seminal descriptions of human T cell markers followed, among which one of the most notable were the reports of Kung, Reinherz, and co-workers

88

EDWARD A. CLARK AND JEFFREY A. LEDBETTER

defining human CD3, CD4, and what later turned out to be the human homologue of murine Lytl, C D 5 (Kung et al., 1979; Reinherz et al., 1979; Reinherz and Schlossman, 1980; Ledbetter et al., 1980). Several investigators resisted the initial focus on T cells (Lake et al., 1979; Ledbetter and Herzenberg, 1979; McMichael et al., 1979; Kung et al., 1979) and attempted to develop MAbs to a smaller and stickier lymphocyte subset in peripheral blood-B cells. Some investigators, exemplified by Lee Nadler, Jerry Ritz, and Stuart Schlossman of the Dana Farber Cancer Center in Boston (Ritz et al., 1980; Stashenko et al., 1980), were oncologists interested in diagnosing and treating hematologic malignancies. They developed MAbs to B lineage cells in part because the majority of lymphoid malignancies are B lineage cells. Another smaller and overlapping group of investigators, exemplified by Bill Sudgen of the University of Wisconsin (Kintner and Sudgen, 1981), were interested in the interaction of Epstein-Barr virus (EBV) and B cells. They attempted to develop MAbs that detected markers expressed on EBV-infected B cells in order to gain insight into EBV-induced B cell transformation and virus-host cell interactions. In 1982 these investigators met in Paris at the First International Workshop on Human Leukocyte Differentiation Antigens to standardize the characterization and classification of human B cell markers (Bernard et al., 1984). By the time a second international workshop was held in Boston, Massachusetts, in 1984, eight B cell-associated markers could be identified clearly (Nadler, 1986), and in the third workshop at Oxford, England, in 1986, four additional B cellassociated markers were characterized (Ling et al., 1987). Whereas only a handful of papers was presented at the first workshop on B cell markers, the second and third workshops resulted in a volume and a publication with 200 pages describing B cell-associated markers (Volume 2, Reinherz et al., 1986; McMichael, 1987). The reader is referred to these volumes for detailed descriptions of B cell markers. It was recognized very early that MAbs to B cell-associated markers are very useful for classifying and treating B cell malignancies (see, e.g., Knapp, 1981; Nadler et al., 1981b; Coda1 and Funderud, 1982). This review does not emphasize possible clinical applications of MAbs to B cell markers; this is described elsewhere (Ledbetter and Clark, 1988). Here we summarize the major B cell markers with an emphasis on recent studies illuminating the structure and possible physiologic function of these molecules. Our review also is limited insofar as we have elected to describe only those markers clearly distinguishable biochemically; without biochemical characterization

89

B CELL-ASSOCIATED SURFACE MOLECULES

it is not possible to determine definitively whether two MAbs detect the same or different structures. For recent succinct summary descriptions, we refer the reader to two exemplary concise reviews (Zola, 1987; Gordon and Guy, 1987). For a discussion on plasma cell markers, the reader should consult Ling et uZ. (1987).

I I . Major B Cell Differentiation Antigens A. OVERVIEW OF EWRESSION AND FUNCTION The major well-defined B cell-associated surface molecules discussed in this review are summarized in Table I. The possible stages in B cell differentiation where these molecules may function are summarized in Fig. 2, and the expression of these molecules on B lineage cells is depicted in Fig. 3. Throughout the review, where possible, we use the World Health Organization (WHO)-established C D nomenclature for the molecules (Ling et al., 1987). The molecular weights listed in most cases are best approximations, except where DNA sequence data are available for predicting the size of the protein cores. Also listed are some of the signals induced by MAbs to these cell surface molecules, including mobilization of intracellular free calcium, [Ca"+li. The current major possible functions of the Antigen

Progenitor B

PreB

I m m a t u r e Mature B B

Activated B

B Blast

Plasma Cell

C l a s s I1

u chain IgD CD19 CD22 CD24 CD 1 O(cALLA) CD20 CD2 1 ( C 3 d r ) CD37 CD39 CDw40 CD23 CD38

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

0

-

0

0

FIG.3. Human B cell differentiation antigens. The B cell-associated surface molecules are expressed at characteristic stages of differentiation. Some are expressed early in ontogeny at the pre-B cell stage or earlier, while another set of markers is found only on Ig' B cells. Shaded portion designates expression only in the cytoplasm. The linear scheme shown does not consider the possible differential expression of these markers on B cell subsets.

TABLE I MAJORHUMAN B CELL-ASSOCIATED SURFACE MOLECULES Antigen (other names)

Molecular mass (kDa)

CD20 (BLBp35) CD19 (B4) CD21 (CR2, EBV) CD22

37135

33/31"

95

CD23 CDw40 (Bp.50) Bgp95 CDlO (CALLA) CD24 (BA-1) CD37 CD39 a

Signal transduction"

Protein core (kDa)

Possible function Competence

52"

c-myc, no [Ca2+],signal, Go-Gl, TPA phosphorylates [Ca2+1,

140

120

C3d receptor, TPA phosphorylates

Regulation of Go to GI entry

130/140

63"

Bridge for surface Ig signal

45 48

36" 28"

Required for anti-Ig [Ca2+],,TPA phosphorylates Fc receptor for IgE, IL-4 regulation No [Ca"],

G1 to S signal, IgE regulation Progression signal, GI to S

95 100

70 86"

Slow [Ca"], Unknown

Competence signal, Go to GI Endopeptidase

No [Ca2+Ii

Unknown

No [Ca"], No [Ca''li

Unknown Unknown

40-45 40-45 80

Unknown 25 55

Inhibition of C" to G-1

and Ig secretion

Indicates molecular mass based on polypeptide predicted from cDNA sequence. [Ca2+Iiindicates whether or not a MAb to the surface polypeptide in question can induce mobilization of intracellular free calcium.

B

CELL-ASSOCIATED SURFACE

MOLECULES

91

molecules in general can be classified as either influencing the movement of resting B cells into the cell cycle (“competence” signal regulation), the movement of activated B cells traversing through the cell cycle (“progression” signal regulation), or the differentiation of mature B cells. It is clear from Fig. 3 that many of the molecules listed in Table I are expressed early in B cell ontogeny. This discussion focuses on the B cell activation pathway, since relatively little is known about how these molecules may function during the development of pre-B and immature B cells. B. CD20: REGULATOR OF B CELLACTIVATION The B lymphocyte specific antigen CD20 (Bl, Bp35) was first identified by Stashenko and co-workers (1980), who termed the antigen B1. They found that CD20 was expressed on almost all B cells from blood and lymphoid tissues, but not on resting or activated T cells, monocytes, or T cell or myeloid malignancies. CD20 could be distinguished from other B cell-associated markers such as surface Ig and class I1 MHC markers. Pokeweed mitogen (PWM)-induced Ig production was shown to require CD20’ cells, and interestingly, already in this early study anti-CD2O alone inhibited immunoglobulin production, a result confirmed and extended later (Tedder et al., 198513; 1986b; Golay et al., 1985). Based on studies with both normal and malignant early B cells, CD20 is thought to be first expressed in pre-B cells: some early cytoplasmic p chains-B leukemias that express other B cell markers such as CD19 and CD22 are CD20- (Nadler et al., 1981b, 1984; Korsmeyer et al., 1983a; Nadler, 1986; Moldenhauer et al., 1986; Pezzutto et al., 1986), implying that pro-B cells express CD19 and CD22 before expressing CD20 and H chain gene rearrangements. In two-color analyses of fetal liver cells, Hokland and co-workers (1985) identified CD19+ CD20- cells, which they interpreted as evidence that CD19 expression precedes CD20 expression in early B cells. Further studies following the maturation of early B cells in culture will help define more precisely just when CD20 is expressed. CD20 is found on both resting and activated B cells but is lost prior to terminal B cell differentiation into plasma cells (Stashenko et al., 1981; Boyd et aE., 1986). Both mantle zone and germinal center B cells in secondary lymphoid follicles express CD20; in some studies, germinal center B cells were reported to express higher levels of CD20 than is found in mantle zone B cells (Bhan et al., 1981; Ledbetter and Clark, 1986),but in other studies mantle zone B cells stained more strongly (Mason et

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CLARK AND JEFFREY A. LEDBETTEH

al., 1986). Consistent with high expression of CD20 on germinal center B cells, the majority of tonsillar B cells in S phase were found to express high levels of CD20 (Valentine et al., 1987). Mason and co-workers (1986) have found that CD20 is not entirely B cell specific, since it is present on dendritic reticulum cells in germinal centers. MacLennan and co-workers (1986; Johnson et al., 1987) found that anti-CD2O stained marginal and germinal center B cells equally well in tissue sections and were B cell specific except that they stained follicular dendritic cells weakly. CD20 was initially described as a 35-kDa nonglycosylated, hydrophobic, phosphoprotein using the Raji or Daudi Burkitt’s lymphoma cell lines as a source of antigen (Oettgen et al., 1983; Clark and Einfeld, 1986; LeBien et al., 1986; Horibe and Knowles, 1986). Although Daudi cells express principally a single 35-kDa phosphoprotein, normal tonsillar B cells express both a major 35-kDa and minor 37-kDa components with a pZ of 7.5 to 8.0 (Valentine et al., 1987; Moldenhauer et al., 1987).CD20 (see Fig. 4a) phosphorylated on threonine and serine residues and not on tyrosine resides (Oettgen et al., 1983). The tumor promoter phorbol myristate acetate (PMA) induces increased expression of class I1 molecules on human B cells (e.g., Godal et al., 1985) and activates protein kinase C (PKC) (Nishizuka, 1984), so it was of interest to assess the effect of PMA on CD20 expression and phosphorylation. B cells exposed for as little as 5 min to PMA had a sixfold increase in phosphorylated CD20 and at the same time induced internalization of CD20 (Valentine et al., 1987).And B cells exposed to PMA for 3 days had markedly reduced levels of CD20 (Tedder et al., 1986a). Both hyperphosphorylation and internalization of CD20 are blocked by inhibitors of PKC, suggesting that hyperphosphorylated CD20 selectively leaves the cell surface (Valentine et al., 1987). M.A. Valentine and K. Meier (unpublished), using purified CD20, recently showed that CD20 can serve as an in vitro substrate for PKC. At the second international workshop in 1984, two groups suggested that CD20 may function in the regulation of B cell activation. Clark and co-workers (1985, 1986b) showed that one anti-CD20 MAb, 1F5 (but not another MAb, 2H7), could stimulate dense tonsillar B cells to proliferate and was costimulatory with either anti-Ig or a lowmolecular-weight BCGF. Concurrently, Golay et al. (1986) showed that 1F5 stimulated increased proliferation on prolymphocytic leukemia (PLL) cells. The 1F5 anti-CD2O MAb also stimulated increased expression of C3d receptors, class I1 MHC molecules, and early activation antigens such as LB-2 (Clark et al., 1986a; see Section V,A),

B CELL-ASSOCIATED SURFACE MOLECULES

93

all within 16 hr (Clark et al., 1986b) F(ab’)zfragments were just as effective as whole anti-CD20 MAb. These results suggested that CD20 functions to regulate an early event in B cell activation, perhaps via a phosphokinase-dependent mechanism; that 1F5 anti-CD2O stimulates an early event in B cell activation was confirmed by groups in Seattle, Washington, Oslo, Norway, and London, England: antiCD20 induced increases in expression of the c-myc protooncogene detectable within 2 hr after activation of blood B cells (Smeland et al., 1985; White et al., 1988) and also induced resting B cells in Go to enlarge, and enter the G1 phase of the cell cycle and express increased levels of class I1 MHC (Golay et al., 1985; Smeland et al., 1985;Clark and Shu, 1987). Because anti-CD20 MAb alone can stimulate resting B cells to enter GI, it can be classified as a “competence” signal (O’Keefe and Pledger, 1984) for B cells along with anti-Ig or PMA (Fig. 2). The initial studies of Stashenko and co-workers (1980) suggested that their anti-CD20 MAb, B1, inhibited B cell differentiation. Subsequent studies confirmed this initial observation and also showed that the B 1 MAb, rather than stimulating B cell proliferation, inhibited proliferation of B cells stimulated with either anti-Ig or EBV (Tedder et al., 1985b; Golay e t al., 1985). Both the stimulatory 1F5 and inhibitory B1 anti-CD2O MAb are IgGz, antibodies and cross-block each other’s binding. This is not unexpected, however, since only 42 amino acids of CD20 are extracellular (see below). It is likely that 1F5 and B1 recognize distinct epitopes of CD20. The pathway of signal transduction induced by anti-CD20 is just beginning to be understood. Anti-CD20 does not induce mobilization of intracellular Ca2+on its own; if added 5 to 15 min before anti-Ig, anti-CD20 does not modulate anti-Ig-induced Caz+ mobilization in resting B cells (Tedder et al., 1986a; Rabinovitch et al., 1987; Smeland et al., 1987a). Recently, we found that within 5 min after B cells are activated by either TPA (Valentine et al., 1987) or anti-Ig (Valentine and Clark, 1988), CD20 is hyperphosphorylated and internalized. This rapid phosphorylation and internalization of CD20 is inhibited in whole cells by several specific inhibitors of PKC, and purified CD20 can serve as substrate for PKC in uitro (Valentine et al., 1989). The protein sequence of CD20 shows that its long carboxyl tail has a number of serine and threonine residues that are potential sites for PKC-medicated phosphorylation (Fig. 4 4 . Thus, the evidence indicates that CD20 may function as a signal in a PKC-dependent activation pathway. Several forms of PKA have been identified as well as other kinases, such as casein kinase 11, so it is still necessary to

b

70

17+

T -

S

S

-

S

SYY-COOH - 386

d

FIG.4. Structure of B cell-associated surface molecules based on protein sequences predicted from cDNAs. (a) CD20; (b) the EBV-encoded latent membrane protein (LMP); (c) CD23; (d) CDw40. The tops of the figures represent the extracellular portions of the molecules. TM, Transmembrane region; CYTO, cytoplasmic region; +, Lys or Arg; -, Asp or Glu. Numbers refer to amino acid residue. N-Linked glycosylation is represented by branches. 94

C +-

COOH321

-

+.-;(

-

+

:\.

i C

-+

I

Y

7

+

+

-

+

L .. G Y +

SOLUBLE CD23 .

.+

.. 48 f -

+ +

.

-+

.

.. .

+

+--

+ +

.

..+

+

TM

d

FIG.4. (Continued) 95

+

96

EDWARD A. CLARK AND JEFFREY A. LEDBETTER

determine more precisely which kinases affect CD20. Furthermore, it is still not clear how internalized phosphorylated CD20 actually functions in modulating activated B cells. Although all of the evidence indicates that CD20 functions to regulate early activation events, it is possible that it functions also at other points in B cell differentiation and activation. CD20 is expressed on pre-B cells and pre-B leukemias (Nadler et al., 1981b; Hokland et al., 1983) and is elevated on B cells in S phase (Valentine et al., 1987); this suggests it may function to regulate more than Go to GI transitions. Agonistic anti-CD20 is most effective at stimulating proliferation if added 12 hr before anti-Ig, and for blood B cells is not costimulatory with BCGF or anti-Ig unless added well before anti-Ig (Clark and Shu, 1987; Smeland et al., 1987a). Blood B cells which express lower levels of class I1 MHC molecules compared to tonsillar B cells may be at a less activated stage than are tonsillar B cells (Clark and Shu, 1987). These results suggest that CD20 may function to regulate B cells at a very early step in activation that can occur prior to surface Ig receptor cross-linking. Using inhibitory anti-CD20, Tedder and co-workers (1986a) found that anti-CD20 was only effective if added in the first 24 hr of culture, which also suggests that CD20 regulates an early event. However, while that B1 MAb inhibited DNA synthesis, it did not inhibit B cells from enlarging, from expressing IL-2 receptors, or from synthesizing RNA. Their results suggest that CD20 may function to provide a signal necessary for activated B cells to traverse the cell cycle. This interpretation is not necessarily inconsistent with findings that the binding of agonistic anti-CD20 MAb to B cells has rapid detectable effects, e.g., a rapid induction of c-myc expression (Smeland et al., 1985). Recent studies with missense mutants have shown that although the protooncogene c-rnyc is elevated early after cell activation, c-rnyc may actually function to regulate G1 to S transitions (Heikkila et al., 1987). A similar mechanism could be evoked for CD20 action. Recently, cDNAs encoding for the CD20 protein were isolated independently by three independent groups (Tedder et al., 1988; Einfeld et al., 1988; Stamenkovic and Seed, 1988a). Using the Agtll expression system and a murine CD20-specific heteroantiserum, we isolated a clone that bound specifically to mRNA from B cell lines (Einfeld et al., 1988). This clone was used to isolate subsequent larger clones from a AgtlO library, and one of these clones in an in oitro translation system expressed a 33-kDa protein specifically precipitated by anti-CD20 MAb. Northern blot analyses of RNA from a number of lymphoid and nonlymphoid cell lines also showed that the

B CELL-ASSOCIATED SURFACE MOLECULES

97

expression of the CD20 antigen correlated with the expression of the 2.1- and 2.8-kb mRNAs detected with the cDNA clone. About the same time, Stamenkovic and Seed (1988a) isolated two full-length cDNAs encoding CD20 using a powerful new expression vector, isolation of transfected COS cells expressing CD20 by panning, and subsequent detection of CD20 expression in transfected cells with immunofluorescence (Aruffo and Seed, 1987; Seed and Aruffo, 1987). Long cDNAs were put into a vector having a powerful promoter, the ability to replicate in bacteria, and a range of mammalian cell types. They also have used this ingenious method to isolate cDNAs encoding for the CD19, CD22, CD37, CD39, and CDw40 molecules described below. The cDNA sequences for CD20 obtained from Einfeld et al. and Stamenkovic and Seed were identical. The amino acid sequence predicted from the open reading frame of the cDNA contains 291 residues and has a molecular weight of 33,097. Analysis of the hydropathy of CD20 revealed three major hydrophobic stretches of about 53 (hydrophobic region a), 25 (region b), and 20 (region c ) amino acids. The lengths of the hydrophobic regions b and c are typical of transmembrane-spanning segments, while the hydrophobic region a could cross the membrane twice. The primary structure of CD20 does not have an N-terminal signal peptide. A likely model of CD20 in the cell membrane is shown in Fig. 4a; as to the polarity ' o f the protein, the C-terminus is placed in the cytoplasm, since our anti-CD2O heteroantiserum reacted with a h g t l l clone containing the 28 C-terminus residues and precipitated CD20, yet did not bind to intact cells expressing CD20 on the cell surface (Einfeld et al., 1988).The N-terminus has also been placed within the cell, assuming that the first hydrophobic region a acts as an internal membrane insertion signal in the absence of a signal peptide. Hydrophobic region a is shown spanning the membrane twice without exiting from the membrane, while hydrophobic regions b and c each span the membrane once. The only part of the protein exposed to the cell surface is a small stretch of 42 amino acids between two transmembrane regions. Surface CD20 can be externally labeled with radioactive iodine, and this region contains tyrosines for labeling sites. CDZO is known to be phosphorylated at serine and threonine residues (Oettgen et al., 1983). The cytoplasmic domains of CD20 contain 15 serine and threonine residues in the highly charged C-terminal tail and 11 of these residues in the N-terminal tail. CD20 has five cysteine residues-two in the proposed extracellular region, one in the transmembrane region, and two cysteines in the proposed cytoplasmic region. There is no immunochemical evidence yet that

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CD20 forms intermolecular disulfide bonds with itself or any other molecules. However, reduced CD20 migrates slightly faster than nonreduced CD20 (Valentine et al., 1987), suggesting the presence of intrachain disulfide bonding. Thus we have included possible intrachain disulfide bonds in the model. The gene encoding CD20 is not rearranged (Stamenkovic and Seed, 1988a). RNA blot analysis of B cell line-derived RNA showed one major transcript of about 2.6 to 3.0 kb and a second less abundant transcript of about 3.3 to 3.8 kb (Einfeld et al., 1988; Stamenkovic and Seed, 1988a). Stamenkovic and Seed also detected a third RNA transcript of 1.5 kb expressed at low levels in Raji cells. The only detectable difference found between the two larger RNA species is a difference at a consensus splice site. Both RNAs encoded for a single 33-kD species in a reticulocyte lysate system. Thus, alternate splicing of the two different RNA species probably does not contribute to the two forms of CD20. An ATG encoding Met 23 is preceded by a G at the 3 position, showing some homology to initiation consensus sequences, and would initiate a protein 2.3 kDa smaller (30.8 kDa) than a protein starting with Met 1(33.1kDa). CDZO-encoded proteins of about 33 and 31 kDa are expressed in COS cells (Stamenkovic and Seed, 1988a). Thus, it is possible that use of alternative translational initiation sites may account for the two protein forms of CD20. The amino acid sequence of CD20 shows no strong homologies with currently available protein sequences. However, there are several receptors that have multiple membrane-spanning regions. One group includes rhodopsin, p-2 adrenergic receptor, and the muscarinic acetylcholine receptors which transmit signals through GTPbinding (G) proteins (for discussion, see Einfeld et al., 1988; Stevens, 1987). Each of these proteins spans the membrane seven times and, like CD20, lack N-terminaI signal sequences. Another more heterogeneous group consists of proteins which act as ion channels, such as the Ca", Mg2+-ATPase,which may cross the membrane eight times, and the larger sodium channel, which has repeated units that cross the membrane six times, and the units of the nicotinic acetylcholine receptor, which are thought to cross four to six times. These proteins have amphipathic helices, which could be involved in forming an ion channel, and have a predominance of negatively charged amino acids, which might direct cation flow. Hydrophobic transmembrane region b of CD20 has amino acids that could give it amphipathic character, region a of CD20 has a very short stretch with charged amino acids, and the C-terminal cytoplasmic domain is highly negatively charged with 25 acidic residues versus 8 basic residues. Thus, CD20 has some

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similarities to ion channels. A recently described synaptic vesicle protein, synaptophysin, also is about 33 kDa in size and has four transmembrane domains and intracytoplasmic N- and C-termini (Sudhof et al., 1987). The CD20 protein also shares some structural similarities with the EBV-encoded latent membrane protein (LMP) (Liebowitz et al., 1986, Fig. 4b). LMP, like CD20, has a short cytoplasmic N-terminal domain, a long, highly negatively charged serine/threonine-rich cytoplasmic C-terminal domain, and multiple transmembrane regions. Both proteins also do not have N-linked glycosylation sites, have a transmembrane cysteine residue, and have relatively few amino acids exposed on the cell surface. LMP has fewer residues exposed on the cell surface than does CD20, and a much longer C-terminal tail. Six of 17 amino acids (35%) in the C-terminus of CD20, which is rich in glutamic acid (positions 265-282), are identical with a region in the C terminal tail of LMP, and five of seven amino acids in the N-terminal tail of CD20 (position 26-33) are identical with an N-terminal tail region in LMP. Even these minor similarities are of interest, since both CD20 and LMP have been implicated in the regulation of human B cell proliferation. The LMP gene is one of a small number of EBV-encoded genes expressed in transformed B lymphoblastoid cell lines (Hennessy et al., 1984; Rowe et al., 1987) and can transform established rodent cells (Wang et aZ., 1985).At this juncture CD20 and LMP are the only lymphocyte-associated surface molecules with multiple transmembrane domains. An intriguing and testable hypothesis is that LMP exerts its transforming activity by mimicking and/or causing dysregulation of the signal transduction pathway normally regulated by CD20. C. CD19: REGULATOR OF PRE-BAND RESTING B CELLFUNCTION The CD19 antigen is one of the most broadly expressed markers for B cells yet discovered (Nadler et al., 1983; Meeker et al., 1984). All resting B cells appear to b e CD19 positive, and CD19 continues to be present on B cells activated in vivo (buoyant tonsillar B cells) or activated in vitro with anti-Ig, protein A, EBV, or TPA (Boyd et al., 1986; Freedman et al., 1986; Schwartz et al., 1986). Pre-B cells in fetal liver also express CD19 (Hokland 1983,1985; Uckun et al., 1988a). In fact, a population of normal B cells that is CD19 negative has not been described, although in the latest stages of differentiation into plasma cells, CD19 is probably lost (Nadler, 1986).Normal cells other than B cells d o not react with C D 19 antibodies except for kidney tubule cells

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(Nadler, 1986) and possibly dendritic reticular cells (Mason et al., 1986). Bone marrow progenitor cells (CFU-GM, CFU-E, CFUGEMM) are CD19 negative, implying that CD19 expression does not begin before the pro-B cell stage of differentiation (Uckun et al., 1988b). CD19 is also strongly expressed in EBV-transformed cytoplasmic p chain-pro-B cell lines (Muraguchi et al., 1988)and cyto-p-ALL (Nadler, 1986; Pezzutto et al., 1986), implying that it is expressed prior to H chain rearrangement. I n addition to its expression on all normal B cells, CD19 is also expressed on almost all malignancies of B cell origin. CD19 is more reliable marker fro B lineage cells than CDlO (CALLA)or CD9 (p24), and the presence of p gene rearrangements establishes that B cell origin of the CD19' ALL (Nadler et al., 1984). The density of CD19 expression is highest on B cell ALLs (we estimate 1-5 x lo5 molecules/cell), but is considerably lower on normal B cells and B cell chronic lymphoctyic Ieukemias and B cell lymphomas (we estimate lo4 to 10' molecules/cell). CD19 is not present on malignancies of T cell origin; thus CD19 is the most B cell-restricted and the most expressed B cell antigen. However, some acute myelomonoblastic and monoblastic leukemia cells express CD19 (Campos et al., 1987), indicating that CD19 may be expressed at certain stages of' monocyte differentiation. CD19 is a heavily glycosylated glycoprotein of approximately 95 kDa. Only one epitope or region of the molecule seems to be recognized by CD19 MAbs, since all CD19 MAbs cross-block each other (Clark and Einfeld, 1986; Uckun et al., 1988a). The CD19 epitope recognized may be carbohydrate, since treatment with endoglycosidase F eliminated reactivity of all CD19 MAbs (Moldenhauer et al., 1986).A complementary DNA for the CD19 gene has recently been isolated by I. Stamenkovic and B. Seed (personal communication), which predicts a protein 51.8 kDa in size and containing one transmembrane region and a long (150 residues) serine/threonine-rich cytoplasmic tail. The extracellular portion of CD19 has five potential N-linked glycosylation sites and more than 70 serines and threonines for potential O-linked glycosylation. CD19 is a member of the Ig supergene family because its three extracellular Ig-like domains have strong homologies with the human Ig C or V regions. The CD19 antigen internalizes readily after being bound by MAb (Uckun et al., 1988). Thus, immunotoxins constructed from CD19 MAbs are highly effective in elimination of clonogenic tumor cells in bone marrow. Their activity has been shown to be potentiated further by cytotoxic drugs (cyclophosphamide derivatives) (Uckun et al.,

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1985a,b). Effective immunotoxins have been made using either ricin or pokeweed antiviral protein conjugated to a CD19 MAb (B43) (Uckun et ul., 1985a,b). CD19 appears to represent an attractive target antigen for drug or toxin delivery in vivo because of its widespread expression on B cell malignancies and lack of expression outside the B cell compartment (Pezzutto et al., 1986). CD19 is likely to have an important functional role, since CD 19 expression seems to be obligatory for B cells. Little was known of the function of CD19 until CD19 MAbs were found to block proliferation of normal resting B cells in response to stimulation of surface Ig (Pezzutto et al., 1987b,c). The inhibitory signal required CD19 cross-linking, and was not dependent upon the Fc region of the CD19 MAb used (HD37). The inhibitory activity of anti-CD19 correlated with its ability to modulate cytoplasmic calcium concentration ( [Ca"] i ) . Although cross-linking of CD19 alone increases [Ca2+Ii,B cells are not activated to progress from Go to GI. CD19 cross-linking also blocks subsequent [Ca"], increases in response to anti-p stimulation (Pezzutto et al., 198713). In addition, cross-linking CD19 simultaneously with anti-p and antid shows that the CD19 and the antigen receptor do not give additive signals (Fig. 5). Thus CD19 functions as a receptor that regulates cytoplasmic calcium concentrations, and the signal is closely related to the signal

i

0

5

10

Time ( m i d

FIG.5. Cytoplasmic calcium mobilization through CD19 and surface 6. Peripheral blood mononuclear cells were indo-1 loaded and B cells were analyzed for [Ca"]], response by gating on CD20' cells using phycoerythrin-2H7 staining (Ledbetter et al., 1988). The signal from anti4 stimulation was seen immediately after addition of 2.5 pg 6TA4-1 M.4b at 2 min. The signal from CD19 stimulation was seen after 10 p g B43 MAb addition at 2 min, followed by additional cross-linking with 40 +g ofanti-rc MAb 187.1 at 5.5 min. Simultaneous stimulation of CD19 and 6 with 10 e g B43 at 2 min was followed by 2.5 p g 6TA4-1 and 40 p g 187.1 at 5.5 min.--, anti-6; * * * , anti-CDlg+anti-K; ---, anti-CD 19+anti4 + anti-rc.

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from surface antigen receptors. In the absence of extracellular calcium, CD 19 cross-linking still increased [Ca2+Ii, indicating that calcium mobilization from cytoplasmic sources was affected; the [Ca2+J increase was much greater when extracellular calcium was present, showing that calcium channels also open after CD19 crosslinking (Pezzutto et al., 1987b,c). Although inhibitory for normal B cells, the B43 CD19 MAb stimulates proliferation of malignant pre-B cell progenitors (Ledbetter et al., 1988).CD19 cross-linking increases [Ca2'Ii on many Ig-negative pre-B cell ALLs, suggesting that CD19 is a receptor for an important growth regulatory signal at the very earliest stages of B cell development. Further support for this idea comes from observations that CD19 expression correlated with the cloning efficiency of B lineage lymphoid progenitor cells and was more highly expressed on B cell ALLs that had higher percentages of S phase cells (Uckun et al., 1988). CD19 expression is also upregulated by growth-promoting stimulation of ALL cells with B cell growth factors (Uckun et al., 1988). The requirement for cross-linking for CD19-mediated inhibition or stimulation of proliferation and for CD19-mediated effects on [Ca2+fIi suggests that these are functionally related responses. Figure 5 shows the effects of CD19 cross-linking with a monoclonal anti-light-chain second-step MAb on the kinetics of [Ca2+li response in normal peripheral blood B cells. We have observed that there are differences among normal or malignant B cell populations in the degree of CD19 cross-linking required for signal transmission (Uckun and Ledbetter, 1988). D. CD21: RECEPTORFOR C3d

AND

EPSTEIN-BARR VIRUS

The CD21 antigen was first identified with the B2 MAb (Nadler et al., 1981a). CD21 expression is restricted to B cells and is present in highest amounts on mature B cells in lymphoid tissues. B cells in peripheral blood and in bone marrow express CD21 in very low levels (Nadler, 1986). After activation in uitro by, e.g., anti-Ig or TPA, B cells lose CD21 simultaneously with loss of IgD (Stashenko et al., 1981; Aman et al., 1984; Boyd et al., 1986; Freedman et al., 1986). B cell subpopulations with the phenotypes CD20' CD21' and CD20' CD21- were isolated from lymphoid organs and were shown to be functionally distinct (Anderson et al., 1985). The CD20+ CD21-t B cells were IgM and IgD positive and could proliferate in response to anti-p plus growth factors, whereas the CD20+ CD21- B cell

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population was IgM and IgD negative but did contain higher frequency of IgG-positive cells. Thus, the CD20' CD21- B cell is more activated or differentiated, and the CD21 antigen appears to be lost during in vivo activation. Because CD21 is not detectable on pre-B cell ALLs (see below), it is possible that it first appears about the time surface Ig is expressed, remained on the surface only for a fairly narrow period of time during B cell differentiation. CD21 is a receptor for EBV, and EBV can transform both pro-B and pre-B cells (Hansson et al., 1983; Muraguchi et al., 1988),implying CD21 may be expressed on some early B lineage cells. Frozen sections of lymphoid tissues showed CD21 expression in the mantle zone and strongly in germinal center areas of secondary follicles (Bhan et al., 1981). However, the CD21' cells in follicles are follicular dendritic cells; the germinal center B cells are buoyant, activated B cells that express little or no surface CD21 (Clark and Einfeld, 1986; Mason et al., 1986; Ling et al., 1987). Unlike CD19 and CD20, CD21 is not expressed on the majority of B cell malignancies. CD21 is expressed on only a small subset of non-T ALLs, but is expressed by most B cell CLLs and about half of B cell lymphomas (Nadler, 1986). Interestingly, the CD2l-positive B cell lymphomas are usually poorly differentiated (centrocytic) lymphomas (Anderson et al., 1984). Although normal T cells and myeloid cells do not express CD21, some T cell ALLs reacted with CD21 antibodies. There were differences in reactivity with CD21 antibodies against distinct epitopes, suggesting that CD21 epitopes are differentially expressed by T cell ALLs (Nadler, 1986). The CD21 antigen is a nonphosphorylated 140-kDa glycoprotein (Oettgen et al., 1983) that is 110 kDa in size when N-linked sugars are removed (Bou6 and LeBien, 1988). CD21 was phosphorylated after treatment with PMA, suggesting that CD21 function may be regulated by protein kinase C (Changelian and Fearon, 1986; Bou6 and LeBien, 1988). It has been identified as the C3d receptor (CR2) (Iida et al., 1983; Weis et al., 1984). These studies established identity between the 140-kDa C3d-binding protein and the CD21 antigen and showed that the B2 and HB5 antibodies did not block the C3d-binding region of CD21. Other CD21 MAbs have been described that do block C3d binding to CD21 (Rao et al., 1985). CD21 has also been identified as a receptor for EBV (Fingeroth et al., 1984; Frade et al., 1985a; Nemerow et al., 1985a)because purified CD21 could bind radiolabeled EBV and CD21 MAbs could block EBV binding to B cells. Thus the CD21 glycoprotein binds to C3d and

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to EBV in distinct regions. In fact, EBV binding to cell lines is enhanced in the presence of C 3 and C3d (Wells et al., 1984). However, CD21 may not b e the only or complete EBV receptor, since antibodies against CD21 could not block EBV binding to cells that expressed C3b and C3bi receptors in addition to C3d receptor (CD21) (Wells et al., 1983).Furthermore, class II-specific antibodies can also block EBV binding, suggesting that HLA class I1 molecules may contribute to or influence the EBV receptor (Reisert et al., 1985). The CD21 antigen bears some homology with receptors for C3b and C4b, based upon its predicted sequence from a partial cDNA clone (Weis et al., 1986). This could be relevant to the detection of some CD21 epitopes on T cell ALLs; the antibodies could be cross-reacting with a distinct but related receptor for C3 on these cells. The CD21 antigen internalizes readily when bound by antibodies or by EBV (Tedder et al., 1986b),and thus, like CD19, can deliver immunotoxins efficiently. The function of CD21 has received considerable attention recently, following the discovery that CD21 MAbs can stimulate proliferation of normal resting B cells (Frade et al., 1985b; Wilson et al., 1985; Nemerow et al., 1985b). Similarly, aggregated C3d fragments of complement have growth stimulatory activity for B cells (Melchers et al., 1985; Melchers and Anderson, 1986). However, soluble C3d is inhibitory, implying that microaggregation or cross-linking of CD21/ CR2 may b e required for a positive signal to B cells (Melchers and Andersson, 1986).Not all CD21 MAbs were active, indicating that the precise epitope of CD21 may be important for functional activity (Nemerow et al., 1985a). In addition, one CD21 MAb has been reported to inhibit BCGF-dependent B cell proliferation (Perri et d., 1986). F (ab’)zfragments of OKB7, a stimulatory CD21 MAb, retained the ability to stimulate proliferation. T cells were required for B cell proliferation (Nemerow et al., 1985b; Wilson et al., 1985). Although the transmembrane signaI deIivered by CD21 MAbs has not been well characterized, one report has described membrane potential changes following EBV binding (Rosenthal and Shapiro, 1983) and other reports have described both membrane depolarization and increases in ctyoplasmic calcium following CD21 MAb binding (Kay et al., 1987; Rabinovitch et at., 1987).Thus the 140-kDa CD21 antigen appears to contain discrete functional domains involved in EBV binding, C3d binding, and stimulatory or inhibitory activity for B cell activation. Several of the CD21 MAbs have been shown to have functional activity that appears to be dependent upon the domain that expresses the epitope recognized (Siaw et al., 1986).

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E. CD22: A BRIDGEFOR SURFACEIg? may b e malignancies of immature B cells at a stage just prior to CD22 surface expression. How cytoplasmic versus surface CD22 expression is regulated is not yet known. However, some cells seen in CD22B-CLL can be induced by PMA to express surface CD22 (Campana et al., 1985; Schwartz et al., 1986). CD22' B cells stimulated with anti-Ig or protein A cease to express CD22 within 3 to 4 days after activation (Freedman et al., 1986; Dorken et al., 1986a). Using two-color flow cytometric analysis and labeled MAb to IgD and CD22, Pezzutto et al. (1987e, 1988) detected three major IgM+ B cell subsets in blood or tonsils: an IgD+ CD22+ subset enriched in dense fractions, and IgDCD22+ subset, and an IgD- CD22- subset enriched in buoyant fractions. Their results suggest that after cell activation, IgD is lost first from the cell surface, then CD22 is lost, followed by IgM. This model is consistent with the expression of CD22 in lymphoid tissues, where it is found on follicular mantle and marginal zone B cells and not on germinal center cells (Mason et al., 1986; Dorken et al., 198613). CD22 MAbs have defined at least three epitopes on CD22, all of which differ in their expression (Moldenhauer et al., 1986; Mason et nl., 1986), with epitope A defined by HD39 having a more restricted expression on B cell malignancies than epitope B (HD6) (Moldenhauer et al., 1986). Epitope C, unlike the other epitopes, is found on some T cell and myeloid malignancies (Dorken and Moldenhauer, 1987); this distinct tissue distribution of epitope C, and the finding that COS cells transfected with a vector containing a cDNA for CD22 express only epitope A and not C (I. Stamenkovic and B. Seed, personal communication, see below), suggest that expression of epitope C may b e under distinct gene control. All three CD22 epitopes are destroyed by treatment with endoglycosidase F, suggesting that they are found on the carbohydrate portion of the CD22 molecule. CD22 is a heterodimer with noncovalently bound chains approximately 130 and 140 kDa in size under reducing conditions (Schwartz et al., 1986; Moldenhauer et al., 1986; BOLGand LeBien, 1988) and 120 and 130 kDa in size under nonreducing conditions (Bou6 and LeBien, 1988). These shifts in mobility suggested that the CD22 chains have intrachain disulfide bonds. This is consistent with the protein sequence predicted from the cDNA sequence of CD22 (see below). Although CD22 and CD21 chains are of a similar molecular weight, the protein backbone of CD21 is larger than that of CD22 and the peptide maps of CD21 and CD22 are quite distinct (Boue and

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LeBien, 1988).The origin and relationship of the two chains of CD22 are not yet firmly established. Moldenhauer and co-workers (1987) found that CD22 treated with Endo F to remove N-linked carbohydrate chains migrated as two distinct bands approximately 85 and 100 kDa in size. By contrast, Bou6 and LeBien (1988) found that N glycanase-treated CD22 migrated as one band about 110 kDa in size. Further, they found that (1)V8 protease-digested peptide maps of the two CD22 forms were virtually indistinguishable, and that (2) in pulse chase experiments, the lower molecular-weight form is evident first and then later both forms are detectable. Their results strongly suggest that the two CD22 forms have a precursor/product relationship and are derived from the same chain. Whereas Moldenhauer and co-workers used Endo F to digest CD22 and consistently detected a 140-kDa CD22 chain on a B lymphoblastoid cell line, Bou6 and LeBien used N-glycanase and detected a 140-kDa chain only occasionally on the Raji BL line. The differences between the two groups may be explainable by use of different methods with different cell lines (see below). Bou6 and LeBien also found that CD22 is constitutively phosphorylated and that the level of phosphorylation of CD22 is augmented by PMA. It is not yet clear whether or not one or both chains are selectively phosphorylated. Recently, Stamenkovic and Seed (198813) have isolated a cDNA encoding for CD22 using the IR method described above (see Section 11,B). This CD22 cDNA encodes for a protein 63 kDa in size, containing one transmembrane domain and a short cytoplasmic tail (Stamenkovic and Seed, 1988b). The extracellular portion has five domains with strong characteristic homologies with distinct members of the Ig supergene family. It will be of interest to learn whether each of these domains is encoded by a distinct exon. These regions of CD22 that have characteristic homologies with other proteins may have distinct functions. The extracellular portion of CD22 has 10 potential N-linked and 86 potential O-linked glycosylation sites. The 26-residue cytoplasmic tail has one threonine and one serine, which may be the sites phosphorylated after B cells are exposed to PMA (Bou6 and LeBien, 1988).An approximately 60-kDa product in fact is evident on gels displaying Endo F-treated CD22 (Moldenhauer et al., 1987). Thus, one possibility is that the enzymes used by the Heidelberg and Minnesota groups to cleave N-linked sugars did not completely remove all carbohydrate from the 62- to 63-kDa protein core. The different molecular-weight species of CD22 could have the same protein

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backbone but different amounts and types of N-linked or 0-linked glycosylation. Recent studies with MAbs to CD22 suggest that CD22 may have an initial function in the regulation of B cell proliferation. Some MAbs to CD22, most notably HD6 to epitope B, augment the entry into cell cycle, RNA synthesis, and proliferation of B cells triggered by anti-Ig (Pezzutto et al., 1987a,e). Furthermore, although anti-CD22 MAbs alone do not stimulate increases in intracellular free [Ca2+Iiin mature B cells, once bound to B cells, they do augment the ability of anti-Ig to induce increases in [Ca”li (Pezzutto et al., 1987e, 1988). To test whether or not surface CD22 may play a pivotal role in anti-Igmediated triggering of B cells, B cells were sorted into CD22+ and CD22- subsets and tested for their ability to mobilize calcium in response to anti-p or anti-6 MAb. Only CD22+ and not CD22- B cells could be triggered (Pezzutto et al., 1988). MAbs to CD22 were reported to inhibit induction of B cell differentiation (Golay et al., 1986), but this was not confirmed (Rawle et al., 1987, Forsgren et al., 1987). Why human B cells require CD22 to respond to the cross-linking of antigen receptors is not yet known. One possibility is that CD22 functions as a “bridge” for transmitting a signal to the cytoplasm once surface Ig is cross-linked. Surface Ig has only a three-amino acid cytoplasmic tail and thus probably either transmits its signal via a conformational change after antigen is bound or via interaction with another membrane molecule with a tail, such as CD22, to transmit an intracytoplasmic signal. CD22 has many of the features that might be expected for such a bridge: (1)it is only expressed on the surface of resting IgM+ B cells: (2) it is a membrane phosphoprotein with a cytoplasmic tail containing potential phosphorylation sites (see above); and (3)part of it has strong homology with immunoglobulin domains, an expected finding given that members of the Ig supergene family generally associate physically only with other members of the Ig gene superfamily. One argument against CD22 being a “bridge” is that no clear physical association with CD22 and surface Ig has yet been demonstrable, unlike the T cell receptor interaction with CD3. However, given that B cells and T cells can recognize antigen in rather different ways, it is possible that the bridges for T cells and B cells are quite different. A labile or low-affinity interaction with surface IgM and its bridge might have several important advantages: first, it might enable the bridge to interact with surface Igs of several different

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isotypes. Second, because B cells, unlike T cells, can bind soluble antigen without self-MHC, it may b e advantageous for a bridge molecule to associate with surface Ig for signaling only after antigen is bound by its B cell receptor. Finally, flexibility in surface Ig-bridge interactions potentially could be an important regulatory mechanism. Inappropriate binding/triggering of surface Ig without a CD22 bridge or with a different bridge is one possible mechanism that could lead to B cell tolerance. In sum, whereas T cell triggering is regulated via intercellular intermolecular interactions with self-MHC, antigen, and CD4, B cell triggering could be regulated in part via intracellular intermolecular interactions with, e.g., CD22.

F.

CD23: BOTHA RECEPTOR FOR IgE

AND A

SOLUBLE FACTOR

CD23 is a protein that has both soluble and membrane forms: it is upregulated by IL-4, can bind to IgE, and as a factor may drive activated B cells to divide either as part of a normal immune response or after EBV infection. How it functions to regulate both the IgE response and B cell proliferation currently is one of the major puzzles in B cell immunology. The CD23 story began when Kintner and Sudgen (1981) described a set of MAbs which reacted with a 45-kDa glycoprotein (Bp45) found on EBV-transformed B lymphoblastoid cell lines (B-LCL) and to a lesser extent on EBV+ Burkitt’s lymphomas (BL), but not on normal B cells or B cell tumor lines that were EBV-. About the same time, others described MAbs reacting with what turned out to b e the same antigen (Rowe et al., 1982; Slovin et al., 1982), while two other groups described B cell blast-associated antigens, BB-1 (Yokochi et al., 1982) and B-LAST-1 (Thorley-Lawson et al., 1982, 1985), that were distinct from CD23 (see Section V,B). Both B-LAST-1 and CD23 (BLAST-2) are 45-kDa species, but based on sequential immunoprecipitation analyses are distinct structures (Thorley-Lawson et aZ., 1985). Furthermore, CD23 is induced on B cells within 24 hr after activation, while B-LAST-1 and BB-1 are expressed much later (Thorley-Lawson et al., 1985; Thorley-Lawson and Mann, 1985; Clark et al., 1986a). In the second international workshop of 1984, one of the original anti-Bp45 MAbs, MHM6 (Rowe et aZ., 1982), was clustered into a new C D group, CD23 (see Nadler, 1986), together with two other MAbs, PL-13 and BLAST-2. In the third international workshop, another set of MAbs were identified that reacted with CD23 (Ling et al., 1987). Using the BLAST-2 anti-CD23 MAb, Thorley-Lawson et al. (1986) reported that CD23 is an N-linked glycoprotein with an isoelec-

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tric point of about pH 4.7. And, most interestingly, they found that CD23 is rapidly lost from the cell surface (th= 1-2 hr) and is shed as a 33-kDa polypeptide. Why should CD23 have both membrane and soluble forms? A partial answer came when two groups at the third international workshop reported that CD23 is a low-affinity receptor for IgE (FceR) (Kikutani et al., 1986b; Yukawa et al., 1987; Bonnefoy et al., 1987). This important observation brought together information from two fields, particularly because low-affinity FceR had been extensively characterized (Spiegelberg, 1984). In the first study, Kikutani and co-workers (1986b) isolated and characterized a cDNA for FceR. COS-7 cells transfected with the FceR gene were bound by IgE, anti-FceR MAb, and by known anti-CD23 antibodies. One anti-CD23 MAb blocked the binding of IgE while another anti-CD23 MAb to a different epitope precipitated the same 45-kDa structure precipitated by anti-FceR MAb. Similarly, Bonnefoy et al. (1987) found that anti-CD23 MAbs and their anti-FceR MAbs bound to the same structure although to distinct epitopes. Low-affinity FceR receptors have been detected on B lymphocytes, monocytes, eosinophils, and platelets (e.g., Spiegelberg, 1984; Delespesse et al., 1986). Their expression on T cells is controversial: whereas Yodoi and Ishizaka (1979) initially reported that T cells have FceR, some groups have confirmed this observation (Nakajima and Delespesse, 1986; Prinz et al., 1987), but others have not (Suemura et al., 1986; Kikutani e t al., 1986a; Rao et al., 1987). Recent evidence suggests FceR, while not on resting T cells, are expressed transiently on activated T cells (Prinz et al., 1987; Delespesse et al., 1986). Whether or not T cells have FceR is of particular interest, because several groups have reported that T cells produce IgE regulatory binding factors which in fact are soluble FceR. Two groups have shown that the amino acid sequence of B cell-derived soluble IgE binding factor is encoded by the CD23 gene (Kikutani et al., 1986b; Ludin et al., 1987).Furthermore, Kisaki et al. (1987) have found that IgE-binding factors derived from human T cell hybridomas were bound by an anti-CD23 antibody. These same hybridomas, however, were surface CD23-, implying that although they produce CD23+ material, CD23 epitopes are not expressed on their cell surface. Three groups independently isolated and sequenced cDNAs en1987; Ikuta et al., coding for CD23 (Kikutani et al., 1986a; Ludin et d., 1987). All three groups expressed their isolated cDNAs in appropriate cell lines and showed that the transfected cells bound IgE and anti-FceR-specific MAb. These cDNAs encode for a protein with 321

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amino acids with a molecular weight of 36,281. A hydropathicity plot showed that CD23 has a 21-amino acid hydrophilic N-terminal region which is followed by a hydrophobic stretch of 26 amino acids, typical of transmembrane domains. CD23, like CD20, does not have a signal sequence, implying that its N-terminus is also on the inside of the cell and that its C-terminus is on the outside of the cell (Fig. 4c). The fact that the soluble CD23 shed was found to be encoded by the Cterminal region further supports the notion that the C-terminus is outside the cell. The C-terminus also has one putative N-linked glycosylation site at position 63 on the extracellular side. Since the CD23 gene encodes for a 36.3-kDa polypeptide, and cell surface CD23 has an apparent molecular weight of 45,000 (Thorley-Lawson et al., 1986; Peterson and Conrad, 1985), it is likely that the CD23 is glycosylated at this site. Human CD23 shares about 30-40% of its sequence in common with human, rat, and chicken asialoglycoprotein receptors. In particular, the cysteine residues of CD23 and human asialoglycoprotein receptor are identical (Ludin et al., 1987). However, CD23 has no “homologies” with rat IgE-binding factors (Ikuta et al., 1987), which have quite distinctive structures. The N-terminal amino acid residue of soluble FceR is located at position 150 (Fig. 4c), and the preceding residue, arginine, is a common target for trypsinlike proteases (Kikutani et al., 198613). The region encoding for the soluble CD23 (residues 150-321) has two clusters of four cysteines each, which may form disulfide bridges to produce a tightly folded, protease-resistant structure. H. Kikutani, E. L. Barsumian, and co-workers (E. L. Barsumian, personal communication) recently have produced purified recombinant soluble CD23, and this material binds IgE. Swendeman and Thorley-Lawson (1987) have reported that partially purified soluble CD23 has an autocrinelike B cell growth factor activity. Their preparation also contained material about 12 kDa in size, so either 25-kDa soluble CD23, the 12-kDa protein, or a combination of the two proteins contributed to the BCGF activity. Whether purified recombinant CD23 has BCGFlike activity or binds to B cells is currently being evaluated. Because soluble CD23 may have biological activity, it was important to test how the shedding of soluble CD23 is regulated. Other molecules related to CD23 that have their N-terminus on the cytoplasmic side, including chicken hepatic lectin (Ashwell and Harford, 1982) and transferrin receptor (Hopkins and Trowbridge, 1983),internalize after they bind their ligands, but it should be noted that the affinity of ligand binding to, e.g., transferring receptors is much higher than IgE binding to low-affinity CD23. Indeed, relatively little surface CD23 is

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internalized after IgE is bound even while extensive shedding is underway (Barsumian et al., 1988). Certain protease inhibitors dramatically reduce shedding of soluble CD23, implying that shedding of CD23 may be required b y a protease-dependent pathway (Barsumian et al., 1988).The level of surface CD23 and CD23 shedding is dramatically increased by either IL-4 or IgE (Kikutani et al., 1986a,b; DeFrance et al., 1987; Guy and Gordon, 1987), which is of interest since IL-4 also induces increased levels of IgE (Lee et al., 1986; Paul and Ohara, 1987) and may play a vital role in IgE immunity to parasitic infections (Finkelman et al., 1986; Paul and Ohara, 1987). Together these results suggest that IL-4 regulates the IgE response both by stimulating IgE production and by increased production of low-affinity receptors for IgE, which presumably could bind to and help remove biologically active IgE from serum or tissue fluids (see Gordon and Guy, 1987). Gordon and co-workers (1986a,b, 1987a,b) have presented evidence that anti-CD23 MAbs have BCGF-like activity and have suggested that CD23 may be a receptor for a low-molecular-weight BCGF. Antibodies to one or three epitopes on CD23 were found to be costimulatory with TPA in inducing B cell proliferation (Gordon et al., 1986a, 1987a). Furthermore, anti-CD23 mimicked low-molecularweight BCGF (Mehta et al., 1985) activity, and blocked the absorption of this BCGF by B cells (Gordon et al., 1986b). Whole antibody, F (ab’)zfragments, and even Fab fragments (albeit at very high doses, 10-100 pg/106 cells) were costimulatory. Most interestingly, antiCD23 MAbs on beads or stuck to plastic were not stimulatory, suggesting that the antibody had to be internalized or induce internalization to be effective (Gordon et al., 1987b). Although provocative, these initial studies were not conclusive. Because rather crude BCGF was used, the absorption results were not definitive. More than saturating concentrations of anti-CD23 were also needed to obtain optimal stimulation (e.g., >10 pg/106 cells) for as yet undefined reasons. The apparent requirement for internalization of anti-CD23 contrasts with the results of Barsumian and co-workers, who found that IgE does not induce internalization of CD23. More recently, Guy and Gordon (1987) found that both BCGF and anti-CD23 induce decreased expression of CD23 on activated B cells, presumably by stimulating the shedding of surface CD23 into a soluble form. Since IgE and IL-4 up-regulate CD23 expression, it is possible that IL-4/ IgE and BCGF may function together to increase surface and then soluble CD23 levels, which may then function to regulate B cell growth (Gordon and Guy, 1987). Together, these studies imply that CD23 is not only (1)a receptor for IgE and (2) a soluble factor, but also

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may b e (3) a receptor for a BCGF, which presumably has some homology with IgE Fc domains that also bind CD23. Are not too many functions being ascribed to one surface molecule? One possibility is that anti-CD23 does not in fact act directly but rather promotes, e.g., the modulation of BCGF receptors and/or the shedding of soluble CD23, which has been reported to have BCGF activity (Swendeman and Thorley-Lawson, 1987). These issues can only be decisively answered by using recombinant BCGF and recombinant soluble CD23 material. It is possible that LT may be able to modulate CD23 levels, because high levels of LT are present in partially purified BCGF (Kehrl et al., 1987b). EBV-transformed lymphoblastoid cell lines (B-LCL) can be stimulated to proliferate by autocrine growth factors (Gordon et al., 1984). Swendeman and Thorley-Lawson (1987) reported that purified soluble CD23 stimulates proliferation of B-LCL and that autocrine growth activity in supernatants of B-LCL is specifically removed with antiCD23 MAb. They propose that EBV induces B cells to make autocrine CD23, which may function in EBV-mediated cell transformation. One EBV gene product that can initiate increased expression of CD23 is EBNA-2: The EBV-BL line, Louckes, when transfected with EBNA-2 but no other EBV genes, expresses elevated levels of CD23 protein and mRNA but shows no changes in the expression of other cell surface proteins, such as C3d receptors or activation antigens (Wang et al., 1987). Thus, the EBNA-2 gene product may directly or indirectly activate CD23 gene expression. However, EBNA-2 may not always selectively activate only CD23 and can also activate other B cellassociated molecules. Calender and co-workers (1987) have found that some EBV- lymphoma lines, when infected with EBV, also show changes in expression of, e.g., CD21, C3d receptors, and the B cell activation antigen Bac-1. Furthermore, some EBV+ BL, while expressing EBNA-2, are CD23- (Rowe et al., 1987). Recently, Bonnefoy and co-workers (1988) found that CD23 and class I1 DR molecules are closely associated on the surface of normal B cells or B cell lines. Some MAb specific for DR partially blocked the binding of IgE to CD23. CD23 was associated with DR but not DQ or D P molecules. The results are particularly intriguing since class I1 MHC may function as a transduction signal in B cells (see Cambier and Ransom, 1987).

G. CDw40/BLCa: RECEPTORS FOR B CELLPROGRESSION SIGNALS Recently, we and others defined 50- to 55-kDa polypeptides expressed on both normal B cells and on epithelial-derived malignan-

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cies (Chan et aZ., 1985; Paulie et al., 1985; Clark and Ledbetter, 1986a). Two MAbs, G28-5 and S2C6, were found to recognize the same epitope and to have similar functional activity (Gordon et al., 1987a) and were assigned to form a provisional cluster group, CDw40 (Ling et al., 1987). CDw40 is found on B cells and interdigitating cells but not on a range of nonhematopoietic normal tissues (Ledbetter et d., 1987d; Ling et al., 1987), but is expressed on melanomas and a variety of carcinomas and carcinoma cell lines, e.g., from the lung, colon, or breast (Ledbetter et al., 1987d). CDw40 is an acidic glycoprotein with an pI of 3.2 and is stable at low pHs or relatively high temperatures (Braesch-Anderson et al., 1986). Recently, Stamenkovic and co-workers (1988) isolated a cDNA encoding for the CDw40 protein using the expression vector-panning method described above. The gene encodes for a polypeptide 277 amino acids long containing a 20-amino acid signal sequence at the N-terminus and a 24-amino acid hydrophobic segment typical of transmembrane segments, about 172 amino acids from the N-terminus (Fig. 4d). Paulie and co-workers (1985) independently isolated the CDw40 protein and sequenced the first 35 amino acids at the N-terminus of CDw40. Their protein sequence matches the sequence predicted by the cDNA sequence and begins 20 amino acids after the first methionine, just after the end of the first hydrophobic domain. The fact that CDw40 has an N-terminal signal sequence indicates that the N-terminal region is oriented to the outside of the cell membrane. The predicted amino acid sequence of CDw40 has a highly significant homology with human nerve growth factor (NGF) receptor and a weaker yet significant homology with human growth hormone receptor and the neu oncogene. In particular, 19 of the 20 cysteine residues of CDw40 align with the cysteine residues of NGF receptor. These similarities strongly suggest that CDw40 is a receptor for a soluble factor. T h e approximately 172 amino acids in the N-terminal external region include 20 cysteines, implying that the region is tightly folded and has two potential N-linked glycosylation sites. The overall polypeptide portion of CD40 is about 28.3 kDa, indicating that about 20 kDa of the glycoprotein may be carbohydrate. The 60-amino acid cytoplasmic tail of CD40 has six threonines and serines, but no tyrosines, as potential sites for phosphorylation, and one threonine is a potential phosphorylation site for calmodulin kinase. We have proposed that the expression of CDw40 and MHC class I1 may be under common regulation (Ledbetter et al., 1987a). First, a comparison of serial tissue sections clearly shows that expression of CDw40 and class I1 MHC is at similar levels on both B cells and interdigitating cells (Ling et al., 1987). Second, the same competence

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signals that induce increased levels of class I1 MHC on B cells also induce increased expression of CDw40 (Ledbetter et al., 1987a; Clark et al., 1988c), including TPA, anti-Ig, anti-CD20, anti-Bgp95, and IL-4. This is true even though these same competence signals have very different effects on modulating expression of CD20 and CD23 Furthermore, acute lymphocytic leukemias that (Clark et al., 1988~). express little or no GDw40 are induced by low-molecular-weight BCGF to express high levels of both class I1 MHC and CDw40 (Ledbetter et al., 1987a; Cheerva et al., 1988). Finally, normal epithelial cells do not express CDw40 (Ledbetter et al., 1987d) but activated, dividing epithelial-derived tumors do. This suggests that, like class I1 MHC, both constitutive expression and the overall level of CDw40 expression is under regulatory control. Thus, it is possible that some of the same transactivating factors that regulate class 11 MHC also regulate CDw40. MAb to CDw40 can deliver a progression signal that augments the proliferation of activated B cells (Clark and Ledbetter, 1986a,b; Ledbetter et al., 1987a; Gordon et al., 1987b). Anti-CDw40 is costimulatory with competence signals including anti-IgM, anti-CD20, anti-Bgp95, and TPA, but is not costimulatory with IL-4 or lowmolecular-weight BCGF (Ledbetter et al., 1987a; Valentine et al., 1988; Clark et al., 1988~).The effects of anti-CDw40 can be distinguished from those of low-molecular-weight BCGF. For instance, some B cell malignancies, in particular follicular center cell lymphomas, can be stimulated with anti-CDw40 but not BCGF (Ledbetter et al., 1987a; Beiske et al., 1988). The properties of anti-CDw40 suggest that CDw40 may normally function as a receptor for a cell-cell or soluble growth signal. The tissue distribution of CDw40 does not correspond to any known growth factor receptors, but the CDw40 gene has clear homology with the NGF receptor. Inui and co-workers (1988) transfected the CDw40 gene isolated by Stamenkovic et al. (1988) into a murine pre-B cell line M12. A MAb to CDw40 stimulated CDw40' cells but not CDw40- pare i t M 12 cells to stop proliferating. In this system, the cytoplasmic tail of CDw40 was required for signal transduction (S. Inui, personal communication). The responsive transformant provided us with an indicator system for testing whether any existing growth factors could stimulate the CDw40+ transformant. No soluble factor yet tested (human IL-1, IL-2, IL-3, IL-4, IL-5, GM-CSF, IFN-a, IFN-P, IFN-.)I,PHA supernatants, and neuroleukin effected the proliferation of the transformant. Thus, our working hypothesis is that CDw40 is a receptor for a soluble factor not yet described.

