Advances in Cancer Research Volume 55

ADVANCESINCANCERRESEARCH VOLUME 55

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ADVANCES IN CANCERRESEARCH Edited by

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

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

Volume 55

ACADEMIC PRESS, INC. Hamourt Brace Jovanovlch, Publishers

San Diego New York Boston London Sydney Tokyo Toronto

T COPYRIGHT 0 1990 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy. recording, or any information storage and retrieval system, without permission in writing from the publisher.

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ISBN 0-12-006655-6 (ak. paper)

PRINTED IN THE UNITED S T A W OF AMERICA 90919293

9 8 1 6 S 4 3 2 1

CONTENTS

CONTFUBUTORS TO VOLUME 55 ....................................................................

ix

jun: Oncogene and Transcription Factor

.

PETERK VOCTAND TIMOTHY J . Bos I. I1. I11. IV. V. VI . VII . VIII . IX.

X. XI.

jun Is the Oncogene of Avian Sarcoma Virus 17 (ASV 17)....................... A Conserved DNA-Binding Motif Relates the Jun Protein to GCN4. A Transcriptional Activator in Yeast .............................................................. Jun and the Human Transcription Factor AP-1: Identity of Key Properties ..................................................................................................... jun Belongs to a Family of Related Genes ................................................. The Cellular jun Gene Lacks Introns; Its Regulatory Regions Contain Interesting Signals ....................................................................................... The Jun and Fos Proteins: Dimerization and Cooperativity .................... The Leucine Zipper Mediates Dimerization ofJun and Fos ................... Regulation of jun: Response to Incoming Signals..................................... The Oncogenicity ofjun: Increased Dosage or Qualitative Change of the Jun Protein? ........................................................................................... The Main Functions of Jun Form a Hierarchical Order: A Hypothesis .. Jun Is a Signal Converter ............................................................................ References ....................................................................................................

2

4 6

8 11 14 17 21 25 28 30 31

Proto-Oncogene c-fos as a Transcription Factor

ROBERTJ . DISTEL AND BRUCEM . SPIECELMAN I . Introduction .................................................................................................. I1. Thefos Gene and Its Expression ............................................................... 111. Role offos in Cell Growth and Differentiation ......................................... V

37

38 40

vi

CONTENTS

IV. V. VI . VII . VIII . IX . X. XI .

fos Is a Participant in Sequence-Specific DNA Binding ............. The Connection between fos and jun ........................................................ The Interaction of c-fos and c-jun .............................................................. fos Can Transactivate Gene Promoters via the TRE ................... fos Stimulates the Binding ofjun to DNA ................................... fos May Interact with Other Sequences or Protein Complexes ............... The TRE Is Subject to Regulation by c-fos and Other Factors .. Conclusions .................................................................................................. References ......................................................................................

42 43 44 45 46 49 49 50 51

Studies on the Polyoma Virus Tumor-Specific Transplantation Antigen (TSTA)

TINADALIANIS I. I1. 111.

IV. V. VI . VII . VIII .

Introduction .................................................................................................. Definition of the Polyoma Tumor-Specific Transplantation Antigen (TSTA) .......................................................................................................... Studies on the Immune Response against Pol yoma Virus-Induced Tumor Development and Polyoma Virus-Induced Tumors ..................... The Molecular Biology of Polyoma Virus .................................................. Initial Studies on the Nature of the Polyoma Tumor-Specific Transplantation Antigen .............................................................................. Recent Studies on the Nature of the Polyoma Tumor-Specific Transplantation Antigen .............................................................................. Present View of the Polyoma Tumor-Specific Transplantation Antigen ......................................................................................................... Future Prospects .......................................................................................... References ....................................................................................................

57

58 59 62 64 65 81 82 83

Growth Dominance of the Metastatic Cancer Cell: Cellular and Molecular Aspects

R . S . KERBEL I . Introduction .................................................................................................. I1. The Selective Nature of the Metastatic Phenotype ................................... 111. Clonal Dominance (Growth Preference) of Metastatically Competent Variant Cells in Primary Tumors: Genetic Analysis ................................. IV . Human Malignant Melanoma as a Paradigm for the Growth Dominance of Metastatic Cancer Cells ......................................................

87 90 97 105

CONTENTS

vii

V . The Role of Growth Factors in the Growth Preference and

Dominance of Metastatically Competent Cells .........................................

115

Dominant Metastatic Phenotype ................................................................

123 125 127

VI . Ectopic Gene Expression and the Pleiotropic Nature of the GrowthVII . Conclusions ..................................................................................................

References ....................................................................................................

The Pathogenesis of Burkitt’s Lymphoma

IANMACRATH I . Introduction .................................................................................................. I1. Definition of Burkitt’s Lymphoma.............................................................. 111. Clinical and Epidemiological Features ..................................................... IV. Phenotype of Burkitt’s Lymphoma ............................................................. V . The Nonrandom Chromosomal Translocations Associated with Burkitt’s Lymphoma .................................................................................... VI. Structure and Function of c-myc ................................................................ VII. Timing of the Translocation in Relation to B Cell Differentiation .......... VIII . Mechanism of Translocation ....................................................................... IX. Structural Changes in c-myc Brought About by the Translocations and Their Possible Functional Consequences ................................................. X . Breakpoints on Chromosomes 14.2. and 22 and Their Functional Significance with Regard to c-myc Expression .......................................... XI* Correlation of Breakpoint Location with Geography ................................ XI1. Effect of the Chromosomal Translocation on c-myc Expression .............. XI11. Consequences of Deregulation of c-myc ................................................... XIV. The Role of Other Genetic Abnormalities ................................................. xv . EBV and Burkitt’s Lymphoma .................................................................... XVI. Synthesis....................................................................................................... XVII. CODA-Clinical Significance ...................................................................... References ....................................................................................................

134 137 141 150 154 157 165 171 176 186 192 195 208 216 293 242 250 251

Mechanisms of Signal Transduction to the Cell Nucleus

ERICHA . NICC I . Introduction .................................................................................................. I1. Early Consequences of Plasma Membrane Receptor Stimulation ........... 111. The Nuclear EnveloDe and Pore Comulexes ............................................ IV. Mechanisms of Signal Transduction ofthe Nucleus .................................

271 274 278 284

viii

CONTENTS

V. The Role of Signal Transduction in Oncogenesis: Concluding

INDEX

Remarks ........................................................................................................ References ....................................................................................................

296 300

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

311

CONTRIBUTORS TO VOLUME 55 Numbers in parentheses indicate the pages on which the author’s contributions begin.

TIMOTHY J. Bos, Norris Cancer Center and School of Medicine, University of Southern California, Los Angeles, Calqornia 90033 ( 1 ) TINADALIANIS, Department of Tumor Biology, Karolinska Institutet, 104 01 Stockholm, Sweden (57) ROBERTJ. DISTEL, Department of Biological Chemistry and Molecular Pharmacology, Dana-Farber Cancer Institute, Haruard Medical School, Boston, Massachusetts 02115 (37) R. S. KERBEL, Division of Cancer and Cell Biology, M t . Sinai Hospital Research Institute, Toronto, Ontario M5G 1x5, Canada (87) IAN MAGRATH,Lymphoma Biology Section, Pediatric Branch, National Cancer Institute, Bethesda, Maryland 20892 (133) ERICHA. NIGG,Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges sllausanne, Switzerland (271) BRUCE M. SPIEGELMAN, Department of Biological Chemistry and Molecular Pharmacology, Dana-Farber Cancer Institute, Haruard Medical School, Boston, Massachusetts 02115 (37) PETERK. VOCT,N o d s Cancer Center and Department of Microbiology, School of Medicine, University of Southern California, Los Angeles, Calqornia 90033 (1)

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jun: ONCOGENE AND TRANSCRIPTION FACTOR Peter K. Vogt and Timothy J. Bos Norris Cancer Center and Department of Microbiology, University of Southern California School of Medicine. Los Angeles, California 90033

