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ADVANCESINCANCERRESEARCH VOLUME 57

This Page Intentionally Left Blank

ADVANCES IN CANCERRESEARCH Edited by

GEORGE F. VANDE WOUDE NCI-FrederickCancer Research and Development Center Frederick, Maryland

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

Volume 57

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1991 BYACADEMIC PRESS,INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. San Diego, California 92101 United K i n g d m Edition published by ACADEMIC PRESS LIMITED 2428 Ova1 Road, London N W 1 7 D X

Library of Congress Catalog Card Number: 52-13360

ISBN 0-12-006657-2 (alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA 91929394

9 8 7 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS TO VOLUME 57 .....................................................................................

xi

myc Family Oncogenes in the Development of Normal and Neoplastic Cells RONALDA . DEPINHO.NICOLESCHREIBER.AGUS. AND FREDERICK w . ALT I . Introduction ................................................................................................... I1. Structure and Organization of myc Family Genes and Transcripts .......... 111. General Features of myc Family Proteins ..................................................... Iv. myc Family Protein Structure-Function Relationships .............................. V Additional Members of the myc Family ........................................................ VI. myc Family Gene Expression in Viuo and in Vdtro VII . Regulation of myc Family Gene Expression ................................................. VIII . Oncogenic Activities of myc Family Oncogenes .......................................... IX. Future Prospects ............................................................................................ References ......................................................................................................

.

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

1 3 5 6 13 14 17 24

34 38

Transcriptional and Transforming Activities of the Adenovirus E l A Proteins THOMAS SHENK AND JANE I. I1. 111. N.

FLINT

Introduction ................................................................................................... Organization and Products of the E1A Gene ............................................. Transactivating Activity of E1A Proteins ..................................................... Transforming Activity of E1A Proteins ........................................................

47 48 50

73

vi

CONTENTS

V.

Perspectives .................................................................................................... References ......................................................................................................

78 79

Mitogenic Action of Lysophosphatidic Acid WOUTER H . MOOLENAAR I . Introduction ................................................................................................... I1. Phosphatidic and Lysophosphatidic Acid: Biosynthesis and Cellular Metabolism ..................................................................................................... 111. Lysophosphatidic Acids as Mitogens ............................................................ N . Mechanism of Action ....................... V. Other Biological Effec VI. Possible Biological Function and Future Prospects ................................... References ......................................................................................................

87 88 91 92 99 99

102

Expression and Interactions of the Src Family of Tyrosine Protein Kinases in T Lymphocytes JOSEPH

B . BOLEN.PETERA. THOMPSON. ELISAEISEMAN.AND IVAN D . HORAK

I. Introduction ........................... I1. The Src Family of Tyrosine Pr ................. 111. Patterns of Expression ............ Iv. Involvement of the Src Family in Signal Transduction during T Cell Activation ........................................................................................................ V . Involvement of the Src Family in Signal Transduction during T Cell Proliferation ................................................................................................... ............................................................................... VI. Conclusions References ......................................................................................................

103 104 112

120

131 140 140

implicating the bcrlabl Gene in the Pathogenesis of Philadelphia Chromosome-Positive Human Leukemia GEORGE Q. DALEY AND YINON BEN-NEW I. I1. 111. N. V.

VI.

Introduction and Background ..................................................................... The Molecular Characterization of the Philadelphia Chromosome ........ From v-abE to c-abl and Back Again ............................................................... a61 in Human Malignancy ............................................................................. Biological Activity of Bcr/Abl in Vitro .......................................................... Animal Models of Phl-Positive Leukemias .................................................. References ......................................................................................................

151 154 156 163 169 173 179

CONTENTS

vii

Oncoprotein Kinases in Mitosis

DAVIDSHALLOWAYAND SURESH SHENOY I . Introduction ................................................................................................... I1. Protooncoprotein Tyrosine Kinases (PTKs) in Mitosis ............................. I11. c-Mos. a Serine/Threonine Kinase. in M Phase ......................................... IV Phosphatases and M Phase ........................................................................... V. Discussion ....................................................................................................... References ......................................................................................................

.

185 188 207 211 213 216

Current Status of the BCR Gene and Its Involvement with Human Leukemia MARTIN

I . Introduction

L. CAMPBELL AND RALPH B . ARLINGHAUS

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

11. The BCR Gene ................................................................................................ 111. BCR Complexes in Hematopoietic Cells .....................................................

l-v.

Concluding Remarks and Future Directions .............................................. References ......................................................................................................

227 232 243 252 254

p53 and Human Malignancies VARDA ROTTERAND

MIRONPROKOCIMER

I . Introduction ................................................................................................... I1. Biological Activity of the p53 Protein in Malignant Transformation ....... 111. Proposed Function ........................................................................................ References ......................................................................................................

257 263 269 269

Directed Plasminogen Activation at the Surface of Normal and Malignant Cells JARI POLLANEN. ROSS W

. STEPHENS. AND ANTTI VAHERI

I . Introduction ................................................................................................... I1. Adhesive Interactions of Cell Surfaces with Extracellular Matrices ......... 111 Proteolytic Modulation of the Extracellular Compartment by the Plasminogen Activation System .................................................................... Iv. The Principle of Directed Cell Surface Plasminogen Activation .............. V. Concluding Remarks ..................................................................................... References ......................................................................................................

.

273 275 284 300 313 314

...

vlll

CONTENTS

Epstein-Barr Virus-Associated Lymphoproliferative Disorders in lmmunocompromised Individuals J .ALERO THOMAS. MARTINJ . ALLDAY. AND DOROTHY H . CRAWFORD I. Epstein-Barr Virus ......................................................................................... I1. EBV-Associated B Lymphoproliferative Disorders ..................................... I11. Burkitt's Lymphoma ...................................................................................... Iv. EBV-Associated Lymphoproliferative Disorders in Immunocompromised Individuals .............................................................................. V. The X-Linked Lymphoproliferative Syndrome .......................................... VI. EBV-Related Lymphoproliferative Diseases Associated with Miscellaneous Immunodeficiency States ..................................................... VII . Lymphoproliferative B Cell Disorders in Organ Transplant Recipients ................................................................................... VIII. NHL Associated with Acquired Immunodeficiency Syndrome ................ IX. EBV and Hodgkin's Disease .......................................................................... X . EBV-Associated Tumors in Nonhuman Primates ....................................... XI . Summary and Future Considerations for EBV-Associated Lymphoproliferative Diseases ....................................................................... References ......................................................................................................

330 333 333 336 337 343 345 364 367 371 372 373

ADF. A Growth-Promoting Factor Derived from Adult T Cell Leukemia and Homologous to Thioredoxin: Involvement in Lymphocyte Immortalizationby HTLV-I and EBV JUNJI YODOI AND

THOMAS TURSZ

I . Introduction ................................................................................................... I1. Identification and Characterization of ADF ............................................... 111. Structural and Functional Analyses of ADF ................................................ Iv. Biological Activities of A D F .......................................................................... V Role of ADF in HLTV-I-Induced ATL ......................................................... VI. Role of ADF in EBV-Induced Immortalization of B Cells ......................... VII. Role of ADF in Lymphocyte Activation and Transformation ................... VIII . Interaction between Virus-Infected Cells and Their Environment .......... References ......................................................................................................

.

381 384 392 397 402 403 404 406 407

ix

CONTENTS

The Hunt for Endogenous Growth-Inhibitory and/or Tumor Suppression Factors: Their Role in Physiological and Pathological Growth Regulation OLAV HILMAR IVERSEN I. Introduction ................................................................................................... 11. Regulation of Growth Generally .................................................................. 111. Endogenous Growth Inhibitors .................................................................... IV. Speculations on Chalones and the Treatment of Cancer References ......................................................................................................

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

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

413 415 424

447 449

455


CONTRIBUTORS TO VOLUME 57

Numbers in parentheses indicate the pages on which the authors’ contributionsbegin.

MARTINJ . ALLDAY, Department of Clinical Sciences, London School of Hygzene and Tropical Medicine, London WCl, England (329) FREDERICK W .ALT,Howard Hughes Medical Institute and Dqbartments of Biochemist? and Microbiology, Columbia University College of Physicians and Surgeons, New Yo&, Nau York 10032 (1) RALPH B. AUNGHAUS, Department of MolecularPathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 (227) YINONBEN-NERIAH, The Lautenberg Centerfor General and Tumor Immunology, T h Hebrew University, Hadassah Medical School, Jerusalem 91010,Israel (151) JOSEPH B. BOLEN,Laboratoly of Tumor Virus Biology, National Cancer Institute, Bethesda, Malyland 20892 ( 103) MARTIN L. CAMPBELL, Department of MolecularPathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 (227) DOROTHY H . CRAWFORD, Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London WCl, England (329) GEORGE Q. DALEY,Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 (151) RONALD A. DEPINHO, Departments of Microbiology and Immunology and of Medicine, Albert Einstein College of Medicine, Bronx, New Ymk 10461 (1) ELISAEISEMAN, Laboratmy of Tumor Virus Biology, National Cancer Institute, Bethesda, Maryland 20892 (103) JANEFLINT,Dqbartment of Molecular Biology, Princeton University,Princeton, New Jersty 08544 (47)

’

’ Present address:Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, P. 0.Box 4000, Princeton, NewJersey 08543. xi

xii

CONTRIBUTORS TO VOLUME

57

IVAND. HORAK, Laboratory of Tumor Virus Biology and Clinical Phannacology Branch, National Cancer Institute, Bethesda, Marylund 20892 (103) OLAV HILMAR IVERSEN, Institute of Pathology, University of Oslo,0027 Oslo 1, Norway (413) WOUTER H. MOOLENAAR, Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands (87) JARI P ~ L L ~ N E Department N, of Virology, University of Helsinki, 00290 Helsinki, Finland (273) MIRONPROKOCIMER, Department of Hematology, Beilinson Medical Center, Petah Tikva 491 00, Israel (257) VARDA ROTTER,Department of Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel (257) NICOLE SCHREIBER-AGUS, Departments of Microbiology and Immunology and of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461 (1) DAVID SHALLOWAY, Department of Molecular and Cell Biology, The Pennsylvania State University, UniversityPark, Pennsylvania 16802 (185) T n o M A s SHENK,Department of Molecular Biology and Howard Hughes Medical Institute, Princeton University, Princeton, New Jersq 08544 (47) SURESH SHENOY, Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsyvania 16802 (185) Ross W .STEPHENS, Department of Vimlogy, University of Helsinki, 00290 Helsinki, Finland (273) J. ALEROTHOMAS, Imperial Cancer Research Fund, Royal College of Surgeons Histopathology Unit, London WC2A 3PN, England (329) PETER A. THOMPSON, Labmatory of Tumor Virus Biology, National Cancer Institute, Bethesda, Malyland 20892 (1O?) THOMAS TURSZ, Institut Gustave-Roussy, 94805 Villejuif Cedex, France (381) AN^ VAHERI, Department of Virology, University of Helsinki, 00290 Helsinki, Finland (273) JUNJI YODOI,Institute for Virus Research, Kyoto University, Kyoto, Japan (381)

Present address: Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.

myc FAMILY ONCOGENES IN THE DEVELOPMENT OF NORMAL AND NEOPLASTIC CELLS Ronald A. DePinho,* Nicole Schreiber-Agus,* and Frederick W. Altt Departments of Microbiology and Immunology and of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461

t Howard Hughes Medical Institute and Departments of Biochemistry and Microbiology Columbia University College of Physicians and Surgeons, New York, New York 10032

1. Introduction 11. Structure and Organization of m y Family Genes and Transcripts 111. General Features of m y Family Proteins IV. myc Family Protein Structure-Function Relationships A. Dimerization, DNA-binding, and Transactivation Domains B. Nuclear Localization C. Phosphorylation D. Transforming Activity V. Additional Members of the myc Family VI. m y Family Gene Expression in Vivo and in Vztro A. Correlation of m y Family Gene Expression with Events of Differentiation B. m y Family Gene Expression in Cell Culture Differentiation Systems VII. Regulation of myc Family Gene Expression A. Mechanisms Governing myc Family Gene Expression B. Regulation of myc Gene Expression in Transgenic Mice C. Autoregulation and Cross-Regulation among m y Family Genes VIII. Oncogenic Activities of myc Family Oncogenes A. Mechanisms of m y Gene Deregulation in Spontaneously Arising Tumors B. In V i m Models of Oncogenesis C. Tissue-Preferential Expression of m y Family Genes during Normal Development Correlates with Tumor Distribution IX. Future Prospects A. Production of null myc Mutations in the Mouse Germ Line B. The Use of Transdominant Mutant Proteins for the Study of m y Function C. Identification of Potential Cellular Targets Transactivated by myc Proteins References

I. Introduction

The myc family of cellular oncogenes, c-my, N-myc, and L-myc, encodes three highly related nuclear phosphoproteins. Although the exact function of m y family proteins has not been determined, they are thought to 1 ADVANCES IN CANCER RESEARCH, VOL. 57

Copyright 6 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

RONALD A. DEPINHO ET AL.

be important in the regulation of normal cellular growth and differentiation. Support for this possibility derives from the findings that myc family genes exhibit unique expression patterns during mammalian development and that dramatic changes in their expression coincide with critical developmental transitions in many cell lineages. Moreover, myc family oncoproteins are localized to the nucleus and possess significant homology to known sequence-specific transcription factors andfor differentiation factors-further suggesting that myc-encoded oncoproteins may serve to regulate specific gene expresion during growth and differentiation. T h e study of myc family genes began in the context of malignant disease. T h e first member of this family, c-myc, was identified as the cellular homologue of the retroviral v-myc oncogene responsible for leukemia in chickens (Sheiness and Bishop, 1979; Sheiness et al., 1980). lnterest in the c-myc gene escalated when it was discovered that this protooncogene was translocated and overexpressed in various lymphoid malignancies (Neuberger and Calabi, 1983).A strong causal link between c-myc deregulation and malignancy was subsequently established both by the frequent deregulation of c-myc in many different types of cancers (reviewed by Cole, 1986) and by the ability of c-myc expression vectors to function as potent transforming agents in primary cultured cells and in transgenic mice. Additional myc family members were subsequently identified in human tumors as highly expressed and amplified genes with homology to my. N-myc and L-myc genes were isolated from human neuroblastomas (Schwab el al., 1983; Kohl et al., 1983) and from small-cell lung carcinomas (Nau et al., 1985), respectively, and subsequently were found to be capable of transforming primary cultured cells and producing tumors in transgenic mice. Thus, the aberrant structure and expression of myc family genes in a variety of neoplasias clearly indicate that myc gene activation can play a role in tumorigenesis. In recent years, a predominant focus in the study of myc biology has been the role of myc family genes in normal cellular growth and differentiation. Although myc family genes share many structural and functional features, their conservation as distinct sequences in evolution indicates important and unique biological activities (Battey et al., 1983; Stanton et al., 1984, 1986; Kohl etal., 1986; DePinhoetal., 1986,1987; van Beneden et al., 1986; King et al., 1986; Legouy etal., 1987; KayeetaE., 1988; Collum and Alt, 1990; Sawai et al., 1990). A clear assay for myc functions has remained elusive. Therefore, many current studies have focused on determining the pattern of m y family gene expression during development and characterizing mechanisms that govern differential m y family gene expression. In this article, we review the structural and functional

myc FAMILY

ONCOGENES

3

features of myc family genes and their products and discuss these properties in relation to known o r suspected roles of myc in normal mammalian development and in malignant disease.

II. Structure and Organization of rnyc Family Genes and Transcripts T h e m y family genes have been mapped to different regions in the human and mouse genomes: c-myc is on human chromosome 8 and mouse chromosome 15, N - m y is on human chromosome 2 and mouse chromosome 12, and L-myc is on human chromosome 1 and mouse chromosome 4 (Crews et al., 1982; Dalla-Favera et al., 1982b; Taub et al., 1982; Nee1 et al., 1982; Kohl et al., 1983; Schwab et al., 1983; Nau et al., 1985; Campbell et al., 1989). Each m y family gene is organized similarly into three exons, encoding multiple transcript species generated through the variable usage of transcriptional start sites and polyadenylation signals as well as through alternative mRNA processing (Fig. 1). I n the human and mouse c-myc gene, transcriptional initiation is fixed at either of two distinct promoter sites, designated P1 and P2, which are positioned downstream from classical TATA boxes and are located approximately 150 bp apart (Battey et aE., 1983; Bernard et al., 1983). Transcriptional initiation from either the P1 or P2 promoter has been shown to influence the processivity of RNA polymerase I1 through c-myc exon 1 sequences (see later). Transcription of the N-myc gene proceeds from multiple sites, spanning a region of 10 bp in the mouse gene (DePinho et al., 1986) and of several hundred base pairs in the human gene (Kohl et al., 1986). T h e 5' structure of the human L-myc transcript is less welldefined, but transcription appears to initiate predominantly from one site (Kaye et al., 1988). Multiple myc transcript forms can also result from alternative processing of the primary mRNA. Several human L-myc transcripts derive from the alternative processing of the first intron and/or the differential use of adenylation signals located in the second intron (Kaye et al., 1988; R. A. DePinho and F. W. Alt, unpublished observations). A shortened L-myc transcript generated from the use of the second-intron polyadenylation signal has been detected in normal tissues (R. A. DePinho and F. W. Alt, unpublished observations) and in small-cell lung carcinomas (Kaye et al., 1988). This transcript is structurally similar to that of another myc-related gene, B-myc, in that each lacks sequences encoded by the exon 3 coding domain (Ingvarsson et al., 1988; see Section V). Transcripts of the human N-myc gene can be generated by splicing from either of two donor splice sites within the first exon to a single second-exon acceptor site (Stanton

4

RONALD A. DEPINHO E T AL.

c-myc

N-myc TAA

AUG

N - ~ Y Tgene .

AAAA

3.0-kb transcnpt

MM

2.8-kb transcript

L-myc (AUG)

AUG

alternale polyA+ signals

TAA

L - r n gene

AMA

A-A-AA

3 6-kb transcript

2.0kb transcnpt

V-AAA,~, I .bkb iranscript

FIG. 1. Alternative forms of m y family transcripts.

and Bishop, 1987). With the exception of the c-myc P1- and P2-initiated transcripts, differences in regulation or function of various myc transcript species have not been established. A notable structural feature of myc family genes is the high degree of nucleic acid conservation present in the large 5' and 3' untranslated

myc FAMILY

ONCOGENES

5

regions (Bernard et al., 1983; DePinho et al., 1986, 1987). The 5' and 3' untranslated regions are unique to individual m y family members, but are evolutionarily conserved for a given m y gene (DePinho et al., 1986, 1987) thus suggesting potential regulatory roles. As outlined below for the c-my gene, sequences that comprise the 5' untranslated region appear important for regulation of transcriptional elongation, and the 3' untranslated sequences appear to be involved in post transcriptional control. It is possible that these distinct genetic regions may play a role in regulating differential expression of the c-, N-, and L-my genes in development and in different tumor types (see Section VII,A). Ill. General Features of myc Family Proteins

The myc family proteins are related, short-lived nuclear phosphoproteins that have been shown to exist in several forms (Abrams et al., 1982; Donner et al., 1982; Alitalo et al., 1983a; Hann et al., 1983; Hann and Eisenman, 1984; Ramsay et al., 1984; Dani et al., 1984; Persson et al., 1986; Sullivan et al., 1986). In addition to differential phosphorylation, multiple forms are derived from the alternative use of translational initiation codons that lie in single open reading frames. The c-mycencoded protein occurs in two major forms, each encoded by the same open reading frame and initiated from one of two in-frame codons (Hann et al., 1988). Translational initiation from an AUG codon positioned in exon 2 yields a 64-kDa protein. Initiation from a CUG codon located near the 3' end of exon 1 yields a protein of 67 kDa; its larger size results from additional amino-terminal residues encoded upstream of the AUG. The functional significanceof multiple protein species has not been determined. However, these two forms of the c-myc protein are evolutionarily conserved from Xenopus to human; this finding is consistent with essential and perhaps separable physiological roles (King et al., 1986). Deregulation of the c-myc gene in Burkitt's lymphoma is frequently accompanied by point mutations that alter or eliminate the larger CUG-initiated open reading frame (Hann et al., 1988). A relationship between these findings and the pathogenesis of Burkitt's lumphoma has not been established. The N-myc-encoded protein also exists in two major forms, one 65 kDa and the other 67 kDa (Ramsay et al., 1986; Slamon et al, 1986; Ikegaki et al., 1986). Synthesis of these alternative N-myc species initiates from one of two in-frame AUG codons positioned near the 5' end of exon 2 (Makela et al., 1989).Two L-myc-encodedproteins have been identified in human lung cancer cells (DeGreve et al., 1988).A smaller 60-kDa form of the protein initiates from an AUG codon near the 5' end of the second

6


exon. The basis for a larger 66-kDa species is less clear, but it may result from initiation at one of two in-frame CUG codons in the first intron or from posttranslational modifications such as phosphorylation. Because the first intron is involved in alternative splicing, the production of these larger proteins could be subject to regulation through differential R N A processing of first-intron sequences (DeGreve at al., 1988; Kaye et al., 1988). A smaller, cytoplasmic L-myc-encoded protein, measuring 34/37 kDa in size and resulting from translation of the shortened L-myc mRNA that lacks the third exon, has recently been identified in human small-cell lung carcinoma (SCLC) cell lines (Ikegaki et al., 1989). The role of and regulation of this smaller species have not been determined.

IV. myc Family Protein Structure-Function Relationships

The myc family genes share regions of protein sequence homology throughout their second and third exon protein-coding domains (Bernard et al., 1983; DePinho et al., 1985, 1987). These regions of homology are interspersed with stretches of completely divergent sequences. There exist two highly conserved regions encoded within exon 2 and three within exon 3 of all three myc family genes (Fig. 2). The N- and c-mycencoded proteins share two additional regions of homology that are not present in the L-myc-encoded protein (DePinho et al., 1987). The L - m y coding region lacks a major internal stretch in the downstream portion of the second exon coding domain, a region previously noted to be subject to structural variation between myc-related proteins (Ralston and Bishop, 1983; Kohl et al., 1986). Homologies to other nuclear proteins of known function and various mutagenesis analyses have begun to provide some insight into the potential physiological significance of some of the conserved regions of myc-encoded proteins. The general findings of these studies are outlined below.

A. DIMERIZATION, DNA-BINDING, AND TRANSACTIVATION DOMAINS The most extensive region of homology among c-, N-, and L-myc gene products is found near the carboxy-terminal portion of the various proteins. This region contains two structural motifs previously identified in transcription and differentiation factors, namely, the leucine zipper (Landschulz et al., 1988) and the helix-loop-helixlbasic region motifs

RQRRNELKRSF

l I

t

human c-myc mouse c-myc chicken c-myc v-myc Xenopus c-myc fish c-myc human N-myc mouse N-myc human L-myc mouse ~ - n y c

r

I 1

human c-myc mouse c-myc chicken c-myc v-myc Xennpur c-myc fish c-myc human N-myc mouse N-myc human L-myc mouse L-myc MyoD1, myogenin, daughterless.twist, achaete-scuteTI,T4,T5 and T8,Lyl- 1, Ig enhancer binding proteins E2,E12, E47. pE3 Ig enhancer binding protein

c-Fos.c-Jun, JunB, Fral, CEBP.GCN4, kE3 Ig enhancer binding protein

FIG. 2. The myc family oncoprotein structure-function relationships and sequence comparisons. Domains are numbered 1-4 and correspond to human c-myc residues 1-104, 105-143, 144-320, and 32 1-439, respectively. A potential transcriptional activation domain has been localized in the human c-myc protein residues 1-143. CK-ll sequences were identified in human c-myc residues 240-26 1 and 342-357. Potential CK-I1sequences in the other myc proteins were identified by sequence homology. These sequences were determined to be residues 240-261 and 342-357 in mouse c-myc, residues 217-238 and 319334 in chicken c-my, residues 217-238 and 319-334 in v-myc, residues 211-236 and 323-338 in Xenopus c-myc, residues 203-233 and 312-327 in fish c-myc, residues 248-275 and 369-384 in human N-myc, residues 248-273 and 367-382 in mouse N-myc, residues 269-284 in human L-my, and residues 273-288 in mouse L-myc. Nuclear localization sequences (M1 and M2) were identified in the human c-myc (320-328 and 364-374), mouse c-myc (320-328 and 364-374), chicken c-myc (297-305 and 341-351), v-my (297-305 and 341-35 I), Xenopus c-myc (300-308 and 348-358), fish c-my (288-296 and 334-344), human N-myc (345-353 and 39 1-401), mouse N-myc (343-351 and 389-399), human L-myc (291-301), and mouse L-myc (295-305) proteins. Helix-loop-helix sequences were identified in the human c-myc (364-407), mouse c-myc (364-407), chicken c-my (343-387), v-myc (343-387), Xenopus c-myc (345-389), fish c-myc (334-377), human N-myc (39 1-434), mouse N-myc (389-432), human L-myc (291-334), and mouse L-myc (295-338) proteins. Other proteins possessing a helix-loop-helix motif are listed at the bottom. The leucine repeat sequences were identified in the human c-myc (406-439), mouse c-myc (406-439), chicken c-myc (386-417), v-myc (386-417), Xenopus c-myc (388-419), fish c-myc (376-409), human N-myc (433-464), mouse N-myc 431-462), human L-myc (333-364), and mouse L-myc (337-368) proteins. Other proteins possessing a leucine repeat are listed at the bottom.

8


(Murre et al., 1989b). These structures are found in a contiguous arrangement, with the leucine zipper located at the carboxy terminus and the helix-loop-helix just amino terminal to the leucine zipper. In addition, this portion of the proteins also contains a region rich in basic amino acid residues that overlaps the amino-terminal end of the helix-loophelix motif. These major landmarks in the myc family proteins are diagrammed in Fig. 2. 1. The Leucine Zipper Structure

The leucine zipper motif was first identified in the C/EBP protein, a rat DNA-binding transcription factor, and subsequently was found in additional proteins by inspection of existing sequences (Landshulz et al., 1988). To date, the leucine zipper motif has been found in several nuclear oncoproteins and/or known sequence-specific transcription factors, for example, myc, c-fos, cjun,jun B,fra-l, C/EBP and GCN4. The leucine zipper structure consists of a linear array of four to five leucine residues extending at every seventh position along one side of an amphipathic a helix (Fig. 2). Dimerization appears to be stabilized by hydrophobic interactions between the leucine repeat surfaces of opposing (Y helices and by putative interhelical salt bridging between charged residues positioned more laterally (Landschulz et al., 1988; O’Shea et al., 1989). T h e helices of paired polypeptides are assembled in a parallel configuration (i.e., mirror image), an arrangement that positions the DNA-binding basic region of each polypeptide on the same side of the protein dimer (O’Shea et al., 1989; Gentz et al., 1989). For tested proteins, the leucine zipper appears to be indispensable for polypeptide dimerization and DN A-binding activity (Sassone-Corsi et al., 1988a, b; Rauscher et al., 1988; Halazonetis et al., 1988; Kouzarides and Ziff 1988; Gentz et al., 1989; Turner and Tjian, 1988; Ransone et al., Neuberg et al., 1989; Schuermann et al., 1989). Homodimer o r heterodimer formation can occur between leucine zipper-containing polypeptides, such as those encoded by fos and j u n (Kouzarides and Ziff, 1988). T h e cjun protein and C/EBP and GCN4 have been shown to form homodimers and to bind DNA directly, whereas the c-fos protein appears to associate indirectly with DNA via its leucine zipper-mediated dimerization with the cjun protein or the fosassociated antigen, p39 (Rauscher et al., 1988; Halazonetis et al., 1988; Sassone-Corsi et al., 1988b; Kouzarides and Ziff, 1989; Nakabeppu et al., 1988; Gentz et al., 1989; Turner and Tjian, 1988; Neuberg et aZ., 1989; Schuermann et al., 1989). The DNA-binding activity of the cjun protein is greatly enhanced when associated with the c-fos protein (Kouzarides and Ziff, 1988, 1989; Rauscher et al., 1988; Sassone-Corsi et al., 1988a;

myc FAMILY ONCOGENES

9

Halazonetis et al., 1988; Schuermann et al., 1989). Similarly, the putative interaction between myc proteins and DNA could occur either directly (as in the case of C/EBP) or indirectly through a collaborative DNA-binding factor (as with c-fos protein). The latter types of associations could serve to broaden the range of transcriptional activities of given sets of factors (Landshulz et al., 1988). Mutational analyses have demonstrated that leucine zipper-mediated honiodimerization is required for c- and N-myc transforming activity (Dang et al., 1989; Nakajima et al., 1989). Furthermore, c-myc and N-myc heterodimers have been observed in in vitro cotranslation assays (J.M. Bishop, personal communication). However, not all leucine zipper regions cross-mix. C/EBP will not combine withfos, jun, or myc proteins, suggesting that there is specificity within the leucine repeat domain (S. McKnight, personal communication). 2. The Helix-Loop-HelixlBasic Region The helix-loop-helix (HLH)/basic region is positioned immediately amino terminal to the leucine repeat region and consists of two amphipathic helices joined by an intervening loop as well as an adjacent region rich in basic amino acid residues (Murre et al., 1989a) (Fig. 2).This motif, shared by all three myc proteins, is common to a number of proteins involved in cellular differentiation and proliferation, a list of which includes muscle differentiation factors MyoD and myogenin (Davis et al., 1987; Edmunson and Olson, 1989); immunoglobulin K enhancerbinding proteins E l 2 and E47 (Murre et al., 1989a); the product of a gene translocated in some T cell leukemias, Lyl-1 (Mellentin et al., 1989); and Drosophila differentiation factors achaete-scute (T3, T4, T5, and T8), daughterless, and twist proteins (Villares and Cabrera, 1987; Alonso and Cabrera, 1988; Caudy et al., 1988; Thisse et al., 1988). The HLH region element has been shown to play a crucial role in the dimerization activity of the MyoD1 factor (Lassar et al., 1989) and E l 2 and E47 (Murre et al., 1989a, b) proteins. The basic residues adjacent to the HLH region appear to generate the DNA contact surface in the MyoD1 factor (Lassar et al., 1989); the comparable organization suggests that this region may function similarly in the myc family proteins. Cellular target sequences of the myc family proteins have not yet been identified. However, a recently developed in uitro technique, known as “selected and amplified binding sequence imprinting” (Blackwell and Weintraub, 1990), has determined that a potential c-myc homodimer core binding sequence is CACGTG (Blackwell et al., 1990). Difficulties in defining the sequence-specific interactions may be the consequence of interactions mediated by associated proteins (as is the

10


case with the c-fos protein) or of requirements for associated proteins to promote DNA-binding activity. I n the past, the characterization of noncovalently associated proteins has failed to uncover specific protein interactions (Eisenman, 1989). However, a novel protein, encoded by max, has been found to oligomerize with the helix-loop-helix/leucine repeat region of the c-myc protein ( R . N . Eisenman, personal communication). Further characterization of max-myc protein interactions as well as identification of additional associated proteins represent important steps in the elucidation of myc family function. 3. Possible Significance of Two Dimerization Mot+ an the myc Protein The myc proteins and an immunoglobulin enhancer binding protein, TFE3 (Beckman et al., 1990), are the sole examples of proteins that contain apparently functional (and adjacent) leucine zipper and HLH motifs. T h e presence of both of these dimerization motifs in myc proteins raises the interesting possibility that myc dimers joined at the leucine zipper may simultaneously couple with accessory proteins via the HLH. These additional interactions could serve to modulate the activities or specificities of myc proteins. Another, not mutually exclusive, possibility is that binding via one domain may hinder the dimerization activity of the other. Recently, an HLH protein devoid of a functional basic region, termed Id, was found to dimerize with the MyoDl-encoded protein and abrogate its sequence-specific binding and transcriptional activities (Benezra et al., 1990). 4. Transcsptzonal Activation Properties of c-myc Amino-Terminal Residues The amino-terminal region of the c-myc protein, previously shown to be required for neoplastic transformation, appears to possess transactivation activities (Kato et al., 1990). When fused to the DNA-binding domain of the yeast GAL4 protein, three regions derived from the amino-terminal portion of the c-my protein (residues 1-41,41-103, and 103-143) were capable of activating transcription of a reporter gene bearing GAL4-binding sites. Taken together, these studies support the concept that myc family proteins function to regulate the transcriptional activities of cellular genes important in the growth of malignant cells. B. NUCLEAR LOCALIZATION T h e myc family proteins are localized to the nucleus. Amino acid residues that direct nuclear targeting and retention of the c-myc protein

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have been identified through deletion analysis and gene fusion experiments (Stone et al., 1987; Dang and Lee, 1988).Two domains, designated M1 (residues 320-328) and M2 (residues 364-374), seem to be involved (Fig. 2). The M I region, which consists of only eight amino acids, appears to be the dominant nuclear-targeting signal and bears homology to the SV40 large T and polyomavirus large T nuclear signals (Kalderon et al., 1984; Richardson et aE., 1986).The M1 region is sufficient for targeting a cytoplasmic muscle pyruvate kinase protein to the nucleus (Dang and Lee, 1988). Deletion of these residues from the c-myc protein results in weak nuclear targeting. A similar sequence is present in the N-myc protein and appears to function as a strong nuclear-targeting signal (Dang and Lee, 1989). The L-myc protein does not contain M1homologous sequences; thus, it may be directed to the nucleus through entirely novel signal sequences or through associated proteins containing nuclear-targeting signals. The M2 sequence is conserved in all three myc proteins and exhibits some nuclear-targeting ability, but does not appear to be a primary determinant of myc nuclear localization (Dang and Lee, 1988). M2 sequences can only partially target the pyruvate kinase protein to the nucleus, and deletion of these sequences from c-myc results in mutant proteins that are still retained predominantly in the nucleus (Dang and Lee, 1988). Because M2 residues are contained within the basic region, the weak nuclear-targeting capacity could be secondary to protein/DNA interactions. C. PHOSPHORYLATION The myc family proteins are heavily phosphorylated, yet little is known about the structural sites and functional ramifications of their phosphorylation. Reversible phosphorylation of m y proteins could serve as an imporant link between signal transduction pathways and differential m y protein activity. The identification of signal transduction processes that lead to changes in myc protein phosphorylation and activity would provide insight into how stimuli acting at the cell surface bring about pleiotropic cellular responses. Characterization of kinases that phosphorylate myc proteins and identification of myc protein residues subject to this modification have been recent undertakings. These studies focused on the cytoplasmic protein kinase, casein kinase I1 (CK-II), which is capable of generating shortterm reversible signals within the nucleus and could potentially serve as a link between receptorltransducer signals and the m y protein. Avian and human c-myc proteins can serve as substrates for reversible phosphorylation by CK-I1 at two different sites (Luscher et aE., 1989). One CK-I1 site

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in the human c-myc protein is located immediately amino terminal to the basic region (residues 342-357) and is conserved in the N- and L-my proteins. The proximal location of this CK-I1 site could potentially influence basic region-substrate interactions. The second CK-I1 site (residues 240-262) is only weakly conserved in N- and L-my. Similar CK-I1 substrate motifs shared by other nuclear oncoproteins (including c-myb and c-fos proteins, ElA, and SV40T antigen) may point to common pathways that influence the activity of a network of nuclear regulatory proteins (for reviews, see Hunter, 1987; Eisenman, 1989). For example, recent findings (Luscher et al., 1990) indicate that CK-IImediated phosphorylation of c-myb protein inhibits its sequence-specific binding activity in a reversible manner. This CK-I1 phosphorylation site, which lies immediately amino terminal to the DNA-binding domain, has been deleted in nearly all oncogenically activated myb proteins. Deletion of the myb-CK-I1 phosphorylation region resulted in DNA binding that was independent of CK-I1 activity and consequently uncouple4 from growth factor-dependent CK-I1 requirements. Although the I c-myc protein contains a CK-I1 domain in an equivalent position relative to its putative DNA-binding domain, the deletion of this domain does not appear to result in enhanced transforming activity in vitro (Street et al., 1990). D. TRANSFORMING ACTIVITY The m y family genes have been shown to possess oncogenic activity in uitro in the rat embryo fibroblast (REF) cooperation assay (Land et al., 1983; Schwab et al., 1985b; Yancopoulos et al., 1985; DePinho et al., 1987; Legouy et al., 1987; Birrer et al., 1988) and the Rat-1 assay (Stone et al., 1987; Small et al., 1987). In the REF assay, c- N- or L-myc expression constructs are cotransfected with an activated H-ras gene into primary REFSand assayed for appearance of transformed foci in the REF monolayer. In the Rat- 1 assay, immortalized but nontransformed Rat- 1 fibroblasts are neoplastically transformed by transfection with a m y expression construct alone. Differential activity of various mutant c-myc proteins in these assays has uncovered four functionally distinct domains: domain 1, residues 1-104; domain 2, residues 105-143; domain 3, residues 144-320; and domain 4, residues 321-439 (Fig. 2) (Stone et al., 1987). These studies demonstrated that whereas domains 2 and 4 (the latter contains the leucine zipper and HLH motifs) were indispensable for transforming activity in both assays, domains 1 and 3 were less essential. Contrasting activities in the two assays were observed with the domain 3 mutants; these mutants were quite active in the cooperation assay but

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nonfunctional in the Rat-1 assay. These data suggest that regions of c-my important for cotransformation may not be identical to those necessary for transformation of established cell lines. Alternatively, they may reflect some unknown inconsistencies in the different assays. These findings have been noted to be in accord with the observation that v - m y mutants in a domain 3-equivalent region become incompetent in macrophage transformation but can still transform fibroblasts (Ramsay and Hayman, 1982; Heaney et al., 1986).

V. Additional Members of the myc Family

The myc family contains a number of less well-characterized genes and pseudogenes that bear significant sequence homology to the c-myc, N-myc, and L-myc genes. Two human L-myc pseudogenes designated L-myc-psi (DePinho et al., 1987)and p-myc (Alt et al., 1986)have been isolated on the basis of third exon homology to the L-myc gene. Both genes have been mapped to the X chromosome (Huebner et al., 1991). L-myc-psi is a processed pseudogene equivalent of the L-myc gene and has been shown to be nonfunctional in the REF assay. The p-my gene contains only a portion of the 3’ untranslated region of the L-my gene and is also nonfunctional in the REF assay. Neither the L-myc-psi nor the p-myc gene is expressed in normal developing tissues or in many tumor tissues analyzed to date (R.A. DePinho, unpublished results). Recently, two myc-related genes, namely, B-myc (Ingvarsson et al., 1988) and s-myc (Sugiyama et al., 1989), have been identified in the rat genome. The B-myc gene structure appears to contain second but not third exon sequences and is very similar in organization to the shortened L-my transcript described above. Notably, the putative B-myc protein contains highly conserved exon 2-encoded regions but lacks the leucine zipper and HLH motifs. The B-myc gene bears significant sequence homology to the c-my gene throughout its coding and intron sequences. B-myc is expressed in adult tissues, particularly the central nervous system. Its physiological significance remains to be established.The rat s-myc gene contains significant homology to the second and third exon of the N-myc gene (Sugiyama et al., 1989).The s-myc coding domain appears to consist of a single processed exon. Although expression of the endogenous s-my gene has not yet been detected, an s-my transcript has been detected following the transfection of the s-my gene into a rat tumor cell line. More importantly, the expression of the transfected s-myc gene seems to suppress the tumorigenic phenotype of the tumor cell line (Sugiyama et al., 1989).

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VI. myc Family Gene Expression in Vivo and in Vitro OF myc FAMILY GENEEXPRESSION A. CORRELATION WITH EVENTS OF DIFFERENTIATION

The myc family genes exhibit distinct patterns of expression with respect to cell type and developmental stage. Dramatic and distinctive changes in their expression are associated with key developmental transitions in many cell lineages. Their differential expression supports the view that each myc gene plays important and unique roles in cell growth and differentiation. 1. Early Embryogenesis

During gastrulation, changing patterns of c- and N-myc expression coincide with both the emergence and the differentiation of major embryonic structures (Downs et al., 1989). For instance, the emerging primitive streak contains high c-myc expression and barely detectable N-myc expression. As the primitive streak matures, c-myc expression becomes reduced and N-myc expression increases significantly. N-myc is also abundantly expressed in the undifferentiated mesoderm but is downregulated in structures derived from the primitive mesoderm, such as the heart and somites. These findings raise the possibility that the onset of enhanced expression of c-myc in the emerging primitive streak or of N-myc in undifferentiated mesoderm may be involved in the development of these germ layers and that their subsequent down-regulation may serve to guide further differentiation in these tissues (Downs et al., 1989).One of the most unexpected findings in early embryogenesis was the observation that high-level c-myc expression does not appear to be a necessary correlate of cellular proliferation. Only a limited subset of actively dividing embryonic cell types possessed abundant c-myc transcripts in the first trimester of human embryonic development (PfeifferOhlsson et al., 1985). Similarly, barely detectable c-myc expression was observed in the intensely proliferative primitive ectoderm of the early mouse embryo (Downs et al., 1989). In contrast, rapidly proliferating cell types in older embryos consistently exhibit high c-myc expression (Schmid et al., 1989). Throughout embryogenesis, N-my and L-myc expression has not been found to correlate well with proliferation in many embryonic cell types (Mugrauer et al., 1988; Mahon et al., 1991).

2. Midgestation Contrasting patterns of c-myc, N-myc, and L-myc expression have also been observed throughout early and late organogenesis (Grady et al.,


15

1987; Mugrauer et al., 1988; Schmid et al., 1989; Mahon et al., 1991; DePinho et al., 1991). During histogenesis, the c-myc gene appears to be expressed in a wide variety of cell types, with enhanced c-myc expression generally paralleling high proliferative activity in most, but not all, cell types (Pfeiffer-Ohlsson et al., 1985; Schmid el al., 1989; Downs et al., 1989). In contrast, high-level expression of N-myc and L-myc appears to be limited to a number of undifferentiated cell types and correlates well with active differentiation but poorly with proliferation uakobovits et al., 1985; Grady et al., 1987; Mugrauer etal., 1988; Mahon et al., 1991).These points are typified in the developing forebrain, where postmitotic neuroblasts that have entered proliferative arrest and have commenced differentiation down-regulate c-myc gene expression but continue to actively express N-myc and L-myc genes until differentiation is completed. Enhanced expression of N-myc has also been observed in differentiating postmitotic lens fiber cells (Mahon et al., 1991) and neuronal cells of the sensory retina (Grady et al., 1987; Mugrauer et al., 1988; Mahon et al., 1991). These findings suggest that N-myc and L-myc expression correlates more strongly with differentiative rather than proliferative processes in these cell types. Similar observations have been made in the developing human fetus during the second trimester (DePinho et al., 1991). Although m y family genes have overlapping patterns of expression during early organ formation, differences in their cell type-specific expression become more readily apparent as differentiation progresses. For example, both N-myc and L-my are widely expressed throughout the primitive brain and neural tube but are differentially expressed in the developing central and peripheral nervous system (Mahon et al., 1991). At later stages of differentiation, N - m y is expressed primarily in the forebrain, midbrain, and spinal and sympathetic ganglia, whereas L-myc is expressed primarily in the hindbrain and is undetectable in the spinal and sympathetic ganglia. Differential myc family gene expression has also been observed in the developing lymphoid system (Zimmerman et al., 1986; R.A. DePinho and F.W. Alt, unpublished results). The c-myc gene is expressed at all stages of murine B and T cell development, but expression of the N-my gene is found only in the immature stages of lineage development. These findings suggest that alterations in the expression of specific myc family genes may be important in terminal differentiation processes in certain cell types. This view is supported by the observation that the aberrant expression of myc has been associated with abnormal cellular differentiation in transgenic mice. For example, forced late-stage expression of N-myc in B lymphoid cells (Rosenbaum et al., 1989; Dildrop et al., 1989; Ma et al., 1989) or overexpression of L-myc in lens fiber cells (Bidder et al., 1991) has been shown to affect normal differentiation in these cell lineages.

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B. myc FAMILY GENEEXPRESSION IN CELLCULTURE DIFFERENTIATION SYSTEMS

Several well-defined in uitro differentiation systems have served as models for the study of regulated m y expression during terminal differentiation (Friend et al., 1971; Martin, 1975, Fuchs and Green, 1981; Jones-Villeneuve et al., 1983; Edwards et al., 1983; Reitsma et al., 1983; Gonda and Metcalf, 1984; Lachman and Skoultchi, 1984; Thiele et al., 1985; Dony et al., 1985; Dean etal., 1986; Endo and Nadal-Ginard, 1986). The best-defined systems are the mouse erythroleukemia cell line for erythrocyte differentiation (Friend et al., 197 l), the HL-60 promyelocytic cell line for macrophage differentiation (Reitsma et al., 1983), human neuroblastoma cell lines for neuronal differentiation (Thiele et al., 1985), and the P19 embryonal carcinoma cell line, which has the capacity to differentiate along neuronal or myogenic pathways depending on the inducing agent (Jones-Villeneuve et al., 1983; Edwards et al., 1983). In most of these systems, myc expression decreases following exposure to differentiation stimuli. In the HL-60 and MEL cells, down-regulation of c-myc gene expression appears to be a necessary genetic event for terminal differentiation (Reitsma et al., 1983; Lachman and Skoultchi, 1984). The importance of down-regulation in the establishment of the differentiated phenotype has been demonstrated by the finding that constitutive c-my expression proved effective in blocking induction of terminal differentiation in MEL cells (Coppola and Cole, 1986; Dmitrovsky et al., 1986). In addition, the production or introduction of c-myc antisense nucleotide sequences appeared to accelerate or induce terminal differentiation in MEL (Prochownick et al., 1988) and HL-60 promyelocytic cells (Yokoyama and Imamoto, 1987; Holt et al., 1988). Significantly, the decrease in c-myc expression occurs prior to any measurable changes in cellular proliferation and prior to the appearance of the differentiated phenotype. In some systems, down-regulation of c-myc expression is biphasic. In MEL cell differentiation, c-myc expression decreased rapidly following treatment with inducing agents, returned to near baseline levels, and eventually disappeared as the cells underwent proliferative arrest (Lachman and Skoultchi, 1984). These findings suggest that the early decrease in the expression of c-myc is more closely associated with differentiation rather than proliferation in these cells. Similar observations have been made in 12-0-tetradecanoylphorbol- 13acetate (TPA)-induced differentiation of primary keratinocytes (Dotto et al., 1986) and in EGTA-induced differentiation of the L6E9 myoblast cell line (Endo and Nadal-Ginard, 1986). The cell type-specific pattern of myc family gene expression observed

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during normal embryogenesis is consistent with lineage-specificroles for each myc member. Correspondingly, different patterns of myc family expression have been associated with different developmental pathways in the P19 system. A significant reduction in N-myc expression precedes neuronal differentiation but is not observed during myogenesis (Morgenbesser et al., 1991). Down-regulation of N-myc expression also accompanies retinoic acid-induced differentiation of human neuroblastoma cell lines (Thiele et al., 1985, 1988)and the F9 teratocarcinoma cell line (Finkelstein and Weinberg, 1988). In the F9 cell lines, c-myc and N-myc appear to be differentially regulated. In contrast to the late-stage down-regulation of c-myc, N-myc expression is rapidly and transiently down-regulated (Finkelstein and Weinberg, 1988). A similar pattern is observed in retinoic acid-treated neuroblastoma cell lines (Thiele et al., 1985).The return of N-myc expression to basal levels at advanced stages of differentiation suggests that the early modulation of N-myc expression is specific to the differentiation process in these cell lines. VII. Regulation of myc Family Gene Expression

A. MECHANISMSGOVERNING myc FAMILY GENEEXPRESSION Expression of c-myc is controlled at several levels, including transcriptional initiation, transcriptional attenuation, and posttranscriptional processes. Much of what is known about regulation of myc gene expression has come from studies of c-my expression in tumor cells (for review, see Marcu, 1987),with relatively few investigations into factors that regulate c-myc in vim or into factors that regulate differential expression of the N- and L-myc genes. Investigations into N-myc regulation have been limited by difficulty in reproducing regulated expression in vitro. 1. Transcriptzonal Initiation Transcriptional regulation of the c-myc gene can be accomplished at the level of initiation, transcriptional attenuation, or a combination of the two processes. Positive and negative regulatory elements appear to modulate c-myc promoter utilization in a number of conditions. For instance, an increase in transcriptional initiation occurs in the response of quiescent cells to mitogenic stimuli (Greenberg and Ziff, 1984; Levine et al., 1986),whereas repression of initiation takes place at late stages of HL-60 and MEL cell differentiation (Grossoand Pitot, 1985; Nepveu et al., 1987; Siebenlist et al., 1988).Repression of transcription initiation also accounts for the down-regulation of the unrearranged c-myc allele in Burkitt’s

18


lymphomas and mouse plasmacytomas (Showe and Croce, 1987) and for the down-regulation of c-myc expression in B cell tumors that express high levels of an N-myc transgene (Ma et al., 1991). T h e cis-acting sequence elements and mechanisms that direct regulation of c-my gene expression have been examined extensively. T h e two c-myc transcription initiation sites, P1 and P2, are preceded ty TATA boxes and by consensus binding sites for a variety of known transcriptional activators, including Spl (Asselin et al., 1989),a “CAAT” box factor (NF-l/CTF-l), AP-1 (Hay et al., 1989; Takimoto et al., 1989), and a plasmacytoma-specific c-myc transcriptional repressor factor (myc-PRF) (Kakkis and Calame, 1987). Most of these consensus binding sequences correspond to DNase I hypersensitivity sites in transcriptionally active c-myc genes (Kelly and Siebenlist, 1986).The binding of sequence-specific factors has been documented in the case of the Spl transcription factor, which binds at two sites upstream of the P1 promoter in the c-myc gene (Asselin et al., 1989). Consensus Spl binding sites are also located in the N- and L-myc promoters, but Sp 1 interaction with these sequences has not been proved (Kohl et al., 1986; DePinho et al., 1987; Kaye et al., 1988). Thefos-jun complex and octamer-binding proteins have also been found to bind to upstream c-myc promoter sequences previously shown to function in a negative fashion (Hay et al., 1989; Takimoto et al., 1989). The physiological significance of the fos-jun and octamer-binding protein interactions has yet to be determined. The myc-PRF protein is a plasmacytoma-specific factor that binds to the c-myc promoter (Kakkis and Calame, 1987) and causes a 30-fold reduction in transcriptional initiation (Kakkis et al., 1989). This interaction may account for some of the transcriptional repression of the silent, unrearranged myc allele in plasmacytomas (Kakkis et al., 1989).

2. TranscriptionalAttenuation Measurements of RNA polymerase 11 transcriptional activity across the length of the c-myc gene have established that a block to transcriptional elongation exists near the 3‘ end of the first exon of both the murine and human genes (Bentley and Groudine, 1986a; Mechti et al., 1986; Nepveu and Marcu, 1986; Eick and Bornkamm, 1986). The regulation of this block has been shown to be a key factor with respect to myc family gene expression in cultured cells and in developing tissues. An increase in transcriptional blockage correlated well with the reduction in c-myc expression accompanying HL-60 (Bentley and Groudine, 1986a; Cleveland et al., 1988) and MEL (Eick and Bornkamm, 1986) differentiation in vitro. In contrast, a decrease in attenuation was found to be associated with elevated c-my expression in mitogen-stimulated mono-

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nuclear cells (Eick et al., 1987). A loss of transcriptional attenuation correlated with overexpression of c-myc in Burkitt’s lymphomas (Showe and Croce, 1987; Spencer et al., 1990) and with enhanced c- and L-myc expression in small-cell lung carcinoma cell lines (Krystal et al., 1988). The modulation of transcriptional attenuation appears to contribute to the regulation of all three myc genes during normal mammalian development (Xu and DePinho, 1991). Varying degrees of attenuation have been correlated with the level of steady-state expression of each myc family gene in a number of tissues and developmental stages. For instance, a significant increase in attenuation is associated with downregulation of N-myc expression during mouse forebrain development. In the adult liver, undetectable N-myc expression is associated with active transcriptional initiation and a prominent block to transcriptional elongation near the 5’ end of exon 1. A significant block to transcriptional activity was also present in the L-myc gene but remained unchanged despite large changes in steady-state mRNA levels during forebrain and liver development. Similarly, significant changes in c - m y expression were associated with only mild alterations in transcriptional blocking in these tissues. During B cell differentiation, regulation of N-myc expression appears to result differentially from either control of transcriptional initiation of attenuation, depending upon developmental stage. The downregulation of N-myc expression that occurs when precursor lymphocytes differentiate into mature B cells results from a corresponding decrease in transcriptional initiation (Ma et al., 1991). However, stimulation of precursor B lymphocytes with the pre-B-specific growth factor IL-7 is accompanied by a dramatic increase in N-myc expression that appears to result largely from the removal of a block in transcriptional elongation (Morrow et al., 1991). The DNA sequences and mechanisms responsible for transcriptional attenuation in eucaryotic genes have not been fully identified, but possible candidates include thymidine tracts, secondary structures in the RNA or DNA, and/or specific transcription factors (for reviews, see Yanofsky, 1988; Spencer and Groudine, 1990). The sequences that mediate premature termination of c-myc gene transcription appear to be orientationdependent elements located near the 3’ end of exon 1 (Bentley and Groudine, 1986a, b, 1988; Eickand Bornkamm, 1986;Chungetal., 1986, 1987; Cesarman et al., 1987). Transcriptional attenuation regions in the c-myc gene have been mapped and verified by the Xenopzls oocyte injection system (Bentley and Groudine, 1988), by in vitro transcription assays utilizing purified RNA polymerase I1 (Kerppola and Kane, 1988),and by transfection studies with gene fusion constructs designed to assay attenu-

20


ation activity (Bentley and Groudine, 1988; Wright and Bishop, 1989). These studies indicated that the truncated c-my transcripts terminate in one of two T-rich regions flanking the exon llintron 1 border that lies downstream to potential stem-loop structures; these elements may or may not be essential for termination (Bentley and Groudine, 1988). Similar intragenic transcriptional attenuators have been identified in a number of procaryotic (Platt, 1986) and eucaryotic (Yanofsky, 1988) genes. The c-myc attenuation regions, which closely resemble rhoindependent transcription terminators in procaryotes, have by themselves been capable of blocking processivity of transcription of heterologous genes (Wright and Bishop, 1989).Although these sequences appear to bind nuclear factors (Remmers et al., 1986; Yang et al., 1986; Marcu, 198’71, the potential role of these DNA/protein interactions in modulating transcriptional elongation remains speculative. Recent studies have demonstrated that regulation of c-myc attenuation can be accomplished by the selective utilization of one promoter versus another. Mutagenesis to abrogate transcriptional initiaion at either the P 1 or P2 promoter of the human (Spencer et al., 1990)and murine (Miller et al., 1989) c - m y genes has demonstrated that initiation at P1 results in read-through transcription, whereas initiation at P2 leads to attenuated transcription. That promoter utilization affects the attenuation state of the c-myc gene is supported by the nature of m y deregulation in Burkitt’s lymphoma-elimination of transcriptional blockage was correlated with a shift from P2 to P1 use (Spencer et aL, 1990). However, modulation of attenuation in differentiating cells, such as HL-60, appears to be promoter independent, indicating that multiple factors may govern transcriptional activity through the c-myc gene (Bentley and Groudine, 1986a; Spencer et al., 1990). The site of transcriptional attenuation in the N-myc gene has been localized near the 5‘ end of exon 1, a region that contains a potential stem-loop structure followed by a T stretch (Xu and DePinho, 1991). L-myc attenuation sequences reside near the 3‘ end of exon 1, an area devoid of a significant thymine tract (Krystal et al., 1988; Xu and DePinho, 1991).

3. Posttranscriptional Regulution In addition to transcriptional regulation, mechanisms that modulate the turnover of short-lived myc mRNAs can be a potent factor in controlling steady-statelevels. In most cultured cells, the half-life of c-myc mRNA has been found to be approximately 15-20 min (Hann and Eisenman, 1984; Ramsay et al., 1984; Dani et al., 1984), although half-lives of 1 hr have been observed in several Burkitt’s lymphoma cell lines (Piechaczyket

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al., 1985; Rabbitts et al., 1985). Half-lives for the N- and L-myc full-length mRNAs are 30 and 45 min, respectively (Sejersen et al., 1987; Saksela, 1987). I n some cells, regulation of c-myc mRNA stability proved to be the dominant mechanism governing c-myc expression. For example, Chinese hamster fibroblasts, which actively transcribe the c-myc gene at all times, maintained high levels of c-myc mRNA only after growth factor-induced stabilization of the transcripts (Blanchard et al., 1985). In F9 embryonal carcinoma cells, decreased c-myc expression resulted from destabilization of the c-myc mRNA (Dony et al., 1985). Sequences located in the 3’ untranslated region of c-myc appeared to mediate rapid mRNA turnover (Bonnieu et al., 1988; Jones and Cole, 1987). T h e increased stability of c-myc mRNA following cycloheximide treatment in some cell types suggests that mRNA destabilization may be accomplished by labile protein factors (Dani et al., 1984). Cytosolic fractions derived from erythroleukemia cells specifically mediated the decay of polysome-bound c-myc and c-myb mRNA but not globin o r histone mRNA (Brewer and Ross, 1989). In contrast, steady-state L-myc mRNA levels decreased following treatment with cycloheximide, indicating that labile factors have a stabilizing effect on L-myc turnover (Saksela, 1987).

B. REGULATION OF myc GENEEXPRESSION IN TRANSGENIC MICE Insight into the mechanisms directing myc expression have been derived principally from in vitro studies examining the regulation of myc family gene expression during the growth and differentiation of cultured cells. More recently, transgenic mouse technology has been utilized to examine the mechanisms and sequence elements that govern stagespecific and tissue-specific regulation of myc family genes in the physiological context of a developing animal (Zimmerman et al., 1990; DePinho et al., 1991).These studies have indicated that expression of a transfected rnyc gene in cultured cells can be quite different from its expression in transgenic mice. I n particular, transfected N-my genes have tended to be expressed when transfected into cell lines, regardless of whether the endogenous N-myc gene is expressed (Zimmerman et al., 1990). Yet, the same genomic clones introduced into the germ line of transgenic mice can, in some cases, yield apparently normal expression patterns, with the transgene being down-regulated at appropriate developmental times in various tissues (Zimmerman et al., 1990). T h e nature of the mechanisms that provide the apparently “proper” expression of the transgenic N-myc gene in some mouse lines has not been determined. T h e ability to attain appropriate expression in some

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transgenic lines suggests that most of the cis-acting sequence elements required for regulated expression are present within the N-myc genomic clones employed (those containing 4 kb of 5’ and 3 kb of 3’ flanking sequences). Thus, the inability to readily reproduce N-myc regulation in cultured cells suggests that factors mediating this regulation may only be transiently present during cellular differentiation and that their appearance is an early developmental event (Zimrnerman et al., 1990). Alternatively, the possibility remains that larger genomic clones may contain elements that will permit regulation of the transgene in a manner similar to that of the endogenous gene in all cases. In this regard, the finding that introduced N - m y (Zimmerman et al., 1990) and L-myc (DePinho et al., 1991) genes were properly regulated only in a subset of transgenic mice indicates that the regulation of N- and L-myc transgene expression is highly sensitive to position effects and further suggests that additional elements may be required for consistent proper expression. Such location-dependent effects have been observed with a number of transgenes, including the Hox-1.3, Hox- 1.4, and a 1 acid glycoprotein genes (Dente et al., 1988; Vassar et al., 1989),and have been ascribed to neighboring elements such as nuclear matrix attachment sites and topoisomerase-binding sites, among others (for reviews, see Palmiter and Brinster, 1986;Jaenisch, 1988; Moroy et al., 1990).Thus, further analysis of the regulation of N- and L-myc expression may require either the introduction of larger genomic fragments or the direct manipulation of sequences in endogenous myc loci by gene targeting strategies (see later).

C. AUTOREGULATION AND CROSS-REGULATION AMONG myc FAMILY GENES Deregulated expression of an endogenous c-myc gene in tumors as a result of translocations or retroviral insertions often is associated with lack of expression of the unaltered endogenous c-myc gene (Stanton et al., 1983; Adams et al., 1985). Endogenous c-my expression is also downregulated in lymphoid tumors that arise as the result of deregulated c-my transgene expression in transgenic mice carrying a c-myc gene under the control of the Ig heavy-chain transcriptional enhancer element (Eke-myc mice; see later). In general, such “autoregulation” of endogenous c-my expression has been found to be mediated at the transcriptional level (Farhlander et al., 1985; Bentley and Groudine, 1986a; Kakkis et al., 1986; Mango et al., 1989; Eick and Bornkamm, 1989; Penn et al., 1990). However, attempts to suppress normal c-myc transcription by transfection of c-myc or v-myc constructs into established cell lines have yielded conflicting results; in some cases down-regulation of endogenous genes

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was observed and in others it was not (Keath et al., 1984a,b; Rapp et al., 1985; Coppola and Cole, 1986; Stone et al., 1987; Cleveland et al., 1988; Richman and Hayday, 1989; Penn et al., 1990). Deregulated N-myc expression also is associated with the lack of endogenous c-myc or N-myc expression in a number of tumors that express very high N-myc levels, including neuroblastomas with amplified N-myc genes (Kohl et al., 1984) and lymphoid tumors from EpN-myc transgenic mice (Dildrop et al., 1989; Rosenbaum et al., 1989). The absence of endogenous c-my expression in tumor cells that express very high N-myc levels is referred to as “cross-regulation” of myc gene expression. Crossregulation of c-myc gene expression by deregulated N - m y expression has also been demonstrated to occur at the level of transcriptional initiation in EpN-my lymphoid tumors (Ma et al., 1991). Down-regulation of normal c-myc expression by a deregulated c-myc gene can occur when the deregulated gene is expressed at levels comparable to the normal endogenous expression levels. However, downregulation of c-myc expression in the context of deregulated N-myc expression almost always appears to require highly elevated N-myc expression levels compared to the levels found in normal N-myc-expressing cells (i.e., the levels found in neuroblastomas with highly amplified N-myc genes). The latter finding has been interpreted to indicate that a “threshold” level of N-myc expression is required to effect down-regulation of c-myc expression (Bernards et al., 1986; Penn et al., 1990). However, transfection experiments in which very high N-my levels were achieved from transfected expression vectors did not always lead to c-myc downregulation. Likewise, Abelson murine leukemia virus (A-MuLV)transformed pre-B lines generated from Epc-myc and EpN-myc transgenic mice did not show down-regulation of endogenous myc gene expression despite extremely high EpN-myc expression levels in the EpN-myc A-MuLV transformants (Ma et al., 1991). Another possible explanation for the auto- and cross-regulatory phenomena is that downregulation of normal my gene expression is not a direct effect of deregulated myc gene expression but rather is an indirect effect perhaps related to participation of the deregulated myc gene in the transforming process (which for N-myc would require very high expression levels). In this context, neither the Epc-myc nor EpN-myc gene appeared to participate in the transformation process with respect to generation of A-MuLV transformants from the corresponding transgenic mice (Langdon et al., 1989). A general model for the down-regulation of endogenous myc genes in transformed cells in which myc has participated in the transformation process emerges from the consideration that essentially all known tumors

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express c-myc except those in which a deregulated myc gene was involved in the transformation process. Expression of the endogenous c-myc genes in dividing tumor cells can be explained if activated transforming genes in those tumors must act through a signaling pathway in which myc is a downstream participant. The expression of c-myc in A-MuLV transformants may represent such a case in which signals generated by the deregulated abl gene ultimately are effected in part through myc-related pathways. However, tumors with directly deregulated rnyc genes would not require endogenous myc expression; in such tumors, lack of the unaltered endogenous myc gene expression would result from normal upstream growth regulaton signals from which the deregulated gene escapes. A correlate of this model in the context of cross-regulation is that N-my can replace c-myc function in tumor cells; the much less frequent activation of N-myc and the fact that it usually is accompanied by large increases in expression relative to activated c-myc genes may imply that additional alterations in the N-myc protein and/or its absolute expression levels are required for N-myc to fully mimic c-my activities. A final prediction from this model is that tumors that lack endogenous c-myc expression will likely have been contributed in part from the action of a deregulated rnyc gene or a deregulated gene that can replace rnyc activity. In this context, the lack of endogenous c-myc expression in Wilms’ tumors that have extremely high-level N-myc expression from a single-copy (unamplified) endogenous N-myc gene supports the possibility that deregulated N-myc expression may be involved in the transformation process in that tumor (Nisen et al., 1986).

VIII. Oncogenic Activities of rnyc Family Oncogenes A. MECHANISMS OF myc GENEDEREGULATION IN SPONTANEOUSLY ARISINGTUMORS Spontaneously arising tumors often harbor aberrantly expressed, and hence activated, m y genes. c-myc deregulation is associated with a vast range of neoplasias (Kelly and Siebenlist, 1986). N- and/or L-myc deregulation is confined to a narrower subset (Alt et al., 1986). Mechanisms for deregulated c-myc expression include chromosomal translocation, gene amplification, and proviral insertion (Kelly and Siebenlist, 1986). In spontaneously arising tumors, activation of the N- and L - m y genes has only been clearly implicated to date by the mechanism of gene amplification (Kohl et al., 1983, 1984; Schwab et al., 1983, 1984; Lee et al., 1984; Nau et al., 1986). However, more recently, N-myc activation through viral insertion has been observed in M-MuLV-induced murine T cell lympho-

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mas (van Lohuizen et al., 1989). Each of these mechanisms will be discussed below with regard to specific tumor types and transformed states. 1. Translocations Reciprocal translocations between the c-myc gene and the immunoglobulin loci occur in murine plasmacytomas and human Burkitt's lymphomas (for reviews, see Leder et al., 1983; Croce and Nowell, 1985). Similar translocation events have been documented in human B cell acute lymphocytic leukemia (B-ALL) (Peschle et al., 1984),rat immunocytomas (Sumegi et al., 1983), and various other lymphoid malignancies. Most c-my translocations in these B lymphoid tumors involve the Ig heavy-chain switch regions or regions within the Ig heavy- or light-chain variable region gene segment locus. Therefore, is has been theorized that the translocation events result from low-frequency aberrant attempts at normal Ig variable region gene o r class switch recombination events that lead to activation of the resulting translocated myc gene and the subsequent selection of the cell in which the translocation occurred in the context of transformation. Further support for this theory derives from the finding that the type of tumor observed correlates well with the translocation of c-my into an actively rearranging variable region locus or switch region associated with particular B cell stages-early B cell tumors are often associated with rearrangements to the JH (variable region) locus; B cell tumors often have rearrangements into the p heavy-chain switch regions; plasma cell tumors (representing later stage cells) often have rearrangements to downstream switch regions. Nevertheless, because variant translocations that appear to involve regions very distal to the rearranging portions of light-chain loci have been noted, the exact mechanism of c-myc translocation in some lymphoid tumors requires further study (for review, see Cory, 1986). c-myc translocations can be categorized into three distinct classesthose that truncate or somatically mutate the 5' coding region of the protooncogene (Leder et al., 1983; Gelmann etal., 1983), those that occur within the 5' proximal region of the gene (Leder et al., 1983), and those that involve regions distal to the c-myc locus (Dalla-Favera et al., 1983; Leder et al., 1983; Croce and Nowell, 1985). Though the mechanism by which these various translocations contribute to lymphoid malignancies is not fully understood, proposed possibilities include (1) mutations within the protein-coding regions of the translocated allele, (2) disruptions of regulatory regions, (3) changes in protein expression patterns, and (4) activation of normally silent c-my alleles (Taub et al., 1984a; Rabbitts et al., 1984; Wiman et al., 1984; Hayday et al., 1984; Croce and Nowell, 1985).

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Loss of first-exon sequences or mutations clustered at the 3‘ end of the first exon can result in the altered production of the larger c-myc protein that accompanies a number of Burkitt’s lymphomas (Hann et al., 1988). The mechanism behind and ramifications of this altered protein expression pattern remain to be determined. First-exon sequence mutations or deletions may also affect regions of transcriptional attenuation and subsequently lead to deregulated expression of the translocated c-my gene. Bypassing of transcriptional blocking in various Burkitt’s lymphomas can be the consequence of either a mutated attenuator region (Cesarman et al., 1987) or a switch to the P1 read-through promoter (Taub et al., 1984a; Richman and Hayday, 1989; Spencer et al., 1990). Clustered mutations within the first intron have been associated with decreased binding of a nuclear factor in five of seven Burkitt’s lymphoma lines analyzed (ZajacKaye et al., 1988). However, it is currently doubtful that this factor contributes to the regulation of c-my expression at the level of transcriptional attenuation; its precise role awaits further characterization. Whereas steady-state expression levels of the translocated c-myc gene in Burkitt’s lymphoma and murine plasmacytoma are comparable to those of other actively dividing B cells (Taub et al., 1984a; Keath et al., 1984a), the untranslocated allele of the c-myc gene is not expressed in either of these tumor types (Fahrlander et al., 1985; Kakkis et al., 1986; Cory, 1986). Silent, unrearranged c-my alleles may reflect the normal state in the tumor progenitor cell population, with neoplasia resulting from translocations that lead to deregulation. 2. Gene Amp Lification Gene amplification is another mechanism by which myc expression often is activated in various tumors. Amplification of the N-myc gene is a common feature of neuroblastomas, retinoblastomas, small-cell lung carcinomas, and other tumors of neuronal and neuroendocrine origin (Kohl et al., 1983, 1984; Schwab et al., 1983, 1984; Lee et al., 1984; Nau et al., 1986). A subset of small-cell lung carcinomas harbor amplifications of the L-myc gene (Nau et al., 1985). I n contrast, c-my gene amplification is associated with a much broader range of tumor types and transformed cultured cell lines (Collins and Groudine, 1982; Dalla-Favera et al., 1982a; Little et al., 1983; Alitalo et al., 198313; Nowell et al., 1983; Schwab el al., 1985a; Yokota et al., 1986). Amplification of m y genes generally occurs in tumors representing normal cell types in which the endogenous m y gene is expressed and corresponding amplification is accompanied by a corresponding increase in expression levels of the amplified gene over that of the normal, unamplified endogenous genes. Amplification of


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N-myc genes is invariably associated with down-regulation of endogenous c-myc gene expression in neuroblastomas; yet, the high-level N-myc expression observed in these tumors obviously does not alter expression of the amplified N-myc genes. Therefore, high-level expression of N-myc does not lead to autoregulation of amplified N-myc genes, as is observed for normal endogenous N-myc genes in EpN-myc tumors that express high levels of the N-myc transgene. This finding suggests the possibility of additional alterations in amplified genes (or cells that harbor these) that could allow them to escape such autoregulatory phenomena. Of interest is the finding that myc gene amplification correlates well with clinical prognosis of a number of tumors (Seeger et al., 1985). For example, advanced stage I11 and IV primary neuroblastoma tumors generally exhibit significant N-myc gene amplification, whereas amplification is limited in stage I and I1 tumors and absent in spontaneously regressing stage IV-S tumors (Brodeur et al., 1984).N-myc gene amplification correlates with progression-free survival rates as well (Seeger et al., 1985),and thus provides a useful marker for determining neuroblastoma tumor stage as well as for predicting clinical outcome. Levels of c-myc amplification often correspond with degree of tumorigenic potential. Highly malignant small-cell lung carcinomas bear levels of c-myc 20- to 70-fold greater than those of more benign counterparts (Little et al., 1983;Johnson et al., 1987).Subclones of the murine osteosarcoma SEWA cell line, which display potent tumorigenicity in nude mice, consistently do not lose their amplified copies of c-myc during in vitro growth (Schwab et al., 1985a; Alt et al., 1985; Martinsson et al., 1988). The association of N-myc amplification with advanced-stage tumors is consistent with amplification occurring as the result of a continual selection process, as is observed with respect to gene amplification in the context of drug resistance (Alt et al., 1978).However, it is not yet clear that early-stage neuroblastomas are progenitors of late-stage neuroblastomas.

3. Proviral Insertion Activation of the c-my locus in a number of B and T lymphoid tumors results from proviral insertion into myc-associated regions. Avian leukosis virus integration either upstream or downstream of the c-myc gene results in B cell lymphomas that exhibit steady-state c-myc expression levels 20to 100-fold greater than those of normal bursa1 B cells (Hayward et al., 1981; Payne et al., 1982). Murine leukemia virus insertion 5’ or 3’ to the c-myc gene gives rise to T cell lymphomas that display increases in c-myc expression ranging from 5- to 30-fold times that of unaffected c-myc genes (Corcoran et al., 1984; Selten et al., 1984). MuLV integration

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into the conserved 3' untranslated region of N-myc has been recently identified in a subset of virally induced T cell lymphomas (van Lohuizen et al., 1989). Localized to a 100-bp span within this putative regulatory region, these proviral insertions result in elevated levels of hybrid N-mycl viral long-terminal-repeat (LTR) transcripts (van Lohuizen et al., 1989). These MuLV-induced T cell lymphomas are the sole examples of lymphoid malignancies resulting from deregulated N-myc.

B. INVrvo MODELS OF ONCOGENESIS Transgenic mouse technology has provided an in vivo model system in which to study the function and regulation of many cellular oncogenes in developmental processes and in oncogenesis (for review, see Moroy et al., 1991).T h e principal strategy has invoived khe introduction of cellular or viral oncogenes driven by their own promoters or linked to tissue-specific promoter/enhancer elements that target expression of an oncogene product to specific tissues. Deregulated, tissue-specific expression of oncogene products has generated mouse models that develop spontaneous neoplasms and/or congenital abnormalities in the targeted tissues. For example, introduction of either c-myc, c-neu, or Ha-ras genes fused to the mouse mammary tumor virus (MMTV) promoter/enhancer led to overexpression of the oncogene product in mammary tissue and to increased incidence of mammary adenocarcinoma (Stewart et al., 1984; Sinn et al., 1987; Muller et al., 1988). Preferential expression and tumorigenic activity of these transgenes in female breast tissue resulted from prolactogenic and/or glucocorticoid stimulation of the MMTV element in pregnant or lactating female mice. Analogous transgene experiments have resulted in the tumorigenic conversion of pancreatic exocrine cells with the rat elastase I gene promoter/enhancer fused to the Ha-ras gene (Quaife et al., 1987), the formation of lens tumors with aA crystalline promoter/ enhancer fused to the SV40 large T antigen (Mahon et al., 1987), and the development of lymphoid neoplasia with the immunoglobulin heavychain enhancer (Ep) element fused to m y family oncogenes (Adarns et al., 1985; Suda et al., 1987; Schmidt et al., 1988; Yukawa et al., 1989; Dildrop et al., 1989; Rosenbaum et a[., 1989; Moroy et al., 1991). Tumorigenesis is thought to be a multistep process in which preneoplastic stages precede development into a full tumor phenotype. The multistep process of tumorigenesis progresses from normal, controlled growth to immortalization and finally to complete malignant transformation. Immortalized cells are capable of continued cell growth in culture; but, unlike fully transformed cells, immortalized cells are an-


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chorage dependent and fail to generate tumors in syngeneic hosts. Evidence from gene transfer studies in cultured cells has suggested that the stepwise process culminating in full transformation results from the sequential activation of oncogenes (Land et al., 1983). This phenomenon appears to be partially recapitulated in the cooperation assay system by the demonstration that primary rat embryo fibroblasts were immortalized by the transfection of an activated myc gene (Land et al., 1983) but were malignantly transformed by the cotransfection of both activated myc and mutant H-ras genes (Land et al., 1983; Yancopoulos et al., 1985; Schwab et al., 198513; DePinho et al., 1987; Legouy et al., 1987; Small et al., 1987; Birrer et al., 1988). In addition, several transgenic models systems have examined the oncogenic activities of myc in a variety of cell lineages, particularly lymphoid cells. The kinetics of disease onset and occurrence in transgenic mice that have a heritable form of malignancy confirm that additional genetic or epigenetic factors cooperate with the inherited oncogene to bring about fully transformed phenotypes. This multihit concept is supported by the emergence of tumors following a long latency period, by the common occurrence of clonal tumors, and by the stochastic kinetics of tumor appearance. Correspondingly, “double-transgenic” mice, carrying both myc and ras oncogenes, developed multiple tumors after a greatly shortened latency period in comparison with “single-transgenic”mice (Sinn et al., 1987). However, even in the double-transgenic mice, the stochastic appearance of tumors and the presence of some latency period indicated that activation of more than two oncogenes may be required for the development of a tumor. 1. myc Transgenes and Lymphold Cancer As described above, myc family genes have been examined in the context of both normal and transformed lymphoid cells. N-myc is expressed in pre-B cells, but expression is down-regulated when pre-B cells mature into surface antigen receptor-positive B cells (Zimmerman et al., 1986). Likewise, c- and N-myc appear to be coexpressed in pre-T cells, but while c-my continues to be expressed, N-myc expression ceases as T cells become fully mature (R.A. DePinho and F.W. Alt, unpublished observations). L-myc expression has generally not been found at any stage of lymphoid development except in a tumor line apparently representing an early T cell precursor (R.A. DePinho and F.W. Alt, unpublished observations). To compare the biological roles of c-, N-, and L-myc in lymphoid malignancy and normal development, each gene was placed under control of the immunoglobulin heavy-chain transcriptional

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enhancer (Ep) element and introduced into transgenic mice. The E p element dominantly drives expression of associated genes in B cells and in T cells as well (Grosschedl et al., 1984). T h e Ep-myc studies extended from observations that mouse plasmacytomas and human Burkitt’s lymphomas consistently possessed translocations that often juxtaposed the c-myc gene and the powerful transcriptional enhancers of the immunoglobulin locus, thus resulting in deregulation and overexpression of the translocated c-myc gene (Leder et al., 1983; Croce and Nowell, 1985; Cory, 1986). The high frequency of this genetic aberration in these tumors coupled with the known transforming potential of the c-my gene strongly suggested a correlation between IgH-mediated deregulation of c-myc gene expression and the generation of these malignancies. T o test this hypothesis directly, a construct bearing the c-my gene under Ep element control was introduced into transgenic mice (Adams et al., 1985; Langdon et al., 1986; Alexander et al., 1987). Dramatically, all of the Epc-myc transgenic mice developed pre-B and B cell lymphomas, thereby establishing a causal role for c-my deregulation in the genesis of B lineage malignancies. However, in most animals, tumor onset was variable among littermates and was often delayed into adulthood. In addition, despite the fact that the E p element was active in most cells of the lymphoid system, emerging tumors were monoclonal or oligoclonal. These finding indicated that additional genetic factors beyond myc deregulation were required for full malignant transformation. Genetic background appears to be an important determinant in the duration of tumor latency and in the type of tumor generated by the Epc-myr transgene. While tumorigenesis in SJLiJ mice was so rapid that propagation of the line was difficult, tumor formation in a C57BL/6 background occurred at a significantly slower rate (Harris et al., 1988). In C3H mice, the Epc-myc transgene led to the exclusive production of T cell tumors, despite comparable transgene expression in both T and B cells. It is possible that the C3H strain possesses either a genetic predisposition to T cell tumors or an enhanced resistance to B cell malignancies (Yukawa et al., 1989). Immune function in Epc-myc transgenic mice appeared to be grossly unaffected. During the preneoplastic stage, there was a mild reduction in the number of mature B cells and a reciprocal rise in the pre-B cell population (Langdon et al., 1986; Alexander et al., 1987). Pre-B cells in these mice seemed to cycle more actively in comparison to nontransgenic controls, this evidenced by the significant increase in pre-B cells in the Gz + S phase of the cell cycle and by the absence of typical resting pre-B cells (Langdon el al., 1986; Alexander et al., 1987). These data have led to

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the speculation that myc- induced cycling of pre-B cells can increase the number of cells at risk for additional tumorigenic events that can collaborate with c-myc to effect full malignant transformation. Experimental support for this theory has been derived from transgenic mice carrying both an Epc-myc transgene and a transgene encoding the Ig p heavychain protein (Nussenzweig et al., 1988). These double transgenics exhibited a marked reduction in the incidence of tumor formation in comparison with transgenic mice carrying Epc-myc alone. Normally, maturing B lymphoid cells must first assemble the immunoglobulin heavychain genes and subsequently rearrange the light-chain genes (Alt et al., 1984). It is conceivable that the expression of an already assembled p gene in pre-B cells accelerates the passage of these cells through the B cell differentiation pathway, thus giving rise to a smaller population of pre-B cells. Reduction of the pre-B cell pool may result in fewer targets for genetic events that act synergistically with the activated c-myc transgene to generate tumors. Thus, the absolute number of cycling pre-B cells is seemingly an important factor in tumor development in Epc-myc transgenic mice (Nussenzweig et al., 1988). T h e cooperation of oncogenes has been studied extensively in the Epc-myc transgenic model (Harris et al., 1988). Characterization of spontaneous secondary events in one Epc-myc-inducedtumor cell line demonstrated that activation of the N-ras oncogene by somatic point mutation had taken place (Harris et aL, 1988). In addition, infection of these animals with helper-free retroviruses containing either ras or raf genes resulted in latency periods considerably shorter than those of infected normal mice or of noninfected transgenic mice (Alexander et al., 1989). Abelson murine leukemia virus, in contrast, did not serve to accelerate the onset of tumor development. It is possible that the v-abl gene does not cooperate with c-myc (Alexander et al., 1989; Langdon et al., 1989). Alternatively, this virus may infect very early B cell precursors cells, wherein the Ep element is not yet active. Collaboration between c-myc and v-abl may thus be limited to later differentiation stages, i.e., to 'antibodysecreting plasma cells (Rosenbaum et al., 1990).

2 . D e v e l e e n t a l Consequences of Deregulated N-myc Expression on B Cell Dfferentiation The consequences of deregulated N-myc transgene expression in lymphoid cells seem to be very similar to those observed in Epc-myc transgenic mice (Rosenbaum et al., 1989; Dildrop et al., 1989). Pre-B, B, and rare T cell lymphomas arose in EpN-myc animals as in the Epc-myc animals, albeit at slightly lower rates. The EpN-myc transgene was expressed at high levels in the spleen and low levels in the thymus. In

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EpN-myc tumors, the transgene was expressed at a very high level compared to the endogenous N-myc mRNA levels encountered in pre-B cells. In contrast, only moderate increases in Epc-my transgene expression compared to endogenous c-myc expression in lymphoid cells have been observed. Correspondingly, N-myc expression has been found to be extremely high in most naturally occurring tumors in which the N - m y gene is deregulated (e.g., neuroblastoma, retinoblastoma, and Wilms’ tumor). Therefore, it appears that N-myc requires highly elevated levels of steadystate mRNA to exert its full transforming potential. The above transgenic experiments clearly established that N-myc possesses oncogenic activity in lymphoid cells (Rosenbaum et al., 1989; Dildrop et al., 1989). Because N-myc is normally down-regulated at the pre-B cell to B cell developmental transition (Zimmerman et al., 1986), the EpN-myc transgenic model was utilized to test the consequences of forced late-stage N-myc expression on B cell differentiation (Ma et al., 1989). In EpN-myc mice, the pre-B cell population appeared to be only mildly expanded in comparison to that of Epc-myc mice, suggesting that constitutive expression of the N-myc gene did not block the emergence of B cells (Dildrop et al., 1989; Rosenbaum et al., 1989). However, there were a number of unusual patterns of lymphocyte-specificgene expression in the EpN-mycderived tumor cell lines. Some cell lines had a mixture of pre-B and B cell markers and/or atypical expression and rearrangement patterns of Ig genes (Ma et al., 1989). During normal B cell development, pre-B cells possess active immunoglobulin VDJ recombinase activity whereas B cells do not (Ma et al., 1989). However, EpN-myc B cell tumors exhibited recombinase activity characteristic of the pre-B cell stage (Ma et al., 1989). This finding suggests the intriguing possibility that constitutive N-myc expression in B cells directly or indirectly maintains some pre-B cell properties. 3. Deregulated Expression of the L-myc Gene in Lymphoid Cells of Transgenic Mice

L-my expression has not been clearly defined in any cell of the lymphocytic lineage. Nevertheless, in a manner similar to c- and N-myc, the oncogenic and developmental consequences of L-myc deregulation were examined in transgenic mice. Although the Ep element is active in both B and T cells, and EpL-myc transgene was found to be preferentially expressed in the T cell compartment (Moroy et al., 1990).The preferential expression of L-myc in T cells is consistent with differences in B and T cells in the posttranscriptional regulation of L-myc. The EpL-myc transgene was associated with an expanded thymic cortex and increased T cell areas in the spleen. Though histological and flow cytometric analysis

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pointed to greater numbers of enlarged cells in the thymus, the distribution of subsets bearing surface CD3, CD4, and CD8 markers appeared to be unaffected. Prior to the appearance of tumors, a high proportion of EpL-myc thymocytes were found to possess a surface marker (1C11) normally present on embryonic thymocytes, activated T cells, T cell lymphomas, and preleukemic thymocytes of mice prone to develop thymic lymphomas. These findings are consistent with the possibility that deregulated L-myc expression is associated with abnormal thymocyte development (Moroy et al., 1990). T h e EpL-myc transgene was capable of irrducing thymic T cell tumors, but its tumorigenic potential was less than that of EpN- or Epc-myc transgenes (Moroy et al., 1990). EpL-myc-induced T cell tumors arose at low frequencies following long latency periods. I n addition, the poor tumor transplantability of these rare tumors was consistent with a more mildly transformed phenotype. T o date, deregulation of the L-my gene has not been associated with the genesis of any naturally occurring lymphoid malignancy. These results correlate with the lower transforming efficiency observed for the L-my gene in the REF assay and suggest that the transforming potential of L-myc may be lower than that of the c- or N-myc genes (DePinho et al., 1987; Birrer et al., 1988). An unanticipated finding in EpL-myc mice was the development of a second malignancy, usually manifested in skin tumors composed of monocytic/myeloid tumor cells (Moroy et al., 1990). I n contrast to the less aggressive T cell disease, these skin tumors were highly invasive and lethal in most animals. These tumor cells were found to express high levels of the L-my transgene, a finding in accord with previous observations that the E p element is active in cells of the myeloid and macrophage lineage (Kemp et al., 1980a,b).An intriguing aspect of this disease was the striking age dependency for tumor onset, with tumorigenesis occurring after a latency of one year. The specific age-related events responsible for the generation of EpL-myc-induced skin tumors have not been determined. Notably, similar tumors do not arise in age-matched controls or in mice harboring EpN- or Epc-myc transgenes. C. TISSUE-PREFERENTIAL EXPRESSION OF myc FAMILY GENESDURING NORMAL DEVELOPMENT CORRELATES WITH TUMOR DISTRIBUTION T h e tissue expression of myc family genes during development at least partially correlates with the types of tumors in which these genes are amplified or overexpressed. T h e generalized tissue distribution of c-myc correlates well with its deregulation in many different types of tumors,

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whereas the more restricted tissue expression of N- and L-myc corresponds to the limited types of tumors in which these genes are activated. For example, high N-myc expression in the developing sensory retina, sympathetic ganglia, and kidney correlates well with N-myc gene overexpression in retinoblastomas (Lee et id., 1984), neuroblastomas (Schwab et al., 1983; Kohl et al., 1983, 1984), and Wilms’ tumors (Nisen et al., 1986). Similarly, elevated N - m y expression in thymocytes correlated with the frequent activation by proviral insertion in MuLV-induced T cell lymphomas (van Lohuizen et al., 1989). L-myc expression in the developing lung is associated with L-myc gene amplification and overexpression in SCLC (Nau et al., 1985). The genetic basis for the tissuepreferential deregulation of N- and L-myc genes has not been determined. It is possible that deregulation processes operate preferentially upon actively expressed genes of a given cell lineage, that myc gene products possess tissue-specific activities, or that deregulation of N- or L - m y expression may require additional genetic events to eliminate tissue-specific repression in tissues where these genes are normally silent. IX. Future Prospects

The past decade has witnessed important advances in our understanding of the role and regulation of myc family oncogenes in normal physiology and malignant disease. Among the major achievements in the myc field have been discovery of its multiple members, characterization of their differential patterns of expression and their potential role in cellular growth and differentiation, dissection of the multiple and complex mechanisms that govern their expression, observation that myc-encoded proteins share substantial homology with known sequence-specifictranscription and differentiation factors, and establishmentof a strong causal link between deregulated myc expression and the genesis of malignant disease. Analysis of myc gene function has generally relied upon examining the physiological impact of deregulated myc expression on cell growth and differentiation in cultured cells and in transgenic mice. Accumulated evidence from these studies has generally supported the view that myc family proteins may function as trans-acting transcription factors capable of regulating genes important in cellular growth and differentiation, although other possibilities remain open. Despite significant progress on many experimental fronts, there exists little insight into the specific roles served by each m y member in normal developmental processes and no direct proof that myc indeed functions as a transcriptional regulator. Several recent technological advances offer significant promise in clarifying these issues. These include the produc-


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tion of animal models that harbor germ-line mutations in myc family genes, the development of dominantly acting mutant myc proteins that function to abrogate myc activities, and the establishment of novel strategies that may permit the identification of direct cellular targets transactivated by the myc proteins. A. PRODUCTION OF null myc MUTATIONS IN

THE

MOUSEGERMLINE Advances in mouse molecular genetics have been successfully employed to produce mouse models that harbor null mutations in the c-myc and N-myc genes. T h e production of these models has relied upon the ability to target crippling mutations in endogenous myc loci of pluripotent embryonal stem (ES) cells and the capacity of these mutant ES cells to become established in the mouse germ line (for review, see Moroy et al., 1991). By analyzing the biological consequences of each null mutant, it should be possible to determine the unique roles served by each myc family gene and the precise points at which each function of the rnyc gene becomes critical for normal proliferative and developmental processes. A number of gene targeting strategies have been employed to produce null mutations in the c-myc (A. Bradley, personal communication) or N-myc (Charron et al., 1990; J. Rossant, personal communication) genes of ES cells. Disruption of the N-myc gene was accomplished by the enrichment of targeted ES clones by making the expression of the Neo' gene conditional upon a homologous recombination event into the actively transcribed N-myc gene. The Neo' gene is positioned to interrupt the N-myc open reading frame and thus results in the loss of functional N-myc protein from the targeted allele (Charron et al., 1990). Using this approach, several laboratories have demonstrated that approximately 1 in 5 to 1 in 30 Neo' ES clones possess homologous integrations into an N-myc allele. Identification of these mutant ES clones, termed N-mycn'wt,was achieved by Southern blotting analysis. Similar experiments have resulted in the production of ES clones with null mutations in the c-myc gene (A. Bradley, personal communication). Germ-line chimeras have been successfully generated from ES clones with mutations in the N-myc (J. Rossant, personal communication) and c-myc (A. Bradley, personal communication) genes. Mice homozygous (Nor c-myc"'") for the disrupted N-myc or c-myc allele have been produced by mating two heterozygous mice (N- or c-myc"'wf). Preliminary evidence indicated that the homozygous state for either the N-myc mutation or the c-myc mutation results in an embryonic lethal condition. Detailed analysis of the precise pathophysiological mechanisms of lethality is currently

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underway. These exciting results demonstrate that the N-myc and c-myc gene products appear to be vital to normal embryonic development. Several important goals can now be realized with this genetic approach. First, a comparison of the biological impact of each null genotype will provide insight into the potential differential role of each myc family member-insight both in terms of the organ systems most affected and of the stage of development during which abnormalities arise. Second, and perhaps more important, the mycnlwf mouse can provide a null genetic background in which detailed structure-function analyses can be performed in the physiological context of a developing animal. Complementation with a mutant myc gene can be accomplished by introducing an altered myc transgene into the mycnIwt background. By interbreeding, animals that are null for the endogenous myc gene but possess the introduced transgene can be derived. These mice can thus function as models for the systematic analysis of gene modifications in the various myc proteins.