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BLCa: B Lymphocyte Carcinoma Cross-Reacting Antigen

About the same time that anti-CDw40 MAbs were described, Chan and co-workers (1985; Yip et al., 1987a,b) described two MAbs, MA5 and MA6, specific for human B cells and carcinomas. The MAbs were generated by immunizing mice with B-cell-associated antigens recognized by a serum from a patient with nasollharyngeal carcinoma (NPC). Anti-BLCa MAbs detect carbohydrate-associated epitopes on a 55-kDa glycoprotein. Although BLCa and CDw40 have similar molecular weights and tissue distributions, they appear to be distinct gene products (Clark et al., 1988a): (1)CDw40 is expressed on carcinoma cell lines but BLCa is not; (2) L cells and a pre-B cell line transfected with CDw40 cDNA express CDw40 but not BLCa; (3) and antiCDw40-specific MAbs or heteroantisera, unlike anti-BLCa, do not block the migration of BLCa in gels. Peptide maps should reveal how related CDw40 and BLCa are. Both anti-CDw4O and anti-BLCa can provide progression signals for B cells activated by PMA or anti-Ig, indicating that CDw40 and BLCa have related functions in B cell activation. However, anti-BLCa, unlike anti-CDw40, is not co-stimulatory with anti-CDBO, implying that the CDw40 and BLCa progression signals are distinct. In sum, CDw40 and BLCa show strong similarities in size, function, and expression, and thus may function some way together in regulating cell growth. In 1979, Wang and co-workers, using a heteroantiserum, described a 54-kDa antigen, gp54, expressed on human B cells and B lymphoblastoid cell lines. Antisera to gp54 stimulated tonsillar B cells to proliferate; however, unlike anti-CDw40 or anti-BLCa MAb, which require activation signals to have an effect, anti-gp54 serum could stimulate tonsillar B cells without additional costimulants. This is probably because activated B cells were present in the tonsillar B preparation. In their discussion, Wang et al. mention that anti-gp54, like anti-CDw40 (Ledbetter et al., 1987d), reacts with carcinoma cell lines, indicating that gp54 may indeed be related to CDw40 and/or BLCa.

H. Bgp95: A UNIQUE95-kDa GLYCOPROTEIN ANTIGENINVOLVED IN B CELLACTIVATION We recently produced an antibody, G28-8, that was classified as CD39 by the third international workshop studies (Ledbetter et al., 1987c; Ling et al., 1987). However, our own studies showed that G28-8 recognizes a 95-kDa B cell-associated antigen (designated

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Bgp95) that is not related to the CD39 antigen (Valentine et al., 1988). The Bgp95 antigen contains N-linked carbohydrate, since it was 70 kDa after endoglycosidase F treatment. Furthermore, Bgp95 and CD39 showed different patterns of expression on cell lines, although both markers are not expressed on most BL lines (Valentine et al., 1988). The CD19 antigen, although similar in size to Bgp95, is also distinct because of differences in expression and function (see below). In functional studies, stimulation by G28-8 MAb or its F ( a b ' ) ~ fragments was distinct from that of antibodies binding to CD20, CD19, CD39, and CDw40 proteins. Anti-Bgp95 induced increased class 11 MHC expression on B cells and a Go to GI cell cycle transition, and it was synergistic with IL-4, PMA, anti-p, or antiCDw40 in stimulating proliferation of resting B cells (Clark et al., 1988c; Valentine et al., 1988). Bgp95 also induced an increase in [Ca2+lj in a subpopulation of tonsillar peripheral blood B cells. Although the Bgp95 MAb alone induced a steady increase in [Ca2+]i detectable even 1 to 2 hr after stimulation, cross-linking the G28-8 MAb with a second MAb specific for murine K light chains induced a rapid increase of [Ca2+Iiwhich peaked at 10 to 20 min and then declined (Fig. 6). The same conditions of cross-linking which increased the kinetics of the calcium flux abrogated the proliferative response, which otherwise followed coincubation of the MAb with BCGF or PMA (Valentine et al., 1988). Thus, conditions leading to rapid [Ca"li increase may not be as effective at stimulating B cell proliferation as conditions favoring a slower prolonged [Ca2+li rise. Although the Bgp95 molecule is present on activated buoyant tonsillar B cells, it did not trigger calcium fluxes in these cells. These results suggest that the Bgp95 protein may function in early B cell activation and that its signal mechanisms are altered by the activation state of the cell. Ill. Other Biochemically Defined Surface Molecules on Pre-B and/or B Cells

A. CD10: THECOMMON ACUTELYMPHOCYTIC LEUKEMIA ANTIGEN The common acute lymphoblastic leukemia-associated antigen (cALLA/CD10) was first defined by Greaves et al. (1975), using a rabbit polyclonal antisera, and later by Ritz et al. (1980), using a MAb. The results of initial studies suggested that CALLA expression was restricted to non-T, non-B ALL cells (Brown et d . , 1975; Ritz et al.,

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Time (min)

130

223 [CO'']

338 i

FIG.6. Bgp95 cross-linking regulates calcium concentration in resting B cells. Dense E- tonsillar B cells were indo-1 loaded and analyzed (Pezzutto et al., 1987d). (4) Calcium response in the first 10 min of stimulation. The response to 10 mg of ,MAb G28-8 anti-Bgp95 added at 2 min (-) is compared to the response from 10mg G28-8 at 2 min, followed by cross-linking with 40 mg of MAb 187.1 anti-rc added at 5.5 min. (---). The response to stimulation of surface IgM [lo pg F (ab'), anti-y] added at 2 min is shown (B) Mean calcium concentration of the population at 1 hr after stimulation. Control unstimulated cells (-) compared to 10 pg F (ab'), anti-p (--.), 10 pg G28-8 anti-Bgp95 (---), or 10 p g G28-8 plus 40 p g 187.1 (--) (Valentine et al., 1988). ( e - 0 ) .

1980). Subsequent studies, however, have shown that CALLA is also expressed on the surface of a wide variety of other normal and neoplastic cells types, including fibroblasts, renal epithelium, bone marrow stroma, granulocytes, lymphomas, and nonhematopoietic tumors (Greaves et al., 1980; Braun et al., 1983; Ritz et al., 1981; Carrel et al., 1983; Keating e t al., 1983; Metzgar et al., 1981; Platt et al., 1983; Cossman et al., 1983). The expression of CALLA by these diverse cell types is unlikely to be due to passive uptake of antigen, since cultured normal fibroblasts express CALLA (Braun et nl., 1983) and normal granulocytes synthesize CALLA (McCormaek et al., 1986; Pesando et al., 1986). The CALLA antigen has been characterized biochemically as a glycoprotein of approximately 100 kDa (Newman et al., 1981). The

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precise molecular weight varies, depending upon the source of antigen, most likely due to variations in carbohydrate content (Braun et al., 1983; Pesando et al., 1980; McCormack et al., 1986). CALLAexpression on pro- and pre-B cells but not mature B cells has aided the characterization of early B cell differentiation in fetal liver and bone marrow (Hokland et aZ., 1983). Pro-B cells are CALLA positive, but earlier progenitors measured in colony-forming assays (CFU-GM) are CALLA negative (Hokland et al., 1983). Little is known, however, of the function of the CALLA antigen. One report has indicated that granulocyte chemotaxis was inhibited by a CALLA/ CDlO MAb, whereas no effects on aggregation or degranulation were seen (McCormack et al., 1986). In addition, CALLA readily internalizes (modulates) in response to MAb, and the rate of internalization appears to vary depending on the affinity of the antibody used (Pesando et al., 1983; Braun et al., 1983; LeBien et al., 1982).

B. CD24 The first MAb made to CD24 was BA-1 (Abramson et al., 1981), and the CD24 cluster was established with MAb developed by Tedder and co-workers (1983). CD24 is expressed on B lineage cells and granulocytes but not on T cells or monocytes. CD24 is not restricted to hematopoietic tissues and can be found on, e.g., tonsillar epithelial cells, neuroblastomas cells, and fetal kidney nephron (Platt et al., 1983; Hsu and Jaffe, 1984; Stockinger et al., 1987). Some CD24 epitopes are expressed on plasma cells, vascular endothelium, and some myeloid and T cell leukemias (Nadler, 1986; Ling et al., 1987; Stockinger et al., 1987). Nevertheless, anti-CD24 MAbs have found use in evaluating B cell tumors, since CD24 is expressed on almost all B lineage cells and malignancies, with the notable exception of hairy cell leukemias (e.g., Melink and LeBien, 1983; Nadler, 1986; Dorken and Moldenhauer, 1987). The CD24 molecule is a single-chain sialoglycoprotein that migrates on gels as a broad band at about 40 to 45 kDa (Pirruccello and LeBien, 1986), most likely because of heterogeneity in its glycosylation. Initially, CD24 was thought to be three chains of 45,55, and 65 kDa (Pirruccello and LeBien, 1985), but the two higher molecularweight bands were subsequently shown to be artifactual IgG and IgM heavy chains (Pirruccello and LeBien, 1986).The association of CD24 with Ig suggests that CD24 might be a receptor for immunoglobulin. However, anti-CD24 did not bind to 45-kDa Fc receptors isolated

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from B cells (Pirruccello and LeBien, 1986). CD24 is similar in size to CD37, but has a different tissue distribution (Fig. 3). One possible clue to the still unknown function of CD24 is the fact that CD24 has several epitopes with characteristic expression, for example, expression on myelomas, neuroblastomas, and S6zary cells (HB8, epitope a), expression of S6zary cells and neuroblastomas (VIBES, epitope b), and expression on neuroblastomas (VIBC5, epitope c) (Stockinger et al., 1987; Nathan et al., 1987). Epitope c is lost rapidly after B cells are activated, and MAbs to epitope c do not block differentiation or costimulate with TPA in proliferation assays. In contrast, epitope b is not lost after B cells are activated and MAbs to epitope b inhibit B cell differentiation and costimulate in proliferation assays with TPA (Engle et al., 1987; Rawle et al., 1987; Rabinovitch et al., 1987). Other MAbs to CD24, including OKB2, have also been reported to inhibit pokeweed mitogen-induced B cell differentiation (Mittler et al., 1983; Rawle et al., 1987). Further work defining more precisely the expression and regulation of CD24 epitopes should help to clarify the function of CD24. C. CD37 In the second international workshop, based on cross-blocking studies, three MAbs, H H 1 (Funderud et al., 1983),HD28 (Dorken et al., 1986b), and BL14 (Brochier et al., 1984),were clustered into a new group, but the antigen recognized was not biochemically defined (Clark and Einfeld, 1986). In the third international workshop, the above MAbs and others were used to biochemically define CD37 as a 40- to 45-kDa glycoprotein (Ling et al., 1987). This marker is strongly expressed on mature B cells, B cell lines, and most B cell malignancies tested, but is not on B cell precursor cells or plasma cells (Brochier et al., 1984; Pallensen and Hager,1987; Ledbetter et al., 1987b). In normal hematopoietic tissues, Pallensen and Hager (1987) found CD37 weakly expressed on thymocytes, neutrophils, macrophages, Langerhans cells, and Kupffer cells. It was also weakly expressed on some nonhematopoietic tissues such as astrocytes, some neurons, and bladder epithelium. Others have also reported that CD37 is weakly expressed on some T cells and myelomonocytic cells (Dorken and Moldenhauer, 1987). However, because CD37 is most strongly expressed on mature B cells, it is a useful B cell marker. Within 24 hr after B cells are activated with TPA, CD37 is lost from the cell surface (Schwartz et al., 1987), and thus appears to be a marker for mature resting B cells.

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Schwartz and co-workers (1987) found that under both reducing and nonreducing conditions CD37 migrates over a broad range, from 36 to 52 kDa, depending on the cell line type. However, after treatment with endoglycosidase F to remove N-linked sugars, CD37 was reduced in size to 25 kDa and had no O-linked sugar chains. Previously, Zipf and co-workers (1983) had described a MAb 41H.16 that recognizes a 40-to 45-kDa B-cell-associated surface molecule. The epitope recognized by this MAb is different from that recognized by antiCD37 MAb and, unlike the CD37 epitope, is expressed on thymic epithelial cells and cell lines not expressing CD37 (Pallesen and Hager, 1987; Ledbetter et al., 1987b). Thus, 41H.16 may recognize a distinct epitope or antigen. The function of CD37 is not known. Some anti-CD37 MAbs weakly costimulate with anti-IgM and BCGF (Ledbetter et al., 1987b), but this effect is not dramatic. Also, anti CD-37 MAbs neither induce [Ca2'li fluxes (Rabinovitch et al., 1987) nor affect the induction of antibody-producing cells (Rawle et al., 1987). Fortunately, 1. Stamenkovic and B. Seed (personal communication) have recently isolated the cDNA encoding for CD37, which should be useful for defining the function of CD37.

D. CD39 In 1982 Rowe and co-workers described a MAb, AC2, that reacted with an 80-kDa surface polypeptide expressed principally on B lymphoblastoid cell lines but not on endemic BL lines. In the third international workshop, we found that our workshop MAb, G28-10, cross-blocked the binding of AC2 and also precipitated an 80-kDa molecule (Ledbetter et al., 1987c), enabling a new CD39 group to be formulated. Initially, MAb G28-8 was placed in the CD39 cluster, but further studies revealed that G28-8 recognizes a distinct larger surface polypeptide with similar yet distinct tissue distribution (Valentine et al., 1988; see Section 11,H). CD39 is expressed on all blood B cells, some tonsillar B cells, and weakly on monocytes. CD39' B cells are found in the follicular mantle and marginal zones of lymphoid tissues, but germinal center B cells are CD39- (Ling et al., 1987; Lueders and Feller, 1987). This restricted pattern is of particular interest since CD39, like CD23, is expressed on virtually all B-LCL but is not found on most BL lines (Rowe et al., 1982, 1987; Gregory et al., 1987a). CD39 is also found on some T cell clones, plasma cells, subepithelial macrophages, and on some smooth muscle and endothelium (Ling et al., 1987). CD39 is not expressed on pro-B leukemia, but is expressed

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on some pre-B leukemias and on a range of B-CLL, immunocytomas, centrocytic lymphomas, and plasmacytomas (Lueders and Feller, 1987). CD39 is a heavily glycosylated glycoprotein of about 80 kDa (Rowe et al., 1982; Ledbetter et al., 1987c) with a 55-kDa core when N-linked sugars are removed (Valentine et al., 1988);CD39 MAbs recognize an epitope expressed on the 55-kDa polypeptide. A cDNA encoding for CD39 has been recently isolated by I. Stamenkovic and B. Seed (personal communication) and is currently being evaluated. The function of CD39 is not yet known. G28-10 anti-CD39 cannot stimulate [Ca2'Ii fluxes and has little or no effect on the induction of B cell proliferation or differentiation, while G28-8 anti-Bgp95 stimulates [Ca2+Iiand increased RNA synthesis in B cells and inhibits Ig secretion in some assays (Rabinovitch et al., 1987; Rawle et al., 1987; Valentine e t al., 1988). Rowe and co-workers (1987) have found that EBNA-1' EBNA-2' BL express CD39 and CD23 and grow in clumps, while EBNA-1' EBNA-2- BL do not express CD39 and CD23 and grow as single-cell suspensions. This result implies that CD39 may be regulated by EBV genes and/or may contribute to homotypic adhesion in B cell lines. However, since markers other than CD39 and CD23 are also expressed on BL expression EBNA-2 and growing in clumps, it is also possible that CD39 and CD23 expression simply reflects a change in the overall differentiated state of the transformed B cells.

E. BLA: A GLYCOLIPID GLOBOTRIAOSYLCERAMIDE A MAb, 38.13, was produced by immunization with Burkitt's lymphoma cells and was found to react with Burkitt's lymphomas but not with normal or activated B cells or B cell CLLs or non-Burkitt's B cell lymphomas (Wiels et al., 1981, 1982).The antigen recognized was soluble in chloroform and methanol and was identified as a glycolipid globotriaosylceramide (Lipinski et al., 1982; Nudelman et al., 1983), also known as the rare blood group antigen PK(Fellows et al., 1985). The enzymatic basis for BLA expression on the cell surface is due both to synthesis by a-galactosyl transferase activity and to membrane organization controlled through interaction with sialosyl residues of a second glycoconjugate (Wiels et al., 1984a). Although normal B cells were originally thought to be negative for BLA expression, a small subpopulation of B cells in germinal centers has been identified as BLA+ (Gregory et al., 1987b; Fyfe et al., 1987). These cells have been postulated to be the normal counterpart of the

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EDWARD A. CLARK AND JEFFREY A. LEDBETTER

Burkitt’s tumor cells (Gregory et at., 1987b). immunotoxins prepared with the 38.13 antibody using ricin A chain or gelonin are effective with extremely rapid kinetics (50% inhibition of protein synthesis in 3 hr) (Wiels et al., 198413).The BLA immunotoxin studies also showed in some cell lines that BLA- cells by conventional phenotyping methods were specifically killed by the BLA immunotoxins, most likely reflecting their expression of very low levels of BLA. The expression of BLA on Burkitt’s lymphomas was down-regulated by treatment with phorbol esters, suggesting that BLA represents a differentiation antigen for a very restricted stage of B cell differentiation (Balana et al., 1985).

F. OTHERMOLECULESEXPRESSED ON RESTINGB CELLS In this section we describe a series of interesting B cell-associated surface molecules that have not yet been given CD nomenclature and are not yet as comprehensively studied as the antigens above. We describe only those molecules that are clearly distinct from the major CD markers, and have not discussed those MAbs made to B cellassociated markers not yet biochemically defined. Common leukocyte markers and other markers strongly expressed on both B and non-B lymphocytes are also not discussed. Table I1 lists some of these antigens and their characteristic properties. A number of these markers are also summarized succinctly by Zola (1987). In 1978 Balch and co-workers described with a heteroantiserum a 65-kDa molecule expressed on human B cells. Subsequently, Wang and her co-workers described a B cell-associated polypeptide of a similar size (68 kDa), also found on immature and mature B cells but no on plasma cells (Miki et al., 1982; Knowles et al., 1983; Wang et al., 1984). Of interest was the finding that their MAb induced the B-LCL, CESS, to produce more IgG (Miki et al., 1982). B. T. Huber and co-workers have previously defined in the mouse a 68-kDa B-cellassociated surface marker, Lyb3, and have shown that anti-Lyb3, like anti-BL2, promotes Ig secretion in B cells (see Kemp et al., 1983). Thus, based on both functional and biochemical similarities, it is likely that BL2 recognizes the Lyb3 homologue in humans. Autoantibodies in serums obtained from Wiskott-Aldrich patients also have been reported to promote B cell differentiation in uitro (Brouet et al., 1980), so it would be of interest to determine whether or not these sera recognize this 68-kDa B cell marker. Wang and co-workers, using MAb BL3, have described a 105-kDa B cell-associated polypeptide that has a more restricted B cell distri-

B CELL-ASSOCIATED SURFACE MOLECULES

123

TABLE I1 HUMAN B CELL-ASSOCIATED SURFACEPOLYPEPTIDES NOT YET GIVENCD NOMENCLATURE Size (kDa)

Antibody ~~

~

Antigen expressionlcharacteristics ~~

Reference

~

B Cells, pre-B ALL Early and mature B cells, MAb

Anti-BDA" BL2

65 68

FMC7 BL3 Anti-gp54"

105 105 54

41H.16 KB6 1

39-43 35-45

FMCl OKB4 Ki-B3

95 87 431220

stimulates Ig secretion Mature subset Mature B subset B cells/weak on T cells, antiserum stimulatoly B cells, granulocytes Mantle B cells, monocytes, granulocytes, macrophages Resting/activated B cells Most mature B cells IgD+ B cell subset

L23 L26

205 30133

IgD+M' B cells Pan-B marker

Balch et al. (1978) Miki et al. (1982) Brooks et at. (1981) Wang et al. (1984) Wang et al. (1979)

Zipf et al. (1983) Pulford et al. (1986) Brooks et al. (1980) Mittler et al. (1983) Leuders and Feller (1987) Takami et al. (1985) Ishii et al. (1984)

~~

a

Heteroantiserum and not MAb.

bution than does BL2. Similarly, Brooks and co-workers (1981; Zola, 1987) have described with MAb, FMC7, a 105-kDa B cell marker that is on a restricted B cell subset. Tedder and co-workers (1985a) also have developed a MAb, HB-4, that reacts with a biochemically undefined marker on a restriction subset of B cells. It will be important to compare these MAbs in a future workshop to determine whether or not they recognize the same structure. As already mentioned, Wang and co-workers (1979) developed antiserum to a 54-kDa polypeptide, which, based on its size, tissue distribution, and possible function, is likely to be CDw40 or BLCa (see Section 11,G). Zipf and co-workers (1983)made a MAb, 41H.16, that recognizes a 39- to 43-kDa glycoprotein and has a number of properties in common with CD37 (see Section 111,C). Pulford and co-workers (1986) have also made a MAb, KB61, that also reacts with a heavily glycosylated antigen of about 35-45 kDa and that is also found on granulocytes and resting B cells not activated B cells. Given their similarities, the 41H.16 and KB61 MAbs were compared, and based on sequential immunoprecipitation analyses, have been found to recognize the same molecule (D.Y. Mason, personal communication). Brooks and co-workers (1980) were the first group to describe a

124

EDWARD A. CLARK AND JEFFREY A. LEDBETTER

MAb to a human B cell-associated marker (accepted July, 1979), which they called FMC1. The F M C l marker is expressed on mature B cells but no on pre-B cells or a pre-B ALLs. Zola (1987)reported that F M C l recognizes a 95-kDa glycoprotein, a size similar to that of both CD19 and Bgp95. CD19, unlike FMC1, is expressed early in B cell development, and Bgp95, unlike FMC1, is not expressed on BL lines. Mittler and co-workers (1983) have also described a B cell-associated marker, OKB4, of a size similar (87 kDa) to that of CD19, Bgp95, and FMC1, but without direct comparisons it is difficult to determine the relationship, if any, of these markers. Two groups have described 205- to 220-kDa polypeptides selectively expressed on B cells that share features with the murine B220 marker (Coffman and Weissman, 1981) and the common leukocyterestricted marker CD45R first described by Dalchau and Fabre (1981). The MAb Ki-B3 (Lueders and Feller, 1987) precipitates proteins 43 and 220 kDa in size expressed on IgD' B cells present in mantle zones but not in marginal zones and weakly in germinal centers (Lueders and Feller, 1987; et aE., Ling et al., 1987). Ki-B3 also reacts with monocytes and some non-T All, BL, and B-LCL, but not with mature T cells. Takami and co-workers (1985) described a MAB, L23, that reacts with a similarly sized surface marker (205 kDa) also expressed selectiveIy on mantle zone B cells and only on some BL and B-LCL (Ishii et al., 1986). It is quite possible that L23 and Ki-B3 recognize the same molecule. The Ki-B3 MAb did not induce [Ca2+lifluxes but was a strong costimulant with anti-Ig to induce B cell proliferation (Rabinovitch et al., 1987). Furthermore, Ki-B3 clearly inhibited IgG secretion by the CESS cell line (Rawle et al., 1987). Lueders and Feller claim that Ki-B3 recognizes a form of the leukocyte common family of molecules (Ralph et al., 1987; Streuli et al., 1987). This is of particular interest given that anti-CD45R MAbs also are good costimulants, albeit with anti-CD3 in inducing T cell proliferation (Ledbetter et al., 1985b). Thus, it is possible that different forms of the common leukocyte family are selectively expressed on distinct lymphoid lineages yet have some common function in regulating cell activation. Ishii et al. (1984, 1986) defined a MAb L26 that precipitates two B-cell-associated polypeptides of about the same size as CD20,33 and 30 Kda, but, unlike CD20, the L26-defined polypeptides are expressed in the cytoplasm and not on the cell surface and are expressed in pro-B ALL and in plasmacytomas (Ishii et al., 1986; Lueders and Feller, 1987).

B CELL-ASSOCIATED SURFACE MOLECULES

IV. Receptors on

125

B Cells for Cytokines

As discussed previously, it is unlikely that many of the CD markers described above are receptors for known cytokines (Clark and Ledbetter, 1986b). I n this section we discuss cytokine receptors expressed on B cells and their possible relationship and interaction with B differentiation markers that are expressed at high levels on B cells. We do not describe receptors on B cells for nutrients or hormones. We refer the reader to a review by Plaut (1987). A. IL-2 RECEPTORS

IL-2 receptors are expressed on activated B cells but not on resting B cells (Tsudo et al., 1984; Waldmann et al., 1984; Jung et al., 1984). These receptors bind anti-CD25 (TAC) MAb and radiolabeled IL-2 (Waldmann et al., 1984). The IL-2 receptors on B cells are functionally active, since anti-TAC blocks B cell differentiation in response to PWM (Depper et al., 1983), and IL-2 enhances the growth and differentiation of activated B cells (Muraguchi et al., 1985; Jung et al., 1984; Mittler et al., 1985; Suzuki and Cooper, 1985; Nakagawa et al., 1985). B cell malignancies such as hairy cell leukemia and B cell lymphomas also express functional IL-2 receptors (Korsmeyer et al., 1983b; Laurent et al., 1986). B. IL-4 RECEPTORS B-cell-stimulating factor 1 (BSF-1, IL-4) was originally characterized as a cytokine that is costimulatory with anti-IgM to augment B cell proliferation (Howard et al., 1982), but it is now known that BSF-1 has a number of biological effects on a variety of cell types other than B cells (Paul and Ohara, 1987). Using either radiolabeled recombinant (Park et al., 1987a) or natural murine BSF-1 (Ohara and Paul, 1987), the receptors for BSF-1 have been found on a broad number of tissues at levels ranging from approximately 30 to 3000 receptors/cell. More recently, Park et al. (1987b), using125I-labeled human recombinant BSF-1, have characterized human BSF-1 receptors. Radiolabeled BSF-1 bound rapidly and specifically to a single class of high-affinity receptor (100 to 2500 receptors/cell), with a K , of about 0.5-1.Ox lo-'' M . Human BSF-1 receptors were expressed on a range of cell lines, such as B cell, T cell, monocyte, or bladder-derived tumor cell lines, and also bound to normal fibroblasts and epithelial cells. Blood

126

EDWARD A. CLARK AND JEFFREY A. LEDBETTER

lymphocytes expressed low levels of BSF-1 receptors, and activation induced increased expression. Using affinity cross-linking, the human BSF-1 receptor was found to be about 140 kDa in size, which is different from the 60- to 70-kDa size reported for the murine BSF-1 receptor (Ohara and Paul, 1987; Park et al., 1987a).The reason for this difference is not clear but one possibility suggested by Park et al. (198713)is that the murine BSF-1 receptors isolated were degraded by proteases.

C. IL-6 RECEPTORS The receptor for human BSF-2 (IL-6) has also recently been characterized (Taga et al., 1987) and resembles the BSF-1 receptor in several of its features. Radiolabeled BSF-2 also bound specifically and rapidly to a single class of high-affinity receptors (80 to 11,000 sitedcell), with a K , of about 2 ~ 1 0 M - .~ BSF-2 ~ receptors were expressed at low to moderate levels on EBV-transformed lymphoblastoid cell lines but were not detectable on Burkitt’s lymphomas lines of T cell lines. A plasmacytoma cell line, U266 (22,000 sitedcell), expressed the highest level of BSF-2 receptors, and some nonlymphoid lines expressed detectable levels. Activated B cells expressed BSF-2 receptors but resting B cells did not. In contrast, resting T cells expressed more BSF-2 receptors than did activated T cells. The results suggest that for B cells BSF-2 receptors are only expressed after activation and increase as B cells mature to plasma cells. This is in accord with the known effects of BSF-2, which stimulates activated B cells to mature into antibody-producing cells (Kishimoto and Hirano, 1988). D. RECEPTORSFOR OTHERFACTORS

A number of well-defined factors such as IL-1 (Lipsky et al., 1983; Falkoff et al., 1983), IFN-a (Harfast et al., 1981; Peters et al., 1986), IFN-7 (Romagnani et al., 1986), and transforming growth factor /3 (Kehrl et al., 1987b), have been reported to modulate B cell proliferation or differentiation. High-affinity receptors for IL-1 have been detected at low levels (100-200 sitedcell) on B cells and B cell lines (Dower et al., 1985; Matsushima et al., 1986), and this receptor is about 60 kDa in size. The B cell-associated receptors for the other factors require further characterization. Maize1 and co-workers have characterized a 12-kDa BCGF and purified it to homogeneity (Mehta et al., 1985). This iodinated BCGF

B CELL-ASSOCIATED SURFACE MOLECULES

127

(low) binds to high-affinity receptors expressed on activated B cells but not on resting B cells (Mehta et al., 1986). Other interleukins, including IL-1 and IL-2, and IFN-y did not block the binding of the BCGF (low), and the BCGF (low) receptors were expressed on cells not expressing IL-2 receptors. Since both LT and TNF bind to receptors on activated B cells and have BCGF activity (Kehrl et al., 1987a,b), it is necessary to determine whether the 12-kDa BCGF of Mehta et al. is LT or TNF or binds to the same or a different receptor than LT and TNF. Ambrus and co-workers have characterized a 60-kDa BCGF produced by certain B and T cell lines (Ambrus and Fauci, 1985; Ambrus et al., 1985). This BCGF (high) bound to activated B cells but not to activated T cells or resting T or B cells, and the binding of the BCGF was specifically inhibited by BCGF but not by IL-2 (Ambrus et al., 1985). What might be the relationship between cytokine receptors and the CD receptors discussed in this review? The IL-1, IL-4, and IL-6 receptors are expressed in very low densitites and thus are probably not related to known CD antigens, which are present in higher numbers. However, the high-affinity receptor for IL-2 is composed of two chains (Smith, 1987). Thus, it is possible that some growth factor receptors have one chain expressed at high levels on resting cells, and another chain with more limited expression. Some CD receptors might function as components or subunits of this kind of growth factor receptor. However, we believe it is more likely that many of the known pan-B markers will prove to be receptors of cell surface-bound ligands, particularly since they are expressed at such high levels: high-affinity receptors for soluble factors tend to be expressed at low levels (10' to 103 sitedcell), while surface molecules involved in cell-cell interactions with cell-bound ligands have lower receptor affinities but are expressed at higher levels on cells (lo4 to lo5 sitedcell). V. Other Surface Molecules Expressed on Activated B Cells

IL-2 receptors were found on activated T cells but not on resting T cells (see Cantrell and Smith, 1984), so it was possible that receptors of B cell-stimulating factors might also be selectively expressed on activated B cells. As described above, while this is true for IL-2 and BCGF (low) receptors on B cells, this is not the case for B cell IL-1, IL-4 or IL-6 receptors. Nevertheless, a number of MAbs have been made that react selectively with structures on activated B cells (Table

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EDWARD A. CLARK AND JEFFREY A. LEDBETTER

TABLE 111 SURFACE MOLECULESSELECTIVELY EXPRESSED ON ACTIVATEDB CELLS Antigen Antibody Early markers IgM MAb56“ B7 AB-1

Size (kDa)

60 60 Unknown

Ba

Unknown

Bac-1

Unknown

LB-2

76-85

Expression/characteristics

Activated B cells by 18 hr, IgM binding protein Activated B cells by 24 hr Rapidly expressed on activated B cells, 3-12 hr Rapidly expressed on activated B cells, 6-12 hr Activated B cells, detect at 16-24 hr Increased on activated B cells by 16 hr, I-CAM-1

Reference Sanders et al. (1987) Freedman et al. (1987) Jung and Fu (1984) Kikutani et al. ( 1 9 8 6 ~ ) Suzuki et al. (1986) Clark et al. (1984)

Late markers

B5

75

BB-1

37

BLAST-1

45

HC2

52-63

a

Activated B cells, peak of expression on day 3 Activated B cells, peak at 5-7 days after activation Activated B cells, peak at 3-4 days after activation Some resting B, elevated after activation, day 4

Freedman et al. (1985) Yokochi et aE. (1982) Thorley-Lawson et al. (1982) Posnett et al. (1984)

Various IgM monoclonal proteins bind to this marker.

111). The functions of most of these “activation” antigens are not known. It is possible they function as receptors for signals that drive activated B cells to enter S phase, divide, and/or differentiate into antibody-producing cells. Alternatively, some of these molecules may function to regulate specific adhesion and migration of activated B cells during their interactions with accessory cells of differentiation into plasma cells. In this section we have divided the activation markers into those that are expressed relatively early after B cells are activated and those that appear to be expressed somewhat later, perhaps after cells leave the S phase. It should be noted that “activation” antigen is a somewhat loosely used relative term. Class I1 MHC, CD23, and CDw40 are expressed at lower levels on resting B cells and have dramatically higher expression on activated B cells. Furthermore, many studies describing “activation” antigens not on resting B cells have used relatively insensitive detection techniques, such as cell sorter analyses incapable of detecting
B

CELL-ASSOCIATED SURFACE

MOLECULES

129

molecules/cell. Thus, activation antigens and other markers may differ more in quantitative expression than in absolute qualitative expression. A perhaps more useful classification used by Boyd and co-workers (1986) is to group markers as being (1) expressed on B cells, then increasing after activation (e.g., CD19, CD20, CDw40; (2) expressed on B cells and rapidly lost after activation (e.g., CD21, CD22); ( 3 ) absent on resting B cells and expressed rapidly (e.g., TF and IL-2 receptors, AB-1, B7, Ba) or more slowly after activation (e.g., BB-1, Blast-1). A. MARKERS EXPRESSED EARLY AFTER ACTIVATION Recently, Sanders et al. (1987) defined a 60-kDa surface polypeptide restricted to B cells that binds to mouse or human IgM immunoglobulins. This protein was only clearly detectable on activated B cells and was not found on resting B cells, resting or activated T cells, or monocytes. The IgM-binding protein was detectable within 18 hr after B cells were activated by PMA and in its expression was distinct from the Bac-1 activation marker (see below). Previous studies have shown that IgM antibodies can enhance the antibody response (e.g., Henry and Jerne, 1968), implying that the 60-kDa polypeptide defined by Sanders e t al. might function in the regulation of activated B cells. Freedman and co-workers (1987), using an IgM MAb called B7, also recently identified a 60-kDa marker expressed very early after human B cells are activated. It was possible that Freedman et al. detected the same IgM-binding protein described by Sanders et al. However, other IgM MAbs used in their study, such as BB-1 and Bac-1, had a reactivity different than that of B7 on various cell lines, indicating that B7 defines a unique specificity. If B7 indeed recognizes an IgM binding protein, it will be of interest to test the effect of the B7 MAb on antibody responses. A number of other markers specific for activated B cells and detectable very soon after B cells are triggered have been described, but because these markers have not yet been definitively characterized biochemically, it is not clear whether or not they are the same or related to other markers. These include MAbs AB-1 (Jung and Fu, 1984), Ba (Kikutani 1986c), and Bac-1 (Suzuki et al., 1986). Two of these h4Abs, Ba and Bac-1, are IgM and thus could be binding the 60-kDa protein described by Saunders et al. (1987). However, the 60-kDa protein is not expressed on some BL lines, such as Daudi, that do express Bac-1 and Ba. Both the AB-1 and Ba MAbs were reported to inhibit B cell proliferation induced by anti-IgM plus BCGF, but did

130

EDWARD A. CLARK AND JEFFREY A. LEDBETTER

not inhibit IL-%stimulated T cell proliferation. Both Jung and Fu (1984) and Kikutani and co-workers (1986~)suggest that their MAbs may recognize receptors for growth factors. However, Ba was only effective at very high concentrations and it was not possible to determine the doses of AB-1 needed for inhibition. Furthermore, B cell activation and growth can be inhibited by a number of different mechanisms, e.g., via surface Fc receptor binding (Phillips and Parker, 1984), so the actual function of these molecules remains open. In 1982 we described a B cell blast-associated antigen which we later term LB-2 (for lymphoblast 2) because it expressed on B and T lymphoblasts but not on resting lymphocytes (Clark et al., 1984, 1986a).The LB-2 marker is 76 to 85 kDa in size, depending on the cell type (Clark et al., 1986a; Patarroyo et al., 1987, 1988), and the gene encoding for LB-2 maps to chromosome 19 (Katz et al., 1984). Patarroyo and co-workers (1987, 1988) found that anti-LB-2 inhibited adhesion between B lymphoblastoid cells and lymphocyte-monocyte adhesion. This suggested that LB-2 may be an adhesion molecule distinct from the CD18/LFA-1 adhesion structures (Springer et al., 1987). Springer and co-workers had also described an adhesion molecule, I-CAM-1 (Dustin et al., 1986; Rothlein et al., 1986), that is widely distributed on both hematopoietic cells and other cell types and is expressed at very high levels on activated lymphocytes. A comparison of LB-2 and I-CAM-1 MAbs has revealed that they react with different epitopes on the same molecule (Makgoba et al., 1988), now designated I-CAM-1, since this protein is homologous to the N-CAM-1 proteins (B. Seed, personal communication). The CD18/ LFA-1 B chain appears to be a ligand for I-CAM-1, since the binding of leukocytes to purified I-CAM-1 on solid phase is inhibited specifically by MAb to either CD18/LFA-1 or I-CAM-1 (Makgoba et al., 1988). Thus, what intially was thought to be an activation marker also apparently functions in cell-cell adhesion interactions. Some homing receptors also change expression during B cell activation (Jalkanan et al., 1986), so it is possible that after activation certain adhesion molecules increase expression to facilitate selective migration into germinal centers and/or selective interaction with, e.g., follicular dendritic cells.

B. MARKERSEXPRESSED LATEAFTER ACTIVATION A MAb designated B5 reacts with a 75-kDa polypeptide expressed specifically on B cells 1 to 3 days after activation (Freedman et al., 1985; Boyd et al., 1986; Beverley, 1987). The B5 marker is expressed on some B-CLL and B cell lymphomas, but is not detectabIe on non-T

B CELL-ASSOCIATED SURFACE MOLECULES

131

ALL, T cell or myeloid malignancies, activated T cells, or non-B hematopoietic cells such as monocytes, T cells, or granulocytes. About the same time that MAbs to CD23 were reported (Kitner and Sudgen, 1981; Rowe et al., 1982; Solvin et al., 1982), two other MAbs to B cell blast markers were described, BB-1 (Yokochi et al., 1982)and B-LAST-1 or BLAST-1 (Thorley-Lawson et al., 1982). Both of these MAbs reacted with B-LCL, with most BL, and with activated B cells, with patterns of reactivity different from the anti-CD23 MAb. The BB-1 marker was subsequently shown to be closely associated with EBV receptors on certain BL (Ehlin-Henriksson et al., 1983). However, BB-1 is a 37-kDa polypeptide, Bp37 (Clark et al., 1986a), encoded by a gene located on chromosome 12 (Katz et al., 1984), and thus is quite distinct from the CD21 receptors for EBV. Bp37 may be sterically associated with the EBV receptor after transformation. EBV infection is not required for Bp37 expression, since B cell blasts and some B-CLL and non-T ALL are Bp37' (Clark et al., 1986a; Boyd et al., 1986). Bp37 is first detectable on B cell blasts 2 to 4 days after activation (Clark et al., 1986b; Boyd et al., 1986). The BLAST-1 MAb, like BB-1, reacts with a marker expressed 2 to 4 days after B cells are activated, which is also found on some non-T ALL and B-CLL (Thorley-Lawson et al., 1982; Thorley-Lawson and Mann, 1985; Boyd et al., 1986); unlike BB-1, BLAST-1 reacts with P3HR-1 and does not react with EBV- BL such as Ramos (Thorley-Lawson et al., 1982). BLAST-1 recognizes a 43-kDa O-linked glycosylated polypeptide that is distinct from CD23 (Thorley-Lawson et al., 1985). Recently a cDNA for BLAST-1 has been isoloated which predicts a 28-kDa polypeptide with five potential N-linked glycosylation sites (Staunton and Thorley-Lawson, 1987). The C-terminus of BLAST-1 has a hydrophobic transmembrane-like region without a cytoplasmic tail and is attached to the cell surface via a phosphatidylinositol (PI) linkage (Staunton et al., 1988). As with other PI-linked surface glycoproteins, such as LFA-3 and Thy-1, BLAST-1 is related to members of the Ig supergene family, most notably to CD4 and class I1 MHC. The 43-kDa chain mentioned above is associated with a nonglycosylated 55-kDa chain not recognized by the BLAST-1 MAb. The markers recognized by BB-1 and BLAST-1 are quite similar, but BB-1 (chromasome 12) is located on a different chromosome than is BLAST1 (chromosome 20) (Katz et al., 1984; Staunton et al., 1988). It is possible that these markers are consitutively expressed as a result of infection by EBV and in some way contribute to maintenance of the transformed state. Another MAb, HC2, reacts with four polypeptides, detectable by

132

EDWARD A. CLARK AND JEFFREY A. LEDBETTER

two-dimensional gel analyses in the 52- to 63-kDa range and distinct from the IL-2 receptor (Posnett et al., 1984). The HC2 marker is expressed on a small subset of activated B cells and is increased on B cells stimulated for 4 to 5 days in culture. This is consistent with it being expressed at high levels on hairy cell leukemias, which resemble plasmoblasts. The HC2 marker, while not on activated T cells, is expressed on myelomonocytic lineage cells. A number of other activation markers have been detected on activated B cells, but are also found on activated T cells, including 4F2 (Haynes et al., 1981),LB-1 (Yokochi et al., 1982), AAl (Chiorazzi et al., 1984), and others (see Beverley, 1987), and are beyond the scope of this review. VI. Surface Molecules Found on T Cells and Subsets of 6 Cells

A. CD5

The CD5 antigen is a 67-kDA glycoprotein homologous to the murine L y l antigen (Ledbetter et al., 1981; Jones et al., 1986; Huang et al., 1987).Although CD5 is a pan-T cell marker, it was also found with high frequency on the of surface Ig+ B cell CLLs (Martin et al., 1980; Royston et al., 1980; Boumsell et al., 1980). Since then, a subpopulation of normal human B cells have been shown to express CD5 (Caligaris-Cappio et al., 1982). The expression on B cell malignancies and on a B cell subpopulation is a feature of CD5 expression also seen in the mouse (Lanier et ul., 1981; Manohar et al., 1982; Hardy e t al., 1984). CD5 is found on a higher number of B cells in patients with rheumatoid arthritis (Plater-Zybeck et ul., 1985; Youinou et al., 1987), and is expressed on B cells in fetal spleen (Botill et al., 1985; Antin et al., 1986) and on B cells recovering after bone marrow transplantation (Ault et al., 1985). The CD5' B cells are primariIy responsibIe for autoantibody production (Hayakawa et al., 1983, 1984; Casali et al., 1987; Hardy et al., 1987) and are primarily h light chain in the mouse (Hardy et al., 1986; Hardy and Hayakawa, 1986). There is currently considerable interest in whether CD5+ B cells represent B cell lineage or whether CD5 expression marks a particular stage of €3 cell differentiation or activation. Many of those working on this problem feel that CD5' B cells are a separate lineage, since (1) CD5 B cells are responsible for essentially all spontaneous IgM secretion, including autoantibodies (Hayakawa et al., 1984; (2) CD5 B cells are able to reconstitute the CD5 B cell compartment when

B CELL-ASSOCIATED SURFACE MOLECULES

133

transferred to irradiated recipeints and thus arise from distinct progenitors (Hayakawa et al., 1985); and ( 3 ) CF5 B cells show a distinct localization pattern in that they are enriched in the peritoneal cavity but are rare in spleen and undetectable in lymph node (Herzenberg et al., 1986). On the other hand, CF5 expression on B cells and B cell CLLs is inducible by phorbol esters (Miller and Gralow, 1984; Youinou et al., 1987), and CD5-positive B cells have unusual proliferative capacity, since they can form spontaneously arising cell lines (Braun et al., 1986). Furthermore, CD5 B cells from some mice are enriched in GI and S phases of the cell cycle (Rabinovitch et al., 1986; Torres et al., 1989). The function of CD5 has been investigated on T cells, where CD5 MAbs have been shown to enhance IL-2 production and proliferation . transresponses (Stanton et al., 1986; Ledbetter et al., 1 9 8 5 ~ )The membrane signal delivered by MAb binding to CD5 results in cytoplasmic calcium mobilization and up-regulation of signaling through the CD3 (antigen receptor) pathway (June et al., 1987). Our recent results show that CD5 has an analogous function on B cell CLLs (Ledbetter et aE., in preparation).

B. CD2, CDlc,

AND

CD28

Several markers previously associated mainly with T lymphocytes have been detected on B lineage cells. First, the pan-T marker, CD2, has been detected on a few non-T, non-B ALL, and B-CLL cells (Guglielmi et al., 1983; Boumsell, 1984). CD2 is also expressed on some fetal liver CD20+ CD19' cells and on an EBV-transformed pro-B cell line (A. Muraguchi, personal communication). The C D l family of molecules, C D l a (49 kDa), C D l b (45 kDa), and C D l c (43 kDa), are principally expressed on cortical thymocytes. Recently, C D l c was reported to be expressed weakly on a subset of normal human B cells, but not on BL or B-LCL cells (Small et al., 1987). This is of interest since CDlc, unlike C D l a and CDlb, does not associate with CD8 (Ledbetter et al., 1985a). Furthermore, some structurally similar murine Qa markers are also expressed on thymocytes and B cells (Morse et al., 1987), implying that there may be a Qa homologue for C D l b . Finally, the pan-T cell marker, CD28, for which a cDNA has been recently isolated and characterized (Aruffo and Seed, 1987), is expressed at high levels on bone marrow plasma cells and on plasmacytomas (Kozbor et al., 1987). However, CD28 is not detectable on BL, B-LCL, or activated or resting B cells. The CD28 molecule functions in the regulation of T cell activation (Ledbetter et

134

EDWARD A. CLARK AND JEFFREY A. LEDBETTER

al., 1 9 8 5 ~Clark ; and Ledbetter, 1986b; Ledbetter and Clark, 1988), but its function in plasma cell differentiation is not known.

VII. Concluding Remarks

In 1986 the single most important contribution to the field of human B cell immunobiology was the isolation and characterization of cDNAs encoding two interleukins, IL-4 (BSF-1 and IL-6 (BSF-2) (see Kishimoto and Hirano, 1988). In 1987, the single most important contribution was the isolation in powerful, efficient expression vectors and the characterization of cDNAs encoding the human B cell-associated differentiation antigens, CD19, CD20, CD22, CD37, CD39, and CDw40, by Stamenkovic and Seed (Aruffo and Seed, 1987; Seed and Aruffo, 1987; Stamenkovic and Seed, 1988a,b,c; Stamenkovic et al., 1988). The availability of expression vectors that encode and can express various B cell surface molecules in transfected cells will facilitate the identification of the natural ligands for these surface molecules and will also be useful for investigations of the molecular basis of signal transduction and ligand binding via these molecules. Once the ligands have been identified and the genes encoding the ligands have been isolated and sequenced, it will be possible, using sets of appropriate MAbs and site-specific mutagenesis, to map precisely the functional epitopes on the receptors and ligands. This approach has been started already for CD2 and LFA-3 by Peterson and Seed (1987). Once the three-dimensional structures of the B cell receptors and their ligands have been determined, it may then be possible to construct drugs that mimic or block cell surface ligands and thus modulate normal or malignant B cell growth and differentiation. Animal models will be required to assess the in vizjo functions of B cell surface molecules and to test the effects of drugs or MAb to these polypeptides. Thus, it is critical to identify the homologues of the human CD markers in nonhuman primates, such as macaques, and in rodent species. Many nonhuman primate species express B cell markers detectable by antihuman MAbs and some MAbs, such as anti-CDw40, which stimulate human B cells, also stimulate macaque B cells (see Clark et al., 1983; Clark and Draves, 1987). The epitopes on CD molecules highly conserved in evolution may be most critical for the function of the molecules. With the exception of CD5 (Ledbetter et al., 1981), no mouse counterparts for the major human B cell surface markers have yet been unequivocally identified. As men-

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tioned above, Lyb3 may be homologous to a 68-kDa human B cell marker, mouse and human forms of a B cell-associated common leucocyte marker may be homologous, and CDw40 has striking similarities with the murine Lyb2 marker (see Subbaro and Mosier, 1982). This paucity of information about murine counterparts for the human C D markers is worrisome and merits special attention, since mouse homologues are needed for studies examining the regulation, in u i m function, and possible role of B cell markers in immunologic diseases. The cDNAs encoding CD19, CD20, etc., will also be useful for isolating and characterizing genomic genes. The genomic genes will then be used to identify how the expression of B cell markers is regulated in normal and malignant B cells. The cDNAs encoding for various B cell markers will also be useful for directly advancing cancer research. It will be important to determine whether the genes encoding B cell-associated surface markers are abnormal (e.g., translocated, deleted, abnormally expressed) in B lineage malignancies. Comparison of the regulation and expression of the genes, mRNAs, and B cell-associated proteins in normal and malignant B cells may give new insights into the origins of and possible control of B cell neoplasms.

ACKNOWLEDGMENTS We wish to thank our many colleagues who provided us with unpublished or in-press data used in the review, most notably Dr. Ivan Stamenkovic and Dr. Brian Seed, and also Dr. Edward Barsumian, Dr. Klaus Beiske, Dr. Max Cooper, Dr. Toshio Hirano, Dr. Seiji Inui, Dr. Tsuneyasu Kaisho, Dr. Hitoshi Kikutani, Dr. Tadamitsu Kishimoto, Dr. Tucker LeBien, Dr. Atsushi Muraguchi, Dr. Erlend Smeland, and Dr. Mary Valentine. We give special thanks to Dr. Tadamitsu Kishimoto for support during the initial writing of this review. We also are grateful to Mr. Kevin Draves, Ms. Nancy Morris, Ms. Geraldine Shu, and Mr. Theta Shu, for continued expert technical assistance. This work was supported by NIH Grants CD08229, GM27905, and RR00166; American Cancer Society Grant IM-422; a Japanese Ministry of Education fellowship; and by Oncogen Corporation.

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ADENOVIRUS PROTEINS AND M H C EXPRESSION Svante Paabo Department of Biochemistry. University of California, Berkeley, California 94720

Liv Severinsson Ludwig Institute for Cancer Research, Uppsala Branch, BMC, 5-75123 Uppsala. Sweden

Mats Andersson Department of Cell Research, University of Uppsala. S-75124 Uppsala, Sweden

Ingrid Martens Department of Medical Virology, University of Uppsala, S-75124 Uppsala. Sweden

Tommy Nilsson and Per A. Peterson Department of Immunology. Research Institute of Scripps Clinic, La Jolla. California 92037

I. Introduction 11. Adenoviruses 111. Adenovirus Gene Products Modulating MHC Cell Surface Expression A. Early Region 1A B. The E l 9 Protein IV. Functional Consequences of Adenovirus-Induced Modulation of MHC Class I Expression A. Viral Pathogenicity B. Viral Tumorigenicity V. Summary and Concluding Remarks References

I. Introduction

Cytotoxic T lymphocytes (CTLs) are one of the main immune mechanisms by which virus-infected and transformed cells become eliminated. CTLs recognize their target cells by receptors specific for combinations of viral antigens or modified endogenous proteins and major histocompatibility complex (MHC) class I self-antigens 151 ADVANCES IN CANCER RESEARCH, VOL. 52

Copyright B 1989 by Academic Press, Inc. All rights of reproduution in any form reserved.

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(Zinkernagel and Doherty, 1975). The latter are highly polymorphic glycoproteins, composed of one heavy chain of 45,000 Da, which spans the cell membrane, and one light chain, Pz-microglobulin. The extracellular part of the heavy chain is folded into three domains (Bjorkman et al., 1987a), the two external domains (a1and az) forming an antigen-binding site, which is variable in structure between different class I alleles. This site seems to have the ability to bind and present foreign peptide antigens to T cells (Bjorkman et al., 1987b). The innermost domains of the heavy chain (ag)and Pz-microglobulin are largely invariant in structure and display significant sequence homology to immunoglobulin constant regions (Peterson et al., 1972; Orr et al., 1979), as well to other members of the immunoglobulin superfamily of proteins (Hood et al., 1985). Class I MHC molecules are present on most somatic cells and constitute an absolute prerequisite for the effector phase of the specific cytolytic immune response. Adenoviruses have evolved mechanisms by which they are able to reduce or abolish the T-celldependent cytolytic response by modulating the cell surface expression of class I molecules in infected and transformed cells. This review summarizes our knowledge of these mechanisms.

II. Adenoviruses Adenoviruses are nonenveloped viruses containing a doublestranded DNA genome. They were initially discovered in latently infected adenoids (Rowe et al., 1953) and have since been shown to be widespread in several mammalian and avian species. In humans, more than 40 serotypes have been identified and classified into subgenera based on oncogenicity in newborn hamsters, on antigenic properties, and on DNA homology (for a review, see Wadell, 1984). Thus, members of subgenus A are highly oncogenic in newborn rodents, whereas viruses of subgenus C, for example, are nononcogenic. However, viruses and DNA of all serotypes can transform rodent cells in.uitt-0. Members of the different subgenera have a propensity to cause particular clinical manifestations in humans (see Table I) and many viruses of different serotypes are able to establish latency in the infected host. The genomic organization of all human adenoviruses studied to date is similar (see Fig. 1). The early phase of an adenovirus infection is initiated by the expression of the pre-early transcription unit E1A (Nevins, 1981).Gene products from the E1A gene are required for the induction of transcription of each one of the other early transcription

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TABLE I CLASSIFICATION OF ADENOVIRUSES AND THEIRMAJOR CLINICAL MANIFESTATIONS~ ~~

Subgenus

Serotypes 12, 18, 31 3, 7, 11, 14, 16, 21, 34, 35

A

B

1,2,5, 6 8-10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36 4

C

D E

F

40,41 ~

a

Clinical manifestations Enteric infections? Respiratory infection, infections of urinary tract Respiratory infections Epidemic keratoconjunctivitis Conjunctivitis, respiratory infections Acute gastroenteritis

~~

Data from Green et al. (1979) and vanLoon et al. (1985).

units (for a review, see Berk, 1986). In addition, the E1A gene, together with the E1B gene, is responsible for the oncogenic transformation ascribed to adenoviruses (reviewed by Graham, 1984). The onset of viral DNA replication marks the beginning of the late phase of infection, during which one single major promoter drives the late transcription unit. The different viral transcripition units seem to encode proteins which are related in function. Thus, during the late phase of infection, mainly viral structural proteins are synthesized. During the early

FIG. 1. (Top),a schematic transcription map of adenovirus 2; (center), the major RNA transcripts encoding the 243- and 289-amino acid residue proteins of region E1A and the E l 9 protein of region E3; (bottom), a schematic illustration ofthe structure of MHC class I molecules (left) and of E l 9 protein (right).

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phase of infection, region E2 transcribes genes encoding proteins required for DNA replication, i.e., a DNA-binding protein (Kmijer et al., 1981), the terminal protein (Smart and Stillman, 1982),and a DNA polymerase (Stillman et al., 1982). Early region 3 (E3) contains multiple open reading frames, at least four of which are expressed in cells infected by adenoviruses of subgenus C (Jeng et al., 1978; Wold et al., 1984, 1986; Tollefson and Wold, 1988). Five of the putative E3 proteins contain hydrophobic domains of the membrane-spanning type (Cladaras and Wold, 1985) and at least two of them seem to have immunoregulatory functions (see below). The E3 region differs from the other viral transcription units in that it is dispensable for the virus when grown in vitro (Jones and Shenk, 1978; Groff and Daniell, 1980). Early region 4 encodes proteins of unknown functions, but mutants lacking most of E4 exhibit a phenotype of reduced viral replication and late protein synthesis, as well as only a partial shutoff of host macromolecular synthesis (Halbert et al., 1985; Weinberg and Ketner, 1986). Ill. Adenovirus Gene Products Modulating MHC Cell Surf ace Expression

A. EARLYREGION1A The E l A transcription unit encodes two coterminal mRNAs of 12 and 13 S, which differ with regard to the amount of internal coding sequences removed by splicing (Berk and Sharp, 1978). These two mRNAs encode nuclear proteins of 243 and 289 amino acids, respectively (for a review, see Berk, 1986), which have identical amino and carboxyl termini. They differ by a sequence of 46 internal amino acids unique to the 289 residue protein (see Fig. 1).This sequence of 46 amino acids, when chemically synthesized, is able to induce transcription of the E2 promoter and thus represents an autonomous transcriptional activation domain (Lillie et al., 1987). If this domain of the protein is also sufficient for the induction of the wide variety of viral and cellular promoters which occurs when ElA is cotransfected into cells together with other genes (for a review, see Nevins, 1986) will undoubtedly soon be elucidated. It will also be interesting to learn whether this domain is responsible for the induction of some cellular genes, such as the 70-kDa heat-shock protein gene (Nevins, 1982) and the P-tubulin gene (Stein and Ziff, 1984), where the rates of transcription are elevated during early infection by adenoviruses. In addition to its function as a transcriptional activator, the gene

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products of E 1A have been shown to down-regulate the expression of some genes. This transcriptional repression activity seems to act by inhibiting viral (Borrelli et al., 1984; Velcich and Ziff, 1985) and cellular (Hen et al., 1985) enhancers. It maps to a region in the E1A protein that is common to both the 243- and the 289-amino acid protein, and the same peptide region is required for adenovirus transformation (Lillie et al., 1986). The E1A transcription unit thus directs the synthesis of two related proteins involved in transcriptional regulation. The 289-amino acid protein contains two distinct sequences responsible for transcriptional activation and repression, respectively, and seems to act primarily as a transcriptional activator (Lillie et al., 1986).The 243-amino acid protein lacks the transcriptional activator domain and functions as a repressor of enhancer-dependent transcription. In addition to the functions outlined above, which are common to all adenovirus serotypes, the E1A gene of serotype 12 of subgenus A has been shown to specifically down-regulate the expression of MHC class I antigens in transformed rat cells (Schrier et al., 1983). This function is mediated by the mRNA that encodes the 289-amino acid protein but can be separated from the activating function by a single nucleotide deletion (Bernards et al., 1983). The expression of the 289-amino acid residue E 1A protein reduces the steady-state levels of class I heavy chain mRNA in transformed rat (Schrier et al., 1983), mouse (Eager et al., 1985), and human cells (Vaessen et al., 1986), while leaving the expression of &-microglobulin mRNA unaffected. In contrast, the ElA gene of serotype 5 of subgenus C does not affect the class I antigen expression of transformed cells. The latter protein is dominant in cells expressing the E1A of both serotypes 12 and 5, inasmuch as such cells synthesize normal levels of class I antigens (Vaessen et al., 1986). Also, the E1A-induced repression of class I antigen expression is reversible by treatment of the cells with interferon-? (Eager et al., 1985). This observation establishes that the down-regulation of class I antigen expression by E1A is an active process, which does not irreversibly alter the MHC genes of the affected cells. However, the molecular mechanism by which the shutdown of MHC class I expression occurs in adenovirus 12transformed cells is not elucidated.