I. jun is the Oncogene of Avian Sarcoma Virus 17 (ASV 17) 11. A Conserved DNA-Binding Motif Relates the Jun Protein to GCN4, a Transcriptional Activator in Yeast 111. Jun and the Human Transcription Factor AP-1: Identity of Key Properties IV. Jun Belongs to a Family of Related Genes V. The Cellularfun Gene Lacks Introns; Its Regulatory Regions Contain Interesting Signals VI. The Jun and Fos Proteins: Dimerization and Cooperativity VII. The Leucine Zipper Mediates Dimerization of Jun and Fos VIII. Regulation of jun: Response to Incoming Signals IX. The Oncogenicity ofjun: Increased Dosage or Qualitative Change of the Jun Protein? X. The Main Functions of Jun Form a Hierarchical Order: A Hypothesis XI. Jun Is a Signal Converter References

There are three principal approaches toward discovering the function of a retroviral oncogene and its cellular progenitor. First, with the nucleotide and amino acid sequence of the oncogene in hand it is possible to search for revealing homologies with cellular genes of known function. Second, one can analyze the pattern of protooncogene expression in the hope that some striking developmental or tissue specificity might suggest a physiological role. Third, the oncoprotein can be examined for characteristic biochemical properties, such as enzymatic activity, binding of growth factor, hormone, or lowmolecular-weight ligand, or affinity for a specific nucleotide sequence. These approaches have been applied with varying degrees of success. Landmark discoveries were made with oncogenes coding for cytoplasmic or cell surface proteins: Homology searches have identified the sis oncogene as a gene coding for platelet-derived growth factor, the erb-B andfms oncogenes as derivatives of genes coding for the receptors of epithelial growth factor and colony stimulating factor1, respectively (Doolittle et al., 1983; Waterfield et al., 1983; Downward et al., 1984; Sherr et al., 1985).The study of the src oncogene has been guided largely by the discovery that the Src protein is a tyrosinespecific protein kinase (Collett and Erikson, 1978; Levinson et al., 1 ADVANCES IN CANCER RESEARCH, VOL.55

Copyright 0 190 by Academic Press, Inc. All rights of reproduction in any form reserved.

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PETER K. VOGT AND TIMOTHY J. BOS

1978; Hunter and Sefton, 1980). The dramatic tissue specificity of mos expression has provided the first clues as to its function in germ cells (Probst and Vande Woude, 1985; Goldman et al., 1987; Sagata et al., 1988).These are highlights of a steadily growing and internally consistent body of experimental data that relates cytoplasmic and cell surface oncoproteins to cellular signal transmission. These oncoproteins function as components of signal chains controlling cell growth and differentiation. Nuclear oncoproteins, on the other hand, have been less well understood. Until quite recently neither search for homologies or defined biochemical activities nor patterns of expression have yielded definitive insights into function. They have, however, generated data compatible with the notion that nuclear oncoproteins participate in the control of DNA synthesis or transcription (Eisenman and Thompson, 1986; Eisenman, 1989). These general ideas on the function of nuclear oncoproteins took on a more specific shape with investigations on the oncogenefos. Over the past 2 years several studies have shown thatfos is clearly involved in the control of transcription (Setoyama et aZ., 1986; Distel et al., 1987). The nature of this involvement remained unclear, however, until it was found that the function offos is closely tied to that ofjun, a new member of the class of nuclear oncogenes. By a series of fortuitous discoveries jun has become one of the best known oncogenes. It is identical to a cellular gene coding for a protein component of AP-1, a cellular transcription factor that regulates gene expression in response to incoming signals. In this article we will consider the structure of the cellular and retroviral jun genes and their relationship to other transcriptional regulators, the interaction between the Jun and Fos proteins, and the control and oncogenic potential ofjun.' I. jun Is the Oncogene of Avian Sarcoma Virus 17 (ASV 17)

In 1983 a new sarcoma virus was isolated from a spontaneous sarcoma in an adult chicken in Los Angeles. The isolate, termed avian sarcoma virus 17 (ASV 17),proved to be a retrovirus capable of causing fibrosarcomas in chickens (Cavalieri et al., 1985a,b). Tumors arise at the site of injection after a relatively long latent period of up to 1 month. Most of these growths are polyclonal in origin, as judged by the absence of uniform viraI/celluIar junction fragments in restriction digests of tumor DNA. These tumors also contain infectious ASV 17. Following convention, the abbreviation of a gene will be italicized, and the corresponding protein set in roman.

jUn:

ONCOGENE AND TRANSCRIPTION FACTOR

3

However, in a rare tumor a single proviral integration site may predominate (L. Nagata, 1988 personal communication). ASV 17 also induces oncogenic transformation in chick embryo fibroblast cultures. In uitro-transformed cells have fusiform shape, grow in parallel array in tightly packed formation on solid substrates under agar, and are able to form anchorage-independent colonies in semisolid medium. Like other retroviruses that induce tumors rapidly and transform cells in culture, ASV 17 is defective in replication and is associated with a nontransforming retrovirus that acts as a helper to complement the defects of ASV 17 (Cavalieri et al., 1985a,b). In the absence of a helper virus, ASV 17 transforms cells but these then fail to produce infectious progeny virus. Chick embryo fibroblasts transformed by ASV 17 in oitro are not immortal, regardless of whether they produce virus or not. They become highly vacuolated after 25 to 30 cell generations, stop dividing, and die (Ball et al., 1989).In contrast, cultures derived from ASV 17-induced tumors have a much longer lifespan, suggesting that they have undergone additional genetic changes that are not seen in cell culture (P. K. Vogt, 1988 unpublished observations). The range of cell types that ASV 17 transforms in uitro is limited. Besides chicken embryonic fibroblasts it includes chicken embryo neuroretina cells and myoblasts (H. Su, 1988 personal communication; F. Tato, 1988 personal communication). In the latter cell type ASV 17 arrests the differentiation program. Transformed myoblasts do not fuse into myotubes, and myotubes never express ASV 17. The genome of ASV 17has been cloned in the EMBL 3 vector from a genomic library of ASV 17-transformed chick embryo fibroblasts (Maki et al., 1987). Restriction enzyme and sequence analysis puts the size of the ASV 17 genome at 3.27 kb (Fig. 1).It contains a 0.93-kb nonviral, cell-derived insert that replaces the 3’ end of the retroviral gag gene, all of the pol gene, and the 5’ portion of env. This presumptive oncogene was subcloned and tested for homology with known oncogenes by dot blot hybridization. No homology was found. The ASV 17 insert and presumptive oncogene was therefore given a new name, jun, condensed from “ju-nana,” which is Japanese for 17, alluding to ASV 17 and to the ground-breaking work of a research associate from Japan (Maki et al., 1987).In this article we will follow conventional oncogene terminology and, where a distinction between the viral and the cellular gene is necessary, will refer to the ASV 17 version ofjun as v-jun and to its counterpart in the vertebrate genome as c-jun. In the ASV 17 viral genome the j u n insert forms one long open reading frame together with the 5’ gag sequences of p19 and the partial sequences of p10 to code for a protein of about 65 kDa (Fig. 1). This protein is detected in ASV 17-transformed cells with Jun-specific antisera. It is

4

PETER K. VOCT AND TIMOTHY J. BOS

FIG.1. The proviral genome ofASV 17. In ASV 17 the entire pol gene, the 3’ portion of the gag, and the 5’ portion of the enu genes have been replaced by a 0.93-kb cell-derived insert. ASV 17 has one long open reading frame that encodes a 65-kDa fusion protein in which 220 gag-encoded amino acids (aa, black) are joined to 296 jun-encoded amino acids (shaded). LTR, Long terminal repeat.

concentrated in the nucleus (Bos et al., 1988). Thejun insert in ASV 17 shows very close homology to t h e j u n gene of vertebrate cells; the immediate progenitor of v-jun is probably chicken c-jun. That jzin is the oncogenic effector of ASV 17 has been shown by moving the gene into another, nononcogenic retroviral genome. j u n was excised from the cloned ASV 17 genome and inserted with and without its 5’ gag tail into the avian retrovirus vector RCAS (Ball et al., 1989; Bos et al., 1990). Both RCAS-jun constructs show virtually the same oncogenic properties in vivo and in uitro as ASV 17, while RCAS alone does not transform cells. j u n is therefore a true oncogene, derived from the cell, inserted into the retroviral genome, and responsible for rapid oncogenic transformation in cell culture and in the animal. II. A Conserved DNA-Binding Motif Relates the Jun Protein to GCN4, a Transcriptional Activator in Yeast