B. THEUSE OF TRANSDOMINANT MUTANT PROTEINS FOR THE STUDY OF myc FUNCTION The dominant interference approach represents another potentially useful strategy for elucidation of the role of myc family genes in normal physiology and malignant transformation. This stratagem employs mutant myc proteins capable of physically associating with and functionally inactivating the native rnyc protein (Herskowitz, 1987). As myc family proteins appear to require homodimer or heterodimer formation to become functionally active, activity of a specific myc protein could theoretically be abrogated through association with an engineered m y protein containing a defective transactivating or basic domain. Recent experiments with the c-myc protein appear to have achieved these goals (Dang et al., 1989). Mutant c-myc proteins, deleted in a region necessary for transformation function, but still capable of dimerizing, had a dominant negative effect on transformation of REFS by wild-type c-myc protein, suggesting that mutant :wild-type heterodimers may be functionally incompetent. In contrast, mutant proteins that were incapable of dimerizing with the wild-type protein had no effect on the transforming efficiency of the wild-type protein. Although dominantly acting mutant proteins may provide a means of abrogating myc activity in a number of experimental systems, effective use of this genetic approach will require a more complete understanding of the range of potential dimerization specificities of the myc protein. Once these interactions are defined, the targeting of dominant mutant proteins to specific cell lineage proteins may prove useful in assessing myc function during normal development.


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C. IDENTIFICATION OF POTENTIAL CELLULAR TARGETS TRANSACTIVATED BY myc PROTEINS The slow progress in elucidating myc function in normal development and in cancer rests firmly on the fact that potential target genes regulated by myc have not been identified. The isolation of target genes, regulated directly by myc, requires a system in which rnyc expression could be modulated in a specific and inducible manner. A number of experimental strategies have been developed to regulate myc activity, including (1) antisence neutralization (Heikkila et al., 1987; Yokoyama and Imamoto, 1987; Prochownick et al., 1988; Holt et al., 1988; Griep and Westphal, 1988), (2) interference using dominantly acting mutant proteins as described above (Dang et al., 1989), and (3) induction of chimeric myclhormone receptor proteins (Eilers et al., 1989). Though limitations relating to requirements for high-level antisense expression or poorly defined protein-protein interactions in dominant mutants may limit the applicability of the former two approaches, the myclhormone receptor fusion gene appears to be a viable system in which to define potential cellular target genes that are transactivated by the myc proteins. A c-myclestrogen receptor fusion gene has been engineered in which the hormone-binding region of the estrogen receptor is linked to the c-my protein-coding region (Eilers et al., 1989). This chimeric protein exhibits the functional activities of the normal c-myc protein, although these activities appear to be strictly hormone dependent. Fibroblasts transfected with this chimeric protein will possess a transformed phenotype in the presence of estrogen and will revert to a nontransformed phenotype when the hormone is removed. This genetic approach has produced a conditional allele of the c-myc protein in which activity can be tightly regulated by addition of hormone, thus providing a means for studying the mechanisms by which myc affects the cellular phenotype. For instance, activation of the myclestrogen receptor chimeric protein has been shown to elicit reentry into and completion of the cell cycle in quiescent mouse and rat fibroblasts (Eilers et al., 1991). Furthermore, differential screening of mRNAs present in the induced and noninduced conditions has identified a candidate cellular target for myc transcriptional activity, namely, the a-prothymosin gene. Current evidence suggests that transcription of the a-prothtymosin gene depends directly upon myc activation (Eilers et al., 1991). ACKNOWLEDGMENTS We thank Drs. Robert Eisenman, Allen Bradley,Janet Rossant, Steve McKnight, and J. Michael Bishop for sharing unpublisheddata. We thank Jolaine Lauridsenand Anna Lau for their assistance in the preparation of this manuscript. This work was supported by the

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Howard Hughes Medical Institute and the American Cancer Society (grant #CD397) to R.D., and NIH grants CA42335 and CA23767 to F.A. R.D. is a recipient of the McDonnell Foundation Scholar Award and a Cancer Research Institute Investigator Award. R.D. is aided by Basil OConner Starter Scholar Research Award No. 5-724 through funds received from the Lifespring Foundation to the March of Dimes Birth Defects Foundation.

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TRANSCRIPTIONAL AND TRANSFORMING ACTIVITIES OF THE ADENOVIRUS E1A PROTEINS Thomas Shenk*st and Jane Flint* * Department of Molecular Biology

t Howard Hughes Medical Institute Princeton University, Princeton, New Jersey 08544

I. Introduction 11. Organization and Products of the E1A Gene

111. Transactivating Activity of E1A Proteins

A. Transactivating Domain B. Properties of Transactivation C. Cellular Factors Implicated in Transactivation D. Mechanisms of Transactivation IV. Transforming Activity of E1A Proteins A . Transforming Domains B. Mechanism of Transformation C. Why Is the E I A Gene an Oncogene? V. Perspectives References

I. Introduction

Human adenoviruses replicate and produce progeny virus upon infection of human cells, but their infectious cycle is blocked within most rodent cells. Rodent cells can be oncogenically transformed subsequent to infection. The products of the adenovirus E IA gene play key roles in both the productive and transforming cycles of infection. During productive growth in human cells, the E1A proteins activate expression of adenovirus genes at the level of transcription (Berk et al., 1979;Jones and Shenk, 1979a; Nevins, 1981). The E1A proteins are also oncoproteins that cooperate with the adenovirus E 1B gene products to oncogenically transform rodent cells (Gallimore et al., 1974; Graham et al., 1974, 1978; Flint et al., 1976; Jones and Shenk, 1979b). Several recent reviews have considered aspects of E1A function (Berk, 1986; Moran and Mathews, 1987; Flint and Shenk, 1989). Here we consider the mechanisms underlying the transcriptional and transforming activities of the E 1A proteins. Unless otherwise noted, discussion of the E1A gene and its products will focus on the well studied group C adenoviruses, adenovirus type 2 (Ad2) and type 5 (Ad5). Differences in the details of E1A gene 47 ADVANCES IN CANCER RESEARCH, VOL. 57

Copyright 0 1991 by Academic Press, lnc. All rights of reproduction in any form reserved.

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THOMAS SHENK AND JANE FLINT

organization, expression, and function almost certainly exist among more distantly related adenoviruses. II. Organization and Products of the E1A Gene

T h e E1A gene lies at the extreme left end of the adenovirus genomic map, and it appears to be the first transcription unit expressed after the viral chromosome reaches the nucleus of the infected cell. There are five known E1A mRNAs (Berk and Sharp, 1978; Chow et al., 1979; Perricaudet et al., 1979; Stephens and Harlow, 1987; Ulfendahl et al., 1987) that share common 5' and 3' ends but differ in their splicing patterns. These RNAs are named 13S, 12S, 11S, 10S, and 9 s based on their sedimentation coefficients and they encode polypeptides that are referred to by the number of amino acids they contain: 289R, 243R, 2 17R, 17IR, and 55R, respectively. Nuclear localized polypeptides encoded by the four largest mRNAs have been identified in Ad2- or Ad5-infected cells. T h e 55R product of the mRNA has not been found within infected cells, but can be synthesized in cell-free extracts programmed with 9 s mRNA. Perhaps the 9 s mRNA is poorly translated or the 55R protein has a short half-life in viva The overlapping exons of the four largest mRNAs share the same reading frame, and, as a result, their protein products are highly related. The 55R protein carries a unique C-terminal sequence because its 9 s mRNA switches into a second reading frame in its 3' exon (Dijkema et al., 1980; Virtanen and Pettersson, 1983; Roberts et al., 1985). When the amino acid sequences of the largest ElA polypeptide from a variety of adenovirus serotypes are compared, the protein can be seen to contain alternating conserved and variable domains (Van Ormondt et ul., 1980; Kimelman et al., 1985; Moran and Mathews, 1987). The evolutionarily constant regions have been designated conserved regions 1-3 (Fig. 1A) and they are roughly delineated by splicejunctions at the RNA level. As discussed in Sections 111 and IV, these conserved sequences correspond to important functional domains within the E 1A proteins. The 13s mRNA encodes all three conserved regions, the 12s mRNA contains regions 1 and 2 but not 3, the 11s species contains regions 2 and 3 but not 1, the 10s species carries region 2 but not 1 or 3, and the 9 s mRNA does not encode any of the conserved domains. Why does adenovirus produce four related E 1A mRNAs and proteins when the largest contains all of the information present in the other three? The answer may lie in the temporal order in which the various E1A mRNAs appear. T h e 13s and 12s mRNAs are synthesized very early after infection whereas the other species appear later (Stephens and

49

ADENOVIRUS E 1A FUNCTION

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FIG.1. Diagram of ElA mRNAs, polypeptides, functional domains, and amino acid sequence of conserved region 3. (A) ElA mRNAs and proteins: 125 and 13s mRNA exons are represented by lines; introns occur where the lines peak upward; poly(A)sequences are marked A,,. The 243- and 289-amino acid polypeptides (243R and 289R) encoded by the 12s and 13s mRNAs, respectively, are designated by rectangles. Polypeptide domains that are conserved between different adenovirus serotypes are indicated as CRl, CR2, and CR3 above the rectangles, and the amino acid numbers of the first and last residue comprising each conserved region are indicated below the rectangles. N and C mark the N-terminal and C-terminal ends of the polypeptides, respectively. (B) ElA functional domains are indicated as shaded areas on rectangles representing the 289R polypeptide. The amino acid numbers of the first and last residue comprising each functional domain are indicated below the rectangles. (C) Amino acid sequence of CR3. Cystines comprising the metal-binding region are underlined.

Harlow, 1987; Ulfendahl et al., 1987). If the smaller E1A proteins mediated only a subset of ElA functions, the delay in their expression could regulate which of the multiple E1A functions predominate at various times during the viral growth cycle. Alternatively, the various ElA proteins might be folded differently. This could allow the smaller polypeptides to carry a domain in an optimal configuration or location, and,

50


as a result, mediate a subset of ElA functions more efficiently or with different specificity than larger members of the set. T h e primary translation products of the E1A mRNAs undergo extensive posttranslational modification. As many as 60 E 1A-specific polypeptide species can be resolved by two-dimensional isoelectric focusing and polyacrylamide gel electrophoresis (Harter and Lewis, 1978; S. Yee et al., 1983; Spindler et al., 1984; Harlow et aL, 1985). Much of the heterogeneity is due to phosphorylation (S. Yee and Branton, 1985; Tsukamoto et al., 1986; Stephens et al., 1986). Consistent with this modification, the number of E 1A polypeptide species can be substantially reduced by treatment with phosphatases, and the primary translation products of E 1A mRNAs migrate faster and at a more basic isoelectric point than do the modified forms. As yet, no functional consequence of the multiple phosphorylations has been identified (Dumont et al., 1989; Tremblay et al., 1989) and no other posttranslational modification of the ElA proteins has been described. Some of the ElA proteins form stable complexes with several host proteins that can be detected by their coprecipitation with E lA-specific antibodies (Yee and Branton, 1985; Harlow et al., 1986). Two of these cellular products have been identified: one is the 105-kDa retinoblastoma gene product (Whyte et al., 1988a) and the other is a 60-kDa cdc2associated polypeptide (Giordano et al., 1989). The associations with these cellular proteins have provided important insights to E 1A function (discussed in Section IV). In sum, the ElA gene gives rise to a complex series of products. Five related E 1A polypeptides are posttranslationally modified to generate on the order of 60 separable protein species that can interact with a variety of cellular polypeptides. Given this complexity, it is not surprising that EIA gene products mediate multiple, profound effects on cell growth and gene expression. Ill. Transactivating Activity of E1A Proteins

T h e 243R ElA protein (Fig. 1A and B) has been reported to exhibit transactivation activity in some experimental circumstances (Ferguson et al., 1985; Leff et al., 1984; Winberg and Shenk, 1984; Simon et al., 1987; Zerler et al., 1987), but the mechanism by which this protein stimulates transcription has not been examined. The 289R E1A protein (Fig. 1A and B) is primarily responsible for transactivation of viral gene expression (reviewed in Berk, 1986).

ADENOVIRUS E 1 A FUNCTION

51

A. TRANSACTIVATING DOMAIN T h e results of mutational analyses of adenovirus E 1A proteins emphasize the importance of the 46 amino acids unique to the larger protein in transactivation. This unique portion is included within conserved region 3 (CR3) of the E1A proteins (Fig. 1B). Mutations within CR3 impair or eliminate transactivation, whereas mutations within sequences common to the 243R and 289R E l A products do not appreciably alter activity (see Section IV) (Moran and Mathews, 1987). Moreover, a synthetic 49-amino acid peptide corresponding exactly to CR3 can stimulate expression from adenovirus promoters in vivo and in vitro (Lillie et al., 1987; Green et al., 1988), indicating that CR3 comprises the minimum transactivation domain. On the other hand, some role has been ascribed to C-terminal amino acids encoded by exon 1, common to the 289R and 243R proteins. Using fusion proteins comprising the GAL4 DNA-binding domain and segments of the 289R ElA protein, Lillie and Green (1989) have observed that amino acids 133-138 make a small but significant contribution to activation of several promoters, whether these contain or lack GAL4 DNA-binding sites. However, the core region essential for induced expression from promoters containing GAL4 DNA-binding sites lies between amino acids 141 and 178 (Martin et al., 1990). Most of the transactivation domain appears to be indispensable. Transactivation activity, assessed using transient cotransfection assays, is lost when any one of several residues in the C-terminal portion of CR3, notably Cys 171, Leu 173, Cys 174, Lys 176, Gly 180, or Ser 185, are substituted (Glenn and Ricciardi, 1985; Lillie et al., 1986; Culp et aE., 1988). Similarly, substitution of Cys 171 or Cys 174 by alanine in the transactivating CR3 peptide eliminated activity (Pusztai et al., 1989; Lowenstein and Green, 1989), as did mutation of one or more conserved amino acids (with the exception of Asp 168) between residues 145 and 177 in GAL4-E1A fusion proteins (Martinet al., 1990). Moreover, in uitro and in vivo assays of the activity of a series of N-terminally deleted CR3 peptides indicated that the region essential for transactivation extends from between residues 153 and 157 to the C terminus of CR3 (Pusztai et al., 1989; Lowenstein and Green, 1989). In these assays, deletion of 1 to 12 residues from the N terminus of the peptide resulted in 5- to 50-fold decreases in specific activity, suggesting that the loss of the function fulfilled by this region can be at least partially overcome by high concentrations of peptide. Whether loss of the N-terminal segment of CR3 in the full-length 289R ElA protein results in a similar phenotype is not known. However, analysis of the GAL4-ElA fusion proteins

52


described previously suggested that this portion of the transactivation domain also plays a critical role. In transient cotransfection assays, the parental fusion protein, which contains residues 121-223 of the 289R E 1A protein, activated expression from the adenovirus E 1 B and E4 promoters but did so more effectively when binding sites for the GAL4 DNA-binding domain were introduced into the promoters (Lillie and Green, 1989). Residues 142-149 of CR3 are essential for such activation (Lillie and Green, 1989; Martin et al., 1990). T h e C-terminal portion of CR3, which all assays identify as essential to transactivation function, contains a Zn2+ finger motif of the form Cys(X)&ys(X)l&ys(X)&ys (Fig. 1C). The 289R, but not the 243R, E1A protein binds 1 mol of Zn2+/moleprotein (Culp et al., 1988), raising the question of whether Zn2+ binding and the integrity of the finger structure are necessary for transactivation. In fact, substitution with glycine of any one of the four cysteine residues that define the Zn2+ finger motif of the 289R E1A protein, Cys 154, Cys 157, Cys 171, and Cys 174, destroys the ability of the protein to transactivate (Culp et al., 1988). Substitution of these residues with alanine in the transactivating CR3 peptide similarly reduced or eliminated its ability to stimulate expression from the E2 promoter in vivo (Pusztai et al., 1989) or transcription of the viral major late (ML) promoter in vitro (Lowenstein and Green, 1989). The repeated observations that mutation of the Cys residues that define the Zn2+finger motif, as well as of other amino acids within that motif, drastically reduces o r eliminates transactivation activity in different assay systems are consistent with the idea that transactivation requires its integrity. Indeed, substitution of Cys 157, 171, or 174 with Ser or Gly destroys not only the transactivation function but also the ability of the 289R ElA to bind Zn2+ (L. Webster and R. Ricciardi, personal communication). The properties of proteins carrying a Cys to Ser or Gly substitution at position 154 were initially surprising and emphasize the importance of this Zn2+ finger structure: these mutant proteins retain wild-type Zn2+-binding activity, despite complete loss of transactivation activity (Culp et al., 1988). Zn2+ binds to these mutant proteins via an alternate metal-binding site, in which two histidine residues substitute for the N-terminal cysteine pair of the Zn2+ finger of the wild-type protein (L. Webster and R. Ricciardi, personal communication). Thus, substitution of Cys 154 results in conversion of a functional metal-binding finger to an alternative metalbinding structure that cannot mediate the transactivation function of the protein. Whether the metal-binding domain of the El A protein mediates interaction with DNA or with other proteins has not been directly demonstrated. However, the ability of second site mutation of residues within the Zn2+ finger to abrogate the transdominant negative pheno-

ADENOVIRUS E1A FUNCTION

53

type induced by mutation of residues within the region 183 to 188 has led to the suggestion that the finger region binds a limiting, cellular transcription factor. In sum, sequences between amino acids 142 and 188, which include an essential Zn2+-binding structure, comprise the critical transactivation domain of the 289R E l A protein (Fig. 1B).

B. PROPERTIES OF TRANSACTIVATION T h e ability of the 289R ElA protein to stimulate transcription was discovered because this function is required for efficient expression of viral early genes and thus for successful productive infection (Jones and Shenk, 1979a; Berk et al., 1979; Nevins, 1981). T h e most unusual property of the E1A transactivator is its lack of specificity. Transcription from all adenoviral early promoters is substantially stimulated by the E 1A protein, whether these promoters are introduced into E 1 A proteinexpressing cells in the viral chromosome or in plasmids or are integrated into the cellular genome (Courtois and Berk, 1984; reviewed in Berk, 1986). It is, therefore, clear that the inducibility of these viral early promoters is not determined or limited by their chromosomal organization. T h e response of adenovirus late promoters appears to be more variable. T h e E2 late promoter is activated with the onset of the late phase of infection (reviewed in Berk, 1986; Flint, 1986). This promoter is refractory to 289R E 1A protein transactivation, but is sensitive to repression (see Section IV,B,l) by the 243R ElA protein (Rossini, 1983; Guilfoyle et al., 1985; Leff and Chambon, 1986; Dery et al., 1987). T h e late plX (Venkatesh and Chinnadurai, 1987), IVa2 (Natarajan and Salzman, 1985), and ML promoters (Lewis and Manley, 1985; Natarajan and Salzman, 1985) have been found to be stimulated by the ElA protein in some transient assays but not in others (Matsui et nl., 1986; Leff and Chambon, 1986; Dery et al., 1987). It is possible that in the latter cases the experimental conditions precluded transactivation o r its detection. Indeed, Natarajan and Salzman (1985) observed no effect of the EIA protein when the activity of a IVag-chloramphenicol acetyltransferase (CAT) reporter gene was assessed by CAT assay, but significant stimulation of authentic IVa2 transcription was detected using a nuclease S1 assay. In this particular construct, production of aberrant mRNA species from which the enzyme could be synthesized appeared to mask stimulation of authentic IVa2 transcription. Transcription from the ML promoter appears to respond to E 1A protein transactivation during adenovirus infection (Lewis and Mathews, 1980; Nevins, 1981) and to the 289R ElA protein or the activating CR3 peptide (see Section III,A) in nitro

54


(Leong and Berk, 1986; Leong and Flint, 1986; Spangler et al., 1987; Green et al., 1988). It therefore seems clear that ML transcription, as well as expression from the IVa2 and PIX promoters, can be stimulated by the E 1A proteins under some, but not all, experimental conditions. Variation in the results obtained using different assays of E 1A protein transactivation has not been uncommon. Such variation might in some cases be attributable to specific problems associated with sole reliance on CAT assays, but its general basis has not been established. Relevant experimental parameters might include the “chromosomal” state of the test promoter (e.g., whether it is present in replicating or nonreplicating plasmids, or in the adenoviral or cellular genomes), the nature and growth state of the cells in which transactivation is tested, the concentration of E 1A proteins achieved (Brunet and Berk, 1988), and the absence or presence of other adenoviral proteins that modulate expression of viral genes, such as the 243R ElA protein (see Section IV), the E4 ORF 6/7 protein (see Section III,C), the ElB 19-kDa protein (White et al., 1986; Herrmann et al., 1987; Yoshida et al., 1987), and the E2A DNA-binding protein (Chang and Shenk, 1990; Morin et al., 1989). Adenovirus promoters whose transcription can be stimulated by the ElA protein share no common sequence (see Kingston et al., 1985; Berk, 1986; Flint, 1986). Moreover, ElA products can, in transient assays, stimulate expression from a wide variety of cellular RNA polymerase I1 promoters, including those of globin (Green P t aL, 1983; Allan etal., 1984; Svensson and Akusjarvi, 1984),preproinsulin (Gaynor et al., 1984), heatshock proteins HSP70 (Nevins, 1982; Kao and Nevins, 1983; Wu et al., 1986) and HSP89 (Simon et al., 1987),c-Myc (Sassone-Corsi and Borrelli, 1987; Lipp et al., 1989), c-Fos (Sassone-Corsi and Borrelli, 1987; Simon et al., 1990), the H-2Kb class I major histocompatibility complex (MHC) (Rosenthal et al., 1985; Katoh et al., 1990), and immunoglobulin (Borrelli et al., 1986; Shurman et al., 1989) genes. Other viral promoters that respond to E 1A protein transactivation include those of the herpes simplex type 1 gpD and TK genes (Everett and Dunlop, 1984; Weeks and Jones, 1985), the adeno-associated virus p l 9 and p5 promoters (Tratschin et al., 1984; Chang el al., 1989), the bovine papillomavirus type 1 LCR (Bernard et al., 1990), the human cytomegalovirus 1E gene (Gorman et al., 1989) and the long terminal repeats (LTRs) of HTLV-I and -11, HIV, and Rous sarcoma virus (Chen et a1.,1985; Nabel et al., 1988; Rice and Mathews, 1988).The large number of promoters that can respond to the E 1A protein provides strong evidence that transactivation does not depend upon a unique “El A-response” element. Indeed, mutagenesis of responsive promoters (see Section II1,C) has invariably failed to identify elements that are required for E 1A protein-stimulated, but not for basal,


55

transcription (reviewed in Berk, 1986; Flint and Shenk, 1989). In this respect, the ElA proteins stand in stark contrast to well-characterized eucaryotic activators, which bind to specific sequences within the promoters whose activities they modulate (see Ma and Ptashne, 1987; Johnson and McKnight, 1989; Mitchell and Tjian, 1989). Perhaps the most emphatic illustration of the promiscuity with which the E1A proteins can act is their ability to stimulate transcription by RNA polymerase I11 in both transient expression and in vitro transcription systems (Berger and Folk, 1985; Gaynor et al., 1985; Hoeffler and Roeder, 1985). As E1A protein-responsive promoters share no common sequence elements and can be transcribed by RNA polymerase I1 or RNA polymerase 111, it has been generally concluded that the ElA proteins must act via an indirect mechanism that can increase the activity of an extremely diverse array of cellular and viral promoters. For this reason, efforts to elucidate the mechanism of E 1A protein transactivation have concentrated on identification of cellular transcription factors participating in the response and the delineation of alterations in their activities induced by the ElA protein.

C. CELLULAR FACTORS IMPLICATED IN TRANSACTIVATION

1. Sequence-Specific Factors a. E2F. The cellular factor E2F was discovered by virtue of its ability to bind to elements of the adenovirus E2 early (E2E) promoter. The E2E promoter is efficiently transactivated by the 289R ElA protein and has been the promoter most frequently employed in studies of the mechanism of transactivation. The E2E transcriptional control region is complex and directs initiation of transcription from a major and minor start site, designated + 1 and -26, respectively (Fig. 2). Sequences located approximately 30 bp upstream of the two initiation sites determine their positions (Elkaim et al., 1983; Murthy etal., 1985; Zajchowski et al., 1985). Neither sequence (TTAAGA and TTAAATTT) is a good match to the consensus TATA sequence (See Breathnach and Chambon, 1981; Shenk, 1981), and it is not known whether either binds the TA'TAbinding factor TFIID (see Section 111,C72).However, upstream recognition sites for two cellular factors have been clearly identified by genetic and biochemical methods (Imperiale et al., 1985; Murthy et al., 1985; Zajchowski et al., 1985; SivaRaman et al., 1986; Kovesdi et al., 1986a,b; SivaRaman and Thimmappaya, 1987; Yee et al., 1987): E2F binds to duplicated sites present at positions -71 to -53 and -49 to -33 and an activating transcription factor/cAMP response element-binding protein (ATF/CREB) site is present at position -82 to -66 (Fig. 2). Analysis of

56


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mutated E2E promoters, and of chimeric promoters containing various segments of the E2E promoter, in transient assays has established that transactivation of E2E transcription is not absolutely dependent on any single promoter element. Several combinations of elements, and therefore transcription factors, can mediate transactivation (Imperiale and Nevins, 1984. Kingston et al., 1984; Imperiale et al., 1985; Murthy et al., 1985; Zajchowski et al., 1985, 1987; Boeuf et al., 1987; Jalinot and Kedinger, 1986; Loeken and Brady, 1989; reviewed in Berk, 1986; Flint and Shenk, 1989). However, when the E2E promoter is carried into E l A protein-expressing cells in the adenoviral genome, mutation of any one of the four E2E promoter elements eliminates transactivation (Manohar et al., 1990). This result implies that the ATF site, the two E2F sites, and the TATA-like sequence are each essential to transactivation of E2E transcription within a virus-infected cell, but such an interpretation is


57

complicated by the ability of the E4 ORF 6/7 protein to stimulate the binding of E2F to the E2E promoter and, under certain experimental circumstances, activate E2E transcription (see later). Although mutational studies emphasize the plasticity of E 1A proteinresponsive E2E promoters (at least in transfected cells), much effort has been devoted to elucidation of the role of E2F, for the compelling reason that the discovery of this cellular factor was accompanied by the demonstration that its DNA-binding activity is significantly increased in adenovirus-infected cells (Kovesdi et al., 1986a,b). In these experiments very little E2F activity could be detected in uninfected cell extracts using E2E promoter DNA fragments containing the two E2F-binding sites (Kovesdi et al., 1986a,b; Reichel et al., 1987). However, when a probe containing a single E2F site and appropriate nonspecific DNA are used, E2F activity can be readily detected in uninfected as well as infected HeLa cells (SivaRaman and Thimmappaya, 1987; Hardy and Shenk, 1989). Adenovirus infection induces the appearance of a novel E2F-DNA complex, but no major quantitative change in E2F activity (Hardy and Shenk, 1989). By contrast, a probe containing two E2F sites, orientated and spaced like those of the E2E promoter (a “double-site” probe), detects the appearance of large quantities of a novel complex (Hardy and Shenk, 1989). This infected cell-specific complex is formed by cooperative binding of E2F to the double-site probe: its formation exhibits a nonlinear dependence on protein concentration, its formation is sensitive to both the spacing and orientation of the two E2F binding sites in the probe, and its stability is considerably increased compared to that of E2F bound to a single-site probe (Hardy and Shenk, 1989). Thus, the most striking change in E2F activity following adenovirus infection is the induction of its ability to bind to E2E sites cooperatively. Several lines of evidence initially suggested that the dramatic increase in E2F DNA-binding activity observed in infected cells was a direct effect of the E1A proteins (Kovesdi et al., 1987; Reichel et al., 1987, 1988; Yee et al., 1987,1989). Nevertheless, more recent experiments have established that formation of the infected cell-specific, cooperatively binding E2F activity depends directly on an E4, not an E 1A, product. The cooperative complex is not formed in cells infected by dZ366 (Hardy et al., 1989; Reichel et al., 1989), a virus from which most of the E4 transcription unit has been deleted but which produces normal ElA proteins (Halbert et al., 1985), o r in adenovirus-transformed cells that synthesize only E 1A and E1B proteins (Hardy et al., 1989; Babiss, 1989; Reichel et al., 1989). Consistent with a role in activation of E2F, products of the E4 transcription unit can stimulate E2E transcription in transfected cells (Goding et al., 1985; Reichel et al., 1989; Neil1 et al., 1990).

58


A variety of observations indicate that the 17-kDa protein encoded by ORF 6/7 of the E4 gene is responsible for induction of the cooperative binding form of E2F. Mutation within the E4 segment common to ORFs 6 and 7 , or in a sequence uniquely coding ORF 7, prevented induction of E2F (Huang and Hearing, 1989; Marton et al., 1990); a virus containing ORF 6 / 7 cDNA in place of the E4 transcription unit induced infected cell-specific E2F as efficiently as did wild-type virus (Huang and Hearing, 1989); an infected cell-specific activity capable of converting E2F to the cooperatively binding form copurifies with the E4 ORF 6/7 17-kDa protein during high-resolution chromatography (Marton et al., 1990), and E2F can be activated by the ORF 6/7 protein synthesized in vitro (Neill et al., 1990). Moreover, cooperative binding of E2F to appropriate double-site probes is the result of interaction of the E4 protein with the factor. Addition of an antibody that recognizes the ORF 617 protein to E2F-containing extracts of infected cells either before or after its binding to E2 promoter DNA resulted in the appearance of a novel complex exhibiting reduced electrophoretic mobility, whose formation was blocked by the E4 peptide against which the antibodies were raised (Huang and Hearing, 1989; Marton et al., 1990; Neill et al., 1990). The significant reduction in infected cell-specific E2F DNA-binding activity observed when the same antibodies were used to deplete nuclear extracts of the E40RF 6 / 7 protein, and any proteins bound to it, confirmed that this E4 protein forms a complex with E2F in the absence of DNA (Huang and Hearing, 1989; Marton et al., 1990). It is, therefore, firmly established that interaction of the E4 ORF 617 protein with E2F results in highly cooperative binding of this factor to its sites in the E2 promoter. Because stable, cooperative binding is observed only with DNA fragments that contain precisely spaced and oriented E2F sites (Hardy et al., 1989; Raychaudhuri et al., 1990),it seems likely that cooperative binding is the result of specific protein-protein interactions. It is not, however, known whether interactions between the E4 ORF 6 / 7 protein and E2F or between E2F-bound E4 17-kDa protein molecules are primarily responsible for cooperative binding. The ability of the ORF 6 / 7 protein to increase the DNA-binding activity of E2F suggests that it might play a role in activation of E2E transcription in infected cells. Although expression of E4 proteins in transfected cells significantly stimulates E2E transcription (Goding el al., 1985; Reichel et al., 1989; Neill et al., 1990), E2E mRNA levels were reduced by less than a factor of two in HeLa cells infected by dZ356 (Marton et al., 1990), a virus that does not induce infected ceil-specific E2F because of a deletion in E4 ORF 6/7 (Halbert et al., 1985). However, in S49 cells, which contain very low concentrations of AP-1 (which can

ADENOVIRUS EIA FUNCTION

59

bind to the ATF/CREB site in the E2E promoter; see Section III,C, l,b), E2E mRNA levels were threefold lower in dE356-infected cells than in wild-type Ad5-infected cells, despite identical levels of E 1A mRNA (Marton et al., 1990). Thus, following infection of certain cell types, the E4 ORF 6/7 protein contributes, in an E1A protein-independent fashion (Marton et al., 1990), to maximal activity of the E2E promoter, presumably as a result of its interaction with E2F. The cell type specificity of the requirement for E4 protein activation of E2E transcription is not surprising. In natural infections, the virus encounters a variety of cell types and might, therefore, be expected to have evolved a number of independent mechanisms to maximize expression of viral genes in a variety of intracellular milieus. A consensus has, therefore, emerged that the E4 ORF 6/7 protein is responsible for the appearance of the cooperatively binding form of E2F in infected cells and that this protein can, under appropriate conditions, maximize transcription from the E2E promoter. A far more difficult question is whether the E1A protein plays any direct role in activation of E2F in infected cells and thus whether its ability to stimulate transcription from the E2E promoter is mediated, at least in part, by E2F. The initial experiments leading to the identification of activated E2F in infected cells established that E 1A products were required, but could not distinguish a direct role for the E1A proteins from an indirect mechanism. Under the conditions of infection used (e.g., Kovesdi et al., 1986a, b; Reichel et al., 1987), viruses that cannot express the E1A protein fail to produce any viral gene products efficiently (Jones and Shenk, 1979a; Berk et al., 1979). Thus, the activation of E2F cooperative DNA-binding activity by the E4 ORF 617 protein raises the possibility that the requirement for E 1A proteins is an indirect one. The properties of E2F activation by the ORF 6/7 protein are entirely consistent with an indirect role for the ElA protein in the activation of E2F. Infected cell-specific E2F complexes formed on either a double-site or a single-site probe can be detected in transformed cell lines that synthesize E4 mRNAs, but not in cell lines that express only E1A and E1B mRNAs (Hardy et al., 1989; Babiss, 1989; Reichel et al., 1989); when high-multiplicity infections of long duration are used to permit expression of the E4 genes, infected cell-specific E2F activities are present in cells infected by viruses that do not express the E 1A proteins, and their appearance correlates with the delayed accumulation of E4 mRNA species (Hardy et al., 1989; Huang and Hearing, 1989; Marton et al., 1990). Both the cooperative, double-site DNA-E2F complex and the infected cell-specific complex detected using a DNA probe that contains only a single E2F-binding site contain the E4 ORF 6/7 protein (Marton et al., 1990),and infected cell-specific complexes formed

60


on double-site probes in the absence of ElA proteins exhibit stabilities and spacing requirements identical to those of the complexes formed in extracts containing E1A proteins (Hardy et al., 1989). It has, nevertheless, been proposed that the ElA protein contributes more directly to activation of E2F. Babiss (1989) observed that the infection-specific form of the factor was not present at 32°C in ElA mutant virus H5hrl-transformed CREF cells, which exhibit a cold-sensitive phenotype. As the truncated E l A protein encoded by H5hrl is conditionally stable (Ricciardi et aE., 1981) and because similar levels of E4 RNA were present at 37 and 32"C, this result was interpreted in terms of ElA protein activation of E2F (Babiss, 1989). However, it was not demonstrated that incubation at 32°C does not impair production of either the E4 mRNA species from which the ORF 6/7 product is made or the protein itself, which has a relatively short half-life (Cutt et al., 1987). More importantly, the truncated EIA protein encoded by H5hrl is defective for transactivation (e.g., Berk et al., 1979). Thus, the finding of activated E2F in H5hrl-transformed cells at 37°C (Babiss, 1989) would appear to provide a strong argument against the notion that cooperative DNA-binding activity of E2F requires the 289R E 1A transactivator. Reichel et al. (1989) have proposed that activation of E2F is a two-step process, mediated by both the E1A and E4 ORF 6/7 proteins. These workers observed that efficient expression of E4 products in cells infected at high multiplicity by a virus unable to produce E1A proteins was not sufficient for induction of activated E2F. Moreover, activation of E2F detected using a single-site probe was ascribed to the EIA protein because this form of activated E2F could not be detected in differentiated F9 cells infected with an E1A-negative virus, but was induced upon d1366 (E4 negative) infection of these cells (Raychaudhuri et al., 1990). The discrepancy between this observation and the markedly different results obtained by others using similar experimental protocols has not been explained, but may reduce to differences among cell lines or growth conditions, or in the concentrations of the ORF 6/7 protein attained. Further support for a two-step E2F activation model was obtained using an in nitro activation system. This approach is based on the observation that mock-infected cell E2F can be activated for cooperative binding by incubation with an Ad5-infected cell fraction devoid of E2F (Bagchi et al., 1989). One step in the in nitro activation is the binding of the E4 protein. This reaction is heat resistant and does not require ATP (Marton et al., 1990; Raychaudhuri et al., 1990). A second reaction has been described that is heat sensitive and ATP dependent (Bagchi et al., 1989).


61

This reaction is interpreted to involve phosphorylation of E2F, given the observation that cooperative binding of E3F can be prevented by phosphatase treatment and restored by treatment with CAMP-dependent protein kinase (Bagchiet al., 1989).Thus, in the two-step model, an E l A protein-dependent phosphorylation reaction is postulated to generate an activated form of E2F, whose cooperative binding to DNA is then induced by interaction with the E4 protein. The in vitro activation reaction studied by Marton et al. (1990)was not, however, inhibited by addition of nonhydrolyzable analogs of ATP and, therefore, did not appear to require phosphorylation. These discrepancies might be the result of experimental differences such as the nature of the assays for activated E2F or the cell type employed. Be that as it may, biochemical studies of E2F activation have not yet yielded compelling evidence that the E1A protein plays a direct role in enhancing the DNA-binding activity of this factor. If E2F mediated the transactivation of the E2E promoter by E1A proteins, then the factor’s recognition sites should confer El A protein inducibility on otherwise refractory promoters. The addition of the E2F sites from the E2E promoter to the SV40 early promoter, indeed, was found to result in a dramatic increase in expression from this promoter in wild-type Ad5-infected but not E 1A-negative virus-infected cells (Yee et al., 1989). However, infection with the ElA-negative virus was performed under conditions in which expression of all viral early proteins is severely inhibited. Thus, it was not established whether the transactivation depended on the E l A or the E4 proteins. The same caveat applies to the stimulation of ElB transcription (Pei and Berk, 1989) and rabbit P-globin transcription (Kovedsdi et al., 1987) by addition of E2F sites. Further, Zajchowski et al. (1987) found that an E2E promoter fragment containing only the two E2F sites failed to activate transcription when linked to the rabbit P-globin promoter in transfected cells expressing only the viral E1A proteins. Thus, it is not clear whether E2F participates in transcriptional activation by E l A proteins. While the participation of E2F in transactivation of E2E transcription by the 289R E 1A protein remains controversial, it is worth recalling that the results of numerous mutational analyses indicate that factors binding to elements at -28 to -23 and -82 to -66 should also be considered. As mentioned previously, it is not yet known whether the former element, which appears TATA-like in its position and function, is recognized by the general transcription factor TFIID (see Section III,C,2). The latter element is of considerable interest. It is recognized by the cellular factor ATF/CREB, whose binding sites are also present in the E3, E4, and ElA promoters.

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b. ATFICREB and AP-I. Activating transcription factor was recognized and named by virtue of the presence of its binding sites in the E1A protein-responsive E4, E3, and E2E promoters, as well as in the transcriptional control region of the ElA gene (SivaRaman et al., 1986; Hurst and Jones, 1987; Lee and Green, 1987; K. A. W. Lee et al., 1987). The ATF-binding sites of the E2, E3, and E4 promoters constitute promoter elements important for both basal and E 1A protein-inducible transcription (reviewed in Berk, 1986; Flint and Shenk, 1989). The ATF-binding site sequence motif CGTCA is identical to that present in the elements mediating cAMP inducibility, cAMP response elements (CREs), of many promoters via the CRE-binding protein, CREB (see Roesler et al., 1988). Initial comparisons of their DNA-binding properties suggested that ATF and CREB are very closely related (Hurst and Jones, 1987; Hardy and Shenk, 1988; Lin and Green, 1988; Leza and Hearing, 1988). It is now clear that members of an extensive family of related proteins recognize ATF-CREB-binding sites; purification of ATF yielded several closely related ATF proteins, immunologically related to members of the Jun family of transcription factors (Hai et al., 1988). Eight different ATF cDNA clones, six of which are unrelated except in a region encoding a leucine zipper motif and adjacent N-terminal basic region (Landschulz et al., 1988), have now been identified (Hai et al., 1989). The ATF family is probably significantly iarger than the eight proteins represented by these clones. A human CREB clone (J. P. Hoeffler et al., 1988) exhibits 75% amino acid identity to ATF-1 (Hai et al., 1989), and ArF-1-ATF-6 share significant similarities with members of the Jun protein family, especially among their basic domains (Hai et al., 1989),which mediate DNA binding (e.g., Gentz et al., 1989; Turner and Tjian, 1989). The functional diversity of this protein family is likely to be even greater than the number of individual ATF proteins, because several (but not all) pairs of ATF proteins form heterodimers that bind ATFICREB site-containing DNA efficiently (Hai et al., 1989). Interestingly, ATF proteins also exhibit differences in their interactions with the ATF consensus sequence (Hai et al., 1989),a property that presumably accounts for functional differences among ATF/CREB-binding sites (Lee et al., 1989). These properties of ATF proteins raise the question of whether all members of the family can participate in E 1A protein-mediated transactivation of transcription. Although this question has not been fully addressed, results of experiments with fusion proteins suggest that ATF-2, but not ATF-1, can mediate transactivation by the EIA protein (Liu and Green, 1990). The mechanism by which the E 1A transactivator might modulate the activity of factors binding to the ATR/CREB sites of viral early promoters is not known. N o differences in the DNA-binding activity of ATF from


63

adenovirus-infected as compared to mock-infected cells have been detected (SivaRaman and Thimmappaya, 1987; Lee and Green, 1987; Muller et al., 1989). However, these assays did not distinguish ATF-2 from other ATF proteins that might participate in the transactivation reaction and whose DNA-binding activities might mask changes in the activity of ATF-2. In the meantime, the close relationship of ATFbinding sites and CREs, and of the proteins binding to these elements, has focused attention on the relationship between the mechanisms of E 1A protein transactivation and CAMP-inducible transcription. The latter process depends on the catalytic subunit of CAMP-dependent protein kinase A (Nairn et al., 1985; Grove et al., 1986),perhaps to phosphorylate CREB to the increased levels observed when intracellular cAMP levels are increased (Montminy and Bileszikjan, 1987; Yamamoto et al., 1988; Gonzalez and Montminy, 1989). In fact, transcription from viral early promoters that contain ATF-binding sites is stimulated by increases in the intracellular concentration of CAMP in both infected (Engel et al., 1988) and transfected (Leza and Hearing, 1988; Sassone-Corsi, 1988), CAMP-responsive human or rodent cells. Like induction of CAMPresponsive cellular genes, such transcriptional activation of viral early genes requires protein kinase A (Engel et al., 1988; Sassone-Corsi, 1988). In Ad5-infected mouse cells, activation of transcription from the E4 and E1A promoters by the E1A protein and by effectors that increase the intracellular concentration of cAMP is highly synergistic (Engel et al., 1988), suggesting that these two inducers operate by complementary, rather than independent, mechanisms. The cellular activity AP-1 binds to a site, 5 TGA(C/G)TCA 3’, that differs by only one nucleotide from the consensus ATFICREB site (Angel et aZ., 1987; W. Lee etal., 1987). Thecloningof AP-l-related genes has established that AP- 1, as originally defined by transcription and DNA-binding activities, in fact comprises several proteins belonging to the Fos and Jun protein families, which interact with AP-l-binding sites as homo- and heterodimers (see Curran and Franza, 1988; Vogt and Tjian, 1988). AP-1 can bind to ATFICREB sites in vitro, albeit with reduced affinity (Angel et al.,1987; Hai et al., 1988; Nakebeppu et al., 1988; Miiller et al., 1989). This property, as well as the presence of an AP-1 element important to both basal and E1A protein-induced transcription in the E3 promoter (Fig. 2) (Hurst and Jones, 1987), suggest that AP-1 might be an important player in transactivation of viral early promoters and the synergistic activation of some promoters by the E1A proteins and CAMP. Indeed, cAMP treatment of Ad5-infected S49 cells induces the DNA-binding activity of a specific form of AP- 1 (Miiller et al., 1989). Maximal induction of this activity depends directly on the EIA

64


protein. Under the same conditions, no alterations in the DNA-binding properties of unfractionated ATF/CREB activities could be detected (Miiller et al., 1989). The induced AP-1 activity binds efficiently not only to AP-1 sites, but also to ATF/CREB sites present in a number of viral early or cellular promoters, as judged by both competition assays and partial purification of a c-Fos protein-containing AP- 1 activity that binds to both AP-1 and ATFICREB sites (Muller et al., 1989). These results therefore imply that an AP-1 activity is involved in the activation of transcription by cAMP and the E1A protein observed in S49 cells. T h e mechanism(s) by which cAMP and the E1A protein act synergistically to stimulate transcription is not yet clear. However, induction of AP-1 activity in infected S49 cells required both protein kinase A and transcription, and was accompanied by an increase in the levels of both c-fos and junB, but not of c-jun or j m D , mRNAs (Muller et al., 1989). Although the kinetics of induction of these two mRNAs were similiar in uninfected and infected cells, a significantly greater degree of induction, dependent on synthesis of ElA proteins, was observed in infected cells (Muller et al., 1989). Thus, one role of the ElA protein in this system appears to be to augment the CAMP-induced stimulation of c-fos andjunB transcription. It is possible that the contribution of the ElA protein to transactivation of viral genes in infected, CAMP-treated S49 cells is, in part, an indirect consequence of its ability, in conjunction with CAMP,to induce a dramatic increase in the production of c-fos and junB mRNA species. This may occur through effects on proteins that are modified by protein kinase A, because cAMP is believed to act via this kinase (see Nairn et al., 1985; Grove et al., 1986) and induction of both transcription of viral early genes and AP- 1 activity in S49 cells requires protein kinase A (Engel et al., 1988; Miiller et al., 1989). On the other hand, stimulation of E4 transcription in infected cells was observed within 15 min of CAMP induction (Engel et al., 1988), a period that appears too short to produce AP-1 components and localize them to the nucleus to stimulate transcription. It has, therefore, been suggested that preexisting cellular factors are initially modified, resulting in stimulation of transcription of both adenoviral genes and those encoding the proteins that comprise the induced AP-1 activity (Miiller et al., 1989). In this case, the increased quantities of AP-1 resulting from the increased synthesis of c-fos and junB mRNAs would be expected to amplify both the magnitude of the transcriptional response and its duration (see Muller et al., 1989). Attractive as this scenario might be, it remains to be established that AP-1 activity can be altered in S49 cells by the E 1A protein in the absence of induction of c-fos and junB expression. As part of a series of experiments to identify and characterize cellular


65

proteins that can bind ATF/CREB sites of E1A protein-responsive viral promoters, Buckbinder et al. (1989) collected evidence implicating AP-1 in transactivation. These workers identified a HeLa cell factor that stimulates in uitro transcription from the E3 promoter and binds to both its AP-1 and ATFICREB sites (see Fig. 2). Affinity purification of this activity using the E3 AP-1 site resulted in a preparation that contained 35 to 45-kDa polypeptides, some of which reacted with antibodies recognizing a c-Jun fusion protein. The activity retained the ability to stimulate E3 transcription and bind to both the AP-1 and ATF/CREB sites of the E3 promoter (Buckbinder et al., 1989; Merino et al., 1989). Moreover, an E3 promoter in which the normal ATF/CREB and AP- 1 sites were replaced by four copies of the AP-1 site was transactivated by the ElA protein even more efficiently than the wild-type E3 promoter, whereas promoters containing two or four copies of the ATF/CREB site were stimulated only twofold. These results indicate that, under appropriate experimental conditions, AP-l-binding sites are sufficient to confer E1A protein inducibility. In these experiments, interactions between E 1A protein transactivation and the pathway whereby CAMPcan stimulate transcription were not investigated. However, soon after its discovery, AP-1 was shown to be involved in the pathway resulting in the induction of transcription by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate(TPA), which depends on protein kinase C (Angel et al., 1987; w. Lee et al., 1987). TPA treatment of HeLa cells cotransfected with an E3-CAT reporter gene and an E l A-expressing plasmid resulted in a significantly greater stimulation of CAT activity than is achieved by either the ElA protein or TPA alone (Buckbinder et al., 1989), suggesting that these two inducers act synergistically. A similar synergistic induction of E3 expression was observed in infected cells. The construct containing four AP-1 sites in place of the one ATFICREB and one AP-1 site of the wild-type E3 promoter was also strongly stimulated by TPA or the ElA protein alone, but the two inducers together resulted in a merely additive response (Buckbinder et al., 1989). Promoters comprising only ATF sites responded much more weakly to either inducer, or to the combination. These results suggest that synergistic induction of E3 expression by the ElA protein and TPA requires both the AP-1 and ATF/CREB sites of the E3 promoter, which are presumably recognized by different factors. T h e large sizes of the ATF/CREB and AP-1 protein families, and the ability of at least some members of each family to form heterodimers that bind ATFICREB or AP-1 sites efficiently, suggest that the mechanism underlying the synergy displayed by the ElA protein and activators of cellular protein kinases in activation of transcription is complex.

66


Nevertheless, such synergy raises the possibility that phosphorylation of cellular transcription factors may contribute to E 1A protein transactivation (see Section 111,D). c. Other Sequence-SpeciJic Factors. Although studied in less detail, a number of other sequence-specific cellular factors have been reported to be involved in E1A protein transactivation. Raychaudhuri et al. (1987) have identified and partially characterized a cellular factor, termed E4F, that binds to the -53 to -46 and -63 to - 170 regions of the E4 protein. T h e binding sites for this factor, deduced from the results of methylation interference experiments, overlap two ATF/CREB sites of the E4 promoter (Fig. 2). Nevertheless, binding sites for E4F could not be detected in other viral promoters (Raychaudhuri et al., 1989), suggesting that E4F and ATF/CREB are distinct factors. Similarly, the relationship of E4F to an independently isolated factor, termed EivF, which binds to the E4 promoter with identical specificity (Cortes et al., 1988), has not been established. Preparations of EivF contain 65- and 72-kDa proteins that indepently stimulate E4 transcription in a reconstituted system (Cortes et al., 1988). The DNA-binding activities of these proteins, as well as of 31and 45-kDa proteins that can bind to both ATFICREB and AP-1 sites in the E3 and E4 promoters, have been reported to be modulated by in vitro dephosphorylation or phosphorylation (Merino et al., 1989). Other factors have been implicated in transactivation by analysis of elements within E 1A-responsive promoters that contribute to transactivation. These include an NF-KB site in the immunoglobulin K chain enhancer (Shurman et al., 1989), a tandemly repeated, 10-bp sequence of the adeno-associated virus p5 promoter whose binding protein has not been identified (Chang et al., 1989)and a binding site for USFiMLTF in the adeno-associated virus P5 promoter (Chang et al., 1989). T h e USF/ MLTF-binding site of the Ad2 ML promoter (Sawadogo and Roeder, 1985; Carthew et al., 1985; Moncollin et al., 1986) is also required for maximal transactivation of this promoter in vivo and in vitro (Lewis and Manley, 1985; R. L. Albin, M. L. Harter, and S. J. Flint, unpublished observations).

a.

2. General Transcription Factors TFZZD. T h e ElB promoter (Fig. 2) is one of the simplest pro-

moters in the viral genome, comprising only two principal elements, a TATA element at -30 to - 24 and a consensus Sp 1 recognition sequence at -48 to -39 (Wu et al., 1987; Parks et al., 1988). Only the TATA element appears to be essential for transactivation of the E1B promoter (Wu et al., 1987; reviewed in Flint and Shenk, 1989).

ADENOVIRUS E I A FUNCTION

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The majority of eucaryotic RNA polymerase I1 promoters contain TATA elements 20-35 nucleotides upstream of the sites at which transcription initiates (reviewed in Breathnach and Chambon, 1981; Shenk, 1981). These elements are recognized by the factor termed TFIID (reviewed in Saltzman and Weinmann, 1989). Binding of TFIID to the TATA element of a mammalian promoter appears to be the first, and committing, step in the formatlon of productive initiation complexes (reviewed in Nakajima et al., 1988; Buratowski et al., 1989). Mammalian TFIID is also the target for some cellular proteins that specifically bind to upstream promoter elements (Sawadago and Roeder, 1985; Horikoshi et al., 1988a, b), a property that emphasizes its critical role in initiation of transcription. Elegant in vitro studies have demonstrated that the pseudorabies virus 1E transactivator protein stimulates transcription by facilitating interaction of TFIID with the TATA sequence, enabling this factor to compete with nonspecific binding proteins such as histones, for binding to the template (Abmayr et al., 1988; Workman et al., 1988). It is, in view of these properties, surprising that TFIID does not play a more general role in transactivation by the E 1A protein. One property that could explain the absence of a more general role for the TATA sequence in transactivation would be functional variation among TATA elements. Such variation was initially suggested by analysis of the TATAAA sequence of the hp70 promoter. Mutation of this sequence eliminated E 1A protein transactivation, and its substitution by the TATTTAT sequence of the SV40 early promoter rendered the promoter refractory to stimulation by the E1A protein (Simon et d.,1988). Similarly, substitution of the ElB TATA sequence with that from the E4 promoter significantly increases basal activity and virtually eliminates E 1A protein-dependent stimulation of E 1B transcription in infected cells (Pei and Berk, 1989). More recent studies of the hp70 promoter have, however, presented a quite different picture of its transactivation. In two independent, carefully controlled analyses, mutation of any one of the several h p 7 0 promoter elements, including the TATA element, reduced both basal and E 1A protein-induced transcription, but no mutation significantly altered the induction ratio (Williams et al., 1989; Taylor and Kingston, 1990). I n other words, neither the TATA nor any other element of the hp70 promoter was essential to its transactivation by the 289R E 1A protein. Moreover, the activity of all hp70 promoters tested, regardless of whether they contained the native TATA element or those of the SV40 early or Ad2 E2E promoters, was stimulated by the E1A protein (Taylor and Kingston, 1990). Although TFIID does not play a general role in transactivation by the E1A protein, it has been suggested to participate in transactivation of

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additional viral and cellular promoters. Like the E1B promoter, the adenovirus ML promoter is constructed of two principal elements, a TATA element and a binding site for the cellular factor USF/MLTF at -63 to -52 (Sawadogo and Roeder, 1985; Carthew et al., 1985; Moncollin et al., 1986). T h e ML promoter can be transactivated by the ElA protein or the CR3 peptide in vitro (see Section 111,B). Extracts of adenovirus-infected cells harvested during the late phase of infection transcribe the ML promoter up to 20-fold times more efficiently than mock-infected cell extracts (Leong and Berk, 1986). The factor responsible for such activation of ML transcription cofractionates with TFIID through one chromatographic step (Leong et aE., 1988). This observation, and the fact that stimulation of ML transcription under these conditions does not require promoter sequences upstream of the TATA element (Leong and Berk, 1986), lead to the suggestion that TFIID plays an important role in transactivation of ML transcription under these conditions (Leong et al., 1988). It remains possible, however, that this stimulation requires the ElA protein (Leong and Berk, 1986) only to permit the infection to enter the late phase, when a second, unidentified transactivator of ML transcription is expressed (Mansour et al., 1986;Jansen-Durr et al., 1988). TATA elements have also been ascribed critical roles in transactivation of transcription from the HIV-1 (Nabel et al., 1988; Bielinska et al., 1989) and c-fos (Simon et at., 1990) promoters. Mutation of these TATA elements reduced both basal and E 1A protein-stimulated expression, in some cases to undetectable levels, but it has not been established that alteration of the TATA elements specifically reduced the induction ratio. Thus, at this juncture, the only clear evidence for participation of TATA-binding proteins in transactivation comes from studies of the E 1B promoter in infected cells (Wu et al. 1987).

b. TFIIIC. Transcription of the adenovirus VA RNA genes by RNA polymerase I11 is stimulated, in E 1A protein-dependent fashion, both in vivo and in vitro (see Section 111,B). The activity of TFIIIC, the ratelimiting component in transcription of VA RNA genes, is increased in extracts of adenovirus-infected or -transformed cells (Hoeffler and Roeder, 1985; Yoshinaga et al., 1986). Two complexes formed between VA RNA promoter DNA and TFIIIC can be detected using gel mobility shift assays, and the concentration of the complex containing active TFIIIC increases in response to adenovirus infection, in the absence of an increase in the total level of TFIIIC (W. K. Hoeffler et al., 1988). As these two complexes can be interconverted by dephosphorylation, it has been suggested that the E 1A protein modulates the phosphorylation state of TFIIIC (W. K. Hoeffler et nl., 1988). Using Sarkosyl to permit


69

analysis of single cycles of transcription, Kovelman and Roeder (1990) have demonstrated that stimulation of transcription by RNA polymerase I11 in adenovirus-infected cell extracts is the result of formation of an increased number of transcription complexes. In toto, then, these in vitro studies suggest that adenovirus infection induces an E 1A proteindependent mechanism that results in increased concentrations of transcriptionally active TFIIIC and thus a greater number of transcription complexes containing the active form of this factor. D. MECHANISMS OF TRANSACTIVATION

T h e failure to identify promoter elements required for ElA proteininduced, but not basal, transcription from responsive promoters and the promiscuity of E 1A protein transactivation are most simply explained by an indirect mechanism of transactivation. The view that the E 1A protein induces increased concentrations of cellular transcription factors or increased levels of active factors has therefore received widespread attention (see Berk, 1986;Jones et d.,1988; Nevins, 1989).The ability of E l A proteins to interact with cellular proteins (see Section IV,B,P), including specific transcription factors (K. McGuire and R. Weimann, personal communication), is also consistent with indirect mechanisms of transactivation. The 289R E 1A protein and CR3 peptide stimulate transcription when added to uninfected cell nuclear extracts in vitro (Spangler et aZ., 1987; Green el al., 1988).It is therefore unlikely that transactivation is generally a result of E 1A protein-induced increases in the absolute concentration of cellular transcriptional components. A mechanism invoking E 1A protein-dependent modification of cellular transcriptional components to increase one o r more of their activities has a number of virtues. In particular, this kind of mechanism can account for the ability of the E 1A protein to work via both general transcription factors (TFIID and TFIIIC) and several sequence-specific factors (see Section lII,C.), and regulation of the activities of cellular transcription factors by phosphorylation has several precedents (e.g., Sorger et al., 1987; Gonzalez and Montminy, 1989). T h e notion that the ElA protein might, directly or indirectly, induce phosphorylation of cellular transcription factors has, consequently, received considerable attention. Two kinds of observation appear consistent with such a mechanism. In the first place, increased phosphorylation of both the general factor TFIIIC and the sequencespecific factors E2F, E4F, and EivF has been detected in adenovirusinfected cell extracts, or following in vitro reaction with infected cell components (Kovelman and Roeder, 1990; Raychaudhuri et ad., 1987,

70


1989; Bagchi et al., 1989; Merino et al., 1989).Second, inducers of cellular protein kinases, CAMP and TPA, can act synergistically with the E1A protein to stimulate transcription from several early promoters (Engel et al., 1988; Buckbinder et al., 1989) and to induce AP-1 activity (Muller et al., 1989). In no case, however, has it been demonstrated that the E1A proteins either phosphorylate cellular factors or modify the activity of a cellular protein kinase. Additional experiments are therefore required to determine unequivocally whether the E1A proteins can alter the phosphorylation state of cellular transcription factors. As adenovirus infection induces profound physiological changes, many of which are mediated by E1A proteins (see Section IV), it will also be important to establish that infected cell-specific alterations in phosphorylation of specific factors are direct effectors of transactivation. It has generally been observed that any promoter that retains detectable basal activity can be transactivated by the E1A proteins. Although the absolute levels of both basal and E 1A protein-stimulated transcriptional activity are typically reduced by mutation of promoter elements, mutant promoters generally remain ElA inducible (see Berk, 1986; Flint and Shenk, 1989; Williams et al., 1989; Taylor and Kingston, 1990). The plasticity of E 1A-responsive promoters is further emphasized by properties of artificial promoters. In a compelling experiment, Taylor and Kingston (1990) tested the E l A protein inducibility of 24 promoters, of which all but the parental hsp70 promoter were artificial. This set of 24 promoters represented all combinations of six upstream elements, ATF, Spl, AP-1, OCTA, and CCAAT protein-binding sites or a nonsense element with TATA elements from the hsp70, SV40 early, or E2E promoters and a nonsense TATA. Some of the factors binding to the upstream site had not previously been implicated in E 1A-mediated transactivation (Spl and OCTA), and eight of these promoters contained one or more nonsense elements. Nevertheless, the activity of every promoter tested was stimulated at least 10-fold by the ElA protein. Importantly, no correlation between the presence of binding sites for specific factors and the degree of transactivation could be discerned (Taylor and Kingston, 1990). If certain cellular factors were preferential targets of the E1A protein, then promoters carrying binding sites for those factors (e.g., AP-1 and ATF) would be predicted to be induced more strongly. However, the only correlation observed was an inverse relation between induced strengths and basal strengths of these promoters (Taylor and Kingston, 1990). These results cannot formally exclude the possibility that the E1A protein can transactivate indirectly via a large number of factors, or combinations of factors. The very large number of E1A protein-


71

responsive, artificial promoters is, however, more readily reconciled with models of transactivation in which the function of the E1A protein is independent of any specific promoter element and its cognate sequencespecific factor. Models that exclude interaction of the E1A protein with, or its effect upon, transcription factors that bind to the promoter imply that the E1A protein must either function directly at the promoter and/ or modify the function of general transcription components (Williams et al., 1989; Taylor and Kingston, 1990). The former prediction has received support from a quite different experimental approach. A fusion protein comprising the DNA-binding domain of the yeast GAL4 transcriptional activator and a segment of the E1A protein (amino acids 121-223) that includes the transactivation domain activated expression from the adenovirus E4 and E1B and the mouse mammary tumor virus LTR promoters. Activation was, however, more efficient when binding sites for the GAL4 protein were added to the promoters (Lillie and Green, 1989). Amino acids 142-178 comprise the minimal segment of the E1A protein required for activation of GAL4-binding sitecontaining promoters by such fusion proteins (Lillie and Green, 1989; Martin et al., 1990). Addition of the herpes simplex virus type 1 VP16 protein transcriptional activating region to GAL4-El A fusion proteins from which portions of the ElA activation regron had been deleted restored their ability to activate expression, independent of the presence of GAL4 DNA-binding sites in the promoters (Lillieand Green, 1989). As a GAL4-VP16 fusion protein did not activate transcription from the wild-type E4 promoter, this result implies that amino acids 150-223 of the ElA proteins provide a function that brings the VP16 protein activating region to the promoter (Lillie and Green, 1989). Consistent with this conclusion, mutations altering or removing the C-terminal segment of CR3 destroyed the ability of GAL4-E 1A-VP16 fusion proteins to activate expression from promoters lacking GAL4 DNA-binding sites, but not activation of GAL4-binding site-containing promoters (Lillie and Green, 1989). Thus, this line of experimentation indicates that the ElA protein contains a region, which includes the C-terminal segment of CR3, that, at least when present in artificial fusion proteins, can interact with the promoter. The notion that the ElA protein can recognize promoters raises the questions of how the recognition occurs and how promoter-associated E 1A protein might stimulate transcription. Promoter recognition could be the result of either direct interaction with DNA or interaction between promoter-bound cellular transcription factors and the E 1A protein. Although the ElA protein contains a Zn*+-binding finger within the Cterminal portion of CR3 that has been ascribed a promoter-interaction

72


function (see Section III,A), no evidence has been collected demonstrating that the E l A protein can bind to DNA in a sequence-specific fashion (Chatterjee et al., 1988). Indeed, certain properties exhibited by fusion proteins in transient assays have prompted the suggestion that function of the E 1A protein at the promoter requires an “adaptor” protein. This idea is based on the observation that the wild-type E1A protein can stimulate expression from a promoter comprising a GAL4 DNA-binding site and the E l B TATA element only when cotransfected into cells expressing a GAL4-ATF-2 fusion protein (Liu and Green, 1990). These observations, together with the similar ability of the known E 1A-binding retinoblastoma protein (see Section IV,B,2), when expressed as a GAL4 fusion protein, to support transactivation by the wild-type E 1A protein, and the requirement for ATF-2 sequences that are not part of its DNAbinding domain, led Liu and Green (1990) to conclude that promoterbound GAL4-ATF-2 protein recruits the E1A protein to the promoter via specific interactions between the N-terminal region of ATF-2 and the viral transactivator. T h e hypothesis that the E1A protein is brought to the promoter via a specific adaptor molecule, ATF-2, can, of course, apply only to E1A protein-responsive promoters that contain binding sites for this factor. As we have seen, many transactivatable promoters do not. Liu and Green (1990) suggest that the ElA protein might not, in fact, act as promiscuously as is generally supposed, a proposition that appears to be ruled out by the stimulation of activity of all members of a large set of promoters (Taylor and Kingston, 1990). Other mechanisms that could account for transactivation of promoters that lack ATF-2-binding sites include association of the ElA protein with “adapting” protein motifs in addition to that present in ATF-2, transactivation of some promoters by a mechanism independent of the CR3 segment of the ElA protein, and interaction of the ElA protein with some promoters via its nonspecific binding to DNA (Liu and Green, 1990). The observations of Liu and Green (1990) are, however, compatible with a rather different mechanism of E 1A protein transactivation proposed by Taylor and Kingston (1990). Because all synthetic promoters tested could be transactivated by the E1A protein, these authors proposed that the E1A protein comes to a promoter independently of any sequence-specific transcription factor and stimulates transcription as a result of effects on the general transcription machinery. With the exception of TFIID, whose binding to the TATA element is an early and “committing” step in initiation (see Section III,C,2), the well-characterized general transcription factors, TFIIB, TFIIE, and TFIIF, as well as RNA polymerase 11, enter transcription complexes relatively late in the


73

initiation sequence, after the commitment step (see Nakajima et al., 1988; Saltzman and Weinmann, 1989; Buratowski et al., 1989). Activation of transcription by the EIA protein must be the result of stimulation of the rate-limiting step in initiation on a given promoter. If the EIA protein in fact increased the activity of one of the general factors listed above, it could exert no effect under conditions in which the commitment step were rate limiting. The undetectable basal activity of the weak reporter promoter suggests that the latter condition may well have pertained in the experiments of Liu and Green (1990). Under such circumstances, any alteration in conditions that relieved rate limitation during the commitment step, such as expression of an upstream activating factor that could bind to the promoter [GAL4-ATF-2 in the case of the GAL4-ElB reporter promoter of Liu and Green (I 990)], would render the promoter sensitive to transactivation. Thus, the notion that the E 1A protein exerts its influence at the promoter via the component(s) of the general transcription machinery provides a parsimonious, unifying hypothesis of transactivation. In sum, three kinds of mechanisms have been proposed to account for E 1A protein transactivation: an indirect role via phosphorylationinduced alterations in the activities of sequence-specific cellular transcription factors, a more direct role at the promoter of the E1A protein targeted via its interaction with specific adaptor molecules (such as ATF-Z), and direct effects exerted as a result of interaction with, or effects upon, general transcription factors. As discussed, these models can, with varying degrees of facility, account for the unusual properties of EIA protein transactivation. On the other hand, all must be considered as no more than working models, for none has yet received an unequivocal experimental demonstration of validity. IV. Transforming Activity of E1A Proteins

T h e E 1A gene alone can immortalize but not fully transform primary rodent cells (Houweling et al., 1980; Ruley, 1983; Zerler et al., 1986). It can cooperate with the adenovirus E l B gene, the polyoma virus middle T antigen gene, or the activated Ha-ras oncogene to produce a transformed phenotype in primary rat cells (Ruley, 1983). The exact changes contributing to the transformed phenotype depend on the cell type under study, but generally include changes in cell morphology, growth to high cell density with loss of contact inhibition, growth in medium containing reduced serum levels, and loss of anchorage dependence (Graham and van der Eb, 1973; Meager et al., 1975). The E1A gene has been reported to transform established NIH 3T3 cells in the absence of a cooperating

74


oncogene when it was expressed at high levels under control of a heterologous promoter (Senear and Lewis, 1986). However, results obtained with established cell lines are difficult to interpret because such lines may harbor mutant recessive oncogenes, such as p53 (discussed in Section IV,B,2), which could influence the transforming activity of E 1A proteins. A. TRANSFORMING DOMAINS Using cDNA constructs it has been possible to demonstrate that either the 13s or 12s ElA product can immortalize rodent cells, although distinctive morphological changes result from expression of the two different E1A polypeptides (Haley et al., 1984; Roberts et al., 1985; Zerler et al., 1986). T h e product of either cDNA can also cooperate with Ha-rusencoded protein to induce transformation of primary rat cells (Zerler et al., 1986; Stephens and Harlow, 1987; Ulfendahl et al., 1987). These experiments lead to the conclusion that conserved region 3, which is not present in the 243R product of the 12s mRNA (Fig. 1A), is not essential for either immortalization or transformation of primary cells by E 1A proteins. The products of the 11s and 10s ElA mRNAs are not able to transform primary cells in cooperation with either polyoma virus middle T antigen o r the Ha-rm-encoded protein (Stephens and Harlow, 1987; Ulfendahl et al., 1987). Both of these ElA polypeptides lack conserved region 1 (Fig. lA), so these results argue that this domain is important for E 1A transforming activity. Mutant studies support this view. Mutations localized within the domain extending from amino acid 1 to 85 can interfere with the transforming activity of E 1A protiens (Schneider et al., 1987; Smith and Ziff, 1988; Velcich and Ziff, 1988; Whyte et al., 1988b). Thus, sequences upstream of and including conserved region 1 play a role in E1A-mediated transformation. The role of amino acids 1-14 is not entirely clear, however. Although mutations in these regions inhibited transformation of primary baby rat kidney cells (Whyte et al., 1988b) and established REF52 rat cells (Velcich and Ziff, 1988) in cooperation with activated rm genes, a similar mutant protein was able to transform primary rat embryo cells at near normal levels in cooperation with the adenovirus ElB gene (Osborne et al., 1982). Perhaps this apparent contradiction results from the use of different cell types or different cooperating oncogenes. Conserved region 2 is also required for the oncogenic activity of E1A proteins. Deletions and amino acid substitutions within this region impair the ability of the protein to immortalize and cooperate with activated ras-encoded proteins to transform primary rat cells (Lillie et al., 1986; Moran et al., 1986; Schneider et al., 1987; Whyte et al., 1988b). Conserved


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region 2 extends from amino acid 121 to 139 of the E1A protein (Fig. 1A). The key domain within conserved region 2 apparently corresponds to residues 121- 127; mutant genes lacking residues 128- 139 are capable of cooperating with ras, but at reduced efficiency (Whyte et al., 1988b). I n the E1A proteins, some mutations outside of the 1 + 85 and 12 1 + 139 domains reduce but do not eliminate the transforming ability of E1A proteins (e.g., Whyte et al., 1988b). These mutations may cause global alterations in the structure of E l A proteins and influence their stability or intracellular location. In sum, the domains that are critical for the oncogenic activity of E l A proteins (Fig. 1B) correspond to sequences upstream of and within conserved region 1 (amino acids 1-85) and a portion of conserved region 2 (amino acids 121-127). Interestingly, mutant ElA proteins lacking either conserved region 1 or 2 can complement to transform cells (Moran and Zerler, 1988). This suggests that the two domains can function independently, and that both functions are required for transformation by ElA proteins. B. MECHANISM OF TRANSFORMATION As discussed above, the transcriptional induction activity of conserved region 3 is not required for immortalization o r transformation by the E1A proteins. Conserved regions 1 and 2 mediate their transforming ability and two biochemical activities are associated with these E1A domains. T h e first is a transrepression activity and the second is the ability to bind the retinoblastoma gene product, as well as several other cellular proteins. 1. Transrepression Can Be Uncoupled from Transformation E 1A proteins can inhibit the transcriptional activity directed by the polyoma virus early promoter (Velcich et al., 1986), the SV40 early promoter (Borrelli et d., 1984; Velcich and Ziff, 1985), the immunoglobulin heavy-chain enhancer (Hen et al., 1985), the insulin control region (Stein and Ziff, 1987) and the cytochrome P-45Oc control region (Sogawa et al., 1989). Mutational analyses have implicated both conserved regions 1 and 2 of the E l A proteins in transrepression activity (Lillie et al., 1986, 1987; Schneider et at., 1987),although conserved region 2 generally appears to play a lesser role (Schneider et al., 1987) and in some assays it is not required (Rochette-Egly et al., 1990). The mechanism underlying transrepression is unclear. In the case of the SV40 enhancer domain, ElA products inhibit the transcriptional activity of multiple elements comprising the SV40 enhancer (Rochette-Egly et al., 1990), suggesting that repression is not specific for one or just a few transcription factors.

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Repression seems likely to occur through a more general, and perhaps indirect, mechanism. T h e fact that transrepression and transforming activitiesboth mapped to conserved regions 1 and 2 led to the proposal that oncogenic transformation might result from the ability of E1A proteins to inhibit transcription of one or more cellular regulatory genes. However, Kuppuswamy and Chinnadurai (1987) have described a mutation in domain 2 of the E 1A protein that abolishes its transforming activity without altering its ability to transrepress. Further, Velcich and Ziff (1988)mutated the E 1A protein, rendering it defective for transrepression without blocking its ability to transform cells in cooperation with an activated ras allele. It therefore seems unlikely that transcriptional repression plays an essential role in the transforming activity of ElA proteins.

2. Binding to Cellular Proteins Correlates with Transformation T h e demonstration that E 1A proteins can bind to the 105-kDa cellular retinoblastoma (RB) protein (Whyte et a.l., 1988a; Egan et al., 1990) provided a new insight into the mechanism of E l A-mediated transformation. The RB gene was first identified through its association with inherited predisposition to retinoblastoma. This tumor characteristically is composed of cells that carry mutations in both RB alleles. Function of the RB protein appears to be required on a continous basis to achieve normal regulation of cell growth, and loss of this “tumor suppressor” or “antioncogene” function can lead to tumor formation. Thus, the E 1A proteins could contribute to the loss of cell growth control if, by binding to the RB protein, they interfered with RB protein function. This interference could mimic the situation in RB tumor cells. Several lines of evidence support the notion that ElA binding might interfere with RB protein function. T h e first derives from mutational analysis of the cellular protein. Two domains that have been identified in the RB protein (amino acids 393-572 and 646-772) are required for its interaction with ELA proteins (Hu et al., 1990). These sites roughly correspond to the locations of naturally occurring mutations in the RB gene (Hu et al., 1990). These mutations are likely mark key functional domains in the RB protein, and ElA protein bound at these sites might well block or alter RB function. Further support for the idea that RB protein binding represents a key to E1A-mediated transformation derives from the fact that it has not been possible to produce a mutation within the El A gene that prevents RB protein binding while maintaining the transformation activity of ElA proteins. In fact, the binding of three cellular proteins, the 105-kDa RB

ADENOVIRUS E l A FUNCTION

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protein plus 107- and 300-kDa polypeptides, is strongly correlated with the transforming activity of ElA proteins (Whyte et al., 1989; Egan et al., 1990). Mutational analysis indicates that the 300-kDa protein binding requires amino acids 1-76 (sequences within the upstream of conserved region l), the 107-kDa protein interacts at amino acids 121-127 (within conserved region 2), and the 105-kDa RB protein requires both amino acids 30-60 and 121-127 (conserved regions 1 and 2) to bind E1A proteins (Whyte et al., 1989). These domains of the E1A proteins are precisely the regions required for its transforming activity. Further, it is possible to mutate the E1A protein to block binding of the 300-kDa protein while maintaining 107- and 105-kDa RB protein binding and vice versa. Neither class of mutant E1A protein can cooperate with an activated rus allele to transform cells, suggesting that at least two and perhaps all three cellular proteins must be bound for transformation to occur. A final argument in support of the hypothesis that RB protein binding is important for the oncogenic activity of E1A proteins comes from the fact that a variety of DNA tumor virus oncogene products bind the protein. I n addition to the E1A proteins, the large T antigens of SV40 (De Caprio et al., 1988; Dyson et al., 1989a) and a variety of other polyomaviruses (Dyson et al., 1990) as well as the E7 protein of human papillomavirus type 16 (Dyson et al., 1989b) all bind the RB protein, and the SV40 large T antigen binds to the same sites on the RB protein as do the E l A proteins (Hu et al., 1990). I n addition to the cellular proteins discussed above, the E1A proteins bind a 60-kDa polypeptide that is normally found in a complex with the cdc2 kinase (Giordano et ul., 1989). This kinase is involved in the regulation of the cell cycle in a wide variety of eucaryotes. Although the role of the 60-kDa polypeptide in cdc2 function is obscure, it is possible that the interaction between the 60-kDa protein and E l A proteins could lead to changes in the activity of the cdc2 kinase, and thereby alter the normal regulation of the cell cycle. However, it is not clear whether this interaction might contribute to the transforming activity of ElA proteins. As yet, experiments that test for a correlation between p60 binding and E1Atransforming activity have not been reported. Finally, it is of interest to note that a second tumor suppressor gene product, p53 (Lane and Crawford, 1979; Linzer and Levine, 1979), is also bound by the oncoproteins of DNA tumor viruses. In the case of adenoviruses, its second oncogene, ElB, encodes a protein that binds p53 (Sarnow at al., 1982). Similarly, the second oncogene product of human papillomavirus type 16, E6, binds p53 (Werness et al., 1990), and the SV40 large T antigen binds both the RB protein and p53. Thus, a theme emerges. T h e oncogenes of DNA tumor viruses bind at least two (RB

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protein and p53) tumor suppressor gene products, and perhaps more if the 60-, 107-, and 300-kDa proteins discussed above prove to have similar growth regulatory functions.

C. WHYIs THE ElA GENEAN ONCOGENE? Under normal conditions adenoviruses d o not transform human cells, and they are not known to contribute to tumorigenesis in their normal human hosts. Yet the E1A gene is an oncogene whose transforming acitivity can be easily demonstrated in rodent cells. Why are the E1A products oncoproteins? Adenoviruses, like many viruses, grow more efficiently in rapidly growing cells than in quiescent cells. Because many of the cells that the virus is likely to infect in its normal human host are quiescent, the virus has evolved mechanisms to activate cells, driving them to progress through the cell cycle, synthesize DNA, and secrete growth factors. T h e E1A proteins play a major role in this activation process (Braithwaite el al., 1983; Kaczmarek et al., 1986; Moran and Zerler, 1988; Quinlan et al., 1987; Stabel et al., 1985), and the mechanism underlying the activation almost certainly involves interfering with the normal activity of growth regulatory proteins, such as the RB protein. This line of reasoning suggests that transformation by E 1A proteins is an abnormal manifestation of a normal lytic function. Transformation occurs in response to E1A functions when the host cell is not killed, either because rodent cells are nonpermissive for replication of an infecting adenovirus or because the E 1A gene is introduced under artifical conditions, e.g., DNA transfection in the absence of the remainder of the viral chromosome. V. Perspectives

T h e E1A proteins are able to enter complexes with a variety of cellular proteins, and it seems likely that these interactions are critical to all E 1A-mediated activities. The best supported model for the mechanism of transcriptional activation invokes action of E 1A proteins at the promoter as part of the initiation complex. Definitive proof of this model requires the isolation of an initiation complex that contains the E1A protein, and the demonstration that the transactivator enhances the rate of initiation when it is present in the complex. The most attractive and logical model for the role of ElA proteins in transformation suggests they bind to and interfere with the activity of proteins that regulate cellular growth. Proof' of this model awaits direct assays for biochemical activities of cellular growth regulators, such as the RB protein, and the demonstration that


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the E l A protein modifies these activities. If these models for EIA functions prove to be correct, the next order of business will be the exploration of the molecular events, mediated by E l A proteins, that alter the activity of transcription complexes and the behavior of growth regulatory proteins. ACKNOWLEDGMENTS

We thank our colleagues who kindly provided us with reprints and preprints of their work. We gratefully acknowledge the competent secretarial assistance of Norma Caputo and Lynn Larkin, and the preparation of figures by Christopher Shenk. T. S. is an American Cancer Society Professor. REFERENCES

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MITOGENIC ACTION OF LYSO PH0sPHATIDI C ACID Wouter H. Moolenaar Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands

I. Introduction 11. Phosphatidic and Lysophosphatidlc Acid: Biosynthesis and Cellular Metabolism

111. 1V.

V. VI.

A. Role in a2 Novo Lipid Biosynthesis B. Formation in Stimulated Cells Lysophosphatidic Acids as Mitogens Mechanism of Action A. Signal Transduction Pathways B. Importance of the Early Signals for Cell Proliferation C. Site of Action Other Biological Effects Possible Biological Function and Future Prospects References

I. Introduction

The proliferation of cells in vivo and in culture is tightly regulated by growth factors, the prototype of which is epidermal growth factor (EGF) (Carpenter and Cohen, 1990; Ullrich and Schlessinger, 1990). Growth factors initiate their action by binding to specific, high-affinity receptor molecules on the cell surface, thereby triggering a myriad of complex biochemical changes that ultimately lead to altered gene expression, DNA replication, and cell division. Growth factors are synthesized by both normal and tumor cells and usually act through paracrine and autocrine mechanisms. Cells activated by growth factors share many phenotypic properties with cells transformed by oncogenic retroviruses, and inappropriate expression of the cellular counterparts of viral oncogenes is thought to play a role in the initiation and maintenance of malignant growth. Hence, the study of the mode of action of growth factors will undoubtedly yield important insights into mechanisms underlying oncogenesis. Although many growth factors and their receptors have been identified, cloned, and characterized, the signal transduction pathways that culminate into replicative DNA synthesis and cell proliferation are poorly understood. The identification of novel mitogens acting on the cell surface and the elucidation of their mechanism of action may help to unravel the complex “mitogenic signaling network,” wherein 87 ADVANCES IN CANCER RESEARCH, VOL. 57

Copyright 0 1991 by Academic Press, Inc. All rights of reprduction in any form reserved.

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multiple signal cascades often act in parallel and similar responses may be produced by different means. I n recent years, membrane phospholipids have attracted a great deal of interest in studies on cellular signaling and growth control. It has long been realized that there is a variety of cellular phospholipids and that their metabolism is highly complex. Though a major function of phospholipids is to form bilayer membranes, the breakdown products of several plasma membrane phospholipids appear to act as signal molecules, i.e., as intracellular second messengers o r as agonists that modulate cell function. The best known examples of phospholipid-derived signal molecules include diacylglycerol, inositol trisphosphate, and prostaglandins, which are all rapidly generated when cells are stimulated by certain hormones or growth factors. T h e simplest naturally occurring phospholipids, phosphatidic acid (PA, or diacylglycerol-3-phosphate)and lysophosphatidic acid (LPA, or monoacylglycerol-3-phosphate), are of particular interest in that they not only are critical intermediates in de nouo lipid biosynthesis and are rapidly produced in activated cells (suggesting a possible role as signal molecules), but also can stimulate cell proliferation and induce morphological changes when added extracellularly to cells in culture. Understanding of the growth factor-like action and the normal biological function of these simple, low-abundance phospholipids in normal and abnormal cell growth is obviously an important research goal. This review summarizes the current state of knowledge about the multiple biological effects of PA and LPA, with particular emphasis on their growth factor-like activity. The potent mitogenic action of exogenous LPA raises many questions to which there are currently only partial answers: (1) How do these phospholipids act, what is their primary cellular target, and what is their metabolic fate? (2) What is the spectrum of biological activities of LPA and PA? (3) Are these molecules reteased from activated cells to serve as mitogens for neighboring cells? (4) Is LPA capable of inducing phenotypical transformation in its target cells? In addition to addressing these points, a brief overview is given of the role of endogenous LPA and PA in de nono lipid biosynthesis and their rapid formation in activated cells.

If. Phosphatidic and Lysophosphatidic Acid: Biosynthesis and Cellular Metabolism

A. ROLEIN DE Novo LIPIDBIOSYNTHESIS LPA and PA are key intermediates in the early steps of phospholipid biosynthesis (for review, see Bishop and Bell, 1988, and references

MITOGENIC ACTION OF LYSOPHOSPHATIDIC ACID

89

therein). These early biosynthetic steps are summarized in Fig. 1. T h e starting point is glycerol-3-phosphate, which is acylated by acyl-CoA to yield LPA (1-acylglycerol-3-phosphate). LPA is then further acylated by the same enzyme (glycerol-3-phosphate acyltransferase) to form PA. As a rule, the fatty acyl chain attached to the C1 position of the glycerol backbone is saturated, whereas the one attached to C2 is usually unsaturated. PA is located at a branchpoint in de nouo phospholipid synthesis (Fig. 1). PA can be hydrolyzed by a specific phosphatase to give diacylglycerol, which is then used for the synthesis of more complex phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). PA can also be converted to cytidinediphospho (CDP)-diacylglycerol for the synthesis of acidic phospholipids such as phosphatidylinositol (PI) and phosphatidylserine (PS). These de nouo metabolic reactions occur mainly in the endoplasmic reticulum (ER) membrane. This is in marked contrast to the rapid generation of LPA and diacylglycerol through the hydrolysis

glycerol-3-P

I

6

OH OH

1-acylglycerol-3- P

lER

$2

phosphatidic acid

8 ? A I

CDP-diacylglycerol

diacylglycerol

FIG. 1. Outline of early steps in de novo phospholipid biosynthesis, showing key precursor role of lysophosphatidic acid (1-acylglycerol-3-phosphate) and phosphatidic acid (diacylglycerol-3-phosphate). Cytidinediphospho (CDP)-diacylglyceroland diacylglycerol are utilized to form phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and other phospholipids. These metabolic reactions occur predominantly in the endoplasmic reticulum (ER).

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WOUTER H. MOOLENAAR

of preexisting phospholipids during cell activation, which occurs in the plasma membrane. B. FORMATION IN STIMULATED CELLS

There has been considerable progress in recent years in understanding the generation and metabolism of phospholipid-derived signal molecules. It is now well established that many hormones, neurotransmitters, and growth factors exert their immediate cellular effects, at least in part, through the hydrolysis of phosphatidylinositol-4,5-P2 (PIP2) to inositol1,4,5-P3 (IPJ), the second messenger that releases stored Ca2+,and 1,2diacylglycerol (DG), the natural activator of protein kinase C. Many, if not all, of these agonist also stimulate the breakdown of phosphatidylcholine through the action of phospholipases of the C and D types (PLC and PLD) to yield DG and PA, respectively. The main characteristics of these signaling systems have been the subject of several reviews (Berridge, 1987; Nishizuka, 1986; Exton, 1990). When cells are stimulated by various agonists, the level of PA in the plasma membrane rises rapidly. PA can be generated via phosphorylation of newly formed DG by DG kinase (Kanoh et al., 1990) or, more directly, through the action of a cellular phospholipase D acting on PC and perhaps also PE (Bocckino et nl., 1987; Exton, 1990). In the latter case, the level of PA often rises more rapidly and to a higher level than that of DG. Whatever its mechanism of formation, PA can function not only as a source of DG (the second messenger for protein kinase C) and as a precursor for CDP-DG in de nono synthetic pathways (Fig. l ) , but also as a potential signal molecule in its own right, as will be discussed below. While the rapid appearance of PA in stimulated cells is generally observed as a direct consequence of the receptor-linked activation of PLC and/or PLD, the generation of LPA during cell activation has been much less thoroughly examined. LPA (mainly C16.0and CIS: 0) has been shown to accumulate rapidly in thrombin-stimulated platelets (Lapetina et nl., 1981; Watson etal., 1985; Gerrard and Robinson, 1989). The production of LPA may be secondary to the formation of PA through the action of a PA-specific phospholipase A2 or, alternatively, through phosphorylation of monoacylglycerol generated by a DG lipase. A PA-specific phospholipase A2 activity, which differs from the type that degrades PC and PE, has indeed been found in platelets (Billah et al., 1981) and it therefore seems reasonable to assume the LPA formation is of physiological significance. An outline of PA and LPA metabolism in stimulated cells is illustrated in Fig. 2. It remains to be explored whether LPA formation upon cell activation occurs as ubiquitously as the accumulation of PA.


91

OH I

7-F-C

Q

?-

o=p-oI

9

diacy O=! lglycerol f;O

...........

R

7-F-C 0 OH

?

O=$

i:..........? PLD i ' y l

o=y-o-

I

o=p-oI

phosphatidic acid

lysophosphatidic acid

I

o=c c=o

g$

phospholipid

FIG. 2. Generation of phosphatidic and lysophosphatidic acids from newly formed diacylglycerol or preexisting phospholipids during cell activation. Diacylglycerol is rapidly formed via receptor-linked activation of phospholipase C and is phosphorylated by diacylglycerol kinase to yield phosphatidic acid, which may also be formed through activation of phospholipase D (PLD) acting on phosphatidylcholine and phosphatidylethanolamine (R denotes the phospholipid headgroup). Phosphatidic acid may be hydrolyzed by a phospholipase A2 (PLA2) to lysophosphatidic acid. The contribution of other pathways to generating lysophosphatidic acid remains to be explored.

Ill. Lysophosphatidic Acids as Mitogens I n addition to being rapidly produced in stimulated cells, PA and LPA can exert their own biological effects. In particular, exogenous PA and LPA stimulate DNA synthesis and cell division in fibroblasts and epithelial cells (Moolenaar et al., 1986; Siegmann, 1987; Yu et al., 1988; van Corven et al., 1989; Imagawa et al., 1989), with LPA being a more potent growth stimulant than the corresponding PA species. Both LPA (1oleoyl) and PA (1,2-dioleoyl) stimulate thymidine incorporation in Rat- 1 cells, with a half-maximal effect observed at about 15 pM and a saturating response at about 100 pA4 (Moolenaar and van Corven, 1990).Although PA often contains a few percent of contaminating LPA (Benton et al., 1982; Jalink et al., 1990), the observed dose-response curve of the mitogenic effect of PA cannot simply be explained by LPA impurities in the

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WOUTER H. MOOLENAAR

PA used. LPA is about equally potent as EGF or 10%fetal calf serum in stimulating thymidine incorporation and cell division in Rat- 1 fibroblasts. As with polypeptide growth factors, the mitogenic response to LPA requires long-term presence of the stimulus. When LPA is removed from the culture medium several hours after stimulation, the cells fail to enter S phase. The growth-stimulating activity does not require the presence of peptide growth factors: neither insulin nor EGF was found to act in a synergistic fashion with LPA (van Corven et al., 1989). In this respect, LPA acts differently from mitogenic peptides such as serotonin or vasopressin, which fail to stimulate cell proliferation unless synergizing growth factors are present (Rozengurt, 1986). T h e effect of LPA and PA on cell proliferation appears to be highly specific. All common lipids, other than PA and LPA, are incapable of stimulating DNA synthesis in quiescent fibroblasts (Moolenaar et al., 1986; van Corven et al., 1989). The relative potencies of various LPA and PA analogs on fibroblast proliferation have recently been determined (E. van Corven, unpublished results). Among the LPAs, (218: lLPA (l-oleoyl) and CIS:LPA (1-palmitoyl) show the most potent activity, whereas the potency decreases when the fatty acid chain length is decreased. In fact, the short-chain LPA, C12 : 0 LPA, shows hardly any mitogenic activity on quiescent fibroblasts. A similar order of activity is observed with PA as a stimulus (1,2,-dioleoyl PA = 1,2-palmitoyl PA >> 1,2,-myristoyl PA >> 1,2-lauroyl PA). At present, it is difficult to rule out that the mitogenic activity observed with a long-chain PA is partially due to LPA present as a contaminant or as a PA metabolite generated during the long-term incubations (20-24 hr) required for mitogenic assays. Though LPA is more potent as a mitogen than PA, the reason for this is far from understood. One possibility is that LPA is inserted into the plasma membrane more easily than PA and that only incorporated phospholipid may express mitogenic activity. The finding that long-chain LPA and PA are much more active than the short-chain analogs is consistent with this view. The mechanism of action of LPA and PA will be discussed in further detail in Section 1V.B.