B. THE E l 9 PROTEIN One of the quantitatively dominating proteins synthesized during the early phase of an adenovirus 2 infection is the E l 9 protein (also

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designated E3/19K or E3-gp25k). This protein is encoded in early region 3 and its polypeptide chain has a molecular weight of 19,000. As it usually contains two carbohydrate units, the mature protein is a 25-kDa molecule. The E 19 protein contains a hydrophobic sequence that anchors it in the membrane, and it seems to be confined to the membrane of the endoplasmic reticulum. (Paabo et al., 1987). The structural features of the E 19 protein include a signal sequence of 17 amino acids (Wold et al., 1985) followed by an intralumenal domain of 104 amino acids. A transmembrane segment 23 amino acids long separates the intralumenal domain from a cytoplasmic, COOHterminal tail of 15 amino acids. The intralumenal domain carries two Asn-linked carbohydrate moieties, which always occur in the highmannose form (Kornfeld and Wold, 1981). By immunoprecipitation of the E 19 protein from transformed rat cells (Kvist et al., 1978), as well as from productively infected cells (Signas et al., 1982; Kampe et al., 1983), it has been shown that this viral protein exists in molecular complexes with MHC class I antigens. Such complexes can be isolated by coprecipitation using antibodies directed against the class I molecule heavy chain, µglobulin, and the E l 9 protein. Several class I and E l 9 molecules seem to be involved in the complexes, but no other proteins have been resolved upon S DS-polyacrylamide gel electrophoresis of the immunoprecipitated complexes. This observation attests to the specificity of the interaction between the E l 9 protein and the class I moIecules. Furthermore, expression of the E l 9 protein by a eukaryotic expression vector has demonstrated that the binding to class I molecules is an intrinsic property of the viral protein (Paabo et al., 1983). Pulse-chase experiments have demonstrated that the complexes between the E l 9 protein and the class I antigens form immediately upon synthesis of the reactant proteins in the rough endoplasmic reticulum and that this interaction abrogates the normal transport of the class I molecules to the plasma membrane via the Golgi complex (Severinsson and Peterson, 1985; Burgert and Kvist, 1985; Anderson et al., 1985). The class I molecules thus become trapped in the endoplasmic reticulum, where they exhibit a long half-life. The transport of other cell surface glycoproteins remains unaffected. Since large amounts of the E l 9 protein are synthesized during a lytic infection, all newly synthesized class I molecules become trapped in the endoplasmic reticulum such that the transport of nascent class I molecules to the cell surface is specifically shut off. As a consequence, the amounts of expressed cell surface class I molecules progressively

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decrease as the viral infection proceeds, and at 18 hr after the onset of the infection, the cells display only about 30%of the initial amount of MHC class I molecules on the cell surface. The lumenal portion of E l 9 is able to bind with specificity to the extracellular part of human class I molecules (Paabo et al., 1986a), while the COOH-terminal eight amino acids of the cytopIasmic portion of the molecule contain the signal by which the E l 9 protein is confined to the endoplasmic reticulum (Paabo et al., 1987). By testing a panel of hybrid class I genes, where exons have been shuffled between two murine genes, it has been shown that domains 1and 2 of the class I heavy chain are essential for the binding to occur (Burgert and Kvist, 1987). However, also relatively minor alterations of the amino acid sequence of the cytoplasmic domain of the E l 9 protein affect the efficiency with which the viral protein binds to human class I molecules (Paabo et al., 1987), perhaps by affecting the structure of the lumenal E l 9 domain. In this context, it is interesting to note that the lumenal part of the E l 9 protein displays amino acid sequence homology to the second domain of human MHC class I1 a chains, to µglobulin, and to other members of the immunoglobulin superfamily of proteins (Chatterjee and Maizel, 1984). The E l 9 protein may thus represent a cellular gene of the immunoglobulin superfamily, which the adenoviruses have acquired during their evolution and have adapted to serve the present function of diminishing class I cell surface expression of the infected cell. IV. Functional Consequences of Adenovirus-Induced Modulation of MHC Class I Expression

A. VIRALPATHOGENICITY Adenoviruses of subgenera B, C, D, and E have all been shown to modulate MHC class I expression with a mechanism analogous to the E l 9 protein of adenovirus 2, while viruses of subgenus A lack such a function (Paabo et al., 1986b). However, subgenus A viruses are endowed with the pretranslational mechanism of class I modulation ascribed to ElA. Thus, all adenoviruses are able to modulate MHC class I expression by one or the other of these two mechanisms. The fact that two different molecular strategies have evolved to achieve these ends in adenoviruses seems to indicate that it is of prime importance for the virus to decrease the amounts of class I molecules on the surface of its host cell. The down-regulation of class I molecule expression caused by the

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E l 9 protein has obvious consequences for the ability of CTLs to recognize an adenovirus-infected cell. Thus, adenovirus-infected cells expressing the HLA-B7 antigen were shown to be considerably more resistant to alloreactive CTLs, specific for HLA-B7, than were their mock-infected counterparts (Andersson et al., 1987). Likewise, murine CTL clones specific for human MHC molecules and restricted by murine class I molecules were significantly less efficient in lysing target cells that constitutively express the E3 region of adenovirus 2 than were cells not expressing any viral genes (Burgert et al., 1987). Preliminary data also indicate that adenovirus-specific CTLs are impaired in their ability to recognize adenovirus-infected cells expressing E3, but not cells infected with viral deletion mutants lacking the E3 region (F. Rawle and L. Gooding, personal communication). With regard to the E1A-induced class I antigen modulation, it has similarly been shown that allogeneic CTLs have a drastically reduced reactivity against adenovirus 12-transformed cell lines (Bernards et al., 1983). It is thus clear that the adenovirus-induced modulation of MHC class I expression has profound effects on the ability of CTLs to recognize virally infected cells. However, it is less clear what consequences this has for the virus and its host during lytic and persistent infections. The lack of understanding of the pathophysiology of adenoviruses is largely due to the absence of a suitable animal model for the human adenoviruses. However, this drawback has recently been overcome by the discovery that human adenovirus 5 in cotton rats produces a pneumonia that is similar to that seen in humans (Pacini et al., 1984). In this experimental model system, a viral deletion mutant lacking a large part of the E 3 region produced a profound increase in pulmonary infiltration (Ginsberg et al., 1987), consistent with the view that the E l 9 protein modulates the immune response to the virus. Also, recombinant adenovirus 5 viruses, carrying the hepatitis B virus surface antigen gene in the E3 region, have been shown to persist at titers in syrian hamsters lower than are seen in the wild-type virus (Morin et al., 1987). However, the exact evaluation of the effects of the E l 9 on viral pathogenicity in these animal models will have to await the use of viral mutants in which only the E l 9 gene is affected, because other gene products of the E3 region may also have immunoregulatory functions. Thus, it has recently been shown that the 14.7-kDa protein of adenovirus 2 (Tollefson and Wold, 1988) protects the infected cell against lysis by tumor necrosis factor (Gooding et al., 1988). The pathophysiological role of the pretranslational down-regulation

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of MHC expression by the E1A region of subgenus A viruses is less clear than that of the E l 9 protein. The effect on class I mRNA levels has only been demonstrated in virally transformed cells (Schrier et al., 1983; Eager et al., 1985; Vaessen et al., 1986), and in one report, adenovirus 12-transformed rat cells were found not to be significantly different from adenovirus 5-transformed cells in terms of class I cell surface expression (Mellow et al., 1984). Furthermore, it has been shown that in acutely infected mouse cells, adenovirus 12 and adenovirus 5 increase the amounts of class I mRNA in the cells (Rosenthal et al., 1985). Therefore, it seems likely that the ability of the E1A gene of subgenus A viruses to down-regulate class I gene expression is not operative during the acute viral infection. Instead, it is conceivable that the E1A gene plays a role during persistent viral infections, which may resemble conditions in the virally transformed cell in that early gene functions are continually expressed in the absence of late gene expression. If so, it is of some interest that herpes simplex viruses of types 1and 2, which are analogous to adenoviruses in their ability to establish persistency, have also been shown to decrease the expression of MHC class I antigens on the cell surface of infected cells. The nature of this function is unknown, but is maps to a region of the viral genome which determines the susceptibility of the infected cells to CTLs (Jennings et al., 1985).

B. VIRALTUMORIGENICITY All adenoviruses can transform rodent cells in uitro by functions encoded in early region 1A. However, only subgenus A viruses are oncogenic in uiuo. Because subgenus A is also the only subgenus whose viruses have an E1A region that turns off class I gene expression in transformed cells, it has been suggested that this explains the difference in oncogenicity between subgenus A viruses and other adenoviruses (Bernards et al., 1983). Thus, it is conceivable that MHC-restricted CTLs, considered to play a crucial role in the elimination of virus-induced tumors (e.g., Stutman, 1975; Klein, 1980), would be unable to recognize cells transformed by the preearly region of adenovirus 12, since such cells are deficient in MHC class I antigen expression. In line with this idea it has,been shown that adenovirus 12-transformed cells show a dramatic decrease in susceptibility to allogeneic CTLs (Bernards et al., 1983). Furthermore, highly oncogenic mouse cell lines, transformed by adenovirus 12 and deficient in class I expression, can be rendered nontumorigenic by the expression of a transfected class I gene

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(Tanaka et al., 1985) or by interferon treatment (Hayashi et al., 1985), which turns on the expression of the endogenous class I genes of the cells. On the other hand, this notion has recently been called into question in a study where no correlation was found between tumorigenicity in immunocompetent syngeneic rodents of adenovirus 12and adenovirus 5-transformed cells and the level of class I MHC molecules expressed on the surface of these cells (Haddada et al., 1986). However, since adenovirus 12-transformed cells are only highly tumorigenic in newborn mice, other factors may also be of considerable importance in the defense against these tumors. Thus, it has been suggested that natural killer cells may detect an absence of or reduced expression of class I molecules on their target cells (Ljunggren and Karre, 1985; Karre et al., 1986). If a reduced class I expression is indeed one factor contributing to the oncogenicity of subgenus A viruses as compared to other adenoviruses, it may be significant that the E1A region of subgenus A viruses is responsible both for transformation of cells and for reduction of MHC class I expression, whereas these functions are separated in the E1A and E 3 regions, respectively, in other adenoviruses. Thus, it may be a very rare event that both of those genetic regions become integrated and expressed in one transformed cell. This could explain the difference in tumorigenicity between the different adenovirus subgenera. V. Summary and Concluding Remarks

Adenoviruses are able to specifically down-regulate the cell surface expression of MHC class I antigens. Most viral serotypes achieve these ends by synthesizing a protein that binds to class I antigens in the endoplasmic reticulum (ER) and impedes the transport of these molecules to the cell surface. However, viruses belonging to the highly oncogenic subgenus A do not affect the cIass I antigen expression during acute infection. Instead, they are distinct from other adenoviruses in that they specifically down-regulate the level of mRNAs, encoding MHC class I antigens, in virally transformed cells. The virus-induced reduction of class I antigen expression drastically diminishes the ability of CTLs to recognize cells infected or transformed by adenovirus. A number of issues concerning these viral mechanisms for class I antigen modulation need to be addressed. The molecular mechanism by which the E1A gene product of subgenus A viruses diminishes class I mRNA levels has not been elucidated. Also, the details of the

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interaction between the E l 9 protein and class I molecules should be studied, preferably by X-ray crystallography of the complexes. This would clarify the role of the antigen-binding site as well as other portions of the class I molecule in the binding to the E l 9 protein. Of general importance for our understanding of the sorting and intracellular transport of proteins is the exact delimitation of the signal for E R localization, which is present in the COOH-terminus of the E l 9 protein. The putative interaction of this peptide sequence with components of the ER membrane should also be studied. Finally, the study of the pathophysiological role of the MHC class I downregulation will undoubtedly yield new insights into how the immune system combats virally infected and transformed cells.

REFERENCES Andersson, M., PGbo, S., Nilsson, T., and Peterson, P. A. (1985). Cell (Cambridge, Mass.) 43, 215-222. Andersson, M., McMichael, A., and Peterson, P. (1987).J. Immunol. 138, 3960-3966. Berk, A. J. (1986). Annu. Rev. Genet. 20,45-79. Berk, A. J,, and Sharp, P. A. (1978). Cell (Cambridge, Mass.) 14,695-711. Bernards, R., Schrier, P. I., Houweling, A., Bos, J. L.. van der Eb, A. J., Zijstra, M., and Melief, C. J. M. (1983). Nature (London) 305, 776-779. Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennet, W. S., Strominger, J. L., and Wiley, D. C. (1987a). Nature (London) 329,506-512. Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennet, W. S., Strominger, J. L., and Wiley, D. C. (1987b). Nature (London) 329,512-518. Borrelli, E., Hen, R., and Chambon, P. (1984). Nature (London) 312, 608-612. Burgert, H.-G., and Kvist, S. (1985). Cell (Cambridge, Mass.) 41,987-997. Burgert, H.-G., and Kvist, S. (1987). EMBOJ. 6,2019-2026. Burgert, H.-G., Maryanski, J. L., and Kvist, S. (1987). Proc. Natl. Acad. Sci. U.S.A.84, 1356-1360. Chatterjee, D., and Maizel, J. V., Jr. (1984). Proc. Natl. Acad. Sci. U.S.A.81,6039-6043. Cladaras, C., and Wold, W. S. M. (1985). Virology 140, 28-43. Eager, K. B., Williams, J., Breiding, D., Pan, S., Knowles, B., Appella, E., and Ricciardi, R. P. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 5525-5529. Ginsberg, H. S., Valesuso, J., Horswood, R., Chanock, R. M., and Prince, G. (1987). In “Vaccines ’87,” pp. 322-326. Cold Spring Harbor Lab., Cold Spring Harbor, New York. Gooding, L. R., Elmore, L. W., Tollefson, A. E., Brady, H. A., and Wold, W. S. M. (1988). Cell (Cambridge, Mass.) 53, 341-346. Graham, F. L. (1984). In “The Adenoviruses” (H. Ginsberg, ed.), pp. 339-398. Plenum, New York. Green, M., Mackey, J. K., Wold, W. S. M., and Rigden, P. (1979).Virology 93,481-492. Groff, D. E., and DanielI, E. (1980). Virology 106, 191-194. Haddada, H., Lewis, A. M., Jr., Sogn, J. A., Coligan, J. E., Cook, J. L., Walker, T. A., and Levine, A. S. (1986). Proc. Natl. Acad. Sci. U.S.A.83, 9684-9688. Halbert, D. N., Cutt, J. R., and Shenk, T. (1985).J. Virol 56, 250-257.

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MULTIDRUG RESISTANCE Alexander M. van der Bliek' and Piet Borst Department of Molecular Biology. The Netherlands Cancer Institute. 1066 CX Amsterdam, The Netherlands

I. Introduction 11. 111. IV. V. VI. VII. VIII. IX.

X. XI. XII. XIII. XIV. XV.

Drugs Affected by MDR What Happens to the Drugs in MDR Cells? Pharmacological Reversal of MDR Alterations in MDR cells P-Glycoprotein Overproduction Amplified Genes in MDR Cell Lines The Central Role of P-Glycoprotein Genes in MDR P-Glycoprotein Structure Deduced from Sequence Comparisons Diversity of P-Glycoproteins Mutated P-Glycoprotein Genes with Altered Drug Transport Properties P-Glycoprotein Expression in Normal Tissue and Its Regulation Coamplified Genes and Alterations Elsewhere in the Genome MDR in the Clinic Outlook References

I . introduction Cells selected for resistance against a single cytostatic drug may simultaneously acquire cross-resistance to a range of other drugs, i.e., may become multidrug resistant. This review deals with the classical form of multidrug resistance (MDR), or pleiotropic drug resistance, discovered 20 years ago and characterized by resistance to a wide range of lipophilic cytotoxic drugs that do not share a common structure or cellular target (Kessel et al., 1968; Biedler and Riehm, 1970; Dano; 1972, Ling and Thompson, 1974). MDR results in resistance to major classes of carcinostatic drugs in clinical use, e.g., vinca alkaloids, anthracyclins, podophyllotoxins, and actinomycin D, but drugs such as cis-platinum or bleomycin are not affected at all, and resistance to methotrexate and most alkylating agents is only marginal (Biedler and Peterson, 1981; Ling et al., 1983; Beck et al., 1983). Nevertheless, MDR could make a significant contribution to clinical resistance to carcinostatic drugs, and the

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Present address: Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125. 165 ADVAXCES IN CANCER RESEARCH, VOL. 52

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in a n y form reserved.

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analysis of tumor samples suggests that it does (Bell et al., 1985; Gerlach et al., 1987; Ma et al., 1987; Fojo et al., 1987; Tsuruo et al., 1987). The diverse range of drugs affected by MDR pointed early on at the plasma membrane as the site of alteration. In 1972 Dan0 showed that MDR cells extrude the affected drugs at increased rates and that this extrusion was energy dependent. After considerable controversy, most investigators in the field now agree that this is indeed the major mechanism for resistance (see Gerlach et al., 1986a; Beck, 1987). The search for alterations in MDR cells that could explain the increased drug extrusion led to the discovery of P-glycoproteins by Ling and co-workers (Juliano and Ling, 1976). P-Glycoproteins are large plasma membrane proteins, overproduced in all classical MDR cells analyzed (Kartner et al., 1983a; Gerlach et al., 1986a). The structure of P-glycoproteins, deduced from the corresponding gene sequences, proves highly informative: the gene has substantial homology with bacteria1 membrane proteins that transport metabolites and it also contains an ATP-binding site that could be involved in providing the energy required for active drug extrusion (Ames, 1986b; Chen et al., 1986; Gerlach et al., 198613; Gros et al., 1986d). Indeed, reintroduction of a cloned P-glycoprotein gene (as cDNAs) into a drug-sensitive host cell made the cell fully multidrug resistant, when the gene was expressed (Gros et al., 1986b; Ueda et al., 1987a). As vesicles from MDR cells or P-glycoprotein bind at least some of the drugs extruded from the cells (Cornwell et at., 1986,1987a; Safa et al., 1986), the P-glycoprotein probably acts as a drug pump, an activity which was demonstrated with vesicles from MDR cells (Horio et al., 1988). Here we review classical MDR with emphasis on the genetic alterations that account for resistance and the possible clinical implications of the knowledge gained in recent years. Aspects of this topic have also been discussed in reviews by Beck (1987), Borst (1984), Croop et al. (1988), Fine and Chabner (1986), Gerlach et al. (1986a), Kessel (1986), Moscow and Cowan (1988), Ozols and Cowan (1986), Pastan and Gottesman (1987), Roninson (1987), Stark (1986), and Tsuruo (1988). II. Drugs Affected by MDR

Drugs affected by MDR have diverse cellular targets, for example, tubulin assembly (colchicine, colcemid, and vinca alkaloids), DNA and RNA synthesis (actinomycin D), protein synthesis (puromycin,

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emetine), and topoisomerase I1 (anthracyclins, podophyllotoxins), or they induce DNA damage (melphalan) (see Biedler and Peterson, 1981; Riordan and Ling, 1985; Gerlach et al., 1986a). What do these drugs have in common? They are generally amphiphilic and possess at least one aromatic ring (see Goodman Gilman et al., 1980). They range in mass from 100 to 2000 Da and many have two ring structures linked by a single bond, which allows rotation. Even though MDR may seem relatively indiscriminate, minor differences in drug structure can cause wide variation in relative resistance (cf. colchicine 180 X versus colcemid 16 X in CHRC5cells; Bech-Hansen et al., 1976). More systematic tests with a series of related drugs will be needed to define the features recognized by MDR cells. MDR is best studied in model cell lines displaying high levels of resistance, and this is reached by stepwise selection or by steadily increasing the drug concentration (Ling and Thompson, 1974; Biedler and Peterson, 1981; Kartner et al., 1983b; Howell et al., 1984). The initial selection is facilitated by pretreating the cells with a mutagen or tumor promoter (Ling and Thompson, 1974; Shen et al., 1986a). Different MDR lines display variation in cross-resistance as well as increased sensitivity to some other drugs (collateral sensitivity) (BechHansen et al., 1976; Biedler and Peterson, 1981).This is illustrated by the two examples presented in Table I. Usually the degree of resistance is highest against the selecting drug. This generalization is not without exceptions, however. For example, the CHO cell line CHRC5selected for resistance with colchicine (180 X) is much more resistant to gramicidin D (5000 X) (Gerlach et al., 1986a). This is remarkable, because gramicidin is a peptide not normally considered in drug-resistance spectra. Another example of nonconformity is the Chinese hamster ovary cell line DNRR51, selected for resistance with daunorubicin (12 X), but more resistant to colchicine (41 X) (Kartner et at., 1983b). Likewise, a human small cell lung carcinoma cell line selected with adriamycin is much more resistant to epirubicin and colchicine (Mirski et al., 1987). Crossresistance also varies between cell lines that were selected from the same parent and with the same drug, suggesting that an element of chance is involved in the precise spectrum of cross-resistance obtained (Ling and Thompson, 1974; Bech-Hansen et al., 1976). The sensitivity to hydrophilic compounds such as bleomycin and cis-platinum is usually unaltered (Harker and Sikic, 1985; Mirski et al., 1987) and, vice versa, cell lines selected with alkylating agents are not cross-resistant to adriamycin or vincristine (Teicher et al., 1986). There are, however, also agents for which MDR cells are more

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TABLE I DRUG RESISTANCEOF Two MDR CHINESE HAMSTER OVARY LINES‘ Relative resistance Drug Colchicine Puromycin Daunorubicin Taxol Vinblastine Emetine Doxorubicin Melphalan Deoxycorticosterone 1-Dehydrotestosterone Acronycine Triton X-100

CHRC5” 180‘ 100 76 20 30 29 25

4-15 0.1 0.1 <0.06 0.3

DNRR51

25 38 41 5 22 11 30 NT* NT 1 0.6 NT

From Ling et al. (1983). The CHRC5cell line was selected with colchicine and the DNRR51cell line was selected with daunorubicin. Relative resistance was determined by the concentration of drug needed to inhibit growth or colony formation in the drug-resistant line to the same degree as the parent line (resistance level=l). NT, not tested.

sensitive than the parent cell line. Such collateral sensitivity is observed with local anesthetics, certain steroids, acronycin, and detergents from the Triton X series (Riehm and Biedler, 1972; Bech-Hansen et al., 1976). The response to Tritons is dependent on the number of ethylene oxide groups in the side chain and ranges from collateral sensitivity to cross-resistance (Bech-Hansen et al., 1976). Thus, collateral sensitivity is greatest to the hydrophobic Tritons. Nonspecific membrane perturbation may also underlie collateral sensitivity to local anesthetics, since it is observed at much higher concentrations than needed for specific effects on nerve function (Seeman, 1972; Kessel, 1986). Likewise, the low level of cross-resistance to methotrexate might also be caused by nonspecific changes at the plasma membrane (Meyers et al., 1985; Mirski et al., 1987). This seems an anomaly, because MDR cells were not selected for methotrexate resistance nor does the drug have characteristics associated with MDR (aromatic rings and hydrophobicity). Here, the folate carrier could be inhibited indirectly by the altered properties of the membrane.

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I l l . What Happens to the Drugs in MDR Cells? There is ample evidence that MDR cells avoid cytotoxicity by keeping the intracellular drug concentration low (Kessel and Bosman, 1970; Riehm and Biedler, 1971; Dano, 1973; Bleyer et al., 1975; Carlsen et al., 1976; Skovsgaard and Nissen, 1982; Riordan and Ling, 1985; Sirotnak et al., 1986). The difference between concentrations measured in parent and MDR cell lines is, however, an order of magnitude less than the relative resistance determined by inhibition of growth or by survival in clonogenic assays. This discrepancy, which has generated much controversy, is best explained by assuming that total cellular drug concentration does not reflect the concentration at the drug target. As drugs affected by MDR are hydrophobic and often basic, they may be sequestered in membranes or acidic cellular compartments, like lysosomes, where they would not affect the target. If the drug concentration in these target-irrelevant compartments is a substantial fraction of the total cellular concentration, the intracellular drug distribution should be taken into account in order to decide how MDR cells survive in high drug concentrations. Evidence for an altered drug distribution in MDR cells comes from studies of anthracyclin fluorescence in intact cells (Willingham et al., 1986). At a given external drug concentration the nuclear fluorescence is high in sensitive cells, but not in resistant cells. This supports the interpretation that MDR cells are resistant by reducing the drug concentration in the drug-relevant compartment. The tendency of amphiphilic (basic) drugs to concentrate in hydrophobic (acidic) compartments explains why cells can have a higher drug concentration than that of the surrounding medium (Riordan and Ling, 1985). It is this property that has made it hard to determine whether MDR cells are protected by (1) decreased influx, (2) enhanced efflux, or (3) binding in a compartment separated from the target. To start with the last point, several authors have proposed that the intracellular binding of drugs to specific compartments is altered in MDR cells (Ramu et al., 1983; Beck et al., 1983), but it is unclear how this could ultimately reduce the drug concentration at the target under steady-state conditions. Drug entry does seem reduced in MDR cells, but the results of these influx experiments are difficult to interpret, because of the rapid absorption of drug to the plasma membrane (Dano, 1973; Beck et al., 1983). Saturable influx, which could reflect carrier-mediated transport, has been observed within the first seconds of the addition of

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ALEXANDER M. VAN DER BLIEK AND PIET BORST

anthracyclins (Skovsgaard, 1978) or vinca alkaloids (Bleyer et al., 1975). There is, however, no evidence that competition reduces uptake. Quite the opposite, many of the drugs that are added actually facilitate the accumulation of others (Skovsgaard, 1980). An alternative explanation for uptake saturation at higher drug concentrations is the self-association of anthracyclins via T-T bonds (Dalmark and Storm, 1981). To settle this issue, initial rates of competition must be determined more accurately, for instance, with the newly developed flow cuvette of Lankelma et al. (1988). On the other hand, if influx is not carrier mediated, then changes in passive diffusion must account for the hypothesized reduction of influx (see Riordan and Ling, 1985). With the exception of mouse P388/ADR cells (Ramu et al., 1983), the changes in lipid structural order and membrane fluidity were, however, not consistent with reduced drug diffusion (Wheeler et al., 1982). It is also hard to imagine how a bidirectional decrease of drug flux would account for reduced intracellular drug concentrations in the steady state. The measurement of drug efflux has its own difficulties. When the cells are loaded prior to measurement, the drugs are distributed to different cellular compartments. Thus, efflux entails redistribution between these compartments, followed by transport across the plasma membrane. This presumably reduces the rate measured for whole cells to a level lower than that expected on the basis of the resistance observed. Nevertheless, most authors now agree that efflux is indeed enhanced in MDR cells (Dano, 1973; Inaba et al., 1979). Efflux from preloaded cells is decreased by competition with nontoxic drug analogs so that accumulation in the steady state is promoted (Skovsgaard, 1980; Kessel and Wheeler, 1984). Supportive data come from studies of the energy requirements of transport. Enhanced efflux in MDR cells is abolished by inhibitors of oxidative phosphorylation (e.g., azide), and by glucose deprivation, while efflux from the parent cell lines is unaffected (Wheeler et al., 1982; Broxterman et al., 1988a). Indeed, ATP-dependent drug transport has now been demonstrated using vesicles isolated from MDR cells (Horio et al., 1988). The enhanced efflux also explains the drug competition experiments mentioned in the previous paragraph. If drugs compete for active efflux, rather than for import, it is understandable why one drug may facilitate accumulation of another drug and why this facilitation is affected by energy depletion (see Kessel, 1986). Decreased influx of drugs into MDR cells might be an experimental artifact, because initial rates are technically difficult to measure and so most influx measurements include an efflux component. This interpretation is

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supported by the necessity to expose cells briefly to drugs in order to detect the saturation of influx (Riordan and Ling, 1985).In conclusion, MDR is most likely caused by an energy-dependent mechanism of enhanced drug efflux. Such transport is relatively indiscriminate and subject to competitive inhibition. IV. Pharmacological Reversal of MDR

MDR can be reversed by a variety of substances, including calciumchannel blockers and calcium-calmodulin antagonists, as reviewed by Helson (1984) and Kessel (1986). This observation has generated much interest, because of its potential clinical use (Rogan et al., 1984; Ozols and Cowan, 1986).Reversing agents restore sensitivity to other drugs, but do not kill the MDR cells without these other drugs, which is a crucial difference with collateral sensitivity. Because reversal was first discovered with the calcium-channel blocker verapamil, substances with an effect on calcium metabolism have been studied most intensely. Examples are verapamil, Tiapamil, Bepridil, nifepidine, perhexiline maleate, trifluoperazine, forskolin, and quinidine (Tsuruo et al., 1983a,b; Schuurhuis et al., 1987; Ramu et al., 1984a; Wadler and Wiernik, 1988). Reversal has also been observed, however, with unrelated substances such as the antiestrogen tamoxifen (Ramu et al., 1984b) and cyclosporin A (Slater et al., 1986). Recent work has provided strong evidence that reversal is due to the competition of “reversal drugs” and “cytotoxic drugs” for efflux from the cell (Kessel and Wilberding, 1985; Kessel, 1986), probably competition for the transport mechanism. Verapamil binds specifically to membrane vesicles isolated from MDR cells and this binding is competitively inhibited b y drugs such as vinblastine and daunorubicin (Cornwell et al., 1987a). Reversal by verapamil is greatly enhanced by depletion of the energy supply and efflux of verapamil is also enhanced in MDR cells (Broxterman et al., 1988a). Verapamil and quinidine inhibit ATP-dependent vinblastine transport into vesicles isolated from MDR cells (Horio et al., 1988). Like verapamil, cyclosporin A reduces the difference in vinblastine accumulation seen in resistant and sensitive cell lines (Goldberg et al., 1988). This is the behavior expected of different substrates competing for the same mechanism of enhanced energy-dependent efflux, presumed to be instrumental for MDR. Energy depletion caused by verapamil cycling might also underlie increased cytotoxicity of verapamil (Cano-Gauci and Riordan, 1987). Hence, the distinction made between drugs that cause reversal of MDR and those used to select for resistance may

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ALEXANDER M. VAN DER BLIEK AND PIET BORST

solely reflect the difference in cytotoxicity and not a different role in efflux. It follows that the best agents for reversal are those with little or no toxic effect, but with maximal affinity for the transport mechanism. The nontoxic analogs of adriamycin fit these criteria (Skovsgaard, 1980; Kessel and Wheeler, 1984), but nontoxic verapamil analogs might be even more suitable, as verapamil binds to the membrane vesicles from MDR cells with higher affinity than the carcinostatic drugs tested (Cornwell et al., 1987a). In designing such analogs, it is a handicap that the normal cellular function of the efflux process and thus its natural substrate are still unknown. Thus, it is difficult to predict what side effects noncytotoxic compounds will have in the intact animal. V. Alterations in MDR Cells

The basis of MDR has been sought in altered protein levels with emphasis on membrane proteins, because these could provide a link to altered drug transport. Several changes have been discovered in MDR cells, but by far the most consistent is the overproduction of a 170-kDa membrane glycoprotein, the P-glycoprotein (Gerlach et al., 1986a), which will be considered in detail in the following discussions. An inventory of the sometimes bewildering variety of other changes is given here. The overproduction of smaller membrane proteins, which has been observed with less consistency, includes glycoproteins in the range of 130-150 kDa (Garman and Center, 1982; Roy and Horwitz, 1985).It is, however, not clear that the latter are truly distinct from Pglycoproteins (Greenberger et al., 1987; Gerlach et al., 1986a). In one instance a doubling of the EGF-binding sites was found, which could in principle reflect overproduction of the E G F receptor, but this has no obvious relation to MDR (Meyers et al., 1986). Decreased levels of membrane proteins have also been reported, e.g., of 72 and 75 kDa in MDR KB cells (Richert et al., 1985) and of 100 kDa in MDR human and CHL cells (Beck e t al., 1979; Biedler and Peterson, 1981; Garman and Center, 1982). The latter has, however, been ascribed to altered glycosylation, since the decrease was detectable with carbohydrate labeling but not with protein staining (Riordan and Ling, 1985). This could be an indirect effect of vastly overproducing a competing glycosyl transferase acceptor (P-glycoprotein). Cytosolic proteins that are overproduced in a subset of the MDR cells include an anionic glutathione transferase (Batist et al., 1986), a 20-kDa phosphorylated protein (Fine et al., 1988), and most frequently a 21-kDa protein

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called sorcin or V19 (Meyers and Biedler, 1981; Martinsson et al., 1985; Shen et al., 1986a; Meyers et al., 1987). In addition to altered protein levels, other changes have been reported for MDR cells. The glycosylation patterns of lipids (gangliosides) are simplified in MDR cells (Biedler and Peterson, 1981; Peterson et al., 1983) and, as suggested for the apparent decrease of 100-kDa proteins, this could result from competition for glycosyl transferases (Gerlach et al., 1986a). Likewise, vast overproduction of P-glycoproteins is believed to account for the earlier observations of generally enhanced glycosylation and charge differences (Kessel, 1979), a phenomenon most likely caused by the fact that Pglycoproteins are rich in the negatively charged sialic acids (Wheeler et al., 1982; Kartner et al., 1983a). Thus, most glycosylation differences could be secondary to the overproduction of proteins that are associated with MDR. Several reports have dealt with changes in phosphorylation patterns. For example, the 20-kDa protein of Fine et al. (1988) is constitutively phosphorylated, while sorcin is probably subject to CAMP-dependent phosphorylation (Biedler et al., 1983; van der Bliek et al., 1986b). Some of the glycoproteins, such as the P-glycoprotein, are also phosphorylated and this might be related to the level of resistance (Garman et al., 1983; Roy and Horwitz, 1985; Hamada et al., 1987). An unusual phenotype was described for an HL60 cell line selected for adriamycin resistance (Marsh et al., 1986). This has a phosphorylated 150-kDa glycoprotein that is present at the same level, but is nonphosphorylated in the drug-sensitive parent cell line. The protein does not cross-react with P-glycoprotein antibodies and the phenotype is stable, suggesting a genetic lesion (Marsh and Center, 1987).However, in all these cases the regulatory function of phosphorylation still needs to b e substantiated by direct evidence. There is a single report on increased alkaline phosphatase activity, which is perhaps related to the changes in phosphorylation patterns (Baskin, 1982). This last line of investigation has, however, not been pursued, nor is it readily apparent how this ties in with the MDR phenotype. To complete this resume of cellular alterations, small decreases of lipid structural order and of membrane fluidity have been observed (Wheeler et al., 1982; Siegfried et al., 1983; Ramu et al., 1983), but neither is consistently changed in MDR cells. As with glycosylation, this might be an indirect effect of overproducing membrane proteins (Gerlach et al., 1986a). Finally, changes in the lipid composition (Ramu et al., 1984c), as well as increased triglyceride content (Ramu

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ALEXANDER M. VAN DER BLIEK AND PIET BORST

et al., 1983), have also been reported, but it is yet unknown if these are general properties of MDR cells. Some highly resistant cells lose their capacity to form tumors in mice, grow slower in tissue culture, and have a distinct morphology (Biedler and Peterson, 1981). It seems likely that these effects result from the high overproduction of membrane proteins, either through altered adherence to external substrates, through altered internal rigidity, or through inadvertent export of metabolites. Altered growth characteristics of a different nature are found in some 3% of the MDR CHO cells selected with colchicine (Ling, 1977). These MDR cells are cold sensitive, i.e., the cell cycle appears reversibly blocked in G1, a property shared with other types of drug-resistant cells but with an as yet unknown cause. In summary, many different cellular alterations accompany the MDR phenotype, but the most consistent is thought to be Pglycoprotein overproduction. Some of the other alterations may prove to be side-effects of vast P-glycoprotein overproduction. VI. P-Glycoprotein Overproduction

Overproduction of large membrane glycoproteins in the classical type of MDR was discovered by Juliano and Ling (1976);the proteins

were called P-glycoproteins for permeability. Although several MDR cell lines initially appeared to lack the overproduced P-glycoproteins (Richert et al., 1985), it is now known that their apparent absence is often due to technical difficulties and thus lack of detection (Shen et al., 1986a; Gerlach et al., 1986a). P-Glycoproteins tend to aggregate in the slots of SDS-PAGE gels and their electrophoretic mobility is greatly influenced by the electrophoresis conditions. Once this was realized, P-glycoprotein overproduction proved to be the most consistent feature of MDR cells (Gerlach et al., 1986a), and a large body of evidence has now established that overproduction is indeed the primary cause of the classical type of MDR. The overproduced P-glycoproteins were detected in early experiments by labeling the carbohydrate moiety (Juliano and Ling, 1976; Biedler and Peterson, 1981). Highly resistant cells overproduce such large quantities (up to 10% of the glycoproteins) that P-glycoproteins are readily seen on two-dimensional gels as well (Beck, 1983). P-Glycoproteins have been isolated from membrane vesicles by fractionation on plant lectin columns (Riordan and Ling, 1979).These crude preparations were used to generate monoclonal antibodies directed against denatured P-glycoprotein. Antibody panels were

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then screened with human, mice, and hamster MDR cells to identify antibodies that recognize highly conserved domains of the denatured protein (Kartner et al., 1985). Independently, monoclonal antibodies, directed against the extracellular domains of native P-glycoproteins, have been isolated from mice injected with whole MDR cells (Hamada and Tsuruo, 1986; Scheper et al., 1988). P-Glycoproteins are heterogeneous on two-dimensional gels (Beck et al., 1983). This is largely attributed to microheterogeneity of glycosylation (Riordan and Ling, 1985). The P-glycoproteins from taxol-selected MDR cells, however, may also have a heterogeneous polypeptide backbone; this is suggested by pulse-chase experiments, which show a 120-kDa species, in addition to the common 125-kDa protein, before modification of the oligosaccharide chains takes place (Greenberger et al., 1987). The nature of this variability and whether it affects the specificity of drug transport is still unknown. The activities of isolated P-glycoproteins have not been thoroughly studied yet, because P-glycoproteins are easily inactivated by anything but the mildest detergents (Hamada and Tsuruo, 1988). Nevertheless, drug binding was demonstrated with photoaffinity-activated analogs of vinblastine (Safa et al., 1986).ATP also binds specifically to P-glycoprotein (Cornwell et al., 1987b), and recently the protein was shown to have ATPase activity (Hamada and Tsuruo, 1988). This establishes a direct link between P-glycoprotein overproduction and the energy dependence of MDR. Finally, the monoclonals directed against the extracellular domain of P-glycoproteins can reverse MDR to some extent (Hamada and Tsuruo, 1986; Broxterman et at., 1988b), also indicating a direct role of P-glycoproteins in increased drug efflux. Instability of P-glycoproteins suggests an explanation for the collateral sensitivity of MDR cells to agents such as local anesthetics. These agents may act as detergents affecting the overproduced P-glycoproteins and thus selectively disrupting membrane function in MDR cells. This hypothesis may be tested with transfectants that have P-glycoprotein encoding cDNAs rendered incapable of causing MDR by site-directed mutagenesis. VII. Amplified Genes in MDR Cell Lines

MDR arises by mutations. Cell survival during selection of drugresistant colonies, is in the range expected for mutation at a single genetic locus to lop6; Gerlach et al., 1986a). Hybrids of resistant and sensitive cells are resistant, demonstrating that MDR is a

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ALEXANDER M. VAN DER BLIEK AND PIET BORST

dominant trait (Ling and Baker, 1978). The MDR cells often have karyotypic signs of gene amplification-double minute chromosomes (DMs; Baskin et al., 1981; Howell et al., 1984) or homogeneously staining regions (HSRs; Biedler and Peterson, 1981; Grund et al., 1983; Meyers et al., 1985; Martinsson et al., 1985; Sen et al., 1987). Gene amplification underlies many forms of drug resistance, because it supports high levels of protein overproduction not easily achieved with a single gene copy (Stark, 1986; Schimke, 1988). In MDR cell line series that were selected with increasing drug concentrations, both the length of HSRs and the number of DMs, which reflect the degree of amplification, closely follow resistance levels. Revertant cell lines have usually lost their amplified DNA. Taken together these findings strongly suggest that overproduced proteins derived from amplified genes can cause MDR. Three approaches have been used to isolate nucleic acids encoding the overproduced proteins: (1)cDNAs encoding part of the overproduced P-glycoprotein were isolated by screening an expression library with antibody (Riordan et al., 1985); (2) cDNAs derived from overexpressed mRNAs were isolated by differential screening with radiolabeled RNA from both parent and resistant cell lines (van der Bliek et al., 1986a); ( 3 ) amplified DNA was isolated by virtue of its repetitive character, either with Cot-fractionation (Scotto et al., 1986), or with an in-gel enrichment procedure (Roninson et al., 1984). A genomic walk then led to the gene (Roninson et al., 1986; Gros et al., 1986a; Teeter et al., 1986) and eventually to the isolation of cDNAs derived from the overexpressed mRNA (Chen et al., 1986; Gros et al., 1986b). Probes isolated with each of these approaches detect a 4.5-kb mRNA that is overexpressed in the MDR cell lines. Because one set of cDNAs was isolated with specific antibodies (Riordan et al., 1985), and others cross-hybridize with these (van der Bliek et al., 1986a; Ueda et al., 1986), it seemed most likely that they all encode P-glycoproteins. Later, this was confirmed by sequence analysis. Screening for differentially expressed RNA also yielded cDNAs that were derived from six unrelated genes that are amplified and overexpressed in the MDR CHO cell line CHRC5(Table 11). These other genes are distinct from P-glycoprotein-encoding genes as judged by different transcript lengths, by lack of cross-hybridization, and by their (partial) sequence (van der Bliek et al., 1986a; A.M. van der Bliek, P.M. Kooiman, P. Borst, unpublished). The six gene classes, which include the P-glycoprotein-encoding genes (called Class 2 in this context), are linked within 1500 kb of the hamster genome (van der Bliek et al., 1986a; Jongsma et al., 1987). This MDR domain is

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TABLE I1 GENECLASSES AMPLIFIEDAND OVEREXPRESSED IN THE CHO LINECH"CS" mRNA(s) (nt)

Gene class

750

1 2a

2b 2c 3 4

5 6

Amplification degree

-

10 10

4500 4500 3200 1000/2600 3600 2600

10 30 30 30 30 30

Nature of gene

P-GIycoprotein (Pgp3)b P-Glycoprotein ( p g p 2 ) P-Glycoprotein ( p g p l ) sorcin -

From Borst and van der Bliek (1988). The pgp3 gene (Class 2a) is probably silent in this cell line (W. Ng and \'. Ling, personal communication). a 'I

amplified in CHRC5 cells, which explains how a whole battery of genes is coerced to overexpress RNA. A tentative map of the Chinese hamster MDR domain has been constructed using pulsed-field gradient gel electrophoresis (Fig. 1) (Borst and van der Bliek, 1988; A.M. van der Bliek, T. van der Velde-Koerts, and P. Borst, unpublished). The human MDR domain is probably very similar, because at least part of the same genes are coamplified in some cell lines (van der Bliek et al., 1988a). The wild-type chromosomal loci have been determined with in situ hybridization-human chromosome 7q21 (Trent et aE., 1985; Trent and Witkowski, 1987; Fojo et al., 1986),Chinese hamster chromosome Sac SfL

Sfl

Irl I€

CLASS

1

-

s a c sac sac

Sac

Sac

Sac

2a

P4P3

mdr3

F9P2

PYPl

mdrl

sorcin 5 0 0 kb

FIG.1. Physical map of the MDR domain in Chinese hamster. Large restriction fragments were generated with the infrequently cutting enzymes Sf;I or Sac11 and then analyzed by pulsed-field gradient gel electrophoresis. Details of the genes are given in Table I; pgp and mdr are synonyms, pgp stands for P-glycoprotein-encoding gene, and mdr stands for multidrug resistance gene. This figure was taken from Borst and van der Bliek (1988) and contains a compilation of data from van der Bliek et al. (1986a), de Bruijn et aE. (1986), and unpublished results of A.M. van der Bliek, T. van der Velde-Koerts, and P. Borst.

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ALEXANDER M. VAN DER BLIEK AND PIET BORST

lq26 (Trent et al., 1985; Jongsma et al., 1987), and mouse chromosome 5 (Martinsson and Levan, 1987). Because the series of coamplified genes span such a large area, it has provided a nice model to study the size and structure of the amplified DNA (Borst and van der Bliek, 1988; van der Bliek, 1988). VIII. The Central Role of P-Glycoprotein Genes in MDR

Differential amplification of genes in the MDR domain was observed in a series of MDR CHL cells (de Bruijn et al., 1986) and mouse SEWA tumor cells (Stahl et al., 1988) selected with different drugs. Each cell line has a different pattern of coamplification (Fig. 2). The order deduced from the amplification gradients flanking Class 2 (P-glycoprotein) genes reflects the genomic map determined with PFGE-analysis (A.M. van der Bliek, T. van der Velde-Koerts, and P. Chinese Hamster CHRC5 DC- 3F/ADX

Mouse AMD~-HSR

CL4-DM CL4:6:13:17-HSR COLR-DM

1

1 # I

VCR~-DM

Gene Class

1

2a

i

I

I

2b

2c

I

3

I

I

4

I

100

x

5

FIG.2. Amplification of individual gene classes in nine MDR cell lines. The amplification levels were determined by hybridizing serial dilutions of genomic DNA with probes representing five gene classes from the M D R domain (see Table 11). Gene Class 6 was not part of this analysis, because it was found at a later stage. The degree of amplification is indicated by the bar at the right. Taken from Stahl et al. (1988).

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Borst, unpublished). The central role of P-glycoproteins in MDR is highlighted by the fact that Class 2 genes are the only ones consistently amplified and overexpressed in this panel of cell lines. In some of these, only a subset of the restriction fragments detected by Class 2 probes on genomic blots is amplified. This partitioning suggested that several genes encoding a small family of P-glycoproteins are arranged side by side in the hamster genome. Such differential amplification has also been detected in a variety of other hamster, mouse, and human MDR cell lines (Riordan et al., 1985; Scotto et al., 1986; Roninson et al., 1986; van der Bliek et al., 1988a). It is now known that this division indeed corresponds to separate, but closely related, genes. P-Glycoprotein genes are overexpressed in all MDR cell lines examined thus far. The degree of overexpression follows that of resistance in a series of cell lines selected with increasing drug concentrations and in revertants (Riordan et al., 1985; Shen et al., 1986b). This is not necessarily reflected by the degree of gene amplification. Low levels of amplification with high mRNA levels and high resistance have been observed in one MDR hamster cell line (Scotto et al., 1986; de Bruijn et al., 1986). Many of the human MDR cell lines have high levels of expression with little or no gene amplification (Shen et aE., 1986b; van der Bliek et al., 1988a). The human genes could be more tightly regulated, because the parent cell lines have extremely low, often undetectable, P-glycoprotein mRNA expression (Shen et al., 1986b; van der Bliek e t aE., 1988a),unlike the rodent cell lines (Scotto et al., 1986; de Bruijn et aE., 1986; Stahl et at., 1988). Therefore, transcriptional activation could be a prerequisite for survival of (some) human MDR cell lines, since the amplification of a silent gene is pointless. The low levels of amplification that are observed do, however, present an alternative explanation. DNA rearrangement in the first round of amplification could activate transcription, if it were to introduce new controlling elements near the promoter. Such an event could make further amplification unnecessary. The important role of transcription regulation is emphasized by a revertant of the human K562/VCR cell line that has retained its high gene copy number, but has lost P-glycoprotein gene expression (Sugimoto et al., 1987). Transcription deregulation may prove a more frequent cause of P-glycoprotein overproduction in patients, partly because resistance in patients need not be as high as in model cell lines and partly because of the purported difference between rodents and humans. However, not enough samples have been tested to back up generalizations.

180

ALEXANDER M. VAN DER BLIEK AND PIET BORST

The functional significance of P-glycoprotein overproduction has been tested directly with transfection experiments. To fish for the essential genes, high-molecular-weight genomic DNA from MDR cell lines was transfected into drug-sensitive cell lines, followed by selection for drug resistance (Debenham et al., 1982; Robertson et al., 1984). The frequency of resistant colonies is greatly increased in recipients of DNA from MDR cells, compared to recipients of DNA from drug-sensitive cells. The recipients of DNA from MDR cells overproduce P-glycoproteins and have amplified exogenous copies of a P-glycoprotein gene. The extraneous origin was apparent from species-related differences in restriction fragment sizes as detected with DNA blots (Gros et al., 1986c; Shen et al., 1986c; Deuchars et al., 1987; Sugimoto and Tsuruo, 1987). None of the other genes that are coamplified in the MDR domain was cotransfected (Deuchars et al., 1987). Finally, the simplest and most conclusive test has been transfection with full-length P-glycoprotein cDNAs placed in an expression vector (Gros et al., 1986b; Croop et al., 1987; Ueda et al., 1987a; van der Bliek et al., 1988b). The transfectants were first selected with a cotransfected dominant marker and in a second round with adriamycin. These cells overexpress the exogenous sequences and are highly resistant to the drugs associated with MDR. Resistance acquired by transfection is also reversed with verapamil (Croop et al., 1987). These results demonstrate that a single overproduced Pglycoprotein confers full-blown MDR. IX. P-Glycoprotein Structure Deduced from

Sequence Comparisons The amino acid sequence of P-glycoproteins has been deduced from cDNAs derived from a human gene called m d r l (Chen et al., 1986), a mouse gene called m d r (Gros et al., 1986d), and a hamster gene now called p g p l (Gerlach et al., 1986b). Later, sequences were added from other P-glycoprotein cDNAs, i.e., human mdr3 (van der Bliek et al., 1987,1988b)and hamster p g p 2 (Endicott et al., 1987).The complete sequences show that the genes encode proteins ranging between 1276 and 1280 amino acid residues, which corresponds to a 140-kDa apoprotein. The postulated structure of P-glycoproteins inferred from the amino acid sequence is shown in Fig. 3. The coding sequences comprise two halves that form a direct repeat with long tracts of identical amino acids. The halves are connected by a linker of 60 amino acids, which is poorly conserved between species. Each half can be divided in two domains: an N-terminal domain with compara-

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FIG.3. Proposed structure of a P-glycoprotein as it spans the cell membrane. The nucleotide-binding folds are brought together in this scheme. The potential N-linked glycosylation sites are indicated by the branches sprouting from the polypeptide. Adapted from Chen et al. (1986) and Gros et al. (1986d .

tively little similarity between the halves, and a C-terminal domain which is largely identical. These domains correspond to functional units, as became apparent from further analysis. The most striking feature of P-glycoproteins is their similarity to bacterial transport proteins in the domains that are also conserved between the two protein halves; this is illustrated with a rootless tree in Fig. 4. The transport proteins include proteins that import maltose, phosphate, ribose, histidine, and oligopeptides. Separated by 100 amino acids, there are two short stretches that fit the Walker consensus for nucleotide-binding sites, presumably brought together in a fold of the protein (Walker et al., 1982). These sequences are conserved between ATPases and other enzymes. ATPase activity has indeed been demonstrated with isolated P-glycoproteins (Hamada and Tsuruo, 1988). The number of residues that P-glycoproteins share with bacterial transport proteins is much greater and is spread over large parts of the 250 amino acids, making up the conserved domains. Most bacterial permeases consist of one peripheral membrane subunit, which contains the ATPase sequence that is similar to Pglycoproteins, and two integral membrane subunits, mediating the import of extracellular substrates (Ames, 1986a; Higgins e t al., 1986). One of the bacterial transport proteins, hemolysin B, is self-contained. Hemolysin B shares the largest number of residues with Pglycoproteins and this homology extends into the upstream domain. The function of hemolysin B is to actively extrude another protein that is encoded by the hemolysin operon (hemolysin A). Apart from the nature of extruded substrates, such a role as mediator of energydependent efflux closely resembles the postulated function of Pglycoproteins. The second prominent feature of P-glycoprotein sequences is a

182

ALEXANDER M. VAN DER BLIEK AND PIET BORST

p21 (H-ras-1)

I

7 OPPD

HlyB

P-glycoprotein HisP PstB MalK

I

U

Adenylate kinase ATPase-a chain

I ATPase-p chain

Biotin carboxyl carrier I

0

I

12

I

24

Segment comparison score (sd units) FIG.4. Cluster analysis of the protein sequence homology of some nucleotidebinding proteins. The C-terminal nucleotide binding domain of the hamster Pglycoprotein derived from the p g p l sequence was used here. The other proteins are the human transforming protein p21 (H-rus-1); nucleotide-binding subunits of the bacterial permeases for oIigopeptides (OppD), histidine (HisP), phosphate (PstB), and maItose (MalK); the nucleotide-binding domain of hemolysin B (HlyB); rabbit adenylate kinase; biotin carboxyl carrier from Propionibacterium shermanii; and a and p chains of the Escherichia coli F1-ATPase. Taken from Gerlach et al. (1986b).

series of six hydrophobic segments located upstream of the ATPases in both halves (Fig. 5). These were predicted to span the membrane when calculated with the algorithm of Eisenberg et al. (1984).Parts of these segments are also conserved in hemolysin B. Together, the twelve hydrophobic segments of P-glycoproteins could form a membrane channel through which drugs are extruded (Fig. 3 ) . The structure of P-glycoproteins is therefore fully compatible with their postulated role as ATP-driven pumps, which remove drugs from the cytoplasm, thereby preventing cytotoxicity. In the postulated structure of P-glycoproteins, only small parts are exposed to the extracellular environment. The largest extracellular domain is found between transmembrane segments 1 and 2 of the N-terminal half. This is one of the most divergent parts, but in each

183

MULTIDHUG RESISTANCE

-tms

I

1

2

3 4

-=

-5 6

nbs 1

i.m s . 1 2

2

=

I

nh-

..I"

5 6

3 4

- I I -

1

I I

2

=

I

I

500 C

.-

1

I

I

;'

-50 1hO

260

3hO

4bO

5bO

660

1

I

I

I

I

I

700

800

900

1000

1100

1200

FIG.5. Hydropathy plot of the predicted amino acid sequence of the P-glycoprotein encoded by mdr3. The putative membrane-spanning segments (tms) and nucleotidebinding folds (nbs) are depicted above the plot. The sequence was taken from van der Bliek et al. (1988b) and the plot was calculated with the method of Kyte and Doolittle (1982) for a window of 15 amino acids.

P-glycoprotein it contains the only external recognition sites for N-linked glycosylation (Asn-X-Ser/Thr). This is probably where the carbohydrate moiety of 30 kDa is attached. The proposed structure of P-glycoproteins has not yet been confirmed with biochemical methods. The antigenic determinants of P-glycoproteins detected by monoclonal antibodies on intact cells are also still unknown. On the other hand, a monoclonal selected for highly conserved determinants is known to recognize the C-terminal ATPase (Gerlach et al., 1986b), and this is only possible with ruptured cells (Kartner et al., 1985), in line with a cytoplasmic epitope. Manipulation of P-glycoproteins has been difficult because of their tendency to aggregate. This may result from the peculiar structure having no less than 12 hydrophobic segments. In spite of that, many questions must be answered before understanding how P-glycoproteins actually translocate drugs. For instance, it is still unknown if both halves are needed to form a unit capable of pumping drugs or, for that matter, if hemolysin B also forms a dimer. Structural and functional studies should benefit greatly from the model deduced from sequence and from the potential to manipulate this using recombinant DNA. Hemolysin B and P-glycoproteins have similar sequences over a much greater length than other bacterial transport proteins, which has prompted the suggestion of further analogy. Conceivably

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P-glycoproteins could export another protein, much like hemolysin B mediates the export of hemolysin A (Gerlach et al., 1986b). In this hypothesis, drug efflux is caused by haphazard affinity for the same export pump or by hitchhiking with the exported polypeptide. The fact that transfection with P-glycoprotein cDNA confers complete MDR rules out the need to overproduce other proteins in order to become resistant (Gros et al., 198613; Ueda et al., 1987a; Croop et al., 1987). Hitchhiking with particularly abundant polypeptides is possible, but as yet no such peptides have been identified. Nevertheless, there is one observation suggesting that P-glycoproteins can indeed export peptides. The MDR cell line CHRC5,selected with colchicine, is also highly resistant to gramicidin D (5000 X versus 180 X for colchicine), which is a cyclic peptide ionophore (Gerlach et al., 1986a). Though this has not been tested, gramicidin D could be removed by active efflux like the other drugs involved in MDR. An additional argument for analogy to hemolysin A export has been the unusual sequence of the hemolysin operon. The CG content is lower than expected for genes from Escherichia coli (Felmlee et al., 1985), which together with the extensive sequence similarity raised the possibility of a mammalian origin (Gerlach et al., 1986b). Hemolysin is, however, also found in bacteria from the genus Proteus, and these have a CG content comparable to that in mammals (Koronakis et al., 1987). This suggests a much more mundane cause for the aberrant E . coli sequences: transfer between bacterial genera. The extensive similarity with P-glycoproteins can also be explained without invoking peptide transport. Similarity in transmembrane segments may merely reflect structural requirements inherent in the export process, since hemolysin B is one of the few known ATP-driven bacterial permeases that export their substrate like P-glycoproteins. Finally, evidence summarized in Section VI strongly indicates that at least some drugs directly bind to P-glycoproteins. Therefore, the role of peptides as P-glycoprotein substrates, though an interesting possibility, now seems doubtful. Another remarkable parallel was found in the emergence of multidrug-resistant Plasmodium stains. MDR seriously hampers the treatment of malaria. As yet there are only pharmacological data, but these point to a P-glycoprotein-related mechanism (Martin et ul., 1987): (1) the structures of the widely used chloroquine analogs resemble drugs implicated in mammalian MDR, (2) malarial MDR is also reversed by verapamil, and ( 3 ) vice versa, mammalian MDR is reversed by chloroquine and by quinidine, used for its antiarhythmic effect (Tsuruo et al., 1984; Zamora and Beck, 1986). Quinidine and quinine

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both kill Plasmodium (Goodman Gilman et al., 1980). Thus, a cytotoxic treatment in one organism may merely cause reversal in the other, which is line with the hypothesis that drug accumulation by reversal reflects competition for the same pump. Yeast is also being scrutinized, because the methods for gene manipulation are more powerful in yeast than in most other eukaryotes. Unwittingly, a gene encoding a drug transport protein was cloned while the investigators were searching for regulatory factors (Kanazawa et al., 1988). This gene, named ATRl after the selective drug aminotriazole, does not confer cross-resistance. Although the derived protein shares few sequences with P-glycoproteins, it does have 12 hydrophobic segments and one ATPase. In fact, many other proteins that transport organic molecules ranging from sugars to antibiotics have 12 membrane-spanning segments (Henderson and Maiden, 1987). This probably reflects a favored channel structure, but its spatial arrangement is not known. Other drug-resistant yeast mutants do have a MDR phenotype and one (PDRI)encodes a putative transcription factor, but the regulated process, presumably responsible, for the phenotype, is unknown (Balzi et al., 1987). Summing up, the primary sequence of P-glycoproteins has provided a structure, which unites many of the features learned from other studies. P-Glycoproteins are most likely ATP-driven efflux pumps and when overproduced may keep the intracellular drug concentration low, providing a molecular explanation for MDR (Ames, 198610). X. Diversity of P-Glycoproteins

Interest in P-glycoprotein diversity stems from the observation that MDR cell lines usually have the highest resistance against the selective drug. One explanation was suggested by the detection of differentially amplified DNA with P-glycoprotein probes (van der Bliek et al., 1986a, 1988a; d e Bruijn et al., 1986; Scotto et al., 1986; Roninson et al., 1986; Stahl et al., 1988). This indicates that several closely related P-glycoprotein genes form a small gene family in mammals. If these have different drug specificities, then differential expression could in principle modulate the MDR phenotype. Two sets of amplified fragments are detected in human DNA and three are detected in rodent DNA (Fig. 2). It became evident from differences in cDNA sequences that these are distinct genes. Two genes have been identified in man, called mdrl (Chen et al., 1986) and mdr3 (van der Bliek et al., 1987), and two have been identified in Chinese hamster, called p g p l and p g p 2 (Endicott et al., 1987). Their

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TABLE I11 PUTATIVEP-GLYCOPROTEIN GENES Class ~

Species

2c

2b

2a

Hamster Mouse Human"

PgPl [mdrl] mdrl

PgP2 mdr2 -

tPgP31" mdr3b mdr3

Bracketed genes have not yet been identified with cDNAs, but presumably do exist. The sequence of cDNAs derived from a mouse equivalent of hamster pgp3 and human mdr3 was recently determined by Gros et al. (1988).These authors have used a different numbering, calling this last gene mouse mdr2, and calling the equivalent of hamster pgp2 mouse m drl. There appears to be no human homologue of hamster pgp2 or mouse mdr2.

numbers designate cross-species similarity and gene order as presented in Fig. 1.The status of other homologues, listed in Table 111, is * as follows: 1. In mouse, two mdr genes have been described (Gros et al., 1986d; Gros et al., 1988). The one sequenced by Gros et al. (1986d) is called mdr2 here, because it is most similar to hamster pgp2 (Endicott et al., 1987). Recently, the sequence of a second mouse mdr gene was determined (Gros et al., 1988). This gene is most similar to human mdr3 and is expressed in pre-B lymphocytes (Gros et al., 1986b). 2. In hamster, a homologue of human mdr3 (called pgp3) has been identified in genomic DNA, but no transcripts have been detected yet (W. Ng and V. Ling, personal communication). Figure 1 shows that this hamster pgp3 resides next to the pgpl and pgp2 genes that were identified with cDNA sequences (Endicott et al., 1987). 3. In humans, DNA from a gene called mdr2 was identified b y cross-hybridization (Roninson et al., 1986),but no transcripts could be detected at that time. Because the sequenced parts are identical to mdr3, it is almost certain that mdr2 and mdr3 are actually one and the same gene (I.B. Roninson, personal communication). We have decided to maintain the designation mdr3, because it refers to crossspecies homology. Humans seem to lack a homologue of rodent mdr2/pgp2, although the evidence for this is still circumstantial (van der Bliek et al., 1988b). Long-distance mapping of the P-glycoprotein genes in hamster and man has shown that they are linked within 500 kb (Fig. 1; van der Bliek et al., 1986a, 1987; A.M. van der Bliek, T. van der Velde-Koerts, and P. Borst, unpublished data), which is in line with the detection of

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I

-2-

I

I

3 4

5 6

nbs 1

2

I-

I-

I

I

tms 1 2

3 4

5 6

nbs 1

II

I I

I W

I

2

-

100

2.