The amino acid sequence of Jun, derived from its nucleotide sequence, shows homology to the amino acid sequence of GCN4 (Vogt et al., 1987) (Fig. 2). GCN4 is a yeast transcriptional activator protein (Hinnebusch, 1985; Struhl, 1987a). It is part of a control system that consists of several positive and negative trans-acting effectors and

5

j U n : ONCOGENE AND TRANSCRIPTION FACTOR

206 I I I

216

JUN

277 I I I

i 8 l

PLFPIDMESQERIKAERIAASKSRKRKLERIARIARLEEKVKTLKAQNSELASTANML~QVA

II II

II

I

II II

GCN4

PLSPIWESSDP

JUN

QLKQKVMNHVNSGCQLMLTQQTF

GCN4

RLIMLVGER

II

II

II Ill I

I1 I I

~EARRRsRARKLQRMKQLEDW

I l l

I

271

II

EELLSKNYHLENEVA

272

296

I

281

F1c.2. Homology between v-Jun and GCN4. The homology between v-Jun and GCN4 is limited to the carboxyl-terminalthird of each protein (hatched).The amino acid sequence identity in this region is 44%.

regulates amino acid synthesis in yeast. GCN4 itself is translationally controlled (Hinnebusch, 1984, 1988; Williams et aZ., 1988). Under conditions of amino acid starvation, it binds to upstream regulatory regions of several structural genes coding for amino acid-synthesizing enzymes and activates the transcription of these genes. GCN4 has two major functional domains: a DNA-binding domain, consisting of the 60 to 70 carboxyl-terminal amino acids, and a transcriptional activator domain, located in the amino-terminal half of the molecule and extending for 17 amino acids that form an acidic region (Hope and Struhl, 1986; Hope et al., 1988). The homology between Jun and GCN4 is confined to the 70 carboxyl-terminal amino acids of the two proteins, including the DNA-binding domain of GCN4 (Vogt et d.,1987). This homology is not only structural but also corresponds to functional equivalence (Struhl, 1987b).A cloned GCN4 gene with its DNAbinding domain replaced by the homology region of v-Jun (the 166 carboxyl-terminal amino acids) is functional in yeast. A similar construct in which the DNA-binding domain of GCN4 is replaced by the 99 carboxyl-terminal amino acids of Jun is marginally active in yeast. The construct containing the larger Jun fragment complements a GCN4 defect by activating transcription of GCN4-dependent genes. However, the level of this transcriptional activation is about twofold less than that mediated by GCN4 itself, presumably because of minor differences in DNA binding. The truncated GCN4 without its own

6

PETER K. VOGT AND TIMOTHY J. BOS

DNA-binding domain and without the substitution by the Jun homology region is inactive in the complementation test. Activity also requires part of the bacterial L e d gene at the 5’ end of the construct. This requirement most probably reflects the need for effective protein dimerization, which may be stabilized by the LexA domain. GCN4 must dimerize in order to bind to DNA (Hope and Struhl, 1987). This condition can be expected to hold for the GCN4-Jun chimeric protein as well. The optimal DNA consensus sequence for GNC4, ATGACTCAT, has perfect dyad symmetry, which would correspond to symmetrical contact points in the GCN4 dimer (Hope and Struhl, 1987). Mutants of this consensus sequence show reduced or no binding of GCN4-Jun chimeric protein. There is therefore a qualitative and a quantitative functional equivalence of Jun and GCN4 DNA-binding domains, although there are also suggestions of subtle differences that remain to be defined (Struhl, 1987b). Recent experiments have shown that the complete, unaltered Jun protein without the transcriptional activator domain of GCN4 also can activate transcription in yeast (Struhl, 1988). The transcriptional activator domain of v-Jun appears quantitatively as effective in yeast as that of GCN4, despite the lack of sequence homology. Deletion analysis has located this domain between Jun amino acids 15 and 102. It contains two acidic regions, one between residues 15and 59, which carries a net negative charge of -7, and a second one between amino acids 87 and 102, with a net negative charge of -4. Thus, the complementation tests using either v-Jun fragments or all of v-Jun in yeast have provided the first definitions of functional domains for the Jun protein: a DNA-binding domain, roughly located within the 166 carboxyl-terminal amino acids, and a transcriptional activator domain, situated between amino-terminal amino acids 15 and 102. Although there exist close structural and functional relationships between GCN4 and Jun on the general level of DNA binding and transcriptional activation, there is no indication that the indigenous function of Jun in vertebrate cells is similar to that of GCN4 in yeast, namely control of amino acid synthesis. Rather, in animal cells Jun appears to have evolved into a transcriptional regulator for a conipletely different set of target genes. Ill. Jun and the Human Transcription Factor AP-1: Identity of Key Properties

AP-1 is a transcription factor isolated from HeLa cells (Lee et al., 1987a; Piette and Yaniv, 1987). It consists of a mixture of polypeptides that have the common property of binding directly or indirectly to the

jun:

ONCOGENE AND TRANSCRIPTION FACTOR

7

DNA sequence TGACTCA and to certain closely related variants of this sequence. AP-1 activates the transcription of several genes, including those coding for metallothionein IIA, collagenase, and stromelysin. There are also binding sites for AP-1 in the SV40 early region and in numerous other cellular and viral genes (Fujita et al., 1983; Angel et al., 1987; Lee et al., 198713; Matrisian et al., 1986). The AP-1 activity of several cell types can be increased by phorbol ester tumor promoters, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), acting through the protein kinase C signal transmission chain, by serum, growth factors, and Ca2+ ionophores (Angel et al., 1987; Chiu et al., 1987; Lee et al., 1987b; Rauscher et al., 1988a,b). The AP-1 consensus sequence TGACTCA is contained in the GCN4 target sequence ATGACTCAT. Because of this identity of GCN4-Jun and AP-1 DNA consensus sequences, an inquiry was made into the possible relationship between Jun and AP-1. The results were dramatic and clear cut (Bohmann et al., 1987; Angel et at., 1988a). Two antisera directed against the viral Jun protein strongly cross-reacted with an approximately 40-kDa polypeptide in an AP-1 preparation purified from HeLa cell nuclear extracts by DNA affinity chromatography. Both of the Jun antisera were prepared against synthetic peptides derived from thejun nucleotide sequence. One of the sera was directed against a peptide from the conserved DNA-binding domain, the other against a peptide from the more divergent amino-terminal half of the molecule. Both sera reacted equally well with the 40-kDa AP-1 protein in immunoblots. A partial genomic clone of the humanjun gene, encompassing the carboxyl-terminal DNA-binding domain, was expressed in bacteria and tested for sequence-specific DNA binding in DNase footprint protection experiments. This fragment of the human Jun protein bound to the same consensus sequence as purified AP-1. Mutations in the consensus sequence that altered the binding of AP-1 had a parallel effect on the affinity of the Jun protein. The Jun protein was also shown to activate transcription from constructs containing AP-1 binding sites. Several tryptic peptides of the 40-kDa AP-1 protein were sequenced; they turned out to be identical to peptides predicted from the nucleotide sequence of the humanjun gene. Thus, Jun and AP-1 are closely related antigenically by epitopes located in two different domains of the proteins, they bind to the same DNA sequences with similar affinity, activate transcription through that same consensus sequence, and show structural identity in all of the sequences that have been compared. The 40-kDa AP-1 protein is therefore very closely related and probably identical to the product of the human j u n gene. The ASV 17 viral Jun protein, expressed in a bacterial vector, has also been compared to AP-1. v-Jun binds to the AP-1 consensus se-