IV. Mechanism of Action

A. SIGNAL TRANSDUCTION PATHWAYS At least two types of cellular signaling mechanisms can be distinguished, which are used by different classes of cell surface receptors. Receptors for peptide growth factors such as EGF are transmembrane proteins with a cytoplasmic domain that functions as a tyrosine-specific

93


protein kinase. Ligand binding causes an immediate stimulation of the catalytic domain, resulting in receptor autophosphorylation as well as in the phosphorylation of various cellular substrate proteins (Ullrich and Schlessinger, 1990; Carpenter and Cohen, 1990). The GTP-binding (G)-protein-linked receptors indirectly communicate to membrane-bound effector enzymes that, upon activation, generate various intracellular messenger molecules. The interaction between the receptor and the effector is mediated by regulatory G proteins. Adrenergic receptors, receptors for neuropeptides, some of which are mitogenic, and probably also the receptors for lipid-soluble agonists such as platelet-activating factor and prostaglandins all belong to the class of G-protein-linked receptors. Figure 3 shows a simplified scheme illustrating how receptor proteins may transmit their signals to the cell interior. PA and LPA are potent and reversible mitogens showing similar doseresponse relationships (Moolenaar and van Corven, 1990). It therefore seems plausible to assume that both lipids act through the same signaling cascades, although formal proof for this notion is lacking at present. Three separate signal transduction pathways in the action of LPA have been identified. These are activation of phospholipase C, release of

J.

PG

U

cellular response

FIG. 3. Schematic representation of receptor (R)-linked signal transduction pathways in growth control. Protein tyrosine kinase receptors act by phosphorylating critical substrate proteins (Ullrich and Schlessinger, 1990). The G-protein-coupled receptors control the activity of effector enzymes such as adenylate cyclase (Ac), regulating CAMPlevels; phospholipase C (PLC), generating two second messengers, i.e., inositol 1,4,5-trisphosphate (IPS), which causes the release of Ca2+,and diacylglycerol (DG), the activator of protein kinase C (PKC); and phospholipase A2 (PLA2), acting on phospholipids to yield lysophospholipids (not shown) and free fatty acids, particularly arachidonic acid (AA), a precursor for prostaglandins (PC) and other lipid metabolites. The putative G proteins controlling phospholipase activity (G, and G,) have not yet been identified. Key: G,, stimulatory G protein; Gi, inhibitory G protein; G,, putative C-protein-activating PLC; G,, putative G-protein-activating PLA2.

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WOUTER H. MOOLENAAR

arachidonic acid presumably as a result of phospholipase A2 activation, and inhibition of adenylate cyclase, as will be discussed below. Little is known about the metabolic conversions of LPA and PA, when added exogenously to cells in culture; the major products formed are probably the corresponding monacyl- and diacylglycerols (Pagan0 and Longmuir, 1985; R. van der Bend, unpublished observations). Mechanistic studies have largely focused on the mode of action of LPA, being the most potent and structurally simplest lipid mitogen. In a recent report, Jalink et al. (1990) presented evidence suggesting that previously reported effects of PA on phospholipase C activity are attributable to contamination with lyso derivatives. LPA mitogenicity is selectively blocked by pertussis toxin (halfmaximal inhibition at 0.2 nglml), whereas the mitogenic response to EGF remains unaffected (van Corven et aL, 1989); this bacterial toxin is known to ADP-ribosylate and thereby inactivate the GJG, family of GTPbinding proteins. 1. Activation of Phospholipase C Addition of LPA to various cell types evokes an immediate breakdown of inositol phospholipids as measured by the formation of inositoltriphosphate and diacylglycerol (van Corven et al., 1989; Jalink et al., 1990). LPA-induced activation of phospholipase C results in a rapid but transient rise in Ca2+,which is primarily caused by the release of intracellularly stored Ca2+. Concomitantly, protein kinase C is activated, as is detected by the phosphorylation of an endogenous 80-kDa protein substrate (van Corven et al., 1989). The kinetics and shape of the LPAinduced Ca2+ increase are virtually indistinguishable from those elicited by bradykinin and other hormones. Furthermore, LPA-induced Ca2+ mobilization is subject to homologous desensitization, a common feature of many signaling systems in which agonist-induced attenuation of cellular responsiveness is thought to have an important regulatory role (Benovic et al., 1988). From studies on permeabilized cells it appears that the response is GTP dependent, suggesting the involvement of a G protein [which is insensitive to pertussis toxin in human fibroblasts (vancorven et al., 1989)l. Thus, the early phosphoinositide-breakdown response to LPA has many of the hallmarks of a receptor-mediated event. Yet, the exact mechanism by which externally added LPA activates GTP-dependent phosphoinositide hydrolysis is not yet clear. Using La3+ as a cellimpermeant “LPA chelator,” Jalink et al. (1990) suggested that LPA exerts its immediate effects via a reversible interaction with an external site on the cell surface. This result, together with the rapidity of the onset


95

of the Ca2+signal, argues against a mechanism that requires trans-bilayer “flip-flop” or internalization of the lipid, although later responses, particularly DNA synthesis, may indeed involve this action.

2. Release of Arachidonic Actd Exogenous LPA, in common with many Ca2+-mobilizing agonists, promotes production of free arachidonic acid, the polyunsaturated fatty acid that is the precursor of prostaglandins and other lipid messengers. Arachidonate can be released from lipids through activation of phospholipase A2, or as a consequence of increased cytoplasmic Ca2+ levels following phospholipase C activation. Treatment of the cells with phorbol ester completely blocks LPA-induced phosphoinositide hydrolysis, without significantly affecting arachidonate liberation. Thus, phorbol ester treatment dissociates LPA-mediated arachidonate release from phosphoinositide breakdown. Studies of permeabilized cells suggest that arachidonate liberation by LPA occurs in a GTP-dependent manner, insensitive to pertussis toxin. The precise biochemical mechanisms underlying this early response to LPA remain to be investigated.

3 . Inhibition of Adenylute Cycluse Both LPA and PA have been reported to inhibit cAMP accumulation in intact cells (Pro11 et ul., 1985; Murayama and Ui, 1987; van Corven et al., 1989). When adenylate cyclase is prestimulated by either forskolin or certain receptor agonists (isoproterenol, prostaglandin El, etc.), addition of LPA decreases cAMP accumulation by a 60-70% within 10 min. This decrease in cAMP is dose dependent and is completely blocked by pretreating the cells with pertussis toxin, indicating that LPA acts through the Gi protein that inhibits adenylate cyclase. B. IMPORTANCEOF THE EARLYSIGNALS FOR CELLPROLIFERATION T h e question now arises as to which, if any, of these early biochemical responses accounts for the late mitogenic effect of LPA. Quite unexpectedly, and contrary to prevailing views (Whitman and Cantley, 1988), it seems unlikely that activation of the phosphoinositide hydrolysisCa2+-protein kinase C pathway is of major importance for mitogenesis. When phospoinositide hydrolysis is blocked by phorbol ester, there is no effect on LPA-induced DNA synthesis. Furthermore, when protein kinase C activity is functionally removed by long-term treatment with 12-0-tetradecanoylphorbol-13-acetate (TPA), LPA is still fully mitogenic (van Corven et al., 1989). It thus appears tha LPA does not rely on the

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WOUTER H. MOOLENAAR

phosphoinositide hydrolysis-protein kinase C cascade to stimulate cell proliferation. Furthermore, we found that bradykinin mimics LPA in stimulating phosphoinositide hydrolysis and arachidonic acid release, at least qualitatively, but fails to elicit a detectable mitogenic response (van Corven et al., 1989). The latter finding further argues against the view that activation of the phospholipase C cascade is sufficient for mitogenesis, at least in previously quiescent fibroblasts. It is also unlikely that LPA-stimulated arachidonate release is important for the mitogenic action of LPA. First, addition of exogenous arachidonate or prostaglandins to quiescent cells does not stimulate DNA synthesis. Second, the estimated EC50 of LPA for arachidonate release is at least an order of magnitude greater than for mitogenesis. In this regard, low concentrations of LPA (-5 pM) can elicit a significant proliferative response while arachidonate release is barely detectable (van Corven et al., 1989). The results obtained with pertussis toxin strongly suggest that Gi, the inhibitory G protein of adenylate cyclase, is critical for LPA-induced mitogenesis. This notion is further supported by the finding that the nonmitogenic peptide bradykinin, which activates G, but not Gi, fails to stimulate DNA synthesis. Interestingly, Seuwen et al. (1988) inferred a similar important role for G, from their studies on serotonin-induced mitogenesis. Figure 4 summarizes the various early responses to in the action of LPA that have been identified. An unexplained finding is that LPA is about 100-fold more potent in evoking its immediate effects than in inducing DNA synthesis (Jalink et al., 1990). How could activation of the G; pathway with subsequent inhibition of adenylate cyclase lead to DNA synthesis? cAMP has long been known as a modulator of cell growth in many cell systems (Rozengurt, 1986). In normal fibroblasts, including Rat-1 and HF cells, agents that raise intracellular cAMP levels exert a marked growth-inhibitory effect and (Heldin et al., 1989). Conversely, it seems reasonable to assume that a reduction in cellular cAMP may have a growth-promoting effect in these cells. Alternatively, Gi or a related member of the pertussis toxin-sensitive Gi family could interact with an as yet unidentified effector system that is important for mitogenesis. Finally, the intriguing possibility exists that in addition to G , and G;, the GTP-binding p2 1 ras-encoded protein may be activated by LPA. Ras proteins are reminiscent of the QI subunit of signal-transducing G proteins and are critical during growth factor action; in particular, PA mitogenesis is completely blocked by a neutralizing anti-Ras antibody (Yu et al., 1988). Recently, PA and other lipids were shown to disrupt the interaction between Ras proteins and its GTPase-activating protein (GAP) (Tsai et al., 1989a). This raises the possibility that PA controls


97

6-

4 -

2-

0LPA

LPA

BK

BK

5

.-

I

[3H] arachidonate

CAMP

2 c .-

si T

T

40

30

20

10

0 LPA

BK

cli

LPA

BK

FIG. 4. Mitogenic response and generation of second messengers induced by LPA and bradykinin (BK) in cultured human fibroblasts in the presence (black bars) or absence (open bars) of 100 ng/ml pertussis toxin. For further details, see van Corven et al. (1989) and Moolenaar and van Corven (1990). (From Moolenaar, W. H., and van Corven, E. J., “Proto-oncogenes in cell development,” Ciba Found. Symp. 150,99-110. Copyright 0 1990. Reprinted by permission of John Wiley & Sons, Ltd.)

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biologically the activity of Ras by interacting with GAP. Subsequent experiments, however, seem to indicate that LPA fails to mimic PA in inhibiting GAP activity (Tsai et al., 1989b). It therefore seems unlikely that the observed mitogenic effects of LPA (and PA) can be explained by a direct effect on Ras-GAP activity. Figure 5 illustrates a hypothetical scheme of action of LPA. C. SITEOF ACTION

An unresolved question concerns the mechanism by which extracellular LPA and to a lesser extent, PA, activate certain G proteins at the inner side of the plasma membrane. It seems that both PA and LPA are capable of activating cells by virtue of the unique structure of their polar head groups, and the greater potency of LPA may be a reflection of its ability to be incorporated into the outer leaflet of the plasma membrane more efficiently. Inserted long-chain LPA might diffuse laterally, thereby triggering a G-protein-coupled “receptor” that could recognize the phosphate head group. Short-chain LPA ((212 : oLPA) might not translocate to the proper site on the plasma membrane, thus being incapable of activating the putative “receptor.” The finding that responsiveness to LPA is cell-type specific (see Section V,A) supports the receptor hypothesis. Another possibility is that inserted LPA perturbs the structure of the lipid bilayer in such a way that G proteins are selectively activated in a receptor-independent fashion. To date, however, there are no data to LPA

I

I

- AC

DNA synthesis

?

PLC

PLA2

I

FIG.5. Proposed scheme of early signal transduction pathways in the action of LPA: PTX, pertussis toxin; p21, GTP-bindingprotein product of the rm protooncogene. Other abbreviationsas in Fig. 3. The arrows denote the direction of cause to effect and should not be taken as evidence that LPA has a direct effect on p21 and other GTP-binding proteins. Note that LPA fails to activate G,. (From Moolenaar, W. H., and van Corven, E. J., “Proto-oncogenesin cell development,”Ciba Found. Symp. 150,99-110. Copyright 0 1990. Reprinted by permission of John Wiley & Sons, Ltd.)


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indicate how LPA might alter bilayer structure; it is equally unclear to what extent the structure and composition of the lipid bilayer may affect G protein function. It should be mentioned that the mechanism by which LPA exerts its immediate cellular effects at the level of the plasma membrane may well be unrelated to the later responses such as DNA synthesis and cell proliferation. At present it cannot be ruled out that trans-bilayer flip-flop or internalization of the lipid with subsequent metabolic conversion is required for LPA-induced mitogenesis. Recent data indicate that LPA is partially converted into monoacylglycerol (R. van der Bend, unpublished data), which could have a signaling role by itself. Clearly the importance of metabolic conversion of exogenous LPA for long-term biological responses remains to be addressed. V. Other Biological Effects

As LPA is a potent activator of certain G-protein-coupled effector systems, it may serve as a convenient probe to identify and dissect various biological responses in different cell systems. Indeed, LPA has been used to monitor cellular effects and to activate second-messenger pathways in various cell types. The results of these studies are summarized in Table I. It is seen that cells as diverse as mammalian fibroblasts, platelets, and Xenopzls oocytes share a common rapid response to LPA, i.e., activation of the G-protein-mediated phospholipase C second-messenger cascade. Yet, responsiveness to LPA is not a universal characteristic of all cell types: human neutrophils, monocytes, Jurkat T cells, and peripheral blood lymphocytes fail to show a detectable rise in [Ca2+],even when challenged with high concentrations of the lipid (Jalink et al., 1990, and unpublished observations). Also, rat mast cells, which rapidly degrand a t e in response to Ca‘+-mobilizing agents, fail to do so when stimulated with LPA (€3. Gomperts, unpublished observations). It thus seems as if cells of the immune system are nonresponsive to LPA. Other leukocytes should be examined for their LPA responsiveness before general statements can be made. Nevertheless, the finding that all lymphoid cells that have been tested lack a detectable response to LPA is intriguing and may contribute to identifying the nature of the putative LPA “receptor” or the specific intracellular targets.

VI. Possible Biological Function and Future Prospects The discovery that PA and LPA exert profound effects on many, but not all, cell types suggests that these low-abundance lipids may have a

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TABLE I CELLULAR RESPONSESTO LPA" Tissue

Response

Fibroblasts

Cell proliferation; Ca2+ mobilization; arachidonate release; inhibition of adenylate cyclase

A43 1 epidermoid carcinoma cells

Cell proliferation; Ca2+ mobilization

Osteosarcoma cells COS (kidney) cells

Ca2+ mobilizationb,' Ca2+mobilizationb

Neuroblastoma cells Colon carcinoma cells

Ca2+ mobilization; rapid morphological changesb CI- secretiond

MDCK epithelial cells

CI- secretiond Ca2+mobilization"

Mouse embryonic stem cells Smooch muscle

Ca2+mobilization; contraction (isolated muscle)'; cell proliferation (cultured cells)

Platelets

Ca2' moblization; aggregation

Xenopur oocytes

Cap -dependent C1- current"

References can be found in the text unless specified in the following notes. K. Jalink, unpublished observations. ' L. Tertoolen, unpublished observations. H. de Jonge, unpublished observations. Tokurnura et al. (1980);also see Salmon and Honeyman (1980).

physiological role as agonists or second messengers. The results obtained to date raise the question of whether PA and LPA formed in the inner leaflet of the plasma membrane by diacylglycerol kinase andlor phospholipases D and A2 act in a manner similar to that of exogenously supplied LPA to activate second-messenger systems. Microinjection studies should yield further insight into this issue. An interesting possibility is that LPA (which is fairly water soluble) is secreted by cells in a manner similar to the secretion of platelet-activating factor, prostaglandins, and other lipid agonists. The released LPA may then activate target cells in a paracrine or autocrine fashion. Phospholipid labeling studies will allow a critical test of this hypothesis. Finally, several reports have described a phospholipase D in mammalian plasma that is specific for glycosyl phosphatidylinositol (GPI), serving as a membrane anchor for surface proteins (Davitz et al., 1989; Low and Prasad, 1988). This GPI-specific phospholipase D may function not only to liberate the anchored protein from the cell surface, but also to produce biologically active PA in the outer leaflet of the plasma membrane. T h e precise physiological role of GPI-specific phospholipase D activity remains to be determined.


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I n summary, long-chain LPAs are potent mitogens for various mammalian cell types, and recent evidence suggests that LPA acts, at least in part, by activating a subset of the family of membrane-bound G proteins, including the inhibitory G protein of adenylate cyclase and the stimulatory G protein of phospholipase C. It seems likely, however, that as-yet unidentified effectors are involved in LPA action; these effector systems might include intracellular protein tyrosine kinases and phosphatases. Whether LPA binds to and thereby activates a cell-type-specific cell surface “receptor” awaits further investigation. Another unanswered question concerns the finding that LPA is about two orders of magnitude more effective in activating phospholipase C and inhibiting adenylate cyclase than in inducing DNA synthesis (Jalink et al., 1990; E. van Corven, unpublished observations). It is conceivable that higher concentrations of LPA are required for long-term effects such as DNA synthesis, due to cellular uptake and/or metabolic conversion of LPA. An alternative, but not mutually exclusive, possibility is that the early signaling events and the ultimate mitogenic response are causally unrelated in that they might be generated by different mechanisms with distinctly different LPA requirements. Future experiments, designed to evaluate the metabolic fate and the cellular targets of LPA, should help in resolving the discrepancy between the dose-response curves. Such experiments should also clarify to what extent the reported effects of PA are attributable to contamination with LPA. Whatever the precise site of action and biological function of LPA may be, this simple phospholipid may serve as a potent pharmacological tool to dissect the role of G-protein-mediated signaling pathways, particularly those involved in growth control. The design and synthesis of biologically inactive LPA analogs that inhibit LPA action, and of fluorescent and photoactive analogs, could greatly contribute to the identification of the cellular “receptor(s)” for LPA. Another challenge for future investigations is to examine whether endogenously produced LPA may be released from normal and/or transformed cells to serve as an “autocrine” mitogen that could, in principle, contribute to the maintenance of the transformed phenotype.

ACKNOWLEDGMENTS I am grateful to my colleagues, in particular Emile van Corven (who designed Figs. 2 , 3 , and 4), Kees Jalink, Angelique van Rijswijk, Rob van der Bend, and Wim J. van Blitterwijk, who have made important contributions to the concepts and experiments described in this article. Furthermore,the secretarial assistance of Paulien Sobels is gratefullyacknowledged. Research related to this review was supported by the Netherlands Cancer Foundation

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WOUTER H. MOOLENAAR

(Koningin Wilhelmina Fonds) and by the Netherlands Organization for Scientific Research (NWO).

REFERENCES Benovic,J. L., Bouvier, M., Caron, H. G., and Lefkowitz, R. J . (1988).Annu. Rev. Cell Biol.4, 405-428. Benton, A. M., Gerrard, J. M., Michiel, T., and Kindom, S. E. (1982). Blood 60,642-649. Berridge, M. J. (1987).Annu. Rev. Biochem. 56, 159-193. Billah, M. M., Lapetina, E. G., and Cuatrecasa, P. (198l).J.Bzol. Chem. 256,5399-5403. Bishop, W. R., and Bell, R. M. (1988). Annu. Reu. Cell Biol. 4,579-610. Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987).J.B i d . Chem. 262, 15309- 15315. Carpenter G., and Cohen, S. (199O).J.Biol. Chem. 265,7709-7712. Davitz, M. A., Horn, J., and Schenkman, S. (1989).J. Biol. Chem. 264, 13760-13764. Exton, J. H. (199O).J.Biol. Chem. 265, 1-4. Gerrard, J. M., and Robinson, P. (1989). Biochim. Biophys. Acta 1001,282-285. Heldin, N. E., Paulsson, Y., Forsberg, K., Heldin, C. -H., and Westermark (1989).J. Cell. Physiol. 138, 17-23. Irnagawa, W., Bandyophadhyay, G. K., Wallace, D., and Nandi, S. (1989). Proc. Natl. Acad. SGZ. U.S . A . 86,4122-4126. Jalink, K., van Corven, E. J., and Moolenaar, W. H. (199O).J.Biol. Chem. 265,12232-12239. Kanoh, H., Yamada, K., and Sakane, F. (1990). TrendcBiochem. Scz. 15,47-50. Lapetina, E. G., Billah, M. M., and Cuatrecasas, P. (1981).Nature (London} 292,367-369. Low, M. G., and Prasad, A. R. S. (1988). Proc. Nail. Acad. Scz. U.S. A. 85,980-984. Moolenaar, W. H., and van Corven, E. J. (1990). Ciba Found. Symp. 150,99-110. Moolenaar, W. H., Kruijer, W., Tilly, B. C., Verlaan, I., Bierman, A. J., and de Laat, S. W. (1986).Nature (London} 323, 171-173. Murayama, T., and Ui, M. (1987).J. Biol. Chem. 5522-5529. Nishizuka, Y. (1986). Science 233,305-312. Pagano, R. E., and Longmuir, K. J. (1985).J.Biol. Chem. 260, 1909-1916. Proll, M. A., Clark, R.B., and Butcher, R. W. (1985).Mol. Pharmacol. 28,331-337. Rozengurt, E. (1986). Science 234, 161-166. Salmon, D. M., and Honeyman, T. W. (1980). Nature (London) 284,344-345. Seuwen, K., Magnaldo, I., and Pouysskgur, J. (1988). Nature (London} 335,254-256. Siegmann, D. (1987).Biochem. Biophys. Res. Commun. 145,228-233. Tokumura, A,, Fukuzawa, K., Yarnada, S., and Tsukatani, H. (1980).Arch. fnt.Phurmacodyn. Thw. 245,74-83. Tsai, M. -H., Yu, C. -L., Wei, F. -S., and Stacey, D. W. (1989a). Science 243,522-526. Tsai, M. -H., Hall, A., and Stacey, D. W. (1989b).Mol. Cell. Biol. 9,5260-5264. Ullrich, A., and Schlessinger, J. (1990). Cell (Cambridge, Mass.) 61, 203-212. van Corven, E. J., Groenink, A,,Jalink, K., Eichholtz, T.,and Moolenaar, W. H. (1989). Cell (Cambridge, Mass.} 59,45-54. Watson, L. P., McConnell, R. T., and Lapetina, E. G. (1985). Bi0chem.J. 232,61-66. Whitman, M., and Cantley, L. (1988).Biochim. Biophys. Acta 984,327-344. Yu, C. -L., Tsai, M. -H., and Stacey, D. W. (1988). Cell (Cambridge, Mass.) 52,63-71.

EXPRESSION AND INTERACTIONS OF THE Src FAMILY OF TYROSINE PROTEIN KINASES IN T LYMPHOCYTES Joseph 6. Bolen,* Peter A. Thompson,* Elisa Ekeman,* and Ivan D. Horak*st ' Laboratory of Tumor Virus Biology and tClinical Pharmacology Branch, National Cancer Institute, Bethesda. Maryland 20892

I. Introduction 11. T h e Src Family of Tyrosine Protein Kinases A. From Oncogenes to Signal Transducers B. General Properties Ill. Patterns of Expression A. lck B. c-fp C. hck D. blk E. c-src F. c-yes G. lyn H. fm IV. Involvement of the Src Family in Signal Transduction during T Cell Activation A. CD4 B. CD8 C. CDSITCR D. CD2 V. Involvement of the Src Family in Signal Transduction during T Cell Proliferation A. Interleukin-2 and the Interleukin-2 Receptor Coniplex B. Interleukin-2 Receptor Signal Transduction C. Alterations in T Lymphocyte Malignancies VI. Conclusions References

1. Introduction

The field of research on tyrosine protein kinases has witnessed extraordinary growth in the past decade. Scientists interested in tyrosine protein kinases are currently engaged in an exceptionally broad range of disciplines, and the biochemical pathways controlled by various tyrosine protein kinases regulating cellular growth, differentiation, and mature physiologic functions are diverse, providing compelling support for the view that this field of study represents a rich source for future discoveries 103 ADVANCES IN CANCER RESEARCH, VOL. 57

Copyright 0 1991 by Academic Press, Inc. All rights of reproducrion in any form reserved.

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B. BOLEN

ET AL.

and insights into many biological systems. Perhaps no group of tyrosine protein kinases has contributed as much to the initial progress in this field as the Src family. This article is not intended to provide a comprehensive review of the Src family as a whole, but we will update some of the recent findings concerning the Src family and summarize the progress in this field that has occurred over the past few years as it pertains to our understanding of this family of nonreceptor tyrosine protein kinases in lymphocytes. As much of this progress has occurred utilizing a single type of lymphocyte-the T lyrnphocyte-a major focus of this review will be directed toward our knowledge of T cell activation and proliferative events as they relate to the potential roles of the Src family of tyrosine protein kinases in these cells. Given that our knowledge of potential functions for the nonreceptor tyrosine protein kinases in T cells and other cells is at an early stage, it is likely that we will raise more questions than we answer. II. The Src Family of Tyrosine Protein Kinases

Several reviews of the src gene and related family members have been published (Eiseman and Bolen, 1990; Cooper, 1990; Veillette and Bolen, 1989; Parsons and Weber, 1989; Bolen and Veillette, 1989; Perlmutter et al., 1988); therefore, the purpose of this section will be to provide a brief general history and description of the Src gene family and to update some recent developments in this field. A. FROMONCOGENES TO SIGNAL TRANSDUCERS

Study of the src-like genes originated with the observation of Peyton ROUS,in the early part of this century, that infection of animals with certain viruses can result in the development of malignancies. Through analysis of the original virus found to cause the these malignancies (the Rous sarcoma virus), it was subsequently discovered that a single gene (termed the v-src gene) was responsible for initiating changes in cellular growth and that the v-src gene encoded a 60-kDa protein possessing kinase activity that phosphorylated tyrosine residues on protein substrates (reviewed by Parsons and Weber, 1989; Cooper, 1990). During this period of exciting genetic and biochemical analysis of v-src, it was discovered that this oncogene was in fact a mutated version of a normal cellular gene (c-src)(Stehelin et al., 1976). This observation set the agenda for many scientists in this field, as exploration turned from asking “How does v-src function as an oncogene?” to “What does the c-src normally do?”As it was subsequently learned that c-src was part of an ever-growing

EXPRESSION AND INTERACTIONS OF SRC FAMILY

105

gene family in which only three members (src,yes, andfgr) were found to represent retrovirus oncogenes, the questions concerning the possible normal functions of this group of tyrosine protein kinases became all the more cogent. Because many cellular peptide hormone receptors were also found to be tyrosine protein kinases whose enzyme activity was mediated by ligand binding (Yarden and Ullrich, 1988), thoughts of physiologic functions for members of the Src family turned to the idea that they too may be involved in cellular signal transduction pathways. It is only within the last 2 years that this hypothesis has been verified for a single Src family member (the product of the Ick gene) in a single cell type (T cells), but it is anticipated that more family members will be found that serve in similar capacities in other cell types.

B. GENERAL PROPERTIES The Src gene family is currently composed of eight members: c-src (Parker et al., 1981; Shalloway et al., 1981; Takeya and Hanafusa, 1983), c-yes (Sukegawa et al., 1989), cfgr (Nishizawa et al., 1986; Inoue et al., 1987; Katamine et al., 1988),frn (Semba et al., 1986; Kawakami et al., 1986), lck (Marth et al., 1985; Voronova and Sefton, 1986),hck (Quintrell et al., 1987; Ziegler et al., 1987; Holtzman et al., 1987), lyn (Yamanashi et al., 1987), and blk (Dymecki et al., 1990). The proteins encoded by these genes are membrane-associated tyrosine protein kinases that lack transmembrane and external amino acid sequences, thereby distinguishing this group from the receptor class of tyrosine protein kinases. T h e predicted amino acid sequences of the members of the Src family expressed in human cells are shown in Fig. 1 and are aligned to demonstrate the sequence similarities and differences among family members. T h e exception is the predicted product of the blk gene, which, to date, has been molecularly cloned from rodent cells only. This is an important consideration, as some members of this gene family possess significant sequence diversity among different mammalian species in the region considered to be the unique domain (Yi and Willman, 1989; King and Cole, 1990). Clearly, increased sequence divergence in c-src and related genes in multiple regions is found in more evolutionarily distant organisms such as Xenopus laevis (Steele, 1985; Steele et al., 1989, 1990) and Drosophila melanogaster (Hoffman et al., 1983; Simon et al., 1985; Gregory et al., 1987) and in simple metazoans such as Hydra attenuata (Bosch et al., 1989). Superimposed over the amino acid sequences in Fig. 1 are boxes that outline the genetically defined functional domains that characterize members of this gene family. Figure 2 illustrates the relationship between these domains using a linear model. It is important to note that these

SRC YES FYN FGR LCK HCK LYN bl k SRC YES FYN FGR LCK HCK LYN bl k SRC YES FYN FGR LCR HCK LYN blk

*

SRC YES FYN FGR LCR HCK LYN blk

SRC YES FYN FGR LCK HCK LYN bl k

ESFLEEAQIMKKLKWDKLVQLYAWSKAFLEEAQVMKLLRHDKLVQLYAWSDAFLAFANLMKQLQWPFUXFlLYAVVT-

EAFLAEANVMKTLQHDKLVKLHAVVT

-

QAFLEEAN4MKTLQHDKLVRLYAWT

GEGFiAl!,KIiPNLW)MAAQVAAGMAYI E M Y I

PEGQDLRLPQLVDMAAQVAEGMAYMERMNYI PSGIKLNVNKLLDMAAQIAEGMAFIEEQNYI DEGSKQPLPKLIDFSAQIAEGMAFIEQRNYI DEGGKV44PKLIDFSAQIAEGMAYIERXNYI

SRC YES FYN FGR LCK HCK LYN blk

SRC YES FYN FGR LCK HCK LYN blk

0Myristylation Domain 0SH3 Domain 0Catalytic Domain 0Unique Domain SH2 Domain L_d Regulatory Domain

107


d

P

A GENERIC SRC FAMILY IUJCMBER

PI

B d c m b r m e Localization

t

ATP

300

200

100

t

AWTOPHOSPHOR~TTON

400

500

~

Amlno Acids

FIG.2. Domains of a generic Src family member.

domains have been defined for the most part by analysis of the c-src gene product and it is assumed that other family members will prove to be similar. Even though transcripts of c-STCand presumably the other family members are translated on free ribosomes (Courtneidge and Bishop, 1982; Brugge, 1986), all members of the family have been shown to be membrane associated. This occurs in spite of the fact, as mentioned above, that members of this family lack either hydrophobic signal-like sequences or sequences that could serve as membrane anchors. Membrane association by members of the Src family requires the covalent modification of the amino terminus by cotranslational addition of the 14-carbon fatty acid, myristic acid, to the common glycine residue at position 2 (Buss et al., 1984; Buss and Sefton, 1985; Schultz et al., 1985; Garber et al., 1985; Kypta et al., 1988; Marchildon et al., 1984). For the src FIG. 1. Alignment of amino acid sequences of the Src family of tyrosine protein kinases. The predicted amino acid sequences are aligned from the amino terminus (top) to the carboxy terminus (bottom). The various genetically defined domains are indicated as are the sites of serinehhreonine and tyrosine phosphorylation.

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JOSEPH B. BOLEN E T AL.

gene product, sequences important for myristylation appear to be the first 7-14 amino acids (Kaplan et al., 1988; Cross et al., 1985). However, myrisylation is not sufficient for stable membrane association (Buss et al., 1984; Garber et al., 1985), it appears as though at least three different regions, i.e., amino acids 1-14, 38-111, and 204-259, act to target the Src protein to various membrane locales (J. M. Kaplan et al., 1990). Whether these sequences directly interact with transport proteins in the cytoplasm or are involved in the interactions of the kinase with membrane proteins acting as internal receptors (Resh, 1988, 1989, 1990; Deichaite et al., 1988) or stabilizing elements is not known. T h e region of the Src family that possesses the greatest sequence diversity extends from approximately amino acid 10 to around amino acid 90 (Fig. 1). It is believed that this region is important for interaction of this group of enzymes with specific cellular proteins, some of which may be substrates. This region is also notable for the presence of several phosphorylation sites that respond to the activation of cyclic AMPdependent protein kinases (serine 17) (Collett et al., 1979; Patschinsky et al., 1986), protein kinase C (serines 12 and 48) (Purchio et al., 1985; Gould et al., 1985), and ~34"" (threonines 34 and 46 and serine 72) (Shenoy et al., 1989; Morgan et al., 1989). Of these serinelthreonine modifications, only those accompanying mitosis were observed to induce changes in pp60"" protein kinase activity (Chackalaparampil and Shalloway, 1988),although increased pp60c5'c protein kinase activity has also been noted following treatment of fibroblasts with platelet-derived growth factor (PDGF)-a change that was accompanied by aminoterminal tyrosine and serine phosphorylations (Ralston and Bishop, 1985; Gould and Hunter, 1988). As Fig. 1 shows, the amino acid residues covalently modified by phosphorylation on the c-src-encoded protein in this regon are not present on most of the other family members. However, this fact does not preclude similar types of modifications at other serine, threonine, or tyrosine residues in the remaining family members. Indeed, the lck gene product, ~ 5 6 ' ~has , been shown to undergo extensive serine and potentially some threonine phosphorylations within the amino-terminal half of the protein following treatment of T cells with activators of protein b a s e C and following T cell receptormediated activation (Casnellie and Lamberts, 1986; Casnelli, 1987; Veillette et al., 1988b; Marth et al., 1989; Hurley and Sefton, 1989). The region of the Src family members extending from amino acids 88-250 represents sequences that share striking homology with other nonreceptor tyrosine protein kinases, such as those from the feslfps gene family and the abl gene family (Hoffman et al., 1983; Pawson, 1988; Sadowski et al., 1986; Simon et al., 1985; Henkemeyer et al., 1988; Franz el


109

al., 1989; Jackson and Baltimore, 1989). Additionally, this region has sequences that are also found in other nontyrosine protein kinases, such as phospholipase Cy, the GAP protein, and the protein product of the gag-crk oncogene of the CTlO avian sarcoma virus (Stahl et al., 1988; Vogel et al., 1988; Trahey et al., 1988; Mayer et al., 1988, reviewed by McCormick, 1989). The homology region can be further subdivided into the SH2 (for src homology region 2) sequences (amino acids 140-250), which are found in all known nonreceptor tyrosine kinases, and the SH3 sequences (amino acids 88- 139), which are absent in feslfps-encoded family members. Though the significance of this region to the physiologic functions of the Src family is presently unknown, the fact that mutation of c-src in this region activates pp60"" protein kinase activity and enhances the ability of this protein to induce morphological transformation of cultured cells indicates that this region is important for the regulation of Src family members intrinsic enzymatic activities. As SH2 domains are believed to act as recognition sequences for phosphorylated tyrosine (Mayer and Hanafusa, 1990; Moran et al., 1990), it is possible that this region normally interacts with the "regulatory" phosphotyrosine in the carboxy terminus of the same enzyme (see later), thereby performing an internal regulatory function for the Src family. The observation that mutations in the homology regions of v-src and v-fps profoundly altered their capacity to transform cells and shift their host range (Bryant and Parsons, 1982; Kitamura and Yoshida, 1983; Cross et al., 1985, Raymond and Parsons, 1987; DeClue and Martin, 1987,1989; Wang and Parsons, 1989; Hirai and Varmus, 1990) argues that these regions also may play a significant role in the interaction of nonreceptor tyrosine kinases with some subset of substrates. The common link between these events may be related to the ability of cellular proteins, through common sequence elements, to interact with other proteins at specific locations (perhaps cytoskeletal components) in close proximity to the plasma membrane. T h e region of the Src family encompassing residues 250 to roughly 5 16 represents the catalytic domain of this group of enzymes and is found to represent the region of highest sequence homology both within the family and with other protein kinases (Hanks et al., 1988; Cooper, 1990). T h e catalytic domain contains the site of ATP binding (Kamps et al., 1984), centered around a common lysine residue with a Src family consensus sequence: Gly-X-Gly-X-X-Gly-Glu-X-Trp-X-Gly-X-X-X-XX-X-X-Val-Ala-X-Lys-X-Leu-Lys-X. The motif Gly-X-Gly-X-X-Gly is found in a wide variety of nucleotide-binding proteins and is believed to be part of the nucleotide-binding fold (Hanks et al., 1988; Sternberg and Taylor, 1984; Toner-Webb and Taylor, 1987). Several other short

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JOSEPH B. BOLEN ET A L .

sequences in this region are also found to be homologous with serineJ threonine kinases (Hanks et aL, 1988). Within the catalytic domain also lies the site of autophosphorylation (for c-src, tyrosine residue 416), which is thought to play an important role in activity and interactions of Src family members (Parsons and Weber, 1989; Cooper, 1990). As all tyrosine protein kinases, whether of the receptor or nonreceptor class, possess analogous autophosphorylation sites (Hanks et al., 1988), it has been speculated that the presence of phosphorylated tyrosines in this region may play some role in the signaling process, perhaps by acting as a regulatable site at which potential substrates may interact or as a site at which phosphorylation could induce con formational changes that allow substrates to bind to other sites (Morrison et al., 1988, 1989, 1990; D. R. Kaplan et al., 1990; Kazlauskas et al., 1990). T h e carboxy-terminal 16 to 19 amino acids of the Src family comprise the region that has been shown by extensive genetic analysis to be capable of playing a major role in the regulation of the basal tyrosine kinase activity of this group of enzymes (Parsons and Weber, 1989; Cooper, 1990). T h e general observation is that mutation of a conserved tyrosine residue, which is normally found to be phosphorylated in uivo (Cooper et al., 1986), to some other amino acid results in the constitutive activation of the tyrosine protein kinase activity of the altered enzyme by as much as 10- to 20-fold (Kmiecik and Shalloway, 1987; Piwnica-Worms etal., 1987; Cartwright et al., 1987). If the mutated protein is expressed in an appropriate cell type, this site-specificalteration in the enzyme sequence significantly enhances the protein’s transforming potential (Kmiecik and Shalloway, 1987; Piwnica-Worms et al., 1987; Cartwright et al., 1987). ‘These observations suggest that the phosphorylated tyrosine residue in this carboxy-terminal domain acts as an inhibitor of kinase activity. Currently, it is unclear whether this phosphorylated residue should be classified as an allosteric or competitive inhibitor of kinase activity. It is possible that the phosphorylated tyrosine could act as an allosteric type inhibitor of tyrosine protein kinase activity in which dephosphorylation by a tyrosine phosphatase would induce conformational alterations leading to an activated state. This idea is consistent with the possibility that the phosphorylated tyrosine residue interacts directly with amino-terminal SH2/SH3 sequences to provide an enzymatically suppressed enzyme conformation that would be altered by dephosphorylation. Another view is that the phosphorylated tyrosine residue in the carboxy terminus be classified as a competitive inhibitor of the Src kinases by interacting with the catalytic domain to prevent autophosphorylation and/or phosphorylation of exogenous substrates. Whether or not the normally “inhibited state” of these


111

enzymes can be reversed by high substrate concentrations, thereby providing proof of a classic allosteric or competitive relationship between the carboxy-terminal phosphate status and enzyme activity, has not been evaluated. Regardless of the type of regulation that carboxy-terminal phosphorylation may provide, it is important to leave open the possibility that dephosphorylation of this regulatory site may be only one way in which the activity of this family of enzymes could be regulated. Alternatively, it may be argued that phosphorylationldephosphorylation at the carboxy terminus is not an important physiologic means of enzyme regulation. Indeed, in at least one case in which a member of the Src family ( ~ 5 6 ' ' ~ ) was shown to be activated in response to a stimulus mimicking physiologic agonists, significant levels of steady-state carboxy-terminal tyrosine dephosphorylation were not observed (Veillette el aZ., 1989a). If the physiologic activation of members of this family were more dependent on their ability to autophosphorylate (by intra- or intermolecular mechanisms) rather than on the phosphorylation state of their carboxy termini, several advantages for the range of potential responses by the Src family could be realized. This type of arrangement could allow for more subtle types of regulation of the responses of Src family members to different physiologic stimuli and allow the magnitude of their response to be potentially based upon the requirement of the particular cell type in which they are expressed. Such a model could account for enzyme molecules that retain the regulatory tyrosine phosphorylation, whose activities accompany conformational alterations induced by other posttranslational alterations (such as amino-terminal serine/threonine phosphorylations) or protein-protein interactions. An advantage of this model would be the lack of a requirement for the actions of a tyrosine phosphatase prior to activation. Retention of the carboxy-terminal tyrosine phosphate might also allow for a faster activation-deactivation cycle of this group of kinases if they are to be involved in responses that require rapid suppression of signaling events followed by a requirement for rapid regulatable reactivation. This theory does, however, suffer from the bias that pictures Src family members playing a role in sorting and processing plentiful incoming stimuli when it may be more likely that the job of many of the Src family members is to amplify the intracellular response resulting from a limited number of interactions between ligands and receptors. This situation may apply particularly to those Src family members (such as the c-src,frn, c-yes, and lyn gene products) that are expressed in a wide variety of cells. Giving credence to this idea is the recent finding that the proteins encoded by c-src, Jyn, and c-yes are all capable of binding to

112


ligand-activated PDGF receptors and that each of the PDGF receptorbound Src family members possesses elevated protein kinase activity (Kypta et al., 1990). Ill. Patterns of Expression

Of the eight Src-related tyrosine kinases identified, four of these kinases, encoded by Zck (Marth et al., 1985; Voronova and Sefton, 1986), et al., 1986; Inoue et al., 1987; Katamine et al., 1988),hck c - f (Nishizawa ~ (Quintrell et al., 1987; Ziegler et al., 1987; Holtzman et al., 1987), and blk (Dymecki et al., 1990),are expressed principally in cells of hematopoietic origin, whereas the other four, encoded by c-src (Parker et al., 1981; Shalloway etal., 1981; Takeya and Hanafusa, 1983),c-yes (Sukegawa et al., 1989), lyn (Yamanashi et al., 1987), andfyn (Semba et al., 1986; Kawakami et al., 1986), are expressed in a wide variety of tissue types (Table I). Though these kinases are thought to play a role in the signal transduction events associated with proliferation and differentiation, many of these kinases, including those with a broad expression pattern, are expressed in postmitotic fully differentiated cells, suggesting that they may be involved in cellular functions that are specific for the cell type in which they are expressed. A. lck T h e lck gene codes for the 56kDa protein, p56"', which is normally expressed only in cells of lymphoid origin. It is expressed at high levels in TABLE I EXPRESSIONOF SRC FAMILYMEMBERSIN HUMANHEMATOPOIETIC

HUMANFIBKOBLASTS

CELLS AND

Cell type Protein"

T cell -

B cell -

++ ++ + ++++ -I+

-

-

a

-

++ ++++

NK cell

+ + ++ ++t -

+

Macrophagel monocyte

Granulocyte

+ -I+ -I+

+++ ++++ ++

?

The gene that encodes the protein is indicated by the superscript,

Platelet

? ? ?

+ +

+++

-

? ?

t+++t

+ +i

?

Fibroblast


113

mature peripheral CD4+ and CD8' T lymphocytes (Marth et al., 1985; Veillette et al., 1988b) as well as in immature CD4+ CD8+ thymic T cell populations (Veillette et al., 1989~).p56lckis also expressed at significant levels in normal human peripheral blood-derived natural killer (NK) cells and, to a lesser degree, in some B cell lines (Marth et al., 1985; E. Eiseman, unpublished data). T h e lymphoid-specific expression of the lck gene is mediated through the activity of separate upstream promoters that give rise to two lck transcripts with different 5' untranslated regions (Voronova et al., 1987; Garvin et al., 1988; Perlmutter et al., 1988). These two transcripts arise by alternative splicing of 5' exons, although the translated product for both RNA species is the same (Adler et al., 1988; Garvin et al., 1988). The distal promoter, which is located approximately 35 kb upstream from the first coding exon is used preferentially in normal lymphoid cells, whereas both promoters are used in lymphoid tumors (Adler et al., 1988; Garvin et al., 1988). B. c-fgr The expression of c-fgr mRNA in normal cells is highest in differentiated normal bone marrow-derived myeloid cells, in normal circulating granulocytes, monocytes, alveolar macrophages, and possibly splenic macrophages, and in leukemic cells with differentiated myelomonocytic phenotypes (Ley et al., 1989; Cheah et aE., 1986; Willman et al., 1987). Epstein-Barr virus (EBV)-transformed human B cells also express cfgr mRNA whereas untransformed B cells do not (Tronick et al., 1985; Cheah et al., 1986). It appears that the expression of c$p mRNA and p55'-fq can be regulated in a complex manner during the proliferation, differentiation, and activation of monocytic cells (Cheah et al., 1986; Ziegler et al., 1988; Ley et al., 1989; Notario et al., 1989; Yi and Willman, 1989). Normal bone marrow-derived monocytic cells express undetectable levels of c-fgr mRNA. These cells retain their ability to proliferate in response to colony-stimulating factor (CSF-1) and can be activated by granulocyte/ macrophage colony-stimulating factor (GM-CSF), bacterial lipopolysaccharide (LPS), or interferon-y (IFN-y) to perform more differentiated macrophage functions. In these cells, c f p mRNA is transiently induced approximately 20-fold 4-8 hr following treatment of the cells with CSF- 1 and can also be induced following addition of GM-CSF, LPS, or IFN-y (Cheah et al., 1986; Yi and Willman, 1989). In contrast, peripheral bloodderived monocytic cells, which have lost the ability to proliferate in response to CSF-1 and represent cells of this lineage at a later stage of

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JOSEPH B. BOLEN E T A L .

differentiation than bone marrow-derived monocytic cells, express high levels of c-fgr mRNA (Ziegler et al., 1988). Interestingly, c$p expression is down-regulated approximately eightfold during macrophage activation induced with INF--y plus low-dose LPS (Ziegler et al., 1988). The expression of c-fgr mRNA (Ley et al., 1989) and ~ 5 5 ~ '(Notario et al., 1989) is induced during the differentiation of myelomonocytic cell lines. Differentiation of HL-60 cells to granulocytes by treatment with retinoic acid, to monocytes with 12-O-tetradecanoylphorbol-13-acetate(TPA), or to myelocytes with dimethyl sulfoxide (DMSO) results in an increase in the expression of c-fgr mRNA and ~ 5 5 ~ (Notario fu et al., 1989). In addition agents that induce differentiation, such as TPA, or inhibit new protein synthesis, such as cyclohexamide, increase the expression of c-fgr mRNA in the U-937 monocytoid cell line (Ley et al., 1989). In normal human neutrophils, p55"fq is present in the plasma membrane as well as in the secondary and tertiary granules, but is absent in primary granules (Gutkind and Robbins, 1989). During neutrophil activation, secondary granules fuse rapidly with the plasma membrane and release their contents to the outside of the cell allowing ~ 5 5 ~ fto f l be translocated from its intracellular location to the plasma membrane (Gutkind and Robbins, 1989).

C. hck The expression of hck is restricted to hematopoietic cells of the myeloid lineage (Ziegler et al., 1987; Quintrell et al., 1987). High levels of the 2.2-kb hck transcript are found in monocytes and granulocytes, and low levels are found in human tonsillar B lymphocytes and in some human and mouse B cell lines (Ziegler et al., 1987; Quintrell et al., 1987). T h e hck gene product is also expressed in several leukemia cell lines, such as the B cell line IM-9, but is most abundant in cells representing the lineages that give rise to either granulocytes (ML-1 and HL-60) or monocytes (U-937) (Ziegler et al., 1987; Quintrell et af., 1987). Originally, hck was isolated from a placental cDNA library, however, hck mRNA is not expressed at detectable levels in human placentas, human foreskin fibroblasts, neuroblastoma cells, T cells, erythroid cells, and KG-I cells, which are bipotential precursors to granulocytes and monocytes (Quintrell et al., 1987). Immunoblotting shows that the hck-encoded protein migrates as a closely spaced doublet with an apparent size of 59 kDa in both mouse and human myeloid cell lines (Ziegler et al., 1988). Immune complex kinase assays of peripheral blood-derived normal human monocytes show that the hck-encoded protein migrates as a closely spaced triplet (Eiseman and Bolen, 1990). At least two of these hck-encoded proteins arise through the


115

use of alternative translational start sites within a single mRNA species (A. Dunn, personal communication). One protein initiates at the first ATG within exon 2, while a second, larger form initiates at a CTG located 63 nucleotides upstream, producing a protein with an additional 21 amino acids at the amino terminus. It is interesting to note that this additional 2 1-amino acid sequence includes two cysteine residues that may represent sites of interaction with a cell surface molecule, analogous to the association of ~ 5 6 with " ~ CD4 (see later). However, no equivalent cysteines are present in the smaller hck-encoded protein. In monocytic cells, IFN-.)Iand LPS can act synergistically to induce expression of cytokines such as tumor necrosis factor (TNF) or interleukin-1 (IL-1) (Adams and Hamilton, 1987; Burchett et al., 1988). Combinations of LPS and IFN-.)Ithat maximally stimulate cytokine release a few hours following treatment also induce a sevenfold increase in hck mRNA expression in both cultured human peripheral blood monocytes and fresh bone marrow-derived monocytes (Ziegler et al., 1988; Yi and Willman, 1989). However, the increased expression of hck mRNA was found to occur late in the activation sequence (approximately 12 hr), demonstrating that the increased expression of hck is not required for cytokine release. In contrast, treatment with phorbol myristate acetate (PMA), which results in the production of significant amounts of TNF and IL-lB, decreases the level of expression of hck mRNA (Ziegler et al., 1988). These obervations support the hypothesis that LPS- and PMAmediated stimulation pathways in myeloid cells are biochemically distinct (Fenton et al., 1988). Examination of human leukemic cells suggests that hck is expressed in both immature and mature myeloid cells; however, the abundance of hck mRNA increases as cells express more mature cell surface markers (Quintrell et al., 1987; Perlmutter et al., 1988). For example, TPA induction of ML- 1, HL-60, and U-937 cell differentiation into more macrophage-like cells increases the level of hck mRNA two- to fivefold. Similarly, differentiation of HL-60 cells to more granulocytic-like cells by DMSO treatment causes a sevenfold increase in hck mRNA (Quintrell et al., 1987). D. blk The blk gene encodes a 55-kDa protein, ~ 5 5 (Dymecki ' ~ et al., 1990). A 2.5-kb RNA is detected in murine spleen but not in thymus, brain, heart, lung, kidney, liver, or intestine (Dymecki et al., 1990). The expression of blk is detected in a wide variety of B lymphoid cell lines but not in cell lines of T lymphoid, myeloid, erythroid, fibroblastoid, neuronal, or hepatocel-

116


lular origin, nor in a mammary carcinoma cell line (Dymeckiet al., 1990). The expression of blk is observed in B cell precursors as well as cell lines representative of mature B cells, suggesting that in B cell ontogeny, blk is expressed before the appearance of surface immunoglobulin (Dymeckiet al., 1990). Thus, ~ 5 5 ' 'may ~ function in signal transduction andlor proliferative events s ecific for B cells in a manner similar to that currently thought for p56" in T cells.

r

E. c-src The protein pp60"" has been reported to be involved in both the transduction of growth-controlling signals and in specialized functions in fully differentiated nonproliferating cells. Expression of c-src is detectable in almost every cell type examined, although the level of expression varies widely. However, highest levels of pp60"" protein and tyrosine kinase activity are observed in neuronal tissues (Cotton and Brugge, 1983; L. K. Sorge et al., 1984; Brugge et al., 1985;J. P. Sorge et al., 1985; Lynch et al., 1986; Cartwright et al., 1987; Wiestler and Walter, 1988), and platelets (Golden et al., 1986). Thus, the abundance and activity of pp60'"'" are highest in postmitotic cells. A portion of the pp60"" expressed in normal neuronal tissues represents neuronal-specific isoforms (Brugge et al., 1985, 1987; Cartwright et al., 1987; Wiestler and Walter, 1988) generated through alternative splicing events between the third and fourth coding exons, which give rise to at least three distinct neuronal c-STCmRNA species (Levy et al., 1987; Martinez et al., 1987; Pyper and Bolen, 1989, 1990). The observed tissuespecific splicing of this gene as well as the high level of pp60 csrc found in neuronal tissues has helped to foster the view that pp60"" plays a role in events associated with both neuronal differentiation and maintenance of mature neuronal cell functions (Brugge et al., 1985, 1987; Fults et al., 1985; Cartwright et al., 1987; Levy et al., 1984; Maness et al., 1988; Schartl and Barnekow, 1984; Sorge et al., 1984; Sudol et al., 1988). Platelets contain high levels of pp60"", which represents 0.2-0.4% of total platelet protein (Golden and Brugge, 1989). Platelets also have the highest known cellular levels of pp60'-" kinase activity, which is 3- to 7-fold higher than the levels detected in brain and at least 30- to 50-fold higher than pp60e5" activity found in muscle, spleen, thymus, lymph node, bone marrow, and erythrocyte lysates (Golden et al., 1986). As platelets also have high constitutive levels of phosphotyrosine-containing proteins, it has been suggested that pp60'"c may be important for platelet homeostasis (Ferrell and Martin, 1988; Golden and Brugge, 1989). Though the normal level of phosphotyrosine-containing proteins in


117

platelets is high compared to other cell types, thrombin treatment of platelets raises the overall level of platelet phosphotyrosine severalfold and increases the number of phosphotyrosine-containing proteins (Ferre11 and Martin, 1988; Golden and Brugge, 1989).However, this elevated level of phosophotyrosine-containing proteins observed during platelet activation is not accompanied by detectable alterations in the activity of pp60eSrC(Golden and Brugge, 1989) or other Src family members expressed in platelets (Horak et al., 1990; J. S. Brugge, personal communication). c-src is expressed in both undifferentiated and differentiated myelomonocytic leukemia cells but not in normal myeloid cells (Willman et al., 1987). Differentiation of the promyelocytic leukemia cell line, HL-60, to granulocytic cells with DMSO or to macrophage-like cells with TPA results in a differentiation-dependent increase in pp60"" kinase activity (Barnekow and Gessler, 1986).

F. c-yes

Human c-yes cDNA was originally cloned from a cDNA library derived from cultured human embryo fibroblasts (Sukegawa et al., 1989). Two human c-yes loci have been located, one on chromosome 18q21.3 and another, which may be a pseudogene, on chromosome 6 (Semba et al., 1986; Yoshida et aZ., 1985). A 4.8-kb c-yes mRNA is expressed in cultured human embryo fibroblasts, placenta, human embryonic lung, liver, and kidney, and the cell lines KB, K562, and A431 (Semba et al., 1986). Immune complex kinase assays of platelets (J.Brugge, personal communication), natural killer cells, and T cells from normal peripheral human blood revealed relatively high levels of p62q" kinase activity, whereas monocytes and B cells contain low levels (Eiseman and Bolen, 1990). In addition, murine mast cells have high levels of p 6 F " kinase activity (E. Eiseman, unpublished data). In normal chicken embryo fibroblasts and in chicken kidney, two c-yes mRNA transcripts of 3.7 and 3.9 kb are found (Sudol and Hanafusa, 1986). The highest level of expression of c-yes mRNA is found in the chicken kidney and brain (Shibuya et al., 1982). In the chicken, two proteins of 59 and 62 kDa are precipitated by affinity-purified anti-yes antibodies (Sudol and Hanafusa, 1986; Sudol et al., 1988). Immune complex kinase assays show relatively high expression of these two proteins in brain, retina, kidney, and liver, and low levels of kinase activity in muscle, heart, bone marrow, and spleen (Sudol and Hanafusa, 1986; Sudol et al., 1988).

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JOSEPH B. BOLEN ET AL.

G. lyn

T h e lyn gene is widely expressed in a diverse array of cell types. This gene was originally isolated from a human placental cDNA library and was found to be transcribed at relatively high levels in human fetal liver (Yamanashi et al., 1987). A 3.2-kb mRNA is expressed in human placenta, fetal brain, lung, liver, and kidney, but was not detected in cultured human embryo fibroblasts (Yamanashi et al., 1987). With regard to hematopoietic cells, Zyn mRNA was found to be preferentially expressed in macrophages, monocytes, platelets, and B cells (Yamanashi et aZ., 1989). No expression of Zyn mRNA was detected in normal T cells; however, it is expressed in T cell lines infected with and producing human T cell lymphotropic virus type I (HTLV-I) (see Section V1) (Yamanashi et al., 1989). lyn mRNA is also expressed in cell lines with phenotypic characteristics of B cells (Raji and FL18) and myeloid cells (HL-60 and K562) (Yamanashi et aZ., 1989). Higher levels of lyn transcripts were detected in mature B cell lines than in a pre-B cell line (yamanashi et aZ., 1989). An alternatively spliced lyn transcript has recently been isolated from murine myeloid cells. The predicted product of this transcript is a 53-kDa protein that lacks 21 amino acids within exon 2 (A. Dunn, personal communication; J. Ihle, personal communication). Based upon examination of the abundance and/or activity of the Zyn gene product, ~ 5 6 4 "can be detected in platelets, macrophaged monocytes, and B lymphocytes (Yamanashi et aZ., 1989; E. Eiseman, unpublished data). p56b" is also expressed in normal peripheral bloodderived NK cells, rat basophils, and human primary cultured foreskin fibroblasts (E. Eiseman, unpublished data). These types of experiments have also shown that in macrophages/monocytes, B cells, and NK cells, ~ 5 6 4 "migrates as a closely spaced doublet in sodium dodecyl sulfatepolyacrylamide gels, whereas foreskin fibroblast-derived ~ 5 6 4 "migrates as a single species (E. Eiseman, unpublished data). The doublet found in human hematopoietic cells may represent the products of the two alternatively spliced Zyn transcripts. Indeed, preliminary analysis of lyn cDNA clones from normal human monocytes has revealed the existence of two Zyn transcripts that differ in the 5' coding region of the mRNA (E. Eiseman, unpublished data).

The frn gene was originally isolated from normal human fibroblasts and endothelial cells, and is expressed in a variety of cells (Kawakami et al., 1986; Semba et al., 1986). Human monocytes express ~ 6 0 s "but


119

polymorphonuclear cells do not (Kawakami et al., 1989). p60fr" is also expressed in platelets at levels approximately 20- to 40-fold higher than in human fibroblasts, and approximately 5- to 10-fold lower than the level of pp60"" (Horak et al., 1990). Purified T lymphocytes (Kawakami et al., 1989; Veillette et al., 1988a) and NK cells (E. Eiseman, unpublished data) from normal human peripheral blood also express p6Obn. There are two distinctfyn-encoded transcripts. One is found in thymocytes, splenocytes, and some hematolymphoid cell lines, and a second more common form is found in other tissue types (Cooke and Perlmutter, 1989). DNA sequence analysis has revealed that the thymocyte form of p60fr" differs from that found in other cell types in the kinase domain and is the result of alternative splicing of exon 7 (Cooke and Perlmutter, 1989). The exon 7 region of p60frn contains the sequence GXGXXG, which participates in binding of ATP to the enzyme (Kamps et al., 1984) and is conserved in the nonlymphocyte form of murine p60fr" (Cooke and Perlmutter, 1989). In the hematopoietic form of a different exon 7 is used (exon 7A) in place of the normal exon and contains the sequence GXGXXA at the ATP-binding site (Cooke and Perlmutter, 1989). The significance of this change in sequence is currently unclear, because both forms of p60fr" are enzymatically active (Cooke and Perlmutter, 1989), although it would be interesting to test whether the altered exon used in the lymphocytes might cause different nucleotide triphosphate or cofactor requirements for the enzyme. T h e expression offyn is stimulated in normal murine T cells by treatment with an anti-T3 (anti-CD3) E monoclonal antibody, which is thought to mimic antigen-induced T cell receptor engagement (Katagiri et al., 1989a). This induction is also observed in T cells stimulated with mitogens such as concanavalin A or a combination of TPA and the calcium ionophore A23187 (Katagiri et al., 1989a). Pretreatment of 'I' cells with forskolin, which inhibits anti-CD3 €-induced T cell growth, also inhibits the induction offyn mRNA (Katagiri et al., 1989a). In contrast, elevated amounts offyn transcripts were not detected at any stage of differentiation of normal murine T cells, including negatively selected CD4- CD8- thymocytes, the unfractionated thymocyte population, relatively mature thymocytes resistant to hydrocortisone, and peripheral lymph node T cells, or in four Thy-l+ CD4- CD8- cell lines, EL-4, BW5147, WEHI-3, and Yac-1 (Katagiri et al., 1989a). Recent evidence suggests that a portion of p6Ofr" expressed in T cells may be associated with one or more of the proteins comprising the T cell antigen receptor CD3 complex (Samelson et al., 1990b). Support for this view is provided by evidence that p60fY" is overexpressed in mice homozygous for the autosomal recessive lpr gene (Katagiri et al., 19SYa, b),

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which results in production of large numbers of abnormal CD4- CD8- T lymphocytes possessing high constituitive tyrosine phosphorylation of the T cell receptor 6 subunit (Samelson et al., 1986). Additionally, the thymocytes of transgenic mice that express afrn gene under the control of the Zck promoter have been shown to be hyperresponsvie to T cell receptor-dependent activation stimuli (R. M. Permutter, personal communication). IV. Involvement of the Src Family in Signal Transduction during T Cell Activation

T h e presentation of antigen to mature antigen-specific T cells triggers a complex and highly ordered sequence of biological signals referred to in aggregate as T cell activation (for review, see Sprent et al., 1990). One of the first detectable alterations is the phosphorylation of several T cell proteins on tyrosine residues (June et al., 1990a). This observation has stimulated interest in the responsible protein tyrosine kinases and phosphatases and the identity of their substrates. In this section some of the recent work relating to these issues in cw//3+ T cells is reviewed. The important topic of signal transduction in y / 6 + T cells, which likely represent a separate developmental lineage (Winoto and Baltimore, 1989),will not be addressed in this review. In addition, the important, though still poorly understood, issue of costimulatory signals generated by antigenpresenting cells will not be extensively discussed (for review, see Mueller et al., 1989). T h e physical interaction of an antigen-specific T cell with an antigenbearing, antigen-presenting cell (APC) involves the engagement of multiple receptor systems on both cells (see Fig. 3). Several lines of evidence implicate the involvement of different T cell surface proteins, in addition to the alp T cell receptor (TCR)/CD3 complex, and their cognate ligands on the surface of the APC in this process. Among these T cell surface proteins are the CD4 and CD8 coreceptors and the CD2 receptor. Each of these receptor systems will be considered in light of their demonstrated or possible roles in modulating signal transduction through the three Src family protein tyrosine kinases (p56'"", p62Ye', and p60fy") known to be expressed in peripheral human T cells, in an attempt to delineate some of the early events in T cell activation. A. CD4 The CD4 receptor is a 55-kDa cell surface glycoprotein expressed by a subset of mature peripheral T lymphocytes as well as a subset of differen-


1

ICAM-1

121

WA-1

ANTIGENPRESENTING

CELL

FIG.3. Predicted surface interactions between a CD4+T lymphocyte and an antigenbearing MHC class I1 antigen-presentingcell.

tiating thymocytes, and by nonlymphoid cells (for review, see Bierer et al., 1989) (Fig. 3). On mature a/@+T cells, the presence of the CD4 receptor is well correlated with MHC class I1 restriction of the T cell. CD4 can directly mediate binding to MHC class II-expressing B cells when transfected into CV-1 cells (Doyle and Strominger, 1987), though the binding affinity for CD4 and MHC class I1 is relatively low (Hussey et al., 1988) and adds little to the stability of the T cell MHC class IIf APC interaction (Koyasu et al., 1990). A signaling function for the CD4 receptor in addition to its modest contributions to T celllAPC adhesion has been strongly supported by recent evidence demonstrating a physical association between CD4 and the p56" protein tyrosine kinase (Veillette et d., 1988a; Rudd et al., 1988) in CD4+ T cells. As this association may represent a structural paradigm for the interaction between other Src family members and cell surface receptors, this complex will be considered in some detail. Genetic reconstitution of the p56lCkklCD4heterodimer HeLa cells (Shaw et al., 1990) and murine fibroblasts (Simpson et al., 1989) upon cotransfection of Zck and CD4 cDNAs has demonstrated that lymphoidspecific factors are not required for establishing or maintaining the

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CD4/p56"' interaction. In the HeLa system it has also been shown that a chimeric molecule containin the cytoplasmic tail of CD4 is capable of complex formation with p 5 a k upon cotransfection and that such complexes could be demonstrated before transport to the Golgi apparatus (Shaw etal., 1989). CD4 was found to bind to a chimeric Lck-CAT protein containing only amino acids 1-32 of Lck in the same system. Further genetic mapping in this system (Shaw et al., 1990) has shown that amino acids 15-23 of p56"' and the 30 membrane-proximal residues of the CD4 cytoplasmic domain are critical for this interaction. Within these domains the integrity of cysteine residues at positions 419 and 421 in the CD4 molecule and positions 20 and 23 in ~ 5 6 was " ~ shown to be indispensable for complex formation. These cysteines, which are apparently not disulfide linked, may be involved in binding a metal ion, as has been shown for the human immunodeficiency virus Tat protein dimer (Frankel et aE., 1988). The cysteine-rich amino-terminal domain of ~ 5 6 ' " ~ required for CD4 binding is not shared by any other member of the Src family. Moreover, in contrast to the data for pp60'-"", myristylation of p56" is dispensable for heterodimer formation and, by extension, for membrane association in this system (Shaw et al., 1990). It remains to be seen whether stable physical association of a transmembrane cell surface glycoprotein with a nonreceptor tyrosine kinase is unique to ~ 5 6 or " ~is a general feature of the Src family. CD4 is functionally as well as structurally linked to p56". Antibodymediated cell-surface cross-linking of CD4 leads to rapid enzymatic activation of the associated ~56'"'(Veillette etal., 1989b). Coincident with this change in activity a recent study has demonstrated alterations in the phosphopeptide map of ~ 5 6 ' "(Veillette ~ et al., 1989a). In this study, antibody-mediated cell surface cross-linking of CD4 in a CD4+ niurine T cell clone led to increased "Pi incorporation into the carboxy-terminal tyrosine ( Y ) residue at position 505 as well as increased label incorporation into the major site of in uitro autophosphorylation, Y394. Moreover, whereas the specific activity of CD4-associated p56" in these cells is no greater, and possibly less, than the specific activity of the ~ 5 6 ~ molecules " not associated with CD4, the incorporation of label into Y505 was markedly lower in the CD4-associated population in resting cells prior to cross-linking. Though increased label incorporation into Y394 during enzymatic activation of p56" was expected based on previous studies, the parallel observation of phosphorylation at Y505 is somewhat surprising in light of the genetic evidence reviewed in the prior section, which suggests a simple negative regulatory role for phosphorylation at this position. Several explanations have been put forward to explain this


123

apparent discrepancy. Increased label incorporation into Y505 during ~ ' cell surface CD4 cross-linking enzymatic activation of ~ 5 6 following may represent a competing negative regulatory event also stimulated by CD4 cross-linking. According to this model, the observed increase in enzymatic activity of ~ 5 6 ' would " ~ represent the activity of CD4-associated ~ 5 6 molecules "~ that have yet to be phosphorylated at Y505. Alternatively, as metabolic labeling with 32Piunder the protocol used in this study may not lead to equilibration of "Pi with the total cellular ATP pool, it may be prudent not to view the observed changes as representing changes in phosphate occupancy alone but rather as an integrated function of occupancy and phosphate turnover. Thus if CD4-associated p56lCk were constituitively phosphorylated at position Y505, the ability to incorporate radiolabeled phosphorus into this site would depend upon the rate of turnover at this position. If this rate was slow in resting cells, relative to the period of metabolic labeling, little radiolabel may be incorporated at Y505. If both Y505 kinase(s) and phosphatase(s) were stimulated by CD4 cross-linking, the net effect would be a greatly enhanced rate of phosphate turnover at this position and, consequently, increased incorporation of radiolabel. As prolonged metabolic labeling of lymphocytes with 32P;results in unacceptable cellular death (P. A. Thompson and J. B. Bolen, unpublished), metabolic labeling to equilibrium is probably not a viable experimental option. It is also possible that phosphorylation-independent changes in the conformation of CD4-associated ~ 5 6 'are ~ responsible for the observed increase in enzymatic activity following CD4 cross-linking. Further studies are needed to shed more light on the molecular mechanisms responsible for modulating the enzymatic activity of ~56"' in CD4+ T cells. Antibody-mediated cell surface cross-linking of CD4 in CD4+ T cells leads to rapid alterations in the patterns observed on antiphosphotyrosine immunoblots of whole cell lysates in parallel with the aforementioned activation of CD4-associated p56". This model system represents the first opportunity to study the substrates of an activated Src family tyrosine kinase in normal cells. Two of the proteins that become phosphorylated on tyrosine residues following CD4 cross-linking have been identified in recent publications; the 6 subunit of the CDS/TCR complex (Veillette et al., 1989b), and p56" itself (Veillette et al., 1989a). The implications of tyrosine phosphorylation of the 6 subunit of the CDJ/TCR complex for T cell function will not be extensively dealt with in this review (for review, see Ashwell and Klausner, 1990). Moreover, as 6 phosphorylation is a relatively late event following CD4 cross-linking, and physical association between 6 and p56lCkhas yet to be demonstrated,

124


it is not clear whether 6 represents a substrate for p56” or for an as yet unidentified protein tyrosine kinase activated downstream of ~ 5 6 ”fol”~ lowing CD4 cross-linking. Recent data from this laboratory have led to tentative identification of a -70-kDa protein that becomes phosphorylated on tyrosine residues following CD4 cross-linking in CD4’ T cell leukemia lines, and normal CD4+ human T lymphocytes isolated from peripheral blood, as the product of the c-rufl protooncogene (Thompson et al., 1991a). As this protein can be coimmunopreciptated with the active conformer of ~56”’ following CD4 cross-linking, it may represent a physiologic substrate. It is interesting in light of these observations that previous work has shown that Raf-1 is a substrate for several receptor tyrosine kinases (Morrison et al., 1988; Morrison, 1990).One suggestion arising from this demonstration of functional homology between a receptor tyrosine kinase and a nonreceptor tyrosine kinase is that an active membrane-associated tyrosine kinase may recruit a “cassette” of relevant substrates, perhaps including Raf- 1, a phosphatidylinositol (PI) kinase, phospholipase Cy, and GAP (for review see Ullrich and Schlessinger, 1990), which it shares in common with other, structurally distinct, membrane-associated tyrosine kinases. Antiphosphotyrosine immunoblotting of whole cell lysates following cell surface CD4 cross-linking of normal CD4+ T lymphocytes isolated from peripheral blood reveals the appearance of tyrosine-phosphorylated protein species in the molecular weight ranges reported for these enzymes (P. A. Thompson and J. B. Bolen, unpublished). If this “cassette” of intracellular proteins proves to be substrates for ~ 5 6 ‘in “ ~this scenario, the contention that tyrosine phosphorylation of these proteins implies a cellular committment to mitosis will have to be reexamined, as antibody-mediated CD4 cross-linking, using either antibodies in solution or immobilized on plastic, does not lead to proliferation of normal CD4+ T lymphocytes (P. A . Thompson, K. Horgan, S. Shaw, and J. B. Bolen, unpublished). Oligomerization of CD4 in the absence of TCR/CDS engagement is not thought to occur in the context of antigen presentation to CD4+ T lymphocytes. Thus, activation of CD4-associated p56“ following antibody-mediated cross-linking of cell surface CD4, though useful as a model system in which to examine signal transduction through a Src family member in normal cells, may have little relevance to the signaling events that occur upon antigen presentation to a CD4+ T lymphocyte. Studies that have examined changes in the architecture of the T cell surface during antigen presentation have demonstrated colocalization of CD4 and the TCR/CD3 complex as an antigen-dependent, MHCrestricted event (Kupfer et nl., 1987). Moreover, effecting coapproxi-


125

mation of CD4 and CD3 with heteroconjugate monoclonal antibodies is more effective at stimulating early biochemical events in T cell activation (Ca2+ mobilization and phosphoinositide metabolism) than is engaging either cell surface antigen alone (Ledbetter et al., 1988). Other evidence supporting a coreceptor role for CD4 during antigen presentation has been reviewed (Janeway, 1989). Recently we demonstrated tha.t CD4-associated p56" is enzymatically activated by antigen presentation to antigen-specific, MHC-restricted human cytotoxic T lymphocyte (CTL) clones and that heteroconjugatemediated coengagement of CD4 with the CD3/TCR complex results in a similar profile of enzymatic activation of CD4-associated ~ 5 6 (Thomp" ~ son et al., 1991b). Intriguingly, whereas antibody-mediated cross-linking of CD4 in normal CD4+T lymphocytes leads to transient activation of CD4-associated p56lCk, with a return to basal activity levels within minutes, activation of ~ 5 6 following "~ antigen presentation or coengagement of CD4 with CD3 is sustained at least 15 min. Suggestively, the array of T cell proteins that become phosphorylated on tyrosine residues following antigen presentation in this system is mimicked by heteroconjugatemediated coengagement of CD4 and CD3 and differs from that seen following antibody-mediated cross-linking of CD4 or CD3. Parallel examination of p62r" and p6Wn revealed no detectable changes in enzymatic activity following coengagement of CD4 with CD3, or following engagement of either cell surface antigen alone. Though the presence of significant levels of p6dj" and p62ye5in all antigen-presenting cells examined to date precludes direct assessment of their role in mediating signal transduction in two cell systems, it may prove possible to address this issue using a planar membrane system for antigen presentation. It remains to be seen whether the mechanism for activation of p56" by antigen presentation, or by antibody-mediated coapproximation of CD4 and CD3, is similar to that which drives p56lCkactivation during cell surface cross-linking of CD4. In like fashion, the substrates for CD4-associated p56lCkfollowing activation by cell surface cross-linking of CD4 may differ from those found following activation by coapproximation of CD4 with CD3 or by antigen presentation.

B. CD8 The CD8 antigen is a cell surface glycoprotein of 32-35kDa expressed on the surface of differentiating thymocytes and mature MHC class I-restricted T cells (for review, see Bierer et al., 1989). Three different transmembrane forms of CD8 have been identified. One is the product of the CD8 (Y gene, and the other two forms, referred to as CD8 f?l,and CD8

126


p2, are differentially spliced products of the CD8 p gene and differ only in the sequences of their cytoplasmic domains (Norment, and Littman, 1988). Multimeric forms of CD8 exist, and disulfide-linked heterodimers of CD8 a and either CD8 P l , CD8 /32 (Norment and Littman, 1988), or CD1 (in thymocytes) have been described (Snow et al., 1985). CD8 can also be found linked to HLA class I antigens on the surface of peripheral blood lymphocytes (Blue et al., 1988). Purified CD8 binds to a nonpolymorphic determinant on MHC class I antigens that does not overlap the polymorphic regions recognized by MHC class I-restricted T cell receptors (Salter et al., 1990). A signaling function for CD8, in addition to its role as an adhesion molecule, has been suggested by the demonstration that CD8 is noncovalently associated with ~ 5 6 ' in " ~CD8+ T lymphocytes (Veillette et al., 1988a; Barber et al., 1989). Cotransfection of CD8a and Zck cDNAs into HeLa cells results in a stable C D 8 / ~ 5 6 ' "interaction ~ as judged by coprecipitation (Shaw et al., 1990). This genetic reconstitution has allowed mapping of interactive ~ domains of the two proteins to amino acid residues 15-23 of ~ 5 6 ' "and 191-197 of CD8 a. Indeed, this six-amino acid sequence from the cytoplasmic domain of CD8 a,VCKCPR, when appended to the cytoplasmic domain of VSV G protein, allows the chimeric protein to stably coassociate with ~ 5 6 ' "upon ~ cotransfection into HeLa cells (Shaw et al., 1990). It is interesting to note that the analogous domain of CD4 was unable to confer this property on the chimeric protein in a similar experiment. As discussed above in the case of CD4, the integrity of the closely spaced cysteine residues in the CD8 a interactive domain was critical in allowing the CD8 a/p56lckinteraction and raises the possibility of metal ion involvement in stabilizing the CD8 a / ~ 5 6 interaction. "~ Cell surface cross-linking of CD8 in CD8+ peripheral T lymphocytes leads to rapid activation of CD8-associated ~ 5 6 ' "(P. ~ A. Thompson and J. B. Bolen, unpublished), thus suggesting a functional as well as structural interaction between CD8 and ~ 5 6 " ~ The . aforementioned data for hetero- and multimeric forms of CD8 suggest that some aspects of signal transduction through the CD8/p56'"' heterodimer will differ from those found for the CD4/p56" heterodimer. There are as yet no data on substrates for CD8-associated ~ 5 6 ' " ~ nor , on the mechanism of activation of CD8- associated ~ 5 6 ' " ~ . Current evidence supports the contention that CD8, similar to CD4, functions as a coreceptor with the CD3/TCR complex during antigen presentation. In a manner analogous to that discussed above for CD4, coengagement of CD8 and the CDS/TCR complex, mediated by heteroconjugate antibodies, results in greatly augmented T cell responses compared to that seen with engagement of either antigen alone (Emmrich et


127

al., 1986). Crystallographic analysis of CD8 binding to the HLA-A2 antigen clearly demonstrates that CD8 and HLA-AZ-restricted TCRs bind to nonoverlapping regions of the HLA-A2 antigen (Salter et al., 1990). In an elegant experiment, a target cell expressing a variant HLAA2.1 that did not bind to CD8 but was recognized by the TCR of a CD8+ CTL clone was transfected with the cDNA for HLA-B'7. Whereas the wild-type HLA-AZ' target was lysed by the CTL clone, neither the abovementioned variant nor the variant expressing HLA-B7 (which did support CD8 binding) was lysed. Moreover, purified variant HLA-2.1 was able to inhibit lysis of wild-type targets by the CTL clone upon coincubation (Salter et al., 1990). These data strongly suggest that binding of CD8 and the CD3ITCR complex to the same MHC class I/antigen complex is requisite for T cell activation. Recently we have shown that CD8-associated ~ 5 6 ' "is~enzymatically activated by antigen presentation to CD8+ MHC class I-restricted antigen-specific human T cell clones (Thompson et al., 199lb). Enzymatic activation was noted soon after antigen presentation (within 1 min) and was stable u p to 15 min. In contrast, the activation of CD8-associated ~ 5 6 ' seen " ~ following cell surface cross-linking of CD8, though rapid, is much more transient, with a return to baseline activity levels noted within 5 min. Because of the constraints imposed by the limited potential of these CD8+ clones to expand, we were unable to examine critically the proteins phosphorylated on tyrosine residues following antigen presentation, nor whether heteroconjugate-mediated coengagement of CD8 and CD3 was able to reproduce these early signaling events. It is clear that more information is needed on signal transduction through CD8associated ~ 5 6 ' ~ . C. CDS/TCR T h e antigen- and MHC-specific T cell antigen receptor exists as a complex heterooligomer on the surface of mature T cells and is composed of two highly polymorphic disulfide-linked polypeptide chains, TCRa and TCRP, in noncovalent association with at least five nonpolymorphic polypeptides, the CD3 complex (see Fig. 3). Recent reviews on the assembly and structure of this receptor system have been published (Ashwell and Klausner, 1990) and will not be discussed here. Compelling evidence exists that ties signal transduction through this receptor system to protein tyrosine kinases. Antibody-mediated ligation of the T cell receptor on mature peripheral T cells results in rapid alterations in the pattern of T cell proteins phosphorylated on tyrosine residues, as monitored by antiphosphotyrosine immunoblotting of whole

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cell lysates (June et al., 1990a). These alterations precede measurable changes in calcium mobilization or PI turnover (June et al., 1990a). Inhibition of phytohemagglutinin (PHA)-induced tyrosine phosphorylation of the 5 component of the CD3 complex with the isoflavone compound genistein has been reported to abolish PHA-stimulated calcium mobilization and PI turnover in T cells (Mustelin et al., 1990), suggesting a functional link between these signaling pathways. In like fashion, treatment of T cells with herbimycin (an inhibitor of noreceptor tyrosine kinases) abrogates T cell antigen receptor-driven stimulation of calcium mobilization and PI turnover (June et al., 1990b). As sequence analysis of the polypeptides comprising the TCR/CD3 complex reveals no consensus sequences for intrinsic tyrosine protein kinase activity, it is assumed that the aforementioned changes are mediated though a functionally coupled protein tyrosine kinase structurally distinct from the receptor complex. T h e description of the C D 4 / ~ 5 6 ' "and ~ CD8/p56z"kinteractions discussed above has engendered speculation that the responsible kinase may be a member of the Src family. Recent work with the murine T cell hybridoma 2B4 has shown that under gentle conditions of detergent lysis, CD3 E antibodies coprecipitate a protein tyrosine kinase that comigrates with p6@" on one-dimensional sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) (Samelson et at., 1990b). One-dimensional Cleveland mapping of the autophosphorylated candidate kinase (Samelson et al., 1990b) and subsequent twodimensional tryptic peptide mapping (L. E. Samelson, personal communication) are consistent with the idea that this coprecipitating tyrosine kinase is p66y". Metabolic labeling of 2B4 cells followed by digitonin lysis and precipitation with anti-Fyn antibodies coprecipitated an estimated 0.1% of the total CD3 E , suggesting a stoichiometry for this interaction much lower than that demonstrated for the p56'"/CD4 and p56"k/CD8 interactions in human T lymphocytes. It remains to be shown that this interaction occurs in normal human T lymphocytes, which contain far less ~ 6 0 6 than % does the 2B4 hybridoma (P. A. Thompson and J. B. Bolen, unpublished). If this proves to be the case, and the estimated stoichiometry from the 2B4 system is valid, the p60n" CD3 E interaction will serve to define a structurally and perhaps functionally distinct subpopulation of T cell antigen receptors. The proposed low stoichiometry of this interaction may also explain why it has been impossible to demonstrate any detectable alterations in ~ 6 0 6 activity % induced by antigen receptor ligation (Samelson et al., 1990b; Thompson et al., 1991b). Previous work suggests that the T cell receptor may be functionally linked to ~ 5 6 ' " ~Antigen . receptor engagement with monoclonal anti-


129

bodies leads to physical approximation of cell surface CD4 (and presumptively the associated ~56'"') with the CD3/TCR complex (Mittler et al., 1989) as determined by fluorescence energy transfer. As heteroconjugate-mediated coapproximation of CD4 with CD3 leads to stimulation of CD4-associated p56" activity (Thompson et al., 199lb), this antigen receptor-driven redistribution of cell surface CD4 may result in activation of the associated ~56'"".If the stoichiometry for this interaction is low, as has been suggested from comodulation experiments (Saizawa et al., 1987), it may prove difficult to demonstrate a change in activity of CD4-associated ~56'" using immune complex kinase reactions of total CD4 immune precipitates, and no such change has been reported. Intriguingly, stimulation of CD4-associated p56"' activity is readily demonstrable in normal human CD4' peripheral T lymphocyes following incubation with various CD3 monoclonal antibodies immobilized on plastic (P. A. Thompson, K. Horgan, S. Shaw, and J. B. Bolen, unpublished). Signals transduced by the T cell antigen receptor may also influence signal transduction through p56" by other means. Cell surface cross-linking of the TCRlCD3 complex on T cells leads, within 30 min, to the appearance of modified forms of p56"' with retarded mobility on SDS-PAGE (Veillette et al., 1988a; Marth et al., 1989). Phosphopeptide mapping of metabolically labeled ~56'"' under these conditions reveals novel amino-terminal sites of serine phosphorylation which can also be generated by incubation of the T cells with phorbol esters (Veillette et al., 1988b).As these phosphorylation changes occur in a region thought to be of potential import in enzyme/substrate interactions, signal transduction through ~ 5 6 ' may " ~ be effected. Intriguingly, the mobility shift of ~56'"' characteristic of serine phosphorylation of a specific tryptic phosphopeptide (Veillette et al., 1988b) is not seen following engagement of the CD3/TCR unless a second cross-linking antibody is added (Veillette et al., 1988b; P. A. Thompson and J. B. Bolen, unpublished), and is also seen following IL-2 treatment of some IL-2-responsive T cells (Horak et al., 1991). Phorbol esters have also been shown to provoke the dissociation of p56" from CD4, but not CD8 (Hurley et al., 1989). T h e emerging information on signal transduction in T cells during activation by antigen presentation suggests the involvement of multiple protein tyrosine kinases in mediating early alterations in tyrosine phosphorylation. If both p56" and p6Obn are implicated in these events, are the signals generated through them antagonistic or complementary? Given the proposed low stoichiometry of the p66mlCD3 E interaction, relative to the p56'"'lCD4 and the p56'"'lCD8 interactions, a shared array of substrates might allow for increased sensitivity under conditions when

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antigen is limiting. Alternatively, the signals generated through one Src family member may fundamentally oppose those generated through the other. Thus, though the effect of enzymatic activation of one Src family member in a T cell may be to contribute to the processes leading to the activated phenotype, activation of a different Src family member in the same cell may serve to attenuate such signals. Recent work with transgenic mice may lend support to the latter hypothesis. Thymocytes from mice expressing high levels of the nonlymphocyte form of p60fy" are hyperresponsive to CD3 engagement whereas those from mice expressing a mutationally activated form of ~ 5 6 ' "were ~ hyporesponsive under similar conditions (R. Perlmutter, personal communication). It may also be that substrate accessibility, and thus the physiologic consequences of activation of a given Src family member, may vary depending upon the nature of the alterations in T cell surface antigen topology that led to its enzymatic activation. The identification of substrates for the active conformers of ~ 5 6 ' " and p60fr" in T cells under normal physiologic conditions will greatly aid in our understanding of this complex issue. D. CD2 The CD2 surface antigen on human T cells is a 50-kDa transmembrane glycoprotein that binds specifically to the CD58 (LFA-3) antigen (Dustin et al., 1987), expressed on the surface of the antigen-presenting cell, during T cell activation (see Fig. 3). The role played by the CD2 antigen in T cell activation has been reviewed (Bierer et al., 1989) and only those issues potentially bearing on signal transduction through the Src family will be discussed here. Cell surface cross-linking of CD2 with stimulatory pairs of CD2 monoclonal antibodies leads to rapid changes in the pattern of T cell proteins phosphorylated on tyrosine residues as monitored by antiphosphotyrosine immunoblotting of whole cell lysates (Samelson et al., 1990a). It is not yet clear that Src family members are involed in mediating these changes. CD2 is not physically associated with any of the Src family members expressed in T cells as judged by immune complex kinase assays on, and immunoblotting of, CD2 immunoprecipitates (P. A. Thompson and J. B. Bolen, unpublished). CD2 may influence the function of Src family members in T cells indirectly via its association with other T cell surface antigens, however. Cell surface cross-linking of CD2 on T cells results in serine phosphorylation of p56"' and a shift in its apparent molecular weight on


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SDS-PAGE reminiscent of that seen under some conditions for CD3/ TCR engagement (Danielianet al., 1989).Antibodies to CD3 E are able to coprecipitate CD2 from digitonin lysates (Brown et al., 1989),suggesting a physical interaction between these two receptors on the surface of resting T lymphocytes. Previous studies demonstrating a requirement for CD3/TCR coexpression for CD2 signaling in human T leukemia cell lines (Bockenstedt et al., 1988; Sreitmeyer et al., 1987) suggest a functional as well as physical association between the two receptor systems, though this functional relationship does not seem to hold for thymocytes (Fox et al., 1985; Pierres et al., 1990)and remains to be shown for normal human T lymphocytes. Chemical cross-linking of T lymphocytes surface labeled with radioiodine followed by detergent lysis and immunoprecipitation has suggested that the CD2 antigen may be physically associated with a protein phosphotyrosine phosphatase, the CD45 antigen, as well (Schraven et al., 1990) (see Fig. 3). It is not yet clear that engagement of CD2 influences CD45 enzymatic activity. Heteroconjugate-mediated coengagement of CD2 and CD45 attenuates CD2-stimulated Ca2+ mobilization and PI metabolism (Samelson et al., 1990a) and results in the dissappearance of tyrosine-phosphorylated proteins on parallel antiphosphotyrosine immunoblots, suggesting that the converse may be true. Murine CD4+ antigen-specific T cell clones deficient for CD45 expression no longer proliferate in response to antigen presentation, and exhibit a diminished proliferative response to IL-2 when compared with the parental CD45+ clone (Pingel and Thomas, 1989). The possible role of CD45 in T cell signal transduction in general, and in modulating the activity of Src family tyrosine kinases specifically,has been the subject of several reviews (Alexander and Cantrell, 1989; Clark and Ledbetter, 1989; Hunter, 1989), and the interested reader is referred to these for a more comprehensive review of this topic.

V. Involvement of the Src Family in Signal Transduction during T Cell Proliferation

In this section the T cell proliferative events will be reviewed and an update is provided concerning recent developments in signal transduction by the interleukin-2 (IL-2) receptor, in which members of the Src family may play a role. Several reviews have been published on T cell proliferation and the IL-2 receptor in different cellular systems (Waldmann, 1986, 1989; Smith, 1988a, b; Schwartz, 1990).