E

050

0

amino acld

FIG.6. Comparison of amino acid sequences predicted for mdrl- and mdr3-encoded P-glycoproteins. The sequences could be aligned with only three gaps of 3-4 amino acids and the percentage identity was determined in blocks of 10 amino acids. The putative membrane-spanning segments (tms) and nucleotide-binding folds (nbs) are depicted above the plot. Sequences were taken from Chen et al. (1986) and van der Bliek et al. (1988b).

only one wild-type locus using in situ hybridization (Trent et al., 1985; Trent and Witkowski, 1987; Jongsma et al., 1987). Sequences of P-glycoproteins are very similar, with few gaps needed for alignment (Endicott et al., 1987; van der Bliek et al., 1987, 1988b). Variation in 3'-untranslated sequences accounts for the slightly different sizes of predominant mRNAs (4100 nt for mdr3 and 4500 nt for others). Protein-coding sequences are largely the same, which suggests that each of the family members functions as active efflux pump. Human mdrl and mdr3, for example, have over 80% identical amino acids (Fig. 6). Divergence is greatest at the N-termini and the linkers connecting the two halves of P-glycoprotein. Not much more than length and hydrophilicity are conserved in these segments. The ATPase domains are at the other end of the scale, being highly conserved in all P-glycoproteins. In several instances gene conversion has reinforced this similarity (van der Bliek et al., 1987, 1988b; Endicott et al., 1987). Sequences that determine the substrate specificity of different P-glycoproteins are probably located in or near the transmembrane domains, because these must come in contact with substances to be exported. However, neither binding sites nor the molecular basis of different substrate specificities are known yet. Evidence for an additional diversification of P-glycoproteins by

1

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ALEXANDER IM. VAN DER BLIEK AND PIET BORST

alternative splicing of pre-mRNAs was obtained with cDNAs from human liver. Three types of cDNAs were obtained. The predominant species has a structure very similar to mdrl cDNAs; another variant has a 7-amino acid insertion in the C-terminal ATPase and a third variant has a deletion of 43 or 47 amino acids, which covers a putative membrane-spanning segment (van der Bliek et at., 1987). A similar deletion of a membrane-spanning segment by alternative splicing was recently observed with chicken anion-transporter mRNAs, but also in this case it is unknown whether the encoded protein is functional (Cox and Lazarides, 1988). Potentially such alterations could affect the substrate specificity of P-glycoproteins, but this remains to be tested. The key question, whether differential expression or alternative splicing influences the MDR phenotype, is still unanswered, Hamster p g p l and pgp2 are expressed simultaneously in an MDR cell line, but their relative contributions to resistance are unknown (Endicott et al., 1987). Mouse mdr2 was successfully used to confer MDR in transfection experiments (Gros et al., 1986b; Croop et al., 1987), while mdrl ( p g p l ) is the sole P-glycoprotein gene amplified in several rodent MDR cell lines (de Bruijn e t al., 1986; Stahl et al., 1988). Thus, differential expression of these two genes could modulate the phenotype, assuming they have different drug specificities; this must still be tested. However, as noted before, humans appear to lack a homologue of rodent mdr2lpgp2. Furthermore, the expression of mdr3 was undetectable in five human MDR cell lines tested in our lab (van der Bliek e t al., 1988a) and in cell lines tested by Roninson et al. (1986) with probes named mdr2 but identical to mdr3 (I.B. Roninson, personal communication). Overexpression of mdrl is the prime cause of MDR here. Variation in cross-resistance patterns, exemplified by the greatly enhanced vincristine resistance of MC-IXC/VCR cells, must result from changes within mdrl or in non-mdr genes. MDR cell lines derived from other cell types still need to be checked for mdr3 overexpression. Of special interest are those derived from tissues, which already have a basal level of transcription (e.g., liver). Finally, transfections with mdr3 constructs are being done to test whether this gene can confer resistance at all. XI. Mutated P-Glycoprotein Genes with Altered Drug Transport Properties

Recently Roninson’s group has shown that mutations in the mdrl gene can substantially affect the spectrum of drugs transported by P-glycoproteins (Choi et al., 1988). During stepwise selection for

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increasing resistance to colchicine, a sudden increase in the relative resistance to colchicine but not to vinblastine or to adriamycin was observed, and this was traced to the appearance of a mutated version of mdrl. A single point mutation changing a glycine into a valine in transmembrane segment 1 (cf. Fig. 3 ) of the P-glycoprotein proved responsible for the approximately threefold increase in the relative resistance to colchicine. This important finding leads to the following picture: during the stringent selection required to obtain highly resistant MDR cells, mutant versions of the gene arise by genetic “noise.” Occasionally a mutant gene copy emerges that codes for a P-glycoprotein, which is more able to extrude the drug used in selection. This altered gene copy confers a selective advantage on the cell that contains it and this advantage can be increased in daughter cells that have preferentially amplified the altered gene copy. Under continued selection the cell population can then change to cells containing only the altered gene. The experiments of Roninson and co-workers have clearly established that the spectrum of resistance in MDR cells can be modulated by mutations in the P-glycoprotein genes. Potentially, such a mechanism could explain the variations in drug resistance in MDR cells generated in the laboratory, and it might also be an important determinant of the cross-resistance spectra in patients, since the intensive treatment required to eliminate large tumor burdens should provide ample opportunity for mutant versions of the P-glycoprotein genes to arise. Further work is required to see whether this mechanism will be able to account for most of the variation in MDR, or whether other mechanisms discussed in the preceding section and in Section XI11 also make a substantial contribution. XII. P-Glycoprotein Expression in Normal Tissue and Its Regulation

The cellular distribution of P-glycoprotein has been determined by electron microscopy with ferritin-labeled monoclonal antibodies (Willingham et al., 1987). Most of the grains are evenly distributed over the plasma membrane, but are absent from coated pits. A few grains are seen in the endoplasmic reticulum and Golgi stacks, as expected for newly synthesized membrane proteins. The rest of the cell is virtually without label, confirming the location at the outer membrane. The antibody used in this study (MRK16) recognizes an extracellular epitope encoded by mdrl (Hamada and Tsuruo, 1986), which is probably not shared with mdr3 because of sequence

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ALEXANDER M. VAN DER BLIEK AND PIET BORST

divergence (van der Bliek et al., 1988b). However, in view of the overall sequence similarity, it seems likely that the protein encoded by mdr3 has a comparable distribution. The expression of P-glycoproteins in normal tissues has been determined by an analysis of RNA levels (Fojo et al., 1987; Baas and Borst, 1988) and by detection with the antibody MRK16 (Thiebaut et al., 1987; Sugawara et al., 1988). P-Glycoprotein mRNAs are detected in all tissues that have been examined, which suggests that the protein is a normal constituent of the cell. In general, intermediate to high levels are found in tissues that have an excretory function: kidney, liver, spleen, colon, jejunum, rectum, and esophagus. However, the studies do not agree fully on the relative expression levels. Variability may reflect species differences, because Baas and Borst (1988) used tissues from Chinese hamster and Fojo et al. (1987) used human tissue. The results are possibly also influenced by variation between sampled individuals and RNA quality. Discrepancies were, for example, in testis, ovarium, and uterus (high in hamster, low in humans), or kidney and liver (high in humans, low in hamster). The most striking difference is the very high level of expression in the human adrenal medulla, but the low levels in hamster. Some human pheochromocytomas (tumors derived from the adrenal gland) also had high expression levels. It is unknown why hamsters and humans differ in this respect. P-Glycoproteins have been localized with more precision, but sacrificing sensitivity with immunoperoxidase staining of human tissue using the MRK16 monoclonal antibody (Thiebaut et al., 1987; Sugawara et al., 1988). Basically, expression followed the patterns detected with RNA, but intense staining was found especially at the surface of the digestive tract, at surfaces of ducts connected to this tract and kidney tubules (Fig. 7), and in placenta. Recent in situ hybridizations also show preferential localization of P-glycoprotein mRNA in the epithelial cells in the villi of small intestine, colon, and stomach in Chinese hamsters (Mukhopadhyay et al., 1988). Thus, a major role of P-glycoproteins seems to be secretion into lumina connected with the external environment. Likewise, most drugs implicated in MDR are excreted via these organs (Goodman Gilman et al., 1980). In the adult adrenal medulla P-glycoprotein staining is distributed evenly, suggesting secretion into the interstitial space. No staining was detected in fetal and neonatal adrenals, indicating that the protein is not required for normal adrenal function (Sugawara et al., 1988). Methods used in both RNA studies could in principle detect each member of the P-glycoprotein family, while the antibody

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FIG.7. Schematic diagram of the organ distribution of P-glycoprotein. The arrows indicate the putative excretory function. The proteins were located with the monoclonal antibody MRK16 directed against an extracellular domain of P-glycoproteins. Taken from Thiebaut et al. (1987).

studies are probably restricted to P-glycoprotein encoded by mdrl . All that is known of mdr3 expression is its presence in human liver and in the hepatoma cell line HepG2 (van der Bliek et al., 1987), whereas the homologous mdr3 cDNA was isolated by Gros et aE., 1986b) from a drug-sensitive pre-B lymphocyte cell line. From the study of MDR cell lines, it is already apparent that mdrl expression does not only depend on gene amplification. Indeed, expression is highly inducible in regenerating liver and (pre)neoplastic hepatic nodules (Thorgeirsson et al., 1987; Fairchild et al., 1987a), in line with earlier studies showing the induction of drug resistance in these cell types (Farber et al., 1976; Judah et al., 1977; Carr and Laishes, 1981; Carr, 1987). There are apparently two types of induction: (1)cell-growth dependent and (2) induction by carcinogens. These include carcinogens such as aflatoxin B 1 , acetylaminofluorene, and diethylnitrosamine, in addition to some less exotic substances (ethanol and CC14). Cross-resistance ranges from MDR-associated drugs to the carcinogens (Carr, 1987). To make matters more complicated, phorbol esters induce MDR in the breast cancer cell line MCF7

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ALEXANDER M. VAN DER BLIEK AND PIET BORST

(Fine et al., 1988), but not in hepatocellular carcinomas or nodules (Carr, 1987).Taken together, these results show that mdrl is regulated by a variety of external factors, not necessarily related to drug selection. Many of the treatments induce the P-450 monooxygenase system as well (see Nebert and Gonzalez, 1987). Therefore, a direct link between mdrl overexpression and carcinogen resistance should be tested separately with a nonhepatic MDR cell line, e.g., a transfectant. The study of P-glycoprotein gene regulation is still in its infancy. Use of alternative mdrl promoters has been observed in different MDR cell lines (Ueda et al., 1987b), which supports the notion that expression is controlled by transcriptional activation. Experiments have been done to test whether protein kinases affect mdr expression or P-glycoprotein activity: MDR MCF7 cells have increased protein kinase C (PK-C) activity (Fine et al., 1988), a mutant of CAMPdependent protein kinase (PK-A) is particularly drug sensitive (Abraham et al., 1987), and many of the MDR-reversing agents (discussed in Section IV) could also affect transcription via PK-C or PK-A. However, none of these studies has directly addressed the question whether mdrl expression is influenced too. P-Glycoproteins can be extensively phosphorylated, which may constitute another level of control (Carlsen et al., 1976; Garman et al., 1983; Roy and Honvitz, 1985). Phosphorylation is partly CAMPdependent in vitro (Mellado and Horwitz, 1987) and it is modulated by a variety of factors in vivo. P-Glycoproteins are phosphorylated at a basal level, which can be increased by phorbol esters or by agents that cause reversal of MDR (verapamil and trifluoperazine), but also influence calcium-dependent phosphorylation (Hamada et al., 1987). With tryptic digests it has been shown that each treatment causes phosphorylation at different sites. Modulation of P-glycoprotein activity by phosphorylation, though not unlikely, has not been demonstrated yet. Especially with the reversing agents, it will be difficult to distinguish between competition for active efflux and downregulation of P-glycoprotein activity. XIII. Coamplified Genes and Alterations Elsewhere in the Genome In addition to P-glycoproteins, there are a number of other proteins with altered expression levels in MDR cell lines. These are discussed here, because they may influence the phenotype, if not by modulating cross-resistance patterns then perhaps by influencing other characteristics. The P-glycoprotein genes are flanked by at least five other

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genes that are coamplified and overexpressed in a subset of the MDR cell lines (Table 11; Fig. 2). Partial sequences of the cDNAs derived from all but Class 3 have been determined (A.M. van der Bliek, P.M. Kooiman, and P. Borst, unpublished), but only Class 4 has significant similarity to other known proteins. Class 4 has been analyzed in detail, because it encodes one of the proteins that were originally deemed important for MDR. Furthermore, high levels of overexpression that are reached by coamplification may inadvertently influence the phenotype. Class 4 encodes sorcin (van der Bliek et al., 1986b), a 21-kDa calcium-binding protein (Koch et al., 1986), formerly called V19, CP22, or the 21-kDa protein (Meyers and Biedler, 1981; Martinsson et al., 1985; Shen et al., 1986a; Meyers et al., 1987). It is overexpressed in some but not all MDR cell lines, a fact that is readily explained by its linkage to P-glycoprotein genes (Fig. 1). The sequence has four putative calcium-binding sites (van der Bliek et al., 1986b), which is common for calmodulin-like proteins. Within the calmodulin family, sorcin is most similar to the regulatory light chain of calciumdependent protease (calpain; Sakihama et al., 1985), but its function remains unknown. It is now very likely that sorcin is only overproduced in (some) MDR cell lines, because the sorcin gene is linked to the P-glycoprotein genes. Nevertheless, simply overproducing a calcium-binding protein at the high levels observed in some MDR cells could alter their phenotype, especially when sorcin elevation is combined with drugs intended to change calcium metabolism. This is currently being tested by cotransfection with mdrl (M.B. Meyers, J.L. Biedler, A.M. van der Bliek, and P. Borst, unpublished). Cross-resistance patterns could be influenced by alterations elsewhere in the genome, either modulating P-glycoproteins directly (factors governing transcription, splicing, modification, etc.), or by distinct but complementary means of resistance. An example is the combination of topoisomerase I1 alterations with classical MDR, first described by Pommier et al. (1986a,b). The topoisomerase I1 alterations only affect drugs that interact with this enzyme, such as anthracyclins and podophyllotoxins, but not drugs with other targets, such as vinca alkaloids or colchicine. Hence, the combination of topoisomerase I1 alterations with MDR leads to cells with relatively low resistance to the latter group of drugs (Pommier et al., 1986a; Zijlstra et al., 1987). Obviously, topoisomerase I1 alterations alone affect the response to several drug classes, but it does not seem helpful to describe this as “atypical inultidrug resistance” (contrast Danks et al., 1987). Likewise, cross-resistance, albeit at a low level,

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ALEXANDER M. VAN DER BLIEK AND PIET BORST

was observed after adriamycin and melphalan selection (Louie et al., 1986; Harker and Sikic, 1985). In these cases the common denominator seems to be the drug target (DNA) and there is no evidence for any relation with classical MDR. A 45-fold overproduction of an anionic glutathione transferase was found in an adriamycin-resistant cell line derived from MCF7 breast cancer cells (Batist et al., 1986; Yeh et al., 1987). However, this cell line also overproduces P-glycoproteins (Fairchild et al., 1987b) and there is no evidence that the glutathione transferase, which is at an unusually low level in the parental cell line, makes any contribution to the resistant phenotype. As discussed in the previous section, cross-talk between P-glycoprotein and P-450 regulation is possible, because they have several inducing agents in common. Finally, the effects of some of the drugs associated with MDR can be countered by a mutation that is specific for that drug. For example, colcemidselected cells can have a tubulin gene mutation or P-glycoprotein overproduction (Keates et al., 1981). In conclusion, stringent selection may allow other mechanisms, in addition to P-glycoprotein overproduction, to modulate the MDR phenotype. However, mutations within a P-glycoprotein-encoding gene has provided the only example of an altered resistance spectrum, which has been demonstrated in transfection experiments. XIV. MDR in the Clinic

Drug resistance occurs all too often in cancer chemotherapy (Curt et al., 1984; Ozols and Cowan, 1986; Sobrero and Bertino, 1986). It is, however, unknown how widespread P-glycoprotein overproduction is. Thus far, the number of reports has been few and far between, probably because the probes have only recently become available for routine use. The ultimate goal is to find out whether the level of P-glycoprotein expression has prognostic value and whether treatment can be adapted accordingly. In a first report by Bell et al. (1985), ascites cells from two out of five patients with clinically drug-resistant ovarian carcinomas were shown to have elevated levels of Pglycoproteins that could be detected with antibodies. Later, 46 specimens representing 12 tumor types were surveyed. Increased levels of P-glycoproteins were detected only in 4 out of the 11 sarcomas that were tested (Gerlach et al., 1987). Screening of 14 additional sarcomas showed 2 more samples with increased Pglycoprotein levels. Of the six patients with elevated P-glycoprotein levels, only three had received prior drug treatment. These did not

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respond to later chemotherapy, nor did one of the patients who already had elevated levels prior to treatment. The remaining two were not treated with chemotherapy. In two patients with acute nonlymphoblastic leukemia, P-glycoprotein was detected after 2 weeks of treatment with drugs, including daunorubicin (Ma et al., 1987). The percentage of peripheral blood cells that stained with P-glycoprotein antibodies increased with subsequent treatment. Similarly, leukemia cells from three out of six patients with chronic myelogenic leukemia had detectable levels of P-glycoprotein after a variety of drug treatments (Tsuruo et al., 1987). Finally, Pglycoprotein expression has been monitored with blotted RNA from tumor samples (Fojo et al., 1987). In one out of four sarcomas and several pheochromocytomas the level of mdrl RNA was increased. It is unknown why there is such variation in expression even without prior treatment. Although recently cells from larger numbers of patients have been screened for P-glycoprotein expression (Fredericks et al., 1988; Shuin et al., 1988), it is clear that insufficient samples have been tested to correlate P-glycoprotein expression with the response to chemotherapy. Verapamil has been used to assist doxorubicin treatment of refractory ovarian carcinomas (Ozols and Cowan, 1986), but its cardiotoxicity has prevented the use of adequate doses. Other agents that reverse MDR are now being considered as alternatives, e.g., Bepridil (Schuurhuis et al., 1987) or cyclosporin A (Nooter et al., 1988), which may be tolerated better. Finally, antibodies may eventually have therapeutic value too. As mentioned before, antibodies that recognize extracellular domains of P-glycoproteins can increase the drug sensitivity of MDR cell lines (Hamada and Tsuruo, 1986; Broxterman et al., 1988b). Antibody-mediated cytotoxicity is also being considered. This has been tested in vitro with P-glycoprotein antibodies coupled to Pseudomonas toxin. The conjugate is capable of selectively killing cells with elevated P-glycoprotein expression (Fitzgerald et al., 1987). In patients, however, the expression of P-glycoproteins in normal tissue could limit the application of such therapy, not to mention the problem of reaching the tumor cells with the conjugate. XV. Outlook

In the past few years MDR lost much of its mystery, but at the same time gained interest, because it has come to rest on solid ground. P-Glycoprotein overproduction has been established as the prime cause, and this results from transcriptional activation, gene

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ALEXANDER M. VAN DER BLIEK AND PIET BORST

amplification, or both. Consistent with enhanced drug efflux, the P-glycoprotein sequence has the features of an ATP-driven pump with two putative nucleotide-binding domains and 12 hydrophobic segments, presumably forming a membrane channel. The most surprising property of P-glycoproteins remains the diversity of substances that they can transport. These substances are all hydrophobic and possess aromatic rings. A more precise definition of the common features of P-glycoprotein substrates remains to be given. The abundance of P-glycoproteins at surfaces facing excretory lumina (gut, liver, spleen, and kidney) does suggest that it rids the body of toxic substances. This is not the whole story, because P-glycoproteins are also found elsewhere. For example, it seems unlikely that human adrenal glands need high levels of P-glycoproteins for waste disposal. The P-glycoprotein transfectants should be useful for identifying the natural substrates, because their phenotype is well defined. Hopefully, such research will lead to compounds with high affinity for P-glycoproteins. These may turn out to be the best agents for reversing MDR. How MDR cell lines adapt to the drug used in selection is not yet fully known, but the stage is set. An important mechanism is undoubtedly the selection in the mdrl ( p g p l ) gene of mutations that have increased the protein’s ability to extrude the selecting drug (Choi et al., 1988). Other elements that could modify the phenotype are differential expression of P-glycoprotein family members, alternative splicing, and augmentation by other resistance mechanisms. Which of these factors contributes to the spectrum of drug resistance in patients remains to b e seen. From a biologist’s point of view, one of the most interesting endeavors will be the search for factors that govern P-glycoprotein expression and splicing. The induction patterns in liver suggest that the genes are tightly regulated and thus require sensors for environmental changes. Phosphorylation of Pglycoproteins may constitute a second level of control. Once the phosphorylated residues are identified, this can be tested with sitedirected mutagenesis. In the same vein, the mechanics of drug binding and pumping can be investigated by manipulating the Pglycoprotein sequence. The frequency of MDR in patients is largely unknown. This situation may improve now that probes are becoming available. What is still urgently required, however, are antibodies able to detect the low level of P-glycoprotein in most normal human tissues and preferably in standard formalin-fixed samples. Eventually it should become clear whether P-glycoprotein expression has prognostic

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value. If trials with agents that reverse MDR are successful, then this may prove to be one of the most tangible benefits coming from the field of MDR research. Testing new agents should be facilitated by the cDNA transfectants. More futuristic approaches to therapy involve the antibodies directed against P-glycoproteins. Finally, several laboratories are trying to infect hematopoietic stem cells with a retrovirus that encodes P-glycoprotein. The goal is to reimplant selected cells that can survive treatment with cytotoxic drugs. This would allow the use of higher drug doses in those cases where bone marrow toxicity is dose limiting. ACKNOWLEDGMENTS We thank Dr. L.A. Grivell, Dr. R. Nusse, Dr. H.M. Pinedo, Dr. L.A. Smets, Dr. H.F. Tabak, and Dr. J.M. Tager for their comments on the manuscript and Mrs. S. Luursema and B. Van Houten for assistance in preparing the manuscript. Unpublished work described in this article was supported by Grant NKI 84-20 from the Queen Wilhelmina Cancer Fund.

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van der Bliek, A. M. (1988). Academic Ph.D. Thesis, University of Amsterdam. van der Bliek, A. M., Van der Velde-Koerts, T., Ling, V., and Borst, P. (1986a).MoZ.Cell. B i d . 6, 1671-1678. van der Bliek, A. M., Meyers, M. B., Biedler, J. L., Hes, E., and Borst, P. (1986b).EMBO J. 5,3201-3208. van der Bliek, A. M., Baas, F., Ten Houte d e Lange, T., Kooiman, P. M., van der Velde-Koerts, T., and Borst, P. (1987). EMBOJ.6, 3325-3331. van der Bliek, A. M., Baas, F., van der Velde-Koerts, T., Biedler, J. L., Meyers, M. B., Ozols, R. F., Hamilton, T. C., Joenje, H., and Borst, P. (1988a). Cancer Res. 48, 5927-5932. van der Bliek, A. M., Kooiman, P. M., Schneider, C., and Borst, P. (1988b). Gene (in press). Wadler, S . , and Wiernik, P. H. (1988). Cancer Res. 48, 539-543. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982). EMBOJ.1,945-951. Wheeler, C., Rader, R., and Kessel, D. (1982). Biochem. Pharmacol. 31, 2691-2693. Willingham, M. C., Cornwell, M. M., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1986). Cancer Res. 46,5941-5946. Willingham, M. C . , Richert, N., Cornwell, M. M.,Tsuruo, T., Hamada, H., Gottesman, M. IM., and Pastan, I. H. (1987).J.Histochem. Cytochem. 35, 1451-1456. Yeh, G. C . , Occhipinti, S. J., Cowan, K. H., Chabner, B. A., and Myers, C. E. (1987). Cancer Res. 47,5994-5999. Zamora, J. M.,and Beck, W. T. (1986).Biochem. Pharmacol. 35,4303-4310. Zijlstra, J. G., De Vries, E. G. E., and Mulder, N. H. (1987).CancerRes. 47,1780-1784.

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GLUTATHIONE TRANSFERASES AS MARKERS OF PRENEOPLASIA AND NEOPLASIA Kiyomi Sat0 Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki 036, Japan

I. Introduction 11. Marker Enzymes of Preneoplasia A. Hepatic Marker Enzymes

B. Nonhepatic Marker Enzymes 111. Molecular Forms of Glutathione Transferases A. General Properties of Glutathione Transferases B. Rat Glutathione Transferases C. Human Glutathione Transferases D. Glutathione Transferases in the Mouse and Other Species E. Species-Independent Classification of Glutathione Transferases IV. Glutathione Transferases as Preneoplastic Markers A. Rat GT-P (7-7) B. Other Forms of Glutathione Transferases in the Rat C. Glutathione Transferase Forms as Preneoplastic Markers in Other Species D. Human GT-T V. Role(s) of Glutathione Transferases in the Mechanisms Underlying Multidrug Resistance VI. Conclusions References

I. Introduction In the past, many tumor-specific or tumor-associated antigens, i.e., tumor markers including alterations in enzyme species, have been reported (see reviews by Schapira, 1973; Ibsen and Fishman, 1979; Law et al., 1980; Levine, 1982; Yogeeswaran, 1983; Sulitzeanu, 1985). More recently, attention has been concentrated on altered phenotype during the early phases of neoplastic development, and new markers, especially enzymes or isoenzymes, have proved useful for detection of preneoplastic lesions in various organs of both animals and man. In addition, description of preneoplastic marker enzymes has also played an important role in characterizing the metabolic patterns of preneoplastic cells, thus providing insights which have facilitated analysis of carcinogenic processes. They have also attracted notice as essential components of new methods for screening of carcinogens and carcinogenic modifiers (promoters and inhibitors) and in the development of 205 ADVANCES IN CANCER RESEARCH, VOL 52

Copvright 0 1989 by Academic Press, Inc AN right\ of reprodu~tionin dny form reserved

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strategies for prevention of carcinogenesis and early treatment of cancer. Since the concept of initiation and promotion was first proposed for skin carcinogenesis (for review, see Berenblum, 1954), it has become generally accepted that the chemically induced carcinogenic process in most if not all tissues can be divided into at least two or three stages; i.e., initiation and promotion, together with further progression steps (see reviews by Diamond et al., 1980; Farber, 1980; Farber and Cameron, 1980; Pitot and Sirica, 1980; Scherer, 1984; Sell et al., 1987; Yuspa and Poirier, 1988; Cerutti, 1988). The initiation stage is generally understood to involve an irreversible process(es), during which alterations of specific genes and, in particular, activation of cellular protooncogenes have been noted to occur (see reviews by Bishop, 1982; Weinberg, 1982; Alitalo and Schwab, 1986; Bister and Jansen, 1986; Sell et al., 1987; Yuspa and Poirier, 1988). During the promotion stage of chemical carcinogenesis, groups of preneoplastic cells (focal lesions) have been observed in many organs prior to the appearance of malignant cancers (Farber and Cameron, 1980; Farber, 1984a,c; Bannasch, 1984, 1986a,b). In rat chemical hepatocarcinogenesis, enzyme-altered foci and hyperplastic or neoplastic hepatocyte nodules have attracted much interest as putative precursor populations (Farber, 1980, 1984a,b,c; Farber and Cameron, 1980; Pitot and Sirica, 1980; Bannasch, 1984, 1986a; Scherer, 1984; Moore and Kitagawa, 1986; Sell et al., 1987). The foci and nodules express alterations in specific enzymes or isoenzymes; i.e., preneoplastic marker enzymes, and, therefore, the smaller lesions have been named enzyme-altered foci (see the review by Pitot and Sirica, 1980). A large number of changes in marker enzyme and isoenzyme phenotypes have been reported for rat hepatic preneoplastic lesions. Drugmetabolizing enzymes form one group that demonstrates alterations (see Table I), and recently certain molecular forms (isoenzymes) of glutathione transferase (GT) (EC 2.5.1.18), one of the most important drug-metabolizing enzymes ( Jakoby, 1978; Chasseaud, 1979; Mannervik, 1985), have been reported as reliable markers for preneoplastic lesions and neoplastic tissues in the liver, as well as in other organs of the the rat and other species, including man. These particular isoenzymes are the rat placental form (GT-P or GT 7-7) and the human placental form (GT-T); the two forms are very similar in physicochemical, enzymatic, and immunological properties and are therefore grouped together in the class pi under the species-independent classification of glutathione transferase proposed by Mannervik et uZ.

(1985). In this article, the known preneoplastic marker enzymes and

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TABLE I MARKER ENZYMES FOR PRENEOPLASTIC HEPATIC LESIONS' Enzymes with decreased activity Carbohydrate-metabolizing enzymes Liver-type isoenzymes of glycolysis (glucokinase, aldolase B, pyruvate kinase L) Glucose-6-phosphatase (GGPase) and other glyconeogenic enzymes Glycogen phosphorylase (liver type; + glycogen storage) Amino acid-metabolizing enzymes Tryptophan 2,3-dioxygenase Serine dehydratase Drug-metabolizing enzymes (in phase I) Cytochrome P-450s Mixed-function oxygenases (aryl hydrocarbon hydroxylase) NADPH P-450 reductase Glutathione peroxidase (Se-dependent) Other enzymes ATPase (Ca2', M$+ dependent) Enzymes with increased activity Carbohydrate-metabolizing enzymes Glucose-6-phosphate dehydrogenase (G6PD) Nonhepatic (fetal-type) isoenzymes of glycolysis Drug-metabolizing enzymes (in phase 11) UDP-glucuronosyltransferase (type I, or fetal type) Epoxide hydrolase (EH) DT-diaphorase (quinone reductase) Aldehyde dehydrogenase (NADP+) Butyryl esterase Glutathione-related enzymes Glutathione transferases (GT) (1-1,3-3, 7-7) y-Glutamyltransferase (GGT) Glutathione peroxidase (Se-independent) (GTs 1-2, 2-2, 7-7) Glutathione reductase (Total glutathione)

'See references in the text for respective enzymes.

molecular forms of glutathione transferase in rat, human, and other species are reviewed. Particular attention is focused on the unique properties of rat GT-P (7-7) and human GT-T and what is known of their gene expression and possible functions (see also reviews by Sato, 1988; Sat0 et al., 1987). II. Marker Enzymes for Preneoplasia

A. HEPATICMARKERENZYMES During the 1960s and 1970s, enzyme (or isoenzyme) alterations in rat hepatomas were extensively studied using transplantable

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hepatomas such as the Yoshida, Novikoff, and Morris types (see reviews by Weinhouse, 1972; Schapira, 1973, 1981; Knox, 1976; S. Sat0 and Sugimura, 1976; Ibsen and Fishman, 1979; Weber, 1977a,b, 1983). Subsequently, interest became focused on primary hepatomas and preneoplastic lesions occurring during rat hepatocarcinogenesis. Hepatic preneoplastic cell populations (foci) can be induced by many carcinogens using different models (see reviews by Farber, 1980, 1984a,b,c; Pitot and Sirica, 1980; Emmelot and Scherer, 1980; Scherer, 1984; Sell et al., 1987), and a large number of marker enzymes useful for their identification; i.e., enzyme-altered foci in rat liver have been reported (Sato et al., 1983a,b; Farber, 198413; Roomi et al., 1985a; Bannasch, 1986a; Moore and Kitagawa, 1986; Sell et al., 1987).The fact that a common metabolic pattern has been established using different protocols has suggested that the changes involved are not random but rather are of physiological significance to the cells involved. Preneoplastic marker enzymes in the rat liver can be divided into two groups, one demonstrating decreased activity and the other demonstrating increased activity (see Table I). In general, the enzymes or isoenzymes related to specific hepatic functions are decreased in foci. These include key liver-type isoenzymes of glycolysis [glucokinase, aldolase B, and pyruvate kinase liver (L) type] and gluconeogenic enzymes such as glucose-6-phosphatase (G6Pase) (Sato et al., 1978; Yanagi et al., 1986). Expression of the amino acid-catabolizing enzymes, serine dehydratase (Kitagawa and Pitot, 1975) and tryptophan 2,3-dioxygenase (pyrrolase),a unique marker for mature hepatocytes (Nakamura et al., 1983), is known to be markedly decreased in foci (Moore et al., 1986d). Among the drug-metabolizing group showing a reduction in amount or activity are enzymes or isoenzymes involved in phase I (oxidation, or hydroxylation and reduction, of drugs) such as certain molecular forms of microsomal cytochrome P-450 and several mixed-function oxygenases such as aryl hydrocarbon hydroxylase (Cameron et al., 1976; Okita et al., 1976; Eriksson et al., 198313; Farber, 1984b; Schulte-Hermann et al., 1984; Schwarz e t al., 1987; Tsuda et al., 1987). In particular, P-450 MCI and MC2 species are markedly decreased in foci and hyperplastic nodules induced by diethylnitrosamine (Buchmann et al., 1985) or by N-ethylN-hydroxyethylnitrosamine (Tsuda et al., 1987). However, certain forms of P-450 are inducible by phenobarbital or 3-methylcholanthrene7 not only in hyperplastic nodules but also in Morris hepatoma 5123D (Schulte-Hermann et al., 1984, 1986; Buchmann et al., 1985; Watanabe et al., 1985), indicating that not all P-450 forms are necessarily

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decreased during hepatocarcinogenesis and that individual isoenzymes exhibit different responses to drugs, even in hepatomas. The induction of phase I species mRNA by phenobarbital in normal and preneoplastic rat livers was also demonstrated using a specific cDNA to phenobarbital-inducible P-450 and by in situ hybridization (Schwarz et al., 1987). The activity of canalicular Ca2+ and M$+dependent adenosine triphosphatase (ATPase), which seems to be related to calcium ion transport (output pump) (Trump and Berezesky, 1987), is also known to be markedly decreased in foci, and the histochemical staining of this enzyme alone (Emmelot and Sherer, 1980) or together with G6Pase has been widely used for detection of enzyme-altered foci deficient in their activities. Pitot and colleagues prefer an approach combining these enzymes together with the positive marker, y-glutamyltransferase (GGT) (Pitot et al., 1978, 1987; Goldsworthy and Pitot, 1985; Hendrich et al., 1987). The second group of rat liver preneoplastic markers consists of enzymes demonstrating increased activity. among these is included the NADPH-generating glucose-6-phosphate dehydrogenase (GGPD), which is markedly increased in altered foci and hyperplastic nodules (Sato et al., 1978; Hacker et al., 1982; Moore et al., 1983, 1986b,f; Enzmann and Bannasoh, 1987). Nonhepatic (prototypic) isoenzymes of glycolysis, such as hexokinase types I1 and 111,aldolase A or C, and pyruvate kinase M2 (K), which are known to be markedly increased in poorly differentiated hepatomas (Weinhouse, 1972; Schapira, 1973, 1981; Yanagi et al., 1974; Sato et al., 1978; Ibsen and Fishman, 1979), have been considered to be increased slightly but significantly in foci and hyperplastic nodules induced by several carcinogens, including N-nitrosomorpholine, as reported by Yanagi et al. (1974) and our laboratory (Sato et al., 1978). However, Reinacher et al. (1986) maintain that the expression of pyruvate kinase M2 cannot be demonstrated in hyperplastic nodules induced by N-nitrosomorpholine followed by phenobarbital using immunohistochemical procedures and specific antibody. This question requires further investigation, since it was observed in our laboratory that some of the foci induced by the Solt-Farber model (Solt and Farber, 1976) are heterogeneously stained using an M2 antibody (I. Hatayama, Y. Inaba, and K. Sato, unpublished results), which might suggest a variation between lesions generated in different systems. Drug-metabolizing enzymes involved in phase I1 (conjugation), such as certain molecular forms of microsomal UDP-glucuronosyltransferase (type I or the fetal type) (Bock et al., 1982; Yin et al., 1982; Astrom et al., 1983; Sat0 et al., 1983a,b; Fischer et al., 1985) and glutathione transferase (the details

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of which will be described later), are included in this group of enzymes that demonstrate increased activity. In addition, a major microsomal form of epoxide hydrolase (EH) is usually also elevated in preneoplastic lesions (Levin et al., 1978; Kuhlmann et al., 1981; Enomoto et al., 1981; Oesch et al., 1983; Wolf et al., 1984; Tsuda et al., 1987). Levin et al. (1978) first identified the microsomal form of epoxide hydrolase (termed epoxide hydrase in their paper) as the preneoplastic (PN) antigen in rat hepatic hyperplastic nodules, which had been reported as a common marker for premalignant and malignant hepatocytes by Farber and his co-workers (see Okita et al., 1975). This report by Levin et al. (1978) was one of the earliest papers which described a role for increased molecular forms of drug-metabolizing enzymes in rat preneoplastic hepatocyte nodules, suggesting their possible use as markers and promoting further studies of enzyme changes in preneoplasia. It was subsequently found that a particular molecular form of epoxide hydrolase (mEHI or EHb, with a broad substrate specificity) was preferentially increased (Batt et al., 1984). The activity of NAD(P)H dehydrogenase (quinone) (also known as DT-diaphorase or quinone reductase, EC 1.6.99.2) (Schor et al., 1978; Astrom et al., 1983; Pickett et al., 1984b) and the activities of certain isoenzymes of aldehyde dehydrogenase (NADP+)(Wischusen et al., 1983; Lindahl and Evces, 1984,1987; Ritter and Eriksson, 1985; Jones et al., 1984; Evces and Lindahl, 1986) and butyryl esterase (carboxylesterase, EC 3.1.1.1) (Kaneko et al., 1979) have also been reported to be elevated in foci and nodules. It was also shown that many glutathione-related enzymes, such as cytosolic glutathione transferase, plasma membrane-bound y-glutamyltransferase (see the review of Hanigan and Pitot, 1985; Tsuchida et al., 1979; Farber, 1980, 1984b; Pitot and Sirica, 1980; Sato et al., 1983a, Roomi et al., 1985a; Bannasch, 1986a), and cytosolic glutathione reductase [NAD(P)H; EC 1.6.4.21 (Kitahara et al., 1983b) are increased in preneoplastic foci, and both total and reduced glutathione levels have been demonstrated to be higher than normal (Deml and Oesterle, 1980; Kitahara et al., 1983b; Roomi et al., 1986). It was reported by Rao et al. (1987a) that the above-mentioned enzymes, included in both positive and negative groups, show little change in hepatic foci, nodules, and hepatomas induced by nonmutagenic (nongenotoxic) peroxisome-proliferating hepatocarcinogens, indicating some variation in enzyme phenotype. It was also pointed out by Farber (1984a, 1984b) and his colleagues (Roomi et d., 1985a) that, in the majority of cases, activity patterns of drug-metabolizing

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211

enzymes are similar in hyperplastic nodules induced by different carcinogens or in different models. While heterogeneity in enzymatic phenotype of foci and nodules, detected histochemically or immunohistochemically, is well known (Pitot et al., 1978; Goldsworthy and Pitot, 1985; Ogawa et al., 1980; Farber, 1984c; Buchmann et al., 1985; Peraino et al., 1986; Enzmann and Bannasch, 1987), Bannasch and his colleagues (1982; Bannasch, 1984, 1986a) have demonstrated that cellular changes involving heterogeneous preneoplastic cell populations (foci) occur during rat chemical hepatocarcinogenesis, from clear glycogen storage and acidophilic cell foci to basophilic lesions through mixed cell foci. This might partly explain heterogeneous enzyme levels, although it appears more likely that individual enzymes are indeed capable of independent expression within foci, the fact of increased conformity with increasing size notwithstanding. Recently, the expression of oncogenes or their products in preneoplastic hepatic foci and in hepatomas has been investigated, and many monoclonal antibodies to enzymes and other proteins, including oncogene products, are becoming available for detection of preneoplastic foci (see the review by Sell et al., 1987; McMahon et al., 1986; Cerutti, 1988; Galand et al., 1988). B. NONHEPATIC MARKERENZYMES Investigation of preneoplastic lesions, including dysplasia and hyperplasia in nonhepatic tissues, has primarily been limited to morphological examination (Farber and Cameron, 1980; Bannasch, 1986b). Only a limited number of potential marker enzymes with increased activity have been described. The induction of ornithine decarboxylase during the promotion stage of skin carcinogenesis is well known, although whether this is limited to focal lesions remains unclear (O’Brien, 1976; Fujiki and Sugimura, 1987; Gilmour et al., 1987). It has been reported that Mg2+-dependentATPase is a marker for detection of two types of precancerous foci in rat pancreas (Bax et al., 1986). Glucose-6-phosphate dehydrogenase appears to be a common marker for neoplastic alteration in the pancreas, thyroid, lung, urinary bladder, kidney, and liver; regardless of the organ, elevated expression is common in the rat, hamster, and even man (see reviews by Weber, 1977a,b; Moore et al., 1986b). Nonspecific cholinesterase (EC 3.1.1.8), P-D-glucuronidase (EC 3.2.1.31), and alkaline phosphatase (EC 3.1.3.1) can be used for differentiation of hyperplastic (dysplastic) urinary bladder (kidney) lesions of rats and mice

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(Bannasch, 1986b). However, new marker enzymes, including GT-P and other glutathione transferase forms, have recently been reported to be increased in lesions of the pancreas, kidney, and lung, as described in detail below.

I l l . Molecular Forms of Glutathione Transferases A. GENERAL PROPERTIES OF GLUTATHIONE TRANSFERASES The glutathione transferases are a family of multifunctional proteins, which act as enzymes and also as binding proteins in various detoxification processes (see reviews by Jakoby, 1978; Chausseaud, 1979; Jakoby and Habig, 1980; Mannervik, 1985; Ketterer, 1986). These enzymes were first identified in rat liver by Booth et aZ. in 1961; for some time afterward their activities were defined according to the chemical structure of the substrates, e.g., glutathione epoxide transferase. However, using l-chloro-2,4-dinitrobenzene(CDNB) as a substrate, multimolecular forms (AA, A, B, and C) were isolated from the rat liver soluble supernatant (cytosolic) fraction by gradient elution from carboxymethyl-cellulose columns (Habig et al., 1974b), and since then it has been recognized that the respective molecular forms have broad substrate specificities and that certain substrates can be utilized by more than one enzyme protein. Thus, it is now generally accepted that individual glutathione transferases cannot be defined according to the chemical nature of the electrophilic center of the substrate. CDNB is regarded as a universal substrate for many glutathione transferase isoenzymes. The notable exception is rat GT-E (5-5), which demonstrates almost negligible activity toward CDNB but high activities toward certain alkyl epoxides and alkyl halides, and also is the most active selenium-independent glutathione peroxidase (Meyer et al., 1984). Many molecular forms of glutathione transferase have been identified from various organs in a variety of species. (Mannervik, 1985; Ketterer, 1986; Mannervik et aZ., 1985, 1987; Hayes and Mantle, 1986a; Hayes et al., 1987). Rat, human, and mouse glutathione transferases, in particular, have been extensively investigated. Although particulate-bound glutathione transferases are known (see below), most of the purified forms have been localized in the cytosol and are homodimeric or heterodimeric proteins. 1. Purification of Glutathione Transferases

Most of the known cytosolic glutathione transferases are adsorbed on S-hexylglutathione-bound Sepharose columns and can be highly purified by a stepwise elution, as first reported by Guthenberg and

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Mannervik (1979).Subsequent isoelectric focusing (Hales et al., 1978; Awasthi et al., 1980; Koskelo and Valmet, 1980) or chromatofocusing (Mannervik and Jensson, 1982) allows separation of individual forms. Although a few forms, such as rat GT-E (5-5) (Meyer et al., 1984) and GT-YkYk (Hayes, 1986), are not adsorbed on the above-mentioned affinity column, the latter, apparently identical with GT 8-8 (Jensson et al., 1986), is adsorbed on glutathione-bound Sepharose columns (Hayes, 1986), as first used for the purification of glutathione transferases by Simons and Vander Jagt (1977). Some molecular forms, such as rat GT-P (7-7), are not easily separable from other forms by isoelectric focusing or chromatofocusing, but can be isolated by ion-exchange chromatography (Satoh et al., 198513). Fast protein liquid chromatography is also useful for the separation of GT molecular forms (Alin et al., 1985; Tateoka et al., 1987). The subunits of glutathione transferases have been separated into monomers under denaturation conditions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Bass et al., 1977); these subunits are named Ya, Yb, and Yc, in order of increasing apparent molecular weight. Subunits are also separable by isoelectric focusing in polyacrylamide gel or by two-dimensional polyacrylamide gel electrophoresis (Kitahara et al., 1983a, 1984; Satoh et al., 1985a). Recently, reverse-phase high-pressure liquid chromatography has been used for isolation of the subunits of this enzyme by Ketterer and colleagues (Ostlund-Farrants et al., 1987). 2. General Functions of Glutathione Transferases Glutathione transferases catalyze the conjugation reactions of electrophilic compounds with reduced glutathione. Many compounds with electrophilic centers are produced from xenobiotics by biotransformation (Chasseaud, 1979), but also from endogenous substances (Mannervik, 1985; Igwe, 1986). The glutathione conjugation reaction is the first step of the mercapturic acid pathway (Booth et al., 1961; Boyland and Chasseaud, 1969; Chasseaud, 1979; Mannervik, 1985), which is one of the most important detoxification processes. The second step of this pathway is catalyzed by GGT, which has been used as one of the markers for putative preneoplastic hepatic foci as mentioned above. In addition, certain forms, such as rat GTs 1-2,2-2, 5-5, and 7-7, are known to possess selenium-independent glutathione peroxidase activity toward lipid peroxides (Kitahara et al., 1983b; Meyer et a1.,1985; see reviews by Ketterer, 1986; Ketterer et al., 1987). Furthermore, since ligandin was identified as a protein that binds steroids, bilirubin, carcinogens, and a number of exogenous

2 14

KIYOMI SAT0

organic anions (Litwack et al., 1971), and then was found to be a basic form of gIutathione transferase (Habig et al., 1974a) (GT 1-1alone, or together with 1-2; see Table 11), this and other forms have similarly been demonstrated to act as binding or carrier proteins for several dyes (bilirubin, bromosulfophthalein, and indocyanine green), cholic acids, steroid hormones, hematin (heme), and carcinogens (Smith et al., 1977; Jakoby, 1978; Maruyama and Listowsky, 1984; Senjo et al., 1985; Homma et al., 1986) and for leukotriene Cq (Sun et al., 1986). Some cytosolic glutathione transferases are also known to be involved in leukotriene and prostaglandin metabolism in the rat (Mannervik et al., 1984; Wu, 1986; Tsuchida et al., 1987; Izumi et al., 1988; Burgess et al., 1987; Chang et al., 1987a,b; Meyer and Ketterer, 1987; Ujihara et al., 1988a,b) (see Table IX) and man (Soderstrom et al., 1985; Ogorochi et al., 1987). Many investigators of chemical carcinogenesis have been attracted by the fact that glutathione transferases, including ligandin, have multipotential detoxifying functions, and therefore have been stimulated to study changes in molecular forms and their roles in metaboTABLE I1 MOLECULARFORMS OF RATGLUTATHIONE TRANSFERASE Subunit pf as dimerb

Name" 1-1 1-2 2-2 3-3 3-4 4-4 5-5 6-6 7-7 8-8 3-6 4-6

Ligandin B AA A C D (X) E

Pf

YaYa YaYc YCYC

YblYbl YblYbz Y bzYbz

-

(YnYn) YPYP (Yf Yf) (YkYk) (YbiYn) (YbzYn) ~

10 (9.8) 9.9 (9.7) 9.8 (9.6) 8.9 (8.8) 8.0 (8.4) 6.9 (8.2) 7.3 5.8 (5.7) 7.0 (8.3) 6.1 (5.8) ? (8.3) ? (6.2)

Molecular weight" 25,000 28,000 26,500 26,500 26,500 26,000 24,000 24,500

~

pP

Classe

7.7 7.6 7.4 6.8

alpha alpha mu

6.6, 6.09 6.8 6.3

~

-

mu mu Pi alpha mu mu ~

References and notes: column 1, from Jakoby et al. (1984); columns 2 and 3, see Mannervik (1985) and Hayes and iMantIe (1986a)for names in parentheses. By isoelectric focusing and, for values in parentheses, by chromatofocusing. Mannervik (1985) and our results. By two-dimensional electrophoresis (our results). Mannervik et al. (1985). f Sato et aE. (1984a). g Yn can be divided into Ynl and Ynz with different PIS(Tsuchida et al., 1987).

GLUTATHIONE TRANSFERASE MARKERS

2 15

lism of carcinogens (see reviews by Smith et al., 1977; Jakoby, 1978; Chasseaud, 1979; Ketterer, 1986; Ketterer et al., 1983, 1985, 1986; Mantle et al., 1987). TRANSFERASES B. RATGLUTATHIONE Rat glutathione transferases have been most extensively investigated, and subsequent to the identification of glutathione conjugation activities in rat liver cytosol by Booth et ul. (1961), many molecular forms were separated by several groups and were classified in a variety of ways. First, Habig e t al. (1974b) named them alphabetically in reverse order of their elution from carboxymethyl-cellulose columns. Most of the rat glutathione transferases are localized in the cytosol of various tissues, with the exception of one form (MW 17,000) (Morgenstern et al., 1982, 1985; Morgenstern and DePierre, 1987), which has been purified from rat liver microsomal fractions. As yet unidentified (unpurified) form(s) involved in the metabolism of leukotrienes have also been reported to be present in particulate fractions from leukemia cells (Jakschik et al., 1982; Bach et al., 1984; Yoshimot0 et al., 1985). Prostaglandin-Hz D-isomerase (EC 5.3.99.2), which demonstrated glutathione transferase activity toward CDNB, was purified from the cytosol fraction of rat spleen (Christ-Hazelhof and Nugteren, 1979; Urade et al., 1987), and was found to differ immunologically from other known cytosolic glutathione transferase forms. At least 11to 13 molecular forms of cytosolic glutathione transferase and eight different subunits have been identified from rat tissues (Tu and Reddy, 1985; Hayes and Mantle, 1986a) (see Table 11), and new forms continue to be reported; e.g., glutathione transferase YcYfetus(PI 8.65) in fetal liver (Scott and Kirsch, 1987) and YnlYnl (PI 6.2) in the brain (Tsuchida et al., 1987). The various forms can be divided into three groups: basic, neutral, and acidic. The basic forms in the liver have been most extensively studied. Rat glutathione transferase forms have been given several different names (see Table 11), but are defined principally by their isoelectric points, subunit molecular weights on SDS-polyacrylamide gel electrophoresis, and immunological properties using the immunoblot method (Sugioka et al., 1985a; Hayes and Mantle, 1986a,b; Soma et al., 1986). The Ouchterlony double immunodiffusion test, however, is still used for discriminating immuno-related forms (Tsuchida et al., 1987). Hybridization between subunits derived from different forms is also useful for detection of structural relationships between GT forms (Kitahara and Sato, 1981; Ishikawa et al., 1988).

2 16

KIYOMI SAT0

Molecular weights of subunits were estimated according to the various marker proteins used; e.g., that of the subunit 1 (Ya) was estimated as 22,000 (Bass et al., 1977), 26,500 (Kitahara et al., 1984), 25,600 (Tu and Reddy, 1985), 25,500 (Hayes and Mantle, 1986a), or 25,000 in the new nomenclature of rat glutathione transferases proposed by Jakoby et al. (1984). The exact value appears to vary, dependent on the acrylamide composition of the resolving gel (Hayes and Mantle, 1986b; J. D. Hayes et al., 1987). Although GT-E (5-5)has a high activity toward epoxides generated by carcinogen metabolism by activating enzymes such as P-450, and therefore seems to be very important in connection with chemical hepatocarcinogenesis, only a limited number of reports have been published regarding its purification (Habig et al., 1974b; Meyer et al., 1984). Tissue-specific expression of rat GT subunits (Mannervik, 1985; Hayes and Mantle, 1986a; Tu et al., 1987; Pickett, 1987) (see Table VII), and sex differences in the subunit composition in the liver (Igarashi et al., 1985), have been described. Whereas Yb is the most abundant form in males (followed by Ya and Yc), it is the least abundant form in females.

Nomenclature of Rat Glutathione Transferases In the nomenclature proposed by Jakoby et al. (1984), rat glutathione transferase subunits have been assigned a number that refers to the order in which they were isolated and characterized (Table 11). However, not all investigators have been using this classification, and one form (GT 6-6) named in this manner might require correction, as suggested by results from our laboratory (Ishikawa et al., 1987, 1988; Tsuchida et al., 1987). Names such as YaYa (see Table 11),which were first used by Bass et al. (1977) to indicate the subunit composition based on separation by SDS-polyacrylamide gel electrophoresis, are still most commonly used. Therefore, the t e d n o l o g y for glutathione transferases used in the references cited will also be used herein. Each GT subunit may perform particular functions as enzymes and binding proteins independently from another subunit consisting of the same molecular form. The reported specific functions of rat GT subunits are summarized in Table 111. cDNAs of rat GT subunits 1 (Ya), 2 (Yc), 3 (Ybl),4 (Ybz), Yb3, which is possibly identical with Ynl (Ishikawa et al., 1988), and 7 (Yp), and genomic DNAs of some subunits have been cloned (see the review by Pickett, 1987 and Table VI). [The primary structure of GT 4-4 was also determined by amino acid sequence analysis (Alin et al., 1986).]

TABLE 111 SPECIFIC FUNCTIONS OF RAT GLUTATHIONE TRANSFERASE SUBUNITS Function

Subunit ~

~~

~

Reference

~

Binding steroid hormone, bilirubin, bile acids, carcinogens (ligandin), and leukotriene C4 Isomerase activity for ketosteroids and prostaglandin Hz (+PGEZ) Reduction of lipid peroxides and prostaglandin Hz (+ PGF2) Reduction of lipid peroxides Conjugation of 4-hydroxyalkenals derived from lipid peroxides Binding bile acids Carrier for heme Binding steroid hormone and bile acids Reduction of nucleic acid hydroperoxide Synthesis of leukotriene Cq (Ynl) Reduction of lipid peroxides and nucleic acid hydroperoxides Conjugation of carcinogens

Jakoby and Habig (1980); Smith and Litwack (1980) Sun et al. (1986) Benson et a1. (1977) Ujihara et al. (1988a) Meyer et al. (1985) Ujihara e$ al. (1988b) Meyer et $1. (1985) Jensson et al. (1986) Hayes and Chalmers (1983) Senjo et (11. (1985) Maruyama and Listowsky (1984); Homma et al. (1986) Hayes and Chalmers (1983) Tan et al. (1986) Tsuchida et al. (1987) Meyer et al. (1985) Tan et al. (1986) Robertson et al. (1986)

218

KIYOLMMI S A T 0

Further advances should resolve the nomenclature among the international scientific community. Rat GT gene expression in various tissues, including preneoplasia, and the elucidation of the mechanism(s), using cDNAs and regulatory elements of genomic DNAs, are topics of current interest (see reviews by Pickett, 1987; Taylor et al., 1987; Tu et aZ., 1987).