8

PETER K. VOCT AND TIMOTHY J. BOS

quence and to two mutants of this sequence with the same affinity as purified AP-1. The DNA-binding properties of v-Jun, c-Jun, and AP-1 appear to be indistinguishable (Bos et aZ., 1988). AP-1, however, is a mixture of proteins, operationally defined by the ability to bind directly or indirectly to the same DNA consensus sequence. AP-1 preparations also contain the product of the oncogenefos and Fos-related proteins as well as several other still unidentified components (Franza et al., 1988; Rauscher et al., 198813: Bohmann et al., 1989; Curran et aZ., 1989). Recent studies illustrate the complexity of this system and also raise the possibility of subtle functional differences between the Jun protein expressed by itself and the AP-1 preparations. These studies were carried out on a transcription factor that binds to the regulatory region of the hamster histone 3 gene (Sharma et al., 1989). The DNA-binding site for this factor has the sequence TGACTCG, which differs by one base from the AP-1 consensus sequence TGACTCA. Interestingly, this site binds bacterially produced viral or cellular Jun, but it does not detectably bind a DNA affinitypurified preparation of AP-1 from HeLa cells. Several explanations can be offered for this difference. The AP-1 preparation may contain a substance that interferes with binding to the site; the bacterially produced proteins may lack appropriate posttranslational modifications that may modulate the properties of HeLa cell AP-1, or there may be a genuine difference in the DNA-binding specificity between AP-1 and Jun. The regulatory site on the hamster histone 3 gene also binds a hamster protein. This hamster transcription factor is neither Jun nor AP-1. It cross-reacts with antiserum directed against the DNA-binding domain of Jun but does not react with an antiserum directed against less conserved amino-terminal epitopes of the Jun protein. IV. Jun Belongs to a Family of Related Genes

If genomic DNA from man or chicken is digested with restriction endonucleases that do not cut within t h e j u n sequence and is then hybridized in a Southern blot to a 32P-labeledjunprobe, a single band becomes labeled, indicating that jun is a single-copy gene and that there are no pseudogenes ofjun in the vertebrate genome. If on such a Southern blot the stringency of hybridization is lowered, additional bands appear and suggest the existence ofjun-related loci (Bohmann et al., 1987; Ball et al., 1989).The humanjun gene has been localized on chromosome 1p3.1,3.2 (Haluska et al., 1988; Hattori et al., 1988). The additional bands appearing in Southern blots at low stringency of hybridization to j u n are not correlated with the presence of human

jufl:ONCOGENE AND TRANSCRIPTION FACTOR

9

chromosome 1 (F. G. Haluska, K. Huebner, and C. M. Croce, 1988 unpublished observations). They are derived from loci residing on other chromosomes. What are these otherjun-related loci? One is jun B, a gene that is structurally and physiologically closely related to c-jun (Ryder et al., 1988).jun B was discovered in a subtractive cDNA library containing genes whose transcription is specifically turned on after growth stimulation in the presence of inhibitors of protein synthesis. Such genes are called immediate early genes of the growth response (Lau and Nathans, 1987). The Jun B protein shows homology to c-Jun in six domains (Figs. 3 and 4). One consists of the carboxyl-terminal94 amino acids of Jun. It contains sequences that are essential for DNA binding and is permissive for a-helical structure. In the aminoterminal direction it is followed closely by a short stretch of homology, characterized by acidic residues and a nonhelical structure. In the approximate center of the molecule is a short homology region rich in prolines.It could be part of a molecular hinge. Excess prolines also continue in the carboxyl-terminal direction. The amino-terminal half of Jun contains a tripartite stretch of homology extending from amino acids 32 to 122 and spanning sequences that are important in transcriptional activation. We propose to refer to this tripartite amino-terminal area of homology as Jun homology region 1, JH-1, consisting of JH-lA, JH-lB, and JH-1C (Fig. 3). The carboxyl-terminal homology region will be JH-2 (consisting of JH-2A and JH-2B) and the center hinge region will be JH-3. The product of more recently discovered gene,jun D, shows homology to c-Jun in similar domains as Jun B (Hirai et al., 1989) (Fig. 3).Close similarity in JH-1 and JH-2 defines the immediate Jun family. The current membership of this family is c-Jun, Jun B, and Jun D. Outside this immediate family there exist more distant relatives of Jun. They show homology to Jun within JH-2 only. Homology in JH-2 then defines the extended Jun family (Fig. 5). To this belong GCN4 of yeast, the protein of the cross-pathway control gene cpc-1 of Neurospora (Paluh et al., 1988), the cyclic AMP response DNAbinding protein CREB of man and rat (Hoeffler et al., 1988; Gonzales et al., 1989), the histone regulatory protein of hamster (Sharma et d., 1989), the Epstein-Barr virus trans-activator BZLF-1 (Farrel et al., 1989), and the Cys-3 protein of Neurospora (Fu et al., 1989). There are also Jun/AP-l-related proteins in plants and Drosophila but sequence information for these is not yet available (Perkins et al., 1988; L. Walling, 1989 personal communication). The recently described lymphomagenic oncogene cbl is distantly related to the Jun family through a partial homology to GCN4 (Langdon et aZ., 1989).

PETER K. VOGT AND TIMOTHY J. BOS

10 V-JUN

CHICKEN

C-JUN

HUMAN

C-JUN

MOUSE

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MOUSE

JUNB

MOUSE

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I Ill I I I I I I I l l Ill1 MCTKMEQPFYHDDSY--------------------~GYGRSPGSLSLHDYKLL~TLALNLA II Ill I II I I I l l 1

D

METPFYGEEALSGLAAGRSSVAGATGAPC%GGTAPPGWPGAPPTSSMLKKDALTLSLA

39 43 43 43

44

60

V-JW

_______-________M------ILTS--PDVGL-LKLASPELERLIIQSSNGLITTTPTPT-QFLCP

82

CHICKEN

C-JUN

II IIII IIIII IlIIlIIIIIllIlIIIIlllIIIIII IIIII DAAS-SLK-PHLRNKNRD------ILTS--PDVGL-LKWSPELERIIQSSffiLITTTPTPT-QFLCP

100

HUMAN

C-JUN

DPVG-SLK-PHLWU(NSD------LLTS--PDVGL-LKLASPELERLIIQSSNGHI~TPTPT-OFLCP

100

IIIII DPVG-SLK-PHLRAKNSD------LLTS--PDVGL-LKLASPELERLIIQSSNGHITTTPTPT-QFLCP II I l l I I I IIIII IIIIII Ill IIIIIII I I DPYR-GLKGPtRRtPGPEGSGAGSYFSMGSDn;RSLKLASLKLASTELERLIWNSffiVITTTPTPP~YFYP Ill I I 11111IIIIll Ill IIIII I II EPW\RCLKPGSATAPSALRPMjAP-DGLLASPD~LLKLASPELERLIIO-SffiLV~TPTST-QFLYP

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IIIIIIIIIIIIIIIIII

I I I I I I I I IIIII

IIIII I I I I I I I I I I I I I I I I I I I I I I I I I I I

100

126

CHICKEN

C-JUN

HUMAN

C-JUN

WSE

C-JUN

WSE

JUN B

MOUSE

JUN D

K N - - - - - - - - - - V T D E Q E G F A E G N R A L A E L H m ) N T L P S V T S M O W S ~ ~ V S S ~ - ~ S F N ~ - - 137 II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I IIIIIII K N - - - - - - - - - - V T D E P E G F ~ G F ~ ~ L H N Q N T L P S ~ S A A Q ~ S ~ ~ V S S ~ - ~ S F N T -155 II IIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIII I I Ill K N - - - - - - - - - - V T D E Q E G F ~ G ~ ~ L H S M J T L P S V T F 159 II I I I I I I l I I I I I l l I I I I I I I l l I I I l I l I l I l I I I I I I I I I I I I I I I I I I1 159 KN----------VTDEQEGFAEGFVRALAELHSONTLPSVTSAAOWSGAG~APAVASVAGA~Y I I I I I I I I I l l I I I1 II I I 1 I l l I 170 RtGGSGtGn;tGVTEEPEGFRDtFVW\LDDLHK---MNHV--Y I Ill1 II I I I I I I IIII I 1 I I I 173 RVA---------ASEEQE-F~G~LEDLHKQSQ~T-AATS------~--A---PPAPMLA