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A. INTERLEUKIN-2 AND THE INTERLEUKIN-2 RECEPTOR COMPLEX Mature peripheral blood T cell populations are maintained at constant numbers and proportions throughout life. Though a portion of the peripheral T cells are considered long-lived cells (Wallis et al., 1979), 30-50% of these cells are characterized as short-lived cell populations (Rocha et al., 1984). In order to replenish peripheral T cells, bone marrow-derived precursor cells travel to and are processed in the thymus, where incoming cells are selected, proliferate, and mature in response to thymic differentiation signals. However, during the maturation process, most cells die and only about 1% of thymocytes leave the thymus per day (Scollay et al., 1980), indicating that thymic lymphocytes alone cannot be responsible for replenishing the peripheral T cell pool. Thus, postthymic expansion of T cells must occur (Stutman, 1986; Rocha, 1987). One cytokine that appears to be critical for this postthymic T cell population expansion is interleukin-2 (Smith, 1988a, b; Robb et al., 1981; Cantrell and Smith, 1986). I n the periphery, activation of T cells during MHC-restricted antigen presentation stimulates the synthesis and secretion of IL-2, a 15.5-kDa glycoprotein (Morgan et al., 1976). T o exert its proliferative effect on T cells, IL-2 must interact with specific receptors (the 1L-2 receptor, or IL-2R) on the cell surface. The cell receptors that are thought to mediate the proliferative signals generated following IL-2 binding are termed the high-affinity receptors (composed of an a and /3 core subunit complexed with several additional putative proteins; see later), which are absent from the surface of resting T lymphocytes. Resting T cells, on the other hand, express an IL-2 receptor (p70-75, or j3) that is considered to possess intermediate affinity for IL-2 (Table 11) (Tsudo et al., 1987a, b; Siegal et al., 1988; Sharon et al., 1988a; Ohashi et al., 1989; Caligiuri et al., 1990; Nagler et al., 1990; Espinoza-Delgado et al., 1990; Waldmann, 1991). The 55-kDa a subunit, or Tac subunit, of the high-affinity receptor is rapidly expressed on the T cell surface during the activation process to form the high-affinity IL-2 receptor core complex. Thus, the growth factor (IL-2) and its high-affinity receptor complex are individually regulated, but both are required to mediate relevant events in this proliferative pathway. The study of human IL-2R was significantly facilitated by the isolation of monoclonal antibodies recognizing the a (anti-Tac)(Uchiyama et al., 1981) and p (anti-p70-75)(Tsudo et al., 1989a, b) core components along with the corresponding cDNAs for these two receptor subunits (Leonard et al., 1984; Nikaido et al., 1984; Tsudo et al., 1987b). Utilizing anti-cY

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TABLE I1 PROPERTIES OF THE HUMAN IL-2 RECEPTORCORECOMPONENTS~

Intermediate affinity (~70-75;P )

lop 14-15 55 kDa Inducible No

22q 11.2-12 70-75 kDa Constitutive? Yes

-

NO

Yes

Yes

5 sec 7 sec

10-9 45 min 5 hr

30 sec 5 hr

Low

Property C:hromosome

Molecular weight Expression IL-2 internalization Signal sequence IL-2 binding & (M) t1,2 association t,,, dissociation

High affinity

affinity (p55; Tac; a )

WP) ?

Inducible Yes

lo-"

' Modified from Creene et al. (1989).

antibody, this receptor component has been biochemically characterized as a 55-kDa transmembrane glycoprotein and sequence analysis of a cDNA encoding p55 revealed a single gene on chromosome 10 capable of encoding a predicted protein of 251 amino acids (Leonard el al., 1984; Nikaido et al., 1984) (see Fig. 4). The a subunit was defined as having a 33-kDa peptide precursor following cleavage of the hydrophobic leader sequence, which undergoes a cotranslastional N-glycosylation to generate 35 and 37-kDa intermediates (Leonard et al., 1982, 1983) and a mature form of 55 kDa as a consequence of additonal O-linked glycosylation events. The first 219 amino acids of the mature protein comprise the extracellular domain, which contains two potential N-linked glycosylation sites (Asn-X-Ser/Thr) and multiple possible O-linked carbohydrate sites (Waldmann, 1989). The transmembrane domain contains a single hydrophobic stretch of 19 amino acids; the cytoplasmic domain is 13 amino acids in length and does not appear to possess an enzymatic function. Sequence analysis of the /3 cDNA IL-2R subunit revealed that the corresponding mRNA is capable of encoding a mature protein of approximately 524 amino acids possessing a 2 14-amino acid extracellular domain, a 25-amino acid transmembrane domain, and a large 285-amino acid cytoplasmic domain (Hatakeyama et al., 1989a). Though the cytoplasmic domain of the /3 subunit is significantly larger than that of the a subunit, to date, no intrinsic enzyme activity has been attributed to this protein, although amino acid residues 267-322 appear to be important

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m? wc

FIG.4. Interleukin-2 receptor complex components. The figure shows the core a and /? subunits along with other proteins that have been suggested to be components of the receptor signaling complex. The potential serine (S), threonine (T), and tyrosine (Y) phosphorylationsites are indicated.

for some portion of the signaling capacity of the /3 subunit (Hatakeyama et al., 1989b). Several recent observations suggest a multi-protein subunit structure of the high-affinity receptor that involves proteins other than the a and /3 subunits (Szollosi et al., 1987; Herrmann and Diamanstein, 1987; Sharon et al., 1988b, 1990; Saragovi and Malek, 1990; Burton et al., 1990; Colamonici et al., 1990). Using coimmunoprecipitation of radiolabeled IL-2, chemical cross-linking, and flow cytometric resonance energy transfer, a series of candidate receptor-associated proteins of molecular weights 22K, 35K, 40K, 75K, and 90K-105K have been found complexed with the and /3 proteins (Waldmann, 1989, 1991). In a recent study (Burton et al., 1990), evidence was presented suggesting that the p90-95 protein associated with IL-2R is an intercellular adhesion molecule (ICAM- 1 or CD54) (see Fig. 4). Intercellular adhesion molecule-1 binds to the /32 integrin lymphocyte function-associated antigen- 1 (LFA-1; or CDl la/ CDl 8), promoting cell adhesion in immune and inflammatory response


135

(Larson and Springer, 1990). Association of ICAM-1 with the IL-2R may facilitate the paracrine IL-2-mediated stimulation of cells expressing I L - ~ R s ,by augmenting T cell/T cell interactions, through receptordirected focusing of IL-2 release by the helper T cell and by concentrating IL-2Rs of the physically linked cells at this site of LFA-lIICAM-lIIL-PR interactions (Burton et al., 1990). Major histocompatibility complex class I antigens have also been suggested as a part of the IL-PRcomplex (Sharon et al., 1988a). As indicated in the previous section, MHC class I molecules play a pivotal role in the immune response as carrier proteins that present peptide antigens to T cells. Interestingly, MHC class I molecules have also been reported to interact with receptors for peptide hormones and growth receptors (Amiot et al., 1988; Due et al., 1986). Saragovi and Malek (1990) have recently reported a 22-kDa protein potentially associated with IL-2R and Sharon et al. (1990)have reported a 95- to 110-kDa protein associated with the IL-2R core proteins (Fig. 4). Whereas the current speculations concerning the structure of the IL-2 receptor complex predict that of a complex multi-subunit structure, none of the putative associated subunits has been shown to be required for the IL-2dependent proliferative events. The recent recognition of sequence similarity among the receptors for growth hormone (GH), prolactin (PRL-R), erythropoetin (EPO-R), IL-2R p , interleukin-6 (IL-GR), interleukin-4 (IL-4R), and granulocyte/ macrophage colony-stimulating factor may facilitate the study of the mechanisms through which this superficially diverse group of receptors may mediate signal transduction events (Patthy, 1990). Comparative analysis revealed significant sequence similarity in the external domains of the cytokine receptors for IL-2 (Hatakeyama et al., 1989a, b), IL-3 and IL-4 (Mosley et al., 1989), IL-6 (Yamasaki et al., 1988; Hirano et al., 1990), and EPO (D’Andrea et al., 1989). The common motifs found in the cytokine receptors may also participate in protein-protein interactions. Interstingly, there is significant conservation among the cytoplasmic domains of EPO-R, IL-3R, and IL-2R fl chains. The observation that IL-2-dependent cell lines can be derived from IL-3-dependent cell lines (LeGros et al., 1985)and that expression of the IL-PR /3 chain in these cell lines conferred IL-2 dependence (Hatakeyama et al., 1989a) raise the possiblity that the IL-2 receptor fl chain interacts with signal transduction machinery similar to that of the 1L-3R.

B. INTERLEUKIN-2 RECEPTOR SIGNAL TRANSDUCTION IL-2 interaction with its high-affinity receptor complex is critical for normal T cell proliferation. T h e immediate consequences of IL-2/IL-2R

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complex interactions are biochemical changes that affect proteinprotein interactions (and potentially protein function) and transmit as yet unknown signals to the nucleus, initiating transcription of specific genes. Several lines of evidence indicate that activation of protein kinase C (PKC) may be important for IL-2-induced proliferation. T h e IL-2R a subunit is also known to be phosphorylated by PKC, predominantly on serine 247 and to a lesser degree on threonine 250 (Shackelford and Trowbridge, 1986; Gallis et al., 1986). Both IL-2 and PMA have been reported to exert overlapping effects on IL-2-sensitive cells, including stimulation of IL-2R expression, stimulation of cellular protein phosphorylation on serinehhreonine residues, PKC translocation to the plasma membrane, induction of cell proliferation, and activation of the Na+/H+ antiporter (Mills et al., 1985; Farrar et al., 1986; Heckford et al., 1986; Beckner and Farrar, 1987; Evans and Farrar, 1987). In addition, H7, a putative PKC-specificinhibitor, was reported to prevent IL-2induced T cell proliferation (Clark et al., 1987). However, the studies by Farrar and Anderson (1985) and Bonvini et aE. (1987) indicated that PKC activation, Ca'+ mobilization, and PI hydrolysis are early consequences of IL-2 interaction with the IL-2R; this has not been concluded by others (Kozumbo et al., 1987; Altman et al., 1990). Recently, an IL-2-inducible B cell line was used to study potential mechanisms of IL-2 signal transduction. Analysis of the signaling pathway showed that IL-2 stimulated rapid hydrolysis of an inositol-containing glycolipid to yield two possible second messengers-a myristylated diacylglycerol and an inositol phosphateglycan (Eardly and Kosland, 1991).These observations thus implicate the glycosyl-phosphatidylinositol system in the intracellular relay of the IL-2 signal. Furthermore, the capacity of mutant T lymphocytes that lack PKC expression to proliferate in response to IL-2 (Mills et al., 1988), as well as the observation that PKC-depleted T cells retain their ability to proliferate in response to IL-2 (Valge et al., 1988), suggests that PKC does not play a major role IL-2 proliferative responses in T cells. Similar results were documented in studies using a B lymphoma cell line possessing an inducible IL-2R (Tigges et al., 1989). IL-4, which is also known to serve as a growth factor for both T and B lym hocytes, has been reported not to stimulate PKC, PI hydrolysis, or Cay+ mobilization (Mizuguchi et at., 1986). Thus, whether IL-2 shares some unique signaling pathway(s) with other lymphokines remains to be determined (Ferris et al., 1989; Patthy, 1990; D'Andrea et al., 1989). Signal transduction by the IL-2R has been shown to involve the activation of both serinelthreonine protein kinases distinct from PKC and tyrosine protein kinases. IL-2-dependent phosphorylation of multiple membrane proteins has been documented (Gaulton and Eardley, 1986;


137

Shackelford and Trowbridge, 1986; Gallis et al., 1986; LeGrue, 1988). Additional observations provide growing evidence of protein tyrosine kinase involvement in IL-2-dependent proliferative responses of T lymphocytes and NK cells (Saltzman et al., 1988, 1990; Ishii et al.,1988; Ferris et al., 1989; Sharon et al., 1989; Asao et al., 1990; Mills et al., 1990; Zu et al., 1990; Merida and Gaulton, 1990; Horak et al., 1991). Given the precedence of the primary role of tyrosine protein kinases in signal transduction in a wide variety of cellular responses, it is tempting to speculate that activation of one or more tyrosine protein kinases is the initial event in signal transduction of the IL-2R. Support for this view is provided by experiments showing that treatment of the murine IL-2dependent cytotoxic T cell line CTLL-2 with IL-2 results in a rapid increase in the tyrosine phosphorylation in several proteins ranging from 38 to 120 kDa. The tyrosine phosphorylation of these proteins reached a maximum level in 15 min and required concentrations of IL-2 that correlated with the proliferation of the CTLL-2 cells (Saltzman et al., 1988). T h e stimulation of activated human T lymphocytes results in rapid phosphorylation of multiple proteins of approximately 32, 33, 55, 56,60, 70,75,92, 100, and 116 kDa (Ferris et al., 1989). Adding 1L-2 to CTLL-2 cells has also been shown to induce a rapid increase in the tyrosine phosphorylation of the approximately 70-kDa c-raf gene product; c-raf is a protooncogene encoding a serine/threonine protein kinase previously noted to be capable of binding to and being phosphorylated by activated CD4-associated ~ 5 6 ' ~Of . particular note is the observation that tyrosine phosphorylation of the Raf- 1 protein following IL-2 treatment increased Raf- 1 kinase activity in a time-dependent manner and correlated well with IL-2-stimulated proliferation of the CTLL-2 cells (Turner et al., 1991). Using antiphosphotyrosine antibodies to immunoprecipitate the IL-2R /3 subunit following IL-2 treatment of leukemic lines, Sharon et al. (1989) and Mills et al. (1990) provided the first indications that the IL-2R /3 subunit can be phosphorylated on tyrosine residues in response to IL-2 [the potential sites for tyrosine (Y) as well as other serine (S) and threonine (T)phosphorylations on the /3 subunit as well as on other potential IL-2R components are shown in Fig. 41. These experiments showed in an IL-2-dependent T cell line stimulated with IL-2 that p70-75 was phosphorylated on tyrosine as early as 1 min and that the phosphorylation rapidly increased within 5 min of IL-2 stimulation, with a maximum phosphorylation level at 15 min. A similar pattern of IL-2R /3 phosphorylation was observed with human peripheral blood lymphocytes (Asao et al., 1990). In this system, tyrosine phosphorylation of IL-2R /3 was found to be IL-2 concentration dependent, in contrast to serinelthreonine

138

JOSEPH B. BOLEN ET A L .

phosphorylation of the same protein, which was found to be largely independent of IL-2 concentration. These results correlate with the recent observation that ~ 5 6 is" rapidly ~ activated (within 1 min) in human 1L-2-dependent T cells in response to physiologic levels of IL-2 (Horak et al., 1991). These observations are consistent with the idea that tyrosine phosphorylation of the IL-2R /3 subunit may be one of the earliest events induced by IL-2-IL2R interaction. Furthermore, this model implies that signaling involves one or more tyrosine protein kinases and that at least one of these kinases may be ~ 5 6 " The ~ . lack of proliferative response to IL-2 and absence of ~ 5 6 ' "in~ several IL-2-independent adult T cell leukemia cell lines also provide support for this idea (see Section V,C) (Horak et al., 1991).However, no ~ 5 6 activity " ~ was found to be coimmunoprecipitated with any known or suspected component of the IL-2R in the normal T cells (I. D. Horak et al., unpublished). Thus, it is possible that as yet other undefined tyrosine protein kinases (or potentially tyrosine phosphatases) may associate with one or more subunits of the IL-2R. Interestingly, it was recently found using cell-free phosphorylation assays with IL-2/IL-2R complexes that tyrosine activity could be detected (Merida and Gaulton, 1990),although the kinase was not identified. C. ALTERATIONS IN T LYMPHOCYTE MALIGNANCIES

Adult T cell leukemia and lymphoma (ATLL) is an aggressive T cell malignancy first described in an adult population in southwestern Japan (Uchiyama et al., 1977). This disease is characterized by the presence of malignant T lymphocytes with a mature helper (CD4+) phenotype but functioning as suppressor cells. ATLL has a heterogeneous clinical presentation that can be broadly divided in four stages based on the extent of disease. These include preleukemia, smoldering ATLL, chronic ATLL, and the most common form, subacute or acute ATLL (Yamaguchi et al., 1983). About 50% of patients with preleukemia have long-lasting spontaneous regression of their disease occurring within months to years after the original diagnosis. The other half have persistent lymphocytosis and some will progress to acute leukemia presentation. Patients with smoldering leukemia have a gradual onset of skin lesions with 0-2% abnormal peripheral blood lymphocytes. Closely related to this group are patients with chronic ATLL, who have a higher number of abnormal lymphocytes in peripheral blood. The fourth group of patients have the typical acute form of ATLL, which is a rapidly progressive disease affecting skin, with generalized lymphadenopathy and hepato-splenomegaly.


139

T h e unusual geographic distribution of ATLL helped to predict an infectious origin for this disease. Independently, two groups found a unique virus, later called HTLV-I (human T cell lymphotrophic virus type I), which is considered the etiologic agent of this disease (Poiessz et al., 1980). HTLV-I was first implicated as the agent responsible for ATLL when in vitro studies showed incorporation of its proviral sequences into the DNA of ATLL cells and when cocultivation of HTLV-Iinfected T cells and normal T lymphocytes resulted in rapid transformation of the normal T cells (Miyoshi et al., 1981; Popovic et al., 1983). Since the identification of HTLV-I, extensive seroepidemiologic surveys have shown that the virus exists not only in Japan, but also in other parts of the world. The leukemic cells from ATLL patients express high- and low-affinity IL-2Rs. Unlike normal T cells, they d o not require prior stimulation for a subunit expression and characteristically express 5- to 10-fold more highaffinity receptors per cell than do maximally stimulated normal T lymphoblasts. An additional alteration is that the IL-PR is not downmodulated in the ATLL cells following addition of IL-2 (Uchiyama et al., 1985). HTLV-I encodes a 40-kDa trans-acting regulatory protein termed TAX (Lee et al., 1984). In addition to stimulating transcription from the HTLV-I long terminal repeat (LTR) elements flanking the virus genome, TAX shares with T cell mitogens the ability to induce transcription of various cellular genes, including the IL-2R a gene, the IL-2 gene, and the GM-CSF and c-fos genes, but decreases expression of j3 polymerase (Felber et al., 1985; Siekevitz et al., 1987). These effects of TAX may bypass the normal signal transduction pathway and contribute to the sustained 'expression of surface IL-2R a and promote autocrine proliferation of the infected cells during the early polyclonal phase of growth. Recent studies show deregulation of selected Src family members in ATLL-derived IL-2-dependent and -independent cells (Oh-Hori et al., 1990; Koga et al, 1988). As previously noted, three members of the Src family are normally expressed in peripheral T cells: p56" (the most abundant) and significantly lower levels of p66'" and p62Y". As well as IL-2-dependent ATLL cell lines, normal human T lymphocytes acutely "~ infected with HTLV-I express 10- to 100-fold decreased ~ 5 6 ' protein (I. D. Horak, unpublished), with proportional coordinate changes in the expression of lck RNA (Koga et al., 1989; J. M. Pyper and J. B. Bolen, unpublished). Decreased IL-P-dependent proliferation in ATLL cells has been found to correlate with decreased amounts of ~ 5 6 and " ~ significant increases in the expression of p56'Y" (I. D. Horak, unpublished). The increased expression observed in these cell types correlates with the

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JOSEPH B . BOLEN E T AL.

increased abundance of lyn steady-state RNA levels (Yamanashi et al., 1989; J. M. Pyper and J. B. Bolen, unpublished). Preliminary results from several ATLL patients suggest a correlation between the ratio of p56zcklp56zY" expression and response to anti-Tac monoclonal antibody therapy (1. D. Horak, unpublished). These results indicate that ATLL cells represent a interesting model system to study T cell signal transduction and T cell proliferation events. These observations also serve to underscore the idea that an understanding of basic signal transduction pathways in different cell types may lead to opportunities for unique applications of molecular biology to clinical problems and yield additional previously unexpected scientific insights. VI. Conclusions

Studies of signal transduction pathways in T lymphocytes have provided important information with regard to signaling mechanisms relevant for the performance of the mature differentiated functions of these cells, as well as that for functions more closely associated with proliferative events. T h e wealth of antibodies to various T cell surface antigens, the knowledge of T cell functions, and the ability to obtain large numbers of homogeneous normal cell populations have allowed for experimental opportunities that are not realistically obtainable in many other cell systems. With regard to the Src family of tyrosine protein kinases, T cells proved to be the model cell system in which our first glimpse of a defined regulatable physiologic function for an Src family member was discovered. These cells have also yielded several possible physiologic substrates for Src family enzymes and have provided a new perspective on the way in which we think about the organization of cellular receptors. More importantly, these initial observations in T cells suggest the possibility that the functions of other members of the Src family may be similar to those now under investigation in T lymphocytes.

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IMPLICATING THE bcrlabl GENE IN THE PATHOGENESIS OF PHI LADELPHI A CHROM0s0M E- POSITIV E HUMAN LEUKEMIA George Q. Daley* and Yinon Ben-Neriaht Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142

t The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91010, Israel

I. Introduction and Background A. T h e Nature of Human Chronic Myelogenous Leukemia B. Cytogenetics and the Philadelphia Chromosome (Ph’) C. A Central Hypothesis for the Role of the Philadelphia Chromosome in Leukemia 11. T h e Molecular Characterization of the Philadelphia Chromosome A. Structural Alterations B. Understanding the Role of the bcriabl Gene in Lcukcmogenesis III. From v-abl to c-ubl and Back Again A. The Abelson Murine Leukemia Virus B. Functional Domains of the c-Abl Protein C. Activation of the c-abl Protooncogene IV. abl in Human Malignancy A. The Bcri Ah1 Proteins of Ph’-Positive Leukemias B. Possible Consequences of the bcr Fusion to abl C. Evaluation of the Intrinsic Enzymatic Activity of Abl Proteins V. Biological Activity of BcriAhl in Vztro A. Expression of bcriabl in Fibroblast Cell Types B. Transformation of Hematopoietic Cell Lines by Abl Proteins V1. Animal Models of Ph’-Positive Leukemias A. Transgenic Models of bcrlabl-Induced Leukemia B. Retroviral Transduction of bcrlabl into Murine Bone Marrow C. T h e Utility of the Animal Models References

I. Introduction and Background

A. THENATURE OF HUMAN CHRONIC MYELOCENOUS LEUKEMIA

Chronic myelogenous leukemia (CML) was first described in the midnineteenth century as a “diseaseof the spleen in which death took place in consequence of the presence of purulent matter in the blood” (Bennett, 151 ADVANCES IN CANCER RESEARCH, VOL. 57

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1845; Craigie, 1845; Virchow, 1845).These early studies highlighted the classic features of the disease: splenomegaly and a marked increase in the white blood cell count. Both features of CML result from a massive expansion of the myeloid cell compartment (for a review, see Champlin and Golde, 1985). In the early “chronic” phase of the disease, the myeloid progenitors have a high proliferative capacity but maintain a largely normal differentiation program. This leads to greatly elevated numbers of mature granuloctyes in the circulation. The chronic phase may be stable for a few years, but, in time, the disease undergoes a transformation. T h e appearance of increasing numbers of immature myeloid cells in the peripheral blood signifies an acceleration in the disease process and presages the terminal “blast crisis” stage. In blast crisis, the hematologic picture resembles that of an acute leukemia: there is a block in cell maturation and undifferentiated blast cells predominate. Patients who undergo transformation to the blastic phase are difficult to treat, and bone marrow transplantation offers the only chance for cure. CML in the chronic phase is a disorder of myeloid cells. After transformation, the phenotype of the blast cells may represent any of the multiple hematopoietic lineages. Blastic cells are of the myeloid type in about 70% of the cases, but a significant percentage of patients develop blastic crises of lymphoid cells (25%), and transformations involving erythroid, megakaryocytic, and primitive undifferentiated cell types have been described (Rosenthal et al., 1977). Cytogenetic and isoenzyme analyses established that the leukemic clone in CML patients contributes to both lymphoid and myeloid hematopoietic lineages (Fialkow et al., 1977, 1978). These studies and the multilineage nature of blastic transformation suggest that CML is a clonal disorder arising in the pluripotent hematopoietic stem cell.

B. CYTOCENETICS AND

THE

PHILADELPHIA

CHROMOSOME ( P H ~ )

Cytogenetic studies established CML as the first human neoplasm to be consistently associated with a particular chromosomal abnormality. Working in Philadelphia, Nowell and Hungerford (1960) noted a “minute chromosome” in bone marrow metaphases of patients with CML. The Philadelphia chromosome (Ph’), as it became known, is a foreshortened chromosome 22 resulting from a reciprocal translocation of chromosomes 9 and 22, and is present in the leukemic cells of greater than 90% of CML cases (Rowley, 1973). Nonhematopoietic tissues, in-

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cluding bone marrow fibroblasts, show a normal karyotype in CML patients; thus, the translocation is not inherited through the germ line but rather is an acquired lesion peculiar to the leukemic clone (Fialkow et al., 1977; Maniatis et al., 1969). Certain CML patients who lack cytogenetic evidence of the Philadelphia chromosome (Ph' negative) carry complex translocations involving several chromosomes in addition to 9 and 22 [for a catalog of translocations in CML, see Bernstein (1988)l. In other Ph'negative CML patients, molecular analysis of translocation breakpoints reveals the same gene rearrangements as seen in their Ph'-positive counterparts (Bartram et al., 1985; Dreazen et al., 1988; Ganesan et al., 1986). Thus, virtually all patients with CML harbor a consistent molecular lesion exemplified by the Philadelphia chromosome. The multiphasic nature of CML and its evolution toward acute leukemia reflect the paradigm of multistep carcinogenesis: malignant tumors arise by a multistep process that requires the accumulation of several genetic mutations. CML in its early stages is an indolent disease, considered by some to be preleukemic rather than a fully transformed state. Because virtually all CML patients in the chronic phase are Ph' positive, the translocation is presumed to represent the initial mutational event. Blastic transformation is usually accompanied by additional chromosomal changes, most frequently duplication of the Ph', isochromy 17q, and trisomy 8 (Rowley, 1980). These frequent secondary chromosomal abnormalities may constitute additional mutational events that contribute to progression of the disease toward the acutely leukemic blast crisis stage.

HYPOTHESIS FOR THE ROLEOF THE C. A CENTRAL PHILADELPHIA CHROMOSOME IN LEUKEMIA T h e hypothesis that the Philadelphia chromosome might have a causative role in human CML was validated by the discovery that the human homologue of the v-abl oncogene of Abelson murine leukemia virus (A-MuLV) mapped to chromosome 9q34 (Heisterkamp et al., 1982), precisely the locus disrupted in the formation of the Philadelphia chromosome. The result immediately suggested an etiologic mechanism: the breakpoint of the Philadelphia chromosome might activate the human c-abl homologue to behave as a transforming gene similar to v-abl. It seemed likely that the human homologue of a murine leukemia virus oncogene could be activated to play a direct role in the pathogenesis of human leukemia. Marshalling data to verify this hypothesis has occupied

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the better part of the last decade. It is now widely accepted that the Philadelphia chromosome harbors an activated abl oncogene that drives the dramatic cell proliferation characteristic of Ph’-positive leukemia.

II. The Molecular Characterization of the Philadelphia Chromosome

A. STRUCTURAL ALTERATIONS Deciphering the molecular consequences of the Philadelphia chromosome translocation has provided substantial evidence to implicate an activated c-abl gene in the etiology of Ph’-positive human leukemia. Early studies of rodent-human somatic cell hybrids made with leukemic cells containing the Philadelphia chromosome demonstrated the translocation of human c-abl from chromosome 9 to the Philadelphia chromosome (de Klein et al., 1982).Fine-structure mapping of the c-abllocus in CML tissue from several patients established that the breakpoints occurred 5‘ to the body of the abl gene, and in one patient occurred within 14.5 kb of the v-abl homologous human sequences (Heisterkamp et al., 1983). Cloning and analysis of genomic DNA corresponding to the breakpoint in this patient’s DNA established that chromosome 22 sequences were fused immediately upstream of the c-abl gene. Although the 5’ extent of the c-abl gene was unknown at that time, these results implied that CML breakpoints would indeed interrupt the c-abl locus. As much as 40 kb of human genomic DNA that mapped 5’ to the c-abl sequences was cloned, but probes derived from that genomic DNA failed to detect additional breakpoints in other CML patients’ tissue. Interestingly, probes derived from the chromosome 22 side of the Philadelphia chromosome breakpoint detected rearrangements in virtually all CML tissues tested (Groffen et al., 1984). The availability of DNA probes from the Philadelphia chromosome breakpoint established that the breakpoints on chromosome 9 occurred at variable and sometimes great distances (hundreds of kilobases) upstream of the c-abl locus. The great variability of breakpoints on chromosome 9 explains why few rearrangements can be detected in tumor DNAs by Southern analysis using abl probes, which span only the 3’ end of this large gene. Unlike the variability of breakpoints on chromosome 9, the breakpoints on chromosome 22 occurred in a restricted cluster defined by a small (5.8 kb) BglII fragment. Hence, this genomic region on chromosome 22 was termed the breakpoint cluster region, or bcr. The molecular cloning of genomic DNA from the breakpoint region

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of the Philadelphia chromosome brought to light the noveljuxtaposition of chromosome 22 bcr sequences with chromosome 9 c-abl sequences. CML cells containing this hybrid genetic locus express two novel products: an altered abl-related mRNA of around 8 kb (Collins et al., 1984; Gale and Canaani, 1984), larger in size than the normal c-abl mRNA (5.3 and 6.5 kb in the mouse, 6 and 7 kb in the human) (Wang and Baltimore, 1983), and an altered form of the c-abl-encoded protein with a molecular mass of 2 10 kDa, larger than the normal c-abl-encoded protein of 145 Da (Konopka et al., 1984). The molecular cloning of the cDNA corresponding to the 8-kb abl-related mRNA of CML cells revealed the relationship of these novel products to the hybrid bcrlabl genomic locus: the altered c-abl mRNA was a fused transcript consisting of both bcr and abl sequences (Grosveld et al., 1986; Shtivelman et al., 1985; Stam et al., 1985). Antibodies raised against synthetic peptides predicted by the bcr and abl sequences immunoprecipitated the altered abl-encoded protein of CML cells and confirmed its hybrid nature (Ben-Neriah et al., 1986b). Figure 1 provides a schematic representation of the chromosomal and genomic structures and the novel fusion products of the Philadelphia chromosome. These results provided firm evidence that the gene product of the c-abl locus in CML cells was indeed structurally altered.

B. UNDERSTANDING THE ROLEOF THE bcrlabl GENEIN LEUKEMOGENESIS Molecular analysis established that mutation of c-abl was at the heart of the Philadelphia chromosome translocation, but these studies could not define the biological nature of the bcrlabl hybrid or its precise role in the initiation or maintenance of leukemia. To address these central issues, answers were needed to the following questions: Does bcrlabl act as a dominant transforming oncogene in the manner of v-abl? Can bcrlabl act to induce leukemia in a suitable animal model system? Recent advances seem to have answered these questions, but fundamental problems still remain: What are the contributions of bcr and abl to the transforming activity of the hybrid protein? What role does bcrlabl play in determining the phenotype of the leukemias with which it is associated? Before we focus on bcrlabl, we will review briefly some features of the viral oncoprotein v-Abl, its normal cellular counterpart c-Abl, and what is known about the mechanisms by which c-abl can be mutated to acquire transforming activity. Ultimately, we will return to discuss the data that firmly implicate bcrlabl in the pathogenesis of Phl-positive leukemia.

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abl 9

3‘ Ib

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Ia

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-0

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7.0-kb mRNA 185-khproteln

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-

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FIG. 1. Chromosomal and genomic structures involved in the generation of the Philadelphia chromosome. The Philadelphia chromosome results from a reciprocal translocation of distal segments of chromosomes 9 and 22. The chromosomal structures are depicted at the left with the corresponding genomic structures on the right. The abl gene maps to band q34 of chromosome 9, and the bcr gene to band q l 1 of chromosome 22. The exact exon-intron structures for bcr and abl have not been completely defined, thus the genes are depicted schematically.Both genes have exceptionally large first introns, with that of abl being at least 200 kb and that of bcr at least 68 kb (these distances are represented by the hash marks). Breakpoints of the Philadelphia chromosome map over a large stretch of genomic DNA upstream of the body of the c-abl gene on chromosome 9. In Ph’-positive cases of CML, breakpoints on chromosome 22 cluster within a short stretch of genomic DNA, denoted the breakpoint cluster region, or bcr, from which the corresponding bcr gene derives its name. In Ph’-positive acute leukemias, the breakpoints on chromosome 22 map within the large first intron of bcr. The Philadelphia chromosome in both acute leukemia and CML produces a fusion bcrlabl mRNA and protein. The Bcr/Abl protein in acute leukemias is an internally truncated version of that in CML. The genomic counterpart of 9qf is not shown. (Adapted from Adams, 1985.)

Ill. From v-abl to c-abl and Back Again

A. THEABELSON MURINELEUKEMIA VIRUS The elucidation of the role of Abl proteins in leukemia harks back some 20 years to the isolation of the Abelson murine leukemia virus. A-MuLV arose in a prednisolone-treated mouse infected with the Moloney murine leukemia virus (Abelson and Rabstein, 1970). Unlike typical Moloney disease, which involves the thymus and arises after a long latency period (4-6 months), Abelson disease is a nonthymic lymphoma that arises after a brief latency period (3-4 weeks). A-MuLV is defective for viral replicative functions and requires the presence of the Moloney helper virus (Scher and Siegler, 1975; Shields et al., 1979). The target cell for A-MuLV infection appears to be an early cell of the lymphoid lineage

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(Tidmarsh et al., 1989).Lymphoid cells transformed by the Abelson virus are representative of the early stages of B cell differentiation prior to immunoglobulin light-chain gene rearrangement (Alt et al., 1981; Whitlock et al., 1983). A-MuLV is capable of transforming a variety of other hematopoietic cell types under defined experimental conditions, including mast cells (Pierce et al., 1985), macrophages (Raschke et al., 1978), and T cells (Cook, 1982). In culture, A-MuLV infection can abrogate the requirement for the multipotential colony-stimulating factor interleukin-3 in factor-dependent cells of both myeloid and lymphoid lineage (Chung et al., 1988; Cook et al., 1985; Mathey-PrCvot et al., 1986; Oliff et al., 1985; Pierce et al., 1985). A-MuLV can also transform fibroblast cell types in vitro (Scher and Siegler, 1975). A-MuLV carries the v-abl oncogene, a fusion of retroviral gag sequences with c-abl sequences derived from the mouse genome (Goff et al., 1980). T h e Gag/v-Abl fusion protein has a tyrosine-specific protein kinase activity that is indispensible for its capacity to transform cells. Temperature-sensitive mutants in the kinase function are conditional for transformation (Engelman and Rosenberg, 1987; Kipreos et al., 1987; Takemori et al., 1987); likewise, mutants defective in kinase activity are transformation defective (Rees-Jones and Goff, 1988; Witte et al., 1980). B. FUNCTIONAL DOMAINS OF THE c-Abl PROTEIN The identification of the Abelson virus provided a means to characterize the normal mouse abl gene. Using probes derived from cloned Abelson virus DNA, workers in the field isolated the normal c-abl gene and began to study its products. They found that the c-abl gene is expressed as two distinctively sized mRNAs in virtually all tissues (Ben-Neriah et al., 1986a; Renshaw et al., 1988; Wang and Baltimore, 1983). In the mouse, the two mRNA sizes correspond to at least four mRNA species (types I through IV) that carry distinct 5‘ sequences derived from alternative first-coding exons. Only the murine mRNA types I and IV are conserved in humans (their homologues are called types Ia and Ib, respectively). These two principal mRNAs are derived from alternative promoters (Bernards et al., 1988; Shtivelman et al., 1986).The levels of expression of the type IV mRNA are constant among tissues, but the type I mRNA varies in abundance (Renshaw et al., 1988). Each of the alternative 5’ exons is spliced onto a common body of c-abl exons, the first of which is called the “common exon,” or abl exon 2. The alternative cDNAs predict two predominant forms of abl-encoded protein with distinct N termini and presumably different functions (Ben-Neriah et al., 1986a; Shtivelman et al., 1986). The full-length cDNA clones for the c-abl-encoded

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proteins have been expressed, and recent work has begun to define various functional domains of c-Abl. The N-terminus of c-Abl type IV (type IB in humans) contains a consensus myristoylation sequence (with glycine at position 2 and lysine at position 7; Towler et al., 1987). As shown convincingly for the viral and cellular Src proteins, modification of the N-terminus by addition of a myristoyl fatty acid moiety is responsible for the association of the Src protein with the plasma membrane (Krueger et al., 1980; Cross et al., 1984; Kamps et al., 1985; Pellman et al., 1985). The c-Abl type IV protein is myristoylated (Jackson and Baltimore, 1989), and myristoylation is responsible for targeting at least a portion of the protein to the plasma membrane (Van Etten et al., 1989). The c-abl gene is a member of the family encoding the nonreceptor class of tyrosine kinases (Hunter and Cooper, 1985). These tyrosinespecific kinases share extensive homology throughout the domain responsible for catalytic activity, a domain called SH 1, signifying the primary region of Src homology (Pawson, 1988). Along with homology, mutations that yield kinase-defective proteins have helped to delineate the kinase domain (Prywes et al., 1983, 1985a; Rees-Jones and Goff, 1988). Certain members of the kinase family share homology beyond the kinase domain. The c-abl gene bears homology to the src family of genes over a stretch of sequence that lies N terminal to the kinase domain (Ben-Neriah et al., 1986a). These domains appear to be involved in regulation of the kinase function in nonreceptor tyrosine kinases and have been denoted SH2 and SH3 (for Src homology regions 2 and 3) (Pawson, 1988). The SH2 domain appears to positively regulate the kinase and transforming activity of this class of proteins, as several mutants in SH2 have normal or decreased kinase activity and are transformation defective (Prywes et al., 1985a; Sadowski et al., 1986). T h e SH3 domain plays a negative regulatory role. Mutations in the SH3 domain result in activation of the protein kinase function, as evidenced by the increased specific enzymatic activity and appearance of phosphotyrosine on the protein in vzvo. SH3 mutations activate the transforming capacity of src and abl (Kato et al., 1986; Sadowski et al., 1986; Potts et al., 1988; Jackson and Baltimore, 1989). Interestingly, the SH2 and SH3 domains are found in an avian retroviral oncogene, v-crk, which lacks a kinase domain (Mayer et al., 1988).v-Crk may work by modulating the function of a cellular tyrosine kinase that is found in association with v-Crk in cells. Alternatively, v-Crk may function by competing for regulatory factors that negatively modulate the activity of tyrosine kinases, or may serve to bridge SHS-containing enzymes to their substrates. The SH3 domain is also found in a variety of other proteins, including phospholipase C y

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(Stahl et al., 1988) and the GTPase-activating protein (GAP) (Vogel el al., 1988), suggesting that a class of regulatory proteins that interacts with SH3 domains may integrate diverse pathways of signal transduction. The SH3 domain is present in spectrin (Lehto et al., 1988) and in an actinbinding protein in yeast (Drubin et al., 1990), suggesting a possible link between the cytoplasmic tyrosine kinases and elements of the cytoskeleton. The presence of a consensus myristoylation signal at the N terminus of c-Abl type IV distinguishes it from type I and suggests that the alternative c-Abl protein types might differ in subcellular localization. Localization studies reveal that c-Abl proteins have a complex distribution. A portion of the type IV c-Abl protein associates with the plasma membrane, and some is found in association with actin microfilaments or diffusely throughout the cytoplasm; however, the majority of type IV c-Abl protein is found in the nucleus (Van Etten et al., 1989). The membraneassociated fraction of type IV c-Abl protein is myristoylated, but not all myristoylated proteins are membrane bound (Buss et al., 1984; Magee and Courtneidge, 1985; Olson and Spizz, 1986), and it is not yet known whether the nuclear fraction of Abl protein is myristoylated. A combination of metabolic labeling with [‘H]myristate and subcellular fractionation is needed to address this issue. These experiments have been exceedingly difficult to perform because of the low efficiency of incorporation of the [3H]myristate label into Abl proteins, and the tendency of Abl protein to be easily extracted from the nucleus during cell disruption (G. Q. Daley, unpublished observations). The c-Abl type I V protein is too large to diffuse passively through the nuclear pores, and thus must actively accumulate in the nucleus. The sequences responsible for targeting the c-Abl protein to the nucleus have been mapped using an immunofluorescence assay to localize mutant c-Abl proteins expressed transiently in COS cells (Van Etten et al., 1989). Mutation of a C-terminal pentalysine motif disrupts the accumulation of c-Abl protein in the nucleus. The KKKKK motif is reminiscent of the canonical nuclear localization signals of other nuclear proteins, such as SV40 large T (Kalderon et al., 1984). The distribution of the type I protein is still under investigation. It appears to be highly unstable and thus is difficult to express at levels high enough to analyze easily. Unlike the other members of the nonreceptor tyrosine kinases that have carboxy-terminal catalytic domains, the kinase domain of c-Abl occurs within the N-terminal half of the protein (Hunter and Cooper, 1985; Reddy et al., 1983). The distinctive large carboxy-terminal domain of c-Abl is encoded in a single exon, and its function is poorly defined. Deletion of this domain does not activate a transforming function for

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c-Abl (P. K. Jackson and D. Baltimore, unpublished results). However, the presence of this domain does appear to enhance the capacity of A-MuLV to transform pre-B cells in vitro and to cause lymphoid disease in vzvo (Murtagh etal., 1986;Rosenberg and Witte, 1988).The C terminus is also implicated in the toxicity of Abl protein products in fibroblast cell types (Goff et al., 1982;Ziegler et al., 1981),which makes it difficult to overexpress nontransforming variants. The presence of this unique domain may differentiate Abl function from the other members of the tyrosine kinase family.

C. ACTIVATION OF THE c-abl PROTOONCOGENE Given the sequence of c-Abl and several transforming counterparts, and information regarding the function of particular domains within the Abl protein, can we understand in what ways c-Abl can be mutated to acquire transforming activity? For the transforming Abl variants that arose by retroviral transduction, recombination of c-abl with either feline or murine retroviral gag sequences yielded a new N terminus for the Abl protein (Reddy et al., 1983;Laprevotte et al., 1984).In the case of AMuLV, recombination of murine c-abl with the parental Moloney virus gag gene sequences occurred within the third exon of the c-abl gene (Fig. 2)(Wang et al., 1984; Ben-Neriah et al., 1986a),and in the independent retrovirally transduced abl gene isolated from a feline fibrosarcoma (fv-abl) (Besmer et al., 1983),recombination of retroviral gag sequences occurred into the second exon of the c-abl gene (Fig. 2) (Bergold et al., 1987). Which alteration is critical-the addition of the novel gag sequence, or the deletion of the N-terminal c-abl sequence? This question can be answered by analyzing the transforming properties of various mutations in c-Abl that mimic v-Abl structure. Overexpression of the type IV c-Abl protein fails to transform NIH 3T3 cells (Franz et al., 1989; Jackson and Baltimore, 1989),but attaching gag sequences to c-abl in a manner that approximates v-Abl protein structure results in a transforming protein (Ben-Neriah and Baltimore, 1986);thus, recombination with gag is sufficient to activate the transforming potential of c-Abl. However, simple deletion of sequences corresponding to the SH3 domain, or linker insertion between the SH3 and SH2 domains, will activate c-abl in the absence of gag sequences (Franz et al., 1989;Jackson and Baltimore, 1989).Therefore, gag sequences are not required for transforming function. Taken together, these results suggest that alterations of the c-Abl N terminus are critical to the activation of the transforming potential of Abl proteins, most likely due to the disruption of the negative regulatory function of the SH3 domain.

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KKKKK

SH3 SHZ

KINASF

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I

I Ib

I

I Ia

P 1 4 5 c-abl

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Ph+ CML bor

I Uu

Ph+ ALL

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J

P I 8 5 bcr/abl

-mmmrmm myrislate

abl exon 2

abl exw 3

FIG. 2. The structure of Abl proteins associated with disease. The c-Abl protein has appeared in an oncogenic form in four distinct contexts: in two retrovirally induced malignancies, a murine lymphoma (the v-Abl protein of Abelson murine leukemia virus) and a feline fibrosarcoma (the fv-Abl protein of Hardy-Zuckerman feline sarcoma virus); and in two human leukemias associated with the Philadelphia chromosome, chronic myelogenous leukemia (the P210 Bcr/Abl protein) and acute leukemia of either myeloid or lymphoid lineage (the P185 Bcr/Abl protein). The structures of the various activated Abl proteins are shown schematically along with the normal protein types for comparison. Human type Ia corresponds to murine type I, and human type Ib corresponds to murine type IV.

Activation of the c-Abl transforming potential does not necessarily entail deletion or mutation of N-terminal SH3 sequences. Indeed, c-Abl can be a transforming protein while retaining the SH3 domain. The gag fusion in the fv-Abl protein preserves the SH3 domain (Bergold et al., 1987), and the attachment of retroviral gag sequences upstream of the SH3 domain will result in a fully transforming protein (Jackson et al., 1991). Likewise, the addition of retroviral gag sequences to the N terminus of Bcr/Abl creates a Gag/Bcr/Abl fusion protein that retains SH3 sequences, but is nonetheless fully capable of transforming NIH 3T3 fibroblasts (Daley et al., 1987). It appears that the addition of a bulky heterologous protein domain to the N terminus of Abl will result in a fusion protein with transforming activity, most likely because the heterologous domain folds in a manner that sterically disrupts the negative regulatory function of SH3. Point mutation within the kinase domain is yet another mechanism for activating the transforming potential of c-Abl (Jackson et al., 1991). Thus, c-Abl can be mutated to become a transforming protein in numerous ways: by deletion or mutation of critical regula-

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tory domains, by point mutations within the kinase domain, or by Nterminal fusion with bulky heterologous sequences. In this regard, Abl is similar to Src in being activated by a variety of mechanisms. Another similarity between the transforming nature of activated Abl and Src proteins is the requirement for N-terminal myristoylation for transformation of fibroblast cell types. It is well-established for transforming Src proteins that the attachment of an N-terminal myristoyl fatty acid is required for the association of the protein with the plasma membrane and for transforming function. Mutants of Src that are defective for myristoylation d o not localize to the plasma membrane and will not transform fibroblasts (Cross et al., 1984; Kamps et al., 1985). Myristoylation also appears critical for the capacity of Abl proteins to transform NIH 3T3 cells (Prywes et al., 1983; Mathey-Prkvot and Baltimore, 1988; Daley et al., 1991). The normal c-Abl type IV protein is myristoylated, and activating N-terminal mutations must preserve the myristoylation function in order for the resulting protein to transform NIH 3T3 fibroblasts. In addition to displacing the SH3 domain, retroviral gag sequences in the v-Abl protein provide an N-terminal myristoylation site. Myristoylation-defective versions of v-Abl and the SH3 mutant of c-Abl fail to transform NIH 3T3 fibroblasts (Daley et aE., 1991). The myristoylation function targets a portion of the c-Abl protein to associate with the plasma membrane, but the majority of c-Abl type IV protein localizes to the nucleus. All fibroblast-transforming variants of Abl that have thus far been characterized are predominantly cytoplasmic in distribution, with a portion remaining at the plasma membrane (Van Etten et al., 1989). Alterations near the N terminus appear to disrupt the normal nuclear localization of Abl and result in redistribution of the protein to the cytoplasmic compartment. These cytoplasmic Abl proteins retain the pentalysine nuclear localization motif. The capacity of this motif to function as a nuclear-targeting signal must be facilitated or modulated by the N terminus. Cytoplasmic relocalization of the Abl protein alone is neither sufficient for transformation nor simply a consequence of the transformed state of the cell. Mutations in the pentalysine motif relocalize the c-Abl protein without activating its transforming function. Furthermore, NIH 3T3 cells that overexpress the c-Abl type IV protein in their nucleus can be transformed by superinfection with an H-ras virus, and in these transformed cells, the c-Abl protein remains primarily nuclear (Van Etten et al., 1989). Most transforming c-Abl variants share several features with v-Abl, including myristoylation (required for the transformation of NIH/3T3 fibroblasts), alterations at the N terminus (most likely acting to disrupt

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SH3 function), and a deregulated tyrosine-specific protein kinase activity. Whereas the endogenous c-Abl proteins lack phosphotyrosine in vivo, transforming variants can be recognized by the presence of phosphotyrosine in viva Transforming Abl variants cause an increase in total cellular phosphotyrosine content and have increased specific activities for certain exogenous substrates in vitro (Davis et al., 1985; Konopka et al., 1984). T h e correlation of the deregulated enzymatic activity with transformation is well established, but substrates relevant to transformation have been difficult to identify experimentally. Antibodies specific for phosphotyrosine residues detect potential substrates in cells transformed by Abl proteins (Huhn et al., 1987); however, establishing that phosphorylation of these substrates is either necessary or sufficient to induce transformation requires that the substrates be isolated and more fully characterized-a daunting and as-yet unrealized task. IV. abl in Human Malignancy

A. THEBcr/Abl PROTEINS OF PHI-POSITIVE LEUKEMIAS T h e presence of a Philadelphia chromosome characterizes the leukemic tissue in over 90% of patients with chronic myelogenous leukemia. Virtually all of these patients will harbor the molecular hallmark of CML, a detectable rearrangement of the bcr gene on chromosome 22 (for a review, see Kurzrock et al., 1988). The leukemic cells in these patients express an altered Abl protein of 210 kDa (P210) with structural and enzymatic properties reminiscent of v-Abl (Davis et al., 1985). The P210 protein is a N-terminal fusion of bcr-encoded sequences to c-abl-encoded sequences beginning at abE exon 2 (Fig. 2) (Konopka et al., 1985; Stam et al., 1985; Ben-Neriah et al., 1986b). A subgroup of patients for whom cytogenetic evidence of the Philadelphia chromosome is lacking have the molecular lesions typical of the Philadelphia chromosome: a rearranged bcr gene with juxtaposition of bcr- and abl-encoded sequences, and expression of the P210 Bcr/Abl protein (Bartram et al., 1985; Dreazen et al., 1988; Ganesan et al., 1986). The remaining few percent of patients who lack both cytogenetic and molecular evidence of the bcrlabljuxtaposition have a distinctly poor prognosis (Pugh et al., 1985). The etiology of the disease in this subgroup of patients may be distinct from that of typical CML. In addition to its association with CML, the Philadelphia chromosome has been observed in about 20% of adults and 5% of children with acute lymphoblastic leukemia (ALL), and 2% of adults with acute myeloid

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leukemia (AML; Look, 1985). About half of these patients harbor the same genomic rearrangement as that found in CML cells, and may represent patients with previously undiagnosed CML who present in blast crisis. The remainder of the patients with Ph*-positiveacute leukemias d o not show evidence of rearrangement within the breakpoint cluster region typical of CML (Erikson et al., 1986; van der Feltz et al., 1988). Leukemic cells from patients with Ph’-positive ALL express a novel abl-related mRNA of 7 kb and an abl-related protein of 185 kDa (called “190 kDa” by some groups), different from the 8-kb bcrlabl mRNA and P210 Bcr/Abl protein typical of CML cells (Chan et al., 1987; Clark et al., 1987, 1988; Kurzrocket al., 1987a; Walker etal., 1987).Cloningof the cDNA for this alternative Philadelphia chromosome product yielded the surprising result that the 7-kb mRNA and its P185 protein product were also a fusion of bcr and abl sequences (ar-Rushdi et al., 1988; Clark et al., 1988; Fainstein et al., 1987; Hermans et al.,1987). The mRNA encoding P185 splices the first exon of the bcr gene to exon 2 of the c-abl gene (Fig. l), resulting in a P185 protein with abl-encoded sequences identical to those in P210, but an internal deletion of the bcr-encoded sequences normally present in P2 10 (Fig. 2). The fact that P 185 is seen in some cases of Ph’-positive AML obviates the notion that P185 exclusively defines lymphoid disease (Kurzrock et al., 1987b). It may be that a particular Philadelphia chromosome breakpoint is generated preferentially in certain hematopoietic progenitors (Secker-Walker et al., 1988). To what extent the differences in the P185 and P210 proteins determine the distinct clinical nature of acute and chronic leukemia is a subject of much speculation and interest.

B. POSSIBLE CONSEQUENCES OF THE bcr FUSION TO abl T h e c-abl gene can be activated to leukemogenicity by a variety of mechanisms, including fusion with gag sequences, internal deletions, and point mutations (see Section 111,C). Given this, it is reasonable to expect that a variety of c-abl-activating mechanisms would appear in the context of human disease, However, only gene fusion of bcr and abl has been implicated in human leukemia thus far. Other mechanisms may account for the small fraction of CML patients for whom either cytogenetic and/or molecular evidence of bcrlabl is lacking. Subtle alterations in c-abl function through small deletions or point mutations may also account for some leukemias that show no evidence of a Philadelphia chromosome. A patient has been described whose leukemic cells showed evidence of bcr rearrangement without juxtaposition of c-abl (Bartram, 1985).The rearrangement in this patient may have juxtaposed bcr sequences with another tyrosine kinase with potential transforming activity, and one may

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speculate that the c-arg gene (Kruh et al., 1986), a close relative of c-abl, might be activated in this manner. Whether the Bcr portion of the altered Abl proteins is critical to the pathogenesis of leukemia is unknown. The almost perfect correlation of the P210 form of Bcr/Abl with chronic leukemia and the P185 form with acute leukemia suggests that the bcr domain is somehow uniquely suited to mediate the activation of c-abl to its oncogenic form. Two mechanisms might account for this: ( 1 ) the bcr domain is critical to the phenotype of the disease, and chronic myelogenous leukemia is the phenotypic consequence of the function of P210 Bcr/Abl, whereas acute leukemia, primarily of lymphoid type, is a phenotypic consequence of the function of P185; (2) the activation of the abl gene is paramount, and bcr is incidentally involved because the bcr locus happens to be a site of frequent recombination with the large first intron of abl on chromosome 9 (Bernards et al., 1987). The first hypothesis is intuitively more attractive, and Witte and his colleagues have presented data that suggest that the P210 and P185 forms of Bcr/Abl differ markedly in their transformation efficiency (see Section V). Moreover, the bcrlabl gene comes under the transcriptional control of the bcr promoter, and the mRNA appears to be more stable than the endogenous abl mRNAs (Collins et al., 1987).This is one mechanism that may explain the overexpression of the P210 protein, but is unlikely to explain the specificity of bcrlabl for promoting myeloproliferative disease. The bcr promoter is constitutively active in a wide variety of tissues and cell lines, including hematopoietic cell types (Collins et al., 1987). T h e addition of a bcr domain may confer on the Bcr/Abl protein aspects of the normal functions of the Bcr protein. Immunoprecipitation of the c-Bcr protein detects a 160-kDa protein (Amson et al., 1989; Dhut et al., 1988; Stam et al., 1987; Timmons and Witte, 1989) or two phosphoproteins of 160 and 190 kDa with associated serinei threonine kinase activity (Ben-Neriah et al., 198613; Li et al., 1989). c-Bcr may be tightly associated with a serine kinase, but is unlikely to be one itself. The sequence of the Bcr protein bears no homology to any of the known serine/threonine kinases (Hariharan and Adams, 1987; Heisterkamp et al., 1985; Mes-Masson et al., 1986). A motif matching a consensus ATP-binding site does occur in the first exon of bcr (Lifshitz et al., 1988), but it is not known whether this has functional significance. In order to fully understand the role of Bcr protein sequences in determining leukemia, various forms of the Bcr/Abl protein must be characterized in an animal model which suitably recapitulates the phenotype of the human disease (see Section VI). With such a model of CML, one can undertake a mutational analysis of the bcr domain in order

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to determine which, if any, bcr sequences are required for generating disease. T h e second hypothesis above is more difficult to test experimentally. A mechanism that promotes the recombination of the bcr and abl genes is unknown, but productive rearrangements may be favored because both genes contain unusually large first introns. The first intron of bcr is some 68 kb in size (Heisterkamp et aZ., 1988), whereas that of c-abl is at least 200 kb (Bernards et aZ., 1987), rendering the target for translocations especially large. In the Philadelphia chromosome of ALL patients, the breakpoints occur in the first intron of the bcr gene and tend to cluster in the 3’ half (Chen et al., 1989; Heisterkamp et al., 1988). Several Philadelphia chromosome breakpoints have been shown to involve homologous Alu sequences on chromosomes 9 and 22, but given that these cases are rare, and the A h repeats so abundant, homologous recombination is unlikely to be a general mechanism for generating the Philadelphia chromosome (Chen et aZ., 1989; Heisterkamp et aZ., 1988). The clustering of the breakpoints within the 5.8-kb CML breakpoint cluster region may be due neither to the presence of a recombination “hot spot” in this stretch of DNA nor to the preservation of a protein domain that is critical to the transforming function of bcrlabl. Rather, there is a favorable sequence of three exons in a row (Heisterkamp et al., 1985) with a splice pattern that preserves the reading frame when fused to the second exon of c-abl. T h e exons immediately upstream and downstream of the CML breakpoint cluster region would not splice in frame to the second c-abl exon, and thus would not produce a functional protein. This may explain the observed tendency for the breakpoints to “cluster.” The first exon of bcr will also splice in frame to the c-abl second exon. Perhaps the first exon of the bcr gene and those within the CML breakpoint cluster region are the only ones that can splice in frame to abl exon 2. We will be able to conclude more when the entire exon-intron structure of the bcr gene is available. The particular chromosomal topology for the bcr and abl genes in humans also accomodates the reciprocal nature of the translocations that give rise to the Philadelphia chromosome; both bcr and c-abl are oriented with their 5’ ends centromeric and their 3’ ends telomeric. The orientations of bcr and abl in mice may not allow for simple reciprocal exchange, which might explain why chronic myelogenous leukemia caused by bcrlabl does not arise spontaneously in mice. How might bcr activate the transforming potential of c-abl? IS the simple loss of the abl sequence sufficient to activate the transforming function of bcrlabl, or does the presence of the bcr domain provide some

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additional function necessary for transformation? The gag sequences of v-abl provide a myristoylation site critical for transformation of NIH 3T3 fibroblasts by A-MuLV. Moreover, gag sequences appear to enhance the stability of Gag/Abl fusion proteins in lymphoid cells, thereby facilitating their transformation (Prywes et al., 1985b). However, gag sequences are clearly not required for Abl proteins to transform lymphoid cells, as mutations in the SH3 domain of c-abl yield altered Abl proteins capable of inducing Abelson disease in mice (Jackson and Baltimore, 1989).T h e SH3 mutations mimic v-abl in enzymatic and biological activity and are presumed to define a domain of c-abl that regulates critical abl functions involving the tyrosine kinase activity. SH3 sequences may mediate the binding of factors that negatively modulate the kinase activity of c-Abl. Alternatively, the SH3 domain may regulate the kinase domain directly. Mutations that activate c-abl might disrupt the capacity of SH3 to bind accessory cellular factors or may negate its intramolecular regulatory role. The sequences defined by the SH3 mutation are maintained in the bcrlabl fusion, but the addition of a bulky N-terminal domain such as Bcr o r Gag may alter the function of SH3 by means of steric hindrance. The magnitude of this effect may be different for the P2 10 and P185 forms of Bcr/ Abl and may account for the differences in intrinsic kinase activity and transforming function of these two forms. Analyzing deletion mutants of bcr would be valuable for determining the critical determinants of bcr function. I n addition to deregulation of the enzymatic activity, alterations in the N terminus of c-Abl appear responsible for redistributing the c-Abl protein from the nuclear compartment to the cytoplasm (Van Etten et al., 1989). The P210 Bcr/Abl protein, like other activated forms of c-Abl (e.g., Gag/Abl or SH3 mutants) is principally cytoplasmic (R. A. Van Etten, G. Q. Daley, and D. Baltimore, unpublished data). The altered N-terminus of the Bcr/Abl protein may disrupt the nuclear localization of Abl, leading to the accumulation of P210 Bcr/Abl in the cytoplasm. The large heterologous Bcr domain may provide signals for relocalization of the c-Abl protein, redirecting the fusion protein to the normal cytoplasmic distribution of Bcr (Amson et al., 1989; Dhut et al., 1988). Alternatively, the Bcr domain may interfere with the nuclear translocation mechanism by masking the nuclear localization signal. However, no dominant function need be invoked for either Gag/Abl or Bcr/Abl fusion proteins, because simple deletion within the c-Abl N terminus is sufficient to redistribute the protein from the nucleus to the cytoplasm (as in the SH3 mutation) (Van Etten et al., 1989) (see also Campbell and Arlinghaus, this volume).

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C. EVALUATION OF THE INTRINSIC ENZYMATIC ACTIVITY OF Abl PROTEINS Both the P210 and P185 forms of the Bcr/Abl fusion protein have an activated tyrosine-specific kinase activity like that of v-Abl. T h e activated forms of c-Abl contain phosphotyrosine in vim, unlike the normal c-Abl proteins, which are not detectably phosphorylated on tyrosine in vivo, even though they have tyrosine kinase activity in vitro (Konopka and Witte, 1985; Ponticelli et al., 1982). Like v-Abl, the human leukemiaspecific Abl proteins demonstrate an enhanced specific activity both for autophosphorylation and for exogenous substrates in immune complex kinase assay (Konopka et al., 1984; Pendergast et al., 1987; Lug0 et al., 1990). T h e deletion or substitution of N-terminal c-Abl sequences alone is sufficient for the release of the c-Abl kinase from negative regulation (Wang, 1988; Franz et al., 1989;Jackson and Baltimore, 1989). However, the determination of specific enzymatic activity for these proteins may be complicated because of protein-protein interactions occurring in vivo, as well as in partially purified preparations (including immune complexes). Multiple phosphorylated protein species are detected upon immunoprecipitation of many cellular protein tyrosine kinases, and some of these have been recently identified. The platelet-derived growth factor (PDGF) receptor is found in association with phospholipase C y , PI-3 kinase, GAP, and perhaps with the c-raf protooncogene (Coughlin et al., 1989; Kaplan et al., 1990; Kazlauskas and Cooper, 1989; Kazlauskas etal., 1990; Meisenhelder et al., 1989; Molloy et al., 1989; Morrison et al., 1989). Immunoprecipitates of the Src and Fps cytoplasmic kinases contain at least two additional prominent phosphoproteins of 62 and 190 kDa (Elis et al., 1990). Immunoprecipitates of v-Abl and P210 associate with multiple phosphorylated proteins (Huhn et al., 1987), one of which is the PI-3 kinase (Varticovski et al., 1991). It is likely that some of these associated proteins are more than substrates or innocent bystanders in the immune complexes, but exist to modulate the enzymatic activity of the kinases in the immune complex kinase assay. Most likely, some of these associations occur in vivo and have a physiologic role in regulating kinase activity. The increased levels of phosphotyrosine in cells transformed by Abl proteins, and the existence of phosphotyrosine modifications on the Abl protein itself, have been regarded as evidence of enhanced intrinsic kinase activity of transforming Abl variants in vivo. However, no difference in intrinsic kinase activity need be postulated to explain these results. Structural changes in the Abl protein might affect one or more of the following: the association of accessory proteins that modulate the kinase function; the substrate specificity, perhaps serving to activate

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other kinases in the cell; o r the localization of the protein, thereby rendering the kinase accessible to novel, illegitimate substrates. Any of these could lead to the changes observed in cells transformed by the Abl kinase, including the increased phosphotyrosine content of the cells and the presence of phosphotyrosine modifications on the Abl protein.

V. Biological Activity of Bcr/Abl in Vitro Although it can cause a variety of hematologic malignancies under defined experimental conditions, the Abelson virus is best characterized as an agent that induces acute lymphoid malignancy upon passage in newborn mice (for a review, see Rosenberg and Witte, 1988). The Abelson murine leukemia virus exerts its transforming influence through the actions of the v-abl oncogene. The chimeric structure and enzymatic properties of the P210 and P185 forms of Bcr/Abl are similar to v-Abl, suggesting that Bcr/Abl also has leukemogenic potential. P2 10 Bcr/Abl is associated with chronic myelogenous leukemia whereas P185 Bcr/Abl is most often found in acute lymphoid malignancies that more closely resemble Abelson virus-induced disease in mice. T h e distinct disease phenotypes may result from the nature of the proteins. Alternatively, the disease may merely reflect the particular lineage in which the fusion protein arises. T o directly assess the biological activity of the Bcr/Abl proteins, both forms of the Bcr/Abl fusion protein have been expressed in different cell types. These studies, reviewed here, are beginning to determine the similarities and differences of v-Abl and Bcr/Abl function. OF bcrlabl IN FIBROBLAST CELLTYPES A. EXPRESSION

The v-abl gene causes the morphologic transformation of N I H 3T3 cells in culture (Scher and Siegler, 1975). Surprisingly however, expression of cDNAs encoding the P210 Bcr/Abl protein in NIH 3T3 cells fails to transform them (Daley et al., 1987; Lug0 and Witte, 1989). This result was the first indication of a difference in the biological activities of v-Abl and P210 Bcr/Abl. The difference is most likely due to the lack of a N-terminal myristoyl fatty acid moiety on the BcrlAbl protein. Recombination of Bcr/Abl with helper retroviral sequences creates a transforming variant of Bcr/Abl that has acquired N-terminal Gag sequences, and is thus myristoylated (Daley et al., 1987). This Gag/Bcr/Abl protein is like v-abl in that it efficiently transforms NIH 3T3 cells in culture, causes Abelson disease in mice, and associates in part with the plasma membrane (G. Q. Daley, R. A. Van Etten, and D. Baltimore, unpublished).

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Membrane association of myristoylated Src proteins is determined by a specific membrane receptor that recognizes the N-terminal myristoyl fatty acid (Resh, 1989). The myristoylation modification is critical for transformation of chick embryo fibroblasts by the src oncogene (Cross et al., 1984; Kamps et al., 1985; Pellman el al., 1985).Membrane association also appears to be critical for transformation of NlH 3T3 fibroblasts by Abl protein variants (see Section II1,C) (Daley et al., 1991).Mutants in the myristoylation function are transformation defective in both systems. The nonmyristoylated Bcr/Abl protein, unlike the GagiBcrlAbl protein, presumably cannot bind the putative myristate receptor. It therefore remains entirely cytoplasmic in distribution and does not transform K I H 3T3 cells. The inability of cytoplasmic forms or Src or Abl to transform fibroblasts suggests that critical substrates reside at the plasma membrane, where they are less accessible to the nonmyristoylated forms. These substrates could be responsible for mediating adherent cell properties such as anchorage dependence of cell growth and contact inhibition, and may include the integrins (Hynes, 1987) or other components of cell substratum attachment sites (Burridge et al., 1988). Furthermore, the lack of membrane association may sequester the Abl protein away from membrane-associated signal transduction systems through which the Abl kinase might act. Abl proteins activate a phosphatidylinositol kinase with specificity for the 3' carbon of the inositol ring (PI-3' kinase), and lipid kinase activity can be demonstrated in immunoprecipitates of cell extracts using antisera directed against Abl determinants (Varticovski et al., 1991). Steady-state levels of the 3'-phosphorylated lipid, PIP3, correlate with the mitogenic activity of the cell. PIP3 levels are elevated only in NlH 3T3 cells transformed by myristoylated, membrane-associated Abl variants, but not in nontransformed cell lines expressing nonmyristoylated, cytoplasmic forms of Abl. By lacking membrane association, the PI-3' kinase associated with nonmyristoylated Abl variants may not have access to phospholipid substrates. Although the P210 Bcr/Abl protein does not transform NIH 3T3 fibroblasts, it can partially transform another fibroblast cell line, Rat- 1 (Lug0 and Witte, 1989). Stable expression of P210 BcriAbl in Rat-1 fibroblasts causes a subtle morphologic transformation. The cells form small colonies when plated in soft agar (implying rather indolent anchorage-independent growth) and form well-encapsulated, noninvasive tumors in nude mice, These effects are enhanced by coexpression of the v-my oncogene, which cooperates in producing cells that exhibit a floridly transformed phenotype: they form large colonies in soft agar (implying robust anchorage-independent growth) and yield aggressive, invasive tumors in nude mice. Coexpression of P210 Bcr/Abl and v-Myc does not

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cause transformation of NIH 3T3 cells. The absence of a transformed phenotype in NIH 3T3 cells could have several explanations. Rat-1 cells may be more tolerant of higher levels of expression of the normally toxic P2 10 Bcr/Abl protein, or may have a lower threshold of protein expression required for eliciting certain features of the transformed phenotype. A precedent for species-specific transformation exists in the human Fps/ Fes kinase, which is transformation-defective in rat cell lines because of a restricted kinase activity (Greer et al., 1988). In comparison to P210, the PI85 Bcr/Abl protein is more efficient at transforming Rat-1 cells (Lugo et al., 1990), though both contain identical Abl sequences and differ only in the size of their N-terminal Bcr moiety. Rat- 1 cells that express P185 Bcr/Abl grow more readily in an anchorageindependent manner. T h e P185 Bcr/Abl protein appears to have a higher kinase activity than P210 Bcr/Abl as assayed by autophosphorylation activity or phosphorylation of exogenous substrate, using protein that is either translated in nitro or expressed transiently in Rat-1 cells (Lugo et al., 1990). It is tempting to speculate that the enhanced transformation efficiency of PI85 Bcr/Abl for Rat- 1 fibroblasts (which correlates with the apparently higher activity of the P185 Bcr/Abl kinase) may underlie the association of the P185 Bcr/Abl protein with more aggressive acute leukemias. B. TRANSFORMATION OF HEMATOPOIETIC CELLLINES B Y Abl PROTEINS In addition to transforming fibroblast cell types in nitro, the Abelson virus can transform a variety of hematopoietic cell types both in uitro and in nzuo, including hematopoietic cell lines dependent on interleukin-3 for growth and proliferation (Chung et al., 1988; Cook et al., 1985; MatheyPrevot et al., 1986;Oliff et al., 1985; Pierce etal., 1985; Rovera et al., 1987). These studies demonstrate that v-Abl will transform cells of diverse myeloid and lymphoid character to growth factor independence and tumorigenicity. Growth factor independence in these systems does not result from the autocrine production of growth factors by transformed cells but appears to result from a constitutive proliferative signal provided by the Abl kinase. Transformation of an IL-3-dependent myeloid line by a temperature-sensitive mutant of v-Abl is reversible and temperature dependent, suggesting that the IL-3-independent phenotype induced by v-Abl requires the continuous function of the v-abl gene product (Kipreos and Wang, 1988). Myristoylation is not required for transformation of hematopoietic cells by Abl proteins. Myristoylation-defective v-Abl and c-Abl SH3 mutants are capable of

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efficiently transforming the IL-3-dependent hematopoietic cell line Ba/F3 (Daley et al., 1991). Cells already established in cell culture may be more susceptible to transformation than are primary tissues. Transformation of hematopoietic cells from primary bone marrow with Abelson virus involves an initial period when cells remain dependent on feeder layers of bone marrow stromal cells or cloned feeder cell lines. During this period, additional genetic events are likely to occur to obviate the need for the supportive feeder cells, and the result is a fully growth factor-independent, tumorigenic phenotype (Rosenberg and Witte, 1988;Wong et al., 1987).Transformation of growth factor-dependent hematopoietic cell types is a property of the P210 and P185 BcrlAbl proteins (Daley and Baltimore, 1988; Hariharan et al., 1988; McLaughlin et al., 1989). These results establish that bcrlabl can function in the manner of a dominant transforming gene in lymphoid and myeloid cell types but not in fibroblasts. The dominant transforming effect of the nonmyristoylated P185 and P210 Bcr/Abl proteins as well as myristoylation-defective mutants of v-Abl for hematopoietic cell types suggests that whereas transformation of fibroblasts is mediated through membrane-associated substrates, critical substrates for hematopoietic cell transformation are localized elsewhere. Contrary to established cell lines transformed by Bcr/Abl, Ph'positive hematopoietic progenitors in CML patients remain dependent on growth factors (Champlin and Golde, 1985).That Bcr/Abl will render established hematopoietic cell lines growth factor independent and tumorigenic in a single step likely depends on the preexistence of cooperating mutations in these immortalized cell lines. Studies involving cell lines already established for growth in culture may not accurately reflect the biological activity of Bcr/Abl in primary tissue. When explanted bone marrow is plated under conditions for longterm marrow culture of either the B lymphoid or myeloid lineage, infection with retroviruses encoding P2 10 Bcr/Abl results in a clonal expansion of cells that express P210 (McLaughlin et d., 1987;Young and Witte, 1988).Interestingly, only immature B lymphoid cells grow out, even in myeloid-enriched culture conditions. In these experiments, the target cell of infection and stimulation by the P210 retrovirus appears similar to that transformed by the Abelson virus. Although P210 promotes the proliferation of cells in primary bone marrow culture, the clones of cells remain dependent on the feeder layer and are not fully tumorigenic. Some clones that express P2 10 progress to tumorigenicity upon continuous in vitro culture, suggesting that additional mutational events must occur to produce the fully tumorigenic phenotype. The evolution of the malignant phenotype in these clones may reflect the process of progres-

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sion that occurs in the human disease. Comparison of the expression of P210 versus P185 in the long-term marrow culture suggests that P185 promotes more vigorous proliferation of the immature lymphoid clones, but the cells nonetheless require maintenance in culture before acquiring a fully tumorigenic phenotype (McLaughlin et al., 1989). In cultures infected with retroviral stocks of equivalent titer, cells infected with virus carrying the P185 gene tend to outgrow those infected with viruses specifying P210. In this assay, the P185 protein provides a stronger growth stimulus than does P2 10. These provocative results establish that the two forms of the Bcr/Abl protein can be distinguished from one another in biological activity, and suggests that this difference underlies the association of the two forms with distinct human leukemias. Modifications of the standard WhitlockiWitte culture conditions have allowed for the maintenance of very early lymphoid progenitor cell lines that maintain their capacity to differentiate into immunoglobulinproducing cells in vivo (Scherle et al., 1990). These clonal lines have been infected with a retrovirus specifying the P2 10 Bcr/Abl protein, and grow to higher density than do uninfected control cultures. Nevertheless, they maintain their full differentiative function. In these lymphoid progenitor cells, as in the myeloid progenitors of CML, expression of the P210 Bcr/Abl protein does not block the differentiation program of the cells. The clonal lymphoid lines progress to tumorigenicity only after extended passage in culture, presumably allowing for the accumulation of mutations in other oncogenes that are needed to cooperate with Bcr/Abl to render a fully tumorigenic phenotype. Establishing similar conditions for the analysis of primitive myeloid progenitors would likely provide an excellent in vitro system for discerning the effects of Bcr/Abl on myeloid cell proliferation.

VI. Animal Models of Ph'-Positive Leukemias Studies in various in vitro culture systems have shed light on the capacity of bcrlabl to promote hematopoietic cell proliferation. However, none of the tissue culture models have demonstrated effects of bcrlabl on primary myeloid cell types, and none have faithfully modeled the interesting and complex biology of CML. To approach this, several labs have attempted to express the bcrlabl gene in vivo in order to assess the biological effects of the fusion gene in a variety of hematologic contexts. T w o distinct strategies have been employed: the creation of transgenic strains of mice that carry the bcrlabl gene, and the transduction of bcrlabl into murine bone marrow cells using retroviral vectors and bone marrow transplantation. The results of these studies establish unequivocally that

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the bcrlabl gene can exert a potent transforming effect on a variety of hematopoietic lineages, and provide valuable model systems for studying the biology of bcrlabl-induced disease. A. TRANSGENIC MODELSOF bcrlabl-INDUCED LEUKEMIA Adams and co-workers established transgenic strains of mice carrying a facsimile of the human bcrlabl gene by fusing bcr to v-abl sequences (Hariharan et al., 1989). T h e bcrlv-abl gene in these animals was expressed under the control of either the imunoglobulin heavy-chain enhancer or the relatively tissue-non-specific long terminal repeat of the myeloproliferative sarcoma virus (MPSV). The transgenic bcrlv-abl mouse strains show a predisposition to develop clonal lymphoid tumors primarily of the T cell type, but no tumors of myeloid lineage were observed. One might have predicted lymphoid disease to arise because of the tissue-specific expression of bcrlabl as directed by the immunoglobulin enhancer; however, the appearance of lymphoid disease in the context of expression from the MPSV long terminal repeat is rather perplexing. Prior data comparing the ability of activated src, fps, and abl oncogenes to transform lymphoid cells in YZVO suggested that lymphoid specificity may derive from the v-Abl kinase (Mathey-Prevot and Baltimore, 1988). I n all of the transgenic strains of mice, abl transgene expression is detected only in leukemic tissue, implying that transcription is activated by somatic mutations. There may be a selection against efficient expression of the abl transgene, owing to the potentially toxic effect of the expressed Abl kinase on embryonic development. Transgenic mice carrying the PI85 BcriAbl protein associated with Ph'-positive acute leukemias develop acute leukemias of lymphoid type in most instances, and in a few cases develop acute myeloid leukemias (Heisterkamp at., 1990). The promoter element in these animals was derived from the mouse metallothionein gene, and expression was noted in a variety of tissues (including muscle, brain, and spleen) without induction with heavy metals. However, only malignancies of hematopoietic tissues were observed, even though the bcrlabl gene appeared to be widely expressed in other tissues. This may imply some hematopoietic specificity to the transforming activity of the BcrlAbl protein. Although some of the mice in this study appeared to demonstrate varying degrees of differentiation toward granulocytes, the mice did not exhibit the granulocytosis chracteristic of CML. These experiments argue strongly for a specific role of P185 in the generation of acute leukemia, implying that the expression of this form of the BcrIAbl protein is incompatible with the

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normal differentiation program of lymphoid and myeloid hematopoietic progenitors typically seen in the chronic phase of CML. The available transgenic strains of mice provide interesting model systems for the study of acute lymphoid and myeloid leukemias, and firmly establish a causal link between the Philadelphia chromosomethrough the action of the bcrlabl gene-and leukemia. However, the transgenic strains do not mimic the complex biology of chronic myelogenous leukemia, in which the leukemic cells maintain their capacity to differentiate. Attempts to recapitulate CML in mice through the generation of transgenic strains are complicated by the choice of promoter used to express the bcrlabl transgene. In human CML, the hybrid bcrlabl gene is expressed under the control of the bcr promoter, which is not tissue specific (Collins et al., 1987). Use of tissue-nonspecific promoters to create transgenic mice may result in a bias against effective expression because of the potentially lethal effect of the Abl kinase during early fetal development. Indeed, in one study, the frequency of mice carrying the bcrlvabl transgene under the control of the MPSV long terminal repeat was much lower than for the immunoglobulin heavy-chain enhancer (3/7 1 versus 12/42pups resulting from microinjection) (Hariharan et al., 1989), and, in another, it is claimed that expression of the P185 BcrlAbl construct under the control of the tissue-nonspecific bcr promoter is lethal during embryogenesis (Heisterkamp et al., 1990).

B. KETROVIRALTRANSDUCTION OF bcrlabl BONEMARROW

INTO M U R I N E

T h e particular association of P2 10 BcrlAbl with CML may depend on the Philadelphia chromosome translocation occurring in the hematopoietic stem cell. Transgenic methodology for creating a strain of mice with a predisposition to CML may require the use of a promoter element with relative specificity for hematopoietic tissue. An alternative approach toward establishing an animal model for CML would be to introduce the bcrlabl gene into the hematopoietic stem cells of mice using retrovirally mediated gene transfer, and indeed this strategy has proved successful. A myeloproliferative syndrome much like human CML can be created by transplanting mouse bone marrow infected with a retrovirus specifying bcrlabl into lethally irradiated recipients (Daley et al., 1990).T h e resulting mice develop the cardinal signs of CML: high peripheral white blood cell counts with a differential increase in the mature granulocyte lineage, the presence of immature myeloid cell types in the peripheral blood, and splenomegaly. The bcrlabl provirus can be detected in DNA from the

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spleen or peripheral blood. Early myeloid cells from the bone marrow, spleen, and peripheral blood express the BcrlAbl protein as determined by immunofluorescence or immunoblotting. This system clearly demonstrates that the bcrlabl gene is capable of initiating a CML-like disease. However, in order for the disease in mice to represent a true model of human CML, certain criteria must be met. Because the translocation that generates the Philadelphia chromosome occurs in the hematopoietic stem cell, the bcrlabl retrovirus must target the pluripotential heniatopoietic stem cell, and thus be detected in pluripotential hematopoietic progenitors and in both lymphoid and myeloid lineages. Fourteen-day spleen colonies arising after transplantation of marrow from affected animals into secondary recipients show the same proviral integration site as the leukemic clone in the donor animal, suggesting that a primitive pluripotential cell equivalent to or possibly more primitive than the colony-forming unit of spleen (CFU-S) has been infected. Because the retrovirus used in these experiments is free of helper virus, the infected cell clones can be transplanted to secondary recipient mice without concern for the spread of the virus. Thus, the leukemic clone can be studied over time to investigate whether the clone will progress toward an acute leukemia, as in the human disease. Secondary recipients transplanted with marrow from primary animals with CML develop either the CML disease or acute lymphoid or myeloid variants that derive from the original leukemic clone. This further establishes that the retrovirus has gained access to a pluripotential hematopoietic stem cell. The progression of the myeloid chronic phase of the disease to a stage mimicking blast crisis in humans further strengthens the similarity of the mouse model to the human disease (Daley et al., 1991). Expression of the bcrlabl gene in murine bone marrow is sufficient to induce CML, and creates a mouse model that should facilitate further study of the disease. In a separate series of experiments using a similar bone marrow reconstitution protocol, Rosenberg, Witte, and their colleagues used retroviral stocks that contained helper virus to transduce the P2 10 bcrlabl and P160 v-abl genes into murine bone marrow (Kelliher et al., 1990). In these experiments, some animals infected with the bcrlabl retrovirus displayed a CML-like granulocytosis with pathologic features reminiscent of human CML. Others developed tumors of monocytic cells that more closely resembled human chronic myelomonocytic leukemia, which is not typically associated with the Philadelphia chromosome. Animals receiving marrow infected with the v-abl virus developed the myelomonocytic disease, and did not seem to exhibit the striking peripheral granulocytosis that characterizes CML. Thus, it is unclear whether the granulocytic lineage was truly involved in the myeloproliferative process induced by

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v-abl. Cell lines of both lymphoid and macrophage lineage were derived from these animals. Some of the cells had been infected to give rise to the myeloproliferative disease. Because of the presence of helper virus in this system, viral spread complicates the study of the transformed clones in secondary transplant recipients, but it introduces the interesting possibility that the helper virus might serve to promote disease progression to acute leukemia by acting as an insertional mutagen. It will be interesting to investigate more animals reconstituted with v-abl-infected marrow, to determine whether a CML-like granulocytosis can result from an activation of the Abl protein by domains other than bcr. CML arises in only a minority of mice reconstituted with bcrlablinfected bone marrow. Indeed, distinct malignancies involving lymphoid as well as other myeloid lineages predominate in the studies reported thus far (Daley et al., 1990; Elefanty et al., 1990; Kelliher et al., 1990). Cory’s group has analyzed the largest number of mice receiving retrovirally infected marrow (Elefanty et al., 1990). In their experiments, the bcrlabl retrovirus induced lymphoid, erythroid, macrophage, and mast cell tumors, establishing that the Bcr/Abl protein can transform a wide spectrum of hematopoietic cell types. Moreover, they observed marked differences in disease distribution and kinetics in different strains of mice (DBAl2 and C57BL/6), suggesting that host factors contribute to disease susceptibility. The same bcrlabl cDNA (172/2 15)(Mes-Masson etal., 1986; Daley et al., 1987) was used in the cloned retroviruses for all of the above experiments. Whereas all three of the above groups report transformation of both lymphoid and myeloid cell types by bcrlabl, Cory’s group did not observe mice with the classic hallmarks of chronic myelogenous leukemia. Strain susceptibility, subtle differences in retroviral structure or titer, or variations of experimental protocol may account for the different disease spectrum by affording bcrlabl expression in a distinct range of cells (e.g., early pluripotential stem cells versus lineagecommitted progenitors). Defining the target cell for the induction of specific diseases via the retroviral infection strategy could help to explain the association of certain translocations with lineage-restricted disease (Secker-Walker et al., 1988). Using purification techniques that enrich for the pluripotent hematopoietic stem cell (Jordan and Lemischka, 1990; Spangrude et al., 1988; Szilvassy et al., 1989), it should be possible to ask whether particular forms of the Abl protein determine a given disease phenotype when introduced directly into the stem cell. Will expression of the P185 Bcr/Abl protein that is typically found in the context of Ph’positive acute leukemia induce ALL o r CML? Likewise, defining lineagecommitted progenitors (Tidmarsh et al., 1989) and infecting these with retroviruses should address the transformation spectrum of various Abl

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proteins for particular lineages of hematopoietic cells. T h e transformation of a defined stem cell or progenitor cell type will provide a more precise system for evaluating the contribution of the transformed cell to hematopoiesis in a transplanted animal.

C. THEUTILITY OF THE ANIMAL MODELS T h e studies reviewed here establish bcrlabl as an oncogene capable of inducing a complex pattern of hematologic diseases, and provide direct evidence that bcrlabl is central to the pathogenesis of the Ph'-positive human leukemias. Murine model systems will be extremely useful tools for answering some of the questions that remain about the biology of Ph'-positive leukemia. One can carefully study the progression of the disease from chronic to acute phase, and test the influence of other oncogenes implicated in progression. Recently, rearrangements in the p53 gene have been detected in a significant proportion of Phl-positive patients in blast crisis, but only rarely in patients in the chronic phase (Ahuja et al., 1989; Kelman et al., 1989). The inactivation of p53, or the acquistion of dominant transforming mutations other than abl, may represent a second genetic event contributing to progression of the malignant clone. Interestingly, p53 maps to chromosome 17, and isochromy 17 is a frequent chromosomal feature associated with progression to blast crisis (Rowley, 1980). T h e role of p53 in promoting disease progression could be probed in the mouse system, where dominant transforming mutants of p53 could be engineered into a retrovirus for transduction into chronic phase bone marrow. Other oncogenes may be tested for their capacity to induce disease progression, or a random mutagenesis protocol taking advantage of Moloney virus as an insertional mutagen could be attempted. In this way, induced mutations could be tagged and rescued through molecular cloning. Through the use of inducible or conditional mutants of bcrlabl, animal models might also address whether bcrlabl is needed for the maintenance and progression of the disease. Chemotherapeutic regimens might be more readily tested for efficacy in mouse model systems. Inhibitors of tyrosine kinase function, such as the class of molecules called tyrophostins (Yaish et al., 1988), or differentiating agents, such as interferon-a (Yoffe et al., 1987) or bryostatin (Kraft et al., 1989) could be tried in the mice. Ultimately, the availability of animal model systems will greatly advance our understanding of the biology of CML, and holds promise for the development and testing of new therapies.

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ACKNOWLEDGMENTS We thank Richard Van Etten, Martin Scott, and Asne Bauskin for critical comments on the manuscript, and Peter K. Jackson, Naomi Rosenberg, and Owen Witte for communicating results prior to publication. Y.B.-N. holds a Career Development Award from the Israel Cancer Research Fund.

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ONCOPROTEIN KINASES IN MITOSIS David Shalloway' and Suresh Shenoy' Department of Molecular and Cell Biology, The Pennsylvania State University University Park, Pennsylvania 16802

I. Introduction A. Oncoprotein Kinases and the Cell Cycle B. Background 11. Protooncoprotein Tyrosine Kinases (PTKs) in Mitosis A. pp60"", a Cytoplasrnic/Mernbrane-AssociatedPTK B. Other Src Family PTKs C. ~ 1 5 0 ~ &a 'Cytoplasmic/Nuclear , PTK D. PTK Growth Factor Receptors E. Potential Targets of PTKs in M Phase 111. c-Mos, a SenneiThreonine Kinase, in M Phase A. c-Mos and Oocyte Maturation B. Interaction of c-Mos and MPF C. Transforming Mos Proteins and Fibroblast Mitosis IV. Phosphatases and M Phase V. Discussion A. Src, Differentiation, and Mitosis B. Relationships between M Phase Regulation, Gl/S Regulation, and Neoplastic Transformation C. Conclusion References

I. Introduction

A. ONCOPROTEIN KINASESAND THE CELLCYCLE Cancer is the preeminent aberration of animal cell cycle control. It reflects a violation of the rule observed by most normal cells: do not divide in the absence of specific growth or stimulatory factors. Normal growth arrest does not occur randomly, but rather at specific cell cycle restriction points. Most adverse conditions, such as deprivation of serum, nutrients, or substrate adherence, cause arrest at Gl/S restriction points that occur shortly before entry into S phase (Pardee, 1989),but growth arrest can also occur in G2 (Gelfant, 1977; Yaoi et al., 1972; Melchers and Present address: Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.

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DAVID SHALLOWAY AND SURESH SHENOY

Lernhardt, 1985; Durkin and Whitfield, 1987). These growth control mechanisms are defeated in neoplastically transformed cells. In this context, the central problem has been to determine the mechanism by which oncoproteins release cells from restriction point arrest. Some general observations suggest that oncoprotein kinases may play an important role: (1) because of their fast turnover, phosphorylation/ dephosphorylation reactions can provide the rapid control needed to regulate transient cell cycle events; (2) most peptide growth factor receptors involved in growth factor-induced release from Gl/S restriction are protooncogene-related tyrosine kinases (Ullrich and Schlessinger, 1990; (3) a large number of proteins are transiently phosphorylated during passage through mitosis and meiosis (Maller et a,L,1977; Westwood et al., 1985; Ozon et al., 1987); (4)~ 3 4 " ' ~ ,a critical regulatory element for passage through at least two domains of the cell cycle (see Section LB), is a kinase. Because resting but not neoplastically transformed cells are predominantly arrested in G1 or Go, most studies have focused on the function of protooncoproteins in serum- or growth factor-induced reentry into the cell cycle at the GI/S transition and on the ability of oncoproteins to bypass this restriction point. However, studies showing that microinjected v-src-encoded (Spivack et al., 1984) or v-ras-encoded (Birchmeier et al., 1985) proteins accelerate progesterone-induced meiotic maturation of X e n o w oocytes provided early hints that oncoproteins might be involved in regulation at the G*/M boundary. Recently, multiple lines of investigation have coalesced to demonstrate that oncoprotein kinases are temporally regulated and/or regulatory in mitosis and meiosis. This review will focus on the experimental basis for these new insights and the resultant shift in thinking about potential mechanisms of neoplastic transformation.