C. HUMANGLUTATHIONE TRANSFERASES Human glutathione transferases can also be divided into basic, neutral, and acidic groups of differing molecular weights (Warholm et al., 1983) (see Table IV), corresponding to the classes alpha, mu, and pi, respectively. In the basic group, five forms (a+) were reported by Kamisaka et al. (1975), but the subunit structures were not clarified. Stockman et al. (1985,1987) identified two basic homodimers and one heterodimer consisting of two subunits, B1 and Bz, with the same molecular weights, but with different PISand unique immunological and enzymatic properties. These two subunits were also described by Soma et al. (1986)and were named Y1 and Yq. Vander Jagt et al. (1985) isolated 13 forms from human liver; these forms had pls ranging from 4.9 to 8.9, but appeared to be identical immunologically with the basic forms reported earlier. Tu et al. (1986), using immunoprecipitation of in vitro human liver poly(A) RNA translation products, described three classes of human GT subunits with molecular weights of 26,000 (Ha), 27,500 (Hb), and 31,000, but they could not detect any subunits with mobility equivalent to that of rat liver Yc. However, a novel and more basic form (PI9.9, MW 26,500), homologous with rat GT 2-2 (YcYc), has been identified in human skin (Del Boccio et al., 1987) (Table IV). GT-p (PI 6.6), a homodimer with a subunit molecular weight slightly higher than those of basic forms, has been reported as a neutral form (Warholm et al., 1981a, 1983), and is present in about 60% of adult human livers. A near-neutral form (PI6.0 by chromatofocusing) similar to GT-p was also identified as GT I11 (Y3Y3) by Soma et al. (1986).GT-$, which was found to be immunologically related to GST-p but more acidic (PI5.5),was reported by Singh et al. (1987a). An acidic form, GT-T, which was very similar to the acidic GT-p isolated from human erythrocytes (Marcus et al., 1978), was purified from term placenta and was found in high levels in human fetal liver but in low levels in adults by Mannervik and his colleagues (Guthenberg et al., 1979, 1986; Guthenberg and Mannervik, 1979, 1981; Warholm et al., 1980, 1981b), Koskelo and Valmet (1980), and

PROPKRTIES OF

TABLE IV HUMAN GLUTATHIONE TRANSFERASES ~

Alpha (basic)

Class

Mu (neutral)

~~

Pi (acidic) ~

Molecular form

Human 2-2"

(Y--E

(9.0-7.5';

GT-2)

;',I./

Subunit structure Molecular weight ( x lo3) PI Immunocrossreactivity Anti-a-c antibody Anti-p antibody Anti-.n antibody Tissue distribution

=a+

Homodimer (Y3Y3) 54 6.6 -

-

Liver

Liver, kidney

Del Boccio et al. (1987).

'Kamisaka et al. (1975).

' Warholm et a2. (1981a, 1983). Hussey et al. (1986). Awasthi et al. (1980). fSingh et al. (1987a). g Guthenberg and Mannervik (1981). Marcus et ul. (1978). I Hayes (1986). Stockman et al. (1985, 1987). Soma et al. (1986). I53 kDa in the original (Del Boccio et al., 1987). '"Board (1981). " GT-4 has been suggested by Laisney et al. (1984) and Singh et al. (1988). J

Acidic pd (@) TI (=e,ep,h A'; GT-3'"J) Homodimer (YsYs)k 54 49 5.5 4.8

+ Skin

GT-1

+

-

+ Placenta, kidney, lung (trace in liver)

220

KIYOMI S A T 0

Polidoro et ul., (1980). Human fetal lung, kidney, brain, and intestine contain only an acidic form (GT-m) (Polidoro et al., 1982; Pacifici et al., 1986), and this is the major type in adult lung (Koskelo et al., 1981), brain (Olson et al., 1983), and spleen (Koskelo, 1983). Developmental tissue-specific expression of glutathione transferases was also noted in human tissues by Faulder et al. (1987). GT-.rr is related to rat GT 7-7 (GT-P) in many properties and is grouped into the class pi in the species-independent classification of Mannervik et ul. (1985) (Table V). Singh et ul. (1987c, 1988) suggested the presence of multiple forms related to GT-.rr in human kidney and skeletal muscle, indicating a possibility of polymorphism at the the human GT-3 locus, on chromosome 11 (Laisney et al., 1984), which, however, should be confirmed. D. GLUTATHIONE TRANSFEZUSES IN THE MOUSEAND OTHERSPECIES

So far three or four glutathione transferase forms have been separated in mouse liver cytosol (Mannervik et al., 1985; Warholm et al., 1986; Hatayama et al., 1986),and two other forms were reported to be present in mouse lung cytosol (Singh et al., 1987b). The subunit structures of three hepatic forms have been clarified-glutathione TABLE V SPECIES-INDEPENDENT CLASSIFICATION OF RAT,HUMAN, AND MOUSE CYTOSOLIC GLUTATHIONE TRANSFEAASE SUBUNITS' Class

Rat

Human

Mouse

alpha

1 (Ya)b(25) 2 (Yc) (28) 8 (Yk or Ya') (24.5) 3 (Yb,) (26.5) 4 (Ybz) (26.5) 6 (Yn, Ynl, or Yb3) (26) ? (Ynz) (26) 7 (Yp or Yf) (24)

BI (YI), BP 0'4)c (26) Subunit 2 (28.5)

ml (25.5) mSd (27) (GT-9.3, GT-8.7)f

mu

Pi

-

Y, (Y3)e (27)

a Relative molecular weights ( x lo3) in parentheses obtained by SDS-polyacrylamide gel electrophoresis, assuming rat Ya MW to be 25 x lo3. Two forms are present for subunit 1 (see Table VI). BZis similar to rat subunit 2 in possessing high activity toward cumene hydroperoxide (Stockman et al., 1987). Mouse m3 is closer to rat subunit 3 than to 4 with regard to N-terminal amino acid sequence and immunological cross-reactivity. Y,, is closer to rat subunit 4 than to 3 with regard to N-terminal amino acid sequence and immunological cross-reactivity. fpearson et al. (1983).

GLUTATHIONE TRANSFERASE MARKERS

22 1

transferase M I, M 11, and M 111-and all three are homodimers with different molecular weights. Mouse GT M I1 is related by immunological and other properties to rat GT 7-7 (GT-P) and human GT-.rr (Mannervik et al., 1985), but GT M I1 is basic, rat GT 7-7 is neutral, and human GT-.rr is acidic. The amino acid composition does not differ greatly among these three forms, and the N-terminal amino acid sequences are quite similar, as pointed out by Mannervik et al. (1985) and as is evident from the whole amino acid sequence of GT-P and GT-7r described by Kano et al. (1987). However, the amounts in liver of these forms were found to vary considerably among the three species. In particular, the adult male mouse liver contains an exceptionally high level of GT M I1 (Hatayama et al., 1986), a finding confirmed by McLellan and Hayes (1987). In rat liver, GT-P (7-7) is usually very low (only in bile ductal cells) and is not easily inducible by drugs (including testosterone) prior to the appearance of preneoplastic nodular hepatocytes. In contrast, GT M 11, though already present in large amounts in adult male liver and also at significant levels in adult female liver, was found to be further inducible by injection of testosterone to females (Hatayama et al., 1986) or by administration of various drugs, including butylated hydroxyanisole (I. Hatayama, and K. Sato, unpublished data). On the other hand, following castration of male mice, the levels were reduced to the lower levels of female mice (Hatayama et al., 1986).The levels of GT M I and I11 were hardly affected by castration or by administration of testosterone, but were influenced by administration of various drugs. The changes of these forms of enzymes in mouse liver, and their roles, especially that of GT M I1 in hepatocarcinogenesis, remain to be clarified. Adams et al. (1987) observed that a hepatic GT form (M 11), related to rat GT YfYf, is induced in female nude (nu/nu) mice by interferon (cYIP)injection, suggesting that this form is inducible by stress. Mouse liver glutathione transferases have, however, attracted attention with regard to the antioxidant-associated chemoprevention of hepatocarcinogenesis reported by Benson and co-workers (1978, 1979, 1984; Pearson et al., 1983), Wattenberg and his colleagues (1986; Wattenberg, 1985) and others (Dock et al., 1984). The form investigated by these groups seems to be GT M 111. Multimolecular forms have also been identified in other species: five forms in Rhesus monkey (Asaoka et al., 1977), three (Smith et al., 1980) or seven (Inaba, 1987) forms in hamster liver, and five (Wiener, 1986) or four (Igarashi et al., 1988) forms in dog liver. Furthermore, hamster liver contains a significant amount of one class pi form

222

KIYOMI S A T 0

immunologically related to GT-P (Inaba, 1987), and dog liver has three major forms, all of which are immunologically related to rat GT-P (Igarashi et al., 1988).

E. SPECIES-INDEPENDENT CLASSIFICATION OF GLUTATHIONE TUNSFERASES Mannervik et al. (1985)demonstrated that the major isoenzymes of cytosolic glutathione transferase from rat, mouse, and man share structural and catalytic properties, which can be used as the bases of a species-independent classification. Isoenzymes from these species were grouped with respect to N-terminal amino acid sequences, substrate specificities, sensitivities to inhibitors and immunological cross-reactivities (by immunodiffusion and immunoblotting) into three classes, alpha, mu, and pi. Each of the three mammalian animal species studied contains at least one isoenzyme of each class. The efficacy of this species-independent classification has been recently confirmed in the laboratory of Del Boccio et al. (1987) and has been principally supported by our group and others (Satoh et al., 198513; Soma et al., 1986, Hatayama et al., 1986; Ishikawa et al., 1988; Hayes and Mantle, 198613; J. D. Hayes et al., 1987a,b; Tu et al., 1986, Tu and Qian, 1987; Tew et al., 1987; Singh et al., 198713, 1988). New forms can also be grouped into this classification (see Table V). Mannervik et al. (1985)suggested that the similarities of isoenzymes in one class reflect evolutionary relationships and that the classification can be applied generally (Mannervik et al., 1987a). Primary structures (whole amino acid sequences) of GT subunits obtained from base sequences of cDNAs are supplying more precise information on homology or diversity among the molecular forms in the same and different species. The cDNAs and genomic DNAs so far defined are listed in Table VI. IV. Glutathione Transferases as Preneoplastic Markers

A. RAT GT-P (7-7) 1. Identification Several groups have investigated the changes in glutathione transferase isoenzymes in rat chemical hepatocarcinogenesis. In our laboratory, an increase of GT-A (3-3) in hyperplastic nodule-bearing rat livers was first noted (Kitahara et at., 1983a; Sato et al., 1983a, 1984b) and subsequently GT-P was identified as a good marker for rat hepatic

TABLE VI cDNAs AND GENOMIC DNAs OF RAT,HUMAN, AND MOUSEGLUTATHIONE TRANSFERASE SUBUNITS ~~

DNA

cDNA Rat

Human

Subunit Ya

25,434 25,474 25,209 25,806 25,802 25,592 25,592 25,549 23,307

a

25,385 (221) 25,425 (221) 23,224 (209) - (222) ?

2 (4 $T

Mouse Genomic DNA Rat Human Mouse

Molecular weighy

M I (Ya) M I11 (GT-9.3)

(221) (221) (220) (217) (217) (217) (217) (217) (209)

~~

Clone

Reference

pGTB38 pGTR261 pGTB42 pGTAIC44 pGTR200 pGTR187 pGTAIC48 no name pGP5 pGSTr7 pGTH 1 pGST2-PvulI pGPi2 pGT55

Pickett et al. (1984a) Lai et a1. (1984) Telakowski-Hopkins et al. (1985) Ding et al. (1985) Lai et al. (1986) Lai and Tu (1986) Ding et al. (1986) Abramovitz and Listowsky (1987) Suguoka et al. (1985) Pemble et (11. (1986) Tu and Qian (1986) Board and Webb (1987) Kano et al. (1987) Daniel et al. (1987) Pearson et al. (1983)

Number of exons introns 7 6 8 7 7 6 7 6 7 6

Number of amino acid residues is given in parentheses.

Telakowski-Hopkins et al. (1986) Morton and Pickett (1988) Okuda et (11. (1987) Cowell et al. (1988) Daniel et al. (1987)

224

KIYOMI SAT0

preneoplastic foci (Sato et al., 1984a,c). Ketterer and his colleagues noted an unidentified form in primary hepatoma induced by N,Ndimethyl-4-aminoazobenzene (Ketterer et al., 1983) and later determined this form to be GT 7-7 (Meyer et al., 1985). Pickett and co-workers (1984b, 1987; Pickett, 1987) have demonstrated three-fold and five-fold increases in the mRNA levels of GT subunits Ya/Yc and Ybl/Ybz in hepatocyte nodules using their respective cDNAs. We first purified a new neutral form (PI6.7 or 6.8) from rat placenta and named it the placental form (GT-P) (Sato et al., 1984a,c), because this isoenzyme was almost the only form of glutathione transferase found in this tissue. This was later confirmed by Di Ilio et al. (198613). Subsequently, we noted that, although GT-P is only present in negligible quantities in normal rat liver, it is markedly increased in hyperplastic nodule-bearing livers (Kitahara et al., 1984), and we were thus able to purify it from such tissue (Satoh et al., 1985b). Mannervik and his colleagues noted a form present in rat lung, which they reported was absent from rat liver (Guthenberg and Mannervik, 1979);later, Guthenberg et al. (1985) and Robertson et al. (1986)demonstrated (using our GT-P antibody) that the new form (GT 7-7) purified from rat kidney and lung is immunologically identical to GT-P. Tu e t al. (1983) also noted a GT subunit (molecular weight 22,000) that was not expressed in normal rat liver but was found to be present in the heart, kidney, and other organs, among in witro translated GT products. GT-P (YpYp)is also related to the GT-A (YfYf) purified from human lung by Hayes and Mantle (1986a), but it differs in subunit composition from the P form (YblYn) named by Hayes (1984). GT-P (7-7)characteristically has the smallest molecular weight and a neutral pl (see Table 11), and while it is not immunologically cross-reactive with any other forms, there is considerable crossreactivity between species, including rat, mouse, hamster, dog, horse, and man (Satoh et al., 1985b; Roomi et al., 1985b). GT 7-7 (GT-P) is now grouped into class pi in the species-independent classification of glutathione transferase (Mannervik et al., 1985) (Tables I1 and VI). 2. Tissue Distribution Single radial immunodiffusion using anti-GT-P antibody revealed that GT-P levels in normal rat tissues, including placenta and fetal and adult livers, are generally very low (Satoh et al., 198513).The kidney (proximal and distal tubules) (Tsuda et al., 1985), lung (bronchiolar epithelial cells) (Yamamoto et al., 1988), pancreas (ductular cells) (Moore e t al., 1985; Obara et al., 1986), small intestine (columnar epithelial cells), skin (epithelial cells), and brain (astroglia cells)

GLUTATHIONE TRANSFERASE MARKERS

225

(Tsuchida et al., 1987) contain significant amounts. The tissue-specific expression of rat GT subunits including Yp is summarized in Table VII. GT-P is ubiquitous but is often found only in small or trace amounts, and especially low levels are found in the liver. 3. Expression during Chemical Hepatocarcinogenesis a. Biochemical Detection. In 1983, double immunodiffusion studies using anti-GT-P antibody prepared in rabbits showed that GT-P is markedly increased in hyperplastic nodule-bearing rat liver (Sato et al., 1984a; Sato, 1988). This was confirmed using two-dimensional electrophoresis. In addition to basic GT subunits such as Ya, Yc, Yb, and Yb’ (subunits 1, 2, 3, and 4, respectively) present in normal rat liver cytosol, two polypeptides (MW 26,000, pZ 6.7 or 6.8, major, and pZ 6.3 minor), were detected in the cytosol from rat liver bearing hyperplastic nodules induced by the Solt and Farber model (Solt and Farber, 1976). These polypeptides were not detectable in appreciable amounts in normal liver and were identified as charge isomers of the GT-P subunit by immunoaffinity column chromatography and later by immunoblotting (Western blotting) using the antibody (Sato et al., 1984a, 1987; Satoh et al., 1985a). In Japan, Muramatsu and his colleagues had already noted polypeptide species of MW 26,000, pZ 6.9 and 6.6, among the polypeptides expressed in rat hyperplastic nodules and primary and transplantable hepatocarcinomas by using two-dimensional gel electrophoreis; these were confirmed to be charge isomers of GT-P by using our GT-P antibody (Sugioka e t al., 1985). In Canada, Farber and colleagues (Roomi et al., 1983; Eriksson et al., 1983a) had also reported the appearance of a polypeptide (named p21) (MW 21,000) detected by SDS-polyacrylamide slab gel electrophoresis in isolated hyperplastic nodules induced by six different models, including the Soh and Farber model. This polypeptide was also immunologically identified as the GT-P subunit (Roomi et al., 1985~). Later, p21 was renamed p26 and was identified as the subunit of a polypeptide of molecular weight 52,000, reconfirmed to be GT-P (Rushmore e t al., 1987, 1988). A GT form named GT 7-7 was also reported to be increased in hyperplastic nodules b y Jensson et al. (1985). The amount of GT-P protein increases 30-fold or more during the early stage of hepatocarcinogenesis induced by the Solt and Farber model, although the quantities of the basic subunits 1and 2 or 3 and 4 are much higher than those of GT-P (7-7), even when GT-P (7-7) is maximally increased (Sato, 1988). The levels of GT-P in hyperplastic nodule-bearing livers and in primary and transplantable (Morris

TABLE VII RELATIVE ORGAN DISTRIBUTION OF RAT GLUTATHIONE TRANSFERASE SUBUNITSO

GT subunit

Liver

+++b

++

+ +++ +++

Kidney

+++ +++ ++

Lung

Heart

Testis

-

-

-

++ ++

++ +

++

+ ++ ++

+ + +

+

++

++ ++ ++ ++ +

+ + +

+

+++

Spleen

++

-

Small intestine

Colon

++ + + +

+ +

-

-

++

++

Brain

+i

Modified from Hayes and Mantle (1986a). of expression. Negative (-), weak (+), moderate (+ +), and strong (++ +), respectively, as detected by two-dimensional electrophoresis followed by immunoblotting. a

* Degree

GLUTATHIONE TRANSFERASE MARKERS

227

5123D) hepatomas, induced by different carcinogens, were found to be 210-fold higher than in normal liver, but were negligible in transplantable Yoshida ascites hepatoma AH 130 (Satoh et al., 1985b), indicating that GT-P tends to decrease with dedifferentiation in hepatomas, as is the case for many drug-metabolizing enzymes. b. Zmmunohistochemical Detection. The peroxidase-antiperoxidase (PAP) or the avidin-biotin-peroxidase complex (ABC) immunohistochemical procedures using anti-GT-P antibody have demonstrated GT-P to be localized in enzyme-altered foci that are also detectable by enhanced GGT activity (Sato et al., 1984a). However, it was established that a proportion of GT-P-positive foci are not recognizable on the basis of GGT staining (Tatematsu et al., 1985, 1987). The foci are inducible by a large number of different protocols using a variety of carcinogens [diethylnitrosamine (DEN), dimethylnitrosamine (DMN), 2-acetylaminofluorene (AAF), aflatoxin B1 (Am1), etc.] and GT-P has been demonstrated to be increased both by immunohistochemical (Tatematsu et al., 1985, 1987, 1988a,b; Moore et al., 1986f, 1987a, 1988a; Manson et al., 1987) and by immunochemical (Roomi et al., 1985c; Satoh et al., 198,513) methods. Hepatomas induced by DEN, AAF, Am,, 3’-methyl-4-dimethylaminoazobenzene (3’-Me-DAB), and other carcinogens were also usually strongly stained. It should be noted that sex differences are observed in the development of GT-P-positive foci induced by some carcinogens. For example, as shown in Fig. 1 and as is evident in the amount of GT-P (Sato, 1988), the areas of GT-P-positive lesions induced in the Solt and Farber model are far larger in male rats than in females, which are under the suppressive effects of estradiol on phenol sulfotransferase (type IV) metabolizing hydroxy-AAF (N. Tateoka, I. Hatayama, S. Tsuchida, K. Satoh, and K. Sato, unpublished results), as was also observed for GGT-positive foci (Blanck et al., 1984, 1986). It is of interest that GT-P is expressed not only in putative preneoplastic hepatic foci induced by chemicals but also in spontaneous lesions, for example, in spontaneous “altered cell foci” in the livers of aged Fisher 344 rats (Mitaka and Tsukada, 1987), or in LEC rats, without administration of any exogenous carcinogens (Oyamada et al., 1988), in both cases particularly in males. Immunohistochemical staining further revealed that very small GT-P positive foci or even single cells, appearing 1or 2 weeks after a single administration of initiator (Moore et al., 1986f, 1987a; Sato, 1988), or in one model even within 48 hr of single doses of DEN, DMN, or AfBl (Moore et al., 1987a), are detectable before an increase in GT-P content or GGT activity is biochemically apparent (Sato,

228

KIYOMI SAT0

FIG. 1. Sex difference in development ofhepatic GT-positive foci induced in the Salt and Farber model (1978). (A) Female rat liver ( ~ 5 )(B) . Male rat liver ( ~ 5 )When . females were ovariectomized 1 week before DEN (200 mg/kg) injection, the resultant foci developed to sizes similar to those observed in males. Subcutaneous injection of estradiol (0.5 mglkg per day) after ovariectomy was associated with a reduction and return to values characteristic for untreated females (N. Tateoka, I. Hatayama, K. Satoh, and K. Sato, unpublished results).

1988). These cells increase with increasing doses of initiator (e.g., DEN) and are not induced by promoters of liver carcinogenesis such as phenobarbital, methylcholanthrene, polychlorinated biphenyls, and isosafrole (Moore et al., 1987a). Therefore, GT-P is considered to be an accurate marker for very early “initiated cells.” GT-P-positive small foci (minifoci) have been observed to persist for a long time (at least 6 months) after a single injection of DEN, 200 mg/kg (Sato, 1988) or even 10 mg/kg (Takahashi et al., 1987), showing that these lesions are to a large extent irreversible, and it has been demonstrated that they develop into large foci after feeding AAF for 2 or 3 weeks

GLUTATHIONE TRANSFERASE MARKERS

229

accompanied by partial hepatectomy (selection pressure) (Sato, 1988). It was shown that, in addition to the cytosol, the nuclei in the foci, especially in the earlier and smaller lesions, are stained by anti-GT-P antibody. However, these nuclei are not stained by antibodies to other enzyme forms, such as GTs 1-2 and 3-4,despite strong binding of the respective antibodies in the cytosol. These results suggest that GT-P or substance(s) immunologically related to GT-P may be present in the nuclei of preneoplastic cells. 4 . Specificity as a Preneoplastic Hepatic Marker a. Inducibility by Short-Term Administration of Drugs. Unlike ordinary drug-metabolizing enzymes, GT-P is not inducible by administration of a large variety, of hepatocarcinogenic promoters or modulators (3-methylcholanthrene, a-hexachlorocyclohexane,carbon tetrachloride, cyproterone acetate, phenobarbital, polychlorinated biphenyls) or even by hepatocarcinogens (DEN, AAF, 3’-Me-DAB, ABI, choline- and methionine-deficient diet, ethionine, clofibrate) in the absence of preneoplastic foci and hyperplastic nodules (Roomi et al., 1985b; Satoh et al., 1985a) or additional drugs (see the review by Ito et al., 1988). GT-P was slightly inducible by the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (Tatematsu et al., 1985, 1987, 1988b) and by ethoxyquin (Thamavit et al., 1985; Manson et al., 1987) in periportal areas; the expression of GT-P after administration of these drugs does not interfere with detection of GT-P-positive foci. On the other hand, GGT is in many cases so strongly induced by these drugs that enzyme-altered foci are no longer recognizable (Tatematsu et al., 1985; Manson et al., 1987).It had previously been pointed out that GGT is increased in the periportal zone I of rat liver under a variety of circumstances not directly related to carcinogenesis (Fischer et al., 1986) and also in primary cultures of normal rat hepatocytes treated with liver tumor promoters and structurally related compounds (Edwards and Lucas, 1985). Among the other drugs examined, lead nitrate proved exceptional in inducing a significant amount of GT-P in rat liver (Roomi et al., 1986, 1987; Sato, 1988; Koo et al., 1988). In this case, expression of GT-P, which was transient and reversible within 2 weeks (Sato, 1988), appeared to be part of a general biochemical pattern involving drug-metabolizing enzymes very similar to those exhibited by hepatic nodules (Roomi et al., 1986). It was demonstrated immunohistochemically that GT-P was induced throughout the liver, although binding was strongest in zone 111. Although lead nitrate is a hepatic

230

KIYOMI S A T 0

mitogen that induces liver hyperplasia (Columbano et al., 1987a,b), and initiation of chemical carcinogenesis is known to require cell proliferation (Cayama et d , 1978), it has been reported that lead nitrate-associated hyperplasia cannot replace compensatory cell proliferation after hepatectomy or carbon tetrachloride (CC14) poisoning in development of GGT-positive or GT-P-positive foci initiated by carcinogens [DEN, N-methyl-N-nitrosourea (MNU)] (Columbano et al., 1987a). Lead nitrate is of particular interest for investigations of the mechanism of GT-P gene expression (Roomi et al., 1986). Many of the known rat hepatic preneoplastic markers, such as GGPase, ATPase, GGT and GGPD, are considerably instable in rapidly induced foci and nodules (Moore et al., 1983; Tatematsu et al., 1988a,b). GGT activity in particular is rapidly lost after withdrawal of carcinogenic agents from the diet. In contrast, GT-P appears to be more stable (Tatematsu et al., 1988a; Koo et al., 1988), although it is slowly reversible. One of the reasons for such interenzyme variation might be differences in protein amounts and intracellular localization. Comparison of the two most commonly used positive markers (see Table VIII) reveals a number of other dissimilarities. The level of GGT expression in hepatocarcinogenesis is oncofetal, whereas it is highest in the normal adult rat kidney; GT-P is not an oncofetal isoenzyme and its highest level of expression is in hyperplastic nodules, far above the levels observed in any normal rat tissues. TABLE VIII A COMPARISON OF GT-P

AND

GGT”

Characteristic

GT-P

GGT

Localization Protein levelb Adult liver Fetal liver Kidney HN, liver Hepatoma Induction in the liver E thionine BHA, BHT Lead nitrate

Cytosol/(nucleus)

Plasma membrane

30

0.1 2 430 2-5 (0.004%)‘ 4-6 (-50)

44

230 500-2000 (1-2%)‘ 400- 1500

(?Id (+I

(+++)” (+ +-+ ++)

(+++)

(++I ~

HN, Hyperplastic nodule; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene. In p g / g tissue. Percentage of total protein. Intensity of expression in protein amount (GT-P) or in activity (GGT)

GLUTATHIONE TRANSFERASE MARKERS

23 1

These advantages have helped establish GT-P as one of the best markers for detection of early liver lesions. b. Preneoplasia Not Expressing GT-P. It has been reported by Reddy and colleagues (see reviews by Reddy and Rao, 1986, and Rao and Reddy, 1987; Rao et al., 1984, 1986a,b, 1987a,b, 1988; Goel et al., 1986) and other groups (Numoto et aZ., 1984; Glauert et al., 1986; Greaves et al., 1986; Hendrich et al., 1987; Wirth et al., 1987) that a new class of carcinogens (nongenotoxic), including the peroxisomeproliferating hypolipidemic agents such as clofibrate, nafenopin, ciprofibrate, Wy-14643, tibric acid and di(2-ethylhexy1)phthalate (Reddy et al., 1980; see also reviews by Reddy and Rao, 1986; Rao and Reddy, 1987), is associated with GT-P- and/or GGT-negative foci and hepatomas. Reddy and his colleagues (Reddy and Rao, 1986; Rao and Reddy, 1987) have therefore suggested that GT-P and GGT expression is not essentially associated with hepatocarcinogenesis. Glutathione transferase activity is known to be inhibited by clofibrate (Foliot et al., 1986), but this cannot account for the preceding observations, because GT-P, GGT, and a-fetoprotein, and the mRNA for each protein, are not expressed in foci and hepatomas induced by peroxisome-proliferating agents (Rao et al., 1988). However, ethionine, which is another proposed nongenotoxic agent, does induce GT-P-positive foci and hepatomas (Ogiso e f al., 1985). The “initiated cells’’ induced by DEN (a mutagenic carcinogen) develop to form GT-P-positive foci by administration of AAF, as observed in the Solt-Farber model, but lead to GT-P-negative lesions after treatment with clofibrate (It0 et al., 1988; I. Hatayama, K. Satoh, and K. Sato; unpublished data) or the hormone dehydroepiandrosterone (DHEA) (Moore et al., 1988b), suggesting that expression of the enzyme may be controlled in the promotion stage, in addition to the initiation stage. Reddy and his colleagues (Lalwani et al., 1987) have identified peroxisome-proliferator binding proteins (apparent MW of 70,000 on SDS-polyacrylamide gel electrophoresis) from rat liver, but they are not detectable in human liver, in which these agents apparently do not induce hepatomas.

5 . Gene Expression The structure of GT-P and the mechanisms underlying regulation of its expression during chemical hepatocarcinogenesis have been investigated by Muramatsu and co-workers (1987). Suguoka et al. (1985) cloned a rat GT-P cDNA (pGP5) from a cDNA library prepared from poly(A)+ RNA of 2-acetylaminofluorene-induced rat hepatocellular carcinoma by screening with synthetic DNA probes

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deduced from a partial amino acid sequence of a GT-P subunit; they were thus able to determine the complete amino acid sequence. The molecular weight of the rat GT-P subunit was calculated to be 23,307. They also showed by Northern blot analysis and dot blot analysis using their cDNA probe (pGP5) that GT-P mRNA (about 750 nucleotides) is abundant in hyperplastic nodules, in Morris 5123D, 7316A, and 7794A, and in chemically induced hepatocellular carcinomas, but is barely detectable in normal liver, in fetal liver, and in an undifferentiated hepatoma, AH 130. Russell et al. (1988) also demonstrated a highly significant correlation between elevations in GT-P mRNA level and hepatocarcinomas induced by DEN and 6-p-dimethylaminophenylazobenzothiazole. Lower levels of the mRNA were detected in lung, testis, kidney, spleen, and placenta, approximately in this order, indicating that the placenta is in fact not a representative tissue in which this gene is normally expressed at high levels (Muramatsu et al., 1987). Muramatsu and his co-workers suggested on the basis of their data that the amount of GT-P in a tissue is, for the most part, if not totally, regulated at the transcription level. Knoll et al. (1986) also cloned a GT-P cDNA from a cDNA library constructed from the mRNAs of a primary tumor induced by DEN. The nucleotide sequence of this cDNA was identical to that reported by Suguoka et al. (1985). Pemble et al. (1986,1987)and Taylor et al. (1987) also cloned a cDNA (pGSTr7) of GT 7-7 by using poly(A)+ RNA isolated from N,N-dimethyl-4-aminoazobenzene-inducedrat hepatoma. The amino acid sequence of the GT 7-7 was also identical with that reported by Suguoka et al. (1985). Okuda et al. (1987)have isolated the GT-P gene from a phage library using their GT-P cDNA clone (pGP5); they demonstrated that it is about 3000 bp long and consists of seven exons and six introns, with the initiator codon being split between the first and second exon. The cap site was mapped 70 nucleotides upstream from the translation initiation site and the promoter “TATA” box was found 27 bp upstream from this putative cap site. Sequences 200 bp upstream from the cap site were rich in G + C residues (61%),and the hexanucleotide sequence 5’-GGGCGG-3’ was found from position -47 to -42. Muramatsu et al. (1987), Okuda et al. (1988), and Sakai et al. (1988) also analyzed the cis-acting regulatory DNA elements of the rat GT-P gene by the chloramphenicol acetyltransferase (CAT) activity assay method. Various regions of the 5’-flanking sequence were fused with a bacterial CAT gene and the transcriptional activity of each construct was determined by transient expression assay after introduction into a hepatoma cell line (dRLh84). Two enhancing elements were found at 2500 and 61 bp upstream from the translation

GLUTATHIONE TRANSFEFIASE MARKERS

233

initiation site (Sakai et al., 1988).The more potent upstream enhancer was divided into two domains; GPE I and GPE 11. Both GPE I and the downstream enhancer sequence contained a 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (TRE) (Chiu et al., 1987) type of sequence (TGATTCAG) (Okuda et al., 1988; Sakai et al., 1988). A silencing element was found at 400 bp upstream from the cap site. Furthermore, factors (AP-1 or a related protein) that were trans-acting on the enhancing and silencing elements were demonstrated (Okuda et al., 1988; Sakai et al., 1988). The GT-P gene is located on rat chromosome 1 at band q43 (Masuda et al., 1986). cDNAs of other rat GST subunits have also been cloned (Table VI) and homologies between the subunits have been studied (see reviews by Pickett, 1987; Tu et al., 1987). Muramatsu and co-workers (1987; Suguoka et aL., 1985) found that the GT-P subunits Y p and Ya had only a 32% amino acid homology, while Ya and Yc had 65% homology (Tu et aZ., 1984). Thus, they speculated that GT-P had evolved from the ancestral gene at a far earlier stage than that at which Ya and Yc separated. Relation to Oncogene Expression. It is very important to clarify whether GT-P expression in hepatocarcinogenesis is related to the activation of specific oncogenes. In this context, it is interesting to note that GT-P becomes highly expressed with malignant transformation in vitro of primary hepatocytes either by transfection with ras oncogenes or by treatment with metabolically activated AfBl (Power et al., 1987). Furthermore, recently Li et al. (1988) demonstrated that the expression of a metallothionein-ras fusion gene (MTrasT24) specifically increases mRNA levels of GGT and GT-P in cultured rat liver cells, in which these genes have been shown to be expressed together. Recently, a short sequence (GTGACTAA) in the polyoma virus enhancer was identified as a ras-responsive element (RRE), which mediates Ha-ras activation and also mediates activation by TPA as a TPA-responsive element (Imler et al., 1988). Similar sequences are observed in the SV40 enhancer, the metallothionein MTIIA gene, the collagenase gene, the interleukin-2 gene, the PEA1 (or AP-1) binding site, and the j u n oncogene product binding site (Imler et al., 1988). The intriguing question of whether GT-P may be regulated by similar mechanism(s) awaits an answer. 6 . Function The actual role(s) and function(s) of GT-P in preneoplastic cells also await clarification. There is no evidence that the carcinogens inducing GT-P-positive foci are detoxified via glutathione conjugation by GT-P,

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KIYOMI SAT0

and although AAF and 3I-Me-DAB are known to be conjugated with glutathione, the process is nonenzymatic (Ketterer et d.,1983, 1986). It has been reported that putative preneoplastic foci are resistant to cytotoxic agents, including hepatocarcinogens; thus they have been termed “resistant cells” (Farber, 1980, 1984b). Among the basic rat enzyme forms, GTs 1-1, 1-2, and 2-2 have large capacities to bind bilirubin, heme, and cholic acids, but they markedly lose glutathione transferase activities through binding to these compounds; the activities of GTs 3-3, 3-4, and 4-4 are less affected, GT-P activity, in contrast, is almost unaffected by binding to such endogenous compounds (Sato, 1988). Therefore, the increased levels of GT-P in foci might allow replacement of basic forms with regard to glutathione transferase activity. Among the substrates so far examined (Table IX), it was pointed out by Mannervik and his colleagues (1985)that GT 7-7 is unique in possessing a high activity toward ethacrynic acid, but other particular substrate specificities have not been noted as yet. It was reported by Robertson et al. (1986) that GT 7-7 purified from rat lung has the highest activity toward the ( +)-7/178-diol-9,10-oxideof benzo[a]pyrene, stereoselectively toward a (+)enantiomer; Hiratsuka et al. (1987) found that the GT-P purified from hyperplastic nodulebearing rat liver stereoselectively conjugates glutathione at the benzylic carbon (C,) of the (-)enantiomer (7S78R) of 9,lO-dihydrobenzo[a]pyrene 7,8-oxide, while other basic forms preferred the (+) enantiomer. These results suggest that the various glutathione transferase molecular forms may have different stereoselectivities toward the same substances, and that GT-P may have unique properties. It was further suggested by Meyer et aZ.(1985) that GT 7-7 purified from hepatomas possesses selenium-independent glutathione peroxidase activity toward lipid hydroperoxides, especially toward arachidonate and linoleate hydroperoxides, and also toward thymine hydroperoxide (Tan et al., 1986) (Table IX). GT-P has significant activity for 4-hydroxynonenal, which is one of the most potent lipid peroxides from arachidonate (Table IX), although GT 8-8, a minor form in both normal and preneoplastic livers, is known to have a higher activity (170 pmollminlmg protein) (Jensson et al., 1986; Danielson et al., 1987). Thus, GT-P (7-7)expression may be related to the prevention of lipid peroxidation, and the latter process has been considered to play an important role(s) during tumor promotion. GT-P may also be concerned with drug-resistant mechanisms of preneoplastic foci (Ketterer, 1986; Ketterei et d.,1986). As shown in Fig. 2, lipid hydroperoxides produced by some promotion regimens can be removed by a series of coupled reactions; glutathione peroxidase

TABLE I X SUBSTRATE SPECIFICITIES OF R ~ GLUTATHIONE T TRANSFERASES

GT forms (pmol/min/mg protein) Substrate Exogenous substrates 1-Chloro-2,4-dinitrobenzene (CDNB) 1,2-Dichloru-4-nitrobenzene

1-1

2-2

3-3

23.1

18.6

47.3

4-4 13.1

truns-4-Phenyl-3-buten-2-one Ethacrynic acid 1,2-Epoxy-3-(p-nitrophenoxy)prupane A5-Androstene-3,17-&oneb Benzo[a~pyrene-7,8-diol-9,10-oxideL Endogenous substrates Linoleate hydroperoxidec Arachidonate hydroperoxide' Crrmene hydroperoxide t-Butylhydroperoxide" Thymine hydroperoxided 4-H ydroxynonenal Leukotriene Af)f Prostaglandin H2R PGH + D isomerase + E isomerase + F reductase g

108

7-7

YnlYn,

18.2

108

0.02

0.02

3.94

0.40

n.d. n.d. 0.08 0.13

n.d. n.d. 1.24 0.06

0.76 0.08 0.08 0.49

0.04 1.62 0.62 0.90

4.29 0.10

0.38 0.08

0.02 0.03

n.d. 0.36

?

0.33

1.6

0.2 0.2 0.2 0.04 0.49 2.2 3.0

0.2 0.2 0.4 0.1 0.27 3.7 140

0.06 n.d. 0.04 n.d. 1.27 2.8 310

1.5 1.5 0.01 0.02 1.25 1.6 67

(DCNB)

Bromosulfophthalein

6-6

3.0 2.6 1.2 0.1 0.13 1.7 5.3

n.d. 1,400 1,250

1.7 9.8 0.5 1.01 0.6 2.9 n.d. 420 390

16 57 24

16 92 28

n.da

0.05

n.d.

0.02 0.05 2.98 0.10

n.d. n.d. n .d. >

43 52 n.d.

?

14 33 14

7.30 n.d. n d 1l.d.

n.d. ? ? ? ? n.d. ? ? 6.6 496

n.d. 63 n.d.

n.d., not detectable; Mannervik (1985); Meyer et al. (1985); Tan et al.(1986); Units, nmoM min/mg protein; f Tsuchida et al. (1987); Ujihara et al. (1988b).

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KIYOMI S A T 0

lipid (membrane)

active oxygen

lo2 , 0;

etc

arachidonic NADPH-cyt P-450 LT o r PC

lipid peroxide(RO0H) (hydroperoxide)

H20

+

ROH

GSSC

FIG.2. Reduction of lipid hydroperoxides formed during carcinogenesis by GT-Pdependent glutathione peroxidase (GSH-Px) activity and a series of coupled reactions (GSSG reductase is glutathione reductase). Enzymes and glutathione (indicated by arrows in parentheses) increase in focal lesions during carcinogenesis. LT, Leukotrienes; PG, prostaglandins.

activity (selenium-independent) of GT-P (7-7) and other forms, followed by reduction of oxidized glutathione b y glutathione reductase [NAD(P)H] using NADPH supplied from the pentose phosphate pathway. The fact that glutathione reduction (Kitahara e t al., 1983b), NADPH-generating C6PD (Sato et al., 1978; Hacker et al., 1982; Moore et al., 1983, 19860, and the total and reduced glutathione levels (Deml and Oesterle 1980; Kitahara et aZ., 198313; Roomi et al., 1986) are all increased in enzyme-altered foci and hyperplastic nodules strongly supports the possibility that the above-described reactions might be operating. GT-P also has a significant LTC4 synthase activity (Tsuchida et al., 1987), though the significance of this remains to be clarified. However, GT-P has no significant activity in prostaglandin metabolism, in contrast to the basic enzyme forms in the cytosol, which were noted to have high activities (Table IX). It is noteworthy that some amounts of GT-P are detectable immunohistochemically in the nuclei of preneoplastic lesions, especially during the early stages of chemical carcinogenesis in rat liver. It can be speculated that GT-P in the nuclei functions in detoxification of genotoxic substances from the cytosol or acts as a carrier protein for exogenous and endogenous substances such as carcinogens and hormones. With regard to this point, it is noteworthy that GT 7-7 has high activity toward thymine hydroperoxide, as reported by Tan et al. (1986). In normal rat liver, subunits 1, 2, 3, and a subunit similar to 5 (5*) (and also GT-p and GT-.rr in human liver) have been detected in free and bound fractions from nuclei and are therefore considered to

GLUTATHIONE T U N S F E U S E MARKERS

237

be of potential importance to the detoxification of DNA peroxides (Ketterer et al., 1988).

7. Application as a Hepatic Preiaeoplastic Marker Ito and his co-workers (1988; Tatematsu et al., 1987) have developed an in vivo medium-term screening test for hepatocarcinogens, which includes DEN initiation (ip injection, 200 mg/kg) followed by a 6-week administration of test compounds starting 2 weeks later and partial hepatectomy performed at week 3. More than 100 chemicals were investigated for their potential to modify GT-P-positive foci development initiated by DEN; of the liver carcinogens, 10 of 11 (90.9%) mutagenic and 11 of 13 (84.6%) nonmutagenic compounds gave positive results (mean, 87%). Carcinogens other than hepatocarcinogens gave less positive results (2 out of 17,11.8%), but none of the compounds reported as noncarcinogenic was positive. GT-P has also been used as a marker to examine modifying or modulating influences of many drugs on the induction of rat liver nodular lesions, since there was no nonspecific induction of GT-P to interfere with the detection of GT-P-positive foci and nodules. Thus dose-dependent inhibitory effects were demonstrated of antioxidant butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethoxyquin (EQ) (Thamavit et al., 1985; Tsuda et al., 1988), ethyl alcohol (Ikawa et al., 1986), dehydroepiandrosterone (DHEA; an inhibitor of G6PD) and diaminopropane (DAP; an inhibitor of ornithine decarboxylase) (Moore et al., 1986d), and acetaminophen and aminophenol (Kurata et al., 1987) on the development of rat liver preneoplastic lesions induced by DEN, dihydroxy-di-n-propyl-nitrosamine, or N-ethyl-Nhydroxyethylnitrosamine. DHEA appears to have some similarities to agents which induce peroxisome proliferation because it induces GT-P-negative or GGT-negative basophilic hepatocellular foci initiwhen administered to ated with dihydroxy-di-n-propyl-nitrosamine, male rats in the diet (0.6%) subsequent to carcinogen exposure (Moore et al., 1986c, 1988b). Yokota et al. (1987, 1988) also used GT-P as a marker for putative preneoplastic hepatocytes transplanted into analbuminemic rats. 8. Expression in Extrahepatic Preneoplasia Whereas GT-P demonstrates tissue-specific expression, it is present in various organs (Table 111). Thus, in cell types normally expressing large amounts of this form, such as the kidney tubular epithelium, a decrease has been noted during chemical carcinogenesis (Tsuda et

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al., 1985, 1987; Kurata et al., 1987) and an increase is often observed in organs normally not expressing GT-P; e.g., GT-P, GGPD, and various P-450 species are elevated in acinar cell lesions of rat pancreas induced by hydroxyamino quinoline l-oxide (Moore et al., 1987b) and in squamous metaplasias and squamous cell carcinomas in rat lung GGT is a marker induced by N-nitroso-bis(2-hydroxypropy1)amine; for adenomatous lesions in rat lung carcinogenesis (Yamamoto et al., 1988). In rat colon carcinomas induced by chemical carcinogens such as N-methyl-N-nitrosourea, GT-P is not expressed, in contrast to positive findings reported for human colonic adenomas and carcinomas (Kodate et al., 1986).

B. OTHERFORMS OF GLUTATHIONE TRANSFERASES IN THE RAT In chemical hepatocarcinogenesis, the basic glutathione transferase subunits Ya/Yc and Ybl/Ybz, in addition to GT-P, are increased in foci and nodules and may perform multiple detoxification functions as enzymes and binding proteins in the resistance of these lesions to cytotoxic agents (see the review by Pickett, 1987; Kitahara et al., 1984; Satoh et al., 1985a,b). However, they are also increased in the surrounding background parenchyma in response to carcinogen treatment, whereas GT-P is not increased. In rat extrahepatic organs such as the kidney, in which glutathione transferase subunits Ya/Yc and Yp are normally predominant and Ybl/Ybz subunits are present only in trace amounts, the YblYbl form (previously named GT-A) becomes markedly increased in preneoplastic and neoplastic lesions induced by N-ethyl-N-hydroxyethylnitrosamine(Tsuda et al., 1985, 1987; Kurata et al., 1987). Determination of the exact significance of changes in individual forms awaits definition of their roles in normal and neoplastic tissues in different organs. C. GLUTATHIONE TRANSFERASE FORMS AS PRENEOPLASTIC MARKERSIN OTHERSPECIES

GT-P is immunocross-reactive among a number of species, and thus the anti-rat GT-P antibody has been used to investigate expression of this enzyme form in preneoplastic lesions and neoplastic tissues of other animals. Lesions of ductal dysplasia and adenocarcinoma induced by N-bis(2-hydroxypropyI)nitrosamine(or methylnitrosourea) in the hamster pancreas stain strongly (Moore et al., 1985; Obara et al., 1986) and are histologically similar to the dysplasia and carcinoma

GLUTATHIONE TRANSFERASE MARKERS

239

lesions observed in human pancreas. Both hepatocellular and cholangiocellular liver lesions (Moore et al., 1986a) and lung preneoplastic foci and nodules (Moore et al., 1987c) induced by propylnitrosamines in Syrian hamsters were also stained, suggesting that a GT-P-like form in the hamster is a possible common immunohistochemical marker and presumably reflecting similarities in biochemical processes involved in preneoplasia in these organs.

D. HUMANGT-T Attempts have been made to stain human preneoplastic tissues immunohistochemically using anti-GT-.rr antibody. GT-T was purified from human term placenta according to the method of Guthenberg and Mannervik (1981), modified in our laboratory using Shexylglutathione column chromatography and chromatofocusing (Soma et al., 1986). The antibody to GT-v was found to be useful for the detection of early precancerous states, or dysplasia, and differentiated carcinomas in several human organs. For example, whereas normal colon mucosa was negative or only slightly stained, human colon adenomas, including cancerous portions, were positive, and colon carcinomas tended to be even more strongly stained than the adenomas (Kodate et al., 1986). In human uterine cervix tissue, normal mucosa did not bind antibody, but mild dysplasia, including koilocytosis (suggesting infection by human papilloma virus), was indicated by light staining and severe dysplasia and squamous cell carcinoma in situ showed strong staining (Shiratori et al., 1987). Similar results have also been obtained for esophageal dysplasia and carcinoma (T. Nishihira, R. Shineha, Y. Sekine, S. Tsuchida, and K. Sato, unpublished data). GT-.rr expression was also demonstrated in Paget’s disease (in situ carcinoma) of the mammary gland, in breast adenocarcinomas, and very strongly in melanoma (K. Hanada, unpublished data). Atypical epithelia and differentiated carcinomas of the stomach also demonstrated binding of anti-GT-.rr antibody (Tsutsumi et al., 1987). Interestingly, stomach epithelium of an 18-week-old fetus aIso expressed GT-T, indicating that the expression of this enzyme form in human stomach carcinoma might be oncofetal in nature (Tsutsumi et al., 1987). Some parts of cirrhotic liver and of differentiated hepatomas were also stainable, albeit weakly (Sato et al., 1987). Similar results were obtained by Hayes et at. (1987).The level of GT-T has been reported to be significantly increased in colon carcinomas and hepatic tumors, especially in cholangiocarcinomas

240

KIYOMI SAT0

and metastases to the liver originating from the gallbladder, stomach, and colon (Soma e t aZ., 1986). Furthermore, the level of GT-.rr in colon adenoma and carcinoma, in esophageal cancer, and in most cell lines derived from various cancers such as esophageal carcinomas was found to be elevated to between 5-fold and 10-fold compared to the respective control tissue values (S. Tsuchida, unpublished data). Other groups of investigators have also reported increased levels of GT-.rr in breast tumors, renal carcinomas, and lung tumors (Di Ilio et al., 1986a, 1987, 1988); in malignant melanomas (Mannervik et al., 1987b);in glioma (Smith et al., 1988); in colon, breast, lung, uterine, and gastric adenocarcinomas and in renal cell carcinomas and melanomas (Shea and Henner, 1987). Small cell human lung cancer cell lines express only low levels of GT-v, as pointed out by Awasthi et al. (1988);indeed the levels of mRNA for GT-.rr in small cell lung cancer cell lines are significantly lower than in lines derived from squamous adenocarcinomas (Nakagawa e t al., 1988).This is of interest in light of the finding that small cell lung cancer cell lines are more sensitive to anticancer agents such as cis-platin and carboplatin than their nonsmall cell counterparts, suggesting that the low levels of glutathione transferase mRNA expression in the former might inversely correlate with high sensitivity to drugs (Nakagawa et al., 1988). However, it remains to be confirmed whether GT-v is directly involved in the mechanism of resistance to these agents. In human resting mononuclear leukocytes, glutathione transferase activity toward trans-stilbene oxide was noted to be due to a particular isoenzyme, which was dominantly inherited in some individuals and was proposed as a marker for susceptibility to lung cancer (Seidegard et al., 1985, 1986). Subsequently, expression of this enzyme form, identified as GT-p, was found in only about 50% of the individuals studied (Seidegard et al., 1987). It was also reported that glutathione transferase activity toward benzo[a]pyrene 4,5-oxide by a “p-like” glutathione transferase form is increased 2-fold to 5-fold in interleukin-2-dependent T cells and 4-fold to 10-fold in lymphoblastoid B cell lines (Jone et al., 1988). Investigation of the levels of GT-.rr in sera of patients with tumors such as gastric and esophageal carcinomas revealed significant elevation. I n addition, these high serum values decreased to the normal range after surgical removal of tumors from esophageal cancer patients (S. Tsuchida, Y. Sekine, R. Shineha, T. Nisihera, and K. Sato, unpublished data). Thus, follow-up of elevated serum GT-.rr levels may be of use for monitoring patients with cancers during the course of treatment.

GLUTATHIONE TRANSFERASE ,MARKERS

24 1

V. Role(s) of Glutathione Transferases in the Mechanisms Underlying Multidrug Resistance

As mentioned above, it has been pointed out by Farber and his co-workers (Farber, 1982, 1984c; Farber and Cameron, 1980) and others (Carr, 1987) that altered foci and hyperplastic nodules induced by different carcinogens in rat liver have pleiotropic resistance to structurally unrelated agents, thus the term “resistant foci and nodules” (Farber and Cameron, 1980; Farber, 1 9 8 4 ~ )It . has also been observed that human malignant cells often demonstrate multidrug resistance (Moscow and Cowan, 1988; Tew and Clapper, 1987; Wolf et al., 1987a,b). At least two types of multidrug resistance (MDR) are known; one is intrinsic or natural (de n o w ) and is associated with the malignant transformation observed in tumors such as lung and colon cancers, and the second is acquired through sublethal exposure to anticancer agents. Many mechanisms might be involved: increased drug output due to changes in drug-specific transport mechanisms (in which a P-glycoprotein and the mdr gene are active); decreased activation of prodrugs (in phase I); alterations in the drug’s target enzymes; alterations in cellular metabolism and repair mechanisms (in which topoisomerases have been noted); and increased inactivating enzymes (in phase 11) (Moscow and Cowan, 1988). Cowan et al. (1986) listed several lines of evidence to suggest that biochemical similarities exist between a human breast cancer cell line, after acquisition of MDR, and a carcinogen-induced rat preneoplastic hepatic nodule, indicating that changes in regulation of phase I and phase I1 drug-metabolizing enzymes may play a role in both. Thiols have especially been considered to play an important role in protection against carcinogen damage (Chasseaud, 1979; Chan et al., 1986). Glutathione metabolism has been shown to be a determinant of therapeutic efficacy (see the review by Arrick and Nathan, 1984). In certain cell lines a number of glutathione transferase forms, including members of class pi, such as human GT-.rr and rat GT-P (YfYf; 7-7), have been noticed to increase after acquisition of MDR; breast cancer cells resistant to adriamycin (Batist et al., 1986), Walker 256 cells resistant to nitrogen mustard (Buller et al., 1987), human squamous carcinoma cell lines resistant to cis-diamminedichloroplatinum (Teicher et al., 1987), ovarian adenocarcinomas resistant to cis-platinum and adriamycin (Wolf et al., 1987a,b), a Chinese hamster ovary cell line resistant to nitrogen mustard (Robson et al., 1987), and small cell lung cancer cell lines resistant to cis-platin (Nakagawa et al., 1988) have all been reported to share this characteristic in common.

242

KIYOMI S A T 0

However, it remains to be confirmed whether the increased glutathione transferase forms actually perform important roles in the acquisition of resistance. Rat basic glutathione transferase forms are known to be related to protection from or modulation of hepatocarcinogenesis initiated by carcinogens such as aflatoxin (Lotlikar et ul., 1984; Kensler et al., 1986, 1987; Jhee et al., 1988), leading to the proposal by Wattenberg and co-workers (Wattenberg, 1985) of use of these enzymes in chemoprevention, although glutathione-mediated binding of carcinogens (e.g., dibromoalkanes) to DNA is known to occur via glutathione conjugation by rat GT YaYc (Inskeep and Guengerich, 1984). In this connection, the report of Manoharan et ul. (1987) that transfection of a number of constructs containing SV40 promoter-glutathione transferase Ya cDNA into monkey Cos cells in culture, results in the increased resistance to antidiolepoxide toxicity. The results clearly indicated that overexpression of glutathione transferase isoenzymes induced in mammalian cells is accompanied by significant biological resistance to a known alkylating agent. VI. Conclusions Many marker enzymes, including drug-metabolizing species, have been reported to be increased in preneoplastic rat liver foci. As a result, glutathione transferases have been extensively investigated during neoplastic development in the liver, especially since ligandin, an azo dye-binding protein, was identified as one of the glutathione transferase molecular forms. Rat GT-P (7-7)has recently become established as one of the best positive markers for early detection of putative preneoplastic and neoplastic cells in chemically induced hepatocarcinogenesis. Although it is not expressed in lesions associated with some nongenotoxic (nonmutagenic) hepatocarcinogens, the fact that this isoenzyme form is not induced by many drugs, in contrast to other drug-metabolizing enzyme species, has particular advantage for its use in screening potential carcinogens and modifiers (promoters and inhibitors). GT-P is expressed at high levels in both spontaneous and induced preneoplastic hepatic lesions at early stages of development. Interest has been concentrated on regulation of GT-P gene expression, resulting in proposed possible relationships of expression to oncogenes, such as ras, jun, and fos, or a connection with protein kinase C through TPA stimulation. Glutathione transferases related to rat GT-P are now grouped together in class pi by the new interspecies classification; these

GLUTATHIONE TRANSFERASE MARKERS

243

enzymes, including human GT-T, have been found in increased levels in preneoplastic lesions in a wide variety of organs in man and other species. They are of potential importance in the mechanism underlying multidrug resistance in experimental and human tumors. It is clear that further investigation of this enzyme family is warranted in the interests of better cancer prevention and cancer therapy.

ACKNOWLEDGMENTS The author sincerely thanks Dr. Malcolm A. Moore for valuable suggestions and discussion of the manuscript. Our studies were supported in part by a Grant-in-Aid for the Special Project Research-Cancer Bioscience and Grants-in-Aid for cancer research from the Ministry of Education, Science, and Culture of Japan, a research grant from the Princess Takamatsu Cancer Research Fund, and a grant from the Karoji Memorial Fund for Medical Research at Hirosaki University.

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Wiener, H. (1986).Eur. J. Biochem. 157,351-363. Wirth, P. J., Rao, M. S., and Evarts, R. P. (1987). Cancer Res. 47,2839-2851. Wischusen, S. M., Evces, S., and Lindahl, R. (1983). Cancer Res. 43, 1710-1715. Wolf, C. R., Macpherson, J. S., and Smyth, J. F. (1986). Biochem. Pharrnacol. 35, 1577- 1581. Wolf, C. R., Lewis, A. D., Carmichael, J., Adams, D. J., Allan, S. G., and Ansell, D. J. (1987a). Biochem. SOC.Trans. 15,728-730. Wolf, C. R., Lewis, A. D., Carmichael, J., Ansell, J., Adams, D. J., Hickson, I. J., Harris, A., Balkwill, F. R., Griffin, D. B., and Hayes, J. D. (1987b). In “Glutathione S-transferases and Carcinogenesis” (T. J. Mantle, C. B. Pickett, and J. D. Hayes, eds.), pp. 199-212. Taylor I%Francis, London. Wolf, C. R., Moll, E., Friedberg, T., Oesch, F., Buchmann, A., Kuhlmann, W. D., and Kunz, H. W. (1984). Carcinogenesis (London) 5,993-1001. Wu, C . (1986). Biochem. Biophys. Res Commun. 134,85-92. Yamamoto, K., Yokose, Y., Nakajima, A., Eimoto, H., Shiraiwa, K., Tamura, K., Tsutsumi, M., and Konishi, Y. (1988). Carcinogenesis (London) 9, 399-404. Yanagi, S., Makiura, S., Arai, M., Matsumura, K., Hirao, K., Ito, N., and Tanaka, T. (1974). Cancer Res 34,2283-2289. Yanagi, S., Sakamoto, M., and Nakano, T. (1986).1nt.J. Cancer 37,459-464. Yin, Z., Sato, K., Tsuda, H., and Ito, N. (1982). Gann 73, 239-248. Yogeeswaran, G. (1983).Adu. Cancer Res. 38,289-350. Yokota, K., Ogawa, K., Mori, M., Nagase, S., and Sato, K. (1987). Gann 78, 109-112. Yokota, K., Ogawa, K., Mori, M., and Nagase, S. (1988). Cancer Res. 48, 387-392. Yoshimoto, T., Soberman, R. J., Lewis, R. A., and Austen, K. F. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 8399-8403. Yuspa, S. H., and Poirier, ,M. C. (1988). Ado. Cancer Res. 50, 25-70.