V-JUN

V-JUN

--SLHSEPPWANLSNFNPNALNS---APNYNAN~YA--------PO---HHINP~VOHPR-~

CHICKEN

C-JUN

--SLHSEPPWANLSNFNPNALNS---APNY”tmYA--------PO---HHINP~VQHPR-L~

HLMRN

C-JUN

MOUSE

C-JUN

WSE

JUN B

MCUSE

JUN D

207 IIIIIIIIIIIIIIIII II I II I I I I II IIIIIIIIIII 224 SASLHSEPPWANLSNFNPGALSSGtGAPSYWUGWFPAQPQPM I I I I I I I I I I I I I I l l l l I I I I I I I l I I I I I I I I l I l I l IIIIIIIII IIIIII IIIIII Ill 227 SASLHSEPPWANLSNFNPCALSSUiGAPSYGAAG~PSQP~PPQPPHHLPWIPVQHPR-~ IIIIIII I Ill I I I II I I A G P - E P - P P W T N L S S Y S P A S A P S ~ S G T A V G ~ S - S Y P T A T I S Y L - P ~ P F A f f i H P A O ~ L S ~ S A 235 I I IIIIIII I l l I I II I A T P G A T E T P W A N L S S F A G t R t P P ~ ~ ~ ~ - P V P F P P P P G A - ~ P P P P - - - - - - - - - H P P R ~ 232

IIIIIIIIIIIIIIIIIIIIII

IIIIIII I I I I

II

189

IIIIIIIIIIIII Ill

V-JUN

256

CHICKEN

C-JUN

214

H

W

C-JUN

291

MOUSE

C-JUN

294

MOUSE

JUN B

304

MOUSE

JUN D

301

V-JUN

CHICKEN

C-JUN

HUMAN

C-JUN

MOUSE

C-JUN

MWSE

JUN B

MCUSE

JUN D

SELASTANMLREWAQLKQKVMNHVNSCCQLMLTWWTF STOP

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII S E L A S T A N M L R E W A Q L X Q ~ ~ S G C Q ~ STOP L~~F IIIIIllIIIIIIIIIIIIIIIIIIIIIIllIlll IIIII SELASTANMLREPVAQLKQ~~SGCOLMLTWWTF STOP I1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I SELASTANMLREWAQLKQKVMNHVNSGCQLMLTWWTF STOP I I I IIIIIIIIIIIII I1 I I I I I I AGLSSAAGLLREQVAQLKQWMTHVSNGCQLLUVKGHAF STOP I I I IIIIIIIIIIIII II IIIII I TEL4STASLLREQVAQLKQKVLSHVNSGQLLPQHQWAY STOP

296 314 331 334 344 341

FIG.3. Sequence comparison of Jun family members. The amino acid sequences (one letter code) of various Jun and Jun family proteins are shown. Vertical lines connect identical residues. Sequence information comes from the following publications: v-Jun (Maki et al., 1987),chicken c-Jun (Nishimura and Vogt, 1988),human c-Jun (Bohmann et al., 1987; Hattori et a!., 1988), mouse c-Jun (Ryseck et al., 1988; Lamph et al., 1988; Ryder and Nathans, 1988),Jun B (Ryder et al., 1988),and Jun D (Hirai et al., 1989).v-Jun amino acid number 1 is the first jun-encoded amino acid in ASV 17.

j U n : ONCOGENE AND TRANSCRIPTION FACTOR

N A

+

B

C

A

JH-1

JH-3

Activator

Hinge

H

f-

11 C

B

JH-2 DNA binding

FIG.4. Schematic representation of Jun homology regions. JH-1 contains the transcriptional activator domain; JH-2 encompasses the DNA-binding domain; and JH-3 is a potential hinge region.

V. The Cellular jun Gene Lacks Introns; Its Regulatory Regions Contain Interesting Signals

The nucleotide sequences of genomic and cDNA clones derived from the human and the chicken jun genes have been published (Hattori et al., 1988; Nishimura and Vogt, 1988). The sequence of the mouse jun cDNA has also become available (Ryseck et al., 1988; Lamph et al., 1988; Ryder and Nathans, 1988).All three genes code for

FIG.5. The Jun family. Jun belongs to a multigene family. Members of the immediate Jun family share homology in two homology regions, JH-1 and JH-2. Each homology region contains several domains which are conserved. An extended Jun family is defined by homology in JH-2 only. It includes, besides GCN4, the CREB protein (HoefRer et al., 1988; Gonzales et al., l989), the histone regulatory protein of hamster cells (Sharma et al., 1989), the cross-pathway control and Cys 3 proteins ofNeurospora (Paluh et al., 1988; Fu et al., 1989), and the Epstein-Barr virus trans-activator BZLF (Farrel et al., 1989).

12,

PETER K. VOGT AND TIMOTHY J. BOS

closely similar proteins (Fig. 3). The mammalian Jun proteins show >95% amino acid sequence identity; the avian protein is approximately 90% identical to its mammalian counterparts. Both genes have two methionine codons, spaced 12 nucleotides apart, close to the 5’ end of the major open reading frame. Only the second of these codons is in a Kozak consensus sequence and is therefore the more likely one to start the Jun proteins. Accordingly, the length of the human Jun protein is probably 327 amino acids, and the lengths of the mouse and chicken proteins 330 and 310 amino acids, respectively. In the figures of this article we have, however, followed established convention and stated the numbering of the Jun amino acids with the first methionine of the open reading frame. The amino acid sequence of the Jun proteins can be divided into several domains (Fig. 6). The aminoterminal 122 amino acids of the c-Jun proteins contain three acidic regions important for transcriptional activation (Struhl, 1988). (In the v-Jun protein two of these regions are fused due to an internal deletion.) In the carboxyl-terminal DNA-binding domain two features stand out: a highly basic region (amino acids 252 to 280, numbering taken from human c-Jun, Fig. 3), conserved in chicken, mouse, and man and an equally conserved periodicity of five leucines, spaced

FIG. 6. Functional domains of Jun. Jun contains two major functional domains. The acidic domain in the amino-terminal half of the molecule is important for transcriptional activation. The DNA-binding domain is located at the carboxyl terminus. It consists of the leucine zipper and a basic domain believed to be the DNA contact surface. Numbering of amino acid residues is from the human Jun protein (Fig. 3). The DNA-binding domain extends from amino acid 252 to the carboxyl terminus. Within the DNA-binding domain are two subdomains, a basic domain thought to be directly involved in DNA contact between 252 and 280 and the leucine zipper domain necessary for protein dimerization between 280 and 308. The net charges in the acidic and basic domains are indicated in parentheses.

jUn: ONCOGENE AND TRANSCRIPTION FACTOR

13

seven residues apart and abutting to the carboxyl border of the basic domain (amino acids 280 to 308 of the human c-Jun). The latter has been termed the leucine zipper (Landschulz et al., 1988). Leucine periodicity region and basic region are part of the DNA-binding domain. The amino terminal border of the DNA binding domain has not been determined accurately; it may be to the left of amino acid 200 (human c-Jun). The central portion of the Jun molecule is highly enriched in prolines. The coding regions of the human and the chicken genes for which genomic sequences have been determined lack introns. RNase protection experiments carried out with the large upstream regulatory region of the human gene indicate that this region also is intronless (Hattori et al., 1988). Although the 5’ regulatory region of the human gene is larger by about 0.6 kb than that of the chicken gene, both genes contain the same conspicuous regulatory signals arranged in identical relative positions (Hattori et al., 1988; Nishimura and Vogt, 1988) (Fig. 7). The key features of the regulatory domains in the human and in the chicken gene are a CAAT box followed by an AP-1-like binding site and two TATA-like elements. These signals are virtually identical in the chicken and in the human gene. There are also extended GC-rich regions upstream of both genes that contain copies of the consensus binding site for the SP-1 transcription factor (seven in the chicken gene) and two copies of an AP-2 binding sequence (Hyman et al., 1989). The transcription start has been determined by RNase protection experiments for the human gene (Hattori et al., 1988).There are five major start sites occurring in two clusters and three minor ones. The main start site in HeLa cells is GGGCGG / 26 hp /

Buman Chicken

GGGCGG / 41 hp / GGGCGG / 24 hp / GGGCGG / 157 tp / GGGCGG / 69 bp /

Buman

CCMTGGGIV\GGCCTTGGGGTGACATCATGGGCTATTTTTAGGGGTTGACTGGTAGC I I I I I I I I I1 I IIIIIIIIIIIIIIIIIIIIIIIIII II I Ill II CCMTGGGGAGCCGC GGGGTGACATCATGGGCTATTTTTAGCGGGCTCCCGGTCGC

Chicken

*

Buman Chicken

t

tl t

t

*

AGATMGTGTTGAGCTCGGGCTGGATAAGGGCTCAGAGTTG IIIIIIII I I I1 I1 II I 1 TGATMGTGARGGCTGCACGCGCGAGCGGGCTCAGAGGCCGGGGCGGGCGGGCGGCAGTGCGACTCAGtl

FIG.7. Thejun promoter. The upstream regulatory elements of chicken c-jun (Nishimura and Vogt, 1988) and human c-jun (Hattori et ol., 1988) are very similar. Both contain SP-1 binding sites, a CCAAT box, an AP-1-like binding sequence, and two TATA-like elements (shown in bold type). The major transcription starts are indicated by + 1. Minor starts are indicated by an *.