B . BACKGROUND Because both protooncogenes and the mechanisms that regulate cell cycle passage are highly conserved over a wide range of species, we will collate information garnered from a variety of experimental systems to uncover the underlying themes. Most oncogene studies have been conducted in mammalian and avian cells, but studies of the fission yeast cell cycle and of meiotic maturation of Xenopus luevis oocytes have provided apposite information. Genetic studies in fission yeasts have revealed a set of cdc genes that control the cell division cycle (Hayles and Nurse, 1986; Lee and Nurse, 1988; Nurse, 1990).The cdc2 gene product, the ~34"" serine/threonine

ONCOPROTEIN KINASES IN MITOSIS

187

kinase, is required for entry into mitosis in fission yeasts (Riabowol et al., 1989), and activation of p34cd'2 by an intricate regulatory network of other cdc-encoded proteins can trigger mitosis (Simanis and Nurse, 1986; Nurse, 1990). Expression of the cdcl3-encoded product, yeast cyclin, oscillates in the cell cycle and is responsible, in part, for regulating ~ 3 4 " ' ~ kinase activity (Moreno et al., 1989). Cyclin expression and p34'd'2 activation are required for the onset of mitosis, and the programmed destruction of cyclin is required for exit (Murray and Kirschner, 1989). ~ 3 4 " ' ~ also participates in regulation of another restriction point, "Start," just before entry into S phase (Lee and Nurse, 1988). Homologues of both ~ 3 4 " and ~ " cyclin ~ are found in all eucaryotes. The ~ 3 4 ' ~sequence "~ is highly conserved across a wide variety of species (Simanis and Nurse, 1986; Dunphy el al., 1988; Gautier et al., 1988; Riabowol et al., 1989; Arion et al., 1988; LabbC et al., 1988; Draetta and Beach, 1988; Morla et al., 1989; Draetta et al., 1989) and human ~ 3 4 " ~ " ~ can functionally replace its yeast counterpart (Lee and Nurse, 1987). In animal cells, as in yeast, ~ 3 4 " is ~ 'required ~ for progression through mitosis (Riabowol et al., 1989). Cyclin sequences are less well conserved, and multiple forms, all characterized by cell-cycle-dependent oscillations of their expression levels, have been identified in animal cells (Evans et al., 1983; Swenson et al., 1986; Westendorf et al., 1989; Minshull et al., 1989; Murray and Kirschner, 1989; Murray et al., 1989; Gautier et al., 1990). The most extensive studies of the interactions between ~ 3 4 " ' ~and cyclins have been conducted with X. Zaevis oocytes. Cultured Xenopus oocytes (in the absence of hormones) arrest at prophase of the first meiotic division (meiosis I). Progesterone induces them to complete meiosis I and to proceed to metaphase of the second meiotic division (meiosis 11). Meiosis 11 is completed after the cell cycle has been reinitiated by fertilization. These natural synchronizations have allowed meiotic maturation to be studied with temporal precision. Meiosis shares a number of biological features with mitosis (e.g., chromosome condensation, dissolution of the nuclear envelope, and cytokinesis), and it is believed that they are regulated by similar biochemical pathways (Sunkara et aE., 1979; Nelkin et al., 1980; Maller, 1987). Thus, interactions among regulatory factors in one system are likely to be operative in the other. We will refer to both without distinction as M phase. Progesterone-induced Xenopus oocyte meiotic maturation is accompanied by the appearance of M-phase-promoting factor (MPF) in the ooplasm (Masui and Clark, 1979; Meijer and Guerrier, 1984; Lohka, 1989). MPF is a dominant-acting agent whose microinjection is sufficient to induce oocyte maturation (Masui and Markert, 1971; Kishimoto and Kanatani, 1976; Maller and Krebs, 1980; Doree, 1982; Wasserman and

1 a8

DAVID SKALLOWAY AND SURESH SHENOY

Masui, 1976) and mitosis in embryo cells (Miake-Lyeet al., 1983; Halleck et al., 1984). Purified MPF from Xenopus oocytes contains roughly equal " and either cyclin B 1 and B2 (Lohka et al., 1988; amounts of ~ 3 4 " ~kinase Gautier et al., 1990).Its kinase activity is high at the onset of M phase and low at anaphase and interphase (Picard et al., 1985, 1987; Capony et al., 1986; Labbe et al., 1988). This provides a biochemical basis for earlier observations showing that fusion of interphase and mitotic cells confers a pseudomitotic phenotype to the interphase nucleus (Rao and Johnson, 1970;Johnson and Rao, 1970). There are a number of different p34cdc2-containingcomplexes that may differ in localization and substrate specificity (e.g., Cisek and Corden, 1989). Some ~ 3 4 " ~ "kinase ' activity is located in the nucleus (Riabowol et al., 1989), but significant activity can be detected in the cytoplasm, particularly in association with the centrosome (Bailly et al., 1989). It is believed that activated MPF phosphorylates key substrates that bring about observable M-phase-specific phenomena such as chromosome condensation, nuclear envelope breakdown, and cytoskeletal reorganization. M phase is associated with a large burst of protein phosphorylation, and it seems likely that MPF acts both by directly phosphorylating end-point effectors and by phosphorylating other kinases to initiate a cascade of activity that phosphorylates a wider range of substrates.

II. Protooncoprotein Tyrosine Kinases (PTKs) in Mitosis

A. pp60'"", A CYTOPLASMIC/MEMBRANEASSOCIATED PTK 1. Mitosis-Specific Phosphorylatzon and Activation of pp60"" Chicken pp60"", expressed in genetically modified NIH 3T3 cells, is transiently phosphorylated at serine and threonine residues during fibroblast mitosis (Chackalaparampil and Shalloway, 1988). These novel phosphorylations retard its electrophoretic mobility and are associated with an increase in its specific kinase activity. The amount of pp60"" does not appear to change significantly between interphase and mitosis. Similar retardations of electrophoretic mobility and increases in specific kinase activity have been observed with endogenous mouse (Chackalaparampil and Shalloway, 1988; P.-H. Lin and D. Shalloway, unpublished results) and human (Morgan et al., 1989) pp60"". These mitosis-specific modifications are most easily observed with cells that have been arrested in metaphase following treatment with nocodazole (Zieve et al., 1980;

ONCOPROTEIN KINASES I N MITOSIS

189

Nusse and Egner, 1984) or colcemid (Otto et al., 1981), inhibitors of microtubule (and hence mitotic spindle) formation, but can be observed in mitotic cells that have been isolated without the use of drugs by mechanical shakeoff alone (Tobey et al., 1967).The modifications are not observed with cells that are still in interphase following inhibitor treatment. Nocodazole appears to be the superior reagent for such studies because its actions are more completely reversed following its removal (Chackalaparampil and Shalloway, 1988). T h e phosphorylation, electrophoretic mobility, and specific kinase activity of pp60"" all revert to their interphase state about 15-25 min after release from nocodazole arrest, in parallel with cytokinesis (Chackalaparampil and Shalloway, 1988). The time of onset of the mitosisspecific phosphorylations has not been determined, but they can occur no earlier than about 1 hr before mitosis; otherwise the mitosis-specific phosphorylations would be detectable in unsynchronized populations. These stringent temporal correlations suggest that pp60"" may play a role in regulating passage through mitosis.

2. p34cdc2Catalyzes the Mitoszs-Specific Phosphorylation of pp6OcSrc Three mitosis-specific phosphorylation sites, T h r 34, T h r 46, and Ser

72, have been identified in the amino-proximal region of chicken pp60" (Shenoy et al., 1989).All three sites are located within the consensus sequence charged/polar-S/T*-P-X-basic (the phosphorylated residue is identified by an asterisk). Residues (within conserved consensus sequences) corresponding to Thr 34 and Ser 72 (but not to Thr 46, which is not conserved) are also phosphorylated in mouse pp60"" (P.-H. Lin and D. Shalloway, unpublished results), and indirect evidence suggests that at least the residue corresponding to T h r 34 is phosphorylated in human pp6Ocsrc (Morgan et al., 1989). T h e consensus sequence is closely related to sites in histone H 1 that are phosphorylated by ~ 3 4 " ' ~during mitosis (Langan et al., 1980). This suggests that ~ 3 4 " ' ~is responsible for the mitosis-specific phosphorylations of pp60"" as well. Direct evidence supports this hypothesis: (1) purified Xenopw MPF (containing p34"" and cyclins) phosphorylates chicken pp60"" at all three sites in vitro (Shenoy et al., 1989); (2) a partially purified fraction from mitotic HeLa cells containing MPF activity phosphorylates human pp60"" at the two sites that are phosphorylated during mitosis in vivo (Morgan et al., 1989); (3) mitotic HeLa cell lysates, but not mitotic lysates that have been precleared with anti-p34"'* antibody, can phosphorylate pp60"" at the mitosis-specific sites (Morgan

190


et al., 1989); and (4) neither protein kinase A nor protein kinase C,

serinehhreonine kinases that phosphorylate pp60"" at other amino region sites, phosphorylates the mitosis-specific sites (Roth et al., 1983; Gould et al., 1985; Purchio et al., 1985, 1986; Gentry et al., 1986; Patschinsky et al., 1986; Gould and Hunter, 1988). ~ 3 4 is"present ~ ~ in multiple complexes, and it is not known which one(s) is (are) responsible for phosphorylating pp60"" in uivo. If pp60"" is phosphorylated prior to nuclear envelope breakdown, the active complex must have access to the cytoplasm. pp60"" is located in the perinuclear region as well as at the plasma membrane (Resh and Erikson, 1985), and significant amounts of both it (David-Pfeuty and Nouvian-Dooghe, 1990; K. Kaplan, H. Varmus, and D. Morgan, Personal communication) and ~ 3 4 " " (Bailly ~ et al., 1989; Riabowol et al., 1989) are concentrated in the centrosomal region, which could be a site of their interaction. Myristylation, which is required for membrane localization of pp60"", is not required for its mitosis-specific phosphorylation (S. Bagrodia and D. Shalloway, unpublished results). In vitro phosphorylation of pp60'-'" to high stoichiometry by MPF does not appear to be sufficient to stimulate pp6OTSrckinase activity (Morgan et al., 1989; Shenoy et al., 1989) suggesting that other in vivo events are also required. It is not yet known if there is a causal relationship between the ~ 3 4 " ~ " ~ - m e d i aphosphorylations ted and stimulation of pp6OCflckinase activity.

3. Multiple Forms of pp60""'" in Mitotic Cells At least three different forms of mitosis-specific overexpressed chicken pp60"", differing in electrophoretic mobility, are present in genetically modified NIH 3T3 cells during mitosis (S. Bagrodia and D. Shalloway, unpublished results). Some, if not all, of these forms represent molecules that are phosphorylated at different subsets of the mitosisspecific sites. Phosphorylation at T h r 34 is required for most of the electrophoretic mobility retardation, whereas phosphorylation at only T h r 46 or Ser 72 has little effect on mobility. The reason for induction of a relatively large (apparent shift of -2 kDa) mobility shift in response to phosphorylation at T h r 34 is unknown, but it may result from a phosphorylation-induced conformational change. Phosphatase treatment of the mitosis-specific forms of ~ ~ 6 0 "restores '~ their electrophoretic mobilities to that of phosphatase-treated normal pp60"" (Shenoy et al., 1989), and there is no evidence to suggest that any modifications beyond altered phosphorylation are involved.


191

4. Mitosis-Specific Dephosphorylation of Tyr 527 and

pp60""" Activation pp60c-srckinase activity can be regulated by altered phosphorylation at Tyr 527 and Tyr 416. Tyr 527, near the carboxyl terminus of pp60"'", is phosphorylated to high stoichiometry in unsynchronized cells (Cooper et al., 1986). Abolishing this phosphorylation by mutation (Cartwright et al., 1987; Kmiecik and Shalloway, 1987; Piwnica-Worms et al., 1987; Reynolds et al., 1987) or phosphatase treatment (Courtneidge, 1985; Cooper and King, 1986) dramatically (10- to 20-fold) stimulates pp60"" kinase activity. Tyr 4 16, within the catalytic domain, can be autophosphorylated in vitro (Smart et al., 1981; Patschinsky et al., 1982), but it is not significantly phosphorylated in vivo unless Tyr 527 phosphorylation is abrogated by polyoma middle T antigen binding (Cartwright et al., 1986) or by mutation. In contrast to the effect of Tyr 527 phosphorylation, Tyr 416 phosphorylation stimulates pp60""" kinase activity (although only threefold) (Kmiecik et al., 1988; Harvey et al., 1989; Ferracini and Brugge, 1990). Mitotic activation is not accompanied by a detectable increase in Tyr 416 phosphorylation o r a large decrease in Tyr 527 phosphorylation (Chackalaparampil and Shalloway, 1988). However, the stoichiometry of Tyr 527 phosphorylation in kinase-defective pp60""" (containing a point mutation in the ATP-binding site) expressed in NIH 3T3 cells is significantly reduced (-70%) in nocodazole-arrested cells, and it may also be reduced to a small extent in wild-type pp60"" (Bagrodia et al., 1991). If the stoichiometry of Tyr 527 phosphorylation is sufficiently high during interphase, a small decrease during mitosis could account for the observed threefold increase in specific kinase activity. This hypothesis is supported by the finding that mutation of Tyr 527 inhibits the mitosisspecific increase in src-encoded kinase activity even though it does not interfere with the p34cdc2-mediatedphosphorylations in vivo (Bagrodia ct al., 1991). (Inhibition of an additional mitosis-specific increase in activity is not simply an indication that pp60"" has already been maximally stimulated by the Tyr 527 mutation: the increase is also blocked by a Tyr 527 mutation in a multisite mutant that has submaximal activity.) In contrast, site-specificmutations at Tyr 416 or at the protein kinase A (Ser 17) or protein kinase C (Ser 12) phosphorylation sites have little or no effect (Yaciuk et al., 1989; Bagrodia et al., 1991). These data could be simply explained by the hypothesis that ~34"'~-mediatedphosphorylations sensitize pp60"" to the activities of Tyr 527 phosphatase or desensitize it to Tyr 527 kinase. This could reflect changes in pp60'" to act as a substrate, changes in Src localization, or other effects. In this as-yet

192


untested model, mitotic activation of pp60"" is a two-step process involving the causally linked activities of ~ 3 4 " ~and " ' a tyrosine phosphatase. 5. Tyr 527 Autophospholylation Purification of a 47-kDa protein that can phosphorylate Tyr 527 in uitro has been reported (Okada and Nakagawa, l989), and it or other Tyr 527 kinases may be important regulators of pp6OC"'"activity. pp60'"" does not autophosphorylate Tyr 527 to a significant extent under conventional in uitro assay conditions (Smart et al., 1981),but can phosphorylate it in an intermolecular reaction (trans-autophosphorylation) when chicken pp60"" is expressed in yeast (Cooper and MacAuley, 1989). This suggests that Tyr 527 may be autophosphorylated in animal cells under appropriate in uiuo conditions. However, kinase-defective pp60""", when expressed in genetically modified unsynchronized cell populations, is extensively phosphorylated at Tyr 527 (Jove et al., 1988; Schuh and Brugge, 1988). This hints that autophosphorylation does not significantly modulate the stoichiometry of Tyr 527 phosphorylation, but these experiments do not exclude the possibility that the kinase-defective pp60"" is trans-autophosphorylated by the endogenous wild-type pp60"". T h e hypothesis that Tyr 527 autophosphorylation is significant in uiuo is supported by the fact that the stoichiometry of Tyr 527 phosphorylation in nocodazole-arrested cells is lower in kinase-defective than in wild-type pp60"" (Bagrodia et al., 1991). It is not known why this difference is apparent only during mitosis and not during interphase, although it may be that increased Tyr 527 phosphatase activity during mitosis enhances sensitivity to differences in Tyr 527 phosphorylation rates. While alternative explanations exist (e.g., that a conformational change induced by the kinase-inactivating point mutation impairs the ability of Tyr 527 to be phosphorylated by an exogenous kinase), the possibility that Tyr 527 autophosphorylation regulates pp60"" activity bears consideration. 6. Do the pp60"" Phosphorylation Sites Act as Inputs to an Analog Computer? The pp60"" "unique" region (-75 amino acids), which lies between the amino-terminal sequence required for membrane localization (residues 1-7) (Kaplan et al., 1988) and Src homology 3 region (approximately residues 83-137) (Pawson, 1988),is the most divergent among the different src-encoded family of kinases (Parsons and Weber, 1989; Cooper, 1990). This region contains the three sites of ~34""~-rnediated phosphorylation and at least three other phosphorylation sites (Fig. 1).


T46

193

s72 190 1 9 2

GZ

wlstm?

517

I PIKA @

548

1

PIKC

@

@

K295

1416

L516 1527

ATP

Auto-B

I"

I

~8th

pmtlg

I

Blndlnp

PlKC

8

FIG. 1. Clustering of pp6OC"" phosphorylation sites in the Src-specific region. The numbered residues (except for G2 and K295, which are required for myristylation and kinase activity, respectively)are phosphorylated by the indicated kinases under appropriate conditions; boundaries of known functional domains are marked. SH2, Src homology 2 region; SH3, Src homology 3 region; PrKA, protein kinase A; PrKC, protein kinase C; pmtAg, phosphorylated when bound by polyoma middle T antigen; autophosphorylation site.

Two more phosphorylation sites (at Tyr 90 and 92), and possibly the site of platelet-derived growth factor (PDGF)-stimulated pp60"" tyrosine phosphorylation, are nearby. Together, these clustered sites are targets for at least five kinases (p34"', protein kinase A, protein kinase C, PDGF receptor or receptor-stimulated kinase, and the unknown kinase responsible for amino-region phosphorylation of pp60"" in the Sdpolyoma middle T complex). It has previously been suggested that pp60'"" functions as an "analog computer" (of currently unknown function) that nonlinearly integrates signals from a variety of kinases and transmits its output via its protein tyrosine kinase activity (Fig. 2) (Kmiecik and Shalloway, 1987).In a wider sense, pp60"" may participate in a distributed network of kinase/ phosphatase circuits (Hunter, 1989). If so, the phosphorylations in and near the unique region may constitute a multiinput domain to the Src computer. The model predicts that these phosphorylations interact in a complex, nonadditive manner. There are two hints that this may be the case: (1) an amino acid substitution at Ser 17 that prevents phosphorylation by protein kinase A stimulates phosphorylation by protein kinase C at Ser 12 (Yaciuk et al., 1989)and (2)the p34cdcz-mediatedphosphorylations suppress phorbol ester-stimulated phosphorylation of Ser 48 by protein kinase C (S. Bagrodia and D. Shalloway, unpublished results). With the exception of the site(s)phosphorylated by the PDGF-stimulated kinase (Ralston and Bishop, 1985; Gould and Hunter, 1988),phosphorylations in this region do not appear to directly affect pp60"" kinase activity to a significant extent (Gould et al., 1985; Purchio et al., 1985;

"4°C.

@

194


PHOSPHORYLATION/ DEPHOSPHORYLATlON (INPUTS) CAMP

Y Kinase

Alternate

Phosphalase

TYROSINE PHOSPHORYLATION SUBSTRATES FIG. 2. pp60'"" as an analog cornputet. The one-letter amino acid code is used; DAG, diacylglycerol; mT, middle T antigen; other abbreviations are defined in the text.

Gould and Hunter, 1988; Hirota et nl., 1988; Morgan et al., 1989; Shenoy et al., 1989; Yaciuk et al., 1989; Espino et al., 1990) and are not required for Src transforming activity (Crossetal., 1984; Hirota etaL, 1988; Yaciuk et al., 1989). However, the possibility that some of these phosphorylations may regulate localization or transient effects merits further investigation.


195

7. p34cdc2Substrate Consensus Sequence T h e mitosis-specific phosphorylation consensus sequence charged/ polar-SIT*-P-X-basic at T h r 34 and Ser 72 in chicken pp60""" is almost exactly conserved in human, mouse, and frog pp60"" (Takeya and Hanafusa, 1983; Tanaka et al., 1987; Martinez et al., 1987; Steele et al., 1989). T h e precise conservation of T h r 34 and its flanking consensus sequence is particularly remarkable because it lies within a region that, otherwise, is highly divergent among species (only 60% of the 30 amino acids surrounding T h r 34 are conserved among chicken and mammalian Src; only 42% are conserved among chicken and Xenopus Src). The Drosophilu c-Src homologue, DSrc28, contains the closely related sequence G-To-P-N-S-K [the superscript (") indicates a potential phosphorylation site] that, when sequences are aligned, is offset by one amino acid from Ser 72 (Gregory et al., 1987), and a C. elegans homologue of pp60"" has a related potential phosphorylation site (E-To-P-E-E)at the location of T h r 34 (C. Thacker and M. Capecchi, personal communication). Human Hck, a member of the Src family of PTKs, has a sequence similar to the consensus (N-To-P-G-1-R),in close alignment with chicken c-Src Ser 72 (Ziegler et al., 1987). A number of ~ 3 4 " ~ phosphorylation "' sites in histone H1 match this consensus exactly whereas other match the relaxed consensus S/To-P-Xbasic (where X represents 0-1 amino acids) (Langan et al., 1980). However, even this relaxed consensus is not required for ~34'~'' phosphorylation, as phosphorylation sites in RNA polymerase I1 (Cisek and Corden, 1989), p150"" (Kipreos and Wang, 1990) (see Section II,C), lamins (Peter et al., 1990b; Ward and Kirschner, 1990; Heald and McKeon, 1990), and nucleolin (Peter et ul., 1990a) have been identified as conforming only in the minimal recognition sequence S/T*-P. In addition, neither Xenopus cyclin B1 nor B2 contain any sequences that match the consensus found in pp60"" (Minshull et al., 1989), even though they are phosphorylated by ~ 3 4 " ' within ~ the purified MPF complex (Lohka et al., 1988; Erikson and Maller, 1989) and in M-phase extracts (Gautier et al., 1990). Though not required (nor sufficient) for phosphorylation by ~ 3 4 " ~ " ~ , the charged/polar-S/T*-P-X-basic sequence may have some value in predicting potential substrates. A computer-assisted search of 14,372 sequences in the Protein Identification Resource (Release 23; National Biomedical Research Foundation) for the appearance of the charged/ polar-S/T*-P-X-basic consensus revealed only 1137 sequences (many closely related) that contained the consensus. A subset of the identified proteins, subjectively selected because of their potential for participation

196


in cell cycle regulation or transformation, is shown in Table I. (More detailed information is available on request.) In addition to the Abl and Mos proteins, which will be discussed below, there is already experimental evidence to suggest or demonstrate that a number of the proteins in the list are phosphorylated and regulated by ~34'~''. Many of these are DNA-binding proteins. For example, the SV40 large T antigen is phosphorylated in vivo at Thr 124, which lies in the sequence S-T*-P-X-K (Scheidtmann et al., 1982), and its phosphorylation is required for SV40 DNA replication (Schnieder and Fanning, 1988; Kalderon and Smith, 1984). Phosphorylation of this site by purified ~ 3 4 " has ~ been demonstrated and has been shown to stimulate SV40 DNA replication in vitro (McVey et al., 1989). In addition, the retinoblastoma-susceptibility (RB) protein, which binds SV40 large T

TABLE I SELECTED PROTEINS CONTAINING

CHARGED/POLAR-S/T*-P-X-BASIC p34"1L2

SUBSTRATE CONSENSUS SEQUENCE^ Kinases Src* Abl* Mos* Raf Neu Dbl Bcl Ets Protein kinase A Protein kinase C

Nuclear oncoproteins and antioncoproteins Retinoblastoma-susceptibility (RB) protein* SV40 large T antigen* BK virus large T antigen JC virus large T antigen Mouse polyomavirus large T Hamster polyomavirus large T Lymphotrophic polyomavirus large T Myc* Myb* EIA

Membrane-associated proteins Fibronectin lntegrin Integrin 8, chain Vincluin

Other proteins Protein phosphatase-1 inhibitor Cdc4 Cdc 10 Histones* MAP 2 -4vian Gag Murine Gag HIV and SIV pol HIV transactivating protein

Some experimental evidence exists (see the references given for rhe following substrates) to suggest (not necessarily conclusively)that the substrates marked with an asterisk are phosphorylated by p34'&' either in viuo or in &fro, i.e.,Src (Morgan etal., 1989; Shenoy etal., 1989), Abl (Kipreosand Wang, 1990), Mos (G. F. Vande Woude, personal communication), RB (Chen et al., 1989), SV40 large T antigen (McVey el al., 1989), Myc (S. R. Hann and W. Boyle. personal communication), Myb (B. Luscher and R. Eisenmann, personal communication), and histones (Langan ef al., 1980, 1989).


197

antigen, is transiently phosphorylated shortly before entry into S phase, "' et al., 1989; Chen et al., 1989; DeCaprio probably by ~ 3 4 " ~ (Buchkovich et al., 1989). Binding of the RB protein by SV40 large T antigen and to be alternative mechanisms for stimphosphorylation by ~ 3 4 " "appear ~ ulating entry into S phase (Cooper and Whyte, 1989). T h e c-Myb and c-Myc proteins also contain the consensus sequence and are hyperphosphorylated at serine and threonine residues during mitosis in lymphoid cells, rodent fibroblasts, and HeLa cells. At least some of the mitosis-specific phosphorylation sites in the c-Myb protein can be phosphorylated by ~ 3 4 " in~ vitro, " ~ and c-Myb and c-Myc proteins from cells treated with okadaic acid, an inhibitor of some serine/threonine phosphatases, have retarded electrophoretic mobilities similar to those of the mitotic proteins (B. Luscher and R. Eisenmann, unpublished results). Phosphorylation of c-Myc by purified ~ 3 4 " has ~ " also ~ been reported (S. R. Hann and W. Boyle, personal communication). There does not appear to be any correlation between the sequence flanking a ~ 3 4 phosphorylation ' ~ ~ site and the time of its phosphorylation during the cell cycle. For example, residues within the charged/ polar-SIT*-P-X-basic consensus can be phosphorylated either during mitosis [e.g., in pp60"" (Shenoy et d., 1989) and histone H1 (Langan et d., 1980)] or during interphase [e.g., SV40 large T antigen (McVey et al., 1989)l. This is also the case for residues that are not within the consensus [e.g., phosphorylation at D-T*-P-E-L in p 15OCab'duringmitosis (Kipreos and Wang, 1990) and at T-S*-P-S-Yin RNA polymerase I1 during interphase (Cisek and Corden, 1989)l. While ~ 3 4 has " been ~ ~ shown to be capable of phosphorylating these sites in vitro, it should be noted that other kinases can also phosphorylate the minimal SIT*-P recognition sequence (Vulliet et al., 1989) and could be involved in viva

B. OTHER Src FAMILYPTKs T h e behavior of other members of the Src family of PTKs during mitosis has not been extensively studied, but preliminary evidence suggests that at least some also undergo mitosis-specific regulation. The kinase activities of human p59'-fin and p62q" are higher in nocodazolearrested cells than in similar cells subsequently released from arrest by incubation in nocodazole-free medium (R. M. Kypta and S. A. Courtneidge, personal communication). Also, anti-Lck immunoprecipitates from nocodazole-arrested NIH 3T3 cells that were modified to express p56" have fivefold higher specific kinase activity than p56" from unsynchronized cells (S. Kusick and J. A. Cooper, personal communication). However, no new phosphorylations have been detected in

198


these cases. T h e Fyn, Yes and Lck proteins all have residues homologous to c-Src Tyr 527 (Marth et al., 1985; Kawakami et al., 1986; Sukegawa et al., 1987) and, like Src, might be activated by partial transient dephosphorylation of these residues. For example, Tyr 505 negatively regulates p56lCkkinaseactivity (Marth et al., 1988) and thus provides a control mechanism that might potentially be involved in ~ 5 6 mitotic '~ activation. If so, there may be mechanisms for mitosis-specific tyrosine dephosphorylation of substrates that are not phosphorylated by ~34'~''.

C. pl5Oeub', A CYTOPLASMI~NUCLEAR PTK 1. Phosphorylution of Abl Proteins by p34cdc2 Like pp60"" and pp60"', both ~ 1 5 0 " ~(type ' IV c-Abl protein) and 160p%-"b[ proteins from mitotic abl-transfected NIH 3T3 cells have retarded electrophoretic mobilities due to increased mitosis-specific phosphorylation (Kipreos and Wang, 1990). As with pp60"", phosphatase treatment restores normal mobility, suggesting that phosphorylation is the only mitosis-specific modification. p 15OMb' is specifically phosphorylated at at least seven novel serine and threonine residues (Kipreos and Wang, 1990). These sites can all be phosphorylated by p34"''-containing immunoprecipitates, suggesting that ~ 3 4 ' ~is' the in uzvo kinase. These immunoprecipitates also can phosphorylate two additional serines in ~150"~'that are phosphorylated throughout the cell cycle in uivo. All nine phosphorylation sites are located within the carboxyl-terminal noncatalytic domain. Neither the identified cell-cycle-independent phosphorylation site (Ser 588; A-V-S*-P-L-L) nor the mitosis-specific phosphorylation site (Thr 566; D-T*-P-E-L) is flanked by sequences that match the consensus that is phosphorylated in pp60'"". This must also be true for most of the other p34cd"2-phosphorylatedsites because p150t"*' contains only two sequences that match the charged/polar-S/T*-P-Xbasic consensus (Shtivelrnan et al., 1986; Ben-Neriah et al., 1986). In contrast with pp6Ocfl', apparently all the mitotic p15OeUb' migrates within a single retarded-mobility band in SDS-polyacrylamide gels (Kipreos and Wang, 1990). This might result from the large number of mitosis-specific phosphorylation sites in p15OCub'. Alternatively, it could reflect improved access of the p34"" kinase to p150mb', which is located both in the nucleus and in the cytoplasm (Van Etten et al., 1989).

2 . Potential Effects of Mitosis-Specific Phosphorylation of p150rdb1 In contrast with the Src family of PTKs, no evidence of mitosis-specific activation of pl5OcUb' kinase activity has been detected (Kipreos and


199

Wang, 1990). This may reflect the fact that p15Oru6' is not negatively regulated by phosphorylation at a residue homologous to c-Src Tyr 527. However, the mitosis-specific phosphorylations may indirectly affect phosphorylation by altering access to specific substrates. Both of the mapped mitosis-specific phosphorylation sites are in the vicinity of a c-Abl carboxyl nuclear localization signal sequence K-K-K-K-K (Van Etten et al., 1989). This echoes the situation with SV40 large T antigen, in which the site phosphorylated by a p34"cz/cyclin B complex in vitro (Thr 124) lies one residue from a homologous nuclear localization sequence P-K-K-K-R-K (McVey et al., 1989). Thus, it will be interesting to determine if p34"d"2-mediatedphosphorylation alters p 150mb' localization. Because relocalization of pl 50'"b' to the cytoplasm, induced by deletion of a small N-terminal regulatory region, is accompanied by neoplastic transformation (Van Etten et al., 1989), phosphorylation-induced relocalization could have important consequences. Interestingly, serine/threonine hyperphosphorylation of a Bcr/Abl fusion protein during mitosis is correlated with a fivefold reduction in its level of phosphotyrosine (E. T. Kipreos and J. Y. Wang, personal communication). This suggests that this protein is subject to increased tyrosine phosphatase activity during mitosis, possibly by mechanisms related to those acting on kinase-defective pp60""" (see Section I,A,4). Not all Abl variants display this effect and it is not known if it requires p34"dcz-mediatedphosphorylation. D. PTK GROWTH FACTOR RECEPTORS

Meiotic maturation of Xenopzls oocytes is normally induced by progesterone (Masui and Clark, 1979),but can also be induced by insulin (El-Etr et al., 1979; Maller and Koontz, 1981).This insulin effect appears to be mediated by insulin receptor PTK activity because it can be blocked by microinjection of a monoclonal antibody that inhibits the insulin receptor tyrosine kinase (Morgan et al., 1986) or by microinjection of protein tyrosine phosphatase 1 B (Tonks et al., 1990). Microinjection of synthetic human epidermal growth factor (EGF) receptor mRNA into Xenopus oocytes can confer the ability to initiate EGF-dependent meiotic maturation in the absence of progesterone, providing direct evidence for the ability of a PTK to induce M-phase progression (Opresko and Wiley, 1990). Like induction of maturation by progesterone, induction by EGF is associated with MPF activation. The kinetics of response suggest that this activation is indirect (Opresko and Wiley, 1990). This is expected because tyrosine phosphorylation of p34"" by the EGF receptor would be predicted to suppress, not

200


stimulate, its activity (Could and Nurse, 1989; Nurse, 1990). Mutant EGF receptor that is only slowly internalized is fully capable of inducing maturation, suggesting that relevant substrates may reside near the oocyte surface (Opresko and Wiley, 1990). A similar phenomenon has been observed with platelet-derived growth factor, which can induce maturation of oocytes that have been microinjected with PDGF receptor mRNA (H. Ueno and L. T. Williams, personal communication). Though not physiologically relevant, the EGF and PDGF responses attest to the conservation through evolution of tyrosine kinase substrates that are involved in the initiation of meiotic maturation.

E. POTENTIAL TARGETS OF PTKs IN M PHASE The 1O-year search for functionally significant substrates of protein tyrosine kinases has identified many interesting, but no convincing, candidates. A major problem has been that the relaxed specificity of PTKs results in phosphorylation of many functionally unimportant substrates (e.g., Beemon et al., 1982; Cooper et al., 1983; Kamps and Sefton, 1988). PTK promiscuity is most forcefully demonstrated by the fact that even random tyrosine-containing acidic amino acid polymers can be efficiently phosphorylated in vitro (Braun et al., 1984). In addition, most studies have investigated phosphorylation patterns in unsynchronized cell populations in which transient phosphorylations of important substrates may have gone undetected. Another problem for studies involving metabolic radioactive labeling is that ionizing radiation causes cell cycle arrest in S and Gz, probably because of chromosome damage (Marin and Bender, 1963; Chackalaparampil and Shalloway, 1988). Incubation with as little as 25 pCi/ml 32P can cause arrest of NIH 3T3 cells in S and GP;similar arrest occurs with [35S]methionine (S. Bagrodia and D. Shalloway, unpublished results). Because many studies have used much higher amounts of 32Pand long labeling times (usually 3 8 hr), they have actually investigated in vivo phosphorylations in S- and Gn-arrested cells. In such studies, even substrates that were phosphorylated for an extended period during G1 could have escaped detection. This problem has been avoided in more recent studies that have used antiphosphotyrosine antibodies with nonradioactively labeled cells. This may partially explain the ability of these reagents to expose previously undetected substrates (for review, see Hunter, 1989). Little is known about M-phase-specific phosphorylation substrates of PTKs. However, some substrates of constitutively activated oncogenic PTKs may also be transiently phosphorylated by pp60""" and other


20 1

protooncoproteins when these kinases are transiently activated during mitosis. Thus, it is useful to review our knowledge of PTK substrates in transformed cells with an eye to potential connections to M phase. Although most of the experimental results have been obtained in studies with pp60""", PTK signaling pathways appear to be fairly redundant, and the possibility that targets of Src are targets of other PTKs as well should be kept in mind. The relationships discussed here are outlined in Fig. 3. 1. Cytoskeletal and Cell-Cell Adhesion Proteins pp60'"" is concentrated at cell focal adhesion plaques (podosomes) (Rohrschneider, 1980; Marchisio et al., 1988) and can phosphorylate multiple adhesion plaque components, such as vinculin (Sefton et al., 1981; It0 et al., 1983), talin (Pasquale et al., 1986; DeClue and Martin, 1987) and integrins (Hirst et al., 1986; Tapley et al., 1989), which may participate in cytoskeleton-to-membrane linkage. Additional uncharacterized adhesion plaque components are tyrosine phosphorylated in Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts (Glenney and Zokas, 1989). Antiphosphotyrosine antibody staining has shown that adhesion plaques are also tyrosine phosphorylated in untransformed cells (Comoglio et al., 1984; Maher et al., 1985). Other cytoskeletal components may also be important substrates. p36 and p35 [also known as calpactins 1and I1 (Glenney, 1986)l are abundant Src substrates (Erikson and Erikson, 1980: Radke etal., 1980; Cooper and Hunter, 1983) that are localized at the cytoplasmic face of the plasma membrane (Amini and Kaji, 1983; Courtneidge et al., 1983; Greenberg and Edelman, 1983; Nigg et al., 1983), where they associate with the cytoskeletal proteins actin and spectrin (Lehto et al., 1983; Gerke and Weber, 1984; Glenney, 1986). Fodrin, which links actin bundles to the plasma membrane, can be phosphorylated in vitro by Src and other PTKs (Akiyama et al., 1986). The fact that the transforming activity of mutated Src proteins correlates with enhanced association with the cytoskeleton is consistent with the hypothesis that phosphorylation of these or related cytoskeletal components is important for transformation (Hamaguchi and Hanafusa, 1987; Loeb et al., 1987). Recent high-resolution immunofluorescence studies have shown that some pp60'-"" is concentrated at the centrosome, the microtubule organizing center (MTOC) (David-Pfeuty and Nouvian-Dooghe, 1990; K. Kaplan, H. Varmus, and D. Morgan, personal communication). pp60"" can phosphorylate tubulin in nitro (Akiyama et al., 1986) and it is possible that Src may modulate cytoskeletal structure by acting on the MTOC. Another popular speculation has been that PTK-mediated

202


FIG.3. Potential signaling pathways involving oncoprotein kinases in M phase. Only pathways discussed in this review are included. Each type of line represents an experimentally determined relationship: , phosphorylation; - - - , dephosphorylation; . _ _ ,.functional connection demonstrated by antisense oligonucleotide or antibody microinjection experiment; . . . . , other. The graphical implication of multistep signaling is only speculative and should be treated with caution; the temporal relationships between the steps may be inappropriate for sequential signaling.

phosphorylations may induce unlinking of microfilaments and adhesion plaques and play a role in inducing the changes (Wang and Goldberg, 1976) in microfilament organization, cell rounding, and cell-cell adhesion that are observed in transformed cells. This is qualitatively supported by the observations that subtransformation level expression of pp60"-"" reduces epithelial cell-cell adhesion (Warren and Nelson, 1987)


203

and that vanadate treatment of baby hamster kidney fibroblasts induces both increased tyrosine phosphorylation of podosome proteins and cytoskeletal changes typical of transformation (Marchisio et al., 1988). Specific biochemical connections between phosphorylations of known substrates and altered morphology have not been established. The phosphorylation of vinculin does not appear to be important, because it is not tyrosine phosphorylated in all Src-transformed cells (Antler et al., 1985) nor is its tyrosine phosphorylation increased during the release of stress fibers before mitosis in normal cells (Rosok and Rohrschneider, 1983). However, vinculin may not play a central role in cell-substrate adhesion (Brands et al., 1990) and phosphorylation of other adhesion plaque components may be important. For example, tyrosine phosphorylation of integrin appears to decrease its affinity for talin and fibronectin (Tapley et al., 1989). Although it has been most extensively studied, pp60'" is not the only tyrosine kinase that interacts with focal adhesion plaques and the cytoskeleton. In particular, the Abl protein is also located at cellsubstratum contact points (Rohrschneider and Najita, 1984).

2. Gap-Junction Proteins Gap junctional communication is rapidly down-regulated when rodent cells are transformed by either polyomavirus (which activates or Rous sarcoma virus (Atkinson et aZ., 1981; Azarnia and pp60c-5Tc) Loewenstein, 1984, 1987; Chang et al., 1985; Mehta et al., 1986). This reflects the ability of pp60'-" to down-regulate gap junctional communication when overexpressed or activated by site-directed mutagenesis (Azarnia et al., 1988). Reduction is evident within 15 min and complete within 30 min, suggesting that the signaling pathway is direct (Azarnia and Loewenstein, 1984). Connexin43, the main component of fibroblast gap junctions is tyrosine phosphorylated in v-Src transformed cells and can be phosphorylated by pp60""" in vitro (Crow et al., 1990). Furthermore, pp60""" phosphorylates connexin43 when mRNAs for both proteins are coinjected into Xenopus oocytes, and this suppresses junctional conductance (Swenson et al., 1990). Thus it seems likely that pp60"-"" down-regulates gap junctional communication between animal cells by a similar tyrosine phosphorylation mechanism. It has been suggested that changes in gap junctional permeability to CAMPor other compounds may participate in oocyte meiotic maturation (Beers and Olsiewski, 1985) and that downregulation of gap junctions may be causally related to cell disaggregation and the onset of mitosis in mammalian cells (Larsen, 1985). If so,

204


modulation of gap junctional communication by pp60""" could be a pathway for regulation of passage through the GZlM transition. 3. SerinelThreonine Kinases and Phosphatases Oocyte maturation is accompanied by a transient burst of serinel threonine phosphorylation (Maller, 1985).This is echoed by the observations that microinjection of pp60""" into Xenopus oocytes causes a 20-40% increase in cellular protein phosphorylation, mostly at serine and threonine residues (Spivack et al., 1984), and that ligand-induced P T K growth factor receptor stimulation induces serinelthreonine phosphorylation as well as tyrosine phosphorylation (Ullrich and Schlessinger, 1990). A potential pathway for transducing tyrosine phosphorylation signals into serinelthreonine phosphorylation signals has been discovered to involve a family or 42- to 44-kDa serinelthreonine kinases that are hypertyrosine phosphorylated in Src-transformed chick embryo fibroblasts and in cells stimulated by a variety of mitogenic factors, including insulin, EGF, and PDGF (Cooper et al., 1982, 1984; Nakamura et al., 1983; Cooper and Hunter, 1985; Contor et al., 1988;Sturgill et al., 1988).Other representatives of the family are phosphorylated in maturing oocytes and eggs (Lohka et al., 1987; Pelech et al., 1988). One member of the family, designated p42, is closely related or identical to microtuble-associated protein (MAP) kinase (Rossomando et al., 1989), a serinelthreonine kinase that phosphorylates the microtubule-associated protein MAP 2 in insulin-treated cells (Ray and Sturgill, 1987).p42 is hyperphosphorylated at tyrosine, serine, and threonine in M-phase-arrested Xenopus eggs (Lohka et al., 1987). It requires simultaneous tyrosine and threonine phosphorylation for activation and thus can function to integrate signals from both tyrosine and threonine kinase pathways and output a serinel threonine kinase signal (Ray and Sturgill, 1988b; Anderson et al., 1990). The requirement for tyrosine phosphorylation may explain the transient activation of MAP kinase by insulin and EGF via their respective PTK receptors (Ray and Sturgill, 1988a; Rossomando et al., 1989). The requirement for threonine phosphorylation may explain the ability of phorbol esters, which activate protein kinase C, to activate MAP kinase (Rossomando et al., 1989). However, protein kinase C activity is also required for tyrosine phosphorylation of MAP kinase following PDGF (Kazlauskas and Cooper, 1988) or EGF (Vila and Weber, 1988) treatment. An important target of MAP kinase is ribosomal S6 kinase 11, which, in Xenopus, is inactivated by dephosphorylation and reactivated following phosphorylation by purified MAP kinase (Sturgill et al., 1988).S6 k'inase


205

I1 phosphorylates ribosomal S6 kinase, which might increase the rate of protein synthesis. S6 kinase I1 activity and S6 serine phosphorylation increase during Xenopus oocyte maturation (Hanocq-Quertier and Baltus, 1981; Neilsen et d,1982; Cicerelli et d.,1988);this could result from an activated MAP kinase cascade. Though p42 may play an important role in meiotic maturation, it is not tyrosine phosphorylated in nocodazole-arrested NIH 3T3 cells and its role in fibroblast mitosis is uncertain (Cooper, 1989). However, other members of the family may participate in mitosis. For example, sea star oocytes contain a distinct 44-kDa meiosis-activated myelin basic protein kinase (MBPK) that, as judged both by antibody cross-reactivity and proteolytic analysis, is closely related to MAP kinase (S. Pelech, personal communication). MBPK is phosphorylated at both serine and tyrosine residues and is cyclically activated during mitosis in the early cell divisions of sea star embryos (Pelech et ad., 1988). Homologues of MBPK or other members of this family could also play a role in vertebrate mitosis. The Raf-1 serine/threonine kinase, which is activated by pp60'"" and growth factor receptor-mediated tyrosine phosphorylation, is another candidate for a tyrosine-to-serine/threonine signal transducer (Morrison et al., 1988). However, its activity during M phase has not yet been characterized. Another potential pathway toward increased serinelthreonine phosphorylation is via phosphorylation-mediated inhibition of protein phosphatase- 1 (PP-l), a broad-specificity serine/threonine phosphatase (Cohen, 1989). Its catalytic subunit can he phosphorylated in vztro by pp60""; this inhibits PP- 1 phosphatase activity (Johansen and Ingebritsen, 1986). Because PP-1 is an S6 phosphatase, its phosphorylation and inactivation in vivo could result in increased S6 phosphorylation (Andres et aZ., 1987). However, as discussed previously, pp60"" is a promiscuous kinase, and in the absence of evidence for in vivo tyrosine phosphorylation of PP-1, the significance of this finding is unknown. 4. GAPandRas Studies showing that microinjection of anti-Ras antibody blocks both the ability of v-Src to transform fibroblasts (Smith et al., 1986) and to induce differentiation of PC12 cells (N. Kremer, J. S. Brugge, and S. Halegoua, personal communication) have suggested that Ras is a downstream effector of some Src activities. This signaling pathway may also be operative in M phase, because microinjection of activated Ras protein, like microinjection of pp60v-src,can accelerate progesterone-induced maturation of Xmopus oocytes (Birchmeier et al., 1985; Allende et al., 1988). Ras appears to lie downstream in the insulin receptor signaling

206


pathway as well, because microinjection of an anti-Ras peptide monoclonal antibody inhibits insulin-induced meiotic maturation of Xenopw oocytes (Deshpande and Kung, 1987; Korn et QZ., 1987). The Ras protein may also participate in regulating the G2/M transition, because v-Ras can release cells from at least one type of GBarrest (Durkin and Whitfield, 1987)and microinjection of antibodies that inactivate c-Ras inhibits cleavage in axolotl eggs (Baltus et al., 1988). Signaling from PTKs to c-Ras could be direct because both baculovirus-expressed pp60c"'c (Abdel-Ghan y et al., 1990) and the insulin receptor (Korn et al., 1987) can tyrosine phosphorylate p21" in uitro under appropriate conditions. Alternatively, modulation of the GTPaseactivating protein (GAP) that regulates Ras could provide an alternative, indirect, signaling pathway. The PDGF receptor (Kazlauskas et al., 1990; Kaplan et al., 1990) and endogenous pp60'-"" (Brott et al., 1991) form stable complexes with GAP; pp60'*-''', mutation-activated pp60'-s'c, and ligand-activated PDGF and EGF receptors can all phosphorylate GAP (Ellis et al., 1990; Molloy el al., 1989; T. Pawson, personal communication). 5. Nuclear Matrm Proteins

Dissolution of the nuclear membrane, either in mitotic cells or during germinal vesicle breakdown, is associated with increased phosphorylation of nuclear membrane-associated protein components (Gerace and Blobel, 1980; Henry and Hodge, 1983; Ottoviano and Gerace, 1987; Heald and McKeon, 1990; Peter et al., 1990a, b; Ward and Kirschner, 1990; Moreno and Nurse, 1990). Most of these phosphorylations are at serines and threonines, and a number of the components can be directly phosphorylated by ~ 3 4 " ' ~in vitro (Peter et al., 1990a, b; Ward and Kirschner, 1990). However, a twofold to threefold increase in phosphotyrosine in nuclear matrix proteins just before mitosis has been reported (Henry and Hodge, 1983), suggesting a potential role for nuclear PTKs such as p150""". PTKs may also be indirectly involved through kinase cascades. For example, S6 kinase 11, a target of MAP kinase (Section II,E,3), can phosphorylate nuclear lamins in vitro (Erikson and Maller, 1988; Ward and Kirschner, 1988), which are phosphorylated during oocyte maturation (Ward and Kirschner, 1988) and mitosis (Miake-Lye and Kirschner, 1985). Thus, activation of MAP kinase by PTKs may lead to lamin phosphorylation, which may, in turn, contribute to nuclear envelope breakdown (Ottoviano and Gerace, 1987).

6. p3Pdc2 At least two tyrosine residues in mammalian ~ 3 4 " ' ~are phosphorylated to a stoichiometry that continuously increases through the S and G2


207

phases of the HeLa cell cycle (Draetta et al., 1988). These are rapidly dephosphorylated at the start of mitosis in mouse 3T3 cells (Morla et al., 1989). Tyrosine dephosphorylation has been shown to be required for activation of ~34'~''kinase in fission yeast (Gould and Nurse, 1989) and starfish oocytes (Pondaven et al., 1990). In particular, site-directed mutagenesis of the tyrosine phosphorylation site in Schizosaccharomyces pornbe ~ 3 4 " ~ (Tyr " ' 15, located in the ATP-binding region) relieves the negative regulation associated with its phosphorylation. Expression of the Tyr 15 + Phe mutant induces a small-cell phenotype, probably because it bypasses many of the inhibitory influences that control the onset of mitosis (Gould and Nurse, 1989). Baculovirus-expressed pp60'-"", which is activated because of its low stoichiometry of Tyr 527 phosphorylation, can phosphorylate denatured ~ 3 4 " ~in" vitro at some of the tyrosines that are phosphorylated in vivo, and it has been suggested that it may be a physiological regulator of ~ 3 4 ' ~activity " ~ (Draetta et al., 1988). However, normal pp60'-" does not significantly phosphorylate ~ 3 4 " ~ "in' the MPF complex (Morgan et al., 1989; Shenoy et al., 1989) and maximal ~ 3 4 " ~ "tyrosine ' phosphorylation is anticorrelated with the cell-cycle dependent activation of pp6OC-'"'. Thus, a different PTK may be responsible for phosphorylating ~34"". Van Etten et al. (1989) have suggested that p150'-"'', which is present in both the nucleus and cytoplasm, may be responsible. Whatever kinase is responsible, if it is also phosphorylated by p34"", mutual phosphorylations may constitute a feedback loop that confers nonlinear response properties to the mitotic regulatory system. Yeast ~ 3 4 " ~ "is' also phosphorylated at T h r 167, outside the ATPbinding region. In contrast to phosphorylation at Tyr 15, phosphorylation at T h r 167 is required for kinase activity (K. L. Gould and P. Nurse, personal communication). Thus, like MAP kinase, yeast p34"" integrates signals from both tyrosine and threonine kinases. Mammalian ~ 3 4 " ~is" 'also phosphorylated at threonine and possibly to a lesser extent at serine (Morla et al., 1989), but the significance of these phosphorylations has not yet been clearly demonstrated.

Ill. c-Mos, a Serine/Threonine Kinase, in M Phase

Discoveries showing that c-Mos plays an important regulatory role in oocyte meiosis have provided another instance of protooncoprotein kinase involvement in M phase. c-Mos may also play a role in mitosis, but its expression in somatic cells is a subject of dispute (Propst et al., 1987; Herzog et al., 1989). c-Mos protein can be rapidly degraded, at least following oocyte maturation (Watanabe et aE., 1989), and its expression

208


might be temporally regulated in fibroblasts. Whether o r not Mos has a normal function in fibroblasts, the studies reviewed in this section suggest that Mos may induce transformation by perturbing M-phase control pathways. A. c-Mos A N D OOCYTE MATURATION 1. c-Mos Expression Zs Necessaly and Sufficient for Induction of Meiosis The observation that large amounts of c-Mos mRNA are sequestered in oocytes but are not translated until the initiation of meiosis led to the suggestion that c-Mos may participate in meiotic maturation, fertilization, o r other early oocyte developmental processes (Goldman et al., 1988; Keshet et al., 1988; Mutter et al., 1988). Sagata et al. (1988) demonstrated this by showing that inhibition of pp39'-"0sxe expression by microinjection of Xenopw oocytes with antisense c-MosXe oligonucleotides blocks both progesterone and insulin-induced germinal vesicle breakdown. Subsequent experiments extended this result to murine oocytes: microinjection with antisense murine c-Mos oligonucleotides prevented initiation of meiosis I1 (O'Keefe et al., 1989; Paules et al., 1989), and microinjection with antibodies that inhibit murine c-Mos kinase activity blocked germinal vesicle breakdown (Zhao et al., 1990). Conversely, microinjection of c-MosXe RNA into prophase-arrested Xenopw oocytes can induce germinal vesicle breakdown and oocyte maturation (Freeman et al., 1989; Sagata et al., 1989a). Thus, c-Mos expression is both necessary and sufficient for the initiation of oocyte maturation. 2.

Is an Essential Component of Cytostatic Factor

pp39c-mosXe

After completing meiosis I, Xenopus oocytes arrest at metaphase of meiosis I1 until stimulated by fertilization or calcium influx (Masui, 1985). Arrest has been attributed to a calcium-sensitive cytostatic factor (CSF) that was first identified by its ability to arrest cleavage at metaphase when injected into two-cell Xenopw embryos (Masui and Markert, 1971 ; Meyerhof and Masui, 1977,1979; Masui and Shibuya, 1987; Shibuya and Masui, 1988). CSF appears during oocyte maturation and disappears in association with a transient increase in cytoplasmic-free calcium following fertilization. The arrest is associated with high MPF activity, which appears to be stabilized by CSF (Gerhart et al., 1984; Newport and Kirschner, 1984; Lohka and Maller, 1985; Murray et al., 1989). Inactiva-


209

tion of CSF and the concomitant decrease in MPF activity is required for reentry into the cell cycle (Gerhart etal., 1984; Masui and Shibuya, 1987). Recent experiments suggest that pp39'-""sxe is the Xenopus CSF or an essential component of its activity. Microinjection of synthetic rnos RNA into two-cell Xenopus embryos causes cleavage arrest at metaphase, and imniunodepletion of pp39c-"0"e from egg cytosol extracts removes their CSF activity (Sagata et al., 1989b). Like CSF, pp39"-"0sx" is rapidly degraded either at fertilization or soon after the completion of meiosis by calpain 11, an endogenous calcium-sensitive protease (Watanabe et al., 1989), and pp39c-"0sxeis not detected after fertilization, even though its mRNA persists (Sagata et al., 1989b). Thus, the patterns of pp39c'""sxe and CSF expression are identical. Other similarities include their stabilization by ATP and NaF and their sedimentation profiles (Masui and Shibuya, 1987; Shibuya and Masui, 1988; Watanabe el al., 1989). Thus, Mos participates both in the initiation of meiosis I and in subsequent cytostatic arrest. 3. A Signaling Pathway Including c-Mos and Other Protooncoproteins Microinjection of p2 1" can accelerate progesterone-induced germinal vesicle breakdown inXenopw oocytes (Birchmeier et al., 1985; Allende et al., 1988; Sadler et al., 1990). This can be blocked by prior microinjection of c-MosXe antisense oligonucleotides (Barrett et al., 1990), although this effect may depend on the stage of oocyte development (Daar and Vande Woude, 1990). The Ras protein mediates some pp60"" activities, including induction of fibroblast transformation and neurite outgrowth (see Section 11,E,4), and it may also function downstream from Src in M phase. Thus, a potential positive feedback loop, Mos + MPF + Src + Ras + Mos, can be traced (see Fig. 3). These interactions may be involved in meiotic maturation or Mos-induced transformation. In regard to the first possibility, it is interesting that a large amount of mRNA for DSrc28, a Drosophila c-Src homologue, is sequestered in oocytes where, like c-Mos, it is probably translated during meiotic maturation (Gregory et al., 1987).

B. INTERACTIONOF c-Mos AND MPF

The CSF activity of pp39c-""sxesuggests that it stabilizes, and possibly activates, MPF. Indeed, c-MosXe-induced cleavage arrest in two-cell Xenopzrs embryos is accompanied by high MPF activity (Sagata et al., 1989b)

2 10


and the block to oocyte maturation induced by c-MosXe antisense oligonucleotide microinjection is accompanied by inhibition of the MPF activation that would otherwise occur (Barrett et al., 1990). Mos function probably involves its serine/threonine kinase activity (Kloetzer et al., 1983; Hannink and Donoghue, 1985; Maxwell and Arlinghaus, 1985), because a kinase-defective c-MosXe mutant with a substitution in the ATP-binding site does not induce germinal vesicle breakdown (Freeman et al., 1989). Resting oocytes contain inactive pre-MPF that can be stimulated either by hormones or by microinjection of catalytic amounts of active MPF (Gerhart et al., 1984). Activation of MPF is associated with increased phosphorylation of the MPF complex (Cyert and Kirschner, 1988; Meijer et al., 1989). The cyclin components can be phosphorylated at serine and threonine residues within the activated MPF complex (Erikson and Maller, 1989) or by extracts taken from maturingXenopus oocytes at times when MPF activity is predicted to be high (Gautier et al., 1990).Cyclin B2 can be phosphorylated by immunoprecipitated c-MosXe and v-Mos proteins to yield phosphopeptides similar to those generated following in vitro phosphorylation by MPF, and it has been suggested that Mosmediated phosphorylation of cyclin in viva may be involved in the activa(Roy et al., 1990). Experimental evidence to tion of MPF by pp39'~m0sxe support this hypothesis is equivocal: whereas cyclin B2 phosphorylation is decreased by 10-40% in extracts from c-MosXe antisense oligonucleotide-injected oocytes (Roy et al., 1990),the phosphorylation of overexpressed cyclin B2 is not increased following microinjection of c-mosXe mRNA, even though microinjected cyclin B2 and c-mmXe mRNAs act synergistically to stimulate oocyte maturation (Freeman et al., 1990). Alternatively, c-MosXe activity may be mediated via phosphorylation of other substrates. Increased cyclin levels can be sufficient to induce cell cycle transitions in Xenopus egg extracts (Murray and Kirschner, 1989), and Mos may activate MPF by directly or indirectly stabilizing cyclin against proteolytic degradation. It has been proposed that his might involve inactivation of a cyclin protease by pp39'~""sxe-mediatedphosphorylation (Hunt, 1989). An independent mechanism of action is suggested by the finding that pp39C~m0sxe forms a complex with and phosphorylates tubulin in vitro. This raises the possibility that pp39'-m0"x"may directly modify microtubules and/or spindles in viuo (Zhou et al., 1990). In this context, it may be significant that some amounts of both ~34'~'' and pp60'-" are localized in the vicinity of the microtubule organizing center.


21 1

C . TRANSFORMING Mos PROTEINS AND FIBROBLAST MITOSIS Some p37'-"05 in mitotic Moloney murine sarcoma virus (MSV)infected cells is coimmunoprecipitated with p34"" and with unidentified 60-, SO-, and 110-kDa phosphoproteins by anti-p34cdc' antibody (Bai et al., 1990).The 60-kDa component is likely to be the 60-kDa mouse cyclin B (Morla et al., 1989), because peptide mapping experiments have established that it is identical to the 60-kDa protein associated with ~ 3 4 " ~in" mitotic cells. These proteins may be part of a 450-kDa complex containing both ~ 3 4 "and ~ an ~37"-""~ that was detected in lysates of MSVtransformed cells following molecular sieve chromatography (Bai et ul., 1990). Most ~ 3 4 " ' ~in these transformed cells is phosphotyrosine deficient and only this form is found in the 450kDa complex. This is in contrast to untransformed cells, in which most ~ 3 4 " ~contains "' phosphotyrosine, which suppresses its activity (see Section II,E,6) (Morla and Wang, 1986; Morla et al., 1989; Draetta et al., 1988). Mitosis-specific phosphorylation and activation of p85gUg-"", a GagMos fusion protein expressed in some MSV-transformed cells, provide another indication of potential relationships between Mos and mitosis. pg5gag-mos is . hyperphosphorylated at serine and threonine residues in nocodazole- or colcemid-arrested MSV-transformed 6m2 rat kidney cells (Liu et al., 1990). The mitosis-specific phosphorylations cause the appearance of additional Mos-immunoreactive bands of 87 and 90 kDa. The location of the phosphorylated residues is unknown, although the gugencoded region contains a sequence matching the chargedl polar-SIT*-PX-basic consensus (Shinnick et al., 1981).There is no significant change in the amount of ~85g~g-""~ protein present in mitosis, but there is a twofold to fourfold increase in its immune complex-specific autophosphorylating activity, and some of the mitosis-specific phosphorylation sites can be phosphorylated in anti-Mos immune complexes (Liu et al., 1990). It is not known if the mitosis-specificincrease in immune complex kinase activity is catalyzed by ~85g"g-""~or by associated ~34'~''kinase. These findings support the hypothesis that interactions between v-Mos and p34"" may be involved in fibroblast transformation. IV. Phosphatases and M Phase

Regulation by transient phosphorylation presupposes the involvement of phosphatases as well as kinases. Tyrosine phosphatase activity is needed for activation of MPF and (probably) pp60'"" during M phase,

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and serine/threonine phosphatase activity is needed for dephosphorylation of M-phase-specific targets following M phase. Genetic studies in yeast and Aspergillus nidulans have demonstrated that proteins homologous to mammalian serinelthreonine protein phosphatase- 1 are required for the completion of mitosis (Booher and Beach, 1989; Doonan and Morris, 1989; Ohkura et al., 1989).The requirement for both serine/ threonine and tyrosine phosphatase activity in animal cell M-phase progression has been demonstrated using phosphatase inhibitors: ( 1) inhibition of protein serinehhreonine phosphatase- 1 and/or phosphatase-2 can modulate MPF activity in cell-free Xenopw extract systems and in starfish oocytes (Cyert and Kirschner, 1988; Picard et al., 1989; Felix et al., 1990; (2) microinjection of inhibitors of protein phosphatase-1 retards progesterone-stimulated Xenopus oocyte meiotic maturation (Huchon et al., 1981; Foulkes and Maller, 1982); and (3) addition of vanadate, a tyrosine phosphatase inhibitor, prevents entry into mitosis (Morla et al., 1989). Treatment of Xenopus oocytes with okadaic acid, an inhibitor of phosphatase-1 and phosphatase-2A (Cohen et al., 1990), induces maturation and activation of MPF (Goris et al., 1989). Similarly, treatment of interphase extracts with okadaic acid stimulates ~ 3 4 ' ~ kinase ' ~ activity (Felix et al., 1990). The mechanism of stimulation is unknown but may be related to the fact that phosphorylation of T h r 167 is required for activation of yeast ~ 3 4 " ~(K. " Gould and P. Nurse, personal communication). This, in addition to inactivation of serine/threonine phosphatases, may account for the stabilization of the mitosis-specific phosphorylations in p 150'-"b' that occurs in okadaic acid-treated cells (Kipreos and Wang, 1990). Okadaic acid treatment induces cell rounding but not chromosomal condensation or nuclear envelope breakdown (Kipreos and Wang, 1990), whereas microinjection of ~34'~'' into fibroblasts causes not only cell rounding and reduced substrate contact (associated with microtubule and actin microfilament reorganization), but also premature chromatin condensation (Lamb et al., 1990). It will be interesting to determine if any of these effects are involved in the ability of okadaic acid to act as a tumor promoter (Suganuma et al., 1988). T h e salient question is the extent to which M-phase regulation depends on changes in phosphatase activity. Microinjected M-phase phosphoproteins are dephosphorylated at similar rates when microinjected into interphase and metaphase Xenopus eggs, implying that the specificity of most M-phase phosphorylation is not due to phosphatase substrate specificity (Karsenti et al., 1987). However, there is an overall increase in phosphatase activity and, hence, phosphate turnover, during M phase, as predicted by the hypothesis that rapid changes in phosphorylation are


2 13

needed to rapidly control the succession of M-phase events (Karsenti et al., 1987). Some potential mechanisms for mitosis-specific regulation of phosphatases have been identified. For example, protein phosphatase- 1 can be indirectly regulated both by cyclic AMP (via a pathway involving protein kinase A-mediated phosphorylation of a protein phosphatase- 1 inhibitor) and by free Ca2+ (Cohen, 1989); the concentrations of both of these factors change dramatically during M phase (Maller, 1985; Hepler, 1989). In addition, the level of an inhibitor of protein phosphatase-1 and phosphatase-2A oscillates in the cell cycle in a manner that suggests that it may play a role in control of ~34'~''activity (Brautigan et al., 1990). V. Discussion

A. Src, DIFFERENTIATION,

AND

MITOSIS

At first glance, the hypothesis that pp6OC-"' plays a role in mitosis seems discordant with the observation that it is present at high levels in differentiated cells such as neural tissue (Brugge et al., 1985), platelets (Golden et al., 1986), and monocytes (Gee et al., 1986) and that it can induce neurite outgrowth from PC 12 cells (Alema et al., 1985). However, it is possible that the same processes are modulated for different purposes in mitosis and differentiation. For example, altered cytoskeletal architecture, increased cell rounding, and reduced cell-cell adhesion are characteristics of mitosis that may be required in motile regions of neurons (e.g., during axon outgrowth). This may account for the concentration of pp60'"" in the rounded and motile growth cones of differentiating neurons (Maness et al., 1988) and in rapidly proliferating and differentiating neural tissues during embryogenesis (Sorge et al., 1984; Fults et al., 1985; Simon et al., 1985; Maness el al., 1986; Ingraham el al., 1989). In this regard, it is interesting that Ser 72, a mitosis-specific phosphorylation site in fibroblast pp60'"", is constitutively phosphorylated in the neuron-specific form of pp60'-"" (D. Middlemas and T. Hunter, personal communication).

B. RELATIONSHIPS BETWEEN M PHASE REGULATION, GI/S REGULATION, A N D NEOPLASTIC TRANSFORMATION Although the full extent of their involvement has not yet been charted, it is clear that some, if not many, oncoprotein kinases play a role in M-phase regulation. Figure 3 summarizes many of the experimental observations that we have reviewed. The graphical implication that these

214


reflect an interacting network of in vzuo control pathways is only speculative, but some possibilities for both positive and negative feedback loops can already be identified. It is expected that the responses of this cybernetic system to perturbations will be highly complex. The interaction of the Mos and Cdc2 proteins provides a good example. Though pp39'-m0sxe appears to activate ~ 3 4 ' ~ "both ' at meiotic metaphase I and 11, at metaphase I this initiates progression and at metaphase I1 this arrests progression. Furthermore, though low-level expression of v-Mos transforms fibroblasts, high-level expression is cytotoxic (Papkoff et al., 1982). possibly because of cell cycle arrest. Toxicity of the v-Abl (Zeigler et al., 1987) and v-Src (Reddy et al., 1988) oncoprotein kinases may have related bases. A principal question is the relationship between these M-phase pathways and the mechanism of neoplastic transformation. Many similarities of the mitotic and transformed phenotypes are apparent. But oncoproteins that can induce transformation by themselves (e.g., pp60'"'') must be capable of directly or indirectly facilitating passage, not only through G2/M but also through GI/S restriction points. At least three potential paradigms are evident: 1. Central regulatory network: Both GI/Sand G2/M restriction points may be controlled by different efferents from a single biochemical control system. 2. Distributed regulatory network: the oncoproteins may participate in separate biochemical control systems that independently control passage through Gl/S and G2/M restriction points. 3. G2/M -+ GI/S regulatory cascade: untimely induction of components of the mitotic phenotype (e.g., alterations in cytoskeletal architecture) during interphase by a constitutively activated oncoprotein may trigger passage through GI/S restriction points (e.g., via feedback mechanisms that respond to cell morphology). Obviously these possibilities are not completely exclusive. The first two models are suggested by the fact that a number of proteins appear to be involved in the control of progression through both M phase and GI/S: (1) Cdc2 is required for passage both through mitosis and through the Start restriction point preceding the G I / S transition in fission yeast (Nurse and Bisset, 1981); (2) pp60"-'" is activated during mitosis but can also enhance responsiveness to EGF-initiated release from serumstarvation-induced Go arrest (Luttrell et al., 1988); (3) S6 kinase I1 is activated both during M phase and following serum or growth factor stimulation of Go-arrested cells (Blenis and Erikson, 1985; Cobb, 1986; Novak-Hofer and Thomas, 1984; Pelech et al., 1986; Tabarini et al., 1985); (4) the retinoblastoma-susceptibility protein is transiently phos-


215

phorylated, probably by p34'd", both in M phase and before the G,/S transition (Buchkovich et aZ., 1989; Chen et al., 1989; DeCaprio et al., 1989); and ( 5 ) studies with temperature-sensitive v-Ras proteins have shown that Ras activity must be present during the GI and G2 phases to drive serum-starved cells through the cycle (Durkin and Whitfield, 1987). The extensive parallelism among the oncoprotein kinase signaling pathways in animal cells is striking. Not only do most of the kinases belong to structurally related families (e.g., growth factor receptor family, Src family, and p42-44 family), but many of them associate with and/or phosphorylate shared target proteins. This characteristic is reminiscent of plastic, adaptable cybernetic systems [e.g., neural networks (Crick, 1989)]and contrasts with the "hard-wired'' paradigm provided by simple allosteric control mechanisms. The parallelism of this kinasel phosphatase network might only reflect straightforward evolutionary elaboration of a number of simple control loops from lower eucaryotes, and a simple underlying structure may remain. Alternatively, the network may have evolved into a multipurpose information processor that can be linked to afferents and efferents in a variety of ways (cf. central versus peripheral nervous system functions). It seems likely that many transient phenomena involving oncoprotein kinases in GI have yet to be discovered. The fact that few have yet been reported may primarily reflect technical limitations. Unlike M phase, wherein the temporal development of precisely synchronized populations can be studied using reversible inhibitors and hormonal stimulation of meiotic maturation, only populations with poor temporal resolution (>1 hr) have generally been available for studies in G I . If such G1specific modifications are as fleeting as some of those observed in mitosis, they will not be detected until sufficiently accurate temporal analysis methods are devised. C. CONCLUSION

Recent discoveries indicate that at least some protooncoprotein kinases participate in the regulation of meiosis and mitosis. Multiple functional connections to p34rdr2,a critical participant in cell cycle control, have been identified. Other, unelucidated signaling pathways involving tyrosine phosphatases are also likely to be involved. T h e role of master kinase is frequently ascribed to ~ 3 4 " ~ In " . this view, the oncoprotein kinases function as slaves or effectors that amplify and transmit p34'dcz-initiated signals by participating in cascades, which ultimately phosphorylate end-point substrates (e.g., cytoskeletal proteins). However, ~ 3 4 ' ~ " is ' itself modulated both directly and indirectly by

2 16


oncoprotein kinase activity, and it also directly phosphorylates end-point substrates such as histone H 1, nucleolins, lamins, and RNA polymerase. It is becoming difficult to distinguish dominance from subservience, and the master/slave paradigm may prove to be of limited utility for organizing our knowledge of cell cycle control mechanisms. Alternative paradigms, perhaps based on more democratic principles, may have greater utility. Only a small fraction of oncogene studies have explored the temporal dimension, and it is likely that many additional interconnections will be discovered as attention continues to be focused on the relationships between M-phase regulation, G I/S regulation, and neoplastic transformation. We anticipate that the interlocking network of kinases and phosphatases participating in M-phase and cell cycle control embodies a sophisticated control system whose complexity has not yet been significantly plumbed.

ACKNOWLEDGMENTS We thank R. B. Arlinghaus, S. Bagrodia, W.Boyle, B. Brott, J. S. Brugge, K. J. Buchkovich, M. Capecchi. 1. Chackalaparampil, J. A. Cooper, S. A. Courtneidge, R. Eisenmann, K. I.. Could, S. Halegoua, S. R. Hann, E. Harlow, T. Hunter, R. Jove. K. Kaplan, E. T. Kipreos, N . Kremer, S. Kusick, S. Kvpta, P. -H. Lin, B. Luscher. J. L. Maller, D. Middlemas, D. Morgan, P. Nurse, T. Pawson, S. Pelech, E. Racker, C. Thacker. H. Ueno. G. F. Vande Woude, H. Varmus, S. Wadsworth, J. Y. Wang, H. S. Wiley, and L. T. Williams for unpublished (as of June, 1990) data and manuscripts, and we thank all of them, R. F. Frisque, and R. A. Schlegel for many helpful comments o n the manuscript. We thank R. Zauhar and the Penn State Biotechnology Institute for assistance with protein data bank searches. Preparation was supported by Public Health Service Grants CA32317, CA47333 and a Research Career Development Award (to D.S.) from the National Cancer Institute.

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CURRENT STATUS OF THE BCR GENE AND ITS INVOLVEMENT WITH HUMAN LEUKEMIA Martin L. Campbell and Ralph B. Arlinghaus Department of Molecular Pathology. The University of Texas M.D. Anderson Cancer Center Houston, Texas 77030

I. Introduction A. Chronic Myelogenous Leukemia B. The Philadelphia Chrornosome C. Biological Activity of P210 BCR/Abl 11. The BCR Gene A. Organization and Genetic Structure B. Expression of BCR 111. BCR Complexes in Hematopoietic Cells A. Complexes Containing ph-P53 B. BCR Protein Complexes C. BCR/Abl Complexes 1V. Concluding Remarks and Future Directions References

I. Introduction

It seems likely that the BCR gene plays a critical role in the pathogenesis of human leukemias that involve the Philadelphia chromosome (Ph'). This altered chromosome was the first karyotypic evidence found to be specifically, characteristically, and consistently present in a human hematopoietic neoplasm, namely, chronic myelogenous leukemia (CML) (Nowell and Hungerford, 1960; Rowley, 1973). Ph' is a chromosomal abnormality resulting from the reciprocal translocation of chromosomes 9 and 22 and is present in greater than 90% of all CML patients, in 17-25% of adults with acute lymphocytic leukemia (ALL) (Priest et al., 1980; Champlin and Golde, 1985), in 5% of children with ALL, and in 5% of adults and children with acute myelogenous leukemia (AML) (Dreazen et al., 1987). Molecular studies indicate that the t[9;22] translocation results in a chimeric gene formed by joining 5' segments of the BCR gene to a 5' truncated form of the abl protooncogene (deKlein et al., 1982; Gale and Canaani, 1984; Shtivelman et al., 1985). Chromosome 9 breakpoints occur over an extended region of DNA sequences in and around the 5' portion of the abl gene. T h e majority of breakpoints on chromosome 9 occur in the intron between two alternate first abl exons 227 ADVANCES IN CANCER RESEARCH, VOL. 57

Copyright Q 1991 by Academic Press, Inc.

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MARTIN L. CAMPBELL AND RALPH B. ARLINGHAUS

(1A and 1B) and in the intron between exon 1A and exon 2 (Heisterkamp et al., 1988). The chromosome 22 breakpoints occur within a restricted portion of the BCR gene termed the breakpoint cluster region (bcr) (Groffen et al., 1984). The fused gene generates a hybrid BCRlabl mRNA (Shtivelman et al., 1985,1986)with a continuous open reading frame that encodes a functional chimeric protein of about 210 kDa (Knopka et al., 1984; Kloetzer et ak, 1985). In Ph'-positive ALL, the breaks within the BCR gene are observed in t w o different locations. About one-third of the cases have breakpoints typical of CML within the bcr region. The other two-thirds of the cases present a more 5' BCR breakpoint (Dreazen et al., 1987) that juxtaposes the first BCR exon to most of the a6l gene, resulting in a chimeric fusion gene product of 185 kDa. However, both chimeric gene products exhibit increased tyrosine kinase activity (Knopka et aE., 1984; Kloetzer et al., 1985; Hermans et al., 1987; Clark el nl., 1987; Kurzrock et al., 1987; Chan et al., 1987; Walker et al., 1987). The repeated occurrence of these BCRI abl rearrangements in these various types of leukemias led us to investigate the structure, function, and role of the normal BCR gene product in hematopoietic cells. In this article we discuss some of our recent findings concerning the presence of'multimeric BCR protein complexes that exist in both normal and leukemic hematopoietic cells and how alterations in the protein composition of these complexes may function to initiate and/or maintain the leukemic state. (In a related article in this volume by Daley and Ben-Neriah, recent studies performed on the Bcrlabl gene product of Ph' and its implications in the pathogenesis of Ph'-positive leukemias are discussed further.) A. CHRONIC MYELOCENOUS LEUKEMIA CML is a human leukemic disease marked by the clonal expansion of pluripotent hematopoietic stem cells. The initial stage of the disease, the chronic phase, is characterized by increased numbers and increased proliferation of cells of the myeloid lineage, typically granulocytes. However, the chronic phase is inherently unstable and ultimately progresses to a more aggressive and uncontrollable form of the disease stage, termed blast crisis. The mean progression time from chronic phase to the development of blast crisis is approximately 3 years. In blast crisis, the proliferating leukemic cells have lost the ability to differentiate, and the relative proportion of immature blastic cell types in the peripheral blood increases. Analysis of cell populations from bone marrow cultures of CML patients in blast crisis typically shows that 299% of the cells are Ph' positive, Additionally, progression into blast crisis is consistently marked by additional karyotypic alterations, including, but not limited to, dupli-

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cations of Ph', trisomy 8, and isochrome 17 (Spiers and Baikie, 1968; Rowley, 1975). The invariable appearance of additional karyotypic abnormalities upon progression into blast crisis suggests that Ph', or, more specifically, the BCRIab1 gene product, may confer some selective advantage over normal Ph'-negative stem cells, but alone is insufficient for the induction of CML blast crisis. The clinical and therapeutic aspects of CML have been extensively covered in previous reviews (Allan, 1989; Kantarjian et al., 1988; Champlin and Golde, 1985).

B. THEPHILADELPHIA CHROMOSOME T h e t[9;22] chromosomal translocation that generates the Ph' chromosome is the specific karyotypic abnormality associated with CML and certain other leukemias (ALL and AML). Because the chromosomal breakpoints occur within introns or nontranslated sequences in both the abl and B C R genes, the subsequent transcription, splicing, and translation of the hybrid BCRlabl gene generate fused but truncated forms of BCR and Abl proteins. This fusion results in an altered but activated 2 10-kDa chimeric protein composed of about 2000 amino acids (P2 10 BCR/Abl). In one study, the overlapping cDNAs for a BCRlabl mRNA have been sequenced; the predicted protein contains 927 amino acids of 5' BCR protein sequences fused to 1096 amino acids of 3' Abl sequences, yielding a protein of 2023 amino acids (Mes-Masson et al., 1986). The expression of P210 BCR/Abl appears to depend on the presence of Ph' and is not myeloid specific. Figure 1 provides a graphic illustration of this translocation as it has been mapped in CML patients. The 5' exons of B C R become fused to most of the abl gene and transcription of this chimeric gene generates an 8.6-kb mRNA in CML (Gale and Canaani, 1984; Shtivelman et al., 1985; Stam et al., 1985). The currently accepted model of the generation of the 8.6-kb mRNA in CML involves correct splicing of the premessenger RNA to fuse truncated B C R and abl sequences into a continuous long open reading frame. There are two known alternate first exons in the human a61 gene. The first abl exon (or exons, depending on where the breakpoint occurs on chromosome 9) is (are) removed during processing of the pre-mRNA as an intronic sequence along with all B C R and abl introns to generate the final 8.6-kb mRNA composed of 3.3 kb of B C R sequences fused to 5.3-kb of abl sequences (Shtivelman et al., 1985). This model of RNA processing is strengthened by the finding that cells from Ph'-positive CML patients that differ in the breakpoints on chromosome 9 by relatively large distances within the abl gene generate BCRlabl mRNAs that join the B C R and abl sequences at exactly the same abZ nucleotide (Shtivelman et al., 1985).

230

9


22 _-

Pd

qN

abl

1

BCR

d

-

5'

-- _-_._.

4"

+

o c 3

9s' I I

10 kb

0

BCR/abl

-

v-ab/ homology

I

0

3'

c--

I 8.7-kb BCR/ab/ mRNA

COOH 21 OK protein

NH2

kinase

FIG. 1. Schematic illustration of the chromosomal recombination event between the abl locus at hand q34 of chromosome 9 and the BCR locus at q l l of chromosome 22 that generates the Ph'. The translocation event is illustrated on the left and the approximate gene structures of the BCR and abl genes are shown on the right. (Modified from Adams, 1985.)

Breaks on chromosome 22 can occur within the BCR gene at two locations (Fig. 2). The breaks in CML occur in introns bounded by several small exons within the central part of the BCR gene (Heisterkamp et al., 1985).Although the breakpoints in the BCR gene in CML are narrowly localized within the 5.8-kb bcr region of chromosome 22 DNA, the translocation breakpoints characterized in ALL patients are sometimes located within the first intron of the BCR gene (termed the minor breakpoint site), which is 5' of the CML region (termed the major breakpoint site) (Fig. 2). The mRNA generated from this type of translocation fuses the first exon of BCR to precisely the same abl sequence found in the CML4kb Y

E

E

E

E E

3' Minor Breakpoint Region ALL

I 5-kb \

,

fbreakpoinl\\ cluster /region in CML\

I

m Bgl II

Bgl II

Major Breakpoint Region CML.ALL

FIG. 2. Schematic illustration of the genetic structure of the RCR gene showing the locations and sizes of intron and exon sequences. Exons numbered 1-5 have been mapped precisely. Slashes indicate a gap in the cloned sequence. (Modified from Hermans el al., 1987. 0 Cell Press.) The size of the first exon is underrepresented. ALL, Ph'-positive acute lymphocytic leukemia; CML, Ph'-positive chronic myelogenous leukemia; E, EcoRI restriction site.

BCR GENE AND P~'-CHROMOSOME LEUKEMIAS

23 1

specific 8.6-kb mRNA, resulting in a 7.0-kb mRNA and a 185-kDa protein (P185 BCR/Abl) (Hermans et al., 1987; Kurzrock et al., 1987). It has been shown that both the P210 and P185 BCR/Abl proteins and the viral oncogenic product of the Abelson murine leukemia virus, P160 Gag/ Abl, possess an activated tyrosine kinase activity as measured by in nitro kinase assays (Knopka et al., 1985; Kloetzer et al., 1985; Kurzrock et al., 1987; Davis et al., 1985). Recent findings by Lug0 et al. (1990) suggest that foreign upstream sequences may be important in the deregulation of the kinase activity of the Abl product. Furthermore, quantitative analysis of both tyrosine kinase activity and transformative potency indicates that the variations in BCR exon content result in functional differences of the P210 BCR/Abl and P185 BCR/Abl proteins. (See Daley and Ben-Neriah, this volume, for an extensive discussion on the BCR/Abl protein activity in nitro and in nivo.) C. BIOLOGICAL ACTIVITY OF P210 BCR/Abl T h e mechanism by which P2 10 BCR/Abl induces malignant transformation is poorly understood. A retroviral construct of P210 BCR/Abl was able to transform immature B lymphoid cells but only after long-term culture (McLaughlin et al., 1987; Young and Witte, 1988). Further, introduction of the BCRIab1 gene into NIH 3T3 cells did not result in any marked cellular transformation or tumorigenicity of this cell type (Daley et al., 1987). The production of transgenic mice carrying a similar construct of BCRlv-abl has shown that lymphomas of both T and pre-B cell types are induced (Hariharan et al., 1989). Analyses of these lymphomas showed that the tumors were clonal in origin, suggesting that transformation leading to tumorigenesis is not only a rare occurrence but that there is an additional requirement for secondary cellular events (also infrequent in their occurrence) before the lymphoma can develop. Studies performed to date on the product of the Ph' indicate that the t[9;22] translocation results in activation of the cellular protooncogene abl and that this activation is strongly implicated in the pathogenesis of these types of leukemic diseases (reviewed in Mes-Masson and Witte, 1987; Groffen et al., 1987; Rosson and Reddy, 1988; Sandberg et al., 1986). It is also clear that the repeated involvement of the BCR gene in the t[9;22] translocation that generates Ph' implicates the BCR gene in an as-yet not understood, but equally important way, in the pathogenesis of CML and other Ph'-positive leukemias. Animal models that resemble Ph'-positive leukemias have recently been reported. In one system, a retrovirus encoding P210 BCR/Abl was inserted into mouse bone marrow cells that were subsequently transplanted into irradiated syngeneic mice (Daley et al., 1990). Trans-

232


plant recipients developed several hematologic malignancies, including a myeloproliferative syndrome closely resembling the chronic phase of human chronic myelogenous leukemia. Tumor tissue from diseased mice harbored the provirus encoding P2 10 BCR/Abl. These results demonstrate that P2 10 BCR/Abl expression can induce chronic myelogenous leukemia in mice, providing a convenient mouse model for further analysis of BCR/Abl involvement in this disease. In a study by Heisterkamp et al. (1990), transgenic mice were generated that harbor a BCRIab1 construct encoding the P185 BCR/Abl (the form associated with Phi-positive ALL). Progeny mice are either moribund with, or die of, acute leukemia (myeloid or lymphoid) 10-58 days after birth. These findings are evidence for a causal relationship between the Philadelphia chromosome and human leukemia. These animal models provide powerful systems to investigate the role of the BCRlabl gene in human leukemia. Site-directed mutants within BCR and abl coding regions can now be evaluated for their diseasecausing potential in these systems. These animal models are discussed in detail in Daley and Ben-Neriah (this volume). II. The BCR Gene

A. ORGANIZATION AND GENETIC STRUCTURE The humanBCR gene is located on the long arm (4) of chromosome 22 at band qll.21. The gene extends over 90 kbp of DNA and contains multiple exons (Fig. 2). The first exon is large and is separated from the second exon by an intron sequence of approximately 68 kbp (Heisterkamp et al., 1988). The breakpoints on chromosome 22 in Ph'-positive ALL are frequently mapped to areas within this first intron sequence (minor breakpoint region) (Hermans et al., 1987). The chromosomal translocation breakpoints that are typical of CML occur more than 50 kb downstream of the first exon within the major breakpoint region. This bcr region contains four exons, historically termed bl-b4. The 5' end of the BCR gene is oriented toward the centromere of chromosome 22, and it, as well as the BCR promoter and transcriptional regulatory sequences, is retained on chromosome 22 following the t[9;22] translocation. In addition to the original BCR gene, there are three ECR-related genes that map to chromosome 22: BCRB, BCR 3, and BCR 4 (Croce et al., 1987), and a functionally active BCR-related gene termed ABR that maps to chromosome l'ip (Heisterkamp et al., 1989). Furthermore, a cDNA from human brain tissue contains 3' sequences that are homologous to 3' BCR exons. The 5' end of this gene also possesses homology to the C1 regulatory region of protein kinase C (Hall et d.,1990) (Table I).

BCR GENE AND

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The three BCR-related genes on chromosome 22 are believed to have been generated by mechanisms of gene duplication, as all three genes are lacking 5' sequences that are present in the intact BCR gene (Lifshitz et al., 1988). However, the high degree of conservation of both intron and exon sequences within these genes makes it difficult to exclude the possiTABLE I BCR GENES,TRANSCRIPTS, A N D PROTEINS ~~

~

Genes" BCR I (complete) BCR-related genes (functional?) BCR 2 BCR 3 BCR 4 ABR n-Chimaerin

~ _ _ _ _ _

Transcripts

~

~ _ _ _ _ _ _

Proteins'

7.0 kb

1901185 kDa"

4.5 kb ( 4.5 kb (2) 4.0 kb" 1.0 kt6 5.0,4.8 kbk 2.2 kb'

160 kDa 1351125 k D d 108 kDa 83 kDa

-

34 kDaJ

a All BCR genes have been mapped to Chromosome 22 (human) (Croce et al., 1987). Additionally, Heisterkamp and Croffen (1988b) have identified BCRrelated sequences on chromosome 22 that are likely equivalent to theBCR-related genes BCR 2 and ECR3 identified by Croce etal. (1987) and they have identified a functionally active BCR-related gene on chromosome 17p that they have termed ABR (Heisterkamp et al., 1989). T he chimaerin gene has not been localized to a specific chromosome, but encodes a message of 2.2 kb in human brain tissue, the 3' end of which is related to human BCR 3' sequences (Hall etal., 1990). As indentified by Li et al. (1989a). There is no evidence to equate an individual BCR protein with a particular BCR transcript except for P160 BCR, which is derived from one of the 4.5-kb transcripts. Campbell et al. (1990)has evidence for the existence of two forms of the P160 BCR protein. Further studies are required to determine the structural difference between these two proteins. Ben-Neriah el al. (1986) have detected a 190-kDa protein and Stam et al. (1987) have identified a BCR protein of 180 kDa in addition to the PI60 BCR protein. Two types of 4.5-kb cDNAs have been identified to date. One BCR transcript (Hariharan and Adams, 1987) is predicted to encode a protein of 1271 amino acids, and a second somewhat smaller cDNA has been sequenced (Lifshitz et al., 1988) that lacks 44 codons begining at amino acid 960 and is predicted to encode a protein of 1227 amino acids. ' Th e 4.0-kb transcript identified by Lifshitz et al. (1988) is presumed to be identical to the 3.0-kb transcript seen by Collins et al. (1987) in some cell lines. f Dhut et al. (1988) and Amson et al. (1989) have identified a BCR protein of 130 kDa. g Identified by Collins et al. (1987) in chick testes. These transcripts are related to the ABR gene locus described by Heisterkamp e l al. (1989). a This transcript corresponds to the n-Chimaerin cDNA described by Hall et al. (1990). I The 34 kDA protein is the n-Chimaerin brain protein described by Hall et al. (1990) and possesses BCR homology at its C-terminal end.