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ABERRANT GLYCOSYLATION IN TUMORS AND TUMOR-ASSOCIATED CARBOHYDRATE ANTIGENS Sen-itiroh Hakornori The Biomembrane Institute. Seattle, Washington 981 19 and Departments of Pathobiology. Immunology. and Microbiology. University of Washington. Seattle, Washington 98195

I. Introduction and Brief Historical Background (1929-1975) 11. Tumor-Associated Glycolipid Antigens in Experimental Tumors 111. Tumor-Associated Carbohydrate Antigens in Human Cancers: Classification, Mosaicism of Expression, and New Procedures for Generation of Antibodies IV. Oncogenes and Aberrant Glycosylation V. Normal and OncofetaI Features of Glycolipid Antigens A. Background B. Lacto-Series Type 1 Chain Antigens C. Lacto-Series Type 2 Chain Antigens D. Ganglio-Series Antigens E. Globo-Series Antigens VI. Carbohydrate GIycoprotein Antigens A. Background and Classification B. Chemically Defined Lacto-Series Antigens Carried by Mucin-Type and Other Glycoproteins C. T, Tn, and Sialyl Tn Antigens D. Glycoprotein Antigens Whose Epitope Structure is Ill-Defined E. Tumor-Associated Peptide Antigens Whose Epitope Structure IS Influenced by Glycosylation VII. Alteration of Histo-Blood Group and Heterophile Antigens Expressed in Human Cancer A. Histo-Blood Group ABH Antigens B. Histo-Blood Group P Antigens C . Forssman Antigens D. Hangariutziu-Deicher Antigens VIII. Aberrant Glycosylation in Preneoplastic Tissues IX. Requirements for Tumor-Associated Carbohydrate Antigens: Density of Antigens and Organizational Framework in Membranes X. Diagnostic Applications A. Earlier Studies B. Serum Antigen Levels Defined by Monoclonal Antibodies C. Serum Antigen-Antibody Complex D. Imaging of Tumors by Labeled Anticarhohydrate Antibodies XI. Tumor-Associated Carbohydrate Antigens as Targets for Therapeutic Applications A. Effect of Monoclonal Antibodies on Tumor Cell Growth In Vitro and Ztr Vivo B. Effect of Antiidiotype or Anti-Antiidiotype Antibodies on Trimor Growth C. Targeting of Antibody-Drug Conjugates to Tumor Cells 257 ADVANCES LN CANCER RESEARCH, VOL. 52

Copyright D 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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D. Active Immunization with Tumor-Associated Antigen: Effect on Tumor Growth XII. Summary and Perspectives References Note Added in Proof

I. Introduction and Brief Historical Background (1929-1975) The goal of cancer research is, obviously, to find effective ways to prevent, diagnose, and cure human cancer. Although a large number of studies on the molecular basis and genetic control mechanisms of cancer cell phenotypes are being carried out, our knowledge derived from such studies is still highly fragmentary and is far from allowing practical application. However, since essentially all cancer cells are characterized by aberration in structure and function, particularly aberrant glycosylation, of the surface membrane, cancer can be regarded as a “molecular disease” of cell membrane glycans.’ Such aberrant structures at the surface membranes may well be effective targets in prevention, diagnosis, and treatment of human cancer. This idea has become particularly realistic since monoclonal antibody (MAb) techniques have been applied in analysis of tumor-associated membrane antigens. Many of these antigens have been identified as carbohydrates, thus there is increasing evidence that essentially all human cancers are characterized by aberrant glycosylation, whereas some other classical phenomena, including oncogene activation and amplification, are limited to certain types of cancer and cannot be regarded as common denominators of human cancer (see Section IV). Aberrant glycosylation per se may be the basis of inappropriate cell/cell and cell/matrix interactions, which may be reflected in the abnormal cell social behavior of tumor cells, such as uncontrolled cell growth, invasiveness, and metastatic potential. Whatever the genetic or epigenetic basis of expression of aberrant glycosylation, the phenomenon is of crucial importance in understanding the antisocial behavior of tumor cells, as well as in practical applications in diagnosis and treatment of human cancer. The presence of tumor-associated lipid antigens was originally suggested more than 50 years ago by Witebsky (1929),Hirszfeld et al. (1929),and Lehmann-Facius and Toda (1930),who demonstrated that some rabbit antisera obtained after extensive immunization of tumor tissue homogenate and adsorbed on normal tissues showed a preferential complement fixation reaction with the lipid extracts of the In the same sense, Linus Pauling and Harvey Itano referred to sickle cell anemia as a “molecular disease” of hemoglobin (see, e.g., Wintrobe, 1985).

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original tumor tissue used as immunogen, although the chemical nature of the lipid antigen could not be elucidated at the time of the study. During the 1950s, 30 years later, Rapport and his associates tried to elucidate the complement-fixing antigen of lipid extracts, as described above, using transplantable tumors (Rapport and Graf, 1961). In his earlier experiment, rabbit antibodies directed to human epidermoid carcinoma reacted specifically with one glycolipid component, which was identified as lactosylceramide, termed “cytolipin H” (Rapport et al., 1959). In another study, rabbit antisera directed to rat lymphosarcoma reacted specifically with a glycosphingolipid similar, but not identical, to then-known globoside, termed “cytolipin R” (Rapport et al. 1964). A similar approach has been applied to various tissue lipids and has provided the basis for the claim that tissuespecific antigenicity is carried by glycolipids (Rapport and Graf, 1969). Because both lactosylceramide and cytolipin R are found abundantly in normal tissue as well, the immunological basis of tumor specificity claimed in these early studies was ambiguous. Perhaps the earliest chemical evidence indicating the occurrence of aberrant glycosylation in human cancer was the changes in blood groups, either incompatible expression of A antigen (Witebsky, 1929; Hirszfeld et al., 1929), or the reduction of A or B determinants (Oh-Uti, 1949; Masamune et al., 1952, 1953, 1960) (see Section VII,A for discussion of subsequent studies). Related to the change of blood group antigens in human cancer, a large quantity of several fucosecontaining glycolipids was found to be accumulated in various types of human adenocarcinomas, some of which were subsequently identified as Le“, Leb, and Le” antigens (Hakomori et al., 1967, see Section V). In these earlier studies, however, glycosylation patterns of tumor tissues were compared with those of corresponding normal tissues. Since it is difficult to clearly identify the progenitor cells of tumor tissue, a claim for aberrant glycosylation in tumor cells could not be convincingly accepted. During the late 1950s to middle 1960s, technology for cell culture and for oncogenic transformation of cultured cells in vitro by DNA and RNA tumor viruses was established. As a consequence, glycosylation patterns of normal and oncogenically transformed cells in vitro were compared. Transformation-dependent changes of gangliosides and neutral glycolipids were clearly demonstrated by Hakomori and Murakami (1968) and Mora et al. (1969). Subsequently, a number of similar studies were carried out using a large variety of transformation systems. These early studies were reviewed previously (Hakomori, 1973, 1975a; Brady and Fishman, 1975). Aberrant glycosylation

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occurring in glycoprotein carbohydrates, as compared to that in glycolipid carbohydrates, was more difficult to demonstrate in the early days. Techniques for studying glycoprotein carbohydrates have included metabolic double-labeling with 3H- or 14C-labeled sugars applied to normal and transformed cells, respectively, followed b y extensive digestion of cell surface glycoprotein by proteases, and comparison of gel filtration patterns of radiolabeled glycopeptides. Thus, the presence of a high-molecular-weight glycopeptide characteristic of transformed cells, and its absence or low quantity in normal cells, were demonstrated by Meezan et al. (1969) and Buck et al. (1970). The phenomenon has been reproduced in various tumor cells as compared with their progenitor cells in vitro (reviewed by Warren et d., 1978).However, the chemical basis of this phenomenon was not clearly described until Ogata et at. (1976)found that N-linked carbohydrates derived from tumor cells contain more multiantennary structures than do those derived from normal cells. In spite of a number of studies performed on aberrant glycosylation of in vitrotransformed cell systems, the information could not be readily applied to human cancers, since normal counterpart cells of human cancer are not available in sufficient quantity for chemical analysis. Aberrant glycosylation in human cancer was initially suggested by a remarkable accumulation of fucose-containing glycolipids found in human adenocarcinoma (Hakomori and Jeanloz, 1964), some of which were identified as lactofucopentaose-I11 ceramide (Yang and Hakomori, 1971),lactofucopentaose-I1 ceramide (Lea glycolipid), and lactodifucohexaose and lactodifucooctaose ceramide ( Leb glycolipid) (Hakomori and Andrews, 1970). The occurrence of aberrant glycosylation in essentially all human cancers was strongly indicated later on when the MAb approach was introduced in tumor immunology. Interestingly, a number of antibodies selected on the basis of preferential reactivity with tumor cells over normal cells have been identified as being directed to Lex, Lea, Leb, or their analogs. Thus, the initial claim that fucose-containing glycolipids could be the tumorassociated antigen have been justified (Sections V,B and V,C).

11. Tumor-Associated Glycolipid Antigens in Experimental Tumors

The first clear evidence that oncogenically transformed cells contain glycolipid antigens that are absent in their progenitor cells was provided by the presence of lactoneotetraosylceramide (nLc4) in hamster embryonic fibroblasts transformed by polyoma virus, and its complete absence in progenitor NIL cells (Gahmberg and Hakomori,

GLYCOSYLATION IN TUMORS AND TUMOR ANTIGENS

26 1

1975). The same glycolipid accumulates in NIL/py tumors grown in hamsters, although it has been found in only very low quantities in various normal tissues of hamsters. Sera of hamsters bearing NIL/py tumor were shown to have enhanced antibody titer directed to nLc4 (Sundsmo and Hakomori, 1976). In another study, mouse 3T3 cells transformed by Kirsten strain of murine sarcoma virus were found to contain a high quantity of gangliotriaosylceramide (Gg3), and rabbit antibodies directed to Gg3 gave a strong immunofiuorescence in tumors grown in BALBic mice derived from these transformed cells. The same antibodies did not stain normal mouse tissues, except mouse testis, sperm, a few cells in lymph nodes, and spleen (Rosenfelder et al., 1977; L. Halfpap and S. Hakomori, unpublished observations). The sarcoma developing in BALB/c mice from murine sarcoma transformed cells also accumulated a large quantity of Gg3. However, Gg3 was undetectable in various normal tissues and cells (Rosenfelder et al., 1977). Similarly, L-5178 lymphoma grown in DBA/2 mice was characterized by the accumulation of Gg3, whereas various normal tissues and cells did not contain Gg3 in detectable quantities (Young et al., 1981). Concurrently, two MAbs directed to Gg3, one IgM and one IgG3, were established (Young et ul., 1979a). Inoculation of L-5178 lymphoma into DBA/2 mice, followed by administration of IgG3 but not IgM antibodies, completely prevented subsequent lymphoma growth. Subcutaneous lymphoma grown after administration of a low-dose IgG3 antibody was found to lack Gg3 antigen (Young and Hakomori, 1981). Vesicular stomatitis virus (VSV) obtained from SV40 virustransformed cells acquires a tumor-specific transplantation antigen that causes rejection of SV40 tumors. A glycolipid fraction prepared from VSV derived from SV40 tumor incorporated into liposomes was shown to be immunogenic and capable of suppressing tumor growth. Antiserum directed to liposomes containing the polar glycolipid fraction and adsorbed with normal hamster tissue specifically reacted with SV40-transformed cells (Huet and Ansel, 1977; Ansel and Blangy, 1984). A tumor-associated antigen was originally defined as a cell surface component that elicits immune response to syngeneic host cells. Increasing antibody production in NIL/py-bearing hamsters against nLc4, which is a characteristic component of NIL/py tumors, exemplified glycolipid tumor antigen (Sundsmo and Hakomori, 1976). Syngeneic immunization of WKA rats with WKA-derived fibrosarcoma (KMT-17) was used for production of MAbs directed to KMT-17 tumor cells. Two MAbs, derived from spleen cells of immunized rats,

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showed cytotoxicity against KMT-17 sarcoma. One antibody was directed to Gb3 and aGal-nLc4 (IVaGalnLq), the other to nLc4 (It0 et al., 1984). Antibody M2590, which was established after syngeneic immunization of C57/BL mice with B16 melanoma, was found to react with melanoma cells specifically (Taniguchi and Wakabayashi, 1984) and to be directed to GM3 ganglioside (Hirabayashi et al., 1985). These findings all indicate that tumor antigens, in the classical sense, in some experimental tumors, may indeed be glycosphingolipids. This concept has been expanded to and verified in many human cancers, as described below. Ill. Tumor-Associated Carbohydrate Antigens in Human Cancers: Classification, Mosaicism of Expression, and New Procedures for Generation of Antibodies

During the past decade, the MAb approach (Kohler and Milstein, 1975) has been extensively applied in analysis of human cancerassociated antigens. A number of MAbs screened on the basis of preferential reactivity with tumor cell lines and tissues versus normal cell lines and tissues have been found to be directed to carbohydrates bound to lipid (glycolipids) or to protein (glycoproteins). The variety of carbohydrate antigen structures is relatively limited, yet multiple hybridoma antibodies directed to the same antigen show characters, fine specificities, and affinities distinctive from the others. Therefore, the variety of antibodies is practically unlimited. The selection of an antibody with appropriate specificity and affinity for practical application is of primary importance. Tumor-associated carbohydrate antigens can be divided into five classes according to their association with carrier molecules: (1) epitope structures clearly identified and expressed both in glycosphingolipids and glycoproteins; (2) those expressed only in glycosphingolipids; (3)those expressed only in glycoproteins; (4) polypeptide epitopes whose antigenicity is only maintained when a single or multiple threonine or serine residue(s) is glycosylated; (5) antigens whose epitope is poorly defined or undefined. The first class of antigens is characterized by lacto-series type 1or type 2 chain (class 1; Table 1,A). The second class of antigens are mainly globo- or ganglioseries glycosphingolipids (class 2; Table 1,A). The third, fourth, and fifth classes are associated with glycoproteins, particularly mucin-type glycoproteins (classes 3-5; Table 1,A). On the other hand, tumorassociated carbohydrate antigens can also be classified according to their specificity of expression, i.e., (1) antigens highly expressed in

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TABLE I CLASSIFICATION OF TUMOR-ASSOCIATED CARBOHYDRATE ANTIGENS A. Classification according to difference in epitope carrier Class 1: Associated with both glycolipid and glycoprotein (mostly lacto-series type 1 or type 2 chain) Class 2: Associated exclusively with glycolipid (mostly globo-series or ganglio-series structure) Class 3: Associated exclusively with glycoprotein (mostly related to mucin-type 0-linked chains such as T, Tn, and sialyl Tn) Class 4: Associated exclusively with glycoprotein whose structure is ill defined Class 5 : Polypeptide epitopes whose antigenicity is influenced by 0-glycosylation B. Classification according to specificity of antigen expression Class 1: Novel structures highly expressed in tumor cells, absent in their progenitor cells, but expressed in limited numbers in other normal cells. Examples: di- or trimeric Le", sialyl dimeric Le" in various gastrointestinal and lung adenocarcinomas, and GDP and GD3 gangliosides in melanoma and neuroblastoma Class 2: Common structures highly expressed in tumor cells, but present in lower quantity in various types of normal cells and tissues. Examples: GM3 and GM2 gangliosides in melanoma, and Gb3 in Burkitt's lymphoma Class 3: Exclusively expressed in tumor cells, but absent (or present in immunologically undetectable levels) in normal cells. Examples: incompatible blood group antigens such as real A antigen and A-like antigen expressed in 0 tumors. The majority of A-like antigens are now identified as Tn antigen (see Class A 3 above; see also Table V)

tumor cells, not expressed in the progenitor cells, but expressed in other types of normal cells and tissues (class 1; Table 1,B); ( 2 ) those expressed highly in tumor cells but expressed weakly in their normal counterparts (class 2; Table 1,B); and ( 3 ) those expressed exclusively in tumor cells, and not in normal cells (class 3 ; Table 1,B). The majority of tumor-associated antigens belong to category (1)or (B), and only rarely to ( 3 ) . Such a classification of antigens is made for convenience. In fact, each tumor expresses different classes of carbohydrate antigens, under unknown mechanisms. In a recent study, serial tumor sections were sequentially dissected and stained with hematoxylin-eosin, anti-CEA, periodate-Schiff reagent, anti-Le" SH1, antidimeric Le" FH4, antisialyl dimeric Le" FH6, and antisialyl Tn TKHB. The patterns of antigen distribution in each tumor section stained by each antibody were clearly distinctive. Some sections were stained

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exclusively by SH1, others by both FH6 and SH1, others exclusively by TKH2, etc. Thus, a mosaicism in stainability with these different anticarbohydrate antibodies is clearly seen within a single tumor (Fig. 1). The same phenomenon has been observed in essentially all tumor cases so far examined (Nakasaki et al., 1989). This finding clearly indicates the necessity of combined antibodies for practical diagnostic and therapeutic applications. The pathobiological significance of this mosaic expression of carbohydrate antigens by tumors remains unknown. The procedure for establishing MAbs defining tumor-associated carbohydrate antigens routinely practiced in various laboratories is based on immunization of mice with tumor cells or membranes derived therefrom, followed by selection of hybridoma by specific or preferentia1 reactivity of produced antibody with tumor cells or tissues over normal cells or tissues. This requires extensive, laborious work at the stage of selection. Our laboratory has established new procedures based on structural information of aberrant glycolipids. The procedure is shown schematically in Fig. 2. A semipurified glycolipid fraction isolated from tumor tissue or synthetic carbohydrate antigen is used as an immunogen coated on bacteria. Alternatively, the immunogen can be tumor cells or tumor cell membranes. However, hybridomas are selected by chemically defined antigens on the basis of positive reactivity with the desired structure and negative reactivity with nondesired structures (Fukushi et al., 1984a,c; Hakomori et al., 1983; Abe et al., 1984). An example using a synthetic glycolipid antigen was provided in a study by Shigeta et al. (1987). The GgLcl (GalNAcPl+ 4[GlcNAcPl+ 31 GalPl- 4GlcPl- Cer), previously characterized as a minor component of leukemia-associated antigen (Kannagi et ul., 1984b), was chemically synthesized (Ito et al., 1986) and used as imniunogen. Two hybridomas producing antibody specific to G g L q were selected by the synthetic antigen. The same approach can be widely used in making unlimited numbers of antibodies directed to tumor-associated antigens. IV. Oncogenes and Aberrant Glycosylation

Oncogenes, which trigger a series of cascade reactions leading to expression of a number of tumor-associated phenotypes, were found originally in retroviruses, subsequently in normal cells, and are activated and amplified in some human cancers (Land et al., 1983; Bishop, 1983; Varmus, 1984). A positive correlation between

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FIG.1. Mosaicism in expression of tumor-associated carbohydrate antigens in a single tumor. A typical example of mosaic expression as described in the text is illustrated in a series of sections from gastric cancer. (A) Hematoxylin-eosin staining; (B) immunoperoxidase staining with anti-Le' antibody AH6 (Abe et al., 1983); (C) immunoperoxidase staining with antidimeric Le" antibody FH4 (Fukushi et al., 1 9 8 4 ~ ) ; (D) immunoperoxidase staining with antisialyl a2 + 6GalNAca (sialyl Tn) antibody TKHB (Kjeldsen et al., 1983); (E) immunoperoxidase staining with antisialyl dimeric Le" antibody FH6 (Fukushi et al., 1984a); and (F) immunoperoxidase staining with anti-CEA antibody. Note that the area not stained by TKH2 was strongly stained by FH4 and FH6. The area weakly stained by FH4 and FH6 was strongly stained by TKH2. The area not stained by FH4, TKHZ, and FH6 was strongly stained by AH6. Single tumors generally show expression of multiple carbohydrate antigens, with various degrees of mosaicism.

synthetic -tes glycolipids

Isolation an5 characterization of tumo~assaciatedglycolipid antigens

nrmOr cells or membranes I Selection

variety of normal and tumor cells or sections

A large

A pure glymiipid

and

I

coated

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1

NO-

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specificity test

positive with tumor tissue and negative with normdl tissue

positive reactivity with desired structure and nqative reactivity w i t h undesired structure

Mcterization of epitope

Isolation and characterization of antigen

predefined epitope assxiat& with tumr specificity

Fic:. 2. Prcicrcdures for establisliirip, rrioriocrlund aiitilmdies that define ~irricil.-associaleclcnrhhydratc iintigrris. T h e noi-rml p r c i c d n r c for estahlishirig irionwlonal antibodics is show^^ ill the second columri, in which turiior cells or menhrarics arc used as inmunogcns arid selection of hybridorrws i s made by positive reactivity with tumor cells and ncgatiue reactivity with nornial crclls a n d tissucs. In contl+:)st,new prcwetliii-t:s we have de\&ped are shown in the third colunin, in which isolated glycolipids (including synthetic coinporinds) are used :IS immuwgens, and hybridomas are selected by positive reactivity with the desired sh'trcture and negative reactivity with other stnlctures. The selection of hybridomas with the purified glycolipid can be made after tumor cells o r membranes are used as immunogens.

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oncogene activation and aberrant synthesis of glycolipids had been found previously, e.g., GM3 synthesis as related to src activation (Hakomori et al., 1977b), Gg3 synthesis as related to rusk transfection in NIH 3T3 cells from human tumors (Tsuchiya and Hakomori, 1983), and GDD and other ganglioside changes as related to adenovirus oncogenes (Nakamura et al., 1984). Aberrant glycosylation was detectable in essentially all human cancers if various MAb probes were applied in combination. In contrast, the estimated incidence of activated oncogenes in human cancers, detected by presently known oncogene probes, was approximately 2 0 4 0 % (Slamon et al., 1984; Yokota et al., 1986; Kurzrock et al., 1986; Nau et aZ., 1986; Barbacid, 1986; Forrester et al., 1987; Bos et al., 1987). In other words, essentially 100% of human cancers exhibits aberrant glycosylation but not oncogene activation. Therefore, known oncogenes are not correlated with aberrant glycosylation in human cancers. This situation contrasts with that in cells transformed by retroviral infection, in which oncogene activation is well correlated with aberrant glycosylation (Hakomori et aZ., 1977b). V. Normal and Oncofetal Features of Glycolipid Antigens

A. BACKGROUND Aberrant glycosylation can be better defined chemically by analysis of glycolipids rather than glycoproteins, since glycolipids can be isolated in homogeneity, and their differences in normal versus cancer tissues can be studied with relative ease. Aberrant glycosylation in human cancer was initially suggested by the discovery of an accumulation of unusual glycosphingolipids containing fucose, N acetylglucosamine, and phytosphingosine in a large variety of human cancers. Fucose was not recognized as a component of normal tissue glycosphingolipids at that time. These glycosphingolipids were chemically and immunochemically identified as Lex, Lea, and Leb (Hakomori et al., 1967; Hakomori and Andrews, 1970; Yang and Hakomori, 1971). A decade later, when the MAb approach was introduced in analysis of human tumor antigens, it became clear that many of the antibodies were directed to Lex, Lea, and Leb. In addition, other fucolipid analogs, such as Ley, sialyl Lea, disialyl Lea, and sialyl Lex, have been characterized as the tumor-associated structures (see Sections V,B and V,C). Thus, the earlier prediction of fucolipids as the major human tumor-associated antigens (Hakomori, 1971b, 1975b) has been verified.

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SEN-ITIROH HAKOMORI

B. LACTO-SERIES TYPE1 CHAINANTIGENS 1. Normal Synthetic Pathway The type 1 chain (Galpl- 3GlcNAcPl- 3Gal) represents the major carrier for the blood group ABH determinant, and constitutes the backbone of Lea, Leb, and other Lewis antigens (see Fig. 3) (Watkins, 1980). In adult gastrointestinal and colorectal epithelia, the type 1 chain predominates over the type 2 chain, and the majority of type 1 chain is fucosylated: either a1 + 4 fucosylation at the penultimate GlcNAc to form Lea, or a1 + 2 fucosylation at the terminal Gal to form type 1 chain H, i.e., Led (Hanfland and Graham, 1981), or co-occurrence of both types of fucosylation to form Leb. These fucosylation patterns of type 1chain are genetically well defined, and the fucosylation product confers blood group Lea, Leb, or Led status to

( kc)

Gala 1-3 G l ~ m1+3Ga1,8 3 1-.4Gl~-Cer

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(1) al-4 fucosylation

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17

mcosylation

Gala 1- 3GlcNAcp 1- 3-1s

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sA2.~l-Wa??!ql-3GIlpl--Uxc

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( F W antigen) -fetal

pthway

FIG.3. Type 1 chain pathways, normal and oncofetal. Structures shown in bold print represent those accumulated in various tumors. Therefore, a1 -+ 4 fucosylation (for Lea synthesis) combined with a1 42 fucosylation (for Leb synthesis) is enhanced greatly in various tumors irrespective of Lewis status of the host. The a2 3 sialylation was also greatly enhanced combined with a1 + 4 fucosylation, resulting in a2 + 3 sialyl Lea. --j

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erythrocytes (Graham et a]., 1977; Watkins, 1980; Hanfland and Graham, 1981; Le Pendu e t al., 1986). The majority of type 1 chain H in bIood group A and B epithelia is converted to A and B antigens, and is also converted and secreted as Lel' in A, B, and 0 secretors. The type 1chain H is virtually absent in nonsecretors, and the precursor is converted and secreted as Le". Rarely, the type 1 chain is present in an unsubstituted state, which may be present in the rare genetic trait Le' (Graham et al., 1977). Real Le' antigen, however, may include a branched structure having an unsubstituted type 1 chain on one end and Le" structure on the other end (Hanfland et al., 19$6)+Sialylation of type 1 chain is limited in normal adult tissue as compared with fucosylation; thus, the idea proposed by Dische (1960), i.e., that sialylation and fucosylation are mutually exclusive and competitive, is essentially true for type 1 chain occurring in the majority of normal adult gastrointestinal epithelia, except for acinar epithelia of pancreas, salivary glands, and in seminal glands, where a2 + 3 sialylation and a1 -+ 4 fucosylation co-occur, thus forming sialyl Le" (a2-+ 3 sialyl Le") antigen (see below).

2. Oncofetal Pathway In tumors, type 1 chain undergoes various enhanced reactions as follows: 1. Enhanced sialylation at both terminal Gal as well as at the penultimate GlcNAc, thus forming sialyl2 + 3 and disialyl(2 + 3 and 2 + 6) type 1 chain. The former structure was defined by MAbs CA50 (Nilsson et al., 1985) or K21 (Rettig et al., 1985; Fukuda et al., 1986), which were originally prepared by immunization with colonic cancer cells and teratocarcinoma cells, respectively. The latter structure was also defined by MAb FH9 (Fukushi e t al., 1986). Sialyl 2- 3 or disialyl 2 + 3, 2 + 6 type 1 chain glycolipids were found to be accumulated in various types of adenocarcinomas and epitheliomas (Fukushi et al., 1986). The same antigen accumulated in human glioma cells grown in nude mice (Mansson et d.,1986). Although these glycolipids were present in undetectable levels in normal gastrointestinal and colorectal mucosae, they are present in meconium (Nilsson et aZ., 1981; Karlsson and Larson, 1979). 2. Noncompetitive a1 + 4 fucosylation to sialyl2 + 3 type 1chain, resulting in sialyl Lea defined by antibody N-19-9 (Koprowski et al., 1979; Magnani e t a]., 1982) or by antibody CSLEA-1 (Chia et al., 1985). A novel disialoganglioside, disialosyl Led (IV3NeuAcII16NeuA-

270

SEN-ITIROH HAXOMOM

cII14FucLc4) was isolated and characterized, and the antibody FH7 directed to this structure was established (Nudelman et al., 1986a). A sequence of enzymatic reactions involved in synthesis of sialyl Lea proceeds via a2 + 3 sialylation of type 1 chain followed by a1 + 4 fucosylation. The reverse sequence never occurs (Hansson and Zopf, 1985). The antigen sialyl 2 + 3 Lea has been found in various types of gastrointestinal and colorectal tumors. These tumors have a1 + 4 fucosyltransferase, nonrestrictive to the presence of sialosyl residue at the adjacent terminus. Thus, tumors can be characterized by the presence of enzyme violating Dische's rule, which applies to normal tissue. 3. Enhanced fucosylation and coexpression of Lea and Leb. Although the enzymatic basis for enhanced fucosylation of type l chain in tumors has not been elucidated, a1 + 4 fucosylation to the penultimate GlcNAc must be greatly enhanced, since the quantity of Lea antigen is quite high in various types of tumor (Hakomori and Andrews, 1970). On the other hand, the level of Leb antigen in various tumors is also high, leading to the coexpression of Lea and Leb antigens in tumors regardless of histo-blood group Lewis status of the host (Hakomori and Andrews, 1970; Blaszczyk et al., 1985; Temper0 et al., 1987). In fact, several MAbs that were established after immunization with colonic cancer cells and were selected on the basis of preferential reactivity with tumor cells over normal cells were identified as being directed to Lea (Blaszczyk et al., 1984; MBrtensson et al., 1988) or Leb (Brockhaus et al., 1981). Some antibodies directed to breast carcinoma were found to cross-react with both Lea and Le" (Gooi et al., 1985). Interestingly, the antibody 43-9F, established after immunization with and showing a preferential reactivity to squamous cell lung carcinoma (Pettijohn et al., 1987), cross-reacts with Lea, although a real antigen could be Lea bound to Le" (MQrtenssonet al., 1988; see Table 11).The IgG3 antibody ST-421, established against a human gastric cancer xenograft, showed strong cytotoxicity to human cancer cells expressing Lea, but was not cytotoxic to normal Le(a+b-) erythrocytes. The exact epitope defined by this antibody remains to be determined (Watanabe et al., 1988). These results suggest that synthesis of Lea and Leb is greatly enhanced in various types of human cancer.

C. LACTO-SERIES TYPE2 CHAINANTIGENS 1. Normal Synthetic Pathway The type 2 chain (Galpl +-4GlcNAcp1+ 3Gal) is the major carrier for blood group ABH determinants of human red blood cells, but is a

MAJOR MONOCLONAL ANTtBODlES

TABLE I1 TLIMOH-ASMK:IATKU LACTO-SEKIRS T Y P E 1 CttAIN

UIHECTED TO

-

~

Antibody N-19-9

Inimunogen Colonic carcinoma cell line

Isotype

Structure

Refercnve

Calpl -+ ,3CluNAc

IgG,

3

4

t

T

CSIXA-1 CA50

Gastric cancer Human colonic ciincer

-

NsuAcm2 Furlrl GalpI + 3GIcNAc 3

K21

Human emhryonal carcinoma cell PA1

-

NeuAcaZ

IJZM

Kopiowski et a[,(1979);

Maarlarii ul (11. (1982); Chia et 01. ( I 985)

Nilsson et ul. (1‘385) Rrttig et ul. (1985); Fiikuda ei ul.(l986)

t Ne11ricaX

1 FH9

6 GalPl-+ 3GluNAc 3

Disialoganglioside fraction of colonic cancer

Fukushi ef

(12.

(1986)

T NeuAcaZ

FH7

K4 43-9F

NeuAca2 j. 6 GalPl+ 3GlcNAc 3 4

Disialoganglioside fraction of colonic cancer

T

t

NeuAca2 Fwal GalPl 3GlcNAcP1-+3Gal

Human embryonal carcinoma cell PA1 Human squamous lung carcinoma cell line (SLC-Lll)

-f

GalPl-+ 3GlcNAc 4

(Le“)

T

Fucoll Galpl- 3ClclVAcPl-+ 3GalP1- 4GlcNAc 4 3

T

T

Fucal Fucal (Le”-Lt.%c ~ m p l t . ~ )

NCC-ST-421

Gastric cancer

Nudelnun et d.(1986a)

Le”

Retttig et al., (1985); Fukuda et QL, (1986) Pettijohn et 01. (1987) Miitensson et ul. (1988)

G?@l+4Glcmc#31-3Gal/31-4GlCsk 3

3

t

t

sA2

-1

3 t Rml

sialvl dimeric fl (FN6 antigen)

~ 1 - [ 4 G l ~ l - 3 G a l p 1 ] n + ~ l - 4 G l ~ 3 6 t

t

sA2

ACRI18 antiqen

-1

Fie:. 4. Type 2 chain pithways, ~ i o r i m land oncofetal. Structurcs shown i n I)old print are accruniilatctl it1 various hiiniuii cancers. Therefore, their synthesis is enhanced. The major synthetic route eiih;u~cetlin tiinlor cells is chain elongation (to lead to i antigen) followed by a1 + 3 fucosylation or 01 1-2 fucosylation, resulting in di- or trinieric Le' or hifucosyl Le? (route 1 + route 4).If i antigen is 012 + 3 or a2 +6 sialylated, and then a1 3 fucosylated, it is converted to FH6 or ACFHlS antigens (route 1 + route 5 or 6 cotnbined with route 3).

GLYCOSYLATION IN TUMORS AND TUiMOR ANTIGENS

273

relatively minor component in epithelial cells (see Fig. 4). In fact, type 2 chain was virtually absent in normal human adult intestinal mucosae (McKibbin et a]., 1982). In normal adult blood cells and tissues, type 2 chain showed extensive variation: (1)Chain elongation by repeating Galpl + 4GlcNAc, with branching at Gal, with Galpl + 4GlcNAcpl+ 6 substitution. In adult blood cells and tissues, the branched structure (which represents I antigen) is predominant over the unbranched chain-elongated structure (which represents i antigen) (Watanabe and Hakomori, 1976; Hakomori, 1981a; Fukuda et al., 1979; 1980). (2) The terminal Gal of branched or unbranched type 2 chain is a1 + 2 fucosylated to yield branched or unbranched type 2 chain H, which could be subsequently converted to a branched or unbranched A or B structure. Reflecting the proportion of type 2 chain core, branched ABH is predominant over the unbranched ABH structure in normal adult tissue (Hakomori et aZ., 1977a; Hakomori, 1981a). ( 3 ) As compared with type 1 chain, sialylation of type 2 chain takes place extensively, and a series of terminally 2 -+ 3 sialylated structures are produced. (4) Sialylated type 2 chain is rarely fucosylated at the subterminal GlcNAc in normal adult cells or tissues, except for granulocytes, monocytes, proximal convoluted kidney tubules, and a few other cell types (Fukushi et al., 1984b). The enzymatic basis of branching in type 2 chain (Basu and Basu, 1984; van den Eijnden et aZ., 1983) and the process of chain elongation (Holmes et al., 1987) have been studied. However, the mechanism for coordinating enzymatic synthesis to maintain the normal pathway of type 2 chain remains to be explored. The presence of Le" and Ley structures in renal cells and secretions has been known since these structures were found in gastric and ovarian cyst mucin (Lloyd et al., 1966; for a review, see Watkins, 1980). After MAbs directed to these structures were established, the distribution of Le" and Ley in various normal tissues was studied and Le" was found most abundantly in normal granulocytes and proximal kidney tubules; Le" and Leywere found in various epithelial cells and secretions. The important finding from these studies is that Le" is closely associated with nonsecretor status and Ley is associated with secretor status, as are Le" and Le", respectively; i.e., a1 += 3fucosyltransferase may define the presence of Le" in nonsecretor epithelia and the secretion therefrom (Sakamoto et al., 1984). Nevertheless, the quantities of Le" and Ley in normal epithelial tissue were much less than those expressed in various cancer tissues derived from epithelial tissues, as shown in the following section.

274

SEN-ITIROH HAKOMORI

2. Oncofetal Pathway Alterations of type 2 chain constitute the major change in glycosylation in most human cancers, and are of central importance. Many MAbs directed to human cancer have been identified as being directed to type 2 chain-based structures. The following changes have been documented: (1) Predominance of an unbranched elongated chain (i antigen), and accumulation of its sialylated structure (sialyl-i antigen); MAb NCC-1004, established after fusion of regional lymph nodes of lung cancer with mouse myeloma, and showing preferential reactivity with tumor, was identified as being directed to i antigen (Hirohashi et al., 1986). (2) A series of unbranched type 2 chains are a1 -+ 3 fucosylated at the penultimate GlcNAc, followed by internal GlcNAc, thus yielding a series of Le" or di- or trimeric Le" structures (Hakomori et al., 1984a; Holmes et al., 1985). Many human adenocarcinomas contain lactofucopentaosyl-I11 ceramide (II13FucnLc4) (Yang and Hakomori, 1971) or di- or trimeric Le" (II13V3FucznLcfi; II13V3VI13Fuc3nLc~) (Hakomori et at., 1984a) as the major neutral glycolipid component. A large number of MAbs directed to human cancer have been identified as anti-Le". The first anti-Le" MAb was prepared after immunization of syngeneic mice with F9 teratocarcinoma, which defined a then unknown antigen highly expressed at the morula stage and called "stage-specific embryonic antigen 1" (SSEA-1) (Solter and Knowles, 1978). The antibody was found to be directed to a series of glycolipids having the Le" determinant, which was accumulated in human colonic cancer (Hakomori et al., 1981b). Concurrently, the reactivity of the antibody was found to be inhibited by a mixture of lactofucopentaose-I1 and -111 (Gooi et al., 1981), and subsequently by a purified lactofucopentaose-I11 but not lactofucopentaose-I1 (Hounsell et al., 1981). Those antigens isolated from whole blood cell membranes have been identified as unbranched type 2 chains with a1 + 3 fucosyl substitution at the penultimate and internal GlcNAc (Kannagi et al., 1982a). Subsequently, a number of antibodies directed to various types of human cancer were identified as being directed to Le" (Brockhaus et al., 1982; Huang et al., 1983; Hansson et al., 1983; Urdal et al., 1983a; Gooi et al., 1983). The antibody FH4, which was prepared against dimeric Lex, did not cross-react with simple Le" (Fukushi et al., 1984c) and showed better specificity to various types of human adenocarcinoma (Fukushi et al., 1984b), particularly colonic cancer (Itzkowitz et al., 1986a). Antibodies to simple Le" react strongly with normal granulocytes (Urdal et al., l983a; Gooi et al., 1983), whereas FH4 hardly reacted

GLYCOSYLATION IN TUMORS AND TUMOR ANTIGENS

275

with normal granulocytes, albeit it did react strongly with myelogenous leukemia HL60 cells (Symington et al., 1985).A similar antibody preferentially recognizing extended Le" and dimeric Le" was also obtained after immunization with myelogenous leukemia cells (Magnani et al., 1984). Various tumor tissues also accumulate a series of glycolipids with Ley structures, which are presumably synthesized by cooperative action of a1 + 2 fucosyl transferase and a1 + 3 fucosyl transferase on type 2 chains. Antibodies directed to the Ley determinant have been identified among various MAbs directed to gastric (Abe et al., 1983), breast (Brown et al., 1983),and colonic cancer (Lloyd et al., 1983). Ley expression, defined by MAb AH6, was positive in all colorectal carcinomas tested, and was weakly positive in epithelia of ascending to transverse colon, but was essentially negative in epithelia of descending colon and rectum except for a limited number in the crypt area (Abe et al., 1986). A series of type 2 chain-bearing Ley determinants were detected. Of these, three major Ley glycolipids accumulating in colonic adenocarcinoma have been identified: IV2FucII13FucnLc4, V12FucV3FucnLc6, and V12FucV3FucII13FucnLc~ (Nudelman et al., 1986b) (for structures, see Table 111). Some MAbs showed preferential reactivity with extended Ley structure with or without internal a1 + 3 fucosyl residues. The antibody KH1, which is directed to trifucosyl Ley ( II13FucV3FucV12FucnLc6)(Kaizu et al., 1986), and antibodies CC1 and CC2, which are directed to extended Ley (V3FucV12FucnLc~), showed a higher specificity to colonic cancer than did antibody AH6 (Kim et al., 1986). Expression of Ley versus Le" antigen in some tumors may reflect the host's secretor/ nonsecretor status, as in normal secretory glands; however, the majority of tumors strongly coexpress both Le" and Ley, and this coexpression in tumor may not reflect the host's secretor status (E. Nudelman, and S. Hakomori, unpublished observation). A series of sialylated type 2 chains can be converted to a series of sialyl Le" antigens by a1 + 3 fucosylation at the penultimate GlcNAc, as well as at internal GlcNAc (Holmes et al., 1986). These compounds can be recognized by MAb CSLEX-1 (Fukushima et al., 1984) or FH6 (Fukushi et al., 1984a). The latter antibody showed more restricted reactivity and reacted preferentially with extended sialyl Le" with or without internal fucosyl structure, as compared with the former antibody, which has nonrestricted reactivity with all sialyl Le" determinants. Immunohistological studies indicate that FH6 antibody shows specific reactivity with tumors, and highly restricted reactivity with normal tissue except for a clear reactivity with granulocytes

NCC1004 SSEA-1

w29 AH6

Natiirally occurring lung cancer Mouse F9 ccll teratocarci noirra

k M

I luniaii gastric c1i1ic~1r

IgM IgM

H i u ~ i a gastric ~i cancer

IgM

G a l P l - + 4GlcNAcP1-+ [3GalP1+ 4GlcNAcpl],,+ 3Gal (i-antigen) Gal01 + 4GlcNAcP1+ 3Gal 3

r

Fuca I (LC') CdP1 + 4C;lcNAcPl + 3C:d 2 3

MKN74

r

t FH4

Purified antigen

Ftrcal Fucal (Le') GalPl- 4GlcNAcPl- 3GalP1- 4GlcNAchl+ 3Gal 3 3

~ G J

r

FH5

CSLEX-1

Purified antigen and tumor cell membrane Gastric cancer cell membrane

IgM

t -

t

Purified antigen

kM

t CCl and CC2

Colonic cancer cells

kM

2

T KH 1

Purified antigen

IgM

-

t

t

Fukushi et nl. ( 1 9 8 4 ~ )

t

t

Fucal k(Fuca1) 4GlcNAcP1+ 3GalP1+ 4GlcNAcpl3

3Gal

Kim et al. (1986)

t

Fucal Fucal (extended Le') Galj3l- 4GlciVAc~l- 3GalP1- 4GlcNAc/3l+ 3Gal 3 2 3 FucPl

Fukushi et al. (1984a)

Fukushima e t al. (1984)

NeuAcaZ FuccY1 (sialyl Le') GaIPl- 4GlcNAcPl- 3GalPl- 4GlcNAcPl-+ 3Gal 3 3 3 NeuAca2 GaIPl

Fukushi et al. (1984a)

t

Fucal Fucal GalPl + 4GlcNAcPl -t 3Gal 3 3

t FH6

S o h arid Knowlcs (1978); Ilakomori et ul. (1981); C h i et ul. (1981) Hrocklxius et ti/. (1982) Abe ot ol. (1983); Lloyd et 01. (1983); Brown et ul. (1983)

t

Fucal Fucal GalPl- 4GlcNAcpl-+ 3GalPI- 4GlcNAcPl- 3Galpl- 4GlcNAc 3 3 3 Fucal

IIirohashi et d . (1986)

t

Fucal

t Fucal

Kaizu et nl. (1986)

GLYCOSYLATION IN TUMORS AND TUMOR ANTIGENS

277

(Fukushi et al., 1985; Itzkowitz et al., 1986a). The sequence for enzymatic synthesis of sialyl Le" is similar to that for synthesis of sialyl Lea, i.e., a1 + 3 fucosylation occurred only in sialyl type 2 chains, whereas sialylation never occurred for Le" structures (Holmes et al., 1986).

D. GANGLIO-SERIES ANTIGENS 1. Normal Synthetic Pathway Sequential synthesis of ganglio-series gangliosides has been well established as being initiated from conversion of LacCer to GM3, which is further converted to GM2, GM1, GD1,, and GT1, (Kaufman et al., 1968; Roseman, 1970; Basu et al., 1980, 1984). GM3 is also converted to GD3, and subsequently to GD2, GDlb, GTlb, and GQlb. GD3 is converted to GT3, which is subsequently converted to GT2, GTI,, GQI,., and GPI, (Svennerholm, 1964; Holmgren et al., 1980) (see Fig. 5). In addition to the above pathway, which is initiated from GM3, another pathway has been described: LacCer is converted to Gg3 (asialo GM2) and subsequently to GM2 (Handa and Burton, 1969); Gg3 could also be converted to Gg4 (asialo GM1) and GMlb, although this route has not been demonstrated in normal human tissue. A novel series of gangliosides, GDl,, GTI,, and GQlp, has been found in frog brain (Ohashi, 1981). These novel gangliosides (see Fig. 5) have not yet been found in normal mammalian tissue, but have been found in some animal tumors (see below). GM1 or asialo GMI can b e converted to fucosyl GM1 or fucosyl asialo GMI as a minor pathway in normal tissue. Metabolic pathways of ganglio-series ganglioside have been reviewed (Roseman, 1970; Brunngraber, 1979; Kanfer, 1983). 2. Oncofetal Pathway

The major ganglio-series antigens accumulating in human cancer and defined by MAbs (see Table IV), which are present only in trace quantities in normal tissue, are the precursors in synthesis of ganglioseries gangliosides (their structures are indicated by boldface in Fig. 5). Typical examples are GD3 accumulated in melanoma and defined by various MAbs (Nudelman et al., 1982; Puke1 et al., 1982; Cheresh et al., 1985), GD2 in melanoma and neuroblastoma (Cahan et al., 1982; Watanabe et al., 1982), and GM2 in melanoma (Irie et al., 1982; Tai et al., 1983). Accumulation of GM3 in various types of melanoma has been assessed, since a specific antibody directed to melanoma was

I I

SAl I

I

I

c Gal+GalNAwGal+Glc-.Cer

I

I

S A S A

l s d , sd,

GQV

a 4 I Rlc

FIG.5. Ganglio-series ganglioside pathway. Structures shown in bold print are accumulated in various neuroectodermal tumors (e.g., melanoma, neuroblastoma) and in T cell lyniphonlas. Note that most accrlniulated species are precursors, which are present in trace quantity in noimal tissues.

TABLE IV MONOCLONAL ANTIBODIES DIRECTED TO TUMOR-ASSOCIATED GANGLIO-SERIES ANTIGENS Antibody Globo-series 38-13

Immanogen

Burkilt's lymphoma (Daudi)

Isotype

IgM (rat)

2

MBrl

Breast cancer cell line MCF-7 IgM

SSEA-3

Mouse embryo

1309 Human lung cancer" and D579 SSEA-4 Human teratocarcinoma 2102 and mouse embryo Ganglio-series 4.2 Human melanoma R24 Human melanoma 83.6 Hnman melanoma

D .l.l

Rat brain tumor B49

OFA-1-1

EBV bansformation of B lymphocyte melanoma

5-3

C57RL melanoma EBV transformation of B lymphocyte melanoma

IgM

Structure

Reference

-

4GlcP1+ Cer Wiels et al. (1981); Nndelman et al. (1982) Fucal + 2Galpl 3GalNAcPl- 3Ga la l+ 4GalP1--* 4Glcpl- Cer MCnhrd et ul. (1983); Bremer et al. (1984) Galpl + 3GalNAcPl + 3Galal 4GalP1- 4GlcPl- Cer Shevinsky et al. (1982); Kannagi et 01. (1983a) Schrump et 02. (1988) Gala1

4GalP1-

--j

--f

IgM (human) NeuAca2+ 3GalPlIgM

3GalNAcpl-

3Ga la l+ 4GalP1-

4GlcPl-

CerKannagi et a[. (1983b)

Yeh et ul. (1982); Nndelnlan et ol. (1982);

9-0-Ac-NeuAca2

-

8NeuAcu2 -+ 3GalPl- 4GlcP1GalNAcPl

-

4GalPl- 4Glc/313

Dippolcl et cil. (19MJ); Puke1 et nl. (1982); Cheresh et t r l . (19841,); Cer Chercsh et ul. (1984a,c); Thurin et d . (1985) Cer h i e et al. (1982) Tai et al. (1983)

t OFA-1-2

12.6 -

Human neuroblastoma cells Small cell lung carcinoma

NenAca2

NeuAca2

-

(CM,)

Natuli e l ol. (1986) Cahan et ul. (1982)

(GD3)

Cheresh et ul. (19841))

J

t 8NeuAca2

F n c a l + 2Galpl- 3GalNAcp1+ 4GalP1- 4GlcPl--f Cer Nilsson et al. (1984, 1986) 3

t NeuAca2

" EBV-transformed lymphocytes from regional lymph nodes of H lung cancer patient, followed by fusion with NS-1.

280

SEN-ITIROH HAKOMORI

found to be directed to GM3 (Taniguchi and Wakabayashi, 1984; Hirabayashi et al., 1985) but may require specific organization because of high concentrations (Nores et d., 1987) (see Section IX). Before the MAb approach was applied, Portoukalian et al. (1979) clearly documented the accumulation of GD3 ganglioside in melanoma, and suggested it to be a possible melanoma-associated antigen. Gg, (asialo GMZ), which accumulates in mouse sarcoma KIMSV and lymphoma L5178 (see Section 111), is also present in human astrocytoma, melanoma (L. Halfpap and S. Hakomori, unpublished observation), and Hodgkin’s lymphoma (Kniep et al., 1983). Presence of asialo GM2 (Gg3) in various but limited types of human cancer could be due to blocked asialo-core synthesis, initiated by the HandaBurton pathway. Various murine and human hybridomas have been established which secrete antibodies showing preferential reactivity with human melanoma and neuroblastoma; many of these antibodies were found to be directed to GM2 (Irie et al., 1982; Tai et al., 1983; Natoli et al., 1986) and GD2 gangliosides (Cahan et al., 1982; Cheresh et al., 1984a). Accumulation of GD:, and GD2 in association with deletion of higher gangliosides suggests a blocking of the BasuKaufman-Roseman pathway. However, recent studies indicate that synthesis of precursor gangliosides such as GD3 and GD2 is also greatly enhanced (Cheresh et al., 1986).It is therefore assumed that accumulation of precursor gangliosides results from both blocked synthesis of higher gangliosides and enhanced synthesis of the precursors. GD3 antigen in some cells was 9-O-acetylated at the terminal sialic acid residue, and a MAb specific to melanoma recognized GD:, containing 9-O-acetylated sialic acid (Cheresh et al., 1 9 8 4 ~Thurin ; et al., 1985). GDR can be converted to reactive antigen by chemical and enzymatic O-acetylation (Cheresh et al., lYS4b). I n some animal cancers, Gg3 can be converted to Gg4 (asialo GMJ, GMIl,, and fucosyl Gg4 (fucosyl asialo GM1) (Taki et al., 1978; 1979; Hirabayashi et al., 1978; Holmes and Hakomori, 1982), and GMIl, may mask antigenicity of Gg3 at the surface of mouse lymphoma. It is not yet known whether the asialo ganglio pathway initiated b y the Handa-Burton synthetic route is enhanced in certain human cancers. Gg4 (asialo GM1) was claimed to be a marker of human acute lyrnphoblastic leukemia (Nakahara et al., 1980). A MAb prepared against small cell lung carcinoma was found to be directed to fucosyl GMI ( IV2FucI13NeuAcGg4) (Nilsson et al., 1984, 1986; Fredman et al., 1986). Mouse lymphoma L5178 and its variant cell lines showing different metastatic potential contain GMlb and its new analog GDI, (Murayama et d . ,1986). Rat ascites hepatoina also

GLYCOSYLATION IN TUMORS AND TUMOR ANTIGENS

28 1

contains GD,, (Taki et al., 1986).The possible presence of GDI, in human cancer remains to be studied.

E. GLOBO-SERIES ANTIGENS 1. Normal Synthetic Pathway Globo-series antigens are unique in having a1 + 4Gal structure at the internal core (Hakomori et al., 1971a), resulting in an unusual conformational structure distinct from that of other series (see Fig. 6) (Scarsdale et al., 1986). Lactosylceramide is converted to globotriaosylceramide (Gb3), which has a unique Gala1 -+ 4Gal structure that is subsequently converted to globotetraosylceramide (Gb4; globoside) (for a review, see Beyer et al., 1981). Globoside is the major neutral glycolipid in a large variety of human cells and tissues, including erythrocytes, kidney, liver, spleen, lung, and gastrointestinal tissue. It is virtually absent in adult nervous tissue. An a1 + 3GalNAc substitution to pGalNAc of globoside results in the well-known Forssman antigen (Siddiqui and Hakomori, 197!), a major globo-series antigen present in erythrocytes and tissues of Forssman-positive species such as goat, sheep, and horse. This antigen is thought to be absent in Forssman-negative species such as human, monkey, rabbit, and pig (Buchbinder, 1935); however, close examination of human tissues could reveal a small quantity of the antigen. Gastrointestinal and lung tissues in the majority of the human population (80%)do not contain Forssman antigen, whereas such tissues in the remaining 20% of the population do contain the antigen (Hakomori et al., 1 9 7 7 ~ )Con. versely, anti-Forssman antibody is found in the 80% majority, whereas antibody level in the 20% minority is undetectably low. Thus, Forssman antigen expression in human tissue is allogeneic (Hakomori et al., 1977c; Young et al., 197913). For many years, no fiirther globoside substitutions were found in human tissues. More recently, an extended substitution series of globosides has been found in teratocarcinomas, i.e., substitution at the terminal GalNAc ofgloboside with Gal61 + 3, F u c a l + 2 G a l p l 4 3, and NeuAca2 + 3Galp1- 3, which were defined, respectively, b y MAbs SSEA-3, MBr-1, and SSEA-4 (Kannagi et al., 198311,~).The distribution pattern of these extended globo structures in normal adult tissue is unknown. In human erythrocytes, globo-H was found a s an extremely minor component (Kannagi et al., 1984a), despite the fact that globoside is the major component, and human erythrocytes did not react with MBr-I. Perhaps a similar pattern could be found in

SEA-3

antigen

GalNAm1-.3Calp 1-+3~alNAc@ 1-*3Gala1+4Gdlpl+4Gl@ l+Cer (or G a b ) 2

t FUQ.21

G l o b 0 A/or B Frc. 6. Globo-series pathways. Stnichires shown in bold print are accumulated in ceitain tumors. For each example, see the text.

GLYCOSYLATION IN TUMORS AND TUMOR ANTIGENS

283

various normal tissues, except for kidney, in which extended globosries antigens must be present in significant quantity (Blomberg et al., 1982). Interestingly, however, antibody MBr-1, which defines globoH, failed to stain essentially all normal human tissues except breast ductal epithelial cells (Mknard et al., 1983). 2. Oncofetal Pathway A great accumulation of globotriaosylceramide (Gb3) has been observed in various types of Burkitt’s lymphoma (BL). A common BL antigen termed BLA, defined by MAb 13.8 (Wiels et al., 1981), has been identified as Gb3 (Nudelman et al., 1983). Accumulation of Gb3 in BL was due to enhanced activity of a1 .+4Gal transferase, rather than inhibited degradation of Gb3 (Wiels et al., 1984). A breast cancer-associated antigen defined by MAb MBrl was established by Colnaghi and associates (Menard et al., 1983). This antibody was identified as being directed to fucosylgalactosyl globoside, i.e., globo-H antigen (Bremer et al., 1984).The same antigen was previously identified from human teratocarcinoma (Kannagi et al., 1983b). Recently, two human MAbs, J309 and D579, were established by EBV transformation, followed by fusion with the NS-1 mouse myeloma cell line, of regional lymph node lymphocytes from patients with lung cancer. Both antibodies define galactosylgloboside and show a clear reactivity with anaplastic cancer cell lines and a few breast cancer cell lines (Schrump et al., 1988). Thus, synthesis of extended globo structure could be enhanced in some human malignancies. Forssman antigen is a typical globo-series antigen, and its expression in human cancer will be discussed in Section VI1,C. VI. Carbohydrate G lycoprotein Antigens

A. BACKGROUND AND CLASSIFICATION Tumor-associated antigens expressed in glycoproteins fall into four categories. One consists of lacto-series antigens whose epitopes are defined by a large number of MAbs, and are expressed in both glycolipids and glycoproteins (Class 1 antigens in Table 1,A). The epitope structures of these antigens have been characterized mostly on the basis of isolated glycolipids rather than glycoproteins, since it is technically easier to isolate a glycolipid as compared to an oligosaccharide-bearing epitope from a glycoprotein. However, the total quantity of tumor antigens associated with glycoproteins must be higher than that associated with glycolipids; antigens shed in the

284

SEN-ITIROH HAKOMORI

blood and detectable by serum assay are predominantly glycoproteins, particularly mucin type (Magnani et al., 1983; Kannagi et al., 1986). The second group of antigens consists exclusively of mucin-type glycoproteins that do not cross-react with glycolipids. These antigens are often associated with lung, colonic, ovarian, and breast carcinoma, and their epitope structure seems to be related to carbohydrate, since the antigenicity is sensitive to sialidase treatment of antigens extracted from tissue. Still, none of these epitopes (listed in Table V) has been structurally elucidated (Table I,A, Class 4). The third group represents the core structure of 0-linked glycans (e.g., T, Tn, and sialyl Tn antigens; Table I,A, Class 3), most of which are mucin-type glycoproteins. Finally, a new type of epitope was recently found in fibronectins, whose epitope is primarily formed by the peptide chain but whose antigenicity is maintained by 0-glycosylation at the threonine residue of the peptide chain (Table I,A, Class 5). Each of these categories will be discussed in the following sections. The major type of aberrant glycosylation occurring in glycoproteins is the predominance of multiantennary structures due to aberrant and enhanced GlcNAc addition to the core structure of N-linked glycopeptides (Takasaki et al., 1980; Yamashita et al., 1984; Mochizuki et al., 1985). These major changes in N-linked carbohydrates seem to be undetectable with MAbs; changes detectable by MAbs as described below are located at the peripheral region of N-linked or 0-linked side chains.

B. CHEMICALLY DEFINEDLACTO-SERIES ANTIGENSCARRIED BY MUCIN-TYPE AND OTHERGLYCOPROTEINS Sialyl Lea antigen, defined by MAb N-19-9, was present in sera of patients with gastrointestinal cancer, and was initially found to be a high-molecular-weight glycoprotein classified as mucin type (Magnani et al., 1983). Sialyl difucosyl Le" (sialyl Le"-i), defined by MAb FH-6, present in sera of patients with cancer, was also found to be associated with mucin-type glycoprotein. 'This antigen is soluble in perchloric acid and eluted in the void volume through a column of Sepharose 4B. A comparatively low concentration of the same epitope was found in sera of normal subjects. It was, however, insoluble in perchloric acid, and showed a low-molecular-weight component. Interestingly, the same cancer-associated carbohydrate epitope is carried by different glycoproteins, depending on the presence or absence of tumor in the host (Kannagi et al., 1984a).LeXor Leyantigen

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285

was found in CEA (carcinoembryonic antigen) purified from seven different cases of colonic cancer. CEA of some cases carried the Ley epitope defined by MAb AH-6; some cases carried dimeric Le' defined by MAb FH-4 (Nichols et al., 1985). The chemical structure of Le'lLeY-bearing carbohydrate chains at the peripheral region of N-linked chains in CEA has been elucidated (Yamashita et al., 1987). The quantity of sialyl Lea, defined by MAb N-19-9, was very high in seminal plasma (Hanisch e t al., 1984), which was isolated and characterized as a short O-linked chain bearing sialyl Lea (Hanisch et al., 1985). The human squamous cell lung cancer antigen defined by MAb 43-9F was identified as a high-molecular-weight mucin glycoprotein (Pettijohn et al., 1987), and the epitope was identified as Lea, possibly carried on type 2 chain Le" (Mirtensson et al., 1988). Nevertheless, the exact nature of these lacto-series carbohydrate chains in most mucin-type glycoproteins has not been precisely characterized. Interestingly, however, the degree of expression of the antigen defined by MAb 43-9F was correlated with malignancy of 1987). squamous cell lung carcinoma (Due et d., C. T, Tn, AND SIALYLTn ANTIGENS The T (Thomsen-Friedenreich) antigen involved in the panagglutination phenomenon has been demonstrated to be associated with the exposure of the nonsialylated a-linked disaccharide (GalPl- 3GalNAcal+ O-Ser/Thr), which is highly reactive with peanut lectin (for a review, see Prokop and Uhlenbruck, 1969). The selective exposure of T antigen in various cancer cell types was first suggested by Vaith and Uhlenbruck (1978) and Springer et al. (1979). The occurrence of Tn antigen in some human cancers has been detected by the reactivity of Helix pomatia and other GalNAc lectins (Springer e t al., 1974). The A-like antigenicity of a-GalNAc residue linked to serine or threonine is detectable by various GalNAc lectins that preferentially agglutinate A erythrocytes. The antigen is cryptic in nornial glycoproteins, but becomes exposed after desialylation followed by Smith degradation (Dahr et al., 1974). However, neither peanut lectin nor the GalNAc lectins were necessarily specific exclusively to T and Tn structure. Clear evidence for the presence of Tn (and, more recently, sialyl Tn) in human cancer has been provided by the establishment of MAbs specific to each structure. The presence of T antigen in human cancer still remains ambiguous. Hirohashi et al. (1985)established MAbs NCC-Lu-35 and NCC-Lu81 after immunization with human squamous lung carcinoma cell line

286

SEN-ITIROH HAKOMORI

Lu65. The antibodies cross-react with blood group A antigen and react specifically with various types of cancer cells, particularly gastrointestinal and lung, in blood group B and 0 hosts. Distribution of the antigens in non-A’normal tissue is extremely limited. Crossreactivity of NCC-Lu-81 with blood group A antigens is minimal; therefore the antibody is able to detect tumor-associated antigen expressed even in type A tumors. The antigen was identified as a-GalNAc linked directly to Ser or Thr; i.e., Tn antigen. An IgG, antibody directed to Tn antigen and not cross-reactive with the blood group A antigen, Cu-1, was recently established (Takahashi et al., 1988). Over 70% of lung cancer tissue but no normal lung tissue and over 90% of stomach cancer but no normal stomach mucosae react with this antibody. The antibody showed a cytotoxic effect on tumor cells in the presence of mononuclear effector cells. Kurosaka et al. (1987) established a MAb (MSL102) that showed specific immunostaining of various types of human cancer and also showed binding activity with a mucin-type glycoprotein. The epitope was identified as sialyl Tn, i.e., NeuAca2 + 6GalNAcal+ R, by specific inhibition of antibody binding by synthetic disaccharide NeuAca2 + 6GalNAca-O-Ser (Kurosaka et al., 1988). Two MAbs, TKH-1 and -2, directed to the sialosyl Tn structure and displaying a remarkable immunohistological tumor specificity, were generated by immunization of mice with ovine submaxillary mucin (OSM) (Kjeldsen et al., 1988). This procedure was based on the previous finding that OSM contains a large number of sialosyl Tn structures (Tettamanti and Pigman, 1968; Pigman and Gottschalk, 1966). Binding of both TKH-1 and TKH-2 to OSM was specifically inhibited by NeuAca2 -+ 6GalNAcal+ O-Ser. The antigen defined by TKH-1 and TKH-2 was highly expressed in gastric, colonic, and pancreatic cancer, but less in lung or liver cancer. This pattern is similar to that observed for previously established antibodies NCC-Lu-81 (Hirohashi et al., 1985) and B72.3 (Thor et al., 1986; Johnson et al., 1986), which were identified as anti-Tn and anti-sialyl Tn antibodies, respectively. In contrast to Tn and sialyl Tn antigens, clearly identified as described above, the nature of the “tumor-associated T antigen” is still ambiguous. Longenecker et al. (1987) prepared MAbs to synthetic GalPl- 3GalNAc disaccharides with a- or p-anomeric configuration. Only the antibodies directed to p-disaccharide, but not to a-disaccharide, exhibited cancer-associated activity. In our studies, MAbs directed against Gal-A, i.e., the GalPl+ 3GalNAcal- 3Gal sequence (Clausen et al., 1987), did not react with any tumor tissue. In

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287

contrast, MAb against G a l P l + 3GalNAcP1- R showed a strong staining of many human tumor tissues (grntoft et al., 1989). Thus, tumor-associated T antigen may be an unknown structure, including the Gal01 3GalNAcPl- R sequence.