14

PETER K. VOGT AND TIMOTHY J. BOS

located 975 nucleotides upstream from the translation initiation codon. For the chicken gene a single transcription start site has been found by primer extension. Its position at 314 nucleotides upstream from the translation initiation codon and 91 nucleotides downstream from the TATTTTTA element corresponds to one of the minor start sites in the human gene. mRNA of the cellularjun gene comes in two sizes, 2.5 to 2.7 and 3.2 to 3.3 kb. The two appear to differ by the use of different poly(A) addition signals (Ryseck et al., 1988; Hattori et al., 1988). The 3' regulatory regions of the human and the chicken gene contain several poly(A) addition signals. They are also very AT rich and include the sequence element ATTTA that is characteristic of highly unstable messenger RNAs (Shaw and Kamen, 1986). VI. The Jun and Fos Proteins: Dirnerization and Cooperativity

Several lines of evidence have linked the product of the oncogene fos to the control of gene expression (Setoyama et al., 1986; Distel et al., 1987; Lech et al., 1988). In the cell the Fos protein is associated with a cellular protein, termed p39 (Curran et al., 1985).Together with p39 Fos binds sequence specifically to DNA and can trans-activate genes (Franza et al., 1988; Kerr et al., 1988; Lucibello et al., 1988; Rauscher et al., 1988a-c; Sassone-Corsi et al., 1988a-c). By itself, however, the Fos protein does not show specific DNA binding (Rauscher et al., 1988c; Sassone-Corsi et al., 1988b). Complex formation with p39 is required for this activity. Antibody directed against Fos can prevent binding of both Fos and p39 to DNA (Distel et al., 1987; Franza et al., 1987). DNA affinity precipitation and gel retardation assays in conjunction with immunoblots of the bound proteins have shown that the target site for the Fos-p39 complex is the AP-1 consensus sequence (Franza et al., 1988; Rauscher et al., 1988a). It was therefore suspected that the specificity for the AP-1 binding site might be mediated by p39 and that p39 is related if not identical to c-Jun. Experimental data have fully confirmed this suspicion (Chiu et al., 1988; Rauscher et al., 198813; Sassone-Corsi et al., 1988a).The two Jun antisera, directed against peptides from the DNA-binding domain and the amino-terminal half of Jun, respectively, react strongly with p39. p39 precipitated from the cell with anti-Jun sera and p39 precipitated with anti-Fos serum as part of the Fos-p39 complex have identical tryptic peptide maps. Therefore, p39 and Jun are identical. Thus Jun forms a complex with Fos that specifically binds to the AP-1 site. The Jun anti-peptide sera recognize only free p39, but not p39 that is complexed to Fos. The epitopes reacting with these antibodies seem to be inaccessible in the complex.

jUn: ONCOGENE AND TRANSCRIPTION FACTOR

15

Studies with in uitro-translated Jun and Fos, either full length or truncated, have defined the Jun-Fos interaction (Halazonetis et al., 1988; Nakabeppu et al., 1988; Rauscher et al., 1988c; Gentz et al., 1989; Turner and Tjian, 1989). In preparations containing only Jun but not Fos, Jun occurs in the form of homodimers and binds to the AP-1 DNA consensus sequence as a homodimer. Dimerization is a prerequisite for Jun-DNA binding. Addition of Fos to a preparation of Jun displaces a Jun molecule from the dimers, substituting a molecule of Fos for it and leading to the formation of Jun-Fos heterodimers. JunFos heterodimers are therefore more stable than Jun-Jun homodimers. Fos cannot associate with itself to form homodimers. Jun-Fos heterodimers are also generated when Jun and Fos are cotranslated in the same system. Although Jun homodimers bind specifically to DNA, Jun-Fos heterodimers show an increased &nity for the AP-1 binding site if DNA binding is measured by gel mobility shift using a radioactively labeled DNA probe. The heterodimers are also more efficient transcriptional activators (Chiu et al., 1988; Sassone-Corsi et al., 1988a). However, the difference between homo- and heterodimers in the affinity for the AP-1 consensus sequence is not seen if they are compared in DNA affinity chromatography; both complexes elute from DNA at the same salt concentration (Turner and Tjian, 1989). Jun homodimers may therefore interact with DNA more extensively than is suggested by the gel retardation assays. A resolution of this apparent contradiction between data from gel shift assays and DNA affinity chromatography will require additional work. The relevant affinities between transcription factor and DNA will be those that prevail at physiological concentrations in the cell and are likely to be influenced by other, as yet unidentified proteins. The functional significance of Jun-Fos heterodimers is also underlined by the observation that the expression of AP-l-controlled genes is dependent on the expression of the cellularfos gene (Schonthal et al., 1988). Fos-Jun complexes are found only in the cell nucleus; in the cytoplasm the two proteins are not bound to each other (Curran et al., 1984). Since Fos and Jun synthesized separately in vitro associate rapidly and stably, they must either be confined to different cytoplasmic compartments or are transiently modified to prevent heterodimer formation. All members of the immediate Jun family, c-Jun, Jun B, and Jun D, dimerize with each other and with Fos; and the heterodimers with Fos show increased DNA binding in assays of gel mobility shifts (Nakabeppu et al., 1988). Since Fos is also a representative of a multigene family of closely related genes that code for Fos-related antigens (Fras) and for Fos B (Cohen and Curran, 1988; Curran and Franza 1988; Zerial et al., 1989; Cohen et al., 1989), and since the various Jun proteins can also form

16

PETER K. VOGT AND TIMOTHY J . BOS

heterodimers with Fras and Fos B, several trans-acting effector complexes appear possible (Fig. 8). All these complexes are expected to bind to the AP-1 binding site. They are heterodimers with the participating proteins belonging to the immediate Jun family and the Fos family. These proteins are also components of DNA affinity-purified AP-1 preparations (Curran and Franza, 1988; Bohmann et al., 1989; Curran et al., 1989). Because of their different structure, they could have different functions in gene regulation. However, c-Jun, Jun B, and Jun D have so far proved indistinguishable in their DNA-binding properties (Nakabeppu et al., 1988). Their presumed functional differences may be based on physiological activities other than DNA binding and may reside in other domains of the proteins. The transcriptional activator function of these proteins would be a prime candidate for combinatorial specificities that arise from the dimerization of different Jun and Fos proteins. These important aspects of Jun function are certain to involve additional protein-protein interactions about which we are at present entirely ignorant. The fact that the Jun and Fos proteins bind to DNA as dimers is mirrored by the dyad symmetry of the consensus binding site, TGACTCA. Dimerization as a prerequisite for DNA binding has already been demonstrated for GCN4 (Hope and Struhl, 1987), and the ability of the GCN4-Jun chimeric proteins to complement a GCN4 defect in yeast appears to depend on efficient dimerization. dun

Jun B

Jun D

Fos

Fm-1

Fos B

cJun

Jun B

Jun D

FOO

Fm-1

Fos B

FIG.8. Dimer Combinationsof the Jun and Fos family proteins. All homo- and heterodimers appear permitted except for Fos, Fra-1, and Fos B, which do not form homodimers and do not dimerize with each other.

jun: ONCOGENE AND TRANSCRIPTION FACTOR

17

In performing their function of transcriptional activation, the JunFos related dimers probably also interact with other proteins. A possible indication of these additional proteins has been seen in studies of the polyoma virus enhancer regions. Two proteins, PEA-2 and PEA-3, bind in this enhancer region immediately adjacent to the AP-1 site (Martin et al., 1988).They may also interact with AP-1. Vil. The Leucine Zipper Mediates Dirnerization of Jun and Fos