'

234


bility that these three BCR-related gene sequences encode functional gene products. T h e order of these genes on chromosome 22 is centromere, BCK 2, BCR 4, BCR 1 , and BCR 3. Heisterkamp and Groffen (1988)have identified BCR-related sequences within two loci on chromosome 22 generated by independent duplications into DNA sequences of the y-glutamyl transpeptidase-dependent gene family. These sequences were also found to be highly conserved among human, chimpanzee, and gorilla (but not mouse), again suggestive of functionality. The translocation leading to Ph' appears to involve breakpoints only in BCK 1 (Croce et al., 1987). The presence of these multiple 3' BCR-related sequences on chromosome 22 and elsewhere indicates that the BCR gene is susceptible to chromosomal rearrangement events not only at the central major breakpoint region, but also 3' and 5' (minor breakpoint region) to the major breakpoint region. Previous analyses of the known BCK gene cDNA sequence and the predicted amino acid sequence have failed to detect any notable homology between BCR and any gene previously characterized (Fig. 3). Hydropathy analysis of the predicted amino acid sequence does not reveal any likely transmembrane domain that, in conjunction with the absence of an N-terminal myristylation site, suggests that the BCR protein is not associated with the cell membrane. However, a careful examination of the predicted protein sequence reveals a consensus ATP-binding site (Lifshitz el al., 1988) and a near-consensus phosphotransfer domain (Fig. 4) (Arlinghaus and Li, 1988) located within the first exon. Residues 163- 190 are similar in pattern to the consensus ATP-binding sequences, and the lysine (K) at position 190 is suitably positioned within an apparent ATP-binding domain (Lifshitz et al., 1988). However, protein kinases are typically characterized by hydrophobic residues at positions 1 and 7 upstream from the tirst Gly in the consensus sequence and by a Val two residues downstream from the last Gly (S. Hanks of the Salk Institute, personal communication). Downstream of this ATP-binding site-like sequence (163- 190) at residues 306-355 is a pattern of sequences that resembles the consensus phosphotransferase domain of protein kinases. Although the 114-1 18 residue gap in BCR between the putative ATP-binding and the phosphotransferase domain is typical of protein kinases, several sequences conserved among various protein kinases do not appear to be present in the BCR putative phosphotransferase domain, as reviewed by Hanks el al. (1988). For example, the DCG domain in BCR (residues 323-325) differs from the consensus DFG domain. Similarly, the BCR TPD (residues 329-331) differs from the consensus APE domain in protein kinases (S. Hanks, personal communication). Other sequence differences also exist. Nevertheless, the sequence comparisons are intriguing and suggest that

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P~'-CHROMOSOME LEUKEMIAS

235

BCR gene products have structural elements resembling those found in protein kinases. The possibility that P160 BCR is a kinase receives support from the observations of Stam and co-workers (1987). They showed that antibodies directed toward the amino-terminal portion of BCR prevented phosphorylation of PI60 BCR in kinase assays, whereas Cterminal antibodies allowed phosphorylation. More recent work by Li et al. ( 1989a) using site-directed antipeptide antibodies demonstrated that immune complexes of BCR proteins have an associated serine/threonine kinase activity. Timmons and Witte (1989) also detected protein kinase activity associated with PI60 BCR that was overexpressed in mammalian cells. The placement of the kinase domain within the first exon of BCR (suggested by Li et al., 1989b) is also consistent with the observations of Stam et al. (1987), who showed that antisera directed against a large fragment within the 5' portion of the BCR gene inhibited the in vitro kinase activity. T h e position of this putative kinase domain in the first exon of BCR further strengthens the role for this gene in Ph'-positive leukemias, as this domain is retained in the chromosomal translocations that generate both the CML-specific P210 BCR/Abl protein and the ALL-specific P185 BCR/Abl protein. I f BCR does encode a protein kinase, it is an evolutionary offshoot that differs from the consensus patterns characterized to date for either the serinelthreonine or tyrosine protein kinases. In this regard, novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, have been detected in human cells, defining a new class of protein kinase (Wilks et aE., 199 1). B. EXPRESSION OF BCR The normal BCR gene product is expressed in all human cell lines tested thus far, including Ph'-positive leukemic cell lines. Probes from the human BCR gene hybridize to fragments of chimpanzee, gorilla, mouse (Heisterkamp and Groffen, 1988), and chicken DNA when analyzed on Southern blots, indicating a high degree of evolutionary con-

FIG.3 (Overleaf). Nucleotide sequence of the BCR sequence, compiled from published sequences of Hariharan and Adams (1987) and Heisterkamp et al. (1985). The predicted open reading initiates at the ATG codon at position 535. The compiled sequence is nearly identical to the sequence published by Lifshitz et al. (1988). Variations occur at the 5' untranslated end and at selected nucleotides. The sequence shown in this figure includes the nucleotideiamino acid sequence for exon "5"(Heisterkampel al., 1985),which was not present in the cDNA clone sequenced by Lifshitz et al. (1988). (Reprinted by permission from Nature, 315, 758-761, copyright 0 1985 Macmillan Magazines Ltd.; and E M R O J . 6, 115-1 19, copyright 0 1987 Oxford University Press.)

MAAAAI~AAAAAAAAAMAAAAAAAAAAAAAAlUAAAATAGGTAGGAGTAGCGTGGTMGGGCCATCAGTGTGGGCCGGGCGGCAGI

102

GCGGCGAMGCCGGCTGGCTGAGCTTAGCGTCCGAGGAGGCGGCGGCGGCGGCGGCGGCAGCGGCGGCGGCGGGGCTGTGGGGCGGTGCG~GC~~GGC 204 ~GGAGCGCGCGGGCCGTGGCCAGAGTCTGGCGGCGGCCTGGCGGAGCGGAGAGCAGCGCCCGCGCCTCGCCGTGCGGAGCAGCCCCGCACACMIAGCGGC

306

GCGCGCAGCCCGCGCCCTICCCCCCGGCGCGCCCCGCCCCGCGCGCCGAGCGCCCCGCICCGCCTCACCTGCCACCAGGCAGTGGGCGGGCATTGTTCGCCG

408 510

CCGCCGCCGCCGCGCGGGGCCATGGGGGCCGCCCGGCGCCCGGGGCCGGGCCTGGCGAGGCCGCCGCGCCGCCGCTCACACGGGCCCCGCGCG~GCCCGGC

612 SerValGlyAspIleGluGlnGluLeuGluArgCysLysAlaSerIleArgArgLeuCluClffil~alAs~l~lUrgPheAr~etIlelyrLeuCln 714 TCAGTGGGCGACATCMGCAGGAGCTGGAGCGCTGCMGGCCTCCATTCGGCGCCTGGAGCAGMGGTGMCCAGGAGCGCITCCGCATCAICIACCTGCAG

816 SerArgProGlnProAlaProAlaAspGLyAlaAspProProProAlaGluGluPr~luA~aAr~r~s~l~l~l~erPr~lyLysAlaArgPro TCGCGCCCGCAGCCAGCGCCCGCCGACGGAGCCGACCCGCCGCCCGCCGAGGAGCCCGAGGCCCGGCCCGACGGCGAGGGTICICCGGGTMGGCCAGGCCC

918

G 1yThrA LaArgArgProG1yAl aA 1aA 1aSerG 1yG 1uArgAspAspArgGLyProProA 1aSerValA 1aA 1aLeuArgSerAsnPheG1uArgI teArg GGGACCGCCCGCAGGCCCGGGGCAGCCGCGTCGGGGMACGGGACGACCGGGGACCCCCCGCCAGCGIGGCGGCGCTCAGGICCMCTTCGAGCGCATCCGC

1020

LysG 1yH isG 1yG1nProG 1yAl aAspA 1aG 1uLysProPheTyrVa1AsnVal G 1uPheHisH isG 1uArgG1yLeuVa 1LysValAsnAspLysG LuVal AAGGGCCATGGCCAGCCCGGGGCGGACGCCGAGAAGCCCTICTACGTGAACGTCGAGTITCACCACGAGCGCGGCCTGGTGMCGTCMCGACAMGAGGTG

1122

SerAspArgI t eSerSerLeuG1ySerG 1nA 1aMet G l M e t G LuArgLysLysSerG1nHisG 1yAlaG1ySerSerVal GL yAspA1aSerArgProPro TCGGACCGCATCAGCICCCIGGGCAGCCATGCACATGCAGATGGAGCGCAAAAAGTCCCAGCACGGCGCGGGCTCGAGCGIGGGGGATGCATCCAGGCCCCCI

1224

1326

G L yG 1ySerArgProProT rpProProLeuG IuT yrG 1nProTyrG LnSer I1eTyrVa1G l yGl WetMetG 1uG 1yClUG1yLysGlyProLeuLeUrg GGCGGTAGCAGGCCCCCITGGCCGCCCCTGGAGIACCAGCCCIACCAGAGCATCTACGICGGGGGCAIGATGGMGGGCAGGGCMGGGCCCGCTCCTGCGC

1428

SerGlnSerThrSerGluGlnGluLysArgLeuThrTrpProArgArgSerTyrSerPr~rgSerPheGluAs~ysGl~l~lyTyrThrPr~s~ys AGCCAGAGCACCTCTGAGCACGAGAAGCGCCTTACCTGGCCCCGCAGGTCCTACTCCCCCCGGAGTTTTGAGGATTGCGCAGGCGGCTATACCCCGGACTGC

1530

SerSerAsnGluAsnLeuThrSerSerGluGluAspPheSerSerGlyGlnSerSerArgValSerProSerProThrThrTyrAr~etPheAr~splys AGCTCCMTGAGAACCTCACCTCCAGCGAGGAGGACTTCTCCTCTGGCCAGTCCAGCCGCGTGTCCCCMGCCCCACCACCTACCGCATGTTCCGGGACAM 1632

1734 GluAlaThrlleValGlyValArgLysThrGl~lnIleTrpProAsnAspGlyCluGlyAlaPheHisGlyAspAro GAGGCCACCATCGTGGGCGTCCGCAAGACCGGGCAGATCTGGCCCAACCATGGCGAGGGCGCCTTCCATGGAGACGCAGATGGCTCGTTCGGMCACCACCACCT

1836

G 1yTyrG LyCysA 1aA LaAspArgA1aG 1uG 1uG LnArgArgHisG1nAspG lyLeuProTyr11 eAspAspSerProSerSerSerProWisLeuSerSer GGATACGGCTGCGCTGCAGACCGGGCAGAGGAGCAGCGCCGGCACCAAGATGGGCTGCCCTACATTGATGACTCGCCCTCCTCATCGCCCCACCT~GCAGC

1938

LysGlyArgGlySerArgAs~laLeuValSerGlyAlaleuGluSerThrLysAlaSerGluLeuAspLeuGluLysGlyL~l~etArglysTr~al MGGGCAGGGGCAGCCGGGATGCGCTGGTCTCGCGAGCCCTGGAGTCCACTAAAGCGAGTGAGCTGGACTTGGAAAAGGGCTTGCACATGAGAAAATGGGTC

2040

LeuSerGlyIleLeuAlaSerGluGluThrTyrLeuSerHisLeffiluAlaLeuLeuLeuPr~etLysProLeuLysAl~~aAlaThrThrSerG~~ro 2142 CTGTCGGGAATCCTGGCTAGCGAGGAGACTTACCTGAGCCACCTGGAGGCACTGCTGCTGCCCATGMCACGCCTTTGMAGCCGCTGCCACCACCTCTCAGCCG ValLeuThrSerGlffilnlleGluThrILePhePheLysValProCluLeuTyrGLulleHisLysGluPheTyrAspclyLeuPhePr~rgValGlffiln GTGCTGACGAGTCAGCACATCGAGACCATCTTCTTCMAGTGCCTGAGCTCTACGAGATCCACMGGAGTTCTATCATGGGCTCTTCCCCCGCGTGCAGCAG

2244

TrpSerHisGlnGlnArgValGlyAspLeuPheGlnLysLeuAlaSerGlnLeuGl~alTyrAr~laPheValAs~snTyrGlyValAl~etGl~et 2346 TGWGCCACCAGCAGCGGGTGGGCGACCTCTTCCAGAAGCTGGCCAGCCAGCTGGGTGTGTACCGGGCCTTCGTGGACMCTACGGAGTTGCCATGGAMTG

AlaGluLysCysCysGlnAlaAsnAlaGlnPheAlaGlulleSerGluAsnLeuArgAl~rgSerAsnLysAspAlaL~spProThrThrly~s~er 2448 GCTGAGMGTGCTGTCAGGCCMTGCTCAGTTTGCAGAMTCTCCGAGAACCTGAGAGCCAGMCACGCMCAMGATGCCMGGATCCMCGACCMGMCACCTCT LeuGluThrLeuLeuTyrLysProValAspArgValThrArgSerThrLeuValL~Hi~spleuLeulysHisThrPr~laSerHisPr~s~isPro CTGGMACTCTGCTCTACMGCCTGTGGACCGTGTGACCAGGAGCACGCTGGTCCTCCATCACTTGCTCMGCACACTCCTGCCAGCCACCCTGACCACCCC 2550

FIG. 3. (Continued.)

LeuLwGLrrAspAlaLwArgILeSerGLnAsnPheLeuSerSerlLeAsnCluGLuILeThrPrdrgArgGlnSerHetThrValLysLysGlyCluHis TTGCTGCAGGACGCCCTCCGCATCTCACAGMCTTCCTGTCCAGCATCAATGAGGAGATCACACCCCGACGGCAGTCCATCACGGTGMGMGCGACACCAC 2652

ArgGLnLeuLeuLysAspSerPheMetValGLuLeuValGluGlyAlaArgLysLeuArgHisValPheLe~heThrGluLeuLeuLe~ysThrLysL~

2756

CGGCAGCTCCTGAAGGACAGCTTCATGGTGCAGCTGGTGGAGGGGGCCCGCMGCTGCGCCACGTCTTCCTGTTCACCCAGCTGCTTCTCTGCACCMGCTC

LysLysGLuSerGlyClyLysThrGlnClnTyrAspCysLysTrpTyrIleProLeuThrAspleuSerPheGL~etValAspCLuLeffiluAlaValPro MGAAGCAGAGCGGAGGCAAAACGCAGCAGTATGACTCCAAATGGTACATTCCGCTCACGCATCTCAGCTTCCAGATGGTGCATGAACTGGAGGCAGTGCCC 2856

AsnlleProLeuValPrdspGluGluLeuAspAlaLeuLysIleLysILeSerGLuILeLysSerAspIlffilrrArgGLuLysArgALaAsnLysGl~er

2958

MCATCCCCCTGGTGCCCGATGAGGAGCTGGACGCTTTGMCATCMGATCTCCCACATCMCAGTCACATCCACACACAGMGAGGGCGAACMGGGCAGC

LysAlaThrGLuArgLeuLysLysLysLeuSerGLuGL~luSerLeuLeuLeuLeMetSerProSerHetAlaPh~rgValHisSerArgAsffiLyLys

5060

MGGCTACGGAGAGGCTGMGMGAAGCTGTCGCACCAGGAGTCACTGCTGCTGCTTATGTCTCCCAGCATGGCCTTCAGGGTGCACAGCCGCMCGGCMG N

w

cc

SerTyrThrPheLeulleSerSerAspTyrGluArgALaGLuTrpArgGluAsnlleArgGluGlffilnLysLysCysPheArgSerPheSerLeuThrSer AGTTACACGTTCCTGATCTCCTCTGACTATGAGCGTGCAGAGTGGAGGGAGMCATCCGGGAGCAGCAGMGMGTGTTTCAGMGCTTCTCCCTGACATCC

3162

ValGLuLeuGl~etLeuThrAsnSerCysValLysLe~lnThrVaLHisSerIleProLeuThrILeAsnLysGluAspAspGluSerProGlyLeuTyr

3264

GTGGAGCTGCAGATGCTGACCMCTCGTGTGTGMACTCCAGACTGTCCACAGCATTCCGCTCACCATCMTMGGMCATCATCAGTCTCCGGGGCTCTAT

3366 AlaLysThrArgValTyrArgAspThrALaGluProAsnTr~snGluGL~heGluIleGluLwGlffilySerGLnThrLe~rglleLeuCysTyrGlu G

C

M

A

G

A

C

C

C

G

C

G

T

C

T

A

C

A

G

G

G

A

C

A

C

A

G

C

T

G

A

G

C

C

A

A

C

T

LysCysTyrAsnLysThrLysl 1eProLysGL uAspCL yG L uSerThrAspArgLeMetGlyLysG1yC 1nVa LG LnLeuAspProGLrrA L sLeuG LtMsp MGTGTTACAACAAGACGMGATCCCCAAGGAGGACCCCGACAGCACGGACACACTCATGGGGMGGGCCAGGTCCAGCTGCACCCGCAGGCCCTGCAGGAC

3468

3570

ArgAspTrpClnArgThrValILeALaHetAsffilylLeGluValLysLeuSerValLysPheAsnSerArgGL~heSerLeuLysAr~etProSerArg AGAGACTGCCAGCGCACCGTCATCGCCATGAATCGGATCGMGTAMGCTCTCGGTCMGTTCMCAGCAGGCAGTTCAGCTTCMCAGCATGCCGTCCCCA

Mn

LysGl nThrG 1 yVa 1PheG 1yVa 1 Lysl 1eA 1aVa LVa 1ThrLysArgGLuArgSerLysVa1TyrVa 1 I 1eVa LArgG 1nCysVa 1G L f f i l u I 1eG 1uArg ~CAGACAGGGGTCTTCGGAGTCAAGATTGCTGTGGTCACCAAGAGAGAGAGGTCCMGGTGCCCTACATCGTGCGCCAGTGCGTGGAGGAGATCGAGCGC

3774

ArgGLyHetGluGluValGlylleTyrArgValSerGlyValAlaThrAsplleGlnAlaLeuLysALaAlaPheAspValAsnAsnLysAspValSerVal CCAGGCATGGAGGAGGTGGGCATCTACCGCGTGTCCGGTGTGGCCACGGACATCCAGGCACTGMGGCAGCCTTCGACGT~TM~GGATGTGTCGGTG 3876

MetMetSerGluHetAspValAsnALalleAlaGlyThrLeuLysLeuTyrPheArgGluL~r~l~roL~heThrAs~l~heTyrProAs~he ATGATGAGCGAGATGGACGTGAACGCCATCGCAGGCACGCTGMGCTGTACTTCCGTGAGCTGCCCGAGCCCCTCTTCACTGACGAGTTCTACCC~CTTC 3978 AlaGluGLylleALaLeuSerAspProValAlalysGluSerCysMetLeuAsnLeuLeuL~SerL~r~L~L~snL~L~ThrPh~~heL~ GCAGACGGCATCGCTCTTTCAGACCCGGTTGCAAAGGAGAGCTGCATGCTCAACCTGCTGCTGTCCCTGCCGGAGGCCMCCTGCTCACCTTCCTTTTCCTT

4080

LeuAspHisLeuLysArgValAlaGluLysGluAlaValAsnLysMetSerLeuHisAsnLe~laThrValPh~l~roThrLeuL~rgProSerGlu CTGGACCACCTGAMAGGGTGGCAGAGAAGGAGGCAGTCAATMGATGTCCCTGCACAACCTCGCCACGGTCTTTGGCCCCACGCTGCTCCGGCCCTCCGAG

4182

LysGLuSerLysLeuProAlaAsnProSerGlnProlleThrMetThrAspSerTrperLeffiluValMetSerGlnValGlnValLeuL~TyrPheLeu AAGGAGAGCAAGCTCCCTGCCAACCCCAGCCAGCCTATCACCATGACTGACAGCTGGTCCTTGGAGGTCATGTCCCAGGTCCAGGTGCTGCTGTACTTCCTG

4284

GlnLeuGluAlalleProAlaProAspSerLysArgGlnSerIleLeuPheSerThrGluVal CAGCTGGAGGCCATCCCTGCCCCGCACAGCMGAGACAGAGCATCCTGTTCTCCACCGMGTCTAMGGTCCCAGTCCATCTCCTGCAGGCAGACAGATGGC

4386

CTGGAAACCTCTGCCTMTCGGGCCATCCGTAGAGCGGAACCTTCCTGAGGTGTCCTTGGCCCACCCCCMGTGTTGGGCCATCTGCCMGAGACAGC~C 4488 CC.MAGCCGAAGGACAGGTGGCCTGGGCAGATCTCGCCCAGGTCTGGGAGCCCCAGGCTGGCCTCAGACTGTGGTTTTTTATGTGGCCACCCCAGGGCCCCC

4590

CAAGCCAGTTCATCTCAGAGTCCAGGCCTGACCCTGGGAGACAGGGTGAAGGGAGTGATTTTTATGMCTTAACTTAGAGTCTAMAGATTTCTACTGGATC

4692

ACTTGTCAAGATGCGCCCTCTCTGGGGAGAAGGGAACGTGACCGGATTCCCTCACTGTTGTATCTTGMTAMCGCTGCTGCTTCATCCTGTAAAAAAAAM

4794

AAAAAAAAAAAAMAAMAAAAAAAAAAAAAAAAAAM

FIG. 3. (Continued.)

240

MARTIN L. CAMPBELL A N D RALPH B. ARLINGHAUS

I

11

111

IV

V

V II

VI

CONSENSUS:

1G.G.

C-@&:

148 LG.G

m:

163 355 KG.C. . G . D . . (16). . . L . K . . .(103). ... G P L . . .Q. .(22)..DCG.. . . ( 3 ) . ...7PO.. ( 1 1 ) . . E . F . . G . . ( 3 ) . . R . . P ........................................... I92AA .............................................

.c..V. .(9-18)..A.K. .(92-106). . . ~ D L . ..N..(l2)..DFG..(19-25)..r~E..(ll)...D.u. 186-216 AA

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

.....

.G..(8-9)...c..P

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

339

..G . V ...( 12) ...A . K .... (90) ....ROL ...N . . ( 1 2 ) . . O F G .... ( ........................................... 1g1..........

.

.

.

FIG. 4. Comparison of consensus protein kinase domains to putative doinains in the Abl and BCR proteins. The single-letter code for amino acids is used. Numbers in parentheses represent amino acid gaps in the sequence. Small capital letters listed in the C O I I S ~ ~ S U S sequence identify amino acid residues that are not strictly conserved in most protein kinases. The consensus sequence was taken from Hunter (1 987) and Hanks et al. (1988). The Abl sequence was taken from Hunter and Cooper (1986). BCR sequences that resemble ATP and phosphotransferase domains were as described by Li el al. (1989a). (Reproduced from Arlinghaus, Involvement of the abl proto-oncogene in human leukemia. In “Genes and Cancer” [D. Carney and K. Sikora, eds.]. Wiley, New York. 0 1990.

servation, and BCR transcripts have been detected in chicken RNA (Collins et al., 1987). Northern blot hybridizations demonstrated approximately equal amounts of BCR transcripts in cultured human cells of hematopoietic T lymphoid, B lymphoid, myeloid, and erythroid origins and in nonhematopoietic cultures of skin and bone marrow fibroblasts. The expression of BCR transcripts did not vary in blood mononuclear cells whether the cells were resting or actively proliferating. Analyses of chicken tissues for expression of BCR revealed that BCR transcripts were highest in brain tissue and lowest in liver tissue, with a unique truncated BCR transcript found in testes (Collins P t al., 1987). The human BCR gene is predominantly transcribed into major KNA species of 7.0 and 4.5 kb; a minor 4-kb transcript (Lifshitz et al., 1988) has also been detected (Table 1). Although the 4.0-kb transcript appears to lack some central sequences, the differences between the major and the variable minor transcripts are unknown. RCR transcripts of both 7 and 4.5 kb have been seen in cells of hematopoietic (SMS-SB, HL-60, KG-1, Raji, U-937, and Molt-4), fibroblastic (SV80 and Wish), epithelial (HeLa and A43 I), and iieuronal origin (AB-32). These transcripts have been seen in embryonic tissue as well. BCR transcripts of 6 , 4, and 1 kb have been also detected in chicken tissues (Collins et al., 1987). Several groups have cloned and sequenced normal BCR cDNAs (Heisterkamp et al., 1985; Lifshitz et al., 1988; Hariharan and Adams, 1987; Mes-Masson et al., 1986).The results indicate that the 4.5-kb RNA

ECR GENE AND

Ph’-CHROMOSOME LEUKEMIAS

24 1

may be composed of two long open reading frames of slightly different size. T h e larger would code for a protein of 1271 amino acid residues. The smaller would code for a protein of 1227 residues. The latter RNA lacks 44 codons beginning at amino acid 960 (Lifshitz et al., 1988). The sequence of the larger 4.5-kb RNA illustrated in Fig. 3 has been determined. The data indicate a single, long, open reading frame of 3813 nucleotides encoding a protein of 127 1 amino acids. The 5‘ untranslated region of the gene upstream from the presumed translation initiation site is extremely guanine (G)/cytosine (C) rich. Greater than 80% of the nucleotides within this region are either G or C. The transcription initiation codon is more than 500 bases 5’ of the presumed translation initiation codon (Hariharan and Adams, 1987). Three GC-rich regions positioned both 5’ and 3’ to the transcription initiation site (Mes-Masson et al., 1986) match the consensus sequence corresponding to the binding site of the transcriptional regulating factor Spl (Dynan et al., 1986). RNA sequences with greater than 100 bases of untranslated nucleotides having a high GC content are thought to be rare in mammalian systems. mRNAs that have been characterized with this type of RNA structure are believed to encode rare or transient protein products such as protooncogenes and growth factors (Kozak, 1987, 1988). Additionally, sequences that possess a high GC content are capable of forming extremely stable secondary loop structures that have been shown to be inhibitory to translation both in uitro and in uiuo (Kozak, 1986, 1988; Pelletier and Sonnenberg, 1985, 1987; Pelletier et al., 1988). The 5’ untranslated sequence of BCR contains a pair of inverted repeats at positions - l 13 to - 130 and -376 to - 393. These repeats are likely to form stable stem loop structures and could play a translational regulatory role of BCR mRNA in uivo. Muller and Witte (1989) have recently demonstrated the inhibitory effect of the 5’ untranslated sequence on translation of a BCRlabZ construct in an in uitro translation system. BCR gene products of 160 and 180 kDa (Stam el al., 1987), 190 kDa (Ben-Neriah et al., 1986), and 130 kDa (Dhut et al., 1988; Amson et al., 1989) have been observed. Although the 160-kDa protein (termed P160 BCR) has been considered as the principal gene product, several BCR proteins are likely to be expressed in human cells (Table I). Work in our laboratory has provided evidence of the presence of BCR proteins of 190/185 kDa, 155 kDa (P160 BCR), and 135, 125, 108, and 83 kDa in hematopoietic cells (Li et al., 1989a) by using two distinct site-directed anti-BCR antibodies termed anti-BCR(738-753) and anti-BCR(89891 1). The 190/185-, 155-, 125-, and 108-kDa proteins were consistently detected by anti-BCR(898-9 11) by immunoblotting. These proteins can be detected by labeling intact cells with [32P]orthophosphate and in uitro by [ Y - ~ ~ P I A T inPimmune complex kinase assays performed with anti-

242


BCR antibodies, indicating that these proteins are phosphoproteins. Phosphoamino acid analyses performed following labeling in kinase assays detected the presence of both phosphoserine and phosphothreonirie in BCR proteins. In structural studies using one-dimensional peptide maps derived from V8 proteolytic digestion of' these proteins, several fragments were found to be conserved among the lt35-, 155-, 135-, 125-, and 108-kDa BCR proteins, but each contained unique fragments as well. Similarly, two-dimensional maps of proteins labeled in uztro kinase assays exhaustively digested with trypsin revealed homology between the 155-, 135-, 125-, 108-, and 83-kDa proteins (Li et aE., 1989a). The origin of these multiple BCR proteins is still under investigation. T h e detection of several different sizes of BCR transcripts in human cell lines could provide a possible explanation for the presence of multiple BCR proteins. I t is well known that primary transcripts of many genes are processed to produce more than one kind of mRNA. In Drosophila, splicing is used to turn expression of gene products on and off (Bingham et a[., 1988). Similarly. differential splicing of a hierarchy of regulatory genes determines the sex of this insect (Baker, 1989),Thus, transcription of the BCR 1 gene could yield several different BCR mRNAs by the process of alternate splicing. Each mRNA could code for a different size of BCR, protein thereby modulating BCR function. For example, the 7-kb RNA might encode the 190/185-kDa proteins, the 4.5-kb RNA would produce P160 BCR, and the 4-kb RNA would yield the 135/125kDa proteins. The 108- and 83-kDa proteins would then have to be encoded by other BCR mRNAs not yet detected. It is interesting that cDNA sequencing predicts the presence of two forms of P160 BCR, one being 1271 amino acids (Fig. 3) and the other, 1227 residues. In fact, our studies have identified two forms of P160 BCR in the CML cell line K562 (Campbell et al., 1990; see below). However, we have not eliminated the unlikely possibility of either rapid autocatalytic processing of primary BCR translation products, or a unique degradation pattern of the larger BCR proteins (190 and 160 kDa), resulting in a uniform pattern of uniquely sized BCR protein fragments. Further studies are under way to determine which of these several possibilities explains o u r results. Degradation appears unlikely, as inclusion of a variety of protease inhibitors and direct Western blotting has failed to affect the pattern of BCR proteins (Li et al., 1989a; J. Liu, J. Q, Guo, and R. Arlinghaus, unpublished results). Another possible interpretation that would explain the presence of five or more BCR proteins concerns the increasing number of proteins that contain BCR homology. These include n-Chimaerin (Hall et al., 1990) and the ABR gene product (Heisterkamp et al., 1989). Recently, two 85-kDa proteins isolated from bovine brain, p85a and p85P, show homology to the C-terminal domain of BCR (Otsu et al., 1991). These

BCR GENE AND

Ph'-CHROMOSOME

LEUKEMIAS

243

proteins also contain the tyrosine kinase regulatory domains SH3 and SH2 which flank the region of BCR homology. Although the functional significance of the BCR homology region in these proteins is unclear, both p85a and p85p bind to and are substrates for the EGF and PDGF receptor tyrosine kinases, as well as the polyoma virus middle T:pp60c"" protein complex. It is intriguing to speculate that the 83-kDa BCR protein described in our studies (Li et al., 1989a) is in fact the human form of p85a andlor p85p. T h e identification of two types of BCR/Abl fusion proteins in Ph'positive CML and ALL suggests that BCR involvement is critical to the development of these neoplastic diseases, but the function of this evolutionarily conserved gene in these diseases remains unclear. Moreover, the role of the BCR gene in normal cellular physiology is unknown. We have recently produced data that begin to elucidate the role ofBCR in hematopoietic cells. Ill. BCR Complexes in Hematopoietic Cells

T h e existence of multimeric protein complexes has been demonstrated for numerous and diverse cell types. Protein-protein complexes play important roles in the functioning of receptors (Cleavers et al., 1988), G proteins (Weiss et al., 1988), proteases (Rivett, 1989), and transcription factors (Saltzman and Weinmann, 1989; Chiu et al., 1988). The interaction of the multiple protein subunits of these complexes, with each other and with their respective ligands (receptors) or targets (transcription factors), seems likely to modulate the activity and functional forms of these various proteins (i.e., receptors or transcription factors). Protein substitution within the complex would allow for diversity of cellular responses induced by activation of these multimeric protein complexes and allow for a greater capacity of regulation of the cellular responses to this activation. A. COMPLEXES CONTAINING ph-P53 Previous studies on P2 10 BCRlAbl have demonstrated that the protein sediments in glycerol gradients as a complex containing a 53-kDa protein termed ph-P53; the complex of ph-P53 and P210 BCR-Abl sediments between standard 100- and 300-kDa protein markers (Maxwell et al., 1987; Li et al., 1988). The gene that encodes the ph-P53 protein is as yet unknown. Several different approaches have been used to identify the gene origin of ph-P53. In our first attempts, antibodies to several known proteins were used to identify ph-P53. The ph-P53 protein did not react with antibody PAb 421, which has been shown to be reactive

244

MARTIN L. CAMPBELL AND RALPH

B. ARLINCHAUS

with human forms of the nonviral T-p53 protein (encoded by the p53 oncogene) (Rotter and Wolf, 1985). This indicates that ph-P53 from K562 cells and p53 are separate and distinct proteins. In studies by Lubbert et al. (1988), neither the p53 oncogene mRNA nor its protein was detected in K562 cells or HL-60 cells, yet both cell lines contain BCR complexes containing ph-P53 (Li et al., 1988, l989a). Similarly, ph-P53 did not react in immunoblots with anti-BCR(898-91 l), anti-Abl(5 1-64) (Li et al., 1988),or a polyclonal antibody raised against the tyrosine kinase domain of the viral abl gene (J. (2. Guo and R. Arlinghaus, unpublished results). Peptide mapping studies indicated that ph-P53 is not a breakdown product of P210 BCR/Abl (Maxwell et al., 1987; Li et al., 1988). These studies were done by labeling ph-P53 in test tube protein kinase assays performed on cell extracts from the Ph’-positive CML cell line K562 (Lozzio and Lozzio. 1975) by using either anti-Abl antibody made against a mouse Abelson leukemia virus peptide (residues 389-403) (Maxwell et al., 1987) or anti-BCR(898-911). Similar studies performed on BCR protein-containing immunoprecipitates from SMS-SB cells (a normal human B lymphoid tissue cell line) indicate that ph-P53 was not related to BCR proteins (Li et al., 1989a). T h e possibility that ph-P53 is the result of antibody heavy-chain phosphorylation was eliminated by performing a series of experiments using antibody cross-linked to Affi-gel 10 resin (Li at al., 1988). Cross-linked anti-Abl(5 1-64) was used to perform the kinase assay, thus labeling P2 10 BCR/Abl and ph-P53 with ‘*P. Sodium dodecyl sulfate (SDS)-mercaptoethanol treatment at boiling temperatures released ph-P53 and P210, but the antibody heavy chain was not released (Li et al., 1988). In summary, these findings indicate that ph-P53 is not related to the p53 oncogene protein, is not the heavy chain of antibody molecules, is not a breakdown product of P210 BCR/Abl, and appears to be unrelated to either BCR or Abl proteins. Further studies are under way to identify the gene for ph-P53 and its role in BCR/Abl-induced leukemias. B. BCR PROTEIN COMPLEXES We have prepared antipeptide antibodies directed toward a wide range of BCR sequences from the amino terminal to the carboxy terminal of the predicted protein, some of which are listed in Fig. 5. These antibodies were used to immunoprecipitate proteins from cytoplasmic extracts of K562 cells, HL-60 cells (Collins et al., 1977), a Ph’-negative promyelocytic leukemia cell line, and SMS-SB cells (Smith et al., 1981) to analyze the nature of these BCR protein complexes further.

BCR GENE AND

Ph'-CHROMOSOME

P210 BCRhBL

t t PI60 BCR

I

1

l I1 I

l 1 1

t t

Anti-BCR

A P, 1-16 137-152

d I I

245

LEUKEMIAS

ABL

I

I I

t I t ~ n s -, 898-911 1256-1271 1069-1084

FIG. 5 . Schematic representation of peptide sequences within P160 BCR used to raise site-directed antibodies. Numbers represent amino acid positions within the BCR coding sequence (Fig. 3). Peptides BCR(1-16), BCR(137-152), and BCR(898-911) are located within the BCR/Abl P210 fusion protein. Peptides BCR(l069-1084) and BCR(1256-1271) are not present in P210 BCRIAbL. BCR peptide amino acid sequences are taken from the published BCR sequences of Hariharan and Adams (1987) and Heisterkamp etal. (1985) as shown in Fig. 3.

In vitro kinase phosphorylation analyses of immunoprecipitates from all three cell lines showed the presence of ph-P53 in a complex with BCK proteins (Li et al., 1989a). ph-P53 was detected by immunoprecipitation of cytoplasmic extracts with antisera directed against BCR sequences separated by approximately 150 amino acids. Detection was specific in each case because antibody prereacted with homologous peptide failed to detect either BCK proteins or ph-P53. Fractionation of cytoplasmic extracts was performed to characterize further the proteins recognized by anti-BCR antibodies. Previous reports have shown that anti-Abl peptide antibodies detected P2 10 BCK/Abl protein complexes in K562 cells that sediment in the 100- to 300-kDa size range in glycerol gradients (Li el al., 1988; Maxwell et al., 1987). SDSpolyacrylamide gel analyses of immunoprecipitates from K562 cell extracts under nonreducing conditions detected bands of 275 and >500 kDa. T h e gel slices containing these bands were excised from the gel and denatured by boiling in sample buffer containing 2-mercaptoethanol prior to analysis on a second SDS-polyacrylamide gel. Denaturation of the 275- and >500-kDa bands yielded P210 BCR/Abl and ph-P53 under these conditions, indicating that the proteins are tightly complexed (Li el al., 1988). Pendergast et al. (1989) determined, by using a baculovirus expression vector, that P2 10 BCR/Abl expressed in SF9 insect cells eluted in the 800-kDa range on Sephacryl S-300 gel filtration columns. Analyses of cytoplasmic BCR complexes from K562, HL-60, and SMSSB cells by immunoprecipitation of cellular extracts following sedimentation on l0-50% glycerol gradients identified BCR proteins of 190-185, 155, 135/125, 108, and 83 kDa cosedimenting with ph-P53 in gradient fractions corresponding to the 100- to 300-kDa size range. A representative pattern of sedimentation of BCR protein complexes from

246


FIG.6. HL-60 cells (2 x lo8) were pulse labeled with 0.5 mCi/ml of ["'SS]methionine in methionine-free media for 20 min (panel A),chased for 3 hr in complete medium (panel B). lysed in detergent buffer, and sedimented on a l0-50% glycerol gradient for 18 hr. Sedimentation is from left to right. Fractions of gradient were immunoprecipitated with anti-BCR(898-911) prereacted with unrelated peptide (odd number) or cognate peptide (even number). Lane numbers represent fraction number from the 10-50% glycerol gradient. (See Li et al., 1989a, for experimental methods.)

["Slmethionine-labeled HL-60 cells is shown in Fig. 6. Panel A shows the results of a 20-min pulse labeling; panel B shows a 20-min pulse followed by a 3-hr chase in nonradioactive culture medium. The pattern of proteins is only slightly altered, comparing the brief pulse label and the long chase period. This type of experiment indicates that BCR proteins and the complexes that house them are relatively stable. T h e identity of the two proteins migrating between ph-P53 and the 83-kDa BCR protein are of interest because they seem to be removed from the complex during the chase. The expression of BCR and BCR-related proteins varied somewhat among the cell lines tested. Moreover, the size of some proteins varied slightly among cell lines. We detected similar BCR protein com-

BCR GENE AND

Ph’-CHROMOSOME LEUKEMIAS

247

plexes in two nonhematopoietic cell lines, HeLa and A498 (W. J. Li and

R. B. Arlinghaus, unpublished results). Given the association of ph-P53 with both the P210 BCR/Abl and BCR proteins, it was of interest to determine whether P210 BCK/Abl formed a complex with BCR proteins. To answer this question we used the antipeptide antibodies directed toward the amino (N) and carboxy (C) termini of P160 BCR. The antibodies directed toward suquences within the carboxy-terminal domain of BCK cannot directly bind to P2 10 BCK/Abl because these C-terminal BCK sequences are not present in the BCK/Abl fusion protein. The C-terminal BCK sequences are translocated to chromosome 9 as a result of the t[9;22] reciprocal translocation (see Fig. 5). As expected, the N-terminal antisera anti-BCK (1-16) immunoprecipitated P2 10 BCK/Abl in vitro kinase assays of cytoplasmic extracts from K562 cells. Surprisingly, antisera directed toward the C-terminal of P 160 BCR, anti-BCR( 1256-1271), also immunoprecipitated P210 BCRiAbl (Fig. 7, panel A). The detection was specific, because antibody incubated in cognate peptide prior to immunoprecipitation of cell extracts failed to detect P210 BCR/Abl. V8-proteolytic digests were performed on the 2 1O-kDa bands immunoprecipitated with both N-terminal and Cterminal antibodies, anti-BCR(137-152) and anti-BCR( 1256-1271), respectively; and the resulting patterns of fragments were identical, providing independent evidence that the P2 10-sized band in anti-BCR C-terminal immunoprecipitates is in fact &210 BCR/Abl (Campbell et al., 1990). Because our previous studies detected protein complexes by sedimenting cytoplasmic extracts through linear gradients of 10-50% glycerol (Li et al., 1988), we next determined whether BCR/BCK/AbI complexes had sedimentation properties similar to P2 1O/ph-P53 complexes. Gradient fractions were tested in immune complex kinase assays using N- and C-terminal BCR antibodies along with our mouse monoclonal antiBCR(898-911). Figure 8 shows that the overall pattern of sedimentation of BCK proteins was similar insofar as position and density of the BCR proteins in each fraction (Campbell et al., 1990), regardless of the antibody used to process the gradient. However, it is clear that P210 BCR/Abl present in slower sedimenting fractions (fractions 5 and 7) was not detected by C-terminal BCR antibodies. Furthermore, although P160 BCR is not detected by kinase assays with anti-BCR(1-16), this antibody can specifically immunoprecipitate the [35S]methionine-labeled P 160 BCK protein produced in a rabbit reticulocyte translation system in vitro. In control experiments, the P120 BCR/Abl band remained near the top of the gradient (in the first five fractions) following fractionation of [35S]methionine-labeled cytoplasmic extracts from K562 cells heated to 95°C

A

B

FIG. 7. Panel A: Antibodies directed against the C terminus and N terminus of P160 BCR specifically recognize a protein the size of P210 BCRIAbl. Cell extracts of K562 cells were immunoprecipitated with anti-BCR(1-16) (rhc N reriniiius of P160 RCK) and antiBCR(1256-1271) (the C terminus of P160 BCR), and with a combination of anti-BCR(116) and anti-BCR( 1256- 1271). Blocked lanes refer to immunoprecipitations performed with antibodies preincubated with excess cognate peptide. Lanes with labels indicating sera were immunoprecipitated with raw sera; lanes labeled with Ig labels were processed with Ig fractions obtained from ammonium sulfate precipitation of raw sera. Each lane represents the products obtained from an in vifro kinase assay of an immunoprecipitation of 10’ K562 cells. Panel B: The proteins were initially immunoprecipitated with a combination of C-ter~ninalantibodies, anti-BCR( 1069-1084) and anti-BCK(1256-1271). and an zn zdro kinase assay was performed. l h r labeled pellets were suspended in 1 L?o SDS buffet containing 70 m M 2-mercaptoethanol and boiled for 5 min to disrupt protein complexes. The sample was divided in twu portions and each fraction was inmunoprecipitated with either BCR N-terminal antibodies, anti-BCR(1- 16) and anti-BCR(137-152) (left lane). or the original combination of BCR C-terminal antibodies, anti-BCR(1069-1094) and antiBCR(1256-127 1) (right lane). (See Campbell ~t al., 1990, for experimental methods.)

RCR GENE AND

Ph'-CHROMOSOME

LEUKEMIAS

249

FIG.8. Detection of P160 BCRlPPlO BCRiAbl protein complexes by glycerol gradient centrifugation using site-directed antibodies targeted against the N-terminal, middle, and C-terminal regions of P160 BCR. The direction of sedimentation is from left to right. Gradient fractions were immunoprecipitated and subjected to the in uitru kinase assay as described by Li et nl. (1989,). Numbers above lanes indicate gradient fractions. The locations of P120 BCRIAbl and its associated degradation product, P190 BCRiAbl, P160 BCR, and ph-P53 are indicated. Left panel, immunoprecipitdted with anti-BCR(1-16); middle panel, immunoprecipitated with anti-BCR(898-911); right panel, immunoprecipitated with anti-BCR( 1256-127 1). (For experimental details, see Campbell et al., 1990.)

prior to sedimentation. Additionally, no ph-P53 was detected in these heated extracts following immune complex kinase assays with antiBCR(898-911). In recent experiments, we have found that protease and phosphatase inhibitors are key factors in detecting these complexes, and under optimal conditions P210 BCR/Abl, P160 BCR, and ph-P53 cosediment in peak fractions at the central portion of the gradient as detected by C-terminal BCR antibodies (Campbell and Arlinghaus, unpublished results). Heat denaturation is SDS buffers is widely used to dissociate protein complexes prior to immunoprecipitation (Chiu et al., 1988). If P210 BCR/Abl is complexed to PI60 BCR, such treatment should render P210

250


BCR/Abl resistant to immunoprecipitation by anti-BCK C-terminal antibodies. This type of experiment was done by first immunoprecipitating cytoplasmic extracts of K562 cells with a mixture of two anti-BCR Cterminal antibodies, anti-BCR(1069- 1084) and anti-BCR (1256- 1271). Two different antibodies were used to increase the recovery of BCR and BCR/Abl proteins in the first immunoprecipitate. The irnmunoprecipitate was labeled in an in vitro kinase assay and denatured by boiling in a 1% SDS buffer containing 70 mM 2-mercaptoethanol. This solution was then diluted 10-fold and split into two fractions. One fraction was immunoprecipitated with a mixture of two N-terminal antibodies, anti-BCR( 116) and anti-BCR(137- 152), and the other traction was immunoprecipitated with the same mixture of C-terminal antibodies as was done in the initial immunoprecipitation. As expected, the N-terminal anti-BCR antibodies precipitated P210 and PlYO BCR/Abl and P160 BCR. The Cterminal antibodies detected P160 BCR but failed to immunoprecipitate P2 10 BCR/Abl (Fig. 7, panel B). Of interest, in longer exposures of Fig. 7, panel B, the C-terminal BCR antibodies also precipitated a series of smaller BCR proteins from these heat-denatured immunoprecipitates identified previously as P135/P125, P108, and P83. In contrast, the amino-terminal BCR antibodies failed to detect these smaller BCR proteins, indicating that the smaller BCR proteins share the carboxy terminus of PI60 BCR but lack the amino-terminal sequences (M. L. Campbell and R. B. Arlinghaus, unpublished results).

C. BCR/Abl COMPLEXES Our findings provide strong evidence that P210 BCR/Abl and P160 BCR (and the smaller BCR proteins) exist in a tightly associated complex in Ph'-positive K562 cells. Several lines of evidence suggest that ph-P53 is also associated with the P160 BCKiP2lO BCR/Abl complex. First, P160 BCR/ph-P53 complexes are found in Ph'-negative cells (e.g., HL-60 cells). Second, P160/ph-P53 complexes sediment in the same region of the glycerol gradient as either P160 BCR/PP 10 BCR/Abl (Campbell et al., 1990) or ph-P53/P210 BCR/Abl complexes (Li et al., 1988). Third, we have found by using C-terminal BCR antibodies to detect P210 BCR/Abl complexes that high levels of the C-terminal BCR antibody detect ph-P53 in complexes containing P160 BCR/P210 BCR/Abl (Campbell et al., 1990). As noted above, under lysis conditions, in which high levels of several protease and phosphatase inhibitors were used, P210 BCR/ Abl, P160 BCR, and ph-P53 cosediment as a complex (Campbell and Arlinghaus, unpublished results). Therefore, our data strongly indicate that a complex consisting of P210 BCR/Abl, P160 BCR, and ph-P53 proteins exists in K562 CML cells.

BCR GENE AND

Normal Hematopoietic Cell

Ph'-CHROMOSOME

25 1

LEUKEMIAS

-

PhiladelphiaChromosome Positive Leukemic Cell

FIG. 9. Proposed structure of BCR protein complexes in normal and Ph'-positive hematopoietic cells.

What is the possible physiological significance of such a ternary complex? Our results indicate that the BCR/ph-P53 complex functions as a serinelthreonine protein kinase in vitro (Li et al., 1989a). However, the P2 10 BCR/AbI/P160 BCR/ph-P53 complex functions as an activated tyrosine protein kinase (Campbell et al., 1990; Li el al., 1989a).These data form the basis for generating a working model that begins to relate our findings to the cause of Ph'-positive leukemias (Fig. 9). This model predicts that P160 BCR forms a dimer structure; this dimer structure would bind ph-P53 to form a tetrameric protein complex. This structure may be more complicated in that other proteins might also participate in the complex yielding a multimeric protein structure. In any case, the putative complex would function as a serinelthreonine protein kinase. This kinase function would be involved in normal hematopoietic cell growth and/or differentiation. In Ph'-positive leukemia cells, the fused and activated BCR/AM protein, by virtue of its BCR sequence, becomes incorporated into the BCR/ph-P53 tetrameric complex by substituting for one of the BCR proteins. The resulting complex would now house three types of proteins, namely P160 BCR, ph-P53, and the activated P2 10 BCR/Abl tyrosine kinase. The highly active tyrosine kinase would

252


then induce drastic changes in the cell phenotype involving growth and differentiation. We propose that complexes containing the activated Abl tyrosine kinase alter normal cellular targets of the BCR and/or Abl kinases by changing sites of phosphorylation (i.e., tyrosine instead of or in addition to serine). The end result of these phosphorylation changes in rarget proteins provides a mechanism for the establishment of a leukemic cell clone with altered responses to normal cellular regulation of growth and differentiation. Obviously, this model is preliminary and must pass the test of further experimentation. Experiments, conducted both in uitro and within whole cells, are planned to assess the physiological significance of BCR and BCR/Abl protein complexes in hematopoietic cell growth and differentiation. IV. Concluding Remarks and Future Directions

The almost invariable association ofthe Ph' gene product with CML suggests an important role for both BCR and abl genes in the manifestation of this disease as well as other types of Ph'-positive leukemias. However, as the clinical phenotype of CML has been reported prior to the appearance of the Ph' (Lisker et al., 1980), it is possible that the chromosomal translocation that generates Ph' may be a necessary, but not the first, step in the generation of the leukemic phenotype. I n any event, though the molecular aspects of the t[9;22] gene translocation that generates Ph' and the P210 BCRiAbl protein have been widely studied and much new information has been generated, the precise roles of the BCR and abf genes in this leukemic process are yet to be determined. Similarly, the roles of these two genes in normal somatic cell physiology remain unclear. We have demonstrated the association of P160 BCK with ph-P53 and P210 BCRiAbl in Ph'-positive cell lines and the question immediately arises as to whether BCR PI60 associates normally with P145 Abl and, if so, does this association exert some regulatory control over the normal abl gene product in hematopoietic cells? Lug0 et al. (1990) have demonstrated that specific quantitative differences exist between BCRIAb1 gene products with variations in BCR exon content. Thus, PI85 BCKiAbI in ALL has increased tyrosine kinase activity and biological transformation potency compared to P210 BCR/Abl. It seems likely that changes in the length of the BCR sequences are responsible for the differential potency noted between P210 BCR/Abl and P185 BCRIAbl. We predict, therefore, that BCR proteins are involved in the regulation of normal Abl protein function. It will be of interest to determine whether mutations within those BCR sequences that appear to alter the potency of BCRiAbl

BCR GENE AND

Phl-CHROMOSOME LEUKEMIAS

253

proteins will disrupt normal hematopoietic cellular functions attributed to the abl gene (Caracciolo et al., 1989). The function o r functions of the P210 BCR/Abl/P160 BCR/ph-P53 protein complex and its role in maintenance of the leukemic state is also under active investigation. Li et al. (1989b) found that P2 10 BCR/Abl expression is differentially altered in K562 and EM2 cells subsequent to treatment of cell cultures with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The amount of P210 BCR/Abl is sharply decreased in K562 cells treated with low levels of TPA, whereas P2 10 is increased in concentration in Phi-positive EM2 cells following TPA treatment. Is this a consequence of alterations in the formation or composition of BCR protein complexes? Further, does the induction of megakaryocytic differentiation of K562 cells in response to TPA treatment alter the composition of BCR protein complexes? One other important question is whether the kinase activity observed in BCR immunoprecipitates originates from the BCR gene product itself o r whether it is merely an associated activity attributed to other proteins within the complex, namely ph-P53. In summary, we view the following questions important and as needing to be addressed regarding the role of BCR in normal and leukemic processes: 1 . Does BCR encode a protein with serine/threonine kinase activity? 2. Are BCR proteins associated with the normal Abl protein? 3. Does the length of BCR sequences within a BCR/Abl protein modulate its potency? If so, what are the molecular steps responsible for this regulation? 4. Are BCR sequences targets of the Abl tyrosine kinase within BCR/ Abl proteins? If so, what is the physiological significance of this phosphorylation? 5. What gene encodes ph-P53, and what is the biochemical function of ph-P53 within BCR protein complexes? 6. What proteins are specifically involved in the BCR/ph-P53 complex and what is the role of this protein complex in somatic cell physiology? Answers to these questions will increase our understanding of the role of BCR in the leukemic process and should lead to better diagnosis and treatment of Ph'-positive leukemias. ACKNOWLEDGMENTS This work was supported by Public Health Service Grams CA49369 and CAI6672 from the National institutes of Health, a grant from the American Cancer Society (CH482),and a

254


grant from the Robert A. Welch Foundation (G 1 1 18). We thank Tammy Trlicek and Linda Jackson for expert help in manuscript preparation. Ralph B. Arlinghaus is supported by a Professorship horn the Abell-Hanger Foundation. Martin Campbell is a Rosalie B. Hite Foundation predoctoral fellow enrolled in the University of Texas Graduate School of Biomedical Sciences.

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p53 AND HUMAN MALIGNANCIES Varda Rotter* and Miron Prokocimer*t * Department of Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

t Department of Hematology, Beilinson Medical Center, Petah Tikva 49100, Israel I. Introduction A . The Human p53 Structurc B. Heterogeneity of the Human p53 Protein C . Identification of the Translation Initiation Site D. Characterization of Human p53 Promotor Regions 11. Biological Activity of the p53 Protein in Malignant Transformation A. p53 Oncogene B. p53 Antioncogene 111. Proposed Function References

I. Introduction

The p53 nuclear protein, which has been shown to act as a dominant oncogene in tumor cells (1-29), has also been recently shown to function as an antioncogene in normal cells (30-32). This apparent discrepancy may be related to the fact that whereas the wild-type p53 protein functions as a suppressor gene, the mutated p53 protein enhances the outburst of the malignant phenotype. This complexity, however, makes it an attractive model system for investigating in depth the interrelationship between either oncogene or antioncogene activity and the neoplastic processes. In agreement with the concept that malignant transformation is a multistage process, it is plausible to assume that the p53 protein, stimulated by various cellular signals, may in turn induce or suppress thc expression of other cellular genes involved in this chain of events. Expression of the p53 may be mediated by several molecular mechanisms that induce or suppress the expression of the p53 gene. Up-regulation or down-regulation could be mediated by major rearrangements, small deletions, or subtle point mutations in gene structure. A. THEHUMANp.53 STRUCTURE The p53 gene is highly conserved in diverse organisms such as Xenopus Zaevis (33), chickens (34), and the mouse and humans (19-22), suggesting

that the encoded protein plays a central and critical role in the cell and 257 ADVANCES IN CANCER RESEARCH, VOL. 57


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therefore is tightly conserved in evolution. The human genome contains a single functional gene consisting of 11 exons; the first noncoding exon is separated from the cluster of the 10 exons by a large intron (see Fig. 1). Screening of a large number of human primary tumors and established cell lines has revealed the existence of several restriction site polymorphisms in the human p53 gene (35,36). One interesting gene polymorphism is the existence of FnudII restriction sites in exon 4 of the p53 gene. Sequence analysis of various cDNA clones that w e isolated indicated that this site is created by the shift of a C to a G, coding for a proline o r an arginine, respectively, at codon 72; the implication of this with regard to protein structure will be discussed later. We found a random distribution of this gene polymorphism in p53 producer primary tumors and established cell lines, suggesting that the protein properties encoded by this gene polymorphism are not directly associated with any specific malignancy (35-37). Another gene polymorphism we discovered was a BamHI restriction site, found at the 5’ region upstream to the first p53 noncoding exon (38). Again, this polymorphism was randomly distributed, at a rather lower incidence, and was not correlated with any specific malignancy. A third polymorphic site was the polymorphic BglII site in the first noncoding large intron, situated 3 kb downstream to the first BglII site (38). Figure 1 represents the restriction map of the p53 gene and the polymorphic sites are outlined. The gene codes for a 2.6-kb mRNA molecule that contains a large 3‘ noncoding region probably involved in the stabilization of the molecule (20). We and others mapped the p53 gene to chromosome 1 7 ~ 1 (23,24), 3 a frequent site of allele loss in most common human cancers, as will be reviewed in detail in the following discussions.

B. HETEROGENEITY OF THE HUMAN p53 PROTEIN A detailed analysis of the human p53 protein has indicated that, as in mouse tumor cells, it consists o f a heterogeneous population. Most of the human cell lines and primary tumors that we analyzed contained one or

FIG. 1 . Detailed structure of the human p53 gene. The boxes represent the distribution of the esons, which are numbered. ATG represents the initiation site found in exon 2. Arrows point to the p53 pojlinorphic sites.

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more distinct forms of the protein that were evident as slower and faster migrating products (35). It was, therefore, expected that the single p53 gene found in these cells would express more than one mRNA species. In order to study the distribution of the various p53 protein types, our strategy was to isolate full-length cDNA clones and to study the nature of the p53 proteins encoded. A battery of p53 cDNA clones that we have isolated from a hgt 10 SV80 cDNA library (35,391 of the SV80-transformed cells expressing the two discrete p53 protein species were studied in detail. The various clones isolated were subcloned in the transcription vector pSP65 and characterized by the in vitro transcription translation assay (35). Clones p53-H 1, p53-H7, and p53-H8, although each had a different overall DNA size, directed the synthesis of the fast-migrating p53 protein found in SV80 cells. Clone p53-Hl9 coded for the more slowly migrating p53 species. By comparing the migration of the in vitro-synthesized products with the in vivo products, we found that each of the cDNAs contained a complete coding region and represented the corresponding in vivo-expressed protein species (35). Partial peptide analysis indicated that the cDNA isolated clones coded for the production of the authentic p53 species expressed in the SV80-transformed fibroblasts (35). Nucleic acid sequence analysis indicated that these two p53 species varied in a single nucleic acid (G versus C), coding for a proline at codon 72 in one and arginine in the other (35). Arginine, which was expressed in the faster migrating species (clone p53-H1, base 215), has a large side chain and is positively charged under physiological conditions. In contrast, proline, the equivalent residue expressed by the more slowly migrating protein (clone p53-H1Y), has a small, nonpolar side chain. It is, therefore, possible that the tertiary structures of the respective proteins are altered, with attendant changes in detergent binding and charge-tomass ratios. This observation of a single nucleotide change was given further support by results from other laboratories. Lamb and Crawford characterized a genomic p53 clone (40), isolated from human liver DNA, that has a G at the position equivalent to nucleotide 2 15 in p53-H 1, which encoded an arginine. In contrast, p53 cDNA clones isolated from an A43 1 cDNA library by Harlow et al. (20) contained in the same position a C and thus, like p53-H19, coded for a proline. These laboratories also recently isolated the corresponding cDNA clones (36). In view of these findings, we infer that the heterogeneity of the various clones represents an authentic variation in the p53 mRNA population. We would like to suggest that these two individual p53 species could have been generated in the cell most likely by gene polymorphism. p53 is a proline-rich molecule; there is a cluster of 14 proline residues

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situated between amino acids 58 and 98. The single mutation at amino acid 72 may greatly modify the tertiary structure of the protein. This specific domain may have a very important role in the biological function of the protein in the cell. The fact that p53 protein is found in the cell nucleus (1,6,41) strongly suggests that the p53 oncogene functions within this cellular compartment and it may be associated with at least two different physiological activities. One may be related to DNA replication and the second may be involved in regulation of the expression of other cellular genes. In both cases, the p53 protein would be expected to bind directly with DNA sequences or DNA-binding proteins found in the cell nucleus. To permit such activities, the protein should have specific structural properties enabling its active migration into the nucleus and its correct folding to allow direct binding to DNA or DNA-binding proteins (42). Indeed, the secondary p53 protein structure predicted from the DNA sequence by the Chau and Fassmann (43) criteria seems to suggest that this protein has a putative nuclear homing domain and it may be folded in a conformation arranging the contact surface for sequence-specific interaction with DNA. p53 protein structure deduced from sequence analysis suggested that this protein may be folded into a tertiary structure that creates several potential elements that either bind directly to DNA or interact with other DNA-binding proteins. The three potential a-turn a-helical structures in the p53 protein, one situated at the N-terminal part, the second at the C-terminal part, and the third at the center of the molecule, seem to be highly probable candidates for DNA-binding sites. I t should be noted that such elements have been shown to mediate the binding to DNA4of several nuclear proteins. T h e best studied of these is the h repressor protein (44). Another important element that may determine the capacity of the p53 protein binding to D N A is its abundance of proline residues, a frequent property of DNA-binding proteins. An interesting example is the AP-2 product, in which the transcriptional domain was shown to be proline rich (45). Analysis of the human and mouse p53 proteins has revealed a conserved cluster of 14 prolines situated at the N-terminal part of the protein, between amino acids 58 and 98. This cluster could potentially be folded into a poly(pro1ine) structure that may create a specific DNA-binding domain. This assumption is strongly supported by the observation that distortion of this proline stretch by a naturally occurring mutation at codon 72, changing the proline into an arginine, induces a major configurational change, manifested by a significant niodification in the electrophoretic mobility of the p53 protein (35,361.

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c. IDENTIFICATION OF THE 7rRANSLATION INITIATION SITE A comparison of the calculated molecular weight of the p53 protein, predicted from the nucleic acid sequence of both the human and the mouse cDNA, and the actual size of p53 estimated by its apparent migration on gel electrophoresis revealed a discrepancy. This discrepancy made it difficult for us to map the translation start site on the basis of protein size. The aim of our experiments was to define the AUG used for the in vivo translation of the human p53 mRNA transcripts. As described above, we isolated several human p53 cDNA types which in an in vitro transcription-translation assay coded for several different protein types. The types designated p53-H1 and p53-H19, representing the in vivo faster and slower migrating protein species, were the most striking ones (35). In addition to these in vim-expressed proteins, we isolated a third type of p53 cDNA, clone p53-H 13, which codes for yet a smaller size p53 protein (-48 kDa). Sequence analysis of p53-Hl3 indicated no deletion in the 3' coding sequences but showed instead that this clone lacks the 5' first AUG found in other clones. In order to deduce which AUG is used in vivo, we decided to modify, by site-specific mutagenesis, the first AUG of the open reading frame of one of our full-length cDNAs, p53-H 19, thus allowing initiation of translation at the second AUG. The results obtained showed that in the absence of the first AUG, due to a deleted 5' end (clone p53-H 13) o r due to an inserted mutation (clone p53-H 19M), the second AUG is used for translation. Judged by the fiequent incidence of the larger p53 protein, it seems that most of the p53 protein is generated in vivo, utilizing the first AUG codon. However, the possibility that the second AUG codes for a minor low-abundant species cannot be excluded. Recently, we found that in the p53 protein of lower species, such as X. Eaevis and the rainbow trout, the first analogous AUG is conserved and is also used as the main translation initiation site in viuo (46). D. CHARACTERIZATION OF HUMAN p53 PROMOTER REGIONS T o gain insight into the mode of regulation of the human p53 gene, we set out to locate the DNA sequences that control the expression of the gene. We constructed recombinant plasmids containing the CAT gene and all or portions of a 3.8-kb DNA fragment isolated from the transformed human cell line SV80. These constructs were introduced into cells and were assayed for their ability to induce the expression of the CAT gene, either stably from an Epstein-Barr virus (EBV)-oriP-based plasmid or transiently from pSVO-CAT. The pSVO-CAT-derived p53

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vectors were introduced into COS cells and transient expression was assayed after 48 hr. The EBV-orzP-derived vectors were introduced in several human cell lines, including HL-60, a p53 nonproducer cell line. T h e EBV-orzP vector contains the hygromycin B drug-resistance gene, which enabled the selection of clones that have stably integrated the introduced vector. Stable human-derived cell lines obtained by transfection of the CAT-derived vectors into K562, Raji, and HL-60 were obtained using electroporation for gene transfer. Our results indicate that the human p53 gene contains at least two promoters that are capable of initiating transcription of the CAT gene (47). p53pl agrees with the p53 promoter, which has been described previously (2 1,40) and maps immediately upstream to the first noncoding exon. A second, novel promoter, p53p2, is located within the first intron and is up to 20-fold more efficient than p53pl in promoting CAT activity. This higher level of expressed CAT activity from p53p2 in cells stably maintaining the CAT vectors, which is also supported by the presence of a higher steady-state level of CAT RNA (47), is not due to a higher number of plasmids per cell (47). We concluded that the human p53 gene may be controlled by two types of promoter and that the differential regulation of these promoters may play an important role in the altered expression of the gene in both normal and transformed cells (47). Consistent with our findings, a previous study by Bienz-Tadmor et al. (2 l ) , comparing the structures of the murine and human p53 regulatory regions, referring to the p l , showed that these sequences are highly conserved and that the murine p53 promoter also contains an upstream negative regulatory element. The authors showed that these regulatory regions d o not contain elements commonly found in other promoters, such as the TATA and CCAAT boxes. They also identified a highly conserved sequence containing extensive dyad symmetry and suggested that these sequences play a role in the translation efficiency of the mRNA (2 1). Initiation of transcription downstream of these sequences, at p53p2 in the case of the human p53 gene, would eliminate this region of dyad symmetry and possibly, as in c-myc, increase the translation efficiency of the mRNA (47). The control of gene expression by the use of multiple promoters having different strengths has been observed previously for other genes. The expression of the mouse a-amylase gene, Amy-Al, is controlled in a tissue-specific manner by a dual promoter (48). In this gene, one promoter is active in liver cells whereas a second promoter, which is 30-fold stronger, is active in parotid cells. The yeast invertase gene, SUC2, is also expressed from two promoters, one of them being inducible by glucose (49). T h e c - m y gene is controlled by expression of at least four pro-

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moters. I n analogy to c-myc, differential usage of the two promoters of the p53 gene may be involved in its regulation during the cell cycle and in its overexpression in transformed cells (50). II. Biological Activity of the p53 Protein in Malignant Transformation

Neoplastic processes can result from loss or inactivation of both copies of a tumor suppressor gene or from amplification or hyperactivation of one of the two copies of protooncogenes. The p53 gene represents both facets in its biological activity. It was considered to be an oncogene and recently has been shown to function as an antioncogene in normal cells. A. p53 ONCOGENL The mutated p53 tumor antigen was shown to be a functional oncogene product that enhances the malignant process. Employing different experimental approaches, it was shown that overproduction of p53 rendered the cells malignant. Introduction of the mutated p53 gene into primary embryonic rat fibroblasts in conjunction with the ras oncogene induced the appearance of transformed cells that developed tumors in nude mice. Thus mutated p53 complemented the M S membraneassociated oncogene, suggesting, that like my,myb, and ElA, it belongs to the nuclear oncogene family (25-27). The idea that the mutated p53 enhances the malignant process was further supported by results obtained with an Abelson murine leukemia virus-transformed p53 nonproducer cell line, designated L12. In the L12 cell line, we found that the endogenous p53 was inactivated because of a proviral Moloney-related DNA sequence insertion into its first p53 intron. Introduction of a functional genomic p53 clone into these nonproducer cells reconstituted the expression of p53. This gene manipulation changed the phenotype of these cells from cells that develop regressing tumors into cells that develop lethal tumors in syngeneic hosts. It wa3 concluded that L12 cells, which express the abl oncogene but lack p53 expression, are only partially transformed. Expression of a completely transformed phenotype requires concomitant expression of abl and mutated p53 genes (28,29). Most primary tumors and cell lines expressing p53 were found to overexpress the mutated forms of the pi53 protein. Screenings of Burkitt’s lymphoma and acute lymphatic leukemia (T and B) human cell lines have shown overexpression of p53 protein. All patients with B lymphoma and B CLL and majority of patients with acute lymphatic leukemia tested by us and others exhibited elevated levels of p53 synthe-

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sis as well (37,51). Southern blot analysis revealed that the p53 gene seemed intact. It is plausible, however, that high p53 levels are due to the presence of small deletions or subtle point mutations leading to the accumulation of the mutated form in the cell. Mutant p53 proteins are much more metabolically stable than the normal proteins and are commonly associated with proteins of the heat-shock protein family (HSP70) (52). Further evidence supporting the notion that overexpression of the mutated p53 gene may enhance the oncogenic process in vivo was provided by generating transgenic mice carrying murine p53 genomic fragments isolated from a mouse Friend erythroleukemia cell line. Neoplasms developed in 20%of the transgenic mice with a high incidence of lung adenocarcinomas, osteosarcomas, and lymphomas (53). These observations provide direct evidence that mutant alleles of the p53 oncogene have oncogenic potential in vivo, with various tissues susceptible to transformation, and play a causal role in tumor formation. Interestingly, these observations raised at the same time the possibility that wild-type p53 functions as an antioncogene and that its expression interferes with the neoplastic process. Consistent with this hypothesis, it is possible that the elevated tumor incidence results from functional inactivation of wild p53 by binding to mutant p53 or by its deletion and the overexpression of the mutant transgene.

B. p53 ANTIONCOGENE T h e hypothesis that p53 fulfills a role in the normal cell cycle is based on the observation that p53 was shown to be expressed in a number of nontransformed cell types, e.g., normal mouse thymocytes (4), cells of 12to 14-day-old embryos (3,54),and NIH 3T3 fibroblasts (7). Moreover, cells treated with mitogens exhibited high levels of p53 protein synthesis, thus suggesting that it starts functioning early in the transition from Go to GI (55). Mercer et al. (56) showed that microinjection of anti-p53 monoclonal antibodies into the cells inhibited DNA synthesis in quiescent nontransformed NIH 3T3 cells stimulated with serum, suggesting that p53 is synthesized as a late Go protein (56). A similar conclusion was reached by Reich and Levine (57), examining the steady-state levels of p53 mRNA and p53 protein synthesis in a synchronous population of N I H 3'1'3 fibroblasts obtained by releasing a culture from densitydependent growth inhibition. Using the antisense methodology, we recently showed that shut-off of p53 expression mediated by introduction of p53 antisense coding plasmids into either transformed or nontransformed cells led to the inhibition of p53 synthesis, which ultimately

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caused cell death (58). Furthermore, it was recently shown that mouse p53, when transiently expressed in SV40-transformed monkey COS cells, markedly inhibits SV40 origin-directed DNA synthesis in vivo (59-61). Inhibition of SV40 DNA synthesis by mouse p53 in vivo and in vitro was found to correlate with the ability of p53 to bind to T antigen and it was suggested that p53 either blocks a very early step in the initiation process or affects several stages of DNA replication. These are, therefore, the first indications of an active role for p53 in regulation of DNA replication and they suggest that this oncogene acts by perturbing either positively or negatively the recruitment of a subset of cellular DNA replication origins or the process of ongoing DNA synthesis. The hypothesis that the wild-type p53 functions as a growth arrest gene was initially concluded from the observation that this protein failed to enhance malignant transformation in in nitro assay (62,63) and was further supported by the fact that the protein actively suppresses the transforming activity of other oncogenes (30). Comparisons of the transforming activity of the various p53-encoded proteins, as evaluated by their ability to transform primary embryonic cells in conjunction with the rus oncogene, have indicated that whereas the mutated p53 protein forms induce the appearance of foci, the wild-type cDNA codes for an inactive p53 protein (62,63).The hypothesis that wild-type p53 functions as a tumor suppressor gene was initially deduced from the observation that wild-type protein failed to enhance malignant transformation, but rather suppressed the transforming activity of other oncogenes. Comparison of the various p53-encoded proteins, regarding their ability to transform primary embryonic cells in cooperation with the rus oncogene, indicate that mutant p53 induces the appearance of morphologically transformed foci, whereas wild type does not (11,15,21). Finally, it was found that wild-type p53 directly suppresses malignant transformation of primary embryonic fibroblasts induced by cotransfection of the rlls oncogene with either Ela or m y (30,31). It was concluded from these experiments that wild-type p53 protein functions as a repressor gene that arrests cell growth. When the mutant p53 protein is cotransfected with rm into primary embryonic rat fibroblasts, it most likely forms a complex with the endogenous rat wild-type p53 protein. This complex causes inactivation of the endogenous wildtype p53 protein, leading eventually to malignant transformation (30,31).In the L12 system described previously, it was found that mutant p53 enhances malignant transformation in a cell system devoid of any endogenous p53 expression. We would like to suggest that the mutant p53 that facilitates the transformation in these cells functions basically in a similar mode. It is possible that in the L12 cells, p53 mutant forms

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complex with other cell growth regulators found in the normal cell. Formation of such complexes may inactivate or suppress other growth modulators found in the cell. Analysis of established human cell lines of myeloid-derived origin have indicated a high incidence of p53 nonproducers. For example, in HL-60, a cell line established from a patient with acute promyeloctic leukemia, it was found that the p53 gene was mostly deleted and significantly rearranged (39). In other p53 noriproducer cell lines examined we did not detect major gene alterations. It is possible, however, that in these cell lines p53 expression is shut off by minor gene mutations that are not detected by Southern blot analysis. Screening of primary human tumors has also shown that malignancies of myeloid origin frequently seem to lack p5Y expression (37). However, gross rearrangements of' the p53 gene have been detected in one out of seven patients with acute promyelocytic leukemia (63a).We speculate that either rearrangement of the p53 gene in HL-60 was caused by a change in karyotype involving loss of one allele and major alterations in the second allele during the continuous passage of these cells in culture, or, alternatively, that acute promyelocytic leukemia patients exhibit at a low frequency an additional major rearrangement occurring in the short arm of chromosome 17. Therefore, the rearrangement in a single patient with acute promyelocytic leukemia as well as the deletion in the p53 gene in HL-60 cells may be authentic observations. It is also possible that in using Southern blot analysis we underestimate the frequency of p53 abnormalities in patients with acute proniyelocytic leukemia. Compatible with the findings in humans, it was found that the p53 gene in cell lines derived from mouse Friend erythroleukemic tumors exhibit a high incidence of p53 gene rearrangements, which either abolished p53 protein synthesis or induced the expression of aberrant p53 protein forms, suggesting that the development of Friend erythroleukemia in mice depends on p53 shut-off expression (64,65). All these findings seem to support the conclusion that malignant development of myeloid cell lineage may depend on p53 shut-off' expression that is mediated by aberrations in the p53 gene structure. The negligible amounts of p53 mRNA detected in myeloid leukemic cell lines (K562, KCI-22, KG-1, ML-3, U-937, and A-7) (51) favor this thesis. Further support for the idea that p53 may function as an antioncogene comes from previous cytogenetic and restriction fragnient-length protein (RFLP) studies that have shown that allelic deletions of chromosome 17p occur in at least 60% of tumors of the colon, breast, lung, ovaries, cervix, adrenal cortex, and bone, and in at least 30% of brain tumors (66-72). Abnormalities of chromosome 17, including 17q mono-

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somy of chromosome 17 or deletion of 17p, have been reported in lymphoma, leukemia and myelodysplastic diseases as well (73,74). These tumor types account for most of the neoplasms occurring in humans. In colorectal carcinoma it was found that the remaining p53 allele was mutated at the highly conserved region of the p53 gene (32). These mutations coincide with the mutated region found in the murine p53 gene. Moreover, such mutations do not coincide only with colorectal tumors with allelic deletions but also occur in at least some tuniors that have retained both parental 17p allelles (75). In view of these data, it was suggested that pathogenesis of mutations in the p53 gene plays a role in the development of colorectal neoplasia. This, again, may be through inactivation of a tumor suppression function of the wild-type p53 gene. The 17p allele loss was demonstrated in lung cancer as well. Recent evidence suggests that the p53 gene located on 1 7 ~ 1 3is frequently mutated or inactivated in all types of human lung cancer. ‘The genetic abnormalities include gross changes such as homozygous deletions and abnormally sized mRNAs along with a variety of point or small mutations that map to the p53 open reading frame and change the amino acid sequence in a region highly conserved among mice and humans. In addition, very low or absent expression of p53 mRNA in lung cancer cell lines compared with normal lung cells was seen (76). Gene rearrangement was recently reported in breast cancer as well. Similar findings were obtained from the analysis of patients with chronic myelogenous leukemia (CML) (38,77). The molecular structural analysis of the p53 gene in patients with Philadelphia chromosomepositive chronic myelogenous leukemia indicates a significant incidence of gene rearrangements in patients at either the accelerated phase or in blastic crisis (38,77).This is compatible with the observation that progression of CML from the chronic to the acute phase involves frequent aberrations in chromosome 17, to which the p53 oncogene has been mapped. We suggest, therefore, that one of the pathways of development of CML to the acute phase is associated with aberrations in the p53 nuclear oncogene. While it is well established that the chronic phase is directly associated with the rearrangements of the abl oncogene, no consistent aberrations in known oncogenes coincide with the blastic crisis phase. T h e molecular events leading to the inevitable malignant evolution from a chronic to an acute leukemia phenotype remain largely unexplored. However, the frequent and nonrandom cytogenetic changes detected with disease progression (in 80% of patients) suggest that superimposed secondary genetic events account for evolution to CML blastic crisis. Rearrangements detected by us were rather gross and it is possible that

268


the actual incidence of p53 alterations was higher, but that under the resolution conditions used we were unable to detect minor aberrations or mutations occurring in CML patients in the acute phase. At this stage of our study, it is not clear whether rearrangements in this subtype of leukemia indeed include shut-off of p53 expression as previously reported by us in our preliminary study (37) or whether these translocations include the appearance of a novel mRNA product that may account for the progression of the disease. If these rearrangements cause down-regulation, then our findings are compatible with the conclusion that p53 functions as an antioncogene in the chronic phase of the disease. I n agreement with this, we would like to advance the hypothesis that rearrangements in the p53 oncogene may be an important facet in the progression from the chronic phase, in which the typical Ph' chromosomal rearrangement is exhibited, to the more severe phase, with additional genetic aberrations exhibited as rearrangements in the p53 gene (Fig. 2). In summary, a large number of human primary tumors exhibit a rather high incidence of genetic alterations in the p53 gene. Some ofthese are subtle mutations and others represent major realignments in the p53 gene structure. In an effort to map these alterations, it became clear that although there is a certain tendency in some human malignancies to exhibit a higher incidence of mutation in the most conserved coding sequences (32), the overall picture suggests the existence of no "hot spot" for mutations in this gene. Detailed screenings of many tumors in several laboratories have detected mutations at a rather random distribution in exon and intron sequences. Therefore, we would like to suggest that in the case of p53, where an inactivation mutation occurs, there might be no "hot spots" for mutations. Any genetic alteration that interferes with the tertiary structure of the protein may be effective.

1

L First "HIT"

Rearrangements in the "abl" oncogene

0-0 chronic phase

1

Second "HIT"

blastic crisis

Inactivation of the "wild-type'' p53

FIG. 2. Suggested model for the progression of chroiiic myelogenous leukemia from the chronic phase to blastic crisis.

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Ill. Proposed Function

Like virtually all other genes, p53 imposes its effect on the cell through the actions of an encoded protein. We propose that p53 may be an example of a normal regulatory protein, the perturbation of which expression can lead to transformation. However, its exact biochemical and physiological functions are still unknown. The consistency and rapidity with which the p53 protein reaches the nucleus in transformed cells suggest that the principal site of action for this protein must be in this organelle. The most prominent biochemical property of p53 revealed so far is its ability to interact with a variety of proteins of viral and cellular origin: SV40 T antigen, the E l b 58-kDa protein, and HSP72/73. It is of note that activated p53 binds poorly or not at all to the SV40 large T or to Elb. It is highly likely that these interactions are directly related to the biological functions of p53. These proteins are structurally unrelated to one another and have apparently acquired tropism for the p53 protein. It appears that strong selective pressures have worked to evolve a number of tumor viruses oncoproteins that are able to complex with p53. It would seem, therefore, that p53 protein sits at a central and critical point in the cell’s growth regulatory pathways. We suggest, therefore, that inhibition of p53 function may be achieved by these oncoproteins or, alternatively, by gene deletions or alterations. Inactivation of p53 expression may predispose the cell to transformation events, mediated by other genes that are then turned on. In this context, the p53 and the retinoblastoma protein are very similar. The SV40 large ?’ antigen gene encodes two domains, detected by both structural and functional studies, that are involved in transformation of cells in culture; these are required for binding to the retinoblastoma and p53 proteins in cells. Similarly, the adenovirus ElA and E l b gene products may contribute to viral oncogenicity by binding the RB and p53 proteins, respectively, and in this way predispose the cell to additional transformative events.

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DIRECTED PLASMINOGEN ACTIVATION AT THE SURFACE OF NORMAL AND MALIGNANT CELLS Jari Pollanen, Ross W. Stephens, and Antti Vaheri Department of Virology, University of Helsinki, 00290 Helsinki, Finland

1. Introduction

11. Adhesive Interactions of Cell Surfaces with Extracellular Matrices

A. The Pericellular Matrix B. Cellular Contact Sites C. Receptors for Extracellular Matrix Proteins D. Defective Cell-Matrix lntcractions in Transformed Cells 111. Proteolytic Modulation of the Extracellular Compartment by the Plasminogen Activation System A. Plasrninogen and Plasmin B. Plasminogen Activators: Structures, Genes, and Receptors C. The Role of Plasminogen Activators in Cellular lnvasion D. Functions of Plasminogen Activators in Other Physiological Settings E. Plasminogen Activator Inhibitors: Specific Fast-Acting Regulators of Extracellular Proteolysis IV. The Principle of Directed Cell Surface Plasminogen Activation A. Pericellular Distribution of the Components of the Plasminogen Activation System B. Activation of pro-u-PA and Plasminogen Occurs in Situ with Cell Surface-Bound Reactants C. Accessibility of Cell Surface-Bound u-PA to Inhibition by PAI-1 and PAI-2 D. Functional Assembly of the Plasmin-Generating System at Focal Contacts E. Scheme for Cell Surface Plasrninogen Activation F. Prospects for Future Applications V. Concluding Remarks References

I. Introduction

T h e normal phenotypes of cells and tissues provide them with optimal conditions for structural and functional integrity. Complex adhesive interactions of individual cells with extracellular matrices, as well as cell-cell and membrane-cytoskeleton interactions, are instrumental in this process. Specific cell surface receptor proteins for extracellular matrix components, such as fibronectin, laminin, thrombospondin, tenascin, and collagens (Hynes, 1987; Ruoslahti, 1988a; Erickson and Bourdon, 1989; Saunders et al., 1989), have been recently identified and 273 ADVANCES 1N CANCER RESEARCH, VOL. 37

Copyright 62 1991 hv Academic Press, Inc. .411 rights of reproduction in any form reserved.

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shown to be responsible for these adhesive interactions. I n terms of' the cellular microenvironment, the multiple interactions resulting in cellular adhesion are commonly found to be focused on discrete contact sites, formed between individual cells and the substratum. Extracellular proteolysis constitutes one mechanism by which such interactions in the extracellular compartment can be modulated, e.g.. during morphogenetic movements, in the course of embryonic development, or during cellular invasion. T h e normal steady-state rate of matrix turnover is usually slow, but it becomes enhanced in various pathological conditions or when a tissue undergoes rapid remodeling (Hay, 1981). T h e proteolytic modulation involved can be quantitatively significant, because many matrix proteins have a high sensitivity to extracellular proteinases. Considering the destructive potential of these enzymes, the existence of effective control mechanisms that are able to restrict spatially and temporally the expression of their activity is a necessity. A pathophysiological example wherein this regulation appears to fail dramatically is the progressive invasion, dissemination, and metastasis of surrounding normal tissues by malignantly transformed cells. Invasiveness is the distinguishing property of malignant cells, not shared by benign tunior cells. Other characteristic properties include uncontrolled proliferation, morphologic and karyotypic changes, loss of anchorage dependence of growth and of contact inhibition, impaired ability to synthesize and deposit extracellular matrix components, as well as disorganization of their normal cytoskeletal interactions. Malignant cells are very commonly found to secrete plasminogen activators, serine proteinases that by limited cleavage convert the circulating zymogen plasmhogen into plasmin. Plasminogen activators have been associated with various physiological events, e.g., cell migration, inflammation, fibrinolysis, tissue remodeling, tissue involution, and trophoblastic invasion (cf. Dan@ el a/., 1985). T h e activation of plasminogen is potentially able to produce large-scale extracellular proteolytic activity, because of the high concentration of plasminogen in extracellular body fluids (approximately 2 p M ) and the broad spectrum of activity of plasmin on extracellular substrates. However, the extracellular activity of plasmin is under the regulation of its fast-acting primary inhibitor ( ~ 2 antiplasmin, and of as-macroglobulin and other proteinase inhibitors. Plasminogen activators, in turn, are regulated by complex formation with the specific and fast-acting plasminogen activator inhibitors. Highaffinity cell surface receptors for plasminogen activators and binding sites for plasminogen and plasmin (Dan@et al., 1989) serve to localize proteolytic activation to the pericellular area, creating yet another level of regulation. In this respect, the plasminogen activation system bears re-

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semblance to the cascadelike processes of blood coagulation and complement activation, in which sequential proteolytic reactions take place in a highly localized manner in the extracellular compartment (MiillerEberhard, 1988; Maim et al., 1988). In this review, we cover the regulatory mechanisms of plasminogen activation at the cellular level, with a particular emphasis on the events occurring at the cell surface-a compartment where the adhesive and proteolytic interactions are brought together at the cell-matrix interface.

II. Adhesive Interactions of Cell Surfaces with Extracellular Matrices A.

‘THE PERICELLULAR

MATRIX

Extracellular matrices are dense three-dimensional networks of interacting glycoproteins and proteoglycans that determine cell shape and maintain tissue integrity. The composition of extracellular matrices in different tissues in the body-in the interstitial connective tissue matrices o r in the basement membranes-varies to a great extent; they mainly consist of different combinations of collagens, fibronectin, laminin, entactin, elastin, proteoglycans, and hyaluronic acid. The term pericellular matrix is often used to describe the matrix deposited by adherent cells in culture, thereby emphasizing the close association of the matrix with the cell surface and defining it as a subset of the various forms of the extracellular matrix (Hedman and Vaheri, 1989). In ultrastructural analysis, the pericellular matrix is found to consist of filamentous and amorphous material and connecting neighboring cells and their growth substratum. Cell surface-associated fibronectin has been located predominantly in the pericellular matrix material (Hedman et nl., 1978; Chen et d., 1978; Crouch et al., 1978). Moreover, the external fibronectin-procollagen fibers were shown to codistribute and interact with heparan and chondroitin sulfate proteoglycans of the pericellular matrix (Hedman et al., 1982). T h e proteoglycans are a diverse group of proteins, grouped together mainly on the basis of their common sulfated carbohydrate component, glycosaminoglycan (Ruoslahti, 1988b). Located either on the cell surface or in the extracellular matrix, proteoglycans promote or modulate cell adhesion and matrix assembly by binding to other matrix components, and by linking them together (Ruoslahti, 1989). These and multiple other interactions that facilitate cell adhesion and anchorage form the structural basis for the functions of normal tissues. I n addition, certain adhesive protein components of pericellular matrices are able to stimulate cell migration (Ali and Hynes, 1978; Thiery et aE.,

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J A R T P ~ L L K N E N ET 4 ~ .

1985) and to influence cellular proliferation and differentiation. These adhesive interactions and their reversal are considered to be particularly important during the morphogenetic migration of cells in embryonic development (Ekblom et al.. 1986) and during tumor cell invasion arid metastasis (Liotta et al., 1983; Vasiljev, 1985). B.

CELLULAR CONTACT SITES

T h e dynamic interactions between cells and their perirellular matrices depend on the delicate balance between adhesion and cellular detachment, which is manifested at the sites where cultured cells contact each other and the growth substratum. Two approaches have been highly informative in studiey on thc specialized niicroenvironment of the cellular contact sites: electron microscopy (EM) (Abercrombie et al., 197 1) and interference reflection microscopy (IRM) (Curtis, 1964). In cultures of fibroblasts, three types of cell-substratum and cell-cell contact sites may be distinguished on the basis of morphological criteria (Chen and Singer, 1982): 1. Focal contacts (also called focal adhesions or adhesion plaqucs) 2. Close contacts 3. Fibronexus contact sitcs (also called extracellular matrix contact sites)

Of these, focal contacts are the sites of closest apposition between the ventral cell surface and the growth substratum (approximately 15 nm), and an electron-dense cytoplasmic zone consisting of microfilaments is associated with these regions (Abercrombie et al., 1971; Chen and Singer, 1982). Focal contacts appear in IRM as discrete black areas, correlating with the adhesion plaques visualized by electron microscopy (Abercronibia and Dunn, 1975; Heath and Dunn, 1978). A third method used to study focal contacts has taken advantage of the inaccessibility of antivitronectiri antibodies to these sites in immunoHuorescence microscopy (Neyfakh et al., 1983). Focal contacts are frequently observed close to the leading edge of moving cells, especially in newly seeded cultures of fibroblasts, where spreading and migrating cells extend laniellipodia and microspikes in the forward direction (Abercrombie et al., 197 1). As these structures adhere to the substratum of the culture dish, new focal contacts are formed (Izzard and Lochner, 1980). Focal contacts are associated with stress fibers (Abercrombie et al., l971), and an inverse correlation has been demonstrated between stress fibers and cell movement (Herman et nl., 1981; Couchman et al., 1982). In addition, fluorescein-labeled actin mi-


277

croinjected into cells concentrates at newly formed focal contacts (Kreis et al., 1982; Wang, 1984), and probably both assembly and disassembly of the actin filaments occur at these sites, regulated by the migratory status of the given cell. Microtubules and possibly some intermediate filaments contribute to the functional stability of the focal contacts (Geiger et al., 1984; Green and Goldman, 1986; Bershadsky et al., 1987; Rinnerthaler et al., 1988). Close contacts typically exhibit a 30- to 50-nm spacing between the cell surface and the substratum, and they appear as larger greyish surface areas when studied with the IRM technique (Abercrombie and Dunn, 1975; Izzard and Lochner, 1976; Heath and Dunn, 1978; Bereiter-Hahn et al., 1979). Close contacts are considered a more temporary type of adhesion than are focal contacts. Fibronexus contacts (Singer, 1979) are characteristic of well-spread and stationary fibroblasts, and they predominate over the two other types in late cultures of cells (Chen and Singer, 1982). In this type of contact, the ventral cell surface is generally far removed from the substratum but is sporadically connected to the substratum by large and cablelike filamentous aggregates of the components of the extracellular matrix (Chen and Singer, 1982). Very close transmembrane associations of actin microfilaments and fibronectin fibrils, which are typically coaxially distributed, have been demonstrated in these structures (Singer, 1979, 1982). A general criticism can be raised against investigations of the cellular contact sites, namely, that the system includes artifacts originating from the planar configuration of the cell culture plates. However, the contact sites cannot be entirely regarded as products of the somewhat artificial conditions of tissue culture, because structurally similar adhesions have been observed in vivo in, e.g., the dense plaque of smooth muscle cells, the myotendinous junction of skeletal muscle, the zonula adherens junction of adjacent epithelial cells, as well as the focal adhesions of platelets bound to the components of the subendothelial matrix exposed during blood vessel injury (see Burridge et al., 1988). Structural protein components of the cellular contact sites have been studied in great detail, and new components will most certainly continue to be discovered. Focal contacts contain vinculin (Geiger, 1979; Burridge and Feramisco, 1980), talin (Burridge and Connell, 1983), and a-actinin (Chen and Singer, 1982) on the cytoplasmic side, the latter being located farther from the plasma membrane than the other two. Focal contacts apparently do not contain fibronectin on the extracellular side (Chen and Singer, 1980, 1982; Birchmeier et al., 1980), although this issue has been controversial. Moreover, the experimentally observed removal of substratum-bound fibronectin from focal adhesion sites (Avnur and Geiger,

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1981) was shown to be dependent on the nature of the coating used, as well as on the presence or absence of serum in the cultures (Grinnell, 1986). In a study of embryonic neural crest cell adhesion and migration, the highly motile cells showed only the diffusely distributed fibronectin receptor complex, whereas the stationary cells had distinct fibronectin receptor immunolabeling concentrated in focal adhesions (Duband et af., 1986). T h e most mature type of adhesion site in cultured fibroblasts, the fibronexus, features codistribution of fibronectin, its receptor (integrin), vinculin, a-actinin. and actin filaments throughout the entire contact site, thereby providing a transmembrane linkage between the extracellular matrix and the cytoskeleton (Chen et al., 1985b; Damsky et al., 1985).T h e integrins are a large family of receptor proteins with specificity for extracellular matrix ligands, and are thought to play an important role in cell attachment and adhesion (Hynes, 1987; Ruoslahti and Pierschbacher, 1987). Further support for the concept of a transmembrane linkage has emerged from in nitro binding data, which has demonstrated the sequence of interactions between the cytoplasmic domain of the integrin /3 subunit and talin (Horwitz et al., 1986; Buck and Horwitz, 1987), talin and vinculin (Burridge and Mangeat, 1984), vinculin and a-actinin, and a-actinin and actin microfilaments (see Burridge et ul., 1988). The low affinities of some of these interactions, as well as the diverse distribution patterns of the individual components in some cell types, have led to speculations that additional components may also be involved (Burridge el al., 1988). On the cytoplasmic face of focal adhesions, additional proteins have been detected, including a 200-kDa protein of cardiac intercalated discs (Maher and Singer, 1983), an 82-kDa protein of fibroblasts and smooth muscle cells (Beckerle, 1986), 70- and 80-kDa proteins detected by human autoantisera (Seneca1et al., 1987),60- and > 300-kDa proteins discovered by a monoclonal antibody that labels focal adhesions (Oesch and Birchmeier, 1982), and a calcium-dependent protease I1 (Beckerle el al., 1987). Recently, a new 150-kDa protein component of focal adhesions, called tensin, was identified and shown to bind to both vinculin and F-actin (Butler and Lin, 1990). The third type of adhesion site, close contacts, can be distinguished from focal contacts by the absence of intracellular vinculin immunolabeling at these sites and by the presence of a-actinin and fibronectin (Chen and Singer, 1982). The structural components of focal contacts on the extracellular side include heparan sulfate proteoglycan (Woods et af., 1984), fibulin (Argraves et af., 1989), urokinase-type plasminogen activator (Fig. 1; discussed in Section IV), as well as vitroncctin (Neyfakh et al., 1983;


279

FOCAL CONTACT

FIG. 1. A schematic illustration of some of the components of focal contacts (focal adhesions) and their interactions. Urokinase-type plasminogen activator (u-PA), shown bound to its cellular receptor (u-PA-R), and plasminogen activator inhibitor type 1 (PAI-1) are discussed in the text in Section IV. An integrin is depicted here functioning as a vitronectin receptor, whereas other members of the integrin receptor family are involved in fibronectin-, collagen-, and lasninin-mediated cell adhesion (not shown). ECM, Extracellular matrix; FN, fibronectin; VN, vitronectin.