-

D. GLYCOPROTEIN ANTIGENS WHOSE EPITOPE STRUCTURE Is ILL-DEFINED

A number of MAbs prepared against various types of human cancer are directed to mucin-type high-molecular-weight glycoproteins whose epitope structure is ill-defined (See Table V). The IgGZ., MAb L-6 defines the glycoprotein antigen widely expressed in lung, colon, breast, and ovarian cancer, and showed a strong tumoricidal activity and inhibited tumor growth in athymic mice (Hellstrom et d., 1986a,b). The antibody showed a weak cross-reactivity with certain glycolipids, but the major epitope, as yet undefined, was found in glycoprotein (H. Kojima, E. Nudelman, S. Hakomori, I. Hellstrom, and K.-E. Hellstrom, unpublished observation). The antibody DUPAN-2, raised against a human pancreatic cell line (Metzgar et al., 1982), is directed to a mucin-type glycoprotein with M, > lo6 kDa. The DUPAN-2 antigen has been reported to be expressed in pancreatic, stomach, gall bladder, and bile duct carcinomas (Metzgar et al., 1984). Reactivity of the antibody was sensitive to both sialidase and protease, and its epitope structure was assumed to consist of both O-linked carbohydrate and peptide (Lan et al., 1987). The antibody OC 125, established after immunization with an epithelial cell line (OVCA433) isolated from a patient with serous papillary cystadenocarcinoma (Bast et al., 1981), reacted with ovarian carcinoma cell lines and cryopreserved ovarian cancer tissue (12 of 20 cases). The antigen is also susceptible to sialidase, and was claimed to be a mucin-type glycoprotein (Klug et al., 1984).Another MAb, OM-1, directed to ovarian cancer cells, defines the high-molecular-weight glycoprotein ( M , 360 kDa) antigen SGA, which is highly expressed at the surface of ovarian cancer cells and sebaceous gland epithelia (DeKretser et al., 1985). A number of MAbs defining breast cancer-associated antigens have been prepared by two methods, one utilizing breast cancer cell lines or membrane fraction prepared from metastatic breast cancer cells, the other utilizing human milk fat globule membrane as immunogen. Interestingly, antigens defined by these approaches have been identified as high-molecular-weight mucin-type glycoproteins; a part of the epitopes defined by the various antibodies could be associated

TABLE V MONOCLONAL ANrIaoDrcs DIHECTEU TO TUMOR-ASSCXIATEU G1.YCOPROTEIN CAHBOHYUIMTE CIIAINS" ~~

Antigen

Antibody

Immunogen

Tn

NCC-LU-35, NCC-LU-81

Squamous lung carcinoma LU65

Sialyl Tn

B72.3

Breast cancer mernhranc

Lung cancer pleural eihrsion

MLS 102 TKH-I, TKH-2

Colonic Ciinwr Ovinc submarillary mucin

L-6

Lung

adenocarcinomit

cells Pancreatic cancer

DUPAN-2

Pancreatic cancer

Ovarian cancer

OC125

Ovarian epithelial cell OVCA433 Ovarian cancer

OM-1

Specificity

GalNAcal + 0 + Ser/Thr (Tn antigen)

Lung, gastric colonic cancer; cross-react with A antigen Colonic and breast cancer

1

Lung? hrt:;ist,

-

SdThr

Unidentified glynoplntt:in

and

HMFG-142

115D8

D73

Human milk fat glohule membrane Human milk lilt plohiile meinbnne Bredst cilncer rnem1,rarie

~~

Knrosaka et (11. (1987); Kjeldsen et uZ. (1988)

Hcllstrorn et a!. (1986a); IIellshijrrl ut

d.(1986b)

nvarian curcinoma Pancreatic/gastric cancer Ovarian canwr

Sialidase sensitive, nnidentified Sialidase sensitive, nnidentified

Metzgar et al. (1982); L m et al. (1987) Bast et al. (1981); Hug et d.(1984); DeKretser et al.

Breast cancer

Mncin-type glycoprotein

Bnrchell et

Hrrast, colorwtd, Inng canc!cr,

MAM6 rnncin glyctipnitcin

Hilkcns et al. (1986)

-

Ovarian cancel

cell Breast cancer

Hirohashi e t al. (1985)

Johnson et al. (1986)

NeuAca2

6 C.alNAi:oI -+ 0

colon,

Reference

Structure

(1986) (11.

(1983)

Krifr ct 01. (19M)