Jun, Fos, and GCN4 belong to a class of DNA-binding proteins that have a common, conserved structural motif, called the leucine zipper (Landschulz et al., 1988). The leucine zipper is an a-helical domain in which four or five leucines occur at regular, seven-residue intervals. These leucines are thus located at approximately the same rotational position in the helix; their side chains are aligned parallel to the axis of the helix and form a straight linear crest that protrudes from the side of the helix. In Jun the leucine zipper is located at the carboxyl terminus, within the domain necessary for DNA binding. In Fos it is located in the approximate middle of the molecule. The leucine zipper has been postulated to effect Jun- Jun and Jun-Fos dimerization, which would occur by hydrophobic interaction of the leucine crests of two molecules. Analysis of the leucine zipper by deletions and by amino acid substitutions has fully substantiated its role in dimerization. This work has been mainly carried out on the Fos leucine zipper, with a less exhaustive analysis of the Jun zipper (Kouzarides and Ziff, 1988; Nakabeppu et al., 1988; Sassone-Corsi et al., 1988c; Bos et al., 1989; Gentz et al., 1989; Schuermann et al., 1989; Turner and Tjian, 1989). For both Jun and Fos only part of the protein is required for dimerization. The 125 carboxyl-terminal amino acids of Jun are sufficient and residues 159 to 199 of Fos are fully active in dimerization. Deletions extending into the leucine zipper abolish dimer-forming ability of the proteins. For instance, a small deletion removing only the carboxylterminal leucine of the Jun leucine zipper leaves the protein unable to combine with Fos. Small interstitial deletions between the leucbes of the zipper also destroy dimerization potential, presumably because they alter the leucine periodicity and eliminate the linear orientation of leucine side chains that may be essential for zipper function. Substitution of single leucines has either no effect on dimerization or reduces dimer formation depending on the substituting amino acid. An exception are substitutions by proline. These abolish dimerization, presumably because they interrupt the a-helical structure of the zipper. Substitution of two leucines greatly reduces or entirely eliminates dimer

7 BASIC DOlUIN 1 - '

- - I

LEUCINE ZIPPER

C-JU.

JmrB

J m D

TOS

Flu 1

Pos B

CYS-3

CC14

CRLB

I1

CPCl

: A K I E E L I A E R D R W K N - L A - L A f l G A S T E

CllBP

: S D U D R L R K R V E Q L S R E L D

ll Y

B8LFl

C

D

S

E

L

E

I

~

Y

~

U

~

V

DNA CONTACT REGION

A

S

~

~

@

A

~

0

Y R QE VL A L b Q A K S S E H D - - - R L R L ~H @

DIMERIZATION SURFACE

I

j U n : ONCOGENE AND TRANSCRIPTION FACTOR

19

formation. An extensive mutational analysis has also been performed on the leucine zipper of the C/EBP enhancer-binding protein, demonstrating the importance of the leucine zipper in C/EBP dimer formation (Landschulz et al., 1990). These results clearly show that the leucine zipper is essential for dimerization of several DNA-binding proteins. They do not, however, rule out the possibility that structures outside the leucine zipper proper also play an important role in dimer formation. The basic structural parameters of a leucine zipper have been determined with an in vivo synthesized peptide representing the leucine zipper of GCN4 (O’Shea et al., 1989).These studies indicate that the a helices in the zipper are arranged in parallel orientation and that the overall structure of the GCN4 dimeric zipper is that of a coiled coil, resembling the architecture of fibrous proteins such as keratin. In the GCN4 zipper the leucine side chains do not interdigitate. Rather, the main stabilizing hydrophobic interactions take place between leucine and hydrophobic side chains of other amino acids within the leucine repeat that also occur in heptad periodicity (4-3 repeat). These interacting hydrophobic residues of the GCN4 leucine zipper form the dimerization surface. In other leucine zippers, including that of Jun, only some of the 4-3 repeat positions contain hydrophobic residues. The role of amino acids located between the periodic leucines requires further study. In Fos extensive mutagenesis of these interstitial residues is without effect on dimer formation with Jun (Schuennann et al., 1989; Turner and Tjian, 1989). Outside the dimerization surface the leucine zipper helix is predominantly hydrophilic, accounting for solubility in aqueous solutions. In some DNA-binding proteins with leucine zippers, including Jun and Fbs, there is a stretch of basic residues immediately adjacent to the amino terminus of the leucine zipper (Figs. 9 and 10).This region is highly conserved in Jun, Fos, and their relatives. It belongs to the essential DNA-binding domain and probably forms the contact points with DNA. Antibodies directed against this domain of Fos strongly interfere with DNA binding (Distel FIG.9. The DNA-binding domain of several proteins with a leucine zipper. Amino acid sequences of the DNA-binding domains of c-Jun,Jun B, Jun D, c-Fos, Fra-1, Fos B, Cys-3, GCN4, CREB, Cpc 1, C/EBP, and Epstein-Barr virus BZLF 1 starting at amino acids 255, 265, 262. 137, 107, 155, 291, 224, 268, 216, 289, and 171, respectively, are shown. Each consists of two distinct regions, a highly basic region believed to make contact with the DNA and the leucine zipper needed for dimerization and postulated to hold the DNA contact surfaces in the correct three-dimensional orientation. Basic residues in the DNA contact region are circled. Some of the proteins have only a vestigial leucine zipper.

20

PETER K. VOGT AND TIMOTHY J . BOS

FIG.10. Schematic representation of a Jun homodimer binding to DNA. The leucine zipper consisting of two parallel a helices (a coiled coil structure) holds the Jun monomers together and stabilizes the a-helical DNA contact regions of the basic domains in the correct spatial orientation. Details of this drawing are hypothetical, the drawing is not to scale; secondary structure outside the DNA-binding domain is not considered.

et al., 1987; Rauscher et al., 1988a,c).Deletions and amino acid substitutions in this region can abolish DNA-binding activity without affecting the ability to dimerize (Kouzarides and Ziff, 1988; Gentz et al., 1989; Landschulz et al., 1990; Turner and Tjian, 1989). These mutants act as trans-dominant lethals: they are able to bind wild-type protein and tie it up in dimers that no longer bind to DNA and may be physiologically inactive. Such mutants will prove extremely valuable in an analysis of Jun and Fos function. The principle of these trans-dominant lethals is applicable to all DNA-binding proteins that dimerize and have a separate DNA contact region. The DNA-binding domains of Jun, Fos, and related proteins consist thus of two functionally and structurally distinct regions, the leucine zipper and the actual DNA contact surface (Fig. 9). The dimerized zipper is believed to hold the DNA contact surface in the correct three-dimensional orientation and to stabilize this orientation. The twofold symmetry of the dimeric protein is matched by the dyad symmetry of the DNA consensus sequence TGACTCA. The DNA contact

jun: ONCOGENE AND TRANSCRIPTION FACTOR

21

region contributed by each protein monomer would then interact with one palindromic half-site of the DNA consensus sequence. In a speculative way, the dimeric structure of two a helices in parallel orientation could be roughly compared to a pair of boots, their heels touching, the uppers representing the leucine zipper and the soles representing the DNA contact surface planted on DNA (Fig. 10). The leucine zippers of different proteins do not interact indiscriminately to form heterodimers. Myc, for instance, does not dimerize with Jun, Fos, or GCN4 although it appears to form homodimers. On the other hand, neither Fos nor Fra-1 form homodimers (Nakabeppu et aZ., 1988; T. Curran, 1989 personal communication; Dang et al., 1989; Gentz et al., 1989; Turner and Tjian, 1989). Since the position of the leucines in all zippers is invariant, the amino acid residues between the leucines which are not the same in different zippers may determine permissible and nonpermissible protein-protein interactions. This possibility is, however, not supported by current, though still limited, mutant analysis (Schuermann et al., 1989; Turner and Tjian, 1989). An alternative explanation for the selectivity of leucine zipper dimerization would assign a critical role to structural motifs outside the zipper proper in determining compatibility between interacting proteins. Using empirical data and model building it should be possible to predict which leucine zippers and which proteins can and which cannot form heterodimers. Besides Jun, Fos, GCN4, and C/EBP (Landschulz et aZ., 1988)there are numerous other proteins with leucine zippers (Fig. 9). Examples are CREB (Hoeffler et al., 1988; Gonzales et al., 1989), Myc (Landschulz et al., 1988), and Cys 3 (Fu et al., 1989). Not all leucine zipper proteins belong to the extended Jun family; nor do all members of this family possess a leucine zipper. The CPC-1 protein of Neurospora has only a vestigial leucine repeat (Paluh et al., 1988) and the BZLF-1 protein of Epstein-Barr virus (Farrel et al., 1989)has only one leucine repeat yet retains homology to Jun in the presumed DNA contact surface. It also binds to the AP-1 site. A computer search has come up with about 200 proteins that carry the leucine heptad periodicity (O’Shea et al., 1989) but it is unlikely that in all these proteins the leucine periodicity is part of a dimerization surface.