Baetscher et al., 1986).However, the distribution of vitronectin covers the entire ventral cell surface area as a homogeneous carpet and is not restricted to the contact sites. The viral D glycoprotein in herpes simplex virus-infected cells (Norrild et al., 1983) and the actin attachment siteassociated 30B6 antigen, which share many properties with the integrins (Kogalski and Singer, 1985),are other integral membrane proteins localized at focal contacts. Figure 1 summarizes some of the components of focal contacts and their interactions. Given the obvious function of cellular contact sites in cellular anchorage, not surprisingly the membrane-cytoskeleton interactions in-

280

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POLLXNEN ET AL..

volved constitute a dynamic entity affected by various regulatory agents (Geiger, 1983; Vasiljev, 1985). Focal contacts have been shown to be modulated, for example, by agents that elevate intracellular levels of cyclic AMP, by epidermal and platelet-derived growth factor, other competence factors, and tumor promoters, and by viral transformation (see Burridge et al., 1988). Interestingly, the protein products of the oncogenes src, abl, and yes, which are all tyrosine kinases, have been detected in the modified focal adhesions of virally transformed cells (Rohrschneider, 1980; Nigg et al., 1982; Krueger et al., 1984; Rohrschneider and Najita, 1Y84; Gentry and Rohrschneider, 1984). Vinculin, talin, and integrins all contain tyrosine phosphorylation sites (Sefton et al., 198 1; Pasquale et al., 1986; Hirst et al., lY86; Declue arid Martin, 1987), but the exact role of phosphorylation of target proteins in focal adhesion disassembly still remains to be established (Turner et af.,1989). Nevertheless, the plasma membrane-cytoskeleton interactions at the cellular contact sites undoubtedly provide an important route of transmembrane signaling. C . RECEPI-ORS FOR EXTRACELLULAR MATRIXPROTEINS

The knowledge of the molecular mechanisms involved in the adhesive interactions between the cell surface and the matrix has increased vastly over the past few years. A large family of cell surface receptors. coinnionly termed inlegrins, has been identified and characterized as receptors for extracellular matrix proteins, as well as key molecules in the adhesion of platelets and lymphoid and myeloid cells. They also appear to have an important role in the morphogenesis of Drosophila (Hynes, 1987; Ruoslahti and Pierschbacher, 1987; Fessler and Fessler, 1989). Two main approaches have been used to isolate these receptors: immunoselection with antibodies blocking cell adhesion, and affinity chromatography with extracellular ligands. The integrins are heterodimers of two menibrane-spanning subunits, a and p, that are noncovalently bound to one another. Both subunits appear to contribute to the formation of the ligand-binding site. Initially, the integrins were grouped into three major families, each with a common p subunit: the family consisted of the very late antigens (VLAs). as well as receptors for collagens, laminin, and fibronectin; and family consisted of the leukocyte adhesion molecules; and the & family consisted of the vitronectin receptor a,& and the platelet glycoprotein gpIIbiIIIa (see Hynes, 1987). Now, with the discovery of several new p subunits (Table I), the integrin superfamily of adhesion receptors has proved to be much more diversified. To date, at least 11 a subunits and 7 /3 subunits have been reported and are able to


28 1

form at least 17 different heterodimer combinations (cf. Ruoslahti and Giancotti, 1990). Using synthetic peptides covering the amino acid sequence of the cell-binding domain of fibronectin, a tripeptide, Arg-Gly-Asp (RGD),was identified as a cell recognition motif (Pierschbacher and Ruoslahti, 1984). Since then, one or more RGD motifs have been found in a large number of adhesive proteins, and RGD-containing peptides have been found to inhibit their interactions in cell adhesion assays; these proteins include vitronectin, type I collagen, fibrinogen, von Willebrand factor, osteopontin, and possibly thrombospondin and collagens other than type I

TABLE I MATRIX RECEPTORS FOR ADHERENTHUMAN CELLS Keceptor

Ligands"

Effectors

fflP1

LM, COLL I V

RGD

ff2Pl

COLL, LM, FN

RGD

ffQP1

LM, COLL, FN

RGD

ff4P1

.>PI

FN FN

IIICS RGD

ff& I

LM

RGD

ff"P3

VN, vWF, FBG, TSP, OP, BSP

RGD

ffvPx

VN, FN

ff"P. ff,PI

?

FN, VN

RGD RGD RGD

ffaP4

LM?

?

Proteoglycans

Various ECM components LM

Ionic charge

69 kDa

YIGSR

References Dedhard and Saulnier (1990); Forsberg el al. (1990) Kunicki et al. (1988); Kirchhofer et al. (1990); Languino et al. (1989) Wayner and Carter (1987); Wayner et al. ( 1988) Mould el al. ( 1990) Pytela el al. (1985a); Takada et al. (1987); Argraves el al. (1987); Wayner et al. (1988) Sonnenberg et al. (1988); Dedhard and Saulnier (1990) Pytela et al. (1985b); Charo et al. (1987); Cheresh and Spiro (1987); Lawler et al. (1988); cf. Ruoslahti and Giancotti ( 1990) Cheresh et al. (1989) Freed et al. (1989) Vogel el al. (1990); Bodary and McLean ( 1990) Sonnenberg et al. (1987); Kajiji et al. (1989); Suzuki and Naitoh (1990); Hogervorst et al. (1990) cf. Ruoslahti (1989); Bernfield and Sanderson (1990) Wewer et al. (1987); Basson et al. (1990).

Abbreviations: LM, laminin; COLL, collagens; FN, fibronectin; VN, vitronectin; vWF, von Willebrand factor; FBG, fibrinogen; TSP, thrombospondin; OP, osteopontin; BSP, bone sialoprotein I; ECM, extracellular matrix.

282

JAR1 POLLANEN E T A L .

(Ruoslahti and Pierschbacher, 1987).The question therefore arises as to how the mutually exclusive specificity profiles of some of the integrins seen at the protein level can he explained, if' most of these interactions can be inhibited with the same RGD-containing synthetic peptides? Proposed answers to this question include secondary binding sites unique to each protein ligand, or the specificities residing in divergent conformations of the RGD peptides (Ruoslahti and Pierschbacher, 1987). Analysis of the cell-binding domain of fibronectin by site-directed riiutagenesis has established a second recognition site functioning synergistically with the RGD site (Obara el al., 1988). The major receptor for the cellbinding domain of fibronectin is the integrin a5P1 (cf. Kuoslahti and Pierschbacher, 1987), whereas the receptor for a secondary site located in the IIICS domain is the integrin a & ~ (Mould f t d., 1990: see Table 1). In addition to integrins, cell adhesion to an extracellular matrix can be mediated by cellsurface proteoglycans (Rwslahti, 1989), including heparan sulfate proteoglycans and the epithelial proteoglycan syndecan (Bernfield and Sanderson, 1990). The latter consists or chondroitin sulfate and heparan sulfate chains linked to an integral membrane core protein (Saunders et al., 1989; Marynen et af., 1989; Mali P t al., 1990). Syndecan binds via its heparan sultate chains to collagen types I, 111, and V, fibronectin, tenascin. and throriibospondin (ct'. Bernfield and Sanderson, 1990), interacts with the actin cytoskeleton, and has a polarized localization on the basolateral surfaces of epithelial cells (Rapraeger et al., 1986). Proteolytic cleavage of syndecan by extracellular enzymes with trypsinlike specificity results in shedding of the matrix-binding ectodomairi from the cell, leaving only its menibranespanning domain (Saunders et al., 1989; Jalkanen t t af., 1987). Furthermore, the heparin-binding domain of fibronectin is required for the full assembly and organization of focal contacts and the associated microfilaments in some cells (Izzard et a/., 1986; Woods et al., 1986). T h e cell surface heparan sulfate proteoglycans therefore appear to mediate an important auxiliary mechanism that complements integriti-mediatetl adhesion. Finally, a GY-kDa nonintegriri laminin-binding protein has been characterized, which recognizes a different peptide motif, YIGSR (see Table I ) . Comparison of this receptor with laminin-binding integrins in endothelial cell adhesion led to a suggestion that the high-affinity 69-kDa interaction might be involved in long-term contacts, whereas the lower affinity integrins could be more important for the initial adhesive event (Basson et al., 1990).


283

D. DEFECTIVE CELL-MATRIX INTERACTIONS IN TRANSFORMED CELLS A general feature of the transformed phenotype is the decreased ability of the cells to adhere and spread on solid substrates (Vasiljev, 1985). Consequently, the cellular contact sites they form in culture appear less stable and immature, with fewer attached stress fibers (Bershadsky et al., 1985). Transformed cells in culture can also be distinguished from their normal counterparts by their contact-independent manner of growth as multilayers. The well-established loss of pericellular matrix following viral transformation concerns all the major structural components, such as procollagens, fibronectin, laminin, and heparan sulfate proteoglycan, and probably results at least partly from altered biosynthesis and failure in extracellular deposition (Alitalo and Vaheri, 1982). However, the fibronectin synthesized and secreted by transformed cells has been shown to be f-unctionallynormal in cell adhesion assays, and its addition to cell cultures transiently reverses the transformed phenotype (Hayman et al., 1981; Wagner et al., 1981). This was also observed with matrix-derived fibronectin from normal cells (Yamada et al., 1976) and with isolated pericellular matrices of normal fibroblasts (Vaheri et al., 1978). One explanation for the failure of assembly of fibronectin-rich pericellular matrices by transformed cells has been provided by the observation that viruses encoding the rus oncogene are able to induce specific reductions in the levels of the fibronectin receptor a5P1and of two other integrin receptors (Plantefaber and Hynes, 1989). Overexpression of transfected a&, integrin was reported to lead to suppression of the transformed phenotype (Giancotti and Ruoslahti, 1990). The altered adhesive phenotype of some transformed cells can also be due to expression of a new type of integrin with an altered adhesive profile, as demonstrated with the a,& integrin (Cheresh et al., 1989). In addition, chemical transformation of cells was shown to be associated with increased expression of laminin and collagen receptors and decreased expression of vitronectin receptors (Dedhard and Saulnier, 1990). T h e loss of pericellular matrix in malignant transformation could also be due to increased proteolytic degradation. Matrix proteins, especially fibronectin, are in general sensitive to extracellular proteinases and can be cleaved by plasmin, elastase, cathepsin G , and trypsin (Vartio et al., 1981, 1983). Viral transformation of fibroblasts was associated with matrix degradation (Fairbairn el ul., 1985) and limited cleavage of cellular fibronectin by plasminogen activator (Quigley et al., 1987). Degradation

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of fibronectin in cultures of Kous sarcoma virus-transformed fibroblasts occurred at focal contacts, in colocalization with the sites of expression of the transforming gene product p60"" (Chen et al., 1984, 1985a). The fibronectin-degrading enzymes of these cells were later characterized as a 150-kDa metalloproteinase and a 120-kDa serine proteinase (Chen and Chen, 1987). Furthermore, maliganantly transformed cells are generally found to secrete increased amounts of proteolytic enzymes and, in particular, plasminogen activators (Dan@et al., 1985), as discussed in detail below. Ill. Proteolytic Modulation of the Extracellular Compartment by the Plasminogen Activation System

A. PLASMINOCEN A N D PLASMIN Plasminogen is an inactive circulating qmogen of the fibrinolytic enzyme system; it can be converted to the proteolytically active enzyme plasmin through the action of enzymes designated as plasminogen activators. Plasminogen molecules are abundantly distributed: they have been found to be present in most extracellular fluids studied, and in plasma at a concentration of 2.0 pM (Lijnen and Collen, 1982). The principal site for plasminogen synthesis appears to be the liver (Raum et al., 1980), although synthesis in other tissues has been reported as well (Valinsky and Reich, 1981; Isseroff and Rifkin, 1983; Saksela and Vihko, 1986). Native human plasminogen is a single-chain glycoprotein containing 790 amino acids, with a molecular weight of 92,000 (Wiman, 1973, 1977; Sottrup-Jensen et al., 1978). The native form of plasminogen has glutamic acid in the amino-terminal position and is designated Glu-plasminogen (Wallen and Wiman, 1975). It is susceptible to specific cleavage catalyzed by plasmin, at positions Arg 67-Met 68, Lys 76-Lys 77, and Lys 77-Val78, resulting in modified forms termed Lys-plrrsminogen (Wallen and U'iman, 1972; Collen and DeMaeyer, 1975; Lijnen and Collen, 1982). These two forms of plasminogen have profound conformational differences (Violand ei at., 1978), reflected in the enhanced rate of activation of Lys-plasminogen to plasmin by urokinase-type plasminogen activator (LI-PA)(Claeys and Vermylen, 1974). I n the determination of the primary structure of human plasminogen, it was found that five regions had extensive sequence and disulfide bridge pattern homology with tripleloop structures known as kringles. Similar structures have been detected in prothrombin (Sottrup-Jensen et al., 1975), u-PA (Gunzler et al., 1982), tissue-type plasminogen activator (t-PA) (Pennica et al., 1983; Wallen et nl., 19831, and lipoprotein(a) (McLean et al., 1987; Eaton P t al., 1987).


285

The plasminogen molecule contains five so-called lysine-binding sites, which specifically interact with o-aminocarboxylic acids, such as lysine, 6-aminocaproic acid, and tranexamic acid (Markus et al., 1978). All of the lysine-binding sites, one of which has very high affinity, are located in the region containing the five kringles (Sottrup-Jensen et al., 1978; Markus et al., 1981; Fig. 2). A change in conformation is induced by saturation of the lysine-binding sites on Glu-plasminogen (Claeys and Vermylen, 1974; Thorsen et al., 1974; Violand et al.,1975; Markus et al., 1979), and this is accompanied by a stimulatory effect on its activation (Takada and Takada, 1981). Interestingly, both these changes resemble the ones observed during the conversion of Glu-plasminogen to Lys-plasminogen. Plasmin is a trypsinlike serine protease that is formed by cleavage of the Arg 560-Val561 peptide bond in plasminogen (Robbins etal., 1967). Plasmin has a relatively broad substrate specificity. I n addition to cleaving fibrin polymers of the clot in thrombolysis (Astrup, 1978), plasmin is able to degrade several components of extracellular matrices and basement membranes, such as fibronectin, laminin (Liotta et al., 1981), and thrombospondin (Lawler and Slayter, 1981). A significant part of'the actions of plasmin on extracellular matrices is exerted indirectly via the activation of latent procollagenases and other metalloproteases (O'Grady et al., 1981; Tryggvason et al., 1987; Werb et al., 1977). The plasmin molecule contains two polypeptide chains (designated A and B) held together by two disulfide bridges (Fig. 2). The A chain, with a molecular weight of 65,000, contains the kringles and thus all the lysine-binding sites and is responsible for fibrin binding. The B chain, with a molecular weight of 25,000, contains the active site, which is similar to the one in other serine proteinases, i.e., it is composed of the amino acid residues His 602, Asp 645, and Ser 740 (Sottrup-Jensen et al., 1978). According to a proposed activation mechanism of plasminogen, the native form is first activated either to Glu-plasmin by plasminogen activators or converted autocatalytically by plasmin to Lys-plasminogen, both of which are then converted to Lys-plasmin as the final product (Dan@et al., 1985). In the fibrinolytic system, plasminogen is bound to a solid fibrin substrate on the thrombus in a specific interaction through the lysinebinding sites, allowing plasmin to be formed in situ and thus to escape the inhibitory effect of circulating plasma inhibitors (Collen, 1980). Interaction by plasma inhibitors depends on the same kringle interactions as are involved in fibrin or cell binding. Furthermore, recent work has demonstrated that plasminogen specifically interacts with the cell surface of endothelial cells, platelets, and many other types of cells, including human tumor cells (Hajar et al., 1986; Miles and Plow, 1985, 1988; Plow et al., 1986; Burtin and Fondaneche, 1988; Pollanen, 1989).The binding of

286

JARI POLLANEN ET AL.

Plasminogen FIG. 2. A schematic illustration of human plasminogen. The kringle domains of the A-chain are numbered 1 to 5. See text for detalls. (Modified from Sottrup-Jensen el al.. 1978.)

plasminogen to the cell surface involves the lysine-binding sites, and plasmin bound to cells appears to be resistant to a2-antiplasmin (Plow et al., 1986). Additionally, plasminogen has been shown to bind to the extracellular matrix and its specific components (Salonen el al., 1984, 1985; Silverstein et al., 1985; Knudsen el al., 1986). Thus, the pericellular binding sites could provide a means of recruiting plasminogen for focalized extracellular proteolysis.

B. PLASMINOGEN ACTIVATORS: STRUCTURES, GENES, AND RECEPTORS Enzymes that activate plasminogen, found in most tissues and body fluids, exist in two major characterized forms, tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activators, with distinctive structural

287


and functional properties. Other plasminogen activators include bacterial streptokinase and staphylokinase, the so-called intrinsic activation system, and some other vertebrate plasminogen activators (cf. Dan@et al., 1985). The two major activators, t-PA and u-PA, have a 40% homology at the amino acid level (Degen et al., 1986)and are coded by different genes (Ny et al., 1984; Riccio et al., 1985; Degen et al., 1986). The u-PA gene is located on chromosome 8 (Rajput et al., 1985), and that of t-PA, on chromosome 10 (Rajput et al., 1985; Tripputi et al., 1985). The human u-PA gene encodes a 2.5-kb mRNA (Verde et al., 1984), which is translated into a 53-kDa glycoprotein (Gunzler et al., 1982);the corresponding values for human t-PA are 2.7 kb for mRNA and 70 kDa for the protein (Pennica et al., 1983; Fisher e t a l . , 1985).At the protein level, the structure of both PASconsists of distinct functional domains (Fig. 3). u-PA and t-PA have in common (1) a growth-factor-like domain, which bears a resemblance to the receptor-binding regions in epidermal growth factor and 1

kcingle plasmin cleavage site involved i n the conversion o f pro-u-PA to active enzyme

-- --1-y- -----= ---158

-

P r o - u- PA FIG. 3. A schematic model of the inactive one-chain proenzyme forni of’human u-PA. The arrow pointing at the Lys 158-lie 159 peptide bond is cleaved by plasmin during the conversion of proenzyme to two-chain active enzyme. Cleavage at the second site, indicated by another arrow, results in an amino-terminalfragment and “low-molecular-weight u-PA” that retains catalytic activity.

288


transforming growth factor a (Gunzler et al., 1982; Derynck et al., 1984; Komoriya et al., 1984), ( 2 ) one kringle structure, and (3) the catalytic domain in the carboxy-terminal part of the molecule, which is closely related to those of other serine proteinases (Strassburger et al., 1983). T h e structural differences between u-PA and t-PA include the absence in u-Pa of the second kringle and of the 43-residue amino-terminal finger domain present in t-PA. These differences have functional implications, because the second kringle in t-PA accounts for most of its fibrin affinity (van Zonneveld et al., 1986), with some contribution coming from the finger domain. The binding of t-PA to fibrin is essential for its full activity to be expressed (Hoylaerts et al., 1982), whereas u-PA has substantial plasminogen activator activity on its own. Both u-PA and t-PA are produced and secreted as single-chain (sc) polypeptides [termed scu-PA, or proenzyme u-PA (pro-u-PA), and sctPA]. The question whether single-chain u-PA really is a genuine proenzyme, with no intrinsic plasminogen-activating capacity, has given rise to much controversy (discussed in Dan@et al., 1989). This debate emerged from studies reporting considerable plasminogen-activating capability for scu-PA purified from several sources and also for a recombinant scu-PA molecule (Collen et al., 1986; Liljnen et al., 1986; Stump et al., 1986a,b). These studies, however, employed assay technology that did not exclude the activation of pro-u-PA by contaminant or generated two-chain (tc) u-PA (tcu-PA) or plasmin (Petersen et al., 1988). Evidence supporting the concept of scu-PA as an inactive proenzyme has been derived from experiments using synthetic substrates, and macromolecular as well as synthetic inhibitors for u-PA (Nielsen el al., 1982; Skriver el al., 1982; Wun et al., 1982; Gurewich et al., 1984; Kasai et al., 1985; Eaton P t al., 1984; Vassalli et aE., 1984; Andreasen et al., 1986b; Pannel and Gurewich, 1987; Stephens et al., 1987). Using site-directed mutagenesis at the plasmin cleavage site of scu-PA, an approximately 200-fold lower activity was shown for the variant recombinant SCLI-PA molecule when compared with the fully active tcu-PA (Nelles et al., 1987; Lijnen et al., 1990). Moreover, recent kinetic investigations have led to a similar conclusion (Ellis et al., 1987; Peterseri el al., 1988). Clearly, the conversion of the virtually inactive scu-PA to the active tcu-PA must be an important regulatory step in the u-PA pathway of plasminogen activation, although the factor(s) responsible for the initiation of the cascade in viva remain to be discovered. pro-u-PA is the predominant form found in the extracellular compartment in vzvo (Skriver et al., 1984; Kielberg et al., 1985). Interestingly, production of an active u-PA was reported for cultured leukemia cells, in contrast to several adherent tumor cell lines, which all secreted almost exclusively

DIRECTED CELL SURFACE PLASMINOCEN ACTIVATION

289

pro-u-PA (Stephens et al., 1988). Conversion of pro-u-PA to active u-PA by a plasmin-independent extracellular mechanism has been suggested in cultured erythroleukemia cells (Alitalo et al., 1989).Zn vitro, the conversion of scu-PA to tcu-PA may be accomplished by minute amounts of plasmin (Wun et al., 1982; Blasi et al., 1987). Certain other serine proteinases, such as kallikrein and trypsin (but not, e.g., thrombin), are able to activate the proenzyme as well (Eaton et al., 1984; Ichinose et al., 1986). T h e active form of u-PA consists of a 24-kDa light or A chain and a 30-kDa heavy or B chain, connected by one disulfide bridge (Verde et al., 1984; Belin et al., 1985). Further cleavage of tcu-PA by plasmin leads to an active 33-kDa low-molecular-weight form (Barlow et al., 1981) that lacks most of the A chain, including the growth factor domain (see Fig. 3). Low-molecular-weight u-PA therefore cannot bind to cell surfaces. Specific cell surface receptors for u-PA were originally discovered in monocytes and monocyte-like U-937 cells (Vassalli et al., 1985; Stoppelli et al., 1985), and they have now been detected in a variety of normal and transformed cells (reviewed by Blasi, 1988). The receptor-binding site of human u-PA was localized to the growth factor domain in the aminoterminal region of the molecule (Appella et al., 1987). The characteristics of the interaction of u-PA with its cellular receptors include a high affinity ( K d 0.2 nM),lack of internalization, a long half-life of 3-6 hr, and stable enzymatic activity of u-PA when bound (Blasi, 1988). Studies by Stoppelli et al., (1986) demonstrated autocrine occupation of the receptors of human A43 1 cells by endogenous pro-u-PA. Both number and affinity of the receptors can be regulated by various effectors, such as the tumor promoter phorbol myristate acetate, epidermal growth factor, and dimethylformamide (Stoppelli et al., 1985; Blasi et al., 1986; Nielsen et al., 1988; Boyd et al., 1988; Picone et al., 1989). Molecular characterization of the receptor protein has shown that it is a single-chain 55- to 60-kDa glycoprotein (Nielsen et al., 1988; Estreicher et al., 1989).T h e binding of t-PA onto endothelial cells has also been described (Hajjar et al., 1986), although no receptor molecule has been isolated yet. It is therefore likely that PA receptors serve to localize plasminogen activator activity onto the cell surface, generating proteolytic potential for the modulation of the surrounding pericellular compartment. Plasminogen activators are regulated both at the level of transcription and at the level of expression of enzyme activity by several chemical and metabolic effectors (reviewed by Dan@et al., 1985; Saksela and Kifkin, 1988). These include phorbol esters; growth factors such as epidermal growth factor, platelet-derived growth factor, and transforming growth factor p; glucocorticoids and other steroid hormones; and several peptide hormones and cytokines. However, the effects on PA synthesis

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JAW1 POLLANEN ET AL.

appear to be highly variable, mainly depending on the organ and cell culture system studied. C. THEROLEOF PLASMINOCEN ACTIVATOKS IN CELLULAR INVASION

T h e assumption that invasive cells need to express proteolytic enzyme activity is based on the special requirements of such cells for cellular detachment, breakdown of the components of interstitial matrices and basement membranes, reattachment, and subsequent penetration at new sites during the multistep process of cancer metastasis. Experimental evidence supporting this concept has emerged from reports of increased secretion of proteolytic enzymes by malignant versus normal cells, the potential enzyme types including the plasminogen activator-plasmin system, specific collagenases, and other serine, cysteine, and metalloproteinases (cf. Reich, 1978; Dan@et al., 1985). A role for plasniinogen activators in these events is supported by studies measuring and characterizing PA in tumor tksues. Turnor tissue extracts from the common carcinomas of the colon, breast, lung, and prostate all contained significantly more u-PA than did their normal counterparts (Markus et a[., 1980; Corasanti et al., 1980; Camiolo et ol., 1981, 1984). Moreover, malignant tissue samples usually exhibited stronger PA activity than did the benign ones (Dan@et al., 1985). The type of plasminogen activator produced by tumor cells appears to be mostly u-PA, with the exception of melanoma and neuroblastorna cells, which produce t-PA (Wilson et nl., 1980; Rijken and Collen. 1981; Neuman etnl., 1989). Certain leukemia cells also produce t-PA, which has been shown to be of prognostic significance (Wilson P t al., 1983). Cells transformed with oncogenic viruses commonly secrete high levels of PA, correlating with the appearance of other features of the malignant phenotype, including anchorage-independent growth, migration across the edge of a wound, loss of pericellular fibronectin, reduced cytoskeletal organization, arid morphological changes (Ossowski et al., 1973a, b, 1975; Reich, 1973; Unkeless et al., 1973; Pollack etal., 1974). In addition, in an immunohistochemical study of 1,ewis lund carcinoma, u-PA was localized in invading areas of the tumors (Skriver et al., 1984). In other systems this approach has given similar results (Camiolo et al., 1984; Kohga et al., 1985). Some studies failed to demonstrate a correlation between the malignant phenotype and level of PL4activity (Wolf and Goldberg, 1978). A possible reason for this failure could be the assay methods applied, which largely ignored the presence of pro-u-PA and endogenous PA inhibitors, as well as the specific type of PA concerned.


29 1

Second, in many of these studies only the secreted PA activity was measured. In other studies, a clear positive correlation could be demonstrated between PA activity and tumor growth (Mira-y-Lopez et al., 1983, 1985). An enhancement of proliferation of an epidermal tumor cell line was observed following the apparent binding of u-PA to its cellular receptors (Kirchheimer et al., 1987), and the inhibition of the activity of endogenous receptor-bound u-PA led to decreased proliferation in a human melanoma cell line (Kirchheimer et al., 1989). Using enzyme immunoassay, moderately increased levels of u-PA antigen were measured in the plasma of patients with breast cancer (Grflndahl-Hansen et al., 1988a). Moreover, it has also been demonstrated that the high u-PA antigen content of tumor tissue extracts is an independent prognostic factor in human breast cancer, strongly associated with early relapses of the patients (Janicke et al., 1989, 1990). This clinically relevant finding was achieved by applying two selected monoclonal antibodies to u-PA in an immunoassay of the patient samples. In fact, u-PA had the strongest impact on relapse rate after surgery (relative risk, 2 1.2), outweighing such established clinical parameters as hormone receptor status (5.8)and lymph-node involvement (3.0) (Janicke et al., 1989).The role of u-PA as a prognostic marker in breast cancer was confirmed by another group using a catalytic assay for detection of this enzyme (Duffy et al., 1990). Further evidence supporting the role of plasminogen activators in tumor cell invasion has accumulated from studies of various model invasion systems. In a pioneering study of this line of research, Ossowski and Reich (1983) demonstrated inhibition of the invasion of the chick embryo by human tumor cells using anticatalytic antibodies to u-PA. Anti-u-PA antibodies, monoclonal and polyclonal, and also protease nexin were later found to inhibit fibrosarcoma cell-mediated degradation of the extracellular matrix (Bergman et al., 1986), invasion of human amniotic membrane by human melanoma cells (Mignatti et al., 1986), and degradation of the extracellular matrix by Rous sarcoma virus-transformed fibroblasts (Sullivan and Quigley, 1986); to prevent the formation of pulmonary metastases of melanoma cells in mice (Hearing et al., 1988); and to inhibit basement membrane invasion by several cell lines of neoplastic origin (Reich et al., 1988). Plasmin-catalyzed activation of procollagenases (cf. Tryggvason et d., 1987) was shown to be an important part of the total effect of plasminogen activation in some of these studies (Mignatti et al., 1986; Reich et al., 1988). Moreover, invasion of the chick chorioallantoic membrane by human tumor cells was shown to be dependent on the enzyme activity of cell surface-bound u-PA (Ossowski, 1988), and plasminogen-dependent degradation of laminin was strongly associated with the level of u-PA receptors (Schlechte et al., 1989). These two

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JAR’ POLLANEN ET AL.

findings show the functional importance of the expression of specific u-PA receptors on the surface of various normal and malignant cells (Blasi, 1988). Furthermore, expression of the human u-PA gene in transfected cells was associated with the induction of extracellular matrix degradation and invasion of a matrigel basement membrane analog (Cajot et al., 1989). This accumulating body of evidence demonstrates the effectiveness of inhibitors of the plasminogen activation system in various invasion models, and strongly supports the hypothesis that the u-PA pathway has considerable significance in tumor cell invasion. Some investigators have found direct effects of plasminogen activators on extracellular matrix degradation in addition to the major pathway mediated via plasmin (Fairbairn et al., 1985; Sullivan and Quigley, 1986). Plasminogen activator purified from transformed chick cells was also shown to be able to degrade directly cellular fibronectin (Quigley et al., 1987). The migration and proliferation of capillary endothelial cells during angiogenesis represent a biological system closely analogous to tumor cell invasion. The production of plasminogen activator and procollagenase by capillary endothelial cells could be stimulated by several angiogenic agents (Rifkin et al., 1981; Gross et al., 1982, 1983). Moreover, in vitro invasion of the human amniotic membrane by bovine capillary endothelial cells treated with the potent angiogenic agent, basic fibroblast growth factor, was shown to involve both the plasminogen activator-plasmin system and specific collagenases (Mignatti et al., 1989).

D. FUNCTIONS OF PLASMINOGEN ACTIVATORS I N OTHER PHYSIOLOGICAL SETTINGS 1 . Clinical Thrombolysis

Removal of fibrin from the intravascular system after it has completed its hemostatic task is one of the in vzvo functions of the plasminogen activation system, a process called fibrinolysis. The enzyme playing a key role in fibrinolysis is t-PA (Dan@et al., 1985), as indicated by its very high affinity for fibrin (Rijken et al., 1982), the increase by several hundredfold in its enzyme activity when bound to solid-phase fibrin substrates (Hoylaerts et al., 1982), and by its distribution in endothelial cells (Kristensen et al., 1984; Angles-Can0 et al., 1985 a,b; Beebe, 1987) and the intima of the vessel wall. Moreover, t-PA interferes with platelet aggregation (Stricker et al., 1986), thereby potentially interrupting rethrombotic events. As an application of these special properties, intravenous administration of either recombinant t-PA or streptokinase has


293

become a routine therapeutic procedure in the treatment of acute myocardial infarction, and relatively good results have been reported from such therapy (van der Werf et al., 1984; Guerci et al., 1987; Haber et al., 1989). In addition, thrombolytic therapy has become a potentially promising treatment for noncoronary arterial thromboses (Verstraete, 1989). T h e field of clinical thrombolysis may develop in the future toward a new generation of thrombolytic agents with properties engineered by recombinant DNA technology.

2. Inflammation It has been proposed that plasminogen activators play a role in this phenomenon, and especially in the migration of monocytes and macrophages to the site of inflammation (Reich, 1978). Degradation of injured tissue is frequently involved in these processes, and it has further been proposed that u-PA released by macrophages and granulocytes might contribute by degrading protein components of extracellular matrices in a fashion parallel to that proposed for tumor cells (Dan@et al., 1985). Murine peritoneal macrophages were shown to be induced to release a variety of proteinases, including PA, following stimulation in vzvo with thioglycolate (Unkeless et al., 1974). Since this initial finding, PA production in macrophages has been reported to be stimulated by asbestos (Hamilton et al., 1976), lymphokines (Klimetzek and Sorg, 1977; Nogueira et al., 1977; Vassalli and Reich, 1977; Gordon and Cohn, 1978; Greineder et al., 1979), interferon (Hovi et al., 198l), colony-stimulating factor (Lin and Gordon, 1979), concanavalin A and phorbol myristate acetate (Vassalli et al., 1977; Neumann and Sorg, 1983), and endotoxin, lipopolysaccaride, and muramyl dipeptide (Golder and Stephens, 1983). Production of u-PA by stimulated macrophages is inhibited by antiinflammatory steroids, e.g., dexamethasone, arid by cholera toxin, colchicine, and vincristine (Hamilton et al., 1976; Vassalli et al., 1976, 1977; Neumann and Sorg, 1983). Polymorphonuclear leukocytes produce u-PA, and its secretion can be stimulated by concanavalin A and phorbol myristate acetate, and inhibited by glucocorticoids (Granelli-Piperno et al., 1977; Granelli-Piperno and Reich, 1978). Peripheral blood monocytes producing and secreting inactive pro-u-PA (Vassalli et al., 1984) start to express active u-PA extracellularly when treated with muramyl dipeptide (Stephens and Golder, 1984). Furthermore, clinical conditions involving either inflammation and/or tissue destruction associated with the PA-plasmin system include allergic vasculitis, xeroderma pigmentosum, pemphigus, psoriasis, rheumatoid arthritis, chronic inflammatory bowel disease, and corneal ulceration (Dan@et al., 1985; Salonen et al., 1987).

294


3. Wound Healing After tissue injury, repair proceeds through several stages, including the formation of a primary clot, reepithelization by migrating cells, and production of granulation tissue and eventually a scar. Not only have the platelets a role in this process as cells releasing clotting factors, growth factors, and mitogens, they also contribute to the nonproteolytic environment of the clot matrix, in that they express and secrete proteinase inhibitors, namely the type 1 PA inhibitor and the tissue inhibitor of metalloproteinases (Erickson et al., 1984; Kruithof ef al., I986a; Alitalo ef al., 1989). Moreover, migration of fibroblasts into the fibrinogen- and fibronectin-rich plasma clots requires the enzymatic activity of plasmin (Knox et al., 1987). In incised mouse wounds, u-PA immunolabeling was transiently detected at the edge of the migrating keratinocytes until the closure of the wound (GrGndahl-Hansen el al., l988b). The binding of u-PA to its receptors has also been suggested to stimulate the migration of keratinocytes (DelRosso af al., 1990). In addition, capillary endothelial cells wounded in cultures show increased amounts of u-PA activity until the wound has closed (Pepper et al., 1987). In similar cultures, antibodies to basic fibroblast growth factor block movement of the cells and suppress the levels of their PA activity (Sato and Rifkin, 1988). u-PA4mediated proteolysis also appears to release active growth factor-proteoglycan complexes from the pericellrilar matrix (Saksela and Kifkin, 1990). These findings all suggest a role for u-PA in cell niigration associated with wound healing, and an interesting analogy has been made between tumors and wounds that do not heal (Nagy el nf., 1988).

4. The Biology of Reproduction The PA-plasmin system has been proposed to play a role in several events in the biology of reproduction, including ovulation, spermatogenesis, mammary gland involution, blastocyst implantation, and embryonic development (Dan@et al., 1985; Saksela and Rifkin, 1988). The proposed role of the PA-plasmin-collagenase cascade in the rupture of the follicular wall at the time of ovulation is based on the temporal relationship of the PA levels found in the follicular fluid prior to the release of the ovum (Beers el al., 1975) and the ability of serine proteinase and collagenase inhibitors to prevent induced ovulation in mice (Reich el al., 1985a,b). A transient increase in t-PA enzyme activity and mRNA level was observed in cultured granulosa cells in response to


295

follicle-stimulating hormone and luteinizing hormone (Ny et ul., 1985, 1987; Canipari and Strickland, 1985; Canipari et al., 1987),with a simultaneous decrease in the secretion of the specific type 1 PA inhibitor, PAI-1 (Ny et al., 1985). Moreover, an increase in t-PA gene transcription was demonstrated in hypophysectomized rats induced to ovulate by gonadotropin-releasing hormone (Ny et al., 1987). It has also been speculated that t-PA contributes to the decrease in viscosity of the follicular fluid and to the prevention of blood clotting at the time of ovulation. Furthermore, t-PA is produced by secondary and fertilized oocytes but not by primary oocytes (Huarte et al., 1987). Primary oocytes contain t-PA mRNA, which is translated only at the time of the resumption of meiotic maturation, and most of the enzyme is probably released at the time of fertilization (Huarte et al., 1985). During spermatogenesis, PA synthesized by Sertoli cells of the testis is thought to be involved in the release of spermatozoa (Lacroix et al., 1977, 1979; Vihko et al., 1988). The heads of spermatozoa bind u-PA, thus further facilitating their migration (Huarte et al., 1987). Mammary gland involution following the cessation of lactation is another tissuedestructive process accompanied by an increase in u-PA production (Ossowski et al., 1979; Larsson et al., 1984), and the hormones oxytocin, prolactin, and hydrocortisone, which stimulate lactation, suppress the PA production by the mammary gland tissue (Ossowski p t al., 1979). The invasion and degradation of uterine tissue by the blastocyst stage embryo parallels with a transient peak in u-PA activity (Liedholm and Astedt, 1975; Strickland et al., 1976; Sherman, 1980). Using in situ hybridization techniques, transient expression of u-PA mKNA was demonstrated in invading and migrating trophoblastic cells of implanted mouse embryos (Sappino et al., 1989). Cell migration and tissue remodeling during embryonic development has been linked with the occurrence of PAS (cf. Dan@et al., l985), e.g., in the developing nervous system. Induction of t-PA mRNA and enzyme activity closely correlated with the morphological differentiation of neuronal cells (Neuman et al., 1989). 5. Prohormone Procewing The PA-plasmin system has been associated with the conversion of proinsulin to insulin (Virji et al., 1980), formation of active ACTH from its precursor in the pituitary gland (Granelli-Piperno and Reich, 1983), and the activation of latent transforming growth factor p (Lyons e l al., 1988).

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JAR' POLLXNEN ET A L .

E. PLASMINOGEN ACTIVATOR INHIBITORS: SPECIFIC FAST-ACTING RWULATORS OF EXTUCELLULARPROTEOLYSIS In the extracellular compartment, the secreted PASbecome subject to the regulatory effects of the specific fast-acting plasminogen activator inhibitors (PAIs) (reviewed by Andreasen el al., 1990). Currently there are four classes: PAl-1, PAI-2, PAl-3, and protease nexin I (Collen, 1986). In addition, some other uncharacterized PAIs may exist (Yamada et al., 1988).The PAIs belong to the serine proteinase inhibitor (serpin) superfamily (Carrel1 and Travis, 1985), and to the subgroup termed arpiine-se~-pins,according to the residue at their reactive center. Of these four classes, PAI-1 and PAI-2 are highly specific for PAS,whereas PAI-3 and protease nexiri I have broader specificity profiles (see later). 1. PAI-I The production of PAl- 1 was originally described in endothelial cells (Loskutoff and Edgington, 1977); its production has since been reported in a wide variety of cell types, including fibroblasts, vascular smooth muscle cells, hepatocytes, hepatoma cells, mammary cells, malanoma cells, fibrosarcoma cells, and platelets. It is also present in plasma (cf. Aridreasen et ul., 1990). PAI- 1 is a single-chain 46- to 54-kDa glycoprotein (Erickson et al., 1984; Andreasen et al., 1986a)encoded by 3.0- and 2.2-kb mRNAs, the shorter transcript possibly being generated by modified polyadenvlation (Ny et al., 1986; Ginsburgeld., 1986).The human P'4I-1 gene of 12.2 kb is located on chromosome 7 (Loskutoff et d.,1987; Klinger et al., 1987). The amino acid sequence deduced from the PAL-1 cDNA contains 379 residues, three potential glycosylation sites, but no cysteine residues. The reactive center is located in the carboxy-terminal region (Ny et al., 1986; Pannekoek el a[., 1986; Ginsburg et a[., 1986; Andreasen et af., 1986~).PAI-1 inhibits tct-PA, sct-PA, and tcu-PA (Erickson et ul., 1984; Colucci et af., l986), but not scu-PA (Andreasen rt al., 1986a), by forming tight sodium dodeoyl sulfate (SDS)-stable 1 : 1 molar complexes in a very fast reaction invohing the actite site 01 the enzyme (Philips rt uf., 1984; Erickson et al., 1985; Colucci et a[., 1986; Kruithof et al., 1986a; Coleman el al., 1986; Sprengers ei al., 1986). T h e t-PA/PAI-1 complex has no affinity for fibrin, probably because of steric hindrance (Levin, 1983). PAI-1 from HT-1080 fibrosarcoma cells was converted by u-PA and t-PA to an inactive form, with the cleavage of a 4-kDa fragment, and it can thus be considered an "alternative substrate" of PAS(Nielsen et al., 1986b). Upon secretion, P,41-1 is rapidly converted from an active into a latent inactive form (Levin and Santell, 1987),which


297

can be reactivated in vitro by protein denaturants (Hekman and Loskutoff, 1985; Andreasen et al., 198613; Erickson et al., 1985) and some negatively charged phospholipids (Lambers et al., 1987). Vitronectin has been identified as a stabilizing binding protein for PAI-I both in solution and in the extracellular matrix (Declerck et al., 1988; Wiman et al., 1988; Mimuro and Loskutoff, 1989; Salonen et al., 1989a; Wun et al., 1989). PAI- 1 secretion and/or gene expression have been demonstrated to be regulated by several effectors, many of which regulate the expression of PA as well (cf. Saksela and Rifkin, 1988). They include interleukin-1 (Bevilacqua et al., 1986; Gramse et al., 1986; Nachman et al., 1986; Emeis and Kooistra, 1986; Schleef et al., 1987), thrombin (Gelehrter and Sznycer-Laszuk, 1986; van Hinsbergh et al., 1987), bacterial endotoxin (Crutchley and Conanan, 1986), basic fibroblast growth factor (Saksela et al., 1987), phorbol myristate acetate (Alitalo et al., 1989; Oliver et al., 1989), as well as dexamethasone, transforming growth factor @, and tumor necrosis factor a (Schleef et al., 1987; Laiho et al., 1987; Lund et al., 1987; Medcalf et al., 1988; Sawdey et ul., 1989) and platelet-derived growth factor (Prendergast and Cole, 1989). Recently, it has been found that the c-myc-regulated gene mrl encodes PAI- 1 (Prendergast et al., 1990).I n vivo, PAI- 1 appears to be the primary regulator of fibrinolysis in the vasculature, and acts as well as an acute-phase reactant (Juhan-Vague et al., 1984; Kruithof et al., 1988). The increased amounts of both PAI-1 and PAI-2 in plasma during pregnancy probably contribute to the maintenance of the overall balance of the fibrinolytic system, as u-PA and t-PA concentrations are increased as well (Kruithof et al., 1987). In addition, high PA1 activities due to increased PAI-1 levels were detected in the plasma of patients with severe preeclampsia, with a positive correlation to the severity of placental damage (Estelles et al., 1989).PAI-1 was shown to be a marker of trophoblast invasion (Feinberg et al., 1989). PAI-1 also seems to be the principal inhibitor responsible for decreased u-PA activity in patients with adult respiratory distress syndrome (Bertozzi el al., 1990).

2. PAI-2 The production of PAI-2 was original1 reported from two major sources: the placenta (Kawano et al., 1970; stedt et al., 1985, 1986) and cells of the monocyte/macrophage lineage (Golder and Stephens, 1983; Vassalli et al., 1984; Chapman and Stone, 1985; Saksela et al., 1985). PAI-2 is present in plasma during pregnancy (Lecander and Astedt, 1986; Nilsson et al., 1986), but otherwise its levels in circulation are usually quite low. The intercellular unglycosylated form of PAI-2 is 47 kDa (Kruithof et al., 1986b; Collen et al., 1986), and it has been proposed

8:

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to undergo glycosylation upon secretion, so that only the 60-kDa form is found extracellularly (Genton et a f . , 1987; Medcalf et al.. 1988), but a fair amount of the PAI-2 synthesized may accumulate intracellularly. The PAI-2 cDNA encodes a protein containing 415 amino acids residues, the predicted amino acid sequence of the placental and the nionocyte/ macrophage forms being virtually identical (Webb et a/., 1987; Ye et al., 1987; Antalis et al., 1988). The PAI-2 gene is located on chromosome 18 (Webb etal., 1987). The secreted and cytosolic forms of PAI-2 arise from a single mRNA (Belin et al., 1989), and the PAI-2 protein appears to have no typical amino-terminal signal peptide sequence (Ye et al., 1987; Antalis et al., 1988). PAI-2 is primarily a rapid inhibitor of u-PA; its effect on soluble tct-PA is considerably slower than that of PAL-1, and on sct-PA even more so (Stephens et al., 1985; Kruithof et al., 198tib).On its physiologically relevant location at the solid-phase fibrin substrate, t-PA was found to be resistant to inactivation by PAI-2 (Leung el a/., 1987). 'The reaction between u-PA and PAI-2 results in the formation of covalently bonded enzyme-inhibitor complexes with 1 : 1 stoichionietry (Wun anti Reich, 1987) and subsequent cleavage of an arginvl bond, releasing a 35-residue carboxy-terminal fragment (Kiso et al., 1988). A fundamental distinction between PAl- I and PAI-2 is the prolonged functional stability of PAI-2 in the soluble phase. A comparison of the properties of PAI- 1 and PAI-2 i, presented in Table 11. The 3' untranslated region of the PAI-2 cDNL4contains a putative regulatory sequence previously associated with inflammatory mediators (Antalis et al., 1988). The secretion of PAI-2 by monocyte/macrophages

Characteristic

PAI-1

Molecrilar weight

46,000-34,000

Amino acid residues mRNA Chromosomal localization Synthesized hy

379 3.012.2 kb 7 Endothelial cells, many types of cells t-PA and u-PA Vitronectin Yes Reactivates Septic shock

Principal enzyme specificity Binding protein Latent inactive form Effect of sodium dodecyl sulfate lncreased plasma concentrations in

PAI-2 47.000 ( i n t i acellular): 60.000 (glycos\.lated) 4 I 5. 2.0 kb 18 Placental irophoblasts, monoryrea u-PA None k n u n~ No Indctikates Pregiiancy


299

was increased by colony-stimulating factor, phorbol myristate acetate, cholera toxin, bacterial lipopolysaccharide, and muramyl dipeptide (Stephens et al., 1985; Wohlwend et al., 1987; Schwartz et al., 1989). PAI-2 gene transcription was stimulated in cultured U-937 cells by lipopolysaccharide and phorbol myristate acetate (Schleuning et al., 1987; Genton et al., 1987). T h e regulation of PAI-2 gene expression is independent of expression of the PAI-1 gene, as suggested by the opposite effects of dexamethasone on the transcription of the two genes in HT-1080 fibrosarcoma cells (Medcalf et al., 1988). PAI-2 gene expression was specifically induced in cells transfected with ras and v-src oncogenes (Cohen et al., 1989). However, the exact physiological roles of PAI-2 in the modulation of proteolytic activity in the extracellular compartment remain to be clarified. 3. PAI-3 This plasminogen activator inhibitor was identified in human urine, and it is also present in plasma (Stump et al., 1986~). The purified PAI-3 is a single-chain 50-kDa glycoprotein that forms 1 :1 molar complexes with both u-PA and thrombin (Stump et al., 1986~).Its reaction with tct-PA occurs at a much slower rate, and it does not react with scu-PA or plasmin. PAI-3 was shown to be immunologically identical to the heparindependent inhibitor of activated protein C anticoagulant (Heeb et al., 1987). During thrombolytic therapy with urokinase, as the concentrations of u-PA reach high levels, significant complex formation with PAI-3 takes place in the plasma, inhibiting the in uiuo activation of plasminogen (Geiger et al., 1989).

4. Protease Nexin I Protease nexins are a family of protease inhibitors, of which the protease nexin I (PN I) is the only one known to affect PAS. PN I is produced by many types of cells, including fibroblastic cells, fibrosarcoma cells, heart muscle cells, kidney epithelial cells, epidermal carcinoma cells, endothelial cells, glial cells, and activated platelets (Baker et al., 1980; Eaton and Baker, 1983; Guenther et al., 1985; Gronke et al., 1987, 1989). It is a less specific serpin, as it inhibits thrombin, u-PA, plasmin, and trypsin by forming covalent complexes with the proteinases (Baker et al., 1980; Scott and Baker, 1983; Eaton et al., 1984). Complex formation with proteinases is promoted by heparin (Scott and Baker, 1983). Complexes of protease nexin I with thrombin and u-PA bind to and are internalized and degraded by human fibroblasts, the binding occurring via the heparin-binding site on protease nexin (Low et al., 1981). However, PN I bound to the cell surface or to the extracellular matrix primarily inhibits

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thrombin, and not u-PA or plasmin (Wagner et af., 1989). PN I is a 45-kDa glycoprotein with 392 amino acid residues (McGrogan et a/., 1988). I t is active in retarding thrombin-induced cell proliferation as well as extracellular matrix degradation by fibrosarcoma cells (Low et d.,1982; Bergman el al., 1986). In the nervous system, PN I produced by astroglial cells promoted axonal outgrowth of cultured neuronal cells (Guenther ~t al., 1985; Gloor P t al., 1986; Rosenblatt et al., l987), and it inhibited cell migration in the developing mouse cerebellum (Lindner et al., 1986). These are some examples of the proposed role for PN I in regulation of proteinase activity at the cell surface and in the extracellular conipartment (Baker and Gronke, 1986).

IV. The Principle of Directed Cell Surface Plasminogen Activation

A.

PERICELLULAR D I S T R I B U T I O N OF THE C O M P O N E N T S

OF THE

PLASMINOGEN ACTIVATION SYSTEM

In immunofluorescence analysis of cultured HT- 1080 fibrosarcoma cells, RD rhabdomyosarcoma cells, and normal human fibroblasts, u-PA antigen has been detected at focal cell-substratum contact sites at the leading edge of the cells and also in areas of cell-cell contacts (Pollanen et af., 1987). At these specific locations it has a striking colocalization with intracellular vinculin, a specitic marker protein of focal contacts (Pollanen et d ,19811). When WI-38 fibroblasts were growth arrested to induce the maturation of fibronexus contact sites (Singer, 1979), the morphology of the vinculin plaques changed into highly elongated striae that were more centrally located, whereas the focalized u-PA inimunolabeling decreased in intensity to below the detection limit (Pollanen et al., 1988). In immuno-EM analysis of HT- 1080 cells, specific u-PA labeling was predominantly found at sites where the cells made contact with the growth substratum beneath all parts of the ventral cell surface, frequently at the leading lamellae of the cell (Pollanen et d.,1988). Immunoferritin labeling of u-PA was also detected between cross-sectioned cellular extensions (lamellipodia and microspikes) and the substratum that were also enriched in a submembranous patch of electron-dense microfilaments. In addition to the cell-substratum adhesion plaques, the anti-u-PA immunoferritin label had a preferential localization at nonjunctional cell-cell contact sites (Pollanen e&af., 1988). These results have established u-PA as an intrinsic component of focal contacts, in contrast to the two other types o f cell-substratum contact, i.e., close contacts and fibronexus contact sites, in which u-PA has not been detected (Table 111).


30 1

TABLE 111 PERICELLULAR DISTRIBUTION OF THE COMPONENTS OF THE u-PA SYSTEM" Component U-PA PAI-1 PAI-2' Plasminogen

Localization Focal contacts, cell-cell contacts Substratum-attached carpet Focal contacts, cell-cell contacts Ventral cell surface (homogeneous)

Interactions u-PA receptor u-PA, t-PA Vitronectin u-PA (primarily) Various, of low affinity

Effectors

GF domain, 16-mer peptide Peptides? Plasniinogen, txa, antiu-PA antibodies Lysine analogs, kringle

4,Lp(4

Data are taken from references cited in the text. Abbreviations: GF, growth factor (domain of u-PA); txa, tranexamic acid; u-PA, urokinase-type plasinirrogen activator; Lp(a), lipoprotein(a). Based on studies with with exogenous rPAI-2.

The u-PA at the focal contacts is bound to specific cell surface receptors, because a synthetic u-PA peptide covering 16 amino acid residues of the growth factor domain, which competes with u-PA for binding to the receptor (Appella et al., 1987), also competes for the binding to focal contacts of HT-1080 cells (Hebert and Baker, 1988). Kinetic studies have demonstrated that some cells producing pro-u-PA are able to saturate their surface receptors in an autocrine manner (Stoppelli et al., 1986). Certain properties of the u-PA/u-PA receptor interaction suggest that the receptor-bound enzyme at focal contacts is functionally relevant: the growth factor domain acts independently of the catalytic domain, so that bound tcu-PA retains full catalytic activity, is not internalized or degraded, and has a half-life of 3-6 hr (Vassalli et al., 1985; Stoppelli et al., 1985; Blasi, 1988). By exerting a directed proteolytic effect at the adhesion site, receptor-bound u-PA at focal contacts may enable regional detachment from interactions of the cell with the extracellular matrix, especially during cell migration, mitosis, invasion, and tissue remodeling, as has been proposed (Dan@et al., 1985; Goldfarb and Liotta, 1986; Saksela and Rifkin, 1988). PA produced by transformed chick fibroblasts has been reported to cleave fibronectin (Quigley et al., 1987) and focal proteolysis of a fibronectin substrate was observed by other serine proteinases as well (Chen and Chen, 1987). Therefore, a controlled release of the cell surface from the fibronectin-rich extracellular matrix, serving an anchoring function, seems possible. Quiescent fibroblasts do not contain detectable u-PA antigen in their focal adhesions (Pollanen et al., 1988) and express decreased amounts of u-PA mRNA (Grimaldi et al., 1986). In addition, in cultures of endothelial cells, the migratory cells

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covering the a mechanically wounded area were induced to secrete increased amounts of u-PA (Pepper et al., 1987). The immunofluorescence pattern of external PAI-1 antigen in HT1080 and P-HES cells consists of a homogeneous layer on the growth substratum (Pollanen et al., 1987) (Table 111). After saponin detachment of cells, the PAI- 1 immunolabel appeared as a homogeneous carpet under the ventral cell surface interrupted by distinct striae lacking the signal (Pollanen et al., 1987). The difference in distribution patterns between u-PA, being enriched on focal contacts, and PAI- 1, being distributed as an extracellular carpet on the growth substratum, could conceivably permit focal u-PA-mediated proteolysis to occur. The distribution of PAI-1 is very similar to that reported for “mesosecrin” in cultured mesothelial, endothelial, and kidney epithelial cells (Rheinwald et aE., 1987) and partial amino acid sequence data suggest that these two proteins are actually identical. Protease nexin I has been shown to be associated with extracellular matrix fibers in colocalization with fibronectin (Fdrrell et al., 1988). The pericellular space thus appears to serve as a reservoir of functionally active inhibitors (Knudsen et al., 1987; Mimuro et al., 1987; Ldiho et al., 1987; Levin and Santell, 1987). This is particularly interesting, as native free PAI- 1 molecules are very unstable and can quickly lose their activity in the liquid phase (Mimuro et al., 1987; Levin and Santell, 1987). The extracellular distribution pattern of PAI-1 is virtually identical to that of vitronectin, a major cell adhesion protein (Barnes et al., 1983; Hayman et al., 1983; Neyfakh et al., 1983). Indeed, subsequent reports have established that PAI- 1 specifically interacts with vitronectin (Declerck et al., 1988; Wiman et al., 1988; Salonen et al., 1989a; Mimuro and Loskutoff, 1989; Wun el al., 1989), but not with fibronectin (Salonen et al., 1989a). In human plasma, PAI-1 appears to circulate as a complex with a multimeric form of vitronectin (Declerck et al., 1988). The binding site is contained in the 50-kDa part of PAI-1 after cleavage by u-PA (Salonen et al., 1989a).The contribution of the vitronectin interaction seems to be in an improvement of the stability of PAI-1 activity (Declerck et al., 1988; Wun et al., 1989).Equally important may be the localization of PAI- 1 into the pericellular space of tissues via vitronectin, thereby providing a regional regulatory mechanism for plasminogen activator-mediated proteolysis (Mimuro and Loskutoff, 1989). Vitronectin bound to PAI-1 retained its biological activity of promoting cell adhesion and spreading (Salonen et aZ., 1989a), and the cellular vitronectin receptor (~$3 had a preferential distribution at focal contacts (Singer et aZ., 1988). This would suggest that vitronectin, in a complex form with PAI-1, is a primary component promoting cell attachment at focal con-


303

tacts. Consistent with this proposal, vitronectin is more resistant to proteolytic cleavage than is fibronectin, which appears to be absent at focal contacts (Pollanen et al., 1988). The pericellular localization of plasminogen bound to HT- 1080 cells is shown in Fig. 4. Plasminogen is distributed in a polarized manner all over the ventral cell surface, whereas the dorsal surfaces score negative. The lysine analog tranexamic acid abolished this specific immunolabeling (Fig. 4B), as did dexamethosone treatment of the cells (Fig. 4C). In HT- 1080 cells cultured in plasminogen-depleted fetal calf serum (FCS)containing medium, dexamethasone also reduced the cell surface plasmin generation in a dose-dependent manner and caused a loss in plasmin binding. In fact, plasmin binding could be completely abrogated by 10 nM dexamethasone. This effect of glucocorticoids on plasminbinding sites appeared to be mediated by a decrease in affinity for plasmin (Pollanen, 1989). Glucocorticoids are also able to suppress the operation of the u-PA pathway of plasminogen activation in two other major ways: by a suppression of u-PA biosynthesis (cf. Dane et al., 1985; Andreasen et al., 1986a) and by an increase in the biosynthesis (Seifert and Gelehrter, 1978; Andreasen et al., 1987) and extracellular deposition (Pollanen et al., 1987) of PAI- 1. These changes in PA activity or antigen levels have been shown to be associated with respective changes in u-PA mRNA (Busso et al., 1986, 1987; Medcalf et al., 1986), PAI-1 mRNA (Andreasen et al., 1987), or gene template activities of both u-PA and PAI-1 (Medcalf et al., 1988). T h e PAI-1 gene contains at least two elements mediating the glucocortiwid response (Kiccio et al., 1985; van Zonneveld el al., 1988). Untreated HT- 1080 cells deposit few pericellular matrix components (Alitalo et al., 1980), but glucocorticoids are able to cause an increase in the matrix deposition of fibronectin and collagen (Furcht et al., 1979; Oliver et al., 1983; McKeown-Longo and Etzler, 1987). These effects all lead to an environment characterized by low proteolytic activity, which probably facilitates the enhanced matrix deposition, considering the broad spectrum of effects of plasmin on connective tissue components (Werb et al., 1977; O’Grady et al., 1981; Salo et al., 1982; Fairbairn et al., 1985; Quigley et al., 1987). Moreover, the inhibition of u-PA biosynthesis by glucocorticoids has been correlated with retarded tumor growth or reduced cell migration in some cell types (Mira-y-Lopez et al., 1985; Medcalf et al., 1986).A cytostatic effect on human glioma and non-small-cell lung carcinoma cultures and inhibition of the growth of human lung adenocarcinoma xenografts in nude mice were also reported (McLean et al., 1986).

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FIG. 4. Polarized localization of native human plasminogen bound to HT-1080 cells over the ventral cell surface area. The cells cultured in plasminogen-depleted fetal calf serum were incubated with 50 pgirnl plasrninogen (Stephens et al., 19891, followed by inimunofluorescence labeling of live cells. using rabbit antihuman plasminogen immunoglobulins (Dako; Glostrup, Denmark) and a previously described methanol postfixation procedure (Pollanen el al., 1987). (A) Cells created with plasminogen alone; (B) cells treated with plasminogen and 100 j d 4 tranexamic acid; (C) cells pretreated with 10-7M dexarnethasone prior to addition of plasrninogen (Pollanen, 1989). Bar = 15 pm.


305

B. ACTIVATION OF pro-u-PA AND PLASMINOCEN OCCURS in Situ WITH CELL SURFACE-BOUND REACTANTS

After addition of purified preparations of native human plasminogen to HT- 1080 cells cultured in plasminogen-depleted FCS-containing medium, dose-dependent amounts of plasmin activity could be recovered as a bound fraction from the cell layer (Stephens et al., 1989). This cellbound plasmin activity resulted from the activation of plasminogen on the cell surface, and was not derived either from preformed plasmin (present as a trace contaminant in the added plasminogen preparation) or from plasmin formed in the medium and subsequently bound to the cells. Experiments on plasmin uptake and release established a one-way traffic for plasmin activity going from HT-1080 cells into the medium, but not vice versa, maintained by the presence of serum inhibitors (Stephens et ul., 1989). Cell surface plasminogen activation in HT- 1080 cultures was catalyzed by cell-bound u-PA (Stephens et. al., 1989). Using inhibitory monoclonal antibodies, it was shown that inhibition of the enzyme activity of u-PA resulted in virtually no detectable cell-bound plasmin activity, whereas inhibition of t-PA had no effect. Bound plasmin activity was also reduced in HT-1080 cultures treated with purified PAI-2, aprotinin, or an anticatalytic monoclonal antibody to human plasmin. Furthermore, preincubation of the cells with either the anticatalytic u-PA antibody or PAI-2 also caused a significant decrease in the subsequent ability of the cells to generate cell-bound plasmin activity. In another approach to evaluate the role ofcell-bound versus free u-PA in the cell surface plasmin generation, most of the endogenous receptor-bound u-PA on HT-1080 cells was competed out with added DFP (diisopropy1fluorophosphate)-inactivated u-PA. This procedure resulted in a decrease of 70% in surface bound u-PA activity that could be released by acid elution. Concomitantly, there was a comparable decrease in the amount of plasmin that could be generated on the cell surface, indicating that a large part, if not all, of the cell surface plasminogen activation in cells cultured in the presence of serum was indeed catalyzed by the surface-bound u-PA (Stephens et ul., 1989). The enzymatic activity of pro-u-PA is at least 250-fold lower than that of the two-chain u-PA (Petersen et al., 1988), and it does not react with PAI-1 (Andreasen et al., 1986b)or PAI-2 (Stephens et al., 1987; Wun and Reich, 1987).With the use of metabolic labeling, of recovery of receptorbound u-PA by acid treatment, and of immunoprecipitation, it has been demonstrated that receptor-bound u-PA on HT-1080 cells was almost

3 06

JARI POLLANEN ET A L .

exclusively present in the single-chain form in serum cultures without plasminogen, whereas virtually all the receptor-bound u-PA was in the two-chain form when the cells were incubated with plasminogen (Stephens el al., 1989). Moreover, both aprotinin and an anticatalytic monoclonal antibody to human plasmin increased the relative amount of pro-u-PA in the presence of plasminogen, as was the case with 100 ph.1 tranexamic acid, which completely inhibited binding of plasmin to the cells but did not impair the ability of plasmin to activate pro-u-PA in solution (Stephens et al., 1989). These findings are consistent with conversion of the cell surface-bound pro-u-PA to active u-PA by surtacebound plasmin.

OF CELLSURFACE-BOUND u-PA TO C. ACCESSIBILITY INHIBI’TION B Y PAI-1 A N D PAI-2

HT-1080 cells release large amounts of PAI-1 (Neilsen 4t al., 1986a) that binds to active u-PA but not to pro-u-PA (Andreasen et al., I986b). I t has been found that there is a lower amount of total u-PA activity on HT-1080 cells when cultured with plasminogen (Stephens et al., 1989). This difference can be nearly abolished with a monoclonal antibody that neutralizes human PAI-I . The decrease in total u-PA after addition of plasminogen can thus be attributed to the pericellularly deposited PAI- 1 binding to and inactivating tcu-PA on the cell surface. In studies of effectors of pro-u-PA activation and plasmin on the surface of HT- 1080 cells in serum-containing medium, the neutralizing anti-PAI- 1 caused a further decrease, from 50 to 21% in the pro-u-PA index of the cells incubated with plasminogen, whereas this value was 85% in control cultures; this coincided with a clear increase in bound plasmin activities (Stephens et al., lY89). Thus, surface-bound u-PA and plasmin generation are apparently susceptible to regulation by endogenous PAI-I deposits in the pericellular matrix of HT-1080 cells. The cell surface activation of plasminogen on HT-1080 cells can also be inhibited by exogenous purified PAI-2 (Stephens et al., 1989). In RD rhabdomyosarcoma cell cultures, the inhibitory effect of human nionocyte-derived recombinant rPAI-2 (Antalis et al., 1988) on surfacebound u-PA was also found to be strongly dependent on the presence of cell surface-bound plasminogen (Pollanen et al., 1YYO). rPAI-2 did not significantly react with surface-bound u-PA when plasminogen was absent, or when plasminogen binding to cells was competitively prevented by tranexamic acid. T h e time course curve of pro-u-PA activation on R D cells following the addition of plasminogen had a pulselike shape, with a maximum of 67%’ of the total amount of recoverable enzyme in the active


307

form after 60 min. (Pollanen et al., 1990). Furthermore, generation of bound plasmin activity was reduced in RD cultures with rPAI-2, as well as with tranexamic acid, and with an anticatalytic monoclonal antibody to human u-PA (Pollanen et al., 1990). Therefore, rPAI-2 is capable of inhibiting surface plasmin generation, which requires the binding of plasminogen to the cell surface, as well as inactivating surface-bound u-PA (Stephens et al., 1989; Pollhen et al., 1990). T h e results obtained with these two different cell lines demonstrated the ability of endogenous PAI-1 and exogenous PAI-2 to regulate cell surface plasminogen activation. By contrast, in endotoxin-stimulated macrophages capable of plasmin-dependent degradation of fibrin substrates, an induced unspecified PA1 effectively blocked soluble PA activity but not the activity from membrane-rich cell fractions prepared by homogenization (Chapman et al., 1982).This result had earlier led to the hypothesis of the cell surface as a site where PA activity could be protected from soluble proteinase inhibitors. In our experiments, the vast majority of u-PA secreted by HT-1080 and RD cells, and present on the cell surface, was in the scu-PA form (Stephens et al., 1989; Pollanen et al., 1990),recently established by several groups to be a true proenzyme, with little or no intrinsic activity (Ellis et al., 1987; Petersen et al., 1988). Moreover, virtually no inhibition of u-PA took place, unless the plasminogen-dependent pro-u-PA activation was allowed (Stephens et al., 1989; Pollanen et al., 1990). This is consistent with the evidence for the lack of reaction of PA1 and pro-u-PA in free solution (Stephens and Golder, 1984; Andreasen et al., 1986b) and in tumor tissue homogenates (Stephens et al., 1987). It has also been shown with human U-937 cells that binding of exogeneous tcu-PA to the cellular receptor (Vassalli et al., 1985; Stoppelli et al., 1986; Blasi, 1988; Nielsen et al., 1988) did not render the enzyme inaccessible to inhibition by PAI-I (Cubellis et al., 1989). Preformed complexes of u-PA and PAI-1 were able to bind to the u-PA receptor of U-937 cells with the same binding specificity as free u-PA (Cubellis et al., 1989). The accessibility of the receptor-bound u-PA to PAIs can be understood in the light of distinct domains of the u-PA molecule being involved in the receptor binding (growth factor domain) versus reaction with the inhibitors (catalytic domain) (Appella et al., 1987). The endogenous receptor-bound u-PA on human monocytes has also since been shown to be inhibited to some extent by PAI-2 (Kirchheimer and Remold, 1989a). PA1-2 also completely inhibited the invasion of amniotic membrane by interferon-y-stimulated human monocytes (Kirchheimer and Remold, 198913). Kinetic analysis in serumfree cultures of U-937 myelomonocytic cells demonstrated the ability of both PAI-1 and PAI-2 to inhibit surface plasminogen activation (Ellis et al., 1990). However, other interactions with surface-bound u-PA may be

308


important for some leukemia cells that continuously produce active u-PA; recent experiments suggest that a~-macroglobulincan interact with cell-bound active u-PA, thereby protecting it from irreversible inactivation by PAI-2 (Stephens et al., submitted). Another interesting aspect of the interaction between receptor-bound u-PA and the PAIs is that though bound u-PA does not appear to be internalized by endocytosis, bound PAI/u-PA complexes are (Cubellis et al., 1990). D. FUNCTIONAL ASSEMBLY OF THE PLASMIN-GENERATING SYSTEM A T FOCALCONTACTS T h e inability to distinguish in situ between the proenzyme and the active forms ofu-PA, or free u-PA and its inactive complexes with specific PA inhibitors, has hampered an understanding of the functional status of this enzyme in in zdro systems (Dan@et a.l., 1985).We discussed previously the ability of rPAI-2 to inhibit cell surface-bound u-PA and plasniiri formation. T h e extracellular localization of functionally active u-PA on human RD rhabdomyosarcoma cells has been examined by using human monocyte-derived rPAI-2 as a specific high-affinity ( K d 10-l' M ) probe for active u-PA (Leung el al., 1987). Binding of rPAI-2 was detected with rabbit anti-PAI-2 IgG antibodies. This new system had the ability to distinguish between active u-PA and its inactive proenzyme and inhibitor complex forms, because PAI-2 binding requires the active site of u-PA (Wun and Reich. 1987). Moreover, PAI-2 reacts only weakly with tissuetype PA (Leung et al., 1987), and no t-PA was detectable in eluates of the RD cell surface. Using this approach, specific rPAI-2 labeling could be detected at focal contacts on the ventral surface of unpermeabilized RD cells in the presence of native human plasniinogen (Piilliinen el a/., 1990) (Table 111). Some rPA1-2 labeling was also observed at areas of cell-to-cell contacts and the bound rPAI-2 probe colocalized with both endogenous u-P.4 antigen and intracellular vinculin at the focal contacts of KD cells (Pollanen el al., 1990). N o labeling was observed in the absence of added plasniinogen and/or rPAI-2 (Pollanen et al., 1990)?indicating that the bound u-PA was initially as pro-u-PA, and that the activation event is therefore crucial to the initiation of cell surface proteolysis, which is directed toward the breakdown of the adhesive interactions between the cell surface and the pericellular matrix. Furthermore it shows that there were few preexisting pericellular binding sites for either endogenous or added PAI-2. In this respect PAI-2 differs from PAI-1, which binds to the substratum-associated vitronectin (Pollanen et af., 1987; Declerck el al., 1988; Salonen et al., 1989a). These results thus demonstrated the func-


309

tional assembly of the plasmin-generating system at focal contacts, and the accessibility of bound u-PA, on which it depends, to added rPAI-2. Thus rPAI-2 has the potential to both localize functionally active u-PA and simultaneously inhibit cell surface plasminogen activation.

E. SCHEME FOR CELLSURFACE PLASMINOGEN ACTIVATION Based on the results detailed above, a model is proposed for plasminogen activation on cells growing in serum-containing medium (Stephens e! al., 1989), as illustrated in Fig. 5 . In this proposed model, the specific u-PA receptors (u-PA-R) and the cellular plasminogen/plasmin (plgjbinding sites are depicted on the cell membrane. Before exposure to plasminogen, virtually all the cell-bound u-PA is present as pro-u-PA. Plasminogen must first bind to the cell surface via its heavy-chain kringles, a step that can be precluded by the presence of tranexamic acid. Formation of cell-bound plasmin activity from bound plasminogen is presumably initiated by the action of trace amounts of active bound u-PA. This leads to rapid activation of u-PA proenzyme, which in turn enables further activation of bound plasminogen, formation of more plasmin, and so on. The availability of trace amounts of bound active u-PA to initiate plasminogen activation is clearly a critical requirement. In HT1080 cells, a plasmin-independent mechanism for proenzyme activation could not be demonstrated (Stephens et al., 1989). It appears quite possible that other nonplasmin serine proteinases exist that could be important in the initiation of plasminogen activation on the cell surface, e.g., in certain leukemia cells (Stephens et al., 1988; Alitalo et al., 1989; Brunner et al., 1990; Ellis et al., 1990). Once initiated, the exponential process of surface activation becomes susceptible to physiologic regulation by the PAIs (Stephens et al., 1989; Pollanen et al., 1990), and it can also be experimentally inhibited by an anticatalytic antibody to u-PA (anti-u-PAab). T h e surface-bound plasmin thus formed is resistant to inhibition by the aa-antiplasmin present in the fluid phase of the pericellular environment, but is sensitive to inhibition by aprotinin and an anticatalytic monoclonal antibody to plasmin (anti-plasmin-ab). Amplification of the production of surface activity is provided by plasmin-catalyzed pro-u-PA activation, which is inhibited by tranexamic acid (by preventing plasminogen binding), aprotinin, and the anticatalytic antibody to plasmin. Consequently, the binding of the proenzyme components precedes the activation loop consisting of active u-PA and plasmin. The recent protein purification (Behrendt et al., 1990) and cDNA

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JAR1 POLLANEN ET A L .

BEFORE PLASMJNOGEN BINDING active U-PA

pro-u-PA

pr o - u - P A

cell membrane

-

U-PA-R

plg-blnding

-

site

PLASMINOGEN BINDING AND INITIAL ACTIVATION

xIumGua

aprotlnln, antl-plasmin-ab

lyslne analogs (tranexamlc acid)

p l a s m inogen

plasm 1 n

AMPLIFIED ACTIVATION AND ENDOGENOUS INHIBITION

aprotinh tranexamic acid anti-plasmin-ab

PAL1 and PAL2 anti-u-PA-ab PA1

FIG.5. Schematic model for cell surface plasmi~iogen activation. (Modificd from Stephens ct al., 1989.) See text for details.

cloning and expression (Koldan et al., 1990) of the u-PA-R have specified the molecular basis of the interaction between cell surface and u-PA, whereas the molecular nature of the plasminogen/plasmin-bindingsites has remained more elusive. T h e cloned u-PA-R cDNA has a unique sequence encoding an integral membrane protein of 3 13 amino acids to which extensive glycosylation gives a final molecular weight of 55,000 (Behrendt et al., 1990; Roldan et al., 1990). T h e extracellular domain of the u-PA-R contains a high nuniber of cysteine residues with a unique

DIRECTED CELL SUKFACE PLASMINOGEN ACTIVATION

311

pattern of spacing (Roldan et al., 1990). With its high affinity and high specificity, the u-PA-R plays a central role in cell surface plasminogen activation. Interaction of u-PA with its receptor was found to stimulate the binding of plasminogen (Plow et al., 1986) as well as its activation (Ellis et al., 1989). Kinetic evaluation of plasminogen activation with U-937 cells demonstrated that increased plasmin formation in the presence of these cells was primarily due to the increased rate in the conversion of scu-PA to tcu-PA by cell-bound plasmin (Ellis et al., 1989). However, in the presence of-the physiological circulating serum proteinase inhibitors, the activation process was demonstrated to be limited to the cell surface with surface-bound reactants (Stephens et al., 1989), a compartment shielded from the rapid actions of a2-antiplasmin (Plow et al., 1986; Gonias et al., 1989). Moreover, rather than occurring randomly on the cell membrane, the plasmin-generating system was found to be functionally expressed at focal adhesions of individual cells (Pollanen et al., 1990). In a modified chorioallantoic membrane invasion model, Ossowski ( 1988) was able to demonstrate that augmented invasiveness was directly dependent on the saturation of the cell surfaces with bound active u-PA, whereas a corresponding stimulation of t-PA secretion had no effect. The secretion of enhanced amounts of mouse u-PA by transfected human cells [unable to bind the mouse enzyme due to species specificity of u-PA-R (Appella et al., 1987; Huarte et al., 1987)] was also without an affect on the invasion (Ossowski, 1988). This would suggest a pivotal role for the u-PA-R in cellular invasion; an overproduction of the enzyme in the absence of receptor binding was not sufficient to enhance invasion. In agreement with this concept, invasion of the amnion tissue by interferon-ystimulated monocytes was also shown to depend on the activity of endogenous receptor-bound u-PA (Kirchheimer and Remold, 1989b). The degree of plasminogen-dependent degradation of laminin by cultured colon carcinoma cells was found to be in strong positive correlation with the number and saturation of u-PA receptors, whereas a poor correlation existed between the laminin degradation and soluble u-PA (Schlechte et al., 1989).These reports point into a pathway of cell surface plasminogen activation, in which the surfaces of the migrating and invading cells are armed with the biochemically broad, but physically directed, proteolytic activity of plasmin. Specific binding sites for plasminogen were first reported on platelets (Miles and Plow, 1985) and in cells expressing receptor sites for both u-PA and plasminogen (Plow et al., 1986). Presently, the candidates for a plasminoged plasmin-binding protein include fibronectin (Salonen et al., 1985),laminin (Salonen et al., 1984),histidine-rich glycoprotein (Lijnen et

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nl., 1980), tetranectin (Christensen and Clemniensen, 1989), and thrombospondin (Silverstein et al., 1985; DePoli et nl., 1989). T h e binding of' plasminogen to the surface of activated platelets seems to be mediated via fibrinogen bound to the glycoprotein IIb-IIIa complex (Miles and Plow, 1985; Adelman et a.l., 1988). The binding of plasminogen to the surface o f endothelial cells has also been described (Hajar et al., 1986; Hajar and Nachman, l988), and lipoprotein {a), which contains 37 repeats of the kringle 4 domain of plasminogen (McLean et al., 1987), competitively inhibits plasminogen binding to endothelial cells and prevents the generation of active plasmin (Miles et al., 1989; Hajar et al., 1989; GonzalesGronow rt al., 1989; Edelberg et nl., 1989). This finding is particularly interesting, as the suppression of endothelial cell surface fibrinolysis would produce a procoagulant state, possibly linking thrornbogenesis with atherogenesis. On the other hand, lipoprotein(a) also has a serine protease domain (McLean el al., 1987), and serine protease activity with ability to cleave fibronectin has recently been described (Salonen et d., 1989b). Theoretically, the binding of plasminogen would only require a set of optimally presented lysine residues in the right conformation. As the plasma concentration of plasrninogen is high and the reported affinities are low and the number of binding sites very high (Miles arid Plow, 1988), it is possible that multiple binding proteins are acting together.

F.

PROSPECTS FOR

FUTUREAPPLICATIONS

Tumor tissues as well as malignant cells in culture usually express high levels of PA activity, which, as discussed above, is strongly associated with their invasiveness. This general finding has been known for some time, and had earlier led to studies on the effect3 of the inhibitory drugs tranexamic acid, E-aminocaproic acid, and aprotinin, on tumor growth and metastasis in animal models, and also to a few clinical trials on human cancer (cf. Ogston, 1984). For example, tranexamic acid inhibited the growth of three human carcinoma cell lines transplanted into nude mice (Ogawa el al., 1982), whereas in other reports the decreased tumor growth depended entirely on the system studied (Peterson, 1968; Hagmar, 1970). Moreover, e-aminocaproic acid was reported to reduce significantly the number of chemically induced colorectal tumors in mice (Corasanti et al., 1982).By contrast, the results obtained with aprotinin on metastases in the various animal model systems were more contradictory (cf. Ogstori, 1984). In a patient with advanced breast cancer, cerebral metastases, and pleurisy, in whom irradiation and chemotherapy had failed, a symptom-free state was achieved 18 months after the onset of combined heparin and tranexamic acid therapy (Astedt ~t al., 1977a).


313

The influence of tranexamic acid on advanced ovarian carcinoma was also studied in a few series of patients ( h t e d t et al., 1977b; Astedt, 1980; Soma et al., 1980; Sigurdsson et al., 1983), and reduction in tumor size and regression of ascites was observed in a proportion of the patients. It should be noted, however, that these studies were carried out at a time when our understanding of the plasminogen activation system was very fragmentary when compared with present knowledge. This fact may explain some of the occasional conflicting results obtained with the use of these antifibrinolytic drugs. Given the current knowledge of the factors involved in cell surface plasminogen activation, it may now be possible to design approaches that could most appropriately reevaluate the potential usefulness of these agents. V. Concluding Remarks

T h e reports reviewed here have established the concept of directed cell surface plasminogen activation. Activation occurring on the solid phase of either the cell surface, in the case of the u-PA pathway, or endothelial cells and the fibrin substrate of thrombi in the t-PA pathway, has the advantage of escaping from the soluble plasmin inhibitors presented in great abundance in extracellular fluids. Receptor-bound pro-uPA at focal contacts and the more uniformly distributed vitronectinbound PAI-1 on the pericellular substratum are the key determinants in the generation of directed cell surface-bound plasmin activity. Given the generalized distribution pattern of plasminogen, it is clear that the u-PA-R has a dominant role in directing proteolytic activity at the critical sites of contacts between cells and substratum. Regulation of this process is provided at multiple levels, including those of gene expression, translation, and proenzyme secretion, and its binding to the membrane receptor, its activation while bound to the receptor, and inhibition by the PAIs. Receptor-bound active u-PA is clearly accessible to inhibition by PAI- 1 and PAI-2. The mechanism that targets u-PA receptors to the focal contact sites is presently poorly understood and needs further investigations, as does the analysis of interactions between the components of the extracellular matrix and the proteolytic machinery. The activation of pro-u-PA seems a critical even in that it initiates the entire u-PA pathway. Further studies are likely to identify the alternative nonplasmin enzyme mechanisms activating pro-u-PA. As our understanding of the complexity of the regulation of plasminogen activation has progressed and has achieved the state wherein recombinant protein components of this system, with native or engineered properties, and other useful tools are becoming available, the assessment

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AL.

o f the full potential for modulation of this enzyme system in various therapeutic or diagnostic applications has become possible. Future progress is now likely to gather speed. Clinical examples of new applications include to date the topical treatment of corneal ulcers with aprotinin (Salonen et al., 1987), the significant improvement o f intestinal lymphangiectasia with tranexamic acid therapy (Mine P t nl., 1989),and the very strong prognostic association of high u-PA levels in breast carcinoma tissue samples with an early relapse of the patient (Janicke et al., 1989). It is conceivable that a new class of anticancer drugs may be generated? which will be aimed at depriving tumor cells of their proteolytic invasive capacity. Strategies for this development are already evident i n four known properties of the surface proteinase system: (1) the requirement for u-PA binding to cell surface receptors, (2) the requirement for plasminogen binding, (3) the necessity of' pro-u-PA activation, and (4) the ability of PAIs to inhibit bound active u-PA. Synthetic peptides or other agents designed as space-fillers to interfere with the interactions involved in these four properties may be useful in preventing tumor dissemination or metastatic recurrence after diagnosis and surgical removal of primary tumors. At least it may be hoped that these known properties will lead to new diagnostic, if not effective therapeutic, applications. ACKNOWLEDGMENTS We thank Dr. Eeva-Marjatta Salonen for helpful discussions, and Professor Karl Trygguason, Professor Veli-Pekka Lehto, and Dr. Markku Jalkanen for detailed comments on the manuscript. T h e original work by the authors was financially supported by the Finnish Cancer Organizations, the Farmos Foundation. the Finnish Medical Society Duodecim, the Finnish Cultural Foundation, the Ida hiontin Foundation, the Medical Research Council of the Academy of Finland, and the Sigrid Juselius Foundation, Helsinki. We also thank Dr. Graeme Woodrow and Biotech Australia P/L for their collaboration in studies of PAI-2.

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EPSTEIN-BARR VIRUS-ASSOCIATED LYMPHOPROLIFERATIVE DISORDERS IN IMMUNOCOMPROMISED INDIVIDUALS J. Alero Thomas,* Martin J. Allday,t and Dorothy H. Crawfordt Imperial Cancer Research Fund/Royal College of Surgeons Histopathology Unit, London, WCPA 3PN, England

t Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London WC1, England

I. Epstein-Barr Virus A. TheVirus B. EBV Infection and B Cell Immortalization in Vitro C. EBV Infection in Vivo D. Immune Response to EBV E. Persistent EBV Infection in the Immuiiocoinproniised J I . EBV-Associated B Lymphoproliferative Disorders 111. Burkitt's Lymphoma IV. EBV-Associated Lymphoproliferative Disorders in Immuncxompromised Individuals V. T h e X-Linked Lymphoproliferative Syndrome A. Acute Primary EBV Infection in XLPS Males B. Histology of Lesions in Fatal Ilk1 XLPS C. EBV-Associated Antigen Expression in XLPS Lesions D. T Cell Phenotypes in Fatal llLl XLPS E. Postacute EBV Infection in XLPS hlales F. Pathogenesis of XLPS G. Treatment VI. EBV-Related Lymphoproliferative Diseases Associated with Misc:ellaneous Immunodeficiency States VII. Lymphoproliferative B Cell Disorders in Organ Transplant Recipients A. Immunosuppression and Postgraft NHI. B. Cliiiical Features of Postgraft NHL C. Tumor Presentation D. Histology of Postgraft B Cell Tumors E. Clonality of Postgraft B Cell Tumors F. Relationship between EBV arid Postgraft Lymphoproliferative Disorders G. Pathogenetic Mechanisms in Transplantation-Associated EBV-Positive B Cell Tumors H. EBV Serological Status in Transplant Recipients with B Lymphoproliferative Disease 1. Treatment of Transplantation-Associated B Cell Tumors

329 ADVANCES IN CANCER RESEARCH, VOI.. 57

Copyright 0 1991 by Academic Press, Inc All rights of reproduction in any form reserved.

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VI11. k HI, Associated with Acquired Immunodcficiency Syndrorne A . Clinical Features B. EBV Role in Pathogenesis of A1L)S-Kelatcd NHL IX. EBV and Hodgkin's Disease .4. EBV Serology in HD Patients B. EBV Associations in HD X. EBV-Associated Tumors in Nonhuman Primates XI. Summary and Future Considerations for EBV-Associated Lymphoproliferative Diseases References

I. Epstein-Barr Virus

A. 'I'HEVIRUS Epstein-Barr virus (EBV) is a member ot'the herpes family of DNA viruses; it infects 90-9570 oft he world's human population (Niederman et al., 1970). Similar to other herpesviruses, the EBV virion is composed of a core consisting of a double-stranded DNA molecule (about 172 kbp), an icosahedral capsid, and a membrane envelope enclosing the capsid. T h e complete nucleotide sequence of the B95-8 strain of the virus has been determined (Baer el al., 1984). T h e virus infects both B lymphocytes and squamous epithelial cells, resulting in immortalization of the former and in viral DNA replication, cell death, and release of new virus progeny in the latter.