Only muliptiant breast cancer (not benign adenoma) was positive ~~

~~~~~~

~~

" Crowreacts wcakly with some glycolipids, but major epitopc is prcscnt i n glycoprntcin (H. Kojinia, E.Nridellriari, S. Ilakonwri, I. IIellstrijiii, and K.-E. Hellstrom, unpublished observations).

GLYCOSYLATION IN TUMORS AND TUMOR ANTIGENS

289

with oligosaccharides and the other part of the epitopes could be in the protein moiety of mucin glycoproteins, although none of the epitopes has been clearly identified chemically. MAb F36122 was generated after immunization with breast tumor cells, and showed strong reactivity with adenocarcinoma of the breast and ovary. The antigen seems to be associated with a ductular lineage of breast adenocarcinoma, but occurs on a limited number of normal ductal structures (Papsidero et al., 1983). The antigen was purified from malignant effusions and was found to be a high-molecular-weight glycoprotein ( M , > 300 kDa) highly reactive with wheat germ lectin. Its antigenicity was resistant to heat and acid treatment and was insensitive to sialidase, but was highly susceptible to base treatment (Papsidero et al., 1984). The antibody B72.3, established after immunization with metastatic breast cancer cell membrane, was reactive no: only with breast cancer but also a number of other gastrointestinal tumors, particularly colonic cancer; however, it showed no significant reactivity with various norma1 tissues (Colcher et al., 1981; Nuti et at., 1982; Stramignoni et al., 1983). The ar+;gen was identified as a high-molecular-weight glycoprotein with MI 220-400 kDa ( Johnson et al., 1986), sensitive to sialidase. The epitope structure was clearly identified as sialyl 2 + 6 N-acetylgalactosaminyl (Y + O-Ser or -Thr, i.e., sialyl Tn (Kjeldsen et al., 1988). Another antibody, DF-3, was raised against the membrane-enriched fraction of a human breast carcinoma cell line, and the antigen glycoprotein (MI 290 kDa) was detectable at the cell surface of human breast carcinoma. Interestingly, the antibody clearly distinguishes malignant and benign breast lesions. Cytoplasmic staining has been observed with 40 of 51 (78%) breast carcinomas, but only 1 of 13. fibroadenoma or fibrocystic disease specimens, i.e., the expression of this glycoprotein antigen is closely related to malignancy (Kufe et al., 1984). The antibody NCRC11, raised by immunizing dissociated human mammary carcinoma cells, showed a remarkable association with breast cancer cells (positive staining of 119 of 126 tumors tested). Expression of this antigen was claimed to be of prognostic valpe. The patients whose tumors exhibited intense staining had an improved survival compared to those with less intensely stained tumors (Ellis et al., 1985). Based on biochemical similarity between actively proliferating breast epithelial cells in breast carcinoma and human milk fat globule membrane, a number of studies have been focused on the immunization of mice with human fat globule membrane followed by establishment of MAbs. Some of these antibodies showed a remarkable

290

SEN-ITIROH HAKOMORI

association with human breast cancer cells. Antibodies HMFG-1 and -2 react with breast, ovarian, and other carcinomas, but not with tumors from mesenchymal tissues (Burchell et al., 1983), and are directed to a large mucin-type molecule present in human milk and lactating human mammary epithelial cells (Shimizu and Yamauchi, 1982). HMFG-2 recognized an extremely heterogeneous group of glycopi-oteins of M , 80-300 kDa, in contrast to HMFG-1, which recognized only high-molecular-weight ( M , 300 kDa) glycoproteins. Metastatic and malignant potential is closely related to expression of HMFG-2 (Burchell et al., 1983). Similarly, antibody 115D8, raised against milk globule membranes, defined an epithelial membrane marker glycoprotein, MAM-6. This antigen was also defined by other MAbs, and is associated not only with breast carcinoma but also a large variety of human malignancies such as colorectal, lung, and prostate carcinoma, melanoma, and lymphoma (Hilkens et al., 1986). A similar approach was used by the research groups of Ceriani et al. (1983) and Foster et al. (1982). The antibodies established were directed to human mammary epithelial cell membranes, and were highly reactive with an antigen closely associated with breast carcinoma. The antibody BLMRL series established by Ceriani et al. (1983) defines glycoproteins with M , 46, 70, and 400 kDa. MAbs were raised against human epidermoid carcinoma A431 cells. One of the antibodies (AR-3) defines a highly glycosylated glycoprotein antigen (CAR-3). This antigen was highly expressed in gastric adenocarcinoma, pancreatic adenocarcinoma, cervical squamous cell carcinoma, uterine endometrial carcinoma, mucinous cystadenoma, and cystadenocarcinoma. It was weakly expressed in normal epithelium from stomach and breast, but is highly expressed in normal testis. Its epitope structure appears to be carbohydrate but has not been defined (Prat et al., 1985). All these tumor-associated mucin-type glycoproteins show a tendency to be released into serum, and some of them have been claimed to have diagnostic value (see Section X). Because of the extreme complexity and heterogeneity of mucin-type glycoproteins, none of the epitope structures, except Tn and sialyl Tn, has been clearly identified. Recently, however, genes encoding core proteins of mucin-type breast cancer antigen were cloned from a cDNA library isolated from breast cancer cells, and expressed in hgtll. These core proteins without glycosylation show clear reactivities with some of the MAbs described above, e.g., DF3 (Abe et al., 1988; Siddiqui et al., 1988) and SM3 (an antibody to HMFG) (Taylor-Papadimitriou et al., 1988), and with antibodies to MAM-6 antigen (Hilkens et al., 1988).

GLYCOSYLATION I N TUMORS AND TUMOR ANTIGENS

29 1

Undoubtedly, many MAbs defining tumor-associated mucin are directed to the protein core, yet some of the antigenic activities could be fully maintained on glycosylation. A typical example of a polypeptide epitope influenced by glycosylation is described in the following section.

E. TUMOR-ASSOCIATED PEPTIDEANTIGENSWHOSEEPITOPE BY GLYCOSYLATION STRUCTURE Is INFLUENCED Fibronectins consist of multiple isotypes showing different molecular weights and degrees of glycosylation. Plasma fibronectin (pFn) showed lower molecular weight than those released from fibroblasts to their culture media, and those present in pericellular matrix of cultured fibroblasts, which are called cellular fibronectin (cFn). Fibronectin secreted from transformed cells (tFn) showed a molecular weight similar to that of cFn, but showed a different glycosylation pattern (Murayama et al., 1984; Nichols et al., 1986). Recently, MAb FDC6, reacting specifically with cFn and tFn but not pFn, was established. This antibody did not react with fibronectins extracted from normal adult tissues, but did react with those isolated from fetal tissue, placenta, and cancer tissues. Thus, a structure or structures defined by MAb FDCG reflects oncofetal status of fibronectin, and the fibronectins reacting with this antibody were termed oncofetal fibronectins (onfFn), while those not reacting with it were termed normal fibronectins (norFn). The structure carrying the FDCG epitope has been found in the domain between “Hep2” and “Feb2,” and is within the type 111 connecting segment (IIIcs). This region was isolated and further fragmented by proteolysis, and an active fragment was isolated. The minimal structure that reacts with FDCG antibody is valyl-threonyl-histidyl-prolyl-glycyl-tyrosine(VTHPGY) 0glycosylated at threonine (T). The peptide VTHPGY, or any peptide containing this sequence, did not react with FDC-6 antibody. The structure of sugars involved in this O-glycosylation is a regular Gal01 + 3GalNAca linkage, carrying one or two sialic acid residues, i.e., ordinary short O-linked structures (Matsuura et al., 1988). Results of these studies clearly indicate that O-glycosylation is important in defining the conformational structure of a polypeptide that creates a tumor-associated antigen. It has been found that blood group M and N determinants are carried by the N-terminal region of glycophorin A, although conformation of the epitope is properly exposed by glycosylation, specifically at the terminal sialic acid. The epitope structure for onfFn defined by FDCG antibody is the first example indicating a

292

SEN-ITIROH HAKOMORI TABLE VI CHANGES OF HISTO-BLOOD GROUPANTIGENSIN HUMAN CANCER

1. Deletion of A and B determinants (Masamune et al., 1952, 1953, 1960) and associated accumulation and disorganization of precursor (H and N-acetyllactosamine) (Dabelsteen et al., 1983) 2. Expression of incompatible A antigen in 0 or B tumors: a. Identification as Tn antigen (Hirohashi et al., 1985) b. Real A antigen expression (ALeb,ALed) (Clausen et al., 1986) c. Other stnichires (Forssman and fucose-less A) are of minor importance 3. Expression of incompatible PPIPkantigen in small p tumor (Levine et al., 1951; Kannagi et al., 198213; Hattori et al., 1987) 4. Change of carrier isotype in A tumor (Dabelsteen et al., 1988). Type 2 and type 3 chain A antigens are absent in normal adult colonic mucosa, but are expressed in tumors

possibility that expression of polypeptide tumor antigen is regulated by glycosylation. Such examples must be present in a large number of so-called tumor-associated polypeptide antigens whose epitope structure is as yet unknown. VII. Alteration of Histo-Blood Group and Heterophile Antigens Expressed in Human Cancer

In the preceding sections, many examples of modification of carrier structures for histo-blood group2 determinants in human cancer, particularly lacto-series carrier types, have been discussed. On the other hand, histo-blood group determinants per se are greatly altered in many human cancers, as will be discussed in this section (see also Table VI). Because this field of study has been reviewed (Hakomori, 198417; Kuhns and Primus, 1985; Feizi, 1985), only a brief overview is presented here. A. HISTO-BLOOD GROUPABH ANTIGENS Reduction of A or B determinants associated with human cancer, first discovered by Oh-Uti (1949) on a chemical basis, was confirmed by immunohistological analysis of various tumors using the red blood cell adherence test (Davidsohn et at., 1966; 1969), mixed hemagglutinin test (Kay and Wallace, 1961), and immunofluorescence test (Prendergast et al., 1968; Dabelsteen and Fulling, 1971), which The term “histo-blood group” is used to emphasize the predominance of ABII (Lewis) antigens in epithelial tissue and P antigens in mesenchymal tissue. They are minor components in blood (Clarrsen and Hakomori, 1989).

GLYCOSYLATION IN TUMORS A N D TUMOR ANTIGENS

293

indicated that deletion of blood group A and B antigens occurs in a large variety of tumors, as well as in preneoplastic lesions (see Section VILA). The pathobiological significance of the deletion or reduction of A and B determinants, particularly a possible correlation with malignant potential of tumors, has been debated (Grntoft e t al., 1987; 1988; see also Feizi, 1985). Incompatible A antigen expressed in tumors from 0 or B individuals has received a great deal of attention. The chemical basis of this phenomenon was initially studied with glycolipid isolated from type 0 tumor, which inhibited lectin-induced A hemagglutination. In addition, rabbit antisera against the glycolipid showed a preferential reactivity with A erythrocytes (Hakomori et al., 1967). Using anti-A immune serum (not naturally occurring anti-A isohemagglutinin), Hakkinen (1970) detected A antigen by immunofluorescence in gastric cancer of B and 0 individuals. More recently, a glycolipid fraction with blood group A activity was demonstrated in a few cases of gastric cancer (Hattori e t al., 1981) and a case of primary hepatoma (Yokota et al., 1981) from blood group 0 individuals. An A-like glycolipid with obscure reactivity was isolated from tumor of host with blood group B, and was identified as ceramide heptasaccharide with difucosylated A structure (Breimer, 1980). Forssman antigen expressed in tumors derived from Forssman-negative tissue has been a well-accepted candidate for A-like antigen, which will be discussed in Section VI1,C. With development of various monoclonal anti-A antibodies defining type 1 chain A, type 3 chain A, ALeb, and ALeY(Abe et al., 1984; Clausen et at., 1985a; 1985b), the properties of A antigen expressed in type 0 colonic cancer have been thoroughly reinvestigated. The presence of real type 1 chain A antigen, either ALed (defined by MAb AH21) or ALeb (defined by MAb HH3), has been detected in approximately 10-15% of primary colonic cancer cases from histo-blood group 0 patients. The presence of glycolipid antigen was demonstrated by TLC immunostaining, and A transferase activity was detected in typical A-expressing 0 tumors (Clausen et al., 1986). Thus, real A antigen, rather than A-like antigen, is indeed expressed in 0 or B tumors, although expression of such incompatible A antigens is observed in less than 15% of cases. Interestingly, the incidence of various human adenocarcinomas, e.g., gastric, colonic, ovarian, and parotid cancer, is higher in blood group A than in the blood group 0 or B populations (Mourant et al., 1978). Although the immunobiological basis for this epidemiological finding is difficult to explain, it is possible that primary or in situ tumors of blood group 0 and B individuals, if expressing real A antigen, were likely to be recognized

294

SEN-ITIROH HAKOMORI

as foreign, and were immunologically rejected. In hct, the incompatible A antigens studied by Clausen et aZ. (1986) were all from primary colonic cancers. Incidence of incompatible A antigen expression in ovarian cancer is also high, reflecting the high incidence of this disease in A individuals (R. Metoki, Y. Tsuji, and S. Hakomori, unpublished observations). Antibodies directed to Tn antigen showed a cross-reactivity with A antigen, and two anti-Tn MAbs, NCC-Lu-38 and -81, showed anti-A properties (Hirohashi et aZ., 1985). Since the incidence of Tn antigen expression is quite high (70-90%) in some tumors, it is possible that some anti-A antibodies may pick up Tn antigen. The high incidence of A expression as observed by Yuan et al. (1986) and Itzkowitz et al. (1986b; 1987) may well reflect a cross-reactivity with Tn antigen. Incompatible A or A-like antigens expressed in B or 0 tumors are therefore manifold; their detectability depends on the specificity of antibodies applied, since each antibody could recognize a variety of antigens which have aGalNAc as an epitope. Two important antigens are real A antigen and Tn antigen, both of which could have important clinical applicability in diagnosis and treatment of human cancer. Forssman antigen may also be able to contribute A cross-reacting antigen in human cancer. The quantity of Forssman expressed in human cancer is too small to be clinically significant (see Section VI1,C). Although A antigen is deleted or reduced in gastric, esophageal, oral, and bladder cancers, its expression remains in colonic cancer. With application of various anti-A MAbs that distinguish carrier type structures, it has become apparent that A antigens, carried by type 1, 2, 3, and 4 chains, are all detected in colonic adenocarcinoma and in normal fetal mucosae, in striking contrast to the absence of type 2 and type 3 chain A structures in normal adult mucosa. Type 1 chain A and ALeb were expressed in part of the normal colonic proximal mucosa (Dabelsteen et al., 1988). Thus, there are several possible mechanisms for aberrant expression of A or A-like antigen: (1)deletion or reduction of A determinant, (2) incompatible expression of A determinant in B or 0 tumor, and (3)anomalous combination of carrier type and A determinant.

B. HISTO-BLOOD GROUPP ANTIGENS An unusual case of gastric cancer involving the occurrence of incompatible histo-blood group P antigen was reported in 1951

GLYCOSYLATION IN TUMORS AND TUMOR ANTIGENS

295

(Levine et al., 1951).A 66 year old female patient with gastric cancer and apparent blood group 0 was treated surgically. Blood examination before surgery revealed that her serum contained antibodies which agglutinated all random erythrocytes except her own. These antibodies were later identified as anti-PPIPk (also called anti-Tja). This was the first reported case of rare blood group p (genotype p p ) , which has an estimated frequency of 1: 100,000. In the patient’s family, however, the incidence was 1: 4, her parents were double first cousins, and her younger sister was found to also have genotype p p . The sister’s serum also contained anti-PPIPk. Before surgery, the patient received a transfusion (approximately 25 ml) of incompatible blood. This produced a severe hemolytic reaction, with fever and an increase in anti-PPIPk titer from 1: 8 to 1:512. Shortly after the transfusion reaction subsided, subtotal gastrectomy was performed; however, the tumor and metastatic region were not completely removed. The patient recovered, survived for another 22 years, and died of old age in 1973. There was no evidence of recurrence or metastasis of the tumor (for a review, see Levine, 1978). The tumor tissue obtained from the 1951 surgery was lyophilized and the powder was kept at low temperatures. Chemical analysis performed in the laboratory of the present reviewer in 1980 clearly indicated the presence of a glycolipid cross-reacting with P (globoside), i.e., GalNAcpl+ 3GalP1+ 4GlcNAcP1+ 3Galpl+ 4Glcpl+ 1Cer. P1 activity was detected in the glycolipid, as well as in the glycoprotein, fraction (Kannagi et al., 1982b). This was the first clear documentation of the presence of PPIPk antigen in cancer from a type p individual. It is suspected that the transfusion of mismatched blood induced a high level of anti-PPIPk, which actively suppressed growth of tumor remaining after the surgery. A second case of incompatible P antigen expressed in gastric cancer of a p p genotype individual was reported recently by Hattori et al. (1987). The female patient’s blood group was p, 0, Le(a-b+); her serum was reactive with all blood samples except her own, and was shown to contain anti-PPIPk antibody, as in the patient from the 1951 case. The p blood group was confirmed serologically with anti-Pl and anti-Pk. TLC immunostaining of the glycolipid fraction of the patient’s tumor tissue revealed the presence of Gb3 (Pk antigen), Gb4 (P antigen), as well as Forssman and incompatible A antigens. In contrast to the earlier case, this patient did not receive transfusions of mismatched blood. Her subsequent clinical history has not been available.

296

SEN-ITIROH HAKOMORI

C. FORSSMAN ANTIGENS Forssman antigen shows an allogeneic expression in man and is absent in normal lung and gastrointestinal mucosa in the majority of the human population, but is detectable in gastric and colonic cancer (Hakomori et al., 1977c) and lung cancer (Yoda et al., 1980). In the majority of lung cancers, whether squamous cell carcinoma or adenocarcinoma, activity of a1 + 3GalNAc transferase, which is responsible for synthesis of Forssman antigen, is greatly enhanced, whereas the same enzyme is undetectable in normal lung tissue (Taniguchi et al., 1981). Although these and other studies (Kawanami, 1972; Mori et al., 1983)indicate a close association of Forssman antigen expression with human malignancy (for a review, see Milgrom et al., 1973), antiForssman antibody, either monoclonal or polyclonal, does not stain tumor tissue or normal tissue (S.-M. Wang, K. Abe, and S. Hakomori, unpublished observations). Furthermore, no MAbs directed to Forssman antigen were found among various hybridomas established after immunization with human cancer. Therefore, the potential clinical usefulness of Forssman antigen as a human cancer marker is questionable. ANTIGENS D. HANGANUTZIU-DEICHER Heterophile Hanganutziu-Deicher (HD) antibodies were originally detected in sera of patients who had received therapeutic injection o f foreign immune serum, and were found to agglutinate erythrocytes of sheep, ox, horse, rabbit, and other animal species, but not human erythrocytes (Hanganutziu, 1924; Deicher, 1926). Later, patients with various diseases (including cancer) who had not received injection of foreign serum were also found to possess HD-type heterophile antibodies (Kasukawa et al., 1975). The HD antigen was identified as a ganglioside containing N-glycolylneuraminic acid (NeuGc) (Higashi et al., 1977; Merrick et al., 1978). Since sera from cancer patients occasionally contained H D antigen, an association of this type of antigen with human cancer was suspected. A series of extensive studies by Higashi, Naiki, and associates (Higashi et al., 1984; 1985; Hirabayashi et al., 1987) clearly indicated the presence of GM3, sialyl paragloboside, and other gangliosides containing NeuGc in colonic cancer, melanoma, retinoblastoma, ovarian cancer, and seminoma. Recent studies have detected the presence of GM.3

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containing 4-O-acetyl-N-glycolyl neuraminic acid in human colonic cancer (Miyoshi et al., 1986). Since the quantity of NeuGc ganglioside present in cancer is extremely low, the antibody recognition may be difficult. Since no data on immunoreactivity or antibody-dependent cytotoxicity with anti-HD antibodies are available, it is difficult to predict the clinical significance of H D antigens in human cancer. VIII. Aberrant Glycosylation in Preneoplastic Tissues

Preneoplastic cells express the same type of aberrant glycosylation as found in established tumor cells in both experimental and human cancer. Cells in preneoplastic nodules in rat liver contain fucosyl GM1 ( IV2FucI13NeuAcGg4), fucosyl asialo GMI ( IV2FucGg4), and a-galactosylfiicosyl GM (ganglio-B; IV2FucIV3aGalI13NeuAcGg4).These glycolipids are completely absent in normal rat liver but are highly expressed in rat hepatoma, although established hepatoma cell lines express the former two glycolipids (Holmes and Hakomori, 1982). The enzymatic basis for the induction of fucoganglioside synthesis in preneoplastic rat liver and hepatoma has been studied. An a1 -+ 2 fucosyltransferase specific to GM1 was induced after rats were fed 2-N-fluoroacetamide for 3 weeks (Holmes and Hakomori, 1983). Deletion or reduction of A antigen associated with accumulation of H and its precursor antigen were observed in preneoplastic dysplasia lesions of oral epithelia (Dabelsteen and Fulling, 1971; Dabelsteen et al., 1983). Ley expression in colonic polyps was found to be closely correlated with preneoplastic state of polyps. Juvenile polyps without dysplasia, having no malignant potential, did not express Ley, whereas tubulovillous and villous adenomas with severe dysplasia, which have high malignant potential, express Ley antigen (Abe et al., 1986). A similar study with antibodies directed to extended Ley (CC1 and CC2) and trifucosyl Ley (KHl) showed an even clearer correlation between the malignant potential of colonic polyps as determined by histological type, degree of dysplasia, and Ley expression rather than by AH6 antibody (Kim et al., 1986). In the majority of liver cirrhosis tissue, the FH6-defined antigen was highly expressed in a characteristic honeycomb-like pattern. The pattern was particularly remarkable in hepatitis-induced cirrhosis, which has a high potential to induce hepatoma. The FH6-defined antigen is highly expressed in hepatoma (Okada et nl., 1988).All these studies clearly indicate that premalignant lesions are characterized by the same aberrant glycosylation as found in malignant tissue.

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IX. Requirements for Tumor-Associated Carbohydrate Antigens: Density of Antigens and Organizational Framework in Membranes

Only a few tumor-associated carbohydrate antigens are known to be virtually absent in normal cells and tissues. These are incompatible histo-blood group antigens and Forssman and H D antigens. Because the incidence and degree of expression of these specific antigens in tumors are low, the antigens are clinically of minor importance, except for real A and T n antigen as incompatible A antigen. A second class of antigens has a novel structure highly expressed in tumor cells, absent in the progenitor cells, but found in unrelated normal tissues, i.e., ectopic expression in tumors. They include dimeric or trimeric Lex,trifucosyl Ley, sialyl difucosyl Lex,monosialyl and disialyl Lea, GD3 and GD2 ganglioside, and Tn and sialyl Tn antigens. The third class of antigens can be detected chemically in a wide number in normal tissues, but are found in very high levels in some tumor cells, e.g., GM3 in melanoma, Gb3 in Burkitt's lymphoma, and Lex, Ley, Lea, and Leb in a variety of human cancers. Despite the fact that antigens of the second and third class are expressed in normal cells, some antibodies directed to them are clinically useful in diagnosis and treatment of human cancer (see Sections X and XI). These antigens are not only present in high quantity, but may exist with a novel organization at the tumor cell surface. The importance of the organization of glycolipid antigens at the cell surface has been suggested by the following various data.

1. Some glycolipids are chemically present in considerable quantity but are hardly detectable by immunological methods, whereas others are present in relatively small quantity but are conspicuous by immunological methods. The restricted expression of major glycolipids present in various types of normal cells by their antibodies has been well documented; e.g., Gb4 in adult human erythrocytes (Hakomori, 1969) and hamster fibroblast NIL cells (Gahmberg and Hakomori, 1975), and GM3 in baby hamster kidney fibroblasts (Hakomori et uZ., 1968). These major glycolipids are immunologically conspicuous in fetal erythrocytes and virally transformed cells, although the chemical quantity of Gb4 or GM3 in the fetal or transformed cells is even lower than in adult or normal progenitor cells. Gb3 in ARH77 human lymphoblastoid cells was not expressed at the cell surface, although cells contained a quantity of Gb3 comparable to that in Burkitt's lymphoma, which expressed this antigen conspicuously at the cell surface (Wiels et uZ., 1984).

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2. Some antibodies are able to recognize density of glycolipid antigens at the cell surface and on solid phase; e.g., anti-SSEA-1 antibody does not react with Le" ceramide pentasaccharide on liposome lysis assay at concentrations below 10 pmol, but reacts maximally at concentrations above 12-15 pmol (Hakomori et al., 1981b). A melanoma-specific antibody M2590 reacted with GM, only when present at concentrations higher than 10 mol%; it did not react at concentrations below 8 mol% in liposome lysis assay. When melanoma cells were treated with sialidase, reactivity (as determined by immunofluorescence and by flow cytofluorometry) decreased suddenly to a minimal level, although only 10% of cellular GM3 was hydrolyzed. Further incubation of cells with sialidase, which caused increasing degradation of GMS, did not alter fluorescence intensity with anti-GM, antibody. These findings indicate that some, if not all, antibodies show a clear threshold value for reactivity in all-or-none fashion (Nores et al., 1987) (see Table VII). 3. Some glycolipid antigens are highly expressed in certain experimental tumors and are also present at lower levels in some normal cells. Administration of antibodies with the proper isotype and affinity may inhibit tumor cell growth without affecting the function of normal cells expressing the same antigen. Mouse lymphoma L5178, in which Gg, is highly expressed, was completely suppressed by administration of the anti-Gg3 IgG3 antibody DlOG11, and the animals remained healthy for an extended period of time despite the fact that Gg3 is also expressed in a few normal cells (Young and Hakomori, 1981). Similarly, growth of B16 melanoma inoculated in C57BL mice was inhibited by administration of anti-GM3 IgG3 antibody DH2, and the animals remained healthy, despite the presence of GM3 in normal cells of various tissues (Dohi et al., 1988). Differential cell surface recognition of normal versus tumor cells TABLE VII

FACTORS AFFECTING ANTIGENICITY AND IMMUNOCENICITY OF MEMBRANE GLYCOLIPIDS 1. Concentration and density of glycolipids: Glycolipid antigens organized at a concentration higher than a certain threshold value are recognized by a certain antibody. The same antigen at subthreshold concentration is not recognized (Hakomori et al., 1981b;Nores et al., 1987) 2. Membrane proteins surrounding glycolipids: Cryptic glyco-epitopes are exposed on protease treatment (for examples, see text) 3. Sialosyl glycoconjugutes surrounding glycolipids: Cryptic glyco-epitopes are exposed on sialidase treatment (for examples, see text) 4. Cerarnide composition: See Table VIII

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involves not only antibodies (Nores et al., 1987), but also secondary immune responses triggered by the antibody (Herlyn et al., 1985; Welt et al., 1987), i.e., activated effector cells, macrophages, and antiidiotype as well as anti-antiidiotype antibody responses, which are capable of recognizing the organization and density of glycolipid antigens. These secondary mechanisms may show even greater ability to distinguish organizational framework of antigens at the surface of tumor cells versus normal cells. What are the precise chemical and physical bases for organizational differences in the surface membranes of normal versus malignant cells? Current research has not yet been able to answer this question. However, the following factors are certainly involved. 1 . Glycolipid concentration and density at the cell surface menibrane. Some MAbs, showing a preferential or specific reactivity with certain types of tumor cells over normal cells, are capable of recognizing a high density of a common glycolipid organized at the tumor cell surface membrane. These antibodies react with the glycolipid antigen only at high density, in an all-or-none fashion, i.e., display a threshold reactivity. Thus, Le” glycolipid highly expressed in colonic adenocarcinoma (Hakomori et al., 1981b) and GM3 ganglioside in melanoma (Nores et al., 1987) display “tumor-specific” reactivity. It is suspected that these glycolipid antigens at high density may have conformations different than they do at low density. However, final conclusions should be based on direct NMR studies of cell membranes. 2. Crypticity of glycolipid antigens. According to the minimum energy conformation model, the axis of the carbohydrate is perpendicular to that of ceramide; glycolipids are inserted in the lipid bilayer through their ceramide moiety, and their carbohydrates are laid on and fixed to the surface of the lipid bilayer, exposing the hydrophilic surface toward the outside of the membrane (Kaizu et at., 1986; Hakomori, 1986). Considering such a model, crypticity of glycolipid antigens could be controlled by several complex factors as described below. a. Membrane Proteins Surrounding Glycolipids. Accessibility of antibody to glycolipids at the cell surface must be greatly influenced b y surrounding membrane proteins. The reactivity of Gb4 in adult human erythrocytes (Hakomori, 1969) and that of G M I in mouse lymphocytes (Stein et al., 1978) were enhanced when cells were treated with protease. Enhanced agglutinability of erythrocytes by antisera versus blood group ABH, Lewis, and Ii antigen after treat-

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ment with proteases has been known empirically. Lectin-induced agglutinability of fibroblasts and lymphocytes is also known to be enhanced by protease treatment. b. Siulosyl Glycoconjugutes Surrounding Glycolipids. Reactivities of Gg, in L5178 lymphoma (Urdal and Hakomori, 1983b) and Gb] in human lymphoblastoid cells (Wiels et ul., 1984) to their respective MAbs were greatly enhanced when the cells were treated with sialidase rather than protease, although no sialyl derivatives of Ggo or Gb3 susceptible to sialidase were present in the cells. The labeling efficiency of Ggo in L5178 cells by galactose oxidase/NaB3H4 was also greatly enhanced after sialidase treatment (Urdal and Hakomori, 1983b). In this case, GMIl, was hydrolyzed and converted to asialo GMI (Gg4). It is possible that Gg3 or Gb3 was made cryptic by the presence of adjacent GM,,, (Kannagi et ul., 1983a) or some other sialosyl glycoprotein that masks Gg, at the cell surface (Urdal and Hakomori, 1983b). c. Cerumide Composition. Another factor determining the crypticity of glycolipids is differential ceramide composition (see Table VIII). Reactivity of glycolipid is greatly affected by chain lengths of fatty acids, and the presencelabsence of the a-hydroxyl group at the fatty acid component of ceramide (Cer). Differences in Cer composition will affect the organizational state of glycolipids in cell surface membranes that modify the glycolipid crypticity to their antibodies and ligands. The two acyl chains in Cer are almost of equal length in glycolipids with short-chain fatty acids (C14 : 0 to C18 : 0), whereas two acyl chains in glycolipids with longer fatty acids (C22 : 0, C22 : 1, C24 : 0, C24 : 1) are very unbalanced; one of them is approximately 1.2-1.4 times longer than the other. This causes organizational and TABLE VIII EVIDENCE THAT CEPAMIDE COMPOSITION Is IMPORTANT FOR IMMUNOGENICITY AND ANTIGENICITYOF GLYCOLIPIDS

1. FucCer from adenocarcinoma contains C14fatty acids and Cm sphingenine and is poorly irnmnnogenic in rabbits, while synthetic FucCer containing Ce4 htty acids and CLssphingenine is skongly immunogenic (Yoshino et al., 1982) 2. Gg3 in L5178 lymphoma showed the following order of reactivity with its MAb: a-OH CL6fatty acid > Cm-C% fatty acid > C16fatty acid (Kannagi et al., 1983a) 3. LicCer with long-chain fatty acids (Cz0-C7,) showed greater reactivity with MAI) TSA7 than did LacCer with short-chain fatty acids (C14-Clx)(Symington et d., 1984;1

4. Propionibacterium grunulosum binds to LacCer with a-OH fatty acids, while Propionibacterium freudenreichii binds to LacCer with long-chain fatty acids (Hansson et d., 1983)

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stability differences of the glycolipids in the lipid bilayer. The arrangement of glycolipid antigens having hydroxylated Cer in either a-hydroxy fatty acids or 3-hydroxysphinganine must be much different in glycolipid antigens without hydroxyl function at the Cer. Although the exact mechanism is unknown, antibodies directed to carbohydrate epitopes of glycolipids showed significantly different reactivity depending on the ceramide composition. The longer the fatty acid chain length, the greater the antibody reactivity demonstrated to the same carbohydrate epitope. The reactivity and immunogenicity of liposomes containing FucCer having long-chain fatty acids and a-hydroxy fatty acids were significantly higher than those of liposomes containing short-chain fatty acids (Yoshino et al., 1982). GD3 ganglioside in human melanoma was characterized as having Cer with long-chain fatty acids, in a striking contrast to GD3 of normal brain having Cer with short-chain fatty acids. GD3 immunogenicity and antigenicity in human melanoma could be much higher than that of GD3 in normal tissue (Nudelman et al., 1982). The reactivity of lactosylceramide with C20-24 fatty acids to its MAb A5T7 was much higher than that of lactosylceramide with C16-18 fatty acids in solid-phase antibody-binding assays (Symington et al., 1984). A close correlation between antigenicity of Gg3 in mouse lymphoma L-5178Y and their Cer composition was studied. A greater antigenicity was found to correlate with the amount of a-hydroxy C16 : 0 fatty acid. It is suggested that the glycolipid becomes more exposed when Cer has a-hydroxy fatty acids (Kannagi et al., 1983a). X. Diagnostic Applications A. EARLIER STUDIES

There have been a few observations indicating that sera of patients with cancer show elevated levels of tumor antigens or antibodies. In a classic study, Tal et al. (1964) observed that sera from tumor patients and pregnant women contained autoimmune antibodies that agglutinated tumor cells in uitro. This agglutination was specifically inhibited by lactose, and it was suggested that the antigen was lactosylceramide. More recently, Jozwiak and Koscielak (1982) observed an elevated level of antibody directed to lactosyl sphingosine in a number of patients with gastrointestinal cancer. The antibody level was determined by inhibition of '251-labeledantihuman IgG to a lactosyl sphingosine-polyacrylamide conjugate. A similar inhibition was observed with the antibody binding to lactosaminyl glycolipid,

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but not to other types of glycolipid. These early studies indicate the presence of antibody directed to lactosyl or N-acetyl lactosamine glycoconjugates in sera of patients with various types of gastrointestinal cancer.

B. SERUM ANTIGENLEVELS DEFINED BY MONOCLONAL ANTIBODIES Enhanced levels of antigen in sera of patients with gastrointestinal cancer were initially observed by inhibition of MAb N-19-9 binding to colonic tumor cells (Koprowski et al., 1981) and by “sandwich” assay (Herlyn et al., 1984). The antigen was subsequently identified as 2- 3 monosialyl Lea (Magnani et al., 1982). Studies using this antibody have assessed the elevated level of the antigen in sera of patients with various types of gastrointestinal tumors (Herlyn et al., 1982; Chia et al., 1985). Of particular interest is the high incidence of antigen elevation in sera of patients with pancreatic cancer, which is otherwise difficult to diagnose in its early stages (Haglund et al., 1986; Ritts et al., 1984; Satake et al., 1985; del Favero et al., 1986). This tumor marker, however, has the drawback that the antigen is absent in sera of Le(a-b-) patients (the incidence of this trait is 1-3% in the Caucasian population), since tumors in such patients do not express a1 -+4 fucosyl transferase due to the absence of the LA gene (Brockhaus et al., 1985; Temper0 et al., 1987). Another antigen with a similar structure, sialyl Le”, defined by antibody CSLEX-1 (Fukushima et al., 1984), was also found to be elevated in several types of human cancer (including lung and breast) and showed a pattern of high elevation complementary to that of sialyl Lea (Chia et al., 1985). Sialyl Le” antigen has an advantage in that it is expressed in tumors in Le(a-b-) patients. The antigen with a similar epitope, defined by the antibody FH6, having a long-chain sialyl lactosamine with internal fucosylation (sialyl Lex-i or sialyl dimeric Le”), was also found to be elevated in patients with various types of cancer (Fukushi et al., 1985; Kannagi et al., 1986). A large-scale screening (over 3000 patients) utilizing this antibody has been performed. The antibody showed a high incidence of positivity in sera of patients with lung adenocarcinoma (86%) and pancreatic cancer (85%), but relatively low positive incidence in other human cancers (average 55%). Nevertheless, the antibody may have diagnostic value since it rarely gave false positive results (Imura et al., 1987). A high incidence of positive cases was found with another antibody, CA50, which defines the 2- 3 sialyl type 1 chain. The antibody, however, cross-reacts with 2- 3 sialyl Lea (Nilsson et al.,1985; Mansson et al., 1985; Holmgren et al., 1983).

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The antibody was able to detect monosialoganglioside in 60-90% of colorectal or pancreatic adenocarcinomas or their metastases, but was essentially unreactive with normal colonic mucosa (Lindholm et al., 1983).Antibody CA50 was able to detect enhanced level of antigen in sera of patients with various types of cancer, particularly colorectal cancer (50-75%), uterine cancer (75%), and prostate cancer (90%) (Holmgren et al., 1984). More recently, a new sialylated type 1 chain, disialyl Lea (2 + 3, 2 + 6 sialyl Lea), was isolated and characterized from human colonic adenocarcinoma, and its antibody, FH-7, which defined 2 + 6 sialyl Lea, was established (Nudelman et al., 1986a). This antibody was capable of detecting the enhanced level of characteristic antigen in various types of human cancer. The antigen level, determined by inhibition of antibody binding, was particularly high in pancreatic, colonic, and bladder cancer (including stages I and 11), although false positive results were also observed in a few patients with nonmalignant diseases such as chronic hepatitis and liver cirrhosis (Kannagi et al., 1988). An antigen with the Le" determinant was detectable in sera of patients with cancer (25 out of 49; 53%) but not in 16 healthy subjects or in seven patients with nonmalignant diseases, when anti-Le" 29-1 antibody was applied using the sandwich method (Herlyn et al., 1984). When IgGS anti-Lexantibody (SH-1) was used as the "catcher" antibody, followed by application of another IgG3 antibody (SH-2) directed to dimeric Le" and used as the detector antibody, the detectability and specificity of the Le" antigen in sera of patients was found to be as high as 7 0 4 0 % (Singhal et al., 1988). However, some other anti-Le" antibodies such as FH2 and FH3 were unable to detect serum antibody levels in patients with cancer. The level of Ley antigen defined by antibody AH6 was relatively high in sera of some patients with hepatoma (Kannagi et al., 1986). An antibody directed to gastric cancer, NCC-ST-439, established by Hirohashi et al. (1984), is capable of detecting high levels of antigen in sera of patients with pancreatic, colorectal, and breast carcinoma. In studies of sera from 19 patients with colorectal carcinoma, the antigen level decreased sharply in all cases after surgical intervention (Sugano et al., 1988). The antigen is sensitive to sialidase and seems to be a sialylated structure, but is not identical to sialyl Lea or sialyl Le". Hanai et al. (198613)established four MAbs, KM32, KM34, KM52, and KM93, directed to human lung squamous cell carcinoma and adenocarcinoma. They utilized a unique technique to enhance production of hybridoma specific to squamous carcinoma. They initially immunized newborn nude mice with normal lung tissue to which the mice were immune tolerant. The same mice, after reaching adulthood,

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were immunized with lung tumor cells, and the resulting MAbs were established. Antibody KM32, cross-reacting with blood group A, may define an A-like antigen associated with various cancers, particularly lung adenocarcinoma. It was found to be capable of detecting elevated levels of A-like antigen in sera of patients with lung cancer (Hanai e t al., 1986a). Antibody KM93 defines a sialidase-sensitive antigen, and could detect a relatively high antigen level in sera of patients with lung adenocarcinoma (Shitara et al., 1987). The antibody DUPAN-2 (Metzgar et al., 1982; 1984), defining mucin-type glycoprotein, is useful in detecting high levels of antigen in sera of patients with pancreatic and gastrointestinal cancer, and was found to be useful in monitoring patients during the course of the disease. The antibody OC125 (Bast e t al., 1981), defining mucin-type glycoprotein that is highly expressed in ovarian carcinoma, could detect high levels of the antigen in sera of patients with ovarian cancer. The antibody C-12 established by Tsuji et al. (1987), originally raised against an endometrial carcinoma cell line, could detect an antigen that is highly expressed in endometrial carcinoma (24 positive out of 25) and ovarian carcinoma (5positive out of 5). The antibody could also detect high antigen levels in sera of patients with not only gynecological cancer but also hepatoma. The high antigen level was also found in patients with liver cirrhosis, but not in normal subjects or patients with other benign diseases. The antigen is expressed in blood group 0 but not A or B erythrocytes, and is assumed to be closely related to H. However, C-12 antigen in sera showed no association with blood group 0, and is therefore distinctive from the common H antigen which is present in sera of blood group 0 subjects, but is absent in sera of blood group A and B subjects (Tsuji et al., 1987). The C-12 epitope could be an extended unbranched Ley (H) structure (H. Tsuji, H. Clausen, E. Nudelman, S. Isojima, and S. Hakomori, unpublished observations). A number of other MAbs raised against various human cancer cell lines or membranes have been found to be directed to sialidasesensitive epitopes carried by mucin-type high-molecular-weight glycoproteins (see Table V). The antigen defined by MAb B72.3, originally raised against the membrane fraction of breast carcinoma (Colcher et al., 1981), has been identified as a sialidase-sensitive epitope associated with mucin (Johnson et al., 1986). The real epitope has been identified as sialyl Tp as previously described (Kjeldsen et al., 1988). The antibody is useful for detection of the antigen present in sera of patients with colon carcinoma (Paterson et al., 1986). Another mucin-type antigen, defined by MAb KL-6, increased in sera

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from patients with lung adenocarcinoma, pancreatic cancer, and breast cancer (Kohno et al., 1987). Various antibodies directed to breast carcinoma, some of which were raised against human milk fat globule membrane (Burchell et al., 1984; Hilkens et al., 1986; Ceriani et al., 1983), and others using breast cancer cell membranes (Johnson et al., 1986; Papsidero et al., 1984; Kufe et al., 1984), displayed the ability to detect high levels of mucin-type antigens in sera of breast cancer patients, particularly those screened by the ability of antibody to recognize serum antigen elevation in sera of patients with breast cancer. Two antibodies, W1 and W9, both directed to carbohydrates of mucin-type glycoproteins, were capable of detecting 60-75% of breast cancer at stages I1 and 111, and are of high diagnostic value (Linsley et al., 1986). There are a number of carbohydrate antigens present in various tumors that are not released into the bloodstream and thus are undetectable by radiometric assay, e.g., GD3 ganglioside in melanoma, and trifucosyl Ley and dimeric Le" in various types of adenocarcinoma. These antigens may, however, be good markers for tumor imaging. The following general characteristics have been noted in antigens detected in sera of cancer patients (see also Table IX):

1. Detectability can vary widely depending on the method used, TABLE IX DIAGNOSTIC APPLICATIONOF TUMOR-ASSOCIATED CARBOHYDRATE ANTIGENS 1. Detection of serum antigens shed from tumors Target molecules: Sialyl 2 + 3 Le" (Koprowski et al., 1981; Herlyn et al., 1982, 1984; Chia et al., 1985; for others, see text) Sialyl 2 + 6 Le" (Kannagi et al., 1988) Sialyl Le" (Chia et al., 1985), sialyl Le"-i (Imura et ul., 1987; Kannagi et al., 1986) Sialyl type 1 chain (Holmgren et al., 1984), Ley (Kannagi et al., 1986) Le" (Herlyn et al., 1984; Singhal et ul., 1988) 2. Detection of antigen-antibody complex through ex oioo circulatory system Target molecule: Le" anti-Lex complex (Singhal et al., 1987) 3. Targeting of labeled antibodies for immuno-imaging Target molecules: CEA, a-fetoprotein (Keenan et al., 1985; Larson, 1985) HMFG mucin-type glycoprotein (Epenetos et a/., 19824 1986a) 17.1 glycoprotein (37 kda) (Chatal et a/., 1986; Herlyn et d., B72.3 glycoprotein, sialyl-Tn (Colcher et nl., 1984) GD2 ganglioside (Heiner et al., 1987) GM1 ganglioside (Dohi et nl., 1988)

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and on intrinsic properties of the antibodies applied. Sandwich assay is sometimes more convenient because it does not require purified antigen, which is not always available. The method shows a high detection rate for some antibodies. However, competitive binding assay (i.e., inhibition of antibody binding to purified antigen) shows a better detection rate for other antibodies. 2. Many of these antigens showing high levels in circulating blood are sialylated compounds. On gel filtration, each antigen was eluted as a high-molecular-weight glycoprotein soluble in perchloric acid and insoluble in chloroform : methanol, i.e., these antigens are mucin-type glycoproteins rather than glycosphingolipids. 3. Not all antigens with elevated levels in sera of cancer patients are the specific products of tumor tissue. Some are components of normal tissue (particularly glandular tissue), and are normally secreted exocrinously. Many of the epithelial cancers are derived from glandular tissues, and some maintain the secretory function and secrete the same antigen endocrinously. In this way, what was originally an exocrine secretion becomes an endocrine secretion in cancer tissues, and the antigen appears in high levels in circulating blood. A typical example could be sialyl Lea or disialyl Lea antigen, abundantly present in normal exocrine secretions from pancreas and salivary glands (Brockhaus et al., 1985). In pancreatic cancer, the antigen is released into the bloodstream, and the level of this antigen appears greatly elevated in the bloodstream. Production of sialyl Lea in pancreatic cancer cells is not greatly increased; its release into the bloodstream is enhanced as a result of the malignancy.

C . SERUMANTIGEN-ANTIBODYCOMPLEX The presence of anticarbohydrate antibodies in sera of patients with cancer, which was suggested in earlier studies (see Section XII,A), was further elucidated in recent studies with MAbs. Antibodies have been detected as immune complexes which can be adsorbed onto a protein A-sepharose column. Hakansson et al. (1985) obtained such immune complexes by elution from protein A-sepharose columns followed by analysis for gangliosides. They detected various types of gangliosides in the immune complex eluate and reported that ganglioside composition in immune complexes from sera of cancer patients was quite different compared to gangliosides in complexes obtained from sera of normal subjects. In an independent study, Singhal et al. (1987) demonstrated a clear qualitative as well as quantitative difference of Le" antigen in the immune complex. Using ex uiz;o circulatory

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assembly through a protein A-silica gel column, they detected the presence of an immune complex with Le" glycolipid. Remarkably, Le" glycolipid was detectable in 100% of 19 tested cases, and was undetectable in all cases tested for nonmalignant disease (0/7). Such a clear-cut distinction has not been observed in any other type of serum antigen assay. It should be noted, however, that the method was based on treatment of a large volume of ex vivo circulating blood passing through a protein A-sepharose column. In contrast, all other serum assays utilized only a minimal quantity (25 pl) of serum. A preliminary attempt to detect levels of glycolipid immune complex using a small sample of serum has been unsuccessful (Singhal et al., 1987). D. IMAGING OF TUMORS BY LABELED ANTICARBOHYDRATE ANTIBODIES Utilizing the specificity of MAbs for tumor antigens expressed at the tumor cell surface, a number of studies have been performed using radiolabeled antibodies to obtain information on tumor location. Successful radioimaging of tumors depends on (1) properties of antigens at the cell surface; (2) qualities of antibodies such as affinity, immunoglobulin isotype, and derivatized state; and (3) kinds of nuclide introduced. In the initial studies, the antigens targeted for imaging were CEA and a-fetoprotein (for reviews, see Keenan et al., 1985; Larson, 1985). The lZ51-labeled antibody HMFG-2, defining mucin-type glycoprotein (Burchell et al., 1984), has been successfully used in targeting the antibody to ovarian, breast, and gastrointestinal tumors (Epenetos et al., 1982a). The same antibody was also applied in diagnosis of malignancy in serous effusions (Epenetos et a1.,1982b). These antigens were, however, actively shedding from the tumor cell surface, and may not be ideal for targeting and imaging purposes. Nonshedding 37-kDa tumor membrane glycoproteins defined by the antibody 17.1 have been extensively used, although the epitope structure of this antibody is ill-defined (Chatal et al., 1984; 1986; Moldofsky et al., 1984; Mach et al., 1983; Herlyn et al., 1986b). Whole or F(ab)Z fragments were labeled with 1251or 1311 using various human carcinomas grafted in nude mice. Of 63 colorectal carcinoma studies, 34 showed significant accumulation of antibody by external photoscanning and tomoscintigraphy (Mach et al., 1983). Radioimmunoimaging of human tumor xenografts was much improved by a mixture of MAb F(ab)z fragments compared to using a single antibody fragment (Munz et al., 1986). For coupling of a preferable radionuclide (such as ''lIn) to an antibody, diethylenetriaminepentaacetic acid (DTPA) conjugation through cyclic DTPA an-

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hydride was useful. The reaction condition for DTPA coupling was investigated (Paik et al., 1983). For possible N M R imaging of tumors, a 19-9 antibody-gadolinium (Gd) complex was prepared through use of the DTPA conjugate, which was used in N M R of tumors to which the Gd-DTPA-antibody 19-9 complex was added. Addition of the complex decreased T1 relaxation of water protons at 90 MHz in proportion to Gd concentration. When radioactive Gd was used, scintillographic visualization of tumors in nude mice was possible (Curtet et al., 1986). The lZ5I-labeledantibody B72.3, defining sialyl Tn, was successfully used for imaging human colonic cancer xenografts in nude mice. Tumorltissue ratio of the tumor-localized antibody increased greatly during 7 days, and showed a prolonged binding activity over a 19-day period of study (Colcher et uZ., 1984). More recently, ganglioside was used for tumor imaging. Localization of human osteosarcoma grown in nude mice was targeted by whole or F (ab)2 fragments of anti-GDZ ganglioside MAb 3F8. The targeting was highly efficient and specific for the osteosarcoma, but not for Ewing's tumor, which did not express GD2 antigen (Heiner et al., 1987). Interestingly, the same GD2 antigen has been found in human neurobIastoma, melanoma, small cell lung carcinoma, and certain brain tumors (see Section V,D). The antibody 3F8, defining GD2 antigen, is therefore the common imaging reagent for these tumor types. Expression of GD2 ganglioside in normal tissues is highly restricted in humans; in mice, it is found only in the thymus. Another study involved GM3 ganglioside in melanoma as a target. An IgG3 antibody, DH2, showed significant inhibitory activity for melanoma growth in ciao and in vitro. The '251-labeIed DH2 antibody accumulates most in B16 melanoma cells, followed by blood, lung, urinary bladder, thyroid, and adjacent tissues; however, little labeling occurred in brain, bone marrow, or other tissues (Dohi et al., 1988). It is possible that these antibodies, 3F8 and DH2, will be useful for imaging of human cancer in future clinical studies. XI. Tumor-Associated Carbohydrate Antigens as Targets for Therapeutic Applications

Host immune response to tumor, particularly spontaneous tumor, is weak or undetectable. A large number of publications from the past several decades indicate that active immunization by killed or attenuated tumor cells (or antigens extracted therefrom) is fruitless. Hybridoma technology, however, has made available an essentially unlimited supply of reagents with highly specific affinity to tumor

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TABLE X THERAPEUTIC APPLICATIONOF TUMOR-ASSOCIATED CARBOHYDRATE ANTIGENS

1. Effect of systemic administration of mouse monoclonal antibodies (IgC&, IgC3) on human cancer in uiuo: activation of antibody-dependent cytotoxic cells (ADCC) and complement-dependent cytotoxicity (CDCC) Target molecule: GD:, in melanoma (Houghton et al., 1985; Herberman et d., 1985) 2. Effect of lesional application of‘antibody (IgM) on tumor Target molecule: GD2 in melanoma (hie and Morton, 1986) 3. Effect of antiidiotype or anti-antiidiotype antibodies Target molecule: 17.1 MAb (DeFreitas et al., 1985; Herlyn et al., 1986a) 4. Targeting drug conjugates (“magic bullets”) Cytotoxic drug conjugate with anti-Gg, (Urdal and Hakomori, 1980; Hakoniori et al., 1982) Cytotoxic drug conjugate with anti-globo-H (Della Torre et al., 1987) Differentiation inducer conjugate with anti-Le” (M. Otaka, A. Singhal, and S. Hakomori, unpublished data) 5. Active immunization with carbohydrate antigen Glycoprotein antigen (Adachi et al., 1988) GalPl- 3GalNAc-protein complex (Henningsson et al., 1987) Ganglioside-BCG complex (Livingston et al., 1987, 1988)

cells, through which many human tumor antigens have been chemically well defined. Thus, many possibilities have opened for suppression of human tumor growth, as discussed below (see also Table X). A.

EFFECTOF MONOCLONAL ANTIBODIESON TUMOR CELLGROWTH In Vitro AND In Vivo

MAbs defining tumor-associated antigens with suitable isotype and affinity have proved to be useful to some extent in suppressing tumor growth in vitro and in vivo, albeit the tumor growth suppression in vivo is highly variable due to multiple, as yet unidentified, factors. A number of studies performed by groups at the Sloan-Kettering Institute, Scripps Clinic, Wistar Institute, and others indicate that cytotoxic effector cells are activated by MAbs that interact through the Fc receptor of lymphocytes or macrophages in concert with classically known complement-dependent cytolysis. Furthermore, some antibodies are able to inhibit tumor growth by themselves, by an unknown mechanism that does not involve effector cells or complement. In addition, antiganglioside antibody may facilitate lymphokinedependent T cell activation. A detailed review of this area of study is not within the scope of this article, but reviews covering the material have been published (Koprowski, 1984; Baldwin and Byers, 1984; Reisfeld and Cheresh, 1985).

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Effective immunotherapy by IgG3 MAb directed to the Gg3 antigen that is highly expressed on mouse lymphoma L5178 was first described by Young and Hakomori (1981). The growth of L5178 lymphoma was completely inhibited by administration of IgG3 anti-Gg3 antibody in a dose-dependent manner. Growth of the variant lymphoma, which did not express Gg3, was not inhibited by the antibody. Growth of neither Gg3 expressor nor nonexpressor lymphoma was inhibited by the antibody directed to the same epitope with IgM isotype. Since both IgG3 and IgM antibodies directed to Gg3 showed complement-dependent cytolysis, the factor involved in this lymphoma suppression could be an antibody-dependent cytotoxic effector cell. The IgG3 mouse MAb R24, defining the GD3 ganglioside that is highly expressed on human melanoma cell surfaces (see Section V,D), activated human effector cell functions, causing lysis of melanoma cells through complement- and antibody-dependent cytotoxicity (Dippold et al., 1984). Another IgG3 MAb directed to GD3, B3.6, showed a clear antibody-mediated melanoma cell lysis (Cheresh et nl., 1985). Subsequently, a phase I clinical trial of R24 antibody in patients with malignant melanoma was carried out. Of 12 patients tested, 3 showed clear regression of tumors, and 4 patients showed a partial response (Houghton et al., 1985). Only inflammatory reactions (in the form of urticaria at the tumor lesion) were observed. GD3 is expressed highly in retina and moderately in kidney and gastrointestinal tissue, yet patients did not develop any visual, gastrointestinal, or renal disease symptoms. It is highly possible that GD3 in melanoma shows a specific organization which is susceptible to complementdependent cytolysis or to antibody-mediated effector cell attack. On the other hand, some melanoma cases showed no response to R24 antibody treatment, although GD3 was still highly expressed in melanoma of such cases; the reason for this phenomenon is not clear. Further elaborate immunobiological studies suggest that a threshold number of R24 molecules is necessary to initiate complement- and cell-mediated cytolysis; i.e., both mechanisms appear to depend on similar threshold quantities of R24 molecules (Welt et al., 1987). A similar IgG3 anti-GD3 antibody (MB3.6) has also been applied in clinical trials in melanoma patients. Of 12 patients, 3 showed partial regression of melanoma. In contrast, another antimelanoma antibody (9.2.27), directed to proteoglycan, did not show any effect upon administration of >lo0 mg of antibody (Herberman et al., 1985). Hellstrom et al. (1985) reported three mouse monoclonal IgG3 antibodies directed to GD3. All three antibodies mediated antibodydependent cytotoxicity in vitro and inhibited outgrowth of human melanoma xenografts in nude mice.

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Anti-GD3 antibody showed strong growth inhibition of melanoma cells in vitro (Dippold et al., 1984), activated IL-2 dependent natural killer cells, and potentiated mitogen-dependent T cell response in vitro (Hersey et al., 1986). IL-2 dependent proliferative and cytotoxic responses of T cell clones from melanoma patients were also activated by anti-GD3 antibody (Hersey et al., 1987). GD3 is expressed by a small subset of T cells which seems to respond to anti-GD3 antibody stimulation (Welt et al., 1987). Thus, the ganglioside GD3 expressed on human melanoma cells is an effective target in in vitro as well as in in vivo immune responses through complement-mediated and effector cell-mediated cytotoxicity by the IgG3 subclass. In more recent studies, such effect was enhanced by lymphokine IL-2. The IgG3 MAbs 14.18 and 116C4, directed against GD2 and GD3, respectively, when used in combination with human peripheral blood mononuciear cells and stimulated with IL-2, lysed melanoma and neuroblastoma cells; i.e., IL-2 displayed an “arming” effect on antibody-dependent cytotoxicity (Honsik et a&, 1986). Interestingly, however, anti-GD3 antibody R24 did not show any significant effect on human melanoma xenografts in nude mice; only a minimal effect was demonstrated at the earliest stage of melanoma development (Welt et al., 1987). This observation reinforces the important lesson that immune responses in mice are quite different from those in humans, and that the mouse model cannot always be adopted to human malignant phenomena. Melanomas, regardless of species, all express high concentrations of GM3. Antibody M2590, claimed to be specific to melanoma, was found to be directed to GM3 (see Sections V,D and IX). The IgG3 version of anti-GM3 antibody (DH2), originally directed to GM3 lactone, was growth inhibitory to mouse and human melanoma in vitm, displayed antibody-dependent cytotoxicity, and inhibited B 16 melanoma growth in syngeneic mice (Dohi et al., 1988). Thus, the DH2 antibody was similar in function to R24, but displayed a more striking effect on melanoma growth in syngeneic mice. The importance of complement-mediated cytotoxicity is fully demonstrated by the effect of human IgM MAb L72, which is directed to GD2 ganglioside. Irie and Morton (1986) injected this antibody into cutaneous metastasis of eight melanoma patients on a daily or weekly basis. Regression was seen in all tumors except those of two patients whose tumors were shown to have low GD2 expression. One patient with melanoma satellitosis, treated with L72, showed complete regression of tumor with no sign of recurrence. Furthermore, with the exception of mild erythema, no significant side effects were observed in any patient.

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B. EFFECT OF ANTIIDIOTYPE OR ANTI-ANTIIDIOTYPE ANTIBODIESON TUMOR GROWTH A second important approach utilizing MAbs is production and application of antiidiotype or anti-antiidiotype antibodies. Immunization with antiidiotype antibody may induce effective immune response to the antigen recognition site of the antibody, which mimics the antigen surface structure, i.e., the internal image of the antibody. The effective immune response is particularly true for anticarbohydrate idiotype antibody, since this could bypass a complex mechanism to cause anticarbohydrate immune response. Anti-antiidiotype antibody originally directed to a carbohydrate antigen may directly and efficiently attack tumor antigen. This is still a hypothesis, and practical application of anticarbohydrate idiotype has not been applied in inhibition of tumor cell growth in z;iz;o. So far, studies have been performed using MAbs with ill-defined antigen structure. Growing evidence in support of these approaches is again not within the scope of this article, but this area has been reviewed in connection with the antiidiotype antibody directed to the anti-17-1 MAb, which is directed to an intrinsic membrane glycoprotein (DeFreitas et al., 1985; Herlyn et al., 1986a). OF ANTIBODY-DRUG CONJUGATES TO TUMOR CELLS C. TARGETING

A third, but popular and perhaps relevant approach utilizing MAbs is based on the classical idea proposed by Paul Ehrlich (1906), who

envisioned the use of antibodies that possess the particular affinity needed to carry therapeutically active agents to specific cells. A number of studies along this line, utilizing MAbs, are being carried out; the MAbs are popularly termed “magic bullets.” Some work appears quite promising, despite the existence of a number of technical problems and pitfalls. This area of research has been reviewed repeatedly (Baldwin, 1985; Gregoriadis et al., 1986; Baldwin and Byers, 1986). An important factor in targeting antibody conjugates to tumor cells, however, is the intrinsic property of the antigen to which the antibody or its fragment is directed. The antigen (1)should be in high concentration and highly exposed at the cell surface; (2) should be sterically and metabolically stable; and (3)should not be shedding from the cell surface, but should be internalized when ligand binds to it. For this purpose, carbohydrate antigens carried by intrinsic membrane glycoproteins or bound to glycolipids are of particular importance. However, no intensive study with anticarbohydrate antibodies

3 14

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has been made. A study was published using biotinyl anti-Gga, avidin, and biotinyl neocarcinostatin as a targeting kit to kill L5178 lymphoma expressing Gg3 in vitro (Urdal and Hakomori, 1980) and in vivo (Hakomori et al., 1982). Using an avidin gold conjugate, the binding and internalization of the biotinylated antibreast cancer antibody MBr-1 in MCF-7 cells were effective. It is suggested that globo-H antigen defined by MBr-1 could be a good target for a toxin carrier agent (Della Torre et al., 1987). There are major drawbacks to the approach using immunotoxins or antibodies conjugated to drugs or liposomes, since the conjugates are rapidly taken up by cellular components of the reticuloendothelial system before they can reach the tumor cells. In addition, since antibody specificity is not highly restricted to tumor cells, the conjugates are targeted to some normal cell populations in addition to tumor cells. In view of these drawbacks, delivery of noncytotoxic differentiation inducers conjugated with anticarbohydrate antibodies seems to be an interesting approach for suppression of tumor growth. In theory, differentiation inducers could modify tumor cell growth by inhibiting malignant properties, while having little or no effect on normal cells and tissues. In an initial trial of this approach, the differentiation inducer sodium butyrate was encapsulated in liposomes covalently linked to anti-Le” IgG3antibody. The differentiation inducer was successfully targeted to human colonic adenocarcinoma cells expressing Le” antigen in uitro, as well as to in uivo cells grown in athymic nu/nu mice (M. Otaka et d., 1989).

D. ACTIVE IMMUNIZATION WITH TUMOR-ASSOCIATED ANTIGEN: EFFECTON TUMOR GROWTH Active immunization with tumor antigen to suppress tumor growth has had little success in the past. However, since tumor-associated carbohydrate antigens have been well established, the approach using purified glycolipid antigens or synthetic antigens should be reevaluated for possible tumor vaccine development. Immunization of syngeneic mice with the glycoprotein antigen of Lewis lung carcinoma purified by peanut lectin column was found to induce cytotoxic effector cells in vivo against Lewis lung carcinoma cells (as detected by Winn’s tumor neutralization assay), and reduced Lewis carcinoma lesions in the lungs after intravenous inoculation (Adachi et aZ., 1984). Because PNA receptor represents T antigen (Thomsen-Friedenreich antigen; GalPl- 3GalNAc), this epitope disaccharide coupled to

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bovine serum albumin (BSA) was used to immunize CAFl J (BALB x A/J) mice. The immunized animals showed delayed-type hypersensitivity (DTH) reaction against the disaccharide-BSA antigen (but not BSA or other carbohydrate-BSA complexes), and became resistant to growth of TA3Ha tumor cells, which show strong expression of T antigen (Henningsson et al., 1987). These two animal experiments indicate the possibility that carbohydrate antigens such as T or T-like antigens induce immune responses that can suppress tumor growth, possibly through cytotoxic T cell response, although the exact mechanism is still unknown. Recently, a similar PNA receptor glycoprotein (tumor-associated carbohydrate antigen; TCA) was purified from the gastric cancer cell line KATO-3 and was used for active immunization of more than 150 patients with various stages of cancer. Some cases showed a reduction of tumor growth (Adachi et aZ., 1988). Interestingly, the effective dose was as small as 50-200 ng of glycoprotein administered intradermally every 3 days and repeated weekly. All the cases in which the glycoprotein administration was effective showed a clear reduction of granulocytes and an increase of lymphocytes. Thus, the granulocyte/ lymphocyte ratio decreased significantly, while the cases in which the treatment was not effective did not show reduction of this ratio. No measurable antibody response nor DTH was recorded (Adachi et al., 1988). A less well-defined tumor-associated antigen (TAA) has also been used in immunotherapy trials, and Hollinshead and associates recently reported 5 years of accumulated data from a Phase I11 clinical trial. Active TAA immunotherapy was reported to be effective for successful suppression of tumors and significantly increased the survival rate of patients when the protocol was adhered to strictly. The survival rate of a total of 234 lung cancer patients in stage 1 and 2 during 5 years was 49% in the control groups, whereas the survival rate of patients receiving active immunotherapy was 70% (Hollinshead et d., 1987). Wallack and associates developed melanoma vaccines by infecting human melanoma cells grown in vitro with Vaccinia virus. The virus shed in the supernatant was pelleted (30,000 g for 2 hr), and 50-pg protein aliquots of the pellet (“Vaccinia melanoma oncolysate”; VMO) were injected intradermally in melanoma patients (Wallack et al., 1977, 1987; Bash and Wallack, 1988). Patients were divided into two groups, showing either IgM or IgG response, and the incidence of recurrence of the melanoma was correlated with the titer of antibodies directed to gangliosides. The higher the antiganglioside IgG antibody

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titer in sera of patients, the lower the incidence of recurrence (Bash et al., 1988).Therefore, the essential epitopes of VMO vaccine could be gangliosides. Morton et al. (1987)immunized 149 patients with stage I1 melanoma after surgery. Patients were treated in three groups: group 1 (surgery only); group 2 (surgery and adjuvant Bacillus Calmette-Guerin [BCG]); group 3 (surgery and adjuvant BCG with melanoma tumor-cell vaccine containing GD3, GM2 and GD2). A significant number of group 3 (but not group 1 or 2) patients developed antibodies against GD2 (IgM; two patients), GM2 (IgM; 10 patients), and GM2 (IgG; two patients). Because gangliosides GM2, GD2, and GD3 represent potential targets for immunological control of melanoma, a systematic study was performed which attempted to elicit immune response against gangliosides (Livingston et al., 1983). Bacillus Calmette-Guerin (BCG) coated with GM2 elicited anti-GMz antibody response in patients with melanoma, particularly those pretreated with cyclophosphamide. Anti-GMz antibodies in vaccinated patients were of the IgM class, and were cytotoxic for melanoma cells expressing GM2 in the presence of human complement (Livingston et al., 1987). In a preliminary followup study, tumor size was reduced and length of survival was increased in patients showing anti-GMz antibody response to vaccination, as compared with patients who did not show anti-GMz response; however, definite conclusions must await further studies (Livingston et al., 1988). XII. ,Summary and Perspectives

Aberrant glycosylation is the most common phenomenon associated with oncogenic transformation expressed in cell membranes of animal and human cancer cells. Many of the aberrant glycosylation products can be recognized by specific MAbs as tumor-associated carbohydrate antigens. Either incomplete synthesis with precursor accumulation or neosynthesis of aberrant structures results in accumulation of certain carbohydrates in high density at the tumor cell surface. They are present in the form of glycosphingolipids, or are associated with glycoproteins, particularly high-molecular-weight mucin-type glycoproteins. Those antigens whose epitope structure is clearly identified have been discussed according to the conventional classification: lacto-series type 1 and type 2, globo-series, ganglio-series. A few classes of important structures are as follows: (1) Elongated type 2 chain (i antigen) with penultimate and internal a1 - 3 fucosylated structures (Le', di-, tri-, or tetrameric Le") combined with a terminally

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a1 + 2 fucosylated structure (Leyand extended Ley) or a terminally sialylated structure (sialyl Le” or sialyl di- or trimeric Le”). (2) For type 1 chain derivatives, sialyl a2 +. 3 or 2 -+ 6 sialylated structures, with or without an a1 +. 4 fucosyl structure, are of great importance. (3) Incompatible blood group A antigen, or heterophile antigen expression, has now been clearly identified chemically and immunochemically. (4)Tn and sialyl Tn structures as tumor-associated antigens have been well defined by MAbs; the high incidence and intensity of expression of these antigens suggest potential clinical applicability. A unique possibility has been suggested based on oncofetal fibronectin: a common 0-glycosylation may induce conformational changes of polypeptides, which are recognized by specific antibodies. Many of these carbohydrate structures accumulating at the tumor cell surface are absent in progenitor cells, but may be found at low levels in other cell types. Antibodies that recognize tumor-associated antigens have the novel ability to recognize the density of antigen at the cell surface in excess of the threshold value, as well as the specific structure of the epitope. Many such antibodies are able to “ignore” subthreshold concentrations of the antigen present at the normal cell surface. Complement-mediated and effector cell-mediated tumor cell lysis may also depend on the concentration and density of carbohydrate antigen and the bound antibody at the cell surface. Variability of antigen expression can be associated with the stage of cancer development, from in situ, through actively infiltrating, to highly metastatic tumor. A tumor does not express one carbohydrate antigen exclusively; rather, most tumors express multiple tumorassociated antigens to different degrees at different loci within the tumor; this is termed “mosaicism” of antigen expression. Many anticarbohydrate antibodies have been utilized in detection of elevated tumor antigen in sera of patients with cancer, e.g., FH6 and FH7 for adenocarcinoma in general, and 19-9 for gastrointestinal tumors, particularly pancreatic cancer. Those fucosylated or fucosyl/ sialylated type 1 or type 2 chain epitopes useful in serum diagnosis are carried by mucin-type high-molecular-weight glycoproteins. Some tumor-associated glycolipid antigens, e.g., GD3 or GD2 ganglioside or 37-kDa glycoprotein (defined by 17.6 antibody), are not readily released in sera; they may be more suitable for antibody-mediated cytolysis as well as targeting by labeled antibodies or their derivatives. Imaging of tumor location through suitably labeled antibodies (or their fragments) directed to specific carbohydrate markers is another

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promising area of study. Knowledge of tumor location is of great clinical importance for effectively focused radiotherapy and surgical operations. Targeting of labeled antibodies, fragments, and their conjugates to tumors for delivery of antitumor drugs is obviously a popular and important approach. Yet carbohydrate antigens have not been utilized as the target for this approach. Extensive studies are expected to be made. Some carbohydrate antigens are strongly immunogenic if properly presented, and data from a few preliminary studies on the effect of active immunization with tumor-associated carbohydrate antigens on tumor growth are now available. Proper assembly of effective antigen in artificial membranes may produce stronger and more specific response, and may eventually lead to the development of tumor vaccines. Thus, our knowledge of the chemical structure and organization of tumor-associated antigens at the tumor cell surface, and their specific MAbs, is increasingly important for the development of more effective diagnostic methods, as well as various methods for treatment and prevention of human cancer.

ACKNOWLEDGMENTS The author wishes to thank Dr. John Magnani for providing information 011 mucintype antigens and Dr. Stephen Anderson for expert assistance during preparation of the manuscript. The author’s work has been supported by Outstanding Investigator Grant CA 42505 from the National Cancer Institute (National Institutes of Health), and b y funds from The Biomembrane Institute.

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NOTEADDED IN PROOF. Anti-LeX (SSEA-I) antibody labeled with lZ5I has been utilized for determination of antibody accumulation in tumor cells in uiuo.The labeled antibody was preferentially accumulated in various Lex-expressing human cancers grown in nude mice. Interestingly, the antibody did not accumulate in various normal tissues and organs (particularly kidney) which also express LeX (Ballou et al., 1984, 1986, 1987).These results suggest that either the Lex antigen expressed in normal tissues was cryptic, or the anti-SSEA-1 antibody was unable to recognize low density LeX but could recognize this antigen organized in high density at the tumor cell surface (Hakomori et al., 1981).

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A Acetylcholine, B cell-associated surface molecules and, 98 Activation B cell-associated surface molecules and. 127-132 Bgp95, 116 biochemically defined molecules, 119 CDZO, 91-99 CD21, 103 CDw40, 114, 115 expression, 127-132 pathway, 83-85 receptors, 125-127 T cells, 132 glutathione transferase and, 233 glycosylation in tumors and, 258, 312 Acute lymphoblastic leukemia chromosome abnormalities and, 18-22, 25 gene rearrangements and hematological neoplasias. 65, 66 simultaneous occurrence, 71 T cell antigen receptor, 55, 57 T cell receptor, 75 Acute myeloblastic leukemia, gene rearrangements and. 68, 70 Acute myeloid leukemia. chromosome abnormalities and, 9 Acute nonlymphocytic leukemia, chromosome abnormalities and. 9-14, 18 Acute promyelocytic leukemia, chromosome abnormalities and, 12 Adenovirus proteins, 151-154, 160, 161 MHC expression El9 protein, 155-157 early region l A , 154, 155 viral pathogenicity, 157-159 viral tumorigenicity, 159, 160 Adriamycin glutathione transferase and, 241 multidrug resistance and, 167 coamplified genes, 194 P-glycoprotein, 172, 173, 180, 189

Adult T cell leukemia chromosome abnormalities and, 24 gene rearrangements and. 59, 61 Agnogenic myeloid metaplasia, chromosome abnormalities and, 18 Alveolar rhabdomyasarcoma. chromosome abnormalities and, 30 Amino acids adenovirus proteins and, 154-157 B cell-associated surface molecules and CDZO, 93, 97-99 CD22, 107 CDZ3, 109. 110 CDw40. 113 chromosome abnormalities and, 37 gene rearrangements and, 46, 50-53 glutathione transferase and molecular forms, 216, 221, 222 preneoplasia, 208, 232, 233 multidrug resistance and, 180, 181, 187, 188 Anemia, chromosome abnormalities and, 13 Angioimmunoblastic lymphadenopathy, gene rearrangements and, 66, 67, 71 Anthracyclins, multidrug resistance and, 170, 193 Antibodies B cell-associated surface molecules and, 84, 85 Bgp95, 115 biochemically defined molecules, 118, 120 CD19, 99 CD20. 93 CD21, 103, 104 CD23, 109. 111 expression on activated cells, 128, 129 history, 86, 87 receptors, 126 gene rearrangements and, 46 B cell antigen receptor, 46-48 hematological neoplasias, 58, 67 simultaneous occurrence, 69 T cell antigen receptor, 49-51

3 34

INDEX

Antibodies (cont.) glutathione transferase and, 209, 224, 225, 227, 238. 239 glycosylation in tumors and, 259, 260, 317, 318 carbohydrate antigens, 264, 286, 289-291 diagnostic applications, 302-309 glycolipid antigens, 261, 262, 269, 270, 274, 275, 277, 280, 283 Hanganutziu-Deicher antigens, 296, 297 histo-blood group ABH antigens, 294 histo-blood p u p P antigens, 295 preneoplastic tissues, 297 requirements, 298-302 therapeutic applications, 310-316 multidrug resistance and, 195-197 amplified genes, 176 clinic, 194 P-glycoprotein, 175, 189-191 Antigens adenovirus proteins and, 151, 152, 160, 161 MHC expression, 153, 155, 158, 159 B cell-associated surface molecules and, 83, 84, 89-91, 134 Bgp95, 115, 116 biochemically defined molecules, 116-119, 121-123 CD19, 99-102 CD20, 91-99 CD21, 102-104 CD22. 105-108 CD23, 108-112 CDw40, 112-115 expression on activated cells, 128-130, 133 history, 86, 87 receptors, 127 T cells, 132 gene rearrangements and, 46 B cell antigen receptor, 46-49 hematological neoplasias, 58, 62, 63, 68 simultaneous occurrence, 69 T cell antigen receptor, 49-57 glutathione transferase and, 205, 210 glycosylation in tumoxs and, see Glycosylation in tumors multidrug resistance and, 183 Antioncogenes. chromosome abnormalities and, 31-34, 38

myelodysplastic syndromes, 15 solid tumors, 29 Ataxia-telangiectasia, gene rearrangements and. 75

B cell antigen receptor, gene rearrangements and, 46-49 B cell-associated surface molecules, 82, 83, 134, 135 activation. 83-85 biologically defined molecules BLA, 121, 122 CDlO, 116-118 CD24, 118, 119 CD37, 119, 120 CD39, 120, 121 expression, 122-124 differentiation antigens Bgp95, 115, 116 CD19, 99-102 CD20, 91-99 CD21. 102-104 CD22. 105-108 CD23, 108-112 CDw40, 112-115 expression, 89-91 expression on activated cells, 127-129 early, 129, 130 late, 130-132 history heteroantisera, 85-87 monoclonal antibodies, 87-89 receptors, 125-127 T cells, 132-134 B cell growth factors, 84, 85 biochemically defined molecules, 120 CD19. 102 CD20, 92, 96 CD21, 104 CD23, 110-112 CDw40, 114. 116 expression on activated cells, 129 receptors, 126, 127 B cell-stimulating factor 1, see Interleukin-4 B cell-stimulating factor 2, see Interleukin-6

INDEX

B cells chromosome abnormalities and acute lymphoblastic leukemia, 18, 20-22 chronic lymphoproliferative disorders, 22, 23 malignant lymphoma, 26 gene rearrangements and, 46 B cell antigen receptor, 46-49 hematological neoplasias, 58, 59, 64. 65. 68 simultaneous occurrence, 70, 72 T cell antigen receptor, 55, 56 glutathione transferase and, 240 B lymphocyte carcinoma cross-reacting antigen, 115, 120, 124, 131, 133 Bgp95, B cell-associated surface molecules and, 115, 116, 124 Bladder carcinoma, chromosome abnormalities and, 29 Bleomycin, multidrug resistance and, 167 Bone marrow chromosome abnormalities and acute lymphoblastic leukemia, 18 acute nonlymphocytic leukemia, 10, 11 cytogenic data, 7, 8 myelodysplastic syndromes. 13, 16 gene rearrangements and, 58 Burkitt's lymphoma B cell-associated surface molecules and biochemically defined molecules, 120-122, 124 differentiation antipens, 92, 108, 116 expression on activated cells. 129, 131 history, 87 receptors. 126 T cells. 133 chromosome abnormalities and acute lymphoblastic leukemia. 21 chronic myeloproliferative disorders, 23 malignant lymphoma, 25 oncogenes, 34, 35, 37 gene rearrangements and, 49, 64, 73 glycosylation in tumors and, 283, 298

C Calcium B cell-associated surface molecules and, 89 Bgp95, 116

335

biochemically defined molecules, 120. 121, 124 CD19, 101. 102 CDZO, 93, 97 CD21, 104 CD22, 107 expression on activated cells, 133 gene rearrangements and, 50 glutathione transferase and. 209 multidrug resistance and, 171, 193 Carbohydrate adenovirus proteins and, 156 B cell-associated surface molecules and biochemically defined molecules. 118 differentiation antigens, 100. 105, 106, 115. 116 multidrug resistance and, 172, 174. 183 Carbohydrate antigens, glycosylation in tumors and, 258, 260, 262-266. 316-318 background, 283, 284 glycoprotein, 287-291 lacto-series antigens, 284. 285 peptide, 291. 292 requirements, 298-302 T antigens, 285-287 therapeutic applications, 309-316 Carcinoembryonic antigen, glycosylation in tumors and, 285, 308 CDl molecules, B cell-associated surface molecules and, 133 CDZ molecules, B cell-associated surface molecules and, 85, 133, 134 CD3 molecules B cell-associated surface molecules and, 88, 133 gene rearrangements and hematological neoplasias, 60-63, 67 T cell antigen receptor, 50, 52 CD4 molecules B cell-associated surface molecules and, 85, 88, 108 gene rearrangements and hematological neoplasias, 60, 61, 63 T cell antigen receptor, 49, 51 CD5 molecules, B cell-associated surface molecules and, 88, 132, 133 CD8 molecules B cell-associated surface molecules and, 85 gene rearrangements and

336

INDEX

CD8 molecules, gene arrangements and (cont.) hematological neoplasias, 60. 63 T cell antigen receptor, 49, 51 CDlO molecules, B cell-associated surface molecules and, 100. 116-118 CD18 molecules, B cell-associated surface molecules and, 85 CD19 molecules, B cell-associated surface molecules and, 135 biochemically defined molecules, 124 differentiation antigens, 91, 97, 99-102, 116 CDZO molecules, B cell-associated surface molecules and. 135 activation, 91-99 biochemically defined molecules, 124 differentiation antigens, 102, 103, 110, 114 CDZl molecules, B cell-associated surface molecules and, 85, 102-104, 131 CDZZ molecules, B cell-associated surface molecules and, 91, 97. 105-108 CD23 molecules, B cell-associated surface molecules and, 85 biochemically defined molecules, 121 differentiation antigens, 114 expression on activated cells, 128, 131 CD24 molecules, B cell-associated surface molecules and, 118, 119 CD37 molecules, B cell-associated surface molecules and, 97, 119, 120, 123 CD39 molecules, B cell-associated surface molecules and, 97, 116, 120, 121 cDNA B cell-associated surface molecules and. 134, 135 biochemically defined molecules, 120, 121 CDZO, 96, 97 CD21, 104 CD22, 105, 106 CDZ3, 109 CDw40, 113, 115 T cells, 133 gene rearrangements and, 50-52 glutathione transferase and molecular forms, 216, 218, 222 multidrug resistance, 242 preneoplaia, 209, 223, 224, 231-233 glycosylation in tumors and, 290

multidrug resistance and, 197 clinic, 193 P-glycoprotein, 175, 176, 180, 184. 185, 188, 191 CDw40, B cell-associated surface molecules and, 134, 135 biochemically defined molecules. 123 differentiation antigens, 97, 112-116 expression on activated cells, 128 Centromeres chromosome abnormalities and, 6 gene rearrangements and, 55, 75 Ceramide, glycosylation in tumos and, 301, 302 Chloramphenicol acetyltransferase. glutathione transferase and, 232 Chromatin, gene rearrangements and, 72 Chromosome abnormalities, 2-4, 37, 38 acute lymphoblastic leukemia, 18-22 acute nonlymphocytic leukemia, 9-13 antioncogenes. 30-34 chronic myeloproliferative disorders, 16-18 cytogenic data, 7-9 cytogenic nomenclature, 4-7 malignant lymphoma, 24-27 myelodysplastic syndromes, 13-16 oncogenes, 30, 31, 34-37 solid tumors, 27-30 Chromosomes B cell-associated surface molecules and. 130, 131 gene rearrangements and B cell antigen receptor, 49 hematological neoplasias, 58, 64 T cell antigen receptor, 52, 53. 55 T cell receptor, 72-75 glutathione transferase and, 233 multidrug resistance and, 176, 178 Chronic lymphocytic leukemia B cell-associated surface molecules and differentiation antigens, 100. 103, 105 expression, 130, 131 history, 87 T cells, 132, 133 chromosome abnormalities and, 22, 23, 26 gene rearrangements and, 60, 64, 65 Chronic lymphoproliferative disorders, chromosome abnormalities and. 22-24

INDEX

Chronic myeloid leukemia chromosome abnormalities and, 2, 3, 16, 17 acute lymphoblastic leukemia, 21 acute nonlymphocytic leukemia, 10 oncogenes, 36, 37 gene rearrangements and, 73 Chronic myelomonocytic leukemia, chromosome abnormalities and, 13. 15. 16 Chronic myeloproliferative disorders, chromosome abnormalities and, 16-18, 22-24 Chronic myelosis, chromosome abnormalities and, 13 Clones adenovirus proteins and, 158 B cell-associated surface molecules and, 96, 97, 102, 104, 120 chromosome abnormalities and, 38 acute lymphoblastic leukemia, 18 acute nonlymphocytic leukemia, 9 cytogenic nomenclature. 7 myelodysplastic syndromes, 16 solid tumom 29 gene rearrangements and, 46 B cell antigen receptor, 48 hematological neoplasias, 57-59, 63, 64, 66, 68 simultaneous occurrence, 69, 70, 72 T cell antigen receptor, 49-52 glutathione transferase and, 216, 232, 233 glycosylation in tumom and, 290, 312 multidrug resistance and, 185 Cutaneous T cell lymphomas. gene rearrangements and, 59-61 Cyclosporin A, multidrug resistance and, 171, 195 Cysteine, B cell-associated surface molecules and, 97, 99, 110, 113 Cytokines. B cell-associated surface molecules and, 125-127 Cytoplasm adenovirus proteins and, 156, 157 B cell-associated surface molecules and, 83 biochemically defined molecules, 124 CD19, 100-102 CD20, 91, 98, 99 CD21, 104

337

CD22, 105-107 CD23, 110 CDw40, 113 expression on activated cells, 133 gene rearrangements and, 59 multidrug resistance and, 182, 183 Cytosol, glutathione transferase and molecular forms, 212, 214, 215, 220, 222 preneoplasia, 225, 229, 236 Cytotoxic T lymphocytes, adenovirus proteins and, 151, 158, 159

Daunorubicin, multidrug resistance and, 167, 171, 195 Delayed type hypersensitivity, glycosylation in tumors and, 315 Deletion chromosome abnormalities and acute lymphoblastic leukemia, 20 acute nonlymphocytic leukemia, 12 Togenic nomendature, 6 malignant lymphoma, 26 myelodysplastic syndromes, 15 gene rearrangements and, 63-65 Dendrites, B cell-associated surface molecules and, 92, 103 Diethylenetriaminepentaaceticacid, glycosylation in tumors and, 308, 309 Differentiation, B cell-associated surface molecules and, 82, 134 antigens, see Antigens biochemicallydefined molecules, 118. 119. E2 expression on activated cells, 128 history, 86, 87 pathway, 83-85 receptors, 125 T cells, 132 Disulfide bonds, B cell-associated surface molecules and, 98, 105 DNA adenovirus proteins and, 152-154 B cell-associated surface molecules and, 89, 96 chromosome abnormalities and, 3. 38 antioncogenes, 31, 32

338

INDEX

DNA, chromosome abnormalities and (cont.) chronic myeloproliferative disorders, 17 malignant lymphoma, 26 oncogenes, 35 gene rearrangements and, 45 B cell antigen receptor, 47 hematological neopiasias, 57-59, 64, 65 T cell antigen receptor. 52, 53 glutathione transferase and molecular forms, 218, 222 multidrug resistance. 242 preneoplasia, 223, 224, 231, 232, 237 multidrug resistance and, 166, 167 amplified genes, 176, 178 P-glycoprotein, 179, 180, 183, 186

CD19, 100 CDZO. 93, 99 CD21, 102-104 CD23, 108, 112 expression on activated cells, 131, 133 history, 88 receptors, 126 glycosylation in tumors and, 283 Escherichiu coli, multidrug resistance and, 184 Essential thrombocytopenia, chromosome abnormalities and, 18 Eukaryotes adenovirus proteins and, 156 mukidrug resistance and, 185 Ewing’s sarcoma, chromosome abnormalities and, 30

E F Electron microscopy, multidrug resistance and, 189 Endoplasmic reticulum adenovirus proteins and, 156, 157, 160, 161 multidrug resistance and, 189 Eosinophils, chromosume abnormalities and, l2 Epidermal growth factor, multidrug resistance and, 172 Epitopes B cell-associated surface molecules and, 134 biochemically defined molecules, 118-120 CD19, 100 CDZl, 103, 104 CDPP, 105, 107 CD23, 109, 111 CDw40, 113, 115 gene rearrangements and, 47 glycosylation in tumors and, 317 carbohydrate antigens, 262, 286-292, 302 diagnostic applications, 303, 305, 308 glycolipid antigens. 283-285 histo-blood group ABH antigens, 294 therapeutic applications, 311, 314, 316 multidrug resistance and, 183, 189 Epoxide hydrolase, glutathione transferase and. 210 Epstein-Barr virus B cell-associated surface molecules and biochemically defined molecules, 121

Fibroblasts, gene rearrangements and, 52, 59 Fibronectin, glycosylation in tumors and. 291. 317 Fonsman antigens, glycosylation in tumors and, 294-296 French-American-Britishclassification, chromosome abnormalities and, 9, 12, 13, 18 Fucosylation, glycosylation in tumors and, 316, 317 diagnostic applications, 303 glycolipid antigens, 268-270, 273-275, 277

Ganglio-series antigens, glycosylation in tumors and. 277-281, 316 Gangliosides. glycosylation in tumors and, 259, 317 carbohydrate antigens, 298, 300. 302 diagnostic applications, 306. 307, 309 glycolipid antigens, 262, 277-281 histo-blood group ABH antigens, 296, 297 therapeutic applications, 311, 312, 315. 316 Gangliotriaosylceramide,glycosylation in tumors and carbohydrate antigens, 299, 301. 311 glycolipid antigens, 261, 280 oncogenes, 267

339

INDEX

Gene rearrangements, lymphoproliferative disorders and, see Lymphoproliferative disorders Globo-series antigens, glycosylation in tumors and, 281-283, 316 Glucose 6-phosphate dehydrogenase, gene rearrangements and, 58 Glutathione transferase. 205-207, 242, 243 human GT-q. 239, 240 molecular forms, 221, 222 human, 218-220 mouse, 280, 221 properties, 212-215 rat, 215-218 multidrug resistance, 241, 242 preneoplastic markers, 223, 226, 238, 239 hepatic enzymes, 207-211 nonhepatic enzymes, 211, 212 rat GT-P application, 237 extrahepatic preneoplasia, 237, 238 function. 233, 234, 236, 237 gene expression, 231-233 hepatocarcinogenesis, 225, 227-229 identification, 222, 224 specificity, 229-231, 235 tissue distribution, 224. 225 Glycolipid antigens, glycosylation in tumors and, 259, 260, 317 carbohydrate, 264 diagnostic applications, 302, 303, 308 ganglio-series, 277-281 globo-series, 281-283 histo-blood group P antigens, 295 lacto-series, 268-277 oncogenes, 267 preneoplastic tissues, 297 requirements, 298, 300-302 therapeutic applications, 313, 314 Glycoprotein. see also Permeability glycoprotein adenovirus proteins and. 152, 156 B cell-associated surface molecules and biochemically defined molecules, 117, 119, 121, 123, 124

diagnostic applications, 305-308 glycolipid antigens, 267 histo-blood group P antigens, 295 therapeutic applications, 313-315 multidrug resistance and, 172 Glycosphingolipids. glycosylation in tumors and, 262, 267, 307, 316 Glycosylation B cell-associated surface molecules and biochemically defined molecules, 118, 121, 123

differentiation antigens, 100, 106, 107, 110, 113

expression on activated cells, 131 multidrug resistance and, 173, 175. 183 Glycosylation in tumors, 258-260, 316-318 carbohydrate antigens, 262-266 background, 283, 284 glycoprotein, 287-291 lacto-series, 284, 285 peptide, 291, 292 requirements, 298-302 T antigens, 285-287 therapeutic applications, 309-316 diagnostic applications earlier studies, 302. 303 serum antigen-antibody complex, 307, 308 serum antigen levels, 303-307 tumor imaging, 308, 309 Forssman antigens, 296 glycolipid antigens, 260-262 background, 267 ganglio-series, 277-281 globo-series, 281-283 lacto-series type 1 chain, 268-271 lacto-series type 2 chain, 270, 272-277 Hanganutziu-Deicher antigens, 296, 297 histo-blood group ABH antigens, 292-294 histo-blood p u p P antigens, 294-296 oncogenes, 264. 267 preneoplastic tissues, 297 Gramkidin, multidrug resistance and, 167

H

differentiation antigens, 100, 103, 108, 113, 115, 116

gene rearrangements and. 50 glycosylation in tumors and. 260, 316, 317 carbohydrate antigens, 262, 283-291

Hairy cell leukemia, chromosome abnormalities and, 24 Hanganutziu-Deicher antigens, glycosylation in tumors and, 296, 297

340

INDEX

Heat shock, adenovirus proteins and, 154 Hematological neoplasias, gene rearrangements and, 57-68 Hematopoietic cells, gene rearrangements and, 72 Hematopoietic dpplasia, chromosome abnormalities and, 13 Hemolysin, multidrug resistance and, 181-184 Hemopoietic stem cells, gene rearrangements and, 49 Hepatic marker enzymes, glutathione transferase and, 207-211 Hepatitis B virus, adenovirus proteins and, 158 Hepatocarcinogenesis, glutathione transferase and, 206, 242 molecular forms. 216, 221 preneoplasia, 209, 211. 222, 225, 227-234, 237 Herpes simplex virus, adenovirus proteins and, 159 Heteroantisera, B cell-associated surface molecules and, 85-87, 97. 115, 122 Histo-blood group ABH antigens, glycosylation in tumors and, 292-294 Histo-blood group P antigens, glycosylation in tumors and, 294-296 Hodgkin’s disease chromosome abnormalities and, 24, 26, 27 gene rearrangements and, 66-68 Homology, gene rearrangements and, 47, 50, 53. 55 Hormones B cell-associated surface molecules and, 113, 125 glutathione transferase and, 214, 231, 236 HTLV-I retrovirus chromosome abnormalities and, 24 gene rearrangements and. 61 Hybridomas B cell-associated surface molecules and, 109 glycosylation in tumors and, 264, 280, 296. 304 Hybrids adenovirus proteins and, 157 chromosome abnormalities and, 31 gene rearrangements and. 50, 55, 58, 72, 75

glutathione transferase and, 209 multidrug resistance and amplified genes. 175-177 P-glycoprotein, 179, 187, 189

I Idiopathic myelofibrosis. chromosome abnormalities and, 18 Immunoglobulins adenovirus proteins and, 152, 157 B cell-associated surface molecules and, 83, 118, 120, 122, 124, 129 biochemically defined molecules, 118, 120, 122, 124 CD19, 99, 101, 102 CD20, 91, 93, 96 CDZI, 102, 103 CD22, 105-108 CD23, 108-112 CDw40, 114, 115 expression on activated cells, 129 history, 86, 87 T cells, 132 chromosome abnormalities and acute lymphoblastic leukemia, 20-22 chronic lymphoproliferative disorders, 23 malignant lymphoma, 25, 26 oncogenes, 35 gene rearrangements and, 46 B cell antigen receptor, 46-49 hematological neoplasias, 57-68 simultaneous occurrence, 69-72 T cell antigen receptor, 50-56 T cell receptor, 73 glycosylation in tumors and carbohydrate antigens, 299, 311. 314-316 diagnostic applications, 302, 304 glycolipid antigens, 261, 270 Immunoprecipitation. gene rearrangements and, 51 Insulin, chromosome abnormalities and, 18 Interferon adenovirus proteins and, 155, 160 B cell-associated surface molecules and, 126, 127 chromosome abnormalities and, 12 glutathione transferase and, 221 Interleukin, gene rearrangements and, 50

INDEX

Interleukin-1, B cell-associated surface molecules and, 126, 127 Interleukin-2 B cell-associated surface molecules and, 84, 85 expression on activated cells, 127, 130, 132 receptors, 125 T cells, 133 glutathione transferase and, 233, 240 glycosylation in tumors and, 312 Interleukin-4, B cell-associated surface molecules and, 134 differentiation antigens, 108, 111, 114, 116 expression on activated cells. 127 receptors, 125-127 Interleukin-5, B cell-associated surface molecules and, 84 Interleukin-6, B cell-associated surface molecules and, 85, 126, 127, 134 Internalization B cell-associated surface molecules and biochemically defined molecules, 118 CD20. 92, 93, 96 CD21, 104 CD23, 110, 111 glycosylation in tumors and, 313, 314 Inversion, chromosome abnormalities and, 6

K Ki 1' lymphomas, gene rearrangements and, 67. 71

L Lacto-series antigens, glycosylation in tumors and, 283-285 type 1, 316 carbohydrate antigens, 262 glycolipid antigens, 268-271 type 2. 316 carbohydrate antigens, 262 glycolipid antigens, 270, 272-277 Latent membrane protein, B cell-associated surface molecules and, 99 Lectin, glycosylation in tumors and, 285, 289, 293, 301, 314

341

Lennert's lymphoma, gene rearrangements and, 66, 69, 70 Leukemia, see also specific leukemia B cell-associated surface molecules and biochemically defined molecules, 116-118, 120 differentiation antigens, 91, 92, 96. 100, 114 expression on activated cells, 130-132 history, 87 receptors, 125 chromosome abnormalities and, 3, 38 gene rearrangements and hematological neoplasias, 59, 62, 63 simultaneous occurrence, 70-72 T cell antigen receptor, 50, 52, 56 T cell receptor, 74 glutathione transferase and, 215 glycosylation in tumom and, 264, 275, 280 multidrug resistance and, 195 Ligands B cell-associated surface molecules and, 85, 110, 127, 134 chromosome abnormalities and, 37 glycosylation in tumors and, 301, 313 Light microscopy, chromosome abnormalities and, 20 Lipids glutathione transferase and, 213, 234 glycosylation in tumors and, 258, 259, 262, 302 multidrug resistance and. 170, 173 Lipogenic tumors, chromosome abnormalities and. 29, 30 Lung cancer, chromosome abnormalities and, 27 Lymphoblasts, chromosome abnormalities and, 20 Lymphocytes, gene rearrangements and hematological neoplasias, 57, 58, 63, 64, 66, 67 T cell antigen receptor, 54 Lymphoid cells, gene rearrangements and B cell antigen receptor, 47 hematological neoplasias, 57, 58, 66, 68 simultaneous occurrence, 69-72 Lymphokines. gene rearrangements and, 49 Lymphomas, see also specific lymphoma chromosome abnormalities and acute lymphoblastic leukemia, 21, 22

342

INDEX

Lymphomas, chromosome abnormalities and (cont.) cytogenic data, 8 malignant. 24-27 gene rearrangements and hematological neoplasias, 59-62, 64,66-68 simultaneous occurrence, 70, 71 T cell receptor, 74 Lymphomatoid papulosis, gene rearrangements and, 64 Lymphoproliferativedisorders. 45, 46 B cell antigen receptor, 46-49 hematological neoplasias, 57-59 acute myeloblastic leukemia, 68 B cell malignancies, 64, 65 chronic T cell malignancies, 59-61 lymphomas, 66-68 lymphomatoid papulosis, 64 pre-B cell malignancies, 65, 66 T cell acute lymphoblastic leukemia, 62, 63 T cell lymphomas, 61. 62 T lymphoproliferative disorder, 63 immunoglobulin genes, 69-72 T cell antigen receptor genomic organization, 52-56 role, 49 somatic rearrangement, 56, 57 structure, 49-52 T cell receptor chromosomal translocation, 72-75 simultaneous occurrence. 69-72 Lymphotoxin, B cell-associated surface molecules and, 84, 112, 127 Lysosomes, multidrug resistance and, 169

M Major histocompatibility complex adenoviw proteins and, 151, 152. 160, 161 expression, 154-160 B cell-associated surface molecules and, 83 antigens. 91-93, 112-114. 116 expression, 128, 131 gene rearrangements and, 49-51 Meningioma, chromosome abnormalities and, 30 Metallothionein, chromosome abnormalities and, 12 Methotrexate, multidrug resistance and, 168

&-Microglobulin, adenoviw proteins and, 152, 155 Mitogens, chromosome abnormalities and, 22 Monoclonal antibodies B cell-associated surface molecules and. 85, 116, 118-124, 134 Bgp95, 116 CD19. 100-102 CD20, 92, 93, 96 CD21, 102-104 CD22, 105, 107 CD23, 108, 109, 111, 112 CDw40, 113-115 expression. 127, 129, 130 history, 87-89 T cells, 133 glutathione transferase and, 211 glycosylation in tumors and, 260, 316-318 carbohydrate antigens, 262, 264, 284-289, 291, 300 diagnostic applications, 303-308 Forssman antigens. 296 glycolipid antigens, 261, 267, 269. 273-277, 280, 283 Hanganutziu-Deicher antigens, 293, 294 oncogenes, 267 therapeutic applications, 310-313 multidrug resistance and, 174, 175, 183. 189 Monosomy, chromosome abnormalities and, 13, 15, 16, 29, 30 mRNA B cell-associated surface molecules and, 96, 97, 112. 135 chromosome abnormalities and, 20, 36, 37 gene rearrangements and, 62, 63, 72, 75 glutathione transferase and, 209, 224, 231-233, 240 multidrug resistance and, 176, 179, 187, 189 Multidrug resistance, 165, 166 alterations, 172-174 amplified genes, 175-178 clinic, 194, 195 coamplified genes. 192-194 drugs affected. 166-168 events, 169-171 glutathione transferase and, 241-243 outlook, 195-197 P-glycoprotein diversity, 185-188 expression, 189-192 genes. 178-180

343

INDEX

mutation, 188, 189 overproduction, 174, 175 structure, 180-185 pharmacological reversal, 171, 172 Mutation adenovirus proteins and, 154, 158 B cell-associated surface molecules and, 134 chromosome abnormalities and, 2, 4, 34 gene rearrangements and, 47, 50 glutathione transferase and, 237 multidrug resistance and, 167. 196 amplified genes, 175 coamplified genes, 194 P-glycoprotein, 175, 185, 188, 189 Mycosis fungoides, gene rearrangements and, 59-61

Myelodysplastic syndromes, chromosome abnormalities and, 10, 13-17

acute nonlymphocytic leukemia, 12 malignant lymphoma, 25. 26 myelodysplastic syndromes. 15 gene rearrangements and, 49, 57, 73, 75 glycosylation in tumors and, 264, 267, 316 Open reading frames, adenovirus proteins and, 154 Osteosarcoma, chromosome abnormalities and, 33

P Parasites, B cell-associated surface molecules and, 111 Peptides adenovirus proteins and, 152, 155, 161 B cell-associated surface molecules and, 97. 106, 115

N

gene rearrangements and. 50, 51 glycosylation in tumors and, 284, 287. 291, 292

Natural killer cells, gene rearrangements and, 63 Neoplasia chromosome abnormalities and, see Chromosome abnormalities glutathione transferase and, 205, 206, 238 hematological, gene rearrangements and, 57-68

Neoplasms, gene rearrangements and, 70, 72 Nerve growth factor, B cell-associated surface molecules and, 113, 114 NIL cells, glycosylation in tumors and, 260, 261

Non-Hodgkin’s lymphomas, chromosome abnormalities and, 24-27 Nucleotides adenovirus proteins and, 155 chromosome abnormalities and, 36 gene rearrangements and, 47. 53 glutathione transferase and, 232 mulridrug resistance and, 181, 196

Oncogenes chromosome abnormalities and, 31, 33-38 acute lymphoblastic leukemia, 19, 20

multidrug resistance and, 167, 184 Permeability gtycoproteins glutathione transferase and, 241 multidrug resistance and, 166. 195-197 alterations, 172-174 clinic, 195 coamplified genes, 192-194 diversity, 185-188 expression, 189-192 genes, 178-180 mutation, 188. 189 overproduction, 174, 175 structure, 180-185 pH, B cell-associated surface molecules and, 109, 113

Phenobarbitol. glutathione transferase and, 208. 209. 228

Phenotype adenovirus proteins and, 154 B cell-associated surface molecules and, 102, 122

chromosome abnormalities and. 19, 31, 32 gene rearrangements and hematological neoplasias, 59, 62-64 simultaneous occurrence, 69-71 glutathione transferase and, 205, 206. 210, 211

glycosylation in tumors and. 258. 264

344

INDEX

Phenotype (cont.) multidrug resistance and, 185, 188, 196 alterations, 173, 174 coamplified genes, 192-194 Philadelphia chromosome, chromosome abnormalities and. 2. 3, 16, 17, 21 Phorbol myristate acetate, B cell-associated surface molecules and Bgp95, 116 CD20, 92, 93 CD22, 105, 106 CDw40, 115 expression, 129 Phosphatidylinositol. B cell-associated surface molecules and, 131 Plasma cells, B cell-associated surface molecules and, 82 biochemically defined molecules, 119. , 120, 122 differentiation antigens, 91, 99 expression on activated cells, 128 history, 86, 88 T cells, 133, 134 Pokeweed mitogen. B cell-associated surface molecules and, 91, 119. 125 Polycythemia Vera, chromosome abnormalities and, 17, 18 Polypeptides adenovirus proteins and. 156 B cell-associated surface molecules and, 82, 134 biochemically defined molecules, 120, 122-124 differentiation antigens, 109, 110, 112, 113 expression on activated cells, 129-131 chromosome abnormalities and, 35, 37 gene rearrangements and B cell antigen receptor, 46-48 T cell antigen receptor, 49-52, 54 glutathione transferase and, 225 glycosylation in tumors and, 262, 291. 292, 317 multidrug resistance and, 175, 184 Preneoplasia glutathione transferase and, see Glutathione transferase glycosylation in tumors and, 293, 297 Prolymphocytic leukemia B cell-associated surface molecules and, 92

chromosome abnormalities and, 23, 24 Proteases B cell-associated surface molecules and, 106, 110, 126 glycosylation in tumors and, 260, 287. 300, 301 multidrug resistance and, 193 Protein adenovirus, see Adenovirus proteins B cell-associated surface molecules and, 89, 135 biochemically defined molecules, 122, 124 CD19. 99, 101 CD20, 96-99 CD21, 103 CD22, 105. 106 CD23, 108, 110, 112 CDw40, 113 expression on activated cells, 129 chromosome abnormalities and, 21, 32, 35-37 gene rearrangements and, 50, 51, 55 glutathione transferase and, 242 molecular forms, 212-214, 216 P-glycoprotein, 211, 231, 236 glycosylation in tumors and carbohydrate antigens, 262, 290, 291. 300, 301 diagnostic applications. 307, 308 multidrug resistance and, 166 alterations, 172. 174 amplified genes, 176 coamplified genes, 192, 193 P-glycoprotein. 175, 180, 181, 183-185, 187-190 Protein kinase, gene rearrangements and, 50 Protein kinase C B cell-associated surface molecules and. 92, 93, 103 glutathione transferase and, 242 multidrug resistance and, 192 Protooncogenes, chromosome abnormalities and, 21, 22, 34, 35

R Receptors adenovirus proteins and, 151

345

INDEX

B cell-associatedsurface molecules and, 134 biochemically defined molecules, 118 cytokines, 125-127 differentiation antigens, 107, 112-115 expression on activated cells, 127. 130 Reed-Stemberg cells. gene rearrangements and, 66-68 Refractory anemia with excess of blasts, chromosome abnormalities and, 13, 14, 16 Refractory anemia with excess of blasts in transformation, chromosome abnormalities and, 13, 14, 16 Refractory anemia with ringed sideroblasts, chromosome abnormalities and, 13, 15 Refractory anemia without excess of blasts, chromosome abnormalities and, 15-15 Renal cell carcinoma, chromosome abnormalities and, 27, 29 Replication, adenovirus proteins and, 153, 154 Restriction fragment length polymorphism chromosome abnormalities and, 31, 32 gene rearrangements and, 52 Retinoblastoma, chromosome abnormalities and, 31-33 Retrovirus chromosome abnormalities and, 24, 34 gene rearrangements and, 61 glycosylation in tumors and, 264, 267 multidrug resistance and, 197 RNA B cell-associated surface molecules and, 96, 98, 107 chromosome abnormalities and. 36 gene rearrangements and, 70 glutathione transferase and, 218, 231, 232 glycosylation in tumors and, 259 multidrug resistance and, 166, 176, 177. 190

S Salivary gland tumors, chromosome abnormalities and, 27, 29 Sezary syndrome, gene rearrangements and, 60, 61 Sialic acid, glycosylation in tumors and, 280, 291

Signal transduction, B cell-associated surface molecules and, 99, 114, 134 Small cell lung cancer, chromosome abnormalities and, 27 Solid tumors, chromosome abnormalities and, 8, 27-30, 38 Somatic hypermutation, gene rearrangements and, 47, 53 Steroids glutathione transferase and, 213, 214 multidrug resistance and, 168 Synovial sarcoma, chromosome abnormalities and. 30

T T cell acute lymphoblastic leukemia, gene rearrangements and, 62, 63, 66, 73 T cell antigen receptor, gene rearrangements and, 46 genomic organization, 52-56 role, 49 somatic rearrangement, 56, 57 structure, 49-52 T cell chronic lymphocytic leukemia, gene rearrangements and, 59-61 T cell prolymphocytic leukemia, gene rearrangements and, 59, 60 T cell receptor genes, gene rearrangements and, 46 chromosomal translocations, 72-75 hematological neoplasias, 57-68 simultaneous occurrence, 69-72 T cells adenovirus proteins and, 152 B cell-associated surface molecules and, 82, 132-134 biochemically defined molecules, 118, 119, 124 CD20, 91 CD21, 103 CD22, 105, 107, 108 CDZ3, 109 expression on activated cells, 127, 130-132 history, 86-88 receptors. 125, 127 chromosome abnormalities and, 22-24, 26 glutathione transferase and, 240 glycosylation in tumors and, 310, 312. 315

346

INDEX

Terminal deoxynucleotidyltransferase,gene rearrangements and, 47, 54, 68, 73 12-0-Tetradecanoylphorbol-13acetate B cell-associated surface molecules and, 93. 99, 102, 111. 114, 119

glutathione transferase and, 233, 242 Thymocytes B cell-associated surface molecules and, 86 gene rearrangements and, 51, 56, 71, 72 Topoisomerase 11, multidrug resistance and, 167. 193

Transcription adenovirus proteins and, 152-155 B cell-associated surface molecules and, 83, 98

chromosome abnormalities and, 21, 25. 26, 35

glutathione transferase and, 232 multidrug resistance and, 195 amplified genes, 176 P-glycoprotein, 179, 185, 186, 188, 192 Transfemn B cell-associated surface molecules and, 110

chromosome abnormalities and, 10 Transforming growth factor, B cell-associated surface molecules and, 85 Translation, B cell-associated surface molecules and, 96, 98 Translocation chromosome abnormalities and acute lymphoblastic leukemia, 19-22 acute nonlymphocytic leukemia, 10-12 chronic lymphoproliferative disorders, 23. 24

chronic myeloproliferative disorders, 16, 17

cytogenic nomenclature, 6 malignant lymphoma, 25, 26 oncogenes, 34-36 solid tumors, 27. 29, 30 gene rearrangements and, 46 B cell antigen receptor, 49 hematological neoplasias, 57 T cell receptor, 72-75 multidrug resistance and, 183 Tiisomy 3, chromosome abnormalities and, 25 TrisOmv 7, chromosome abnormalities and, 29

Trisomy 8, chromosome abnormalities and, 13, 15, 16

Tubulin, multidrug resistance and, 166, 194 Tumor necrosis factor adenovirus proteins and, 158 B cell-associated surface molecules and, 84. 127

Tumors adenovirus proteins and, 159, 160 B cell-associated surface molecules and biochemically defined molecules, 117, 118, 122

differentiation antigens, 92, 108, 114 history, 86 receptors, 125 chromosome abnormalities and, 2-4, 37. 38 acute lymphoblastic leukemia, 22 antioncogenes. 31-33 malignant lymphoma, 25 oncogenes, 30. 31, 34 solid tumors, 27-30 gene rearrangements and hematological neoplasias, 58, 59, 61, 64, 67

simultaneous occurrence, 71 T cell antigen receptor, 57 T cell receptor, 72 glutathione transferase and, 205. 243 multidrug resistance, 241 preneoplasia, 229, 232, 239, 240 glycosylation in, see Glycosylation in tumors multidrug resistance and, 166. 167 amplified genes, 174 clinic, 194, 195 P-glycoprotein, 178, 189 Tyrosine kinase, chromosome abnormalities and, 21, 37

U Uterine leiomyomas, chromosome abnormalities and, 29

v Verapamil, multidrug resistance and. 171, 172, 180

347

INDEX

Vesicular stomatitis virus. glycosylation in tumoni and, 261 Vinblastine, multidrug resistance and, 171, 175, 189 Vincristine. multidrug resistance and, 167

Y Yeast, multidrug resistance and, 185

Z Zinc, chromosome abnormalities and, 12 Wild-type virus, adenovirus proteins and, 158 Wilms’ tumor chromosome abnormalities and, 31-33 gene rearrangements and, 73

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