B. EBV INFECTION AND B CELLIMMORTALIZATION I N VITRO 111 vitro infection of B cells with EBV occurs via binding of the viral envelope glycoprotein (gp340) to the CR2 receptor (CD2 l ) , which is the physiological ligand for the C3d component of complement and is present on all mature B cells (Fingeroth et of.,1984).This results in B cell immortalization to produce continually growing lymphoblastoid cell lines (LGLs). These cell lines are generally polyclonal; the cells are diploid, have an irregular stellate appearance, and usually grow in tight clumps. 'l'hese cells are immortalized by EBV hut are not transformed bjr the virus, as is shown by lack of growth in soft agar and lack of tumorigenicity after subcutaneous inoculation into nude mice (Nilsson et al., 1977). LCLs express some normal B cell markers, including HLA class I and I1 antigens, CD 19, CD20, and surface immunoglobulin (Ig) with or without cytoplasmic Ig and secretion. Antigens characteristic of activated lymphocytes (such as CD23, CD39, CD30, and CDW70), cell surface adhesion molecules (CAMS) [such as lymphocyte function antigens 1 and 3

EBV-ASSOCIATED LYMPHOPROLIFERATIVE DISORDERS

33 1

(LFA- 1 and LFA-3)], and intercellular adhesion molecule-1 (ICAM- 1) and receptors for interleukin-2 (IL-2) and transferrin are also expressed. CD23 (also known as Blast-2) is of particular interest. It is a 45-kDa membrane-associated glycoprotein (Thorley-Lawson et al., 1985) that is rapidly shed from the surface of EBV-immortalized B cells in a soluble form (sCD23). A complex that includes sCD23 appears to be a major EBV-induced autocrine growth factor necessary for the independent growth of L,CI,s (Swendeman and Thorley-Lawson, 1987). The EBV genome, which is linear in the virus particle, circularizes on infection of B cells and later amplifies so that it is generally maintained as multiple copies (5-500 per cell) of an extrachromosomal circular episome. Viral gene expression is described as a latent infection and is restricted to eight latent proteins that are compatible with continued cell proliferation. These include the latent membrane protein (LMP), the EB viral nuclear antigen (EBNA) complex (EBNAs 1-6) (Dillner and Kallin, 1988), and the products of the terminal protein gene (Laux et al., 1988). The function of these proteins is for the most part unknown, but EBNA 1 is thought to be essential for the maintenance of the viral episome. EBNA 2 is thought to have an essential immortalizing function because EBNA 2 deletion mutants are nonimmortalizing (Dambaugh et al., 1986). Furthermore, the EBNA 2 gene, when transfected into a EBV-negative Burkitt’s lymphoma cell line, up-regulates CD23 expression, thus enhancing autocrine growth (Wang et al., 1987).LMP causes the expression of a tumorigenic phenotype when expressed in Rat-1 cells, and causes increased calcium flux in B cells, also suggesting a role in immortalization (Wang et al., 1985). C. EBV INFECTION I N Vrvo

EB virus infects the majority of individuals world wide and thereafter remains in the body as a persistent infection for life. If primary infection occurs during the early years of life it is usually asymptomatic. Approximately 50% of those who first encounter the virus during adolescence, however, develop infectious mononucleosis (IM). This is generally a self-limiting disease typified by viral replication in pharyngeal epithelial cells and polyclonal proliferation of EBV-infected B cells, followed by the characteristic appearance of “atypical” cytotoxic T lymphocytes. The IM syndrome, which is characterized by fever, lymphadenopathy, pharyngitis, skin rash, and splenomegaly, usually lasts 3 to 6 weeks with complete resolution of symptoms thereafter. During and directly following primary infection, immunoglobulin (IgM and IgG) antibodies to viral capsid antigens (VCAs) and early antigens (EAs) are detectable; however, IgG

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antibodies to one or more of the EBNA species only appear 30-50 days after infection. IgG antibodies against VCAs and EBNAs remain essentially stable over many years. T cell immunity is generally assumed to represent the effective response that limits EBV-driven B cell proliferation. Suppressor T cells, natural killer (NK) cells, and both EBV-specific and -unrestricted cytotoxic T cells, together with interferon, have a role in the control o f primary infection. T h e precise mode of action of these effector mechanism details is, however, unclear (Rickinson, 1986). Following primary infection, EBV persists in the body for life with low level viral replication in squamous epithelial cells of the pharynx and shedding of infectious, immortalizing virus into the oropharyngeal cavity. Persistent infection of B cells must also occur, because "spontaneou~" LCLs can be grown from the cultured B lymphocytes of a proportion of seropositive individuals. I t is thought that these cells become infected while trafficking through the pharyngeal lymphoid tissue, where close contacts are formed with the epithelial cells (Sixbey, 1989). D. IMMUNE

RESPONSE TO

EBV

After initial acute infection with or without clinical manifestations of IM, the majority of individuals develop an asymptomatic persistent infection in which there is no overt sign of EBV-associated lymphoproliferation. 'There is good evidence that EBV-specific cytotoxic T lymphocyte (CTL) memory plays a major role in regulating this viral persistence and preventing uncontrolled proliferation of EBV-infected €3 cells (Moss rt al., 1978; Rickinson el al., 1981a). CTL memory cells can be demonstrated in all normal persistently infected individuals (Rickinson et al., 1981b). This response displays HLA class I restriction (Wallace et al., 1982), but only a few target structures have so far been identified. These include LMP, EBNA 2, and EBNA 3. Neutralizing antibodies directed against membrane antigen (MA) components as well as IgG anti-VCA and EBNA antibodies persist for life and may help to control the persistent infection. E. PERSISTENTEBV INFECTIONI N THE IMMUNOCOMPROMISED

Organ and bone marrow transplant recipients, who are iatrogenically immunosuppressed for long periods, have been studied in detail. Many of these patients have a reactivated infection as judged by their serological profile. This shows raised levels of IgG antibody to VCA and EA and is often associated with increased viral shedding from the pharynx


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and increased numbers of circulating virus-carrying B cells. These findings are particularly marked in cyclosporin-treated patients and can be correlated with decreased or absent EBV-specific cytotoxic T cells in the circulation (Crawford et ul., 1981). This suggests that T cells normally control the level of viral replication in the pharynx, and that a decrease in ‘I‘ cell control tips the virus/host balance in favor of increased virus production, which is reflected 5 y raised antibody titers to lytic cycle antigens. Because only a minority of immunocompromized individuals develop overt disease related to EBV, some level of immunological control must remain in the majority of patients. However, the nature of these controlling mechanisms has not been fully elucidated. II. EBV-Associated B Lymphoproliferative Disorders

T h e close association between EBV and African Burkitt’s lymphoma (BL) is well recognized. Extensive data relating to the seroepidemiology, immunology, and virology of BL provide an important basis for the oncogenic role of viruses in the development of human malignancy. These studies indicate that EBV acts as one of a number of factors, including environmental and genetic elements, which contribute to the pathogenesis of BL. The description of BL in this review will primarily relate to those factors that provide a framework for current understanding and interpretation of the role of EBV in other lymphoproliferative disorders developing in immunocomprornized individuals. Ill. Burkitt’s Lymphoma

Burkitt’s lymphoma is classified as a high-grade malignant lymphoma of small, noncleaved follicle center B cell type (Lukes and Collins, 1974); it frequently involves the jaw and predominantly affects children from Central Africa (Burkitt, 1958, 1962). BL is found at high incidence across equatorial Africa and Papua New Guinea, in areas that are generally at altitudes below 1500 meters. This geographical distribution is coincident with that for holoendemic malaria (Burkitt, 1962). EBV was first discovered in cultured BL biopsies by Epstein and colleagues 25 years ago (Epstein et al., 1964). Subsequently, 96%of all the endemic BL (eBL) cases from the high-incidence regions have been shown to contain EBV DNA and also express EBNA (zur Hausen and Scholte-Holthausen, 1970; Reedman and Klein, 1973; Lindahl et at., 1974). The remaining cases (4%) contain no detectable EBV DNA or EBV-associated antigens and in this respect are similar to sporadic forms of BL (sBL), which occur in other parts of the world. The frequency of

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sBL in tropical Africa is about the same as it is throughout the rest of the world (Lenoir, 1986). Unlike eBL, which is generally restricted to children between 6 and 10 years of age, sBL mainly affects young adults and often localizes to extranodal sites but rarely involves the jaw. A large prospective study from the World Health Organization (WHO) has shown that the risk of developing eBL is 30 times greater in African children with elevated anti-EBV VCA antibody titers than in the normal population (de T h e et al., 1975).Although EBV is always present in eBL, it cannot be considered as the sole etiological agent in BL because it is an extremely ubiquitous virus and is not associated with the development of sBL. Phenotyping studies have shown that eBL tumor cells are monoclonal and almost exclusively express surface immunoglobulin (sIg), usually of IgM type. The use of monoclonal antibodies to study the surface of cells in BL biopsy material and newly cultured BL lines has revealed that the tumor cells express B cell markers CD19 and CD20 as well as common acute lymphoblastic leukemia antigen (CALLA; CD 10) and BLassociated antigen (BLA) (Rowe et al., 1987; Rowe and Gregory, 1988). These cells do not, however, express B cell “activation” antigens found on all LCLs and most activated normal B cells (Rowe et al., 1987; Rowe and Gregory, 1988). Neither do BL biopsies and early (< 20) passage BL cell lines express the adhesion molecules LFA- 1, ICAM- 1, and LFA-3 (Gregory and Rickinson, 1988; Gregory et al., 1988), which are found on most LCLs. Both sBL and eBL cells display the phenotype described above. However, many EBV-positive eBL cell lines have the capacity to “drift” toward the LCL-like phenotype within the first 20 or so passages zn vitro (Rowe et al., 1987). BL cells, unlike LCLs, are generally tumorigenic in nude mice assays (Nilsson el al., 1977). The pattern of EBV antigen expression in latently infected eBL cells is different from that found in LCLs. Significantly, although ERNA-1 is invariably present, EBNAs 2-6 and LMP have not been detected (D. T. Rowe et al., 1986; M. Rowe et al., 1987).EBNA-2 and LMP expressed in B cells act as targets for EBV-specific cytotoxic T lymphocytes. Thus, in relatively immunocompetent individuals there is strong immunoselection against EBNA 2 and LMP expression. Expression of the antigens on tumor cells would result in their elimination and inhibition of tumor development. The f-ailureto detect EBNA 2 and LMP in eBL is therefore consistent with the observed resistance of recently cultured BL cells to the effects of cytotoxic T cells (Rooney et al., 1985; Rowe et al., 1986). It is unclear, however, from currently available data what restricts the expression of EBV latent genes in these cells, although recent studies suggest that methylation of DNA in the control regions of some latent genes may


335

be involved (Masucci et al., 1989; Allday et al., 1990). Interestingly, when BL cells are grown in vitro, and immunoselection removed, cells expressing the full range of latent proteins appear in the culture; these have a selective advantage and outgrow the remainder (D. T . Rowe et al., 1986; M. Rowe et al., 1987). This expansion of EBV gene expression to include EBNA 2 and LMP probably accounts for the phenotypic drift exhibited by BL cells in vitro. In addition to EBNA 1, small, untranslated EBencoded RNAs (EBERs) are always expressed in eBL cells (Rymo, 1979; Rowe et al., 1987). It is not known whether other, as-yet unidentified, EBV gene products are also present in eBL cells and contribute to the malignant phenotype. Despite variations in the clinical manifestations of eBL and sBL, all BL tumor cells contain characteristic reciprocal chromosomal translocations (Manolov and Manolova, 1972; Lenoir, 1986) of which the three most common involve a breakpoint in the long arm of chromosome 8 at the 8q24 locus. The most frequent form transposes the telomeric region of‘ chromosome 8 to chromosome 14 [t(8:14)(q24:q32)], and in the less common “variant translocations,” genetic material is transposed from chromosome 2 to chromosome 8 [t(m)(qll:q24)] and from chromosome 8 to chromosome 22 [t(8:22)(q24:qll)](see Lenoir, 1986). The breakpoint on chromosome 8 is consistently adjacent to or within the c-myc oncogene and chromosomal translocation results in juxtaposition of c-my with one of the lg gene complexes on either chromosome 14 (Ig heavy chain), chromosome 2 (Ig K chain), or chromosome 22 (Ig A chain). Kecent molecular cloning and DNA sequencing analyses show a remarkable heterogeneity of the loci involved at these breakpoints. eBL and sBL exhibit slightly dissimilar chromosomal translocations, which probably occur by different mechanisms. It is likely that the tumors are derived from lymphocytes at different stages in the B cell differentiation pathway. In eBL, translocations predominantly involve J regions of Ig heavy and K light chains and probably occur during early B cell differentiation when functional Ig genes are formed (Haluska et nl., 1986; Neri et al., 1988). In sBL, on the other hand, the breakpoints occur within or near the Ig heavy chain SM switch region; thus sBL tend to secrete Ig and represent more mature B cells (Neri et nl., 1988). Although chromosomal translocation may not directly disrupt the c-myc gene in most cases of eBL, the invariable occurrence of point mutations and/or deletions in the 3’ region of exon 1 may be important in c-my gene activation (Cesarman et al., 1987; Eick et al., 1987; Morse et al., 1989). It therefore appears that both chromosomal translocation to an Ig locus and disruption of transcriptional control signals result in c-myc activation. The precise role that translocation plays in conferring active expression of c-myc is not clear,

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but it has been suggested that the transfer o f c-mny to a region of active chromatin may place it under the influence of an Ig enhancer element or dominant control region (Lenoir and Bornkamm, 1987). Two hypotheses have been proposed for the etiology of BL (Klein, 1979, 1987; Lenoir and Sornkamrn, 1987). In his original model, Klein proposed that children in high-incidence areas carry a large number of EBV-infected cells due to immunosuppression of T cell responses to EBV-infected cells caused by chronic Plasmodiumfa~ciparummalaria infection (Whittle el al., 1984). He argued that the presence of this large pool of mitotically active EBV-carrying B cells increased the possibility of a random second event, such as chromosome translocations involving c-my in the selective expansion of a monoclonal population of B cells. In light of more recent findings, Lenoir and Bornkamm ( 1987), proposed an alternative hypothesis which suggests that c-??zyc/Iggene translocations occur as regular but low-frequency nonrandom events in normal B cells during the process of active Ig gene rearrangement. Polyclonal B cell activation by P . falczPaniin, rather than immunosuppression due to chronic malaria, would then be the critical factor that increases the peripheral pool of recently recruited bone marrow (BM) B cells with actively rearranging Ig genes. These cells would usually die unless subsequently infected by EBV when a deregulated c-myc is complemented by an as-yet unidentified EBV function. This concept also provides an explanation for the pathogenesis of sBL in which another genetic lesion could replace EBV infection and provide the final stimulus for neoplastic transformation. The cooperation of and c-myc oncogenes in the malignant transformation of murine B lymphocytes suggests that ras gene activation may substitute for EBV infection in sBL (Schwartz et al., 1986). Consistent with this hypothesis is the observation that an sBL-derived cell line, Ramos, contains an activated N - r u ~gene (Murray et al., 1983). IV. EBV-Associated Lymphoproliferative Disorders in lmmunocompromised Individuals

Apart from BL, EBV is associated with a wide spectrum of lymphoproliferative diseases that develop in a setting of inherited or acquired immunodeficiency. A common factor in the development of' these lesions is thought to be an underlying deficit in the normal immunoregulatory mechanisms that control EBV infection in uivo. In some individuals, selective impairment of immune responses related to EBV may be unrecognized until after primary exposure to the virus, when untoward clinical complications develop. In other instances, defects in normal EBV control


337

mechanisms coexist with overt clinical manifestations of a generalized or partial immunosuppressed state, which in some cases may be iatrogenically induced. The clinicopathological expressions of the various lymphoproliferative disorders associated with EBV are discussed below. V. The X-Linked Lymphoproliferative Syndrome

The X-linked lymphoproliferative syndrome (XLPS) is an X-linked recessive inherited disorder in which primary EBV infection results in rapidly fatal or chronic IM, hypo- or agammaglobulinemia, or malignant lymphoma (Purtilo et al., 1981, 1982, 1987; Sullivan, 1985). The syndrome exclusively affects males who are either siblings in a single family or maternally related individuals in different families. Mothers of affected offspring, although asymptomatic for EBV infection, are seropositive and have evidence of abnormal serological responses to EBV with raised IgG VCA and anti-EA antibody levels suggestive of persistent active infection (Ando et al., 1986). The genetic locus responsible for uncontrolled B cell proliferation after primary EBV infection in affected males has been attributed to mutation of the putative lymphoproliferative control gene ( X l c ) on the X chromosome (Skare et al., 1987; 1989). The disease was first described in 1975 by Purtilo and colleagues in six male kindred of the Duncan family, from which the syndrome derives its eponymous name. Through recognition of the existence of other similarly affected families, a register was established in 1978 to monitor the various clinical phenotypes associated with XLPS and to identify individuals at risk of developing the disease. Two hundred patients from 50 families world wide are currently registered. XLPS carries a high mortality rate, with survival no longer than 10 years in 70% of cases and with death occurring in all casesby 40 years'of age (Grierson and Purtilo, 1987). A. ACUTEPRIMARY EBV INFECTION IN XLPS MALES Before primary exposure to EBV, patients with XLPS are able to tolerate infection with other agents but have evidence of lowered T cell-mediated and humoral immunity, indicative of defective T cell cooperation with B cells (Lindsten et al., 1982; Purtilo et al., 1982; Sullivan, 1985, 1988). Following infection with EBV, 57-65% of patients develop rapidly fatal IM and have a median survival rate of 32 days (Grierson and Purtilo, 1987; Mroczek et al., 1987). The average age of these patients is 2.7 years, an age when primary infection with EBV in normal immunocompetent children is generally asymptomatic.

338


Serologically, I M in XLPS is associated with polyclonal production of Ig, elevated levels of IgM VCA antibodies, raised or in some cases low anti-EA antibodies, but absence of antibody production to EBNA (Sakamoto et al., 1980; Sullivan et al., 1983). Heterophile antibodies have also been reported to be absent in the majority of cases (Grierson and Purtilo, 1987). I n the early stages, evidence of reversed CD4:CDS T cell ratios and enhanced NK cell activity are not unusual findings for IM. EBNA-positive cells expressing polyclonal Ig are present in 1-507; of circulating peripheral blood cells and have the capacity to form spontaneous B LCLs in culture. However, these patients appear to lack specific cytotoxic T cell activity to EBV in viz~o,because cell lines derived from peripheral blood mononuclear cells from these cases fail to regress after addition of autologous T cells in vitro (Harada et al., 1982). An additional B cell defect has also been identified in some patients. The failure of HLA-restricted EBV-specificcytotoxic T cells to recogize and kill a spontaneously transformed B cell line, derived in vitro from bone marrow cells from an XLPS patient, has been attributed to the absence or altered expression of B cell EBV antigens, which are necessary for effector T cell recognition (Ando et al., 1986). However similar B tell defects have not been confirmed in other studies (Sullivan, 1985). B. HISTOLOGY OF LESXONS I N FATALIM XLPS

Apart from constitutional symptoms typical of IM, patients present with rapidly progressive lymph node enlargement and splenomegaly. T h e histological features in lymphoid tissues during the initial phases of disease are not dissimilar to the changes seen in normal immunocompetent patients with IM. These are characterized by massive infiltrates of reactive, polymorphous niononuclear cell infiltrates containing transformed (small or medium size) lymphocytes, immunoblasts, some plasma cells, and occasional multinucleate giant cells resembling ReedSternberg cells of Hodgkin’s disease (Purtilo et al., 1982; Childs el al., 1987). Typically, the T-dependent areas in lymph node (paracortex) and spleen (periarteriolar lymphoid sheath) are expanded by proliferating cells and often exhibit a starry-sky appearance due to numerous phagocytic histiocytes/tingible body macrophages. In general, splenic B cell areas remain intact whereas lymph node B cell follicles are either inconspicuous or absent. Disease progression is accompanied by a diminution in morphologically activated lymphoblasts and immunoblasts, a concurrent increase in plasma cells and histiocytes, and areas of focal necrosis. Rapid disease dissemination involves other organs, including thymus, bone marrow,


339

liver, lower respiratory tract, small and large intestine, brain, and in some cases, heart, kidney, and adrenals (Weisenburger and Purtilo, 1986). Autopsy samples of these organs from fatal cases may show a range of histological changes corresponding to either the massive lymphoproliferative phase of early disease or varying degrees of tissue necrosis, pleiomorphic cell infiltration, and hemophagocytosis, which are prominent features of progressive disease. The multiple focal or generalized areas of necrosis in these tissues are accompanied by mixtures of atypical lymphoblasts, immunoblasts, and prominent phagocytic histiocytes. Frequent sites of infiltration are hepatic periportal areas, tracheal and gastrointestinal submucosa, peribronchial and pulmonary interstitium, and perivascular aggregates in all organs. A detailed histological study of autopsy samples from 21 XLPS IM cases has shown a relationship between the length of survival and degree of thymic involvement, suggesting that injury to this site may contribute to the immune defects that are present in fatal cases, as well as in XLPS patients who survive I M and later develop complications of hypogammaglobulinemia and malignant lymphoma (Mroczek et al., 1987). Overwhelming hepatic or bone marrow failure due to massive necrosis of these organs is a major cause of death in 22.5% of XLPS patients with acute IM (Grierson and Purtilo, 1987). The cause of this necrotic process is not known but has been attributed to anomalous cytopathic effects of NK cells and non-EBV-infected cells (Sullivan et al., 1983). Patients also succumb to opportunistic infection or severe gastrointestinal or pulmonary hemorrhage resulting from submucosal infiltration and ulceration, secondarily complicated by hemostatic dysfunction due to aplastic anemia and bone marrow failure. C. EBV-ASSOCIATED ANTIGEN EXPRESSION IN XLPS LESIONS The cellular infiltrates in XLPS contain EBNA-positive cells expressing polyclonal Ig (Mroczek et al., 1987; Purtilo et aE., 1981) but lack lytic cycle antigens VCA and EA (Morgan-Capner el al., 1986; Thomas et al., 1990) or viral particles identifiable by ultrastructural examination (Morgan-Capner et al., 1986). EBV DNA has been demonstrated in the lesions of some cases (Purtilo et al., 198 1; Grierson and Purtilo, 1987; Falk et al., 1990). Karyotypic studies reveal no specific translocations in either tissues o r in uitro immortalized B cell lines from these patients (Harris and Docherty, 1988). The immunohistological phenotypes of autopsy samples from several organs have been examined in detail in two XLPS males who died within 2 1 days after onset of acute disease. Both patients

340

J. ALERO THOMAS ET AL.

have been previously documented as individual case reports (Ando et al., 1986; Morgan-Capner et al., 1986). The histological appearances in the primary and secondary lymphoid organs in each case were representative of the morphological spectrum associated with IM. The cellular infiltrates in both cases contained polyclonal Ig-positive B cells that expressed EBNA 1 and LMP but lacked positive staining for EA, VCA, and MA. EBNA 2-positive cells were also identified in the diffuse lymphoproliferative infiltrate in one case (Thomas et al., 1990).Although EBNA-positive B cells in both patients lacked most B cell differentiation and activation markers, including CD23, the presence of CD38 and class I1 major histocompatibility antigens was consistent with the morphologically activated nature of these cells. Furthermore, the expression of CAMS LFA- 1 and ICAM-1 were notably absent, although LFA-3 was detectable in one case only. Similar findings of polyclonal B cell populatiars and/or full EBNA expression in tumors form 4/7 XLPS cases reported by Falk et al. ( 1990) support the conclusion that these lesions phenotypically resemble BLCIs rather than BL. D. T CELL PHENOI'YPES I N FATALIM XLPS Accompanying nonneoplastic small lymphocytes reported in XLPS autopsy lesions (Morgan-Capner et al., 1986) consist of varying numbers of mature T cells mainly of activated, inducer type (class 11+, CD4+), which nevertheless lack specific CD25 activation antigen. Thymic involvement in one patient (Ando et al., 1986; J. A. Thomas unpublished observations) was characterized by diffuse EBNA-positive cell infiltration, severe thymocyte depletion, and loss of normal corticomedullary demarcation. Obvious evidence of thymic involution, which is not an uncommon finding in fatal XLPS (Mroczek et al., 1987; Weisenburger and Purtilo, 1986), was not apparent in this sample by conventional histology, although phenotyping studies showed a conspicuous absence of cortical epithelium. In contrast to other XLPS studies in which thymic T cells have been reported (Mroczek et al., 1987),thymic tissue in this case contained only low numbers of phenotypically mature inducer (CD2+, CD4') and suppressor/cytotoxic (CD2+, CD8+) T cells, which lacked class 11and specific CD25 activation antigens. These cells probably represented residual medullary thymocytes, consistent with their location in areas of antigenically defined medullary epithelium, rather than reactive T cell populations present in the cellular infiltrates of other organs. These findings illustrate the phenotypic discrepancies between circulating and tissue-infiltrating T cell populations that may occur in some patients. T h e increased numbers of circulating suppressor/cytotoxic T


34 1

cells, normally seen in acute IM, may also form a major component of the tissue infiltrates in XLPS-associated IM. On the other hand, the prevalence of inducer T cell populations, particularly in lymphoid organs of some patients, may reflect either an expansion of the normally predominant (CD4+) subset in these sites or tissue depletion of suppressor/ cytotoxic cells due to increased requirements in the periphery. The precise functional role of the inducer T cells is not known, although EBVspecific cytotoxic T cells of inducer type have been reported (Misko et al., 1984). E. POSTACUTE EBV INFECTION IN XLPS MALES

XLPS males who survive acute IM continue to show abnormal serological patterns for EBV and progressively deteriorating levels of humoral and cell-mediated immunity. Typically, these patients have abnormally depressed antibody levels to EBV-associated antigens, particularly EBNA. Sakamoto et al. (1980) have shown that 80% of survivors have either low antibody titers to VCA with no response to EBNA or have no serological evidence of prior EBV exposure. The majority of patients show progressive combined variable immunodeficiency characterized by hypo- o r agammaglobulinemia with EBV-specific and nonspecific defects in T and B cell function (Purtilo et al., 1982, 1987; Weisenburger and Purtilo, 1986; Grierson and Purtilo, 1987; Sullivan, 1988). Persistently inverted CD4:CD8 ratios, increased T suppressor cell activity, depressed lymphocyte responses to mitogenic and mixed lymphocyte stimulation, inability to switch from IgM to IgG antibody responses after bacterophage 4174 challenge in uiuo (Ochs et al., 1983), and deficient NK cell activity are evident in most cases. Defects in cellular responses to EBV in uitro include the failure to generate EBV-specific cytotoxic T cells (Harada et ul., 1982) and absence of regression by autologous EBV-induced BLCLs. Normal HLA-restricted EBV-specific cytotoxic T cells have been reported in two XLPS survivors who developed hypogammaglobulinemia (Rousset et al., 1986). The absence of Ig production by EBVinfected BLCLs and low numbers of circulating B cells (Purtilo et al., 1984) in survivors with hypogammaglobulinemia have not been reported in all cases who develop this clinical “phenotype” (Rousset et al., 1986). Long-term XLPS survivors account for 251 135 registered patients (Weisenburger and Purtilo, 1986) and are more common among patients who develop hypogammaglobulinemia (Grierson and Purtilo, 1987). Between 20 and 35% of XLPS survivors develop malignant lymphoma in 1-17 years after acute IM (Purtilo et al., 1982, 1987). Lymphoma may develop as isolated disease or coexist with hypo- or agammaglobulinemia,

342


or develop concurrently with acute fatal 1M and hypogammaglobulinemia. In 15% of cases, clinical evidence of acute I M is absent (Grierson and Purtilo, 1987; Harrington et al., 1987; Sullivan, 1988). The median age of patients with lymphoma alone is 4.9 years and median survival is 12 months after diagnosis. These tumors, which have all been classed as B cell non-Hodgkin's lymphoma (NHL), are commonly extranodal and present as localized lesions at one or more anatomical sites. T h e ileocecal region is a common site of presentation in 78% of cases (Grierson and Purtilo, 1987), followed by involvement of lungs, liver, kidneys, thymus, colon, tonsils, and muscle in decreasing order of frequency in other cases. In 18 cases studied by Grierson and Purtilo (1987), all tumors showed diffuse, intermediate, or high-grade B cell proliferation histologically classified as small, cleaved (12.5%)or small, noncleaved (45%) in type. In 17.5% of patients, the tumors were immunoblastic or large, noncleaved type. Of all tumors, 6% were histologically unclassifiable. With treatment, median survival was 6 months in 50% of patients. The longest survival times of 7-15 years were seen in patients who developed NHL with hypogammaglobulinemia. Lymphoproliferative lesions from 10 patient with acute I M who died within 20 weeks after disease onset contained EBV DNA and oligoclonal o r monoclonal B cell populations identified by Ig gene rearrangement analysis. Some of these tumors also contained the rearranged m y protooncogene (Brichacek et al., 1987). F. PATHOGENESIS OF XLPS

The predisposition of males with XLPS to develop life-threatening sequelae to primary EBV infection is due to an inherited defect in the immunoregulatory mechanisms that normally control EBV infection in viva Individuals with XLPS are able to withstand infection with other common childhood infectious agents before their first exposure to EBV. Furthermore, these individuals have the capacity to mount normal humoral and T cell-mediated immune responses to EBV in the early stages of acute primary infection. The inherent immunological defect in these patients appears to relate to the presence of' functionally ineffective EBV-specific cytotoxic T cells, which are responsible for the control of EBV-induced B cell proliferation in normal immunocompetent individuals. T h e failure to check the growth of EBV-infected B cells in vivo results in a massive and overwhelming polyclonal €3 cell proliferation, rapidly culminating in death due to irreversible destruction of lymphoid tissues and other body organs. On the other hand, identification of clonal B cell


343

populations with altered oncogene configurations in one series of patients (Brichacek et al., 1987) suggests the selective outgrowth of a malignant B cell population from a polyclonal B cell proliferation possibly caused by multiple genetic events similar to those proposed in the pathogenesis of BL (see Section 111). Similar mechanisms are probably responsible for the development of NHL in XLPS survivors in whom there is a gradual but progressive deterioration in cellular and humoral immunity. The presence of EBV-infected B cells with inadequate and/or ineffective EBV-specific cytotoxic T cell function would inevitably engender uncontrolled EBV-induced B cell proliferation in these patients. By contrast, excessive T suppressor cell activity is thought to abrogate EBV-induced B cell proliferation and induce acquired hypo- or agammaglobulinemia, which contributes to the clinical entity of X-linked recessive combined immunodeficiency disease described in the majority of XLPS IM survivors (Purtilo et al., 1975, 1978, 1984). G. TREATMENT

In view of the rapidly fatal course of acute IM in XLPS patients, supportive therapy with steroids, cytotoxic agents, high-titer EBV gammaglobulin, interferon-a and -7, and leukocyte transfusion has been disappointing (Purtilo et al., 1981; Sullivan, 1985; Andersson and Ernberg, 1988). Alternative treatment with the anti-viral agent acyclovir has also proved unsuccessful (Sullivan et al., 1984; Andersson and Ernberg, 1988; Grierson and Purtilo, 1987). Clearly, measures aimed at detecting at-risk families by pedigree analysis, serological screening for maternal carriers, and antenatal screening for specific XLPS-related genetic mutation would be important in indentifying susceptible individuals who would benefit from specific antiviral or immunoprophylactic therapy to prevent primary infection with EBV. In XLPS survivors who later develop NHL, treatment with conventional chemotherapy, surgery, and irradiation has resulted in tumor resolution and long-term survival in some cases. VI. EBV-Related Lymphoproliferative Diseases Associated with Miscellaneous lrnmunodeficiency States

Polyclonal high-grade (immunoblastic) B cell lymphomas arising in the context of inherited primary immunodeficiency diseases have been reported in patients with severe combined immunodeficiency (Borzy et al., 1979; Kersey et al., 1980) and in u p to 10% of patients with ataxia

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J. ALERO THOMAS ET A L .

telangectasia. Both of these conditions are associated with varying degrees of abnormal T and B cell immunity. A relationship to EBV has been demonstrated by the presence of EBV DNA and/or EBNA in the proliferating cells of B cell tumors developing in patients with severe cornbined immunodeficiency (Gartner et a/., 1987), following thymic epithelial (Reece el al., 1981) or bone marrow (Shearer et al., 1985) grafting as treatment for this condition, and in a B cell lymphoma from a patient with ataxia telangectasia (Saemundsen et al., 198 1). Included within this group of diseases is the chronic EBV syndrome reported in children and adults who have selective humoral and T cell-mediated defects and abnormal serological responses to EBV (Jones, 1987). A third of these patients are prone to develop lymphoproliferative disorders (Editorial, 1985). Sporadic fatal IM (Penman, 1970; Chang and Campbell, 1975; Look et al., 198l), atypical lymphoproliferations associated with persistent active EBV infection (Krueger el ul., 1987), and IM-like illnesses mimicking NHL (Robinson et ul., 1980) have been extensively documented. Although clinically similar to XLPS, sporadic. fatal 1M frequently develops in older children (average age 13 years), affects both sexes equally, and is not usually associated with a family history of complicated EBV infection. However, EBV-associated fatal lymphoproliferations and malignant NHL have been described in related females in single families (Veltri P t al., 1983; Weisenburger and Purtilo, 1986), suggesting that an inherited defect in normal EBV immunity similar to XLPS may also exist in some of these cases. Subtle defects in T cell-mediated immunity (Crawford et ul., 1979) and selective Ig deficiencies have also been reported in some sporadic cases of fatal IM. A minority of patients, following primary exposure to EBV. develop chronic active EBV infection in which a recurrent or persistent IM-like illness is associated with abnormal serological profiles typified by elevated anti-EA and reduced or absent anti-EBNA antibody levels. A small proportion of these patients develop lyniphoprolif erative disease of large granular lymphocytes (LDCL). In a recent survey of seven patients with LDGL, EBV DNA in abnormal granular peripheral blood lymphocytes was identified in five of these cases, including one patient with chronic EBV infection (Kawa-Ha et ul., 1989). It is interesting that high numbers of these cells in patients with detectable EBV express the CD2 T cell differentiation antigen. Evidence is now emerging that a causal association may exist between EBV and certain lymphoproliferative lesions of peripheral T cell origin. Kikuta et al. (1988) have reported the presence of EBV DNA and EBNA in peripheral T cells of a child with Kawasaki-like disease following


345

primary EBV infection. EBV DNA has also been identified in fatal T cell lymphomas arising in children and adults with a complex variety of hematological disorders and dysgammaglobulinemia (Jones et al., 1988). More recently, nasal peripheral T cell lymphomas from five patients with lethal midline granuloma have been shown to contain EBV DNA and EBNA (Harabuchi et aE., 1990).The serological findings in these patients were suggestive of chronic EBV infection and persistent viral activity. T h e presence of EBV DNA (and EBNA when investigated) in these T cell proliferations suggests that under certain circumstances, '1'cells may be additional targets of EBV infection. This is supported by evidence of sustained EBV replication in T cells transfected with EBV DNA in uitro (Stevenson et al., 1986). The mode of EBV infection in T cells in vzvo is unclear because expression of the CR2, CD2 I , EBV receptor is generally perceived to be restricted to B cells and certain squamous epithlelial components. However, EBV-binding molecules have been detected in some phenotypically immature human malignant T cell lines in uztro (Fingeroth et al., 1988). Expression of the same or related molecules on circulating mature T cells, which may serve as a portal for EBV entry, has yet to be determined. The nature of this unusual cellular association with EBV requires further investigation to elucidate precisely the pattern of viral gene expression in these cells. Although EBV-containing peripheral T cells from affected individuals did not appear to support EB viral replication in vitro or in uzvo (Kikuta et al., 1988), tumor cell expression of latent gene products EBNA 2 and LMP, in cases of midline granuloma (Harabuchi et al., 1990), strongly suggests a primary role for EBV in the development of this tumor. VII. Lymphoproliferative 6 Cell Disorders in Organ Transplant Recipients

Advances in immunosuppressive therapy and greater understanding of the mechanisms involved in the induction and control of graft-versushost disease (GVHD) have been major factors in promoting allograft transplantation for definitive treatment of incurable organ disease. However, the profound immunosuppression necessary for graft survival carries a well-recognized predisposition to the development of de now cancer in organ and bone marrow transplant recipients. The overall frequency of all types of cancer in these patients is estimated to be 45- 100 times greater than in the general population (Hardie et al., 1980; Penn, 1981). With the exception of skin and lip cancers, which occur with the highest frequency (38% of all postgraft cancers) (Penn, 1983), NHL represents the second most frequently occurring malignancy in these

346


patients and constit.utes 26% of all cancers in graft recipients, which represents a 28-fold increase relative to age-matched controls (Penn, 1983). The overall incidence of NHL in 19-32% of solid organ transplant recipients is a striking contrast to the 3-4% incidence of NHL in the general population (Penn, 1979, 1981; Sheil, 1986). I n common with other types of de novo cancer associated with transplantation, contributory factors influencing the development of NHL include genetic predisposition, primary immune deficiency, secondary o r iatrogenically induced immunosuppression, chronic immunostimulation by foreign antigens in transplanted allografts, and oncogenic virus activation (Penn, 1979). Recognition that the frequency with cyclosporin (Cs), and the almost invariable demonstration of EBV DNA or EBV-related gene products in these lesions have provided import.ant clues to the pathogenesis of NHL in transplant recipients. T h e failure to adequately control active or latent EBV infection due to inhibition of EBV-specific T cell responses by prolonged or high doses of Cs is thought to be one of the major underlying factors that contributes to the uncontrolled proliferation of EBV-infected B cells in these patients. Cumulative data from several individual case reports and studies of large patient series led Hanto and co-workers to suggest that the unusual clinical features and biological behavior of these lesions constitute a distinct clinicopathological entity (Hanto et al., 1981, 1985) that is distinguishable from NHL arising spontaneously in nontransplanted individuals. Typically, transplantation-associated NHLs are characterized by their rapid onset, aggressive behavior, predilection for extranodal sites of presentation, and often complete or partial regression following reduction or withdrawal of immunosuppressive therapy. Although a majority of these lesions are histologically classifiable as NHLs of a high-grade type, they exhibit a wide range of morphological changes. These have been interpreted to represent different stages of EBV-induced B cell proliferation, from a diffuse polymorphic hyperplasia to diffuse polymorphic B cell lymphoma (Frizzera et al., 1981; Fizzera, 1987). Increasing evidence of nonclonal and/or clonal B cell populations within these lesions existing either separately or concurrently in single lesions, or in several lesions at multiple sites, strongly suggests a progressive sequence of events in which a polyclonal and reactive B cell hyperplasia can evolve to a monoclonal and malignant B cell tumor. A. IMMUNOSUPPRESSION

AND

POSTGRAFT NHL

Following the introduction of Cs for prophylaxis and treatment of GVHD, Calne et 01. (1979) reported an 8.8% incidence of NHL in early


347

clinical trial transplant patients and that approximately one-third of these occurred in patients treated with Cs alone. By contrast, no NHL developed in patients treated with non-Cs-related regimes (Nagington and Gray, 1980). T h e preponderance of Cs-related postgraft NHL is emphasized in recent data from the Cincinnati Transplant Tumor Registry in which NHL represented 30% of all de novo malignancies in transplant recipients treated with Cs compared with 12%in patients treated with conventional immunosuppressive agents (Penn, 1987). The disproportionately high frequency of NHL in Cs-treated transplant recipients has been confirmed in several reports and a compilation of these data is shown in Table I. The relationship between NHL and Cs therapy is likely to be due to a combination of several factors, including high-dose treatment schedules used to induce immunosuppression in some patient groups as well as the effects of incorporating Cs with several different cytotoxic agents. Beveridge et al. (1 984) suggested that the risks of NHL developing in the TABLE I INCIDENCEOF Cs A N D NON-&RELATEDB CELLTUMORS I N TRANSPLANT RECIPIENTS

Organ graft Solid organ grafts

+ bone marrow

Kenal

Cardiac + cardiopulmonary

Cardiac

Cardiopulmonary

Bone marrow Hepatic

Cs-related tumors (%)

Non-Cs-related tumors ($6)

1.4 41 30

0.9 12 12

Hanto el al. (1981) €10et al. (1985) Penn (1987) Penn ( I 988)

6.5 8 2.5 3.9

0 0.3

Nagington and Gray ( 1980) Beveridge et al. (1984) Starzl et al. (1984) Wilkinson et al. (1989)

3.7 7.7 0.6 6.3 13 1.8 33.3 4.6 9.4 0.2 2.2

5 -

Beveridge et al. (1984) Brumbaugh et al. (1985) Wreghitt et al. (1989)

1.5

Starzl et al. (1984) Nalesnik et al. (1988) Randhawa et al. (1989) Penn (1983) Shapiro et al. ( I 988)

-

Nalesnik et al. (1988)

References

Starzl et al. (1984) Hanto el al. (1985) Nalesnik et al. (1988)

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context of Cs therapy may be no greater than with other immunosuppressive agents. These authors noted a dramatic increase of NHL and lymphoproliferative disorders in 8% of patients treated with combinations of Cs and other cytotoxic agents in contrast to 0.4%of patients who developed NHL following treatment with Cs alone or Cs combined with low-dose prednisolone. T h e risks associated with the use of Cs in combination with several different agents have recently been reinforced by reports of significant increases in lymphoproliferative disorders in 1 1.9 and 3.9%, respectively, in cardiac and renal transplant recipients receiving triple therapy with Cs, prednisolone, and azathioprine. By contrast, lymphoid tumors occurred in only 0.3% of renal transplant recipients receiving combined azathioprine and steroids, and no tumors were associated with 189 renal graft operations in individuals conditioned with steroids and Cs o r azathioprine (Wilkinson et al., 1989). The marked variation in tumor frequency among different patient groups also relates to the mode and intensity of itiiniunosuppressinri treatment. The highest incidence of postgraft NHL occurs among heart and h e a r t h n g transplant recipients. A high proportion of these tumors (0.6-33.3%) are Cs related (see Table 1) and develop in regrafted patients (Bieber et d., 1981; Weintraub and Warnke, 1982). These patients are more likely to receive higher cumulative doses of' immunosuppression than are patients with successful first grafts. Quantitative analysis of immunosuppression parameters during the first 3 months after grafting in one series of heart and heart/lung recipients has shown that niean Cs levels, total antithymocyte globulin (ATG) dosage, and duration of'T cell suppression were considerably higher in patients who developed NHL compared with patients who remained tumor free (Brumbaugh et nl., 1985). An increased risk of postgraft lymphoproliferative tumor development is also associated with additional forms of immunosuppression specifically designed to prevent or control GVHD and graft rejection. Among bone marrow transplant recipients, major risk factors relating to therapy include T cell depletion of donor marrow (Lyttelton et ul., 1988; Shapiro et ol., 1988; Zutter el al., 1988; Witherspoon ct al., 1989) and anti-T cell inimunotherapy (Martin et al., 1984; Zutter et al., 1988). Similarly, rapidly progressive lymphoproliferative lesions have been reported in renal transplant recipients receiving monoclonal antibody OKT3 for graft rejection (Canioni et al., 1989). On the basis of available data, it is now generally agreed that the degree of immunosuppression rather than any one specific agent contributes a significant risk to the development of postgraft NHL (Editorial, 1983; Penn, 1988). This is supported by the absence or considerable


349

decrease in incidence of transplantation-associated NHL following adoption of low-dose treatment schedules (Fine et al., 1984; Bia and Flye, 1985; Snyder et al., 1988) adjusted by careful monitoring of drug blood levels (Beveridge et al., 1984). B. CLINICAL FEATURESOF POSTGRAFT NHL

Postgraft NHL and lymphoproliferative disorders can present with a variety of clinical manifestations relating either to the effects of localized tumor o r to widespread disease. These may be accompanied by constitutional symptoms suggestive of an IM-like illness. More frequently, tumor presentation may be masked by concurrent common postgraft complications of infection and graft rejection, which may result in delayed tumor diagnosis. Several criteria have been used to categorize the clinical features of postgraft NHL in different patients groups. On the basis of patient age and postgraft time interval, Hanto et al. (198 1 , 1985) found that tumors developing rapidly, on average within 9 months after grafting, were more frequently associated with young males, of 21 years average age, and commonly presented as localized central nervous system (CNS) disease with systemic illness. Conversely, tumors in older patients, of 47 years average age, were less rapid in onset, developing on average 5.3 years after transplantation, and were more usually localized masses in the gastrointestinal tract. In correlating clinical presentation with tumor location, Nalesnik et oZ. (1988) found that localizing symptoms most frequently related to head and neck, gastrointestinal, or specific organ involvement and were not always indicative of the extent of the disease process. In 50% of cases in this series, tumor diagnosis was made at autopsy. In children less than 18 years of age and recipients of all types of grafts, three distinct presenting syndromes have been described (Ho et al., 1988). Two clinical forms, which are thought to represent different stages of the same disease process, manifest as a self-limited IM-like illness with either localized or widespread tumors. The third clinical form is characterized by presentation of a localized extra nodal mass.

c. TUMOR PRESENTATION Comparative studies of Cs and non-Cs-related forms of immunosuppression appear to have a distinct influence on the mode of clinical presentation of postgraft lymphoproliferative tumors (Penn, 198 1, 1987, 1988). Findings in the most recent of these reports show that Cs-related tumors develop more rapidly, on average within 12 months of grafting,

350


and have an almost equal distribution o f nodal (49%)and extranodal (51%) sites, similar to that seen in the general population. By contrast, tumors developing in treated non-Cs patients develop over a longer postgraft time interval (1-196.5 months) and are extranodal in the majority (78%) of cases. Extranodal lesions associated with Cs predominantly involve the small bowel whereas CNS involvement is more common in treated non-Cs cases. The prevalence of transplantationassociated N H L in extranodal sites has an overall frequency of 78% compared with 2 4 4 8 % in the general population (Penn, 1988). Extranodal lesions may be present at multiple sites or occur in 69%)of cases as a single-organ disease. Frequent sites of involvement are the CNS, lungs, bowel (Patchell, 1988; Penn, 1981, 1987, 1988; Touraine el al., 1989; Tubman et al., 1983), and allografted tissue (Sheil, 1986; Citterio et al., 1987), with more unusual sites reported at presentation or at autopsy in prostate, fallopian tube, lacrymal gland, tongue, and soft tissues at ATG injection sites (Bieber et al., 1981; Penn, 1983; Saemundsen et al., 1982; Thomas et nl., 1990). The high frequency of CNS lymphoma in 39-4970 of postgraft recipients is striking in comparison with the overall 2% incidence of CNS lymphoma in nongrafted individuals (Patchel, 1988). The CNS is the only site of involvement in 85% of transplant recipients with cerebral disease (Penn, 1979). CNS lesions are reported to develop within 50 months after grafting arid are not associated with any specific clinical o r radiological feature that distinguishes them from similar lesions in the general population (Tubman et al., 1983; Day et al., 1987; Patchell, 1988). Altered mental state and personality changes, which are a trequent form of CNS disease presentation, may be related to the usually deep location and multicentric nature of these lesions, although focal signs of localized brain disease are uncommon (Penn, 1983; Patchell, 1988). Causes for the prevalence of primary CNS lymphoma following transplantation are not known, but the paucity of immunological effector cells within cerebral tissue may be a significant factor in allowing uncontrolled tumor growth at this site. Gastrointestinal involvement, which is prevalent among renal graft recipients, can present as bowel obstruction, nonobstructing masses with widespread ulceration, hemorrhage, or perforation (Tubman et ul., 1983; Starzl el af., 1984). Liver lymphoma, which usually occurs as secondary disease in nontransplanted individuals, has been reported in 57% of transplant recipients often in association with Cs immunosuppression (Honda et al., 1989). Hepatic involvement may occur as a single primary tumor or as multiple lesions with evidence of extrahepatic disease in adjacent organs and lymph nodes. Intrathoracic lymphoma is often a manifestation of generalized disease and is usually rapidly progressive.


35 1

Primary pulmonary lymphoma can occur as solitary or multiple nodules, rarely without hilar or mediastinal lymphadenopathy, and develops within grafted tissue in 60%of heart-lung transplant recipients (Yousem P t al., 1989). Primary presentation of NHL in other solid organ grafts has a frequency of 17 and 8.6%in renal and liver graft recipients, respectively, but has not been reported in patients receiving heart grafts alone (Nalesnik et al., 1988). T h e predilection for lesions to develop in grafted organs may relate to specific microenvironmental factors in these tissues that favor EBVinduced B cell proliferation. The origin of the tumor cells has been identified by cytogenetics (Martin et al., 1984; Bloom et al., 1985),by HLA typing (Thomas et al., 1990), and by analysis of leukocyte isoenzyme (Gossett et al., 1979; Martin et al., 1984) o r DNA restriction fragment length polymorphisms (Shapiro et al., 1988) in recipients of bone marrow, renal, and heart-lung grafts. Lesions arising in transplanted solid organs might result from passive transfer of EBV-infected B cells in the grafted tissue. This concept is supported by a recent report in which allografted renal tissue was implicated as the sole vehicle for EBV transmission in a patient who was previously seronegative and subsequently developed an EBV-positive graft-associated lymphoma within 53 days after transplantation (Denning et al., 1989). Other factors are clearly involved in tumors that are host derived (Bloom etal., 1985; Shearer et al., 1985; Zutter et al., 1988). Because postgraft tumors of recipient origin frequently occur in association with mismatched grafts, graft rejection and chronic antigenic stimulation from recurrent GVHD might result in viral activation at these sites. A similar pattern of events has been postulated as a contributory factor in the pathogenesis of BL, in which EBV-induced B cell hyperproliferation is a consequence of chronic antigenic stimulation through repeated malaria infection (see Section 111). D. HISTOLOGY OF POSTGRAFTB CELLTUMORS Though many of the lymphoproliferative tumors arising in the context of organ and bone marrow transplantation are histologically classifiable as high-grade B cell NHL of immunoblastic or undifferentiated large cell type, in many instances these lesions also exhibit striking morphological heterogeneity consistent with the tissue changes associated with acute EBV infection. Observations based on the histological appearances of B cell tumors developing in patients of the Minnesota and Colorado transplant series led Frizzera (1987) and Frizzera et al. (1981) to propose that EBV-associated tumors in transplant recipients represent a distinct clinicopathological entity characterized by either diffuse

352

J . ALEKO THOMAS E T A L .

polymorphic B cell hyperplasia or diffuse polymorphic B cell lymphoma. These patterns incorporate the concept of a malignant lymphoma evolving from a nonmalignant, reactive B lymphoid proliferation. Common histological features in these lesions show polymorphic cellular infiltrates containing several morphological stages of B cell differentiation, extensive nodal obliteration, and evidence of localized o r widespread necrosis. Lesions corresponding to the pattern of diffuse polymorphic B cell hyperplasia exhibit predoniinantly plasmacytoid cell proliferation, single-cell necrosis, and large morphologically reactive lymphocytes. These appearances are not dissimilar to the patterns of polyclonal B cell proliferation seen in acute IM. On the other hand, lesions exhibiting changes of diffuse polyniorphic B cell lymphoma show extensive tissue necrosis and infiltrates of atypical large cells consistent with immunoblasts. A recent detailed review of graft-associated B cell tumors in 83 histologically evaluable tissue samples from 37 patients in the Pittsburgh and Colorado transplant series (Nalesnik et at., 1988) has identified polymorphic lymphocyte proliferations in the majority (47/83) ofsamples. These contained a wide range of morphological types, including cleaved and noncleaved lymphoid cells, immunoblasts, plasrnacytoid forms, and plasma cells. Mononiorphic lesions were the second most frequent finding (in 12/83 samples). These showed features similar to conventional NHL with either small or large noncleaved cell proliferations but without evidence of plasmacytoid differentiation. Samples from a minority of cases showed “minimal polymorphism,” mainly with features of late B cell differentiation characterized by plasniacytoid and plasma cells. In rare instances, this pattern was seen in enlarged lymph nodes, which either preceded or were concurrent with B cell tumors at other sites. The findings in this series contrast with those of Frizzera (1987) in that neither extensive necrosis nor the presence of atypical lymphocytes, defined as large cells with irregular vesicular nuclei and prominent nucleoli, were associated with morphological evidence o f malignant progression. The marked morphological heterogeneity of transplantationassociated lymphoproliferations has been frequently reported in other series. The histological appearances of diffuse high-grade large-cell lymphoma are more frequent among cardiac transplant recipients (Weintraub and Warnke, 1982), whereas diffuse hyperplastic B cell proliferations are more prevalent among bone marrow transplant recipients (Shapiro et al., 1988).The term poJltransplunt ~ ~ ~ m p h o ~ ~ r o l i f e disease r a f i 7 , ehas generally been adopted to encompass the variety of morphological manifestations that accompany these lesions. The term posttrumplunt fymphoprofferative syndrome has also been suggested for B cell hyperplasias of


353

rapid onset that contain substantial numbers of reactive T cells and have been identified in some renal transplant recipients (Canioni et al., 1989). These lesions were associated with anti-T cell monoclonal antibody prophylaxis for graft rejection and completely regressed following reduction in immunosuppression. E. CLONALITY OF POSTGRAFT B CELL‘rUMORS

The proliferating B cell populations in postgraft lymphoid tumors are noted for their striking phenotypic and genotypic heterogeneity with respect to Ig expression (Nalesnik et al., 1988; Hanto et al., 1989). The fact that the clonal characteristics of these lesions are not always consistent with the morphological appearances of the tumor cells has prompted considerable controversy regarding the neoplastic nature of these lesions. On the basis of mixed cellular expression of surface and cytoplasmic Ig, polyclonal B cell proliferations were initially reported in lesions from renal (Frizzera et id., 1981) and thymic transplant (Reece et al., 1981) recipients, whereas light-chain restriction indicating monoclonality was reported in other cases (Hanto et al., 1982). Furthermore, absence of demonstrable Ig isotype expression was frequently found in tumors from allograft recipients (Weintraub and Warnke, 1982; Cleary et al., 1984) Subsequently, the use of highly sensitive DNA hybridization techniques with Southern blot analysis for detection of Ig gene rearrangement has provided an important and highly successful method for distinguishing between clonal and nonclonal populations and for confirming the B cell lineage affiliation of these lesions. Cleary et aZ. (1984) showed that although lesions in 9110 cardiac transplant recipients failed to express Ig as detected by immunocytochemical methods, all tumor cells showed rearranged Ig gene DNA, indicating the presence of a single clone within each lesion. Evidence of B cell monoclonality by DNA hybridization has now been widely reported in tumors from renal (Hanto et al., 1989),cardiac (Cleary et al., 1984) and bone marrow (Shapiro et a1.,1988) transplant recipients. In general, there is good correlation between genotyping analysis and immunocytochemical evidence of niorioclonality (Nalesnik et al., 1988). However, the high sensitivity of DNA analysis, which allows the detection of monoclonal proliferations constituting 1% of the total tumor tissue (Cleary et al., 1984),has been found to be a superior indicator of clonality in many instances in which tumors are either immunocytochemically polyclonal (Nalesnik et al., 1988; Hanto et al., 1989) or lack demonstrable Ig (Cleary et al., 1984). The presence of more than one, and often several,

354


separate and distinct B cell population with unique Ig gene rearrangements has been noted concurrently in the same lesion or at different anatomical sites, or in recurrent tumors developing in the same individual (Cleary and Sklar, 1984; Shearer et al., 1985; Hanto et nl., 1989). These tumors are considered to be multiclonal or oligoclonal, suggesting either the rapid progression of the lesions from a polyclonal to monoclonal proliferation or, less likely, the de 710110 development of multiple autonomous B cell tumors, which may occur simultanteously in the same individual. The possibility has been suggested that these individual clones are related to each other and derived from a single malignant or premalignant progenitor cell that undergoes several stages of transformation, resulting in the production of a number of' B cell clones, each with a distinct rearrangement of Ig gene DNA (Cleary and Sklar, 1984). In support of this hypothesis, Shearer et al., (1985) have identified at the same anatomical site recurrent monoclonal lesions that were genotypically identical apart from the presence of additionally rearranged Ig gene DNA in the recurrent lesion only. I t was suggested that this unique new clone may have developed from the original clonal population in the initial tumor, which had retained the capacity for continued Ig gene rearrangement. The complexity of these tumors is further compounded by evidence of monoclonal progression from a polyclonal proliferation in that both clonal and nonclonal populations can occur simultaneously in the same lesion o r at different anatomical sites. Hanto et al. ( 1989) have identified a subset of renal transplant patients with morphologically malignant tumor that showed no light-chain restriction by Ig phenotyping but contained a small population of B cells with clonally rearranged Ig gene DNA. 'rhe presence of these patterns in a single lesion was interpreted to represent an intermediary stage in the progression of a hyperplastic B cell proliferation to an overtly monoclonal and malignant population. On the other hand, demonstration of several minor B cell clones with nonidentically rearranged Ig gene DNA and monoclonal or polyclonal Ig expression, developing simultaneously in noncontiguous lesions, further endorses the concept of multiclonal primary tumor evolution in some cases. Because multiclonality may be the result of unstable Ig gene rearrangement due to somatic hypermutation, Cleary et al. (1988) have utilized differences in the EBV genome configuration as an alternative and more reliable marker for clonality in these tumors. Using Southern blot analysis with specific DNA probes for fused termini of the EB virus genome, the detection of a single restriction fragment band confirmed the presence of a monoclonal proliferation of EBV-infected B cells in lymphoid tumors from the majority of solid organ allograft recipients.


355

This was further supported by demonstration of clonal Ig gene rearrangements in the same tumor samples. However, multiple concurrent tumors, or recurrent lesions, in the same individual were frequently shown to be multiclonal in origin by the presence of different configurations of both Ig gene DNA and fused EB virus gene termini. Complex oligoclonal patterns were also identified, suggesting multiple transient tumor clones from which a single dominant B cell population might eventually emerge. Karyotyping studies have identified several inconsistent cytogenetic abnormalities in postgraft B cell tumors, including aneuploidy, deletion, and trisomy, often in a single chromosome (Frizzera et al., 1981; Cleary et al., 1984; Hanto et al., 1985). However, none of these lesions has been shown to contain the chromosome 8 translocations or c-myc gene rearrangements that are characteristic of BL. On the basis of available clinicopathological data, correlation between the clonal composition and morphological appearances of' postgraft B cell tumors appears to be consistent with the progression of a polyclonal and reactive B cell proliferation to a monoclonal and histologically malignant population. T h e role of EBV in the pathogenesis of these tumors can be interpreted in the light of these observations and the biological behavior of these tumors. Hyperplastic and polyclonal B cell proliferations that regress after withdrawal or reduction of immunosuppression and treatment with antiviral agents probably represent the early stages of polyclonal B cell activation by EBV. Concurrent polyclonal, oligoclonal, and monoclonal proliferations, which respond to similar therapy, may constitute an intermediate or premalignant stage in the evolution of a monoclonal lymphoma from a benign EBV-induced polyclonal hyperplasia. An established monoclonal population which in many instances is refractory to this form of treatment and may represent the later malignant stages of this pathological spectrum requires other forms of therapy, including conventional cytotoxic agents, irradiation, and surgery for effective eradication. F. RELATIONSHIP BETWEEN EBV AND POSTCRAFT LYMPHOPROLIFERATIVE DISORDERS

The implication of EBV in the pathogenesis of postgraft lymphomas has been based on molecular hybridization evidence of EBV DNA in tumor extracts (Saemundsen et al., 1982; Andiman et al., 1983; Ho et al., 1985) or in tumor cells (Weiss and Movahed, 1989) and on the expression of EBNA and EBV gene products in proliferating B lymphoid populations as detected by tissue section immunocytochemistry (Crawford et al.,

356


1980; Hanto et af.,1982; Starzl rt al., 1984) or on immunoblot studies of extracted tissue proteins (Thomas and Crawford, 1989). EBV-associated B cell tumors occur with an estimated frequency of 14%, among all cancers developing after transplantation (Touraine at al., 1989). AIthough the majority of evaluated postgraft lymphomas show EBV involvement, absence of EBV DNA or EBNA has been reported in some rases investigated for EBV involvement (Bloom EL al., 1985). In sifu hybridization with specific DNA probes provides a highly sensitive method for detecting viral agents at a single-cell level and is readily applicable to conventionally processed histological samples. Posttransplant lymphomas have been estimated to contain between 5 and 20 EBV genome equivalents per cell (Cleary et al., 1984; Weiss and Movahed, 1989) using Southern blot or in situ hybridization techniques. EBNA detection by anticomplement immunocytochemistry (Reedman and Klein, 1973; Guohua el al., 1981) is regarded to be less sensitive and is limited in application to fresh or cryopreserved histological material only, but nevertheless provides rapid and reliable results for diagnosis in many instances. EBNA-containing B cells usually constitute a major proportion of the proliferating cell populations in lesions showing either benign hyperplasia or malignant histological appearances. By contrast, hyperplastic lesions have been shown to contain low amounts of detectable EBV DNA by in situ hybridization (Weiss and Movahed, 1989). The significance of EBV infection in postgraft lymphoproliferative tumors has for the most part remained an enigma. It has been suggested that EBV may have a primary role in the development of these lesions i n that the proliferating cells may be the result of a clonal expansion of latently infected B cells released from the normal immunoregulatory constraints, which prevent uncontrolled cell growth induced by EBV in vivo. 'l'his issue has been partially resolved by two recent immunocytochemical studies demonstrating the presence of full EBV latent gene expression in the proliferating cells of tumors developing in solid organ (Thomas and Crawford, 1989) and bone marrow (Young et al., 1989a) transplant recipients. The recent availability of specific monoclonal antibodies to EBV gene products has provided a unique opportunity to examine these tumors for the cellular expression of EBV-related antigens by immunohistological methods. Phenotyping studies by Thomas and Crawford (1989) and (Thomas at al., 1990) have identified EBNA 1 and EBNA 2 expression in the proliferating B cells of postgraft tumors from three renal and five cardiopulmonary or cardiac allograft recipients (Figs. 1 and 2). Additional expression of EBNA 3 and/or LMP was demonstrated in some samples, but lytic cycle antigens, EA, VCA, and MA, were not detected. Immunoblot analysis of extracted tissue proteins


357

FIG. 1 . Anticomplement immunoperoxidase staining for EBNA on an EBV-positive BL (Raji) cell line (cytospin preparation; X630).

confirmed the presence of EBNA types 1-6 (excluding EBNA 5 ) in a tumor from one renal transplant recipient. It is striking that similar patterns of latent gene expression were identified in all lesions, despite the morphological appearance of high-grade B cell lymphoma in each case and heterogeneous expression of Ig that was monoclonal (four cases) polyclonal (two cases), or inconclusive (two cases) (Figs. 3 and 4). The presence of EBNA 1 and 2 and LMP was also demonstrated by Young et al., (1989a) in B cell tumors from five immunocompromised recipients of allogerieic or autologous bone marrow grafts. All tumors in this study were phenotypically and/or genotypically typed as monoclonal. Additional phenotyping studies in these lesions for expression of cell lineage and LFA antigens showed no consistent pattern of expression with respect to markers of B cell differentiation (Thomas et al., 1990; Young et al., 1989a). However, in both series of patients, tumor cells in

358


FIG.2. EBNA-positive tumor cells in a heart and lung posttransplant B cell lymphoma (cryostat section; x630).

the majority of cases expressed the B cell activation antigen CD23 and CAMS, LFA-1, LFA-3, and ICAM-1.

G. PATHOGENETIC MECHANISMS IN TRANSPLANTATIONASSOCIATED EBV-POSITIVE B CELLTUMORS The cellular expression of latent EBV infection identified in postgraft lymphoproliferative lesions is important because it shows close phenotypic similarity to the EBNA l , EBNA 2, and LMP profile, which is characteristic of EBV-immortalized LCLs derived from normal B cells in vitro. This is in marked contrast to the highly restricted pattern of EBV gene products, confined to EBNA 1, seen in BL tumor cells and BL cell lines early in culture. These distinct phenotypic differences provide new


359

FIG.3. Histological appearance of a high-grade centroblastic B cell lymphoma from a heart and lung transplant recipient (hematoxylinand eosidparaffin section; X400).

insight on the mechanisms involved in the pathogenesis of the two tumor types. Both EBNA 2 and LMP act as targets for EBV-specific T cell cytotoxicity and are thought to be pivotal for effective removal of latently infected cells in vivo. BL usually develops in seropositive individuals in whom the immunoregulatory controls for EBV are relatively normal. In this instance, T cell surveillance would be ineffective in controlling the growth of' EBV-infected cells with restricted viral gene expression through failure to express relevant determinants required for T cell recognition. In transplant recipients, on the other hand, the sustained proliferation of B cells with full cellular expression of EBV latent gene products is likely to arise through the profound inhibition of EBV-specific T cell function by therapeutic agents, required for immunosuppression in these patients. It

360


FIG. 4. Monoclonal Ig K-positive B cell proliferation in the centroblastic B cell tumor from the heart and lung transplant recipient shown in Fig. 3. The poorly differentiatcd tumor cell population exhibits moderate staining for cytoplasmic Ig, in cwitrast with the central plasmacytic form with intense cytoplasmic I!: staining (paraffin section; ~ 4 0 0 ) .

is also salient that the cells in these lesions express the CD23 B cell activation antigen, which is up-regulated by EBNA 2 and, in soluble fbrm, acts as an autocrine B cell growth factor, allowing T-independent B cell growth. T h e pattern of viral gene expression in these tumors is remarkably similar to that seen in B cell tumors induced in cotton-top tamarins following high parenteral doses of EBV (Young e l nl., 1989b; Allday PI ul., 1990). ?'he immunological responses in seronegative animals appear to be unable to control the proliferation of EBV-infected cells. Both transplantation-associated human tumots and experimentally induced tumors in animals are phenotypically similar to LCLs derived from infected normal B cells in ziitro in which EBNA 2 arid LMP are thought to be essential for cell immortalization. These differences


36 1

strongly suggest that EBV may be the sole agent involved in the development of these tumors, whereas it is thought to have a contributory role in the etiology of BL, which appears to involve a number of pathogenetic factors. Differences in the biological behavior of postgraft tumors and BL may also be interpreted in the light of the contrasting patterns of CAM expression in these lesions. Several cell surface molecules mediating cell adhesion are now recognized to be important in many lymphocytemediated immune responses. Non-antigen-specific binding of leukocyte CAMs and corresponding ligands on lymphocytes and other cell types strengthens various intercellular contacts involved in I’cell recognition of specific antigen. B LCLs with latent EBV gene expression constitutively express CAMs, LFA-1, LFA-3, and ICAM-1 and are susceptible to EBV-specific T cell cytotoxicity in vitro (Gregory and Rickinson, 1988). EBNA 2 and LMP regulate the expression of LFA-3 and ICAM-I in these cells. Similar CAM and latent gene expression in postgraft B cell tumors strengthens their phenotypic similarity to LCLs derived from EBV inimortalization of normal B cells in vztro. The restoration of normal host controls following withdrawal of immunosuppression results in rapid tumor regression in many of these patients. However, the restricted expression of EBV latent gene products in fresh BL tumor cells and recent BL cell lines that also poorly express LFA-3 and ICAM-1 may contribute to the resistance of these cells to cytotoxic T cell activity and provide them with a selective advantage whereby they escape immunosurveillance (Gregory et al., 1988).

H. EBV SEROLOGICAL STATUSI N TRANSPLANT RECIPIENTS WITH B LYMPHOPROLIFERATIVE DISEASE Transplant recipients who develop B cell tumors often have serological evidence of primary or reactivated infection (Breinig et al., 1987; Ho et al., 1985, 1988). Primary infection in adults and children is considered to be an important risk factor in the development of EBV-related B cell diseases postgraft and is evident in 70% of cases with these lesions (Ho et al., 1988). Postgraft seroconversion in previously seronegative patients may be clinically associated with a self-limiting IM-like syndrome accompanied by focal or rapidly progressive and fatal lymphoproliferative lesions, or may be associated with isolated extranodal lymphoma (Ho et al., 1988). The frequency of primary infection is, not unexpectedly, higher in children with evidence of seroconversion occurring immediately prior to or, more often, within 12 months of grafting. In a study of 11 pediatric transplant recipients, Ho el al. (1988) emphasize the

362


difficulty in diagnosing primary infection in these cases, because the serological profiles are atypical due to the effects of immunosuppression and can be further obscured by passively acquired antibodies through blood transfusion (Breinig et al., 1987).Absent or low responses to E B N A seen in the majority (711 1) of patients in this study were held to be a more valuable indicator of primary EBV infection than were absent or low antibody responses to IgM VCA and EA, which were not found to be wholly specific or sensitive as parameters of primary infection, as in normal imniunocompetent individuals. Similarly, the presence of heterophile antibodies indicated by positive monospot testing is notably absent in transplant recipients with primary intection (Ho el al., 1988). T h e majority of adults are seropositive for EBV before transplantation. Evidence of reactivation, which is usually asymptomatic, occurs in 22.8-48% of cases after grafting (Snyder et al., 1988; Ho et al., 1988; Wreghitt et ul., 1989). Significant fourfold or greater increases in antibody levels to VCA and EA occur in the first 2 years, and more usually within 2-6 months of grafting (Cheeseman et al., 19130; Dummer et nl., 1983; Wreghitt ~t al., 1989), whereas changes in EBNA antibody titers may be low or undetectable in some patients. The high frequency o f antibodies to EBV lytic cycle antigens is consistent with the increased rate of oropharyrigeal viral excretion reported in 5 0 4 7 % of these patients (Chang et al., 1978; Cheeseman et al., 1980). These findings are more prevalent among patients receiving Cs immunosuppression (Hanto et al., 1982; Nagington and Gray, 1980) and probably results from the suppressive effects of Cs on T cell function and, in particular, the inhibition 01 EBV-specific T cell cytotoxicity (Crawford rt ul., 1981). However, regardless of therapeutic agent, the intensity of inimunosuppressiori may have a greater influence on the debelopment of EBV infection in these patients, rather than the use of Cs per se (Cheeseman et al., 1980; Wreghitt et al., 1989). Evidence of reactivated EBV infection has been reported in association with the development ot EBV-related B cell tumors in small patient series (Starzl ~t al., 1984; Thomas Pf d.,1990). Serological diagnosis of EBV reactivation was indicated by significant rises in antibody levels to IgC VCA and EA in patients who were seropositive prior to transplantation. Although there appears to be no specific serological parameter closely related to lymphoma development, elevated levels of restricted type anti-EA antibodies and high mean Cs levels have predicted a higher risk of lymphoma occurring in some cases (Brumbaugh et al., 1985). In the minority of patients, lymphoproliferative tumors may develop without serological evidence of active EBV infection (Starzl et al., 1984).


363

I. TREATMENT OF TRANSPLANTATION-ASSOCIATED B CELLTUMORS Transplantation-associated B cell tumors carry a high mortality rate of over 80%.Through recognition of the risks inherent in overimmunosuppression, the use of carefully monitored low-dose drug regimes with fewer agents has in many instances reduced the frequency of these tumors. Several different approaches have been applied to the treatment of posttransplantation lymphoma and the varying success of these appears to be largely dependent on tumor type. In many cases, reduction or cessation of immunosuppression alone is sufficient to produce complete tumor regression without recurrence (Starzl et al., 1984). This response is more common in patients with polyclonal lesions of polymorphic histological appearance, but may also include tumors containing clonal or mixtures of clonal and nonclonal B cell proliferations (Nalesnik et al., 1988).On the basis of the putative etiological role of EBV in these lesions, Hanto et al. (1982, 1985)proposed the use the antiviral agent acyclovir as an alternative form of therapy. This agent is a potent inhibitor of viral DNA polymerase. Regardless of changes to immunosuppression, acyclovir induced complete disease resolution in patients with widespread polyclonal lesions that were morphologically benign and were accompanied by an IM-like illness. However, only a transient response to acyclovir was seen in patients with lesions containing mixed polyclonal and monoclonal B cell populations, suggesting incipient malignant progression. In these cases, additional withdrawal or reduction in immunosuppression was recommended. On the other hand, tumors of a prognostically poor category, exhibiting typical histological and phenotypic criteria of malignancy, were refractory to acyclovir and required more conventional forms of therapy with cytotoxic agents and irradiation. T h e variable response to acyclovir has been confirmed in other patient series (Sullivan et al., 1984; Pirsch et al., 19S9), and lymphoproliferative lesions developing during prophylactic acyclovir treatment have been reported in bone marrow transplant recipients (Shapiro et al., 1988; Zutter et al., 1988). The precise mechanism by which acyclovir causes EBV-positive tumor regression is not known, because it principally acts at the lytic cycle level by inhibiting linear DNA synthesis and preventing production of viral particles. Although there is clear evidence of EBV latent gene expression and episomal EBV DNA in the proliferating B cells of these lesions, the majority of patients with postgraft tumors also show increased viral shedding from the oropharynx. Acyclovir may therefore exert an indirect effect to reduce tumor burden by inhibiting augmented

364

J. ALERO THOMAS Er AL.

permissive infection in viva Another agent, ganciclovir, which is more potent as an inhibitor of EBV, has recently been used successfully to treat acyclovir-resistant polyclonal lesions in two patients individually receiving a renal and pancreatic allograft (Pirsch et al., 1989). Beneficial results have also been reported with anti-B cell monoclonal antibodies in bone marrow and solid organ transplant recipients (Blanche et al., 1988; Touraine Pt al., 1989). VIII. NHL Associated with Acquired Immunodeficiency Syndrome

Individuals who are seropositive for human immunodeficiency virus ( H IV) have an increased risk of developing lymphoproliferative disorders, and up to 5%develop histologically classifiable NHL (Ziegler et al., 1982, 1984; Ernberg, 1989). In view of' the high frequency of these tumors in these patients, NHL is now included as one of the diagnostic criteria for acquired immunodeficiency syndrome (AIDS) (Centers for Disease Control, 1985, 1987). Although the majority of these tuniors express surface Ig and/or B cell-associated antigens, they constitute an extremely heterogeneous group of lymphoid neoplasias. A. CLINICAL FEATURES An informative study of 14 immunocompromised homosexual males with aggressive NHL has identified two main categories of lymphoma associated with AIDS and the AIDS related complex (ARC) (Kalter et ul., 1985). The first group of tumors, which showed several of the clinicopathological features associated with lymphoproliferative lesions developing in organ transplant recipients, was identified in six patients and was defined as NHL of a diffuse large-cell or histiocytic type. These tumors were more prevalent in patients with advanced AIDS, characterized by profound T cell dysfunction and multiple opportunistic infection, and were poorly responsive to chemotherapy. Although the tumors in these patients were not investigated for EBV, a more recent study has identified episomal EBV DNA and EBNA in three AIDS-associated cases of NHL, which expressed monoclonal Ig (Knowles et aE., 1989). These lesions also lacked the chromosomal translocations involving the c-my and lg loci that are characteristic of BL. Furthermore, the presence of tumor cells in two of these cases expressing B cell activation markers Ki24 and CD23, which are known to be induced by EBV infection of resting B cells and are specifically associated with the expression of EBNA 2 (Calender et al., 1987; Wang et al., 1987),provides additional evidence of


365

the similarity of these tumors to transplant-associated lymphomas and EBV-induced LCL in vitro. It is likely that the proliferation of these cells is driven by EBV gene products in a manner analogous to LCL. T h e second category of tumors, reported by Kalter et al. (1985), were described as diffuse undifferentiated lymphomas that usually presented as abdominal masses and were considered to be histologically similar to BL. These BL-like tumors were more often associated with generalized chronic lymphadenopathy and less severe immune dysfunction, consistent with early stages of AIDSIARC, and were prognostically more favorable because the tumors responded well to chemotherapy. An EBV relationship was not established in these tumors. A number of subsequent studies have revealed considerable heterogeneity with respect to EBV involvement in BIAke lesions from AIDS/ARC patients. Based on current available data, it appears that lymphomas analogous to EBVnegative sBL, EBV-positive eBL, and unusual sBL types containing EBV all occur at a relatively high frequency in HIV-positive individuals. In a recent study of 16 AIDS-associated NHL cases (Subar el al., 1988), chromosomal translocations analogous to sBL involving c-my locus rearrangements and a breakpoint in the IgH sp switch region were identified in a high proportion (12/16) of tumors. Although the majority of these sBL-like lesions were EBV negative, surprisingly, the presence of EBV DNA was detected in 4 cases. Another recent study has reported an EBV-positive AIDS-BL with the eBL pattern of c-myclIg rearrangement (Haluska et al., 1989).

B. EBV ROLEIN PATHOGENESIS OF AIDSRELATED NHL T h e pathogenesis of the lymphomas with the classical sBL phenotype is unlikely to involve EBV and presumably requires some other genetic lesion to confer a growth advantage to a selected cell clone containing a deregulated c-my gene. On the other hand, in the EBV-positive BL-like tumors, EBV may play a pathogenic role analogous to that in eBL. In support of this, the exclusive expression of EBNA 1 recently reported in a case of AIDS-related NHL (Thomas and Crawford, 1989; Allday et al., 1990) is consistent with the restricted pattern of EBV gene expression seen in immunoblots of tumor extracts from eBL (Rowe et al., 1986). However, the possibility that EBV is a mere passenger in an sBL driven to malignancy by deregulation of c-myc and another genetic lesion(s) cannot be excluded. None of the tumors associated with AIDS/ARC have been found to contain both EBV and HIV, o r any other virus.

366


T h e current picture of B-cell-derived NHL associated with AIDS/ ARC is both complex and incomplete. Table I 1 summarizes the four types of tumors that have been identified: type 1 resembles the EBVrelated N HL in iatrogenically immunosuppressed organ transplant recipients. Assuming that latent EBV gene expression, including EBNA 2 and LMP, is required to drive this type of B cell proliferation, in a manner similar to the in vitro growth of LCLs, it is highly probable that cytotoxic T cell dysfunction is required for the development of these tumors. This would explain why these lesions occur in severely immunosuppressed patients with advanced AIDS and multiple opportunistic infections. Lymphoma types 2-4 (Table 11) are BL-like in that they exhibit c-myrlIg translocations. T h e association of these tumor types with ARC and early A l D S suggests that the reactive lymphadenopathy caused by polyclonal activation of B cells, which is a characteristic of HIV infection (CliffordLane et al., 1983), may be a predisposing factor in the development of such chromosomal translocations. The concomitant deregulation of' c-my could then contribute to the development of the tumor whether or not EBV is involved. I t is currently unclear what role the virus is playing in the EBV-positive BL-like NHL or whether it plays the same role in all cases.

KHL

Type

c-mycllg translocation

IN

EBV

B cell activation antigens

1

None

+

+

2

sBL-like

-

NDb

sBL-like

3

1

eBL-like

TABLE I 1 HIV-POSITIVE INDIVIDUALS

+ +

ND

ND

Histopathology

Rcfcrcnces

Large-cell lymphoma''

Knowlrs rt nl. ( 1989)

Small noncleaved cell NHL; large noncleaved cell NIiL (undifterentiated lymphoma)'

Subar r l n l .

Small noncleaved cell NHL; large noncleaved cell NHL; irnniunoblastic plasmacytoid lymphonia (undifferentiated lymphoma)'

Subar d ul.

BL-like NHL (undifferentiated lymphoma)'

Haluska ef d.

* Type 1 possibly includes the large-cell lymphomas described by Kalter uf al. 1935).

(1988)

( 1988); Neri ('1 ctl.

(1Y88)

(IY8Y)

' ND, Not detected. 'The undifferentiated lymphomas described by Kalter ~t al. (1985) probably fall into one of these g~-otips.


367

IX. EBV and Hodgkin’s Disease

The previously- reported relationship between EBV and HD is currently receiving renewed attention through advances in the histological application of molecular hybridization and the wider availability of probes specific to the EBV genome. HD is an heterogeneous clinical entity of unknown etiology in which progressive lymph node enlargement is frequently but not invariably accompanied by constitutional symptoms. Histological subtypes of HD are defined by the presence of multinucleate Reed-Sternberg cells and their mononuclear variants (Fig. 5 ) , which are pathognomonic for HD, and variable numbers of lymphocytes, plasma cells, and histiocytes. Although generally considered to be a malignant lymphoma requiring treatment with cytotoxic agents and/or radiotherapy, the clinical behavior of HD varies with histological type.

FIG.5. Reed-Sternberg cell in Hodgkin’s disease (hematoxylin and eosiniparaffin section; x 1000).

368


T h e lymphocyte-predominant and nodular-sclerosing forms, which usually develop in younger age groups, carry a good prognosis and show excellent response to treatment, whereas a gradually progressive or rapidly fatal outcome is more often associated with the mixed-cellularity and lymphocyte-depleted subtypes. respectively. The clinical and histological heterogeneity of HD is thought to resemble a chronic infectious process in some subtypes. The direct or indirect involvement of a common viral agent was initially postulated from socioepidemiological studies showing the peak disease incidence among young adults of high socioeconomic groups, or children from less privileged backgrounds (Evans and Gutensohn, 1984). In young adults, delayed infection with many common childhood viruses is often clinically more severe, suggesting one predisposing factor in these individuals. Over the last 20 years, several lines of evidence have established a more specific relationship between EBV and HD. A. EBV SEROLOGY IN HD PATIENTS Patients with IM have a fourfold increased risk of developing HD, usually within a 1- to 4-year period (Munoz ut al., 1978; Rosdahl et al., 1974), although longer intervals of up to 10 years have been reported. Several serological studies have shown significantly high levels of antibodies to EBV-specific antigens in HD cases either before (Evans and Gutesohn, 1984) or at the time of diagnosis (Johansson et a/., 1970) o r during therapy. Elevated antibodies to EBV VCA and EA occur in 2% of HD patients and are more prevalent among older age groups (Masucci et al., 1984). However, 5% of cases show no evidence of previous EBV infection (Mueller et al., 1989). In a recent large retrospective casecontrol analysis of sera from over 240,000 individuals, Mueller et d. (1989) identified altered patterns of EBV antibody responses in 43 HD patients, which in some cases preceded clinical diagnosis by 3-6 years. From this study, significant risk factors were associated with elevated antibody titers to EBNA, IgA, VCA, and IgG VCA, and to a lesser extent with elevated antibodies to diffuse-type EA. No risk association was found for the presence of raised antibodies to the restricted form of EA, and IgM VCA antibodies were significantly decreased. In younger age groups, an increased risk was not evident in patients with elevated IgG or IgA antibodies to VCA. The altered pattern of EBV antibody responses in HD on the one hand suggests a persistent active infection with increased viral replication. This is reflected in the augmented titers of IgG antibodies to VCA and EA that may antecede diagnosis by some years. The additional presence of IgA antibodies to VCA in some cases is


369

reminiscent of the serological profile in EBV-associated nasopharyngeal carcinoma and has been suggested to indicate active viral replication in the epithelium. This is further supported by evidence of increased oropharyngeal shedding of'virus in some cases (Lange et al., 1978). However, impaired responses to EBNA, similar to the pattern seen in XLPS and other immunodeficiency states associated with EBV-related B cell tumors, have been reported (Lange et al., 1978). On the basis of the serological findings alone, it is not clear whether EBV has a direct pathogenetic role in HD. Because elevated antibodies to EBV-associated antigens are not a ubiquitous finding, it is likely that the association exists in only a subset of cases. This is substantiated by the prevalence of high antibody titers among patients with the nodular-sclerosing and mixed-cellularity histological subtypes of HD (Mueller et al., 1989),in which EBV DNA was more frequently demonstrated (see Section IX,B). Elevated levels of anti-EBNA and anti-VCA antibodies have also been reported in the lymphocyte-predominant (Mochanko et al., 1979) and lymphocytedepleted forms, respectively (Johansson et al., 1970). B. EBV ASSOCIATIONS I N HD Data from recent DNA hybridization studies have strengthened the implication of EBV in the pathogenesis of HD. A summary of the findings in these studies is shown in Table 111. EBV-specific DNA sequences, corresponding to internal repeated regions of the genome, have been demonstrated in affected tissues of 1 3 4 3 %of cases using slot blot in situ and/or Southern hybridization. The rate of EBV genome-positive HD is significantly higher among HIV-seropositive immunocompromised males (83%)compared with HIV-seronegative (19%)individuals (Uccini et al., 1989). However, the frequency among non-HIV-infected cases of HD may be higher: Herbst et al. (1990) have recently detected EBV DNA, in 58% of patients, using the highly sensitive polymerase chain reaction for DNA amplification. T h e presence of EBV DNA has been identified more frequently among nodular-sclerosing and mixed-cellularity lesions, compared with other histological subtypes. A recent study of 34 HD cases has shown no correlation between the presence of tumor EBV DNA and patient age, suggesting no aetiological differences relative to the bimodal incidence of the disease (Libetta et al., 1990). In positive samples, the quantity of viral DNA by Southern blotting has been estimated to be in the range of 0.03-2 genome copies per cell (Staal et al., 1989). DNA hybridization studies with probes to terminal repeated sequences of the EBV genome have also indicated the presence of monoclonal populations of EBV-infected cells in lesions from all (Anagnostopoulos et al.,

3 70

J. ALERO THOMAS E T .4L.

TABLE 111 DEMOKSTRATION OF EBV DNA I N HODGKIN'S DISEASE EBV DNA

+ H D cases Method of EBV genome

Histology" Total HL) cascs tested

No. (7%) ___

NS

R3C

LP

LD

detectionh

References

~ _ _ _ _ ~ ~ ~ _ _ _

lU(83) 6(19)

2

8

-

-

-

-

-

-

ISH; S H

Uccini el ai.( 1989)

7(17)

7

-

-

-

SH; ISH

Anagnostopoulos rt al. ( 1989)

39

5(13)

3

Herbst el ai. (198%)

28

7(25)

4

3

16

S(19)

4(19)

3 2

-

21

2

I 3 -

SH

15(44)

4 9

-

34

132d

80 (60)

78

40

81

12(HIV+)' 32(HIV)' 42

ISH; SH

Uccini el (11. ( 1989)

SH

Libetra ct

-

SH

Staal et al. ( 1989)

S H ; SBH; ISH

Weiss rt a/. (1989)

-

SH; SBH PC R

Wciss rt al. (1985)

d.(1990)

Hcrbst ef al. (1990)

NS. Nodular sclerosing; MC. mixed rellularity: LP, lymphocyte predominant: LD, Ivmphocyte depleted. ISH. In sihi hyhridizatiun; S11, Southern hybridization; SRH. slot blot hybridization: PCR polymerase chain rraction. ' Patients seroposiirive or seronegative to,-huniaii immunodeficiency virus (HIV). Five atypical cases in this series were not classified by conventional hihtological criteria for HD. '

1989; Weiss et al., 1989) or a proportion of cases (Staal et al., 1989; Weiss ul af., 1987) examined, and oligoclonal populations in one patient (Weiss et al., 1989).Cell localization studies by in situ hybridization on frozen or conventionally processed tissue sections reveal that EBV-specific sequences specifically targeted to Keed-Sternberg (R-S) cells and their mononuclear variants with a minimal or absent signal over accompanying reactive cells (Anagnastopoulos el al., 1989; Herbst et al., 1989, 1990; Uccini et af.,1989; Weiss et al., 1989). R-S cells have been estimated to contain up to 100 EBV genome copies per cell (Weiss et af.,1989). Fewer patients have detectable EBV DNA in their tumors as assessed by in sztu hybridization compared with other detection methods using tissueextracted DNA (Weiss el al., 1989). This may be related to the relative insensitivity of in situ hybridization despite signal intensification with isotopically labeled probes. Furthermore, EBV-infected cells, other than R-S cells, may be present and these cells may contain copy numbers that are below the level of detection by this method. EBNA-containing R-S cells have been documented in a single case of HD in a patient with chronic EBV infection (Poppema et af., 1985).


37 1

On present available data, two major questions arise regarding the nature of the role of EBV in HD. First, EBV may also be present in B lymphoid components of these lesions. The altered patterns of both humoral and T cell-mediated specific EBV responses in many of these patients is consistent with persistently enhanced viral activity in infected B cell populations. However, the absence of EBV DNA hybridization to lymphocytes in either affected or uninvolved tissue suggests that the role of EBV is inherent to the HD process rather than a generalized phenomenon. Furthermore, the presence of monoclonal EBV-infected cells indicating a single cell origin would counter a coincidental EBV infection in B cells. In this instance, a polyclonal pattern of EBV DNA might be expected (Weiss and Movahed, 1989; Weiss et al., 1989). Second, because R-S cells are thought to be the neoplastic component in HD, the unequivocal presence of EBV DNA in these cells raises the possibility of a direct causal relationship between EBV and HD, at least in a proportion of cases. The histogenesis of R-S cells is not known and remains controversial, although a lymphocyte derivation is currently favored (Cabanillas et al., 1988; Herbst et al., 1989a;Jaffe, 1989).The absence of a demonstrable CD2 1 EBV-binding receptor on R-S cells suggests that viral access may be by a different mechanism from that which occurs in B cells and epithelium. On the other hand, given the multinucleated configuration of the typical R-S cell, a hybrid origin from two distinct cell lineages (Bayliss and Wolf, 1980) has been a suggested possibility (Andreesen et al., 1989).

X. EBV-Associated Tumors in Nonhuman Primates

Two species of nonhuman primates develop lesions after experimental infection with EBV-the owl monkey (Aotus sp., Epstein et al., 1973) and the cotton-top tamarin (Sunpinus oedipus, Shope et al., 1973). The latter has been chosen for experimental studies with EBV in vivo (Epstein and Morgan, 1986). Intraperitoneal and intramuscular EBV inoculations produce tumors in cotton-top tamarins within 2-3 weeks. Histological analysis has shown that these lesions are large-cell malignant lymphomas; they contain EBV DNA (2-25 copies per cell), express all EBNA species and LMP, and are mono- or oligoclonal (Allday et al., 1990; Young, et al., 1989b; Cleary et al., 1985). In all these respects the tamarin lymphomas resemble the tumors that develop in the iatrogenically immunosuppressed transplant recipients and the large-cell lymphomas in severely immunocompromised AIDS patients.

372

J. ALERO THOMAS E T 4 L .

XI. Summary and Future Considerations for EBVAssociated Lymphoproliferative Diseases Since its discovery in 1964 (Epstein el d.), extensive studies of the clinical and biological behavior of EBV have led to a wealth of knowledge regarding the viral and immunological events involved in natural EBV infection. Identification of these processes has led to a greater understanding of the adverse consequences that can arise through inadequate host control of a virus following infection. Through major advances in the field of molecular biology, even further progress has been niade in recent years in elucidating the structure and function of the EBV genome, thereby providing important insights into the oncogenic role of EBV in human neoplasia. I t is now indisputable that EBV is closely associated with two human malignancies, eBL and the epithelial tumor, undifferentiated nasopharyngeal carcinoma (not covered in this review). There is also increasing awareness that EBV may be causally linked to a wide range of lymphoproliferative disorders in which impairment of EBV-specific host immunity appears to be an important underlying factor. Controversy has arisen as to whether these lesions represent a true malignant process in which EBV DNA has a direct developmental role, or whether they are manifestations of an exaggerated hyperplastic response induced by EBV. In many instances, EBV-related lymphoproliferative disorders in immunocompromised individuals fulfill established criteria for malignancy based on histology, evidence of clonality, and rapidly aggressive clinical course. However, a proportion of these tumors developing in the context of iatrogenic immunosuppression for organ and bone marrow transplantation undergo complete resolution following withdrawal of immunosuppressive therapy, either alone or in combination with antiviral agents. This response is thought likely to occur only if EBV is the sole agent involved in producing cell proliferation. A cogent explanation for tumor regression in these cases has been provided by evidence that proliferating cells have full phenotypic expression of EBV latent gene products, thereby retaining the potential to respond to normal EBV immunoregulation following restoration of these responses in the immunocornproniised host. However, uncontrolled cell proliferation induced by EBV would increase the likelihood of an aberrant genetic event by which the tumor cells might escape imtnunosurveillance. The nature of the genetic change(s) responsible for irreversible cell growth, frequently apparent in these lesions, has yet to be determined. Based on present available data, these additional events are different from those associated with eBL in which well-defined chromosomal translocations involving the c-my onco-


373

gene have been identified. The general consensus is that the clinical, morphological, cytogenetic, and clonal heterogeneity of these tumors reflects a spectrum of changes through which a polymorphic, polyclonal hyperplastic process induced by EBV evolves to an oligoclonal and ultimately multiclonal or monoclonal malignant tumor population. The current availability of sophisticated methods for genetic investigation opens up unique opportunities to explore the pathogenesis of EBVrelated diseases at a molecular level. New information in this area could have important potential for clinical application, to identify individuals with untoward susceptibility to EBV infection, for early diagnosis of EBV-related tumors, and for designing new therapeutic approaches to these diseases. In the absence of definitive treatment for EBV-related diseases, EBV continues to be a significant public health problem, because eBL and undifferentiated nasopharyngeal carcinoma occur with high frequency among certain indigenous populations world wide. This, together with the considerable morbidity associated with complicated EBV infection in immunodeficient individuals, draws attention to the need for effective therapeutic measures to prevent or reduce the incidence of these diseases. In parallel with studies on the pathophysiology of EBV, work is progressing on the development of a vaccine to prevent primary EBV infection in humans. The major glycoprotein envelope of EBV (gp340-350/ 320), also known as the EBV MA complex, has been identified as a suitable immunogen for vaccine production. EBV MA is the primary target of the high-titer neutralizing antibodies that help to confer protection from severe disease in infected individuals. Experimental studies with a subunit vaccine based on gp340 have been successful in inducing protective immunity to EBV in cotton-top tamarins as well as in preventing the development of EBV-induced lymphomas in these animals (Epstein and Morgan, 1986; Morgan et al., 1988).These encouraging results in primate animal models afford an optimistic outlook for future clinical trials to assess the protective effects of subunit vaccines in humans and for their practical use in vaccination programs on a wide scale.

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