VIII. Regulation of jun: Response to Incoming Signals

fun is regulated at the transcriptional, posttranscriptional, and posttranslational levels. The basis for the transcriptional regulation may be found in the 5‘ noncoding sequences of the gene (Fig. 7). The SP-1 binding site, CAAT box, and the AP-1 binding site of the human

22

PETER K. VOCT AND TIMOTHY J. BOS

cellular j u n regulatory region bind the respective transcription factors and function in the positive control of transcription from thejun promoter (Angel et d., 1988b). The same upstream regulatory regions, encompassing SP-1 binding sites, CAAT box, and AP-1 binding site, have been identified in the chickenjun gene as essential for positive transcriptional regulation (T. Nishimura, 1988 personal communication). The important feature of this control system is thatjun transcription is autoregulated by Jun/AP-1 itself: binding of Jun/AP-1 to the AP-1 site increases transcription from the jun promoter (Angel et al., 1988b). In serum-starved cells, only low base levels ofjun mRNA and Jun protein are seen. Following growth stimulation by addition of serum there is a rapid increase of the amount ofjun message that can reach 30-fold by 30 min (Almendral et al., 1988;Lamph et al., 1988; Rauscher et al., 1988b; Ryder and Nathans, 1988; Ryseck et al., 1988).jun mRNA levels then decline until about 3 to 4 hr, when they reach near-basal levels. In the presence of the protein synthesis inhibitor cycloheximide, jun is superinduced by growth stimulatory signals. Higher levels of j u n mRNA are reached, and the RNA is more stable. The rapid induction ofjun transcription that is insensitive to inhibition of protein synthesis characterizes c-jun as an immediate early gene of the growth response, similar t o j u n B andfos (Lau and Nathans, 1987). Jun D, on the other hand, is expressed at constant levels in serum-starved and -stimulated cells (Hirai et al., 1989). The Jun protein has a positive regulatory effect on the transcription of its own gene (Angel et al., 1988b). One could speculate that the availability of active Jun-Jun or Jun-Fos dimers able to bind to the AP-1 site of thejun gene may constitute a rate-limiting element in the regulation of jun transcription. Growth signals would then effect an increase injun transcription by activating cellular Jun protein and Fos proteins (Angel et al., 1988b). This activation may result from posttranslational modification of one or of both partners of the Jun-Fos complex. The phorbol ester tumor promoter TPA has been known for some time to elevate AP-1 activity in the absence of protein synthesis through mobilization of protein kinase C (PKC)(Angel et al., 1987; Lee et al., 1987b; Lamph et al., 1988). Cellular mutants that are insensitive to TPA promotion also fail to respond with the induction of AP-1 after TPA treatment (Bernstein and Colburn, 1989).The mechanism of AP-1 activation by TPA is not completely understood. Jun/AP-1 is a phosphoprotein, and phosphorylation can be expected to play some role in regulating AP-1 activity (Angel et al., 1988b). However, the extent of AP-1 phosphorylation is not altered after TPA treatment of the cell

jun: ONCOGENE AND TRANSCRIPTION

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(W. Boyle, 1988personal communication). The posttranslational modification of Jun could be indirect and result from a modification of Fos (Angel et al., 198813). Fos is phosphorylated, but as yet no functional change has been correlated with this modification. Preliminary results suggest that Fos may be poly(ADP) ribosylated in response to oxidants; poly(ADP) ribosylation would have the potential of interfering with DNA binding (C. Cerutti, 1989 personal communication). A qualitative or quantitative change of Fos leading to increased levels of Jun-Fos heterodimers would elevate AP-1 activity. Upregulation of jun transcription, presumably mediated by direct or indirect posttranslational activation of Jun itself, can be achieved with a surprising number of signals, including TPA, serum, growth factors, and oncogenes. TPA inducesjun transcription transiently; so do serum stimulation of starved cells as well as growth factors (epithelial growth factor, transforming growth factor p) added to cell cultures (Quantin and Breathnach, 1988; Pertovaara et al., 1989).Tumor necrosis factor a,on the other hand, induces a long-lasting elevation ofjun transcription in human fibroblasts (Brenner et al., 1989). Continued presence of serum or, alternatively, long-term expression of oncogenes can result in constitutively high levels of AP-1, especially in fibroblast cell lines. Oncogenes exerting a positive regulatory effect on Jun are src, activated c-Ha-ras, polyoma virus middle T,mos, andfos (Imler et al., 1988b; Wasylyk et al., 1988).Cells stably transformed by SV40 virus also show constitutively high AP-1 levels (Piette et al., 1988). In contrast, cells transfected with the myc oncogene or the genes coding for SV40 large T antigen or adenovirus E1A do not show increased AP-1 levels (Wasylyk et aE., 1988). The active oncogenes on this list are commonly referred to as “transforming,” the inactive ones as “immortalizing.” However, transforming and immortalizing activities of oncogenes are not invariant but cell dependent. The significance of these properties with respect tojun induction is not clear. Also uncertain is the effect of elevated CAMPonjun transcription. Published results on this point are not in agreement and may depend on the type of cell and promoter construct that is tested (Angel et al., 1988b; Piette et al., 1988). The growth factors and oncogenes that induce Jun are components of mitotic signal chains. They may be ordered in tentative sequences, e.g., external growth factors + receptor + Src + Ras + Mos . . . + Fos + . . . + Jun or, starting with TPA --+ , , , --* PKC + . . . + Fos + . .+ Jun. Jun induction appears, therefore, to be the response to several converging growth signals. Although there have been no exhaustive studies of the developmental and tissue-specific regulation ofjun, available data suggest that

.

24

PETER K. VOGT AND TIMOTHY J. BOS

jun is expressed at fairly constant levels during embryogenesis and it is distributed ubiquitously in the organism but at low levels (Ball et al., 1989; Ryder and Nathans, 1988). Exceptions are ovary, heart, and lung tissue which contain higher concentrations ofjun transcripts, a situation that also extends to small cell lung carcinoma of man (J. Minna, 1988personal communication; Hirai et al., 1989).Jun B and D differ in their patterns of tissue-specific mRNA expression from c-Jun: Jun B expression is high in testis and ovary, Jun D mRNA is elevated in intestine, thymus, spleen, lung, and heart (Hirai et aZ., 1989). Since the induction of Jun by serum or growth factors is transient, there must also be powerful mechanisms of negative regulation at work. j u n mRNA appears to be highly unstable, as is indicated by its fast decline from the peak reached after growth stimulation. This instability may be mediated by AT-rich 3' untranslated regions of t h e j u n mRNA, containing the ATTTA signal sequence. It can be considered a form of posttranscriptional control ofjun. The fact thatjun is superinduced in the presence of cycloheximide implies that degradation of j u n mRNA involves new protein synthesis. These degradative proteins could possible be controlled by Jun itself. The Jun protein, induced by serum or TPA, appears to be unstable (Lamph et al., 1988),but accurate determinations of the half-life of Jun remains to be made. The cellular and viral Jun proteins also carry a very strong PEST sequence (Fig. 11).

PEST Score

Residues

Human c-Jun

14.1

227-253

Mouse c-Jun

14.1

229-255

Chicken cJun

Protein

Sequence

K E E P ~ T V P E M P G E T P P L S PI D M E S Q E R

14.1

206.23~

K E E P O T V P E M P G E T P P L S P I D M E S Q E R

V-JW

10.8

178-204

H

Jun 8

4.9

249-265

E E

P

a

T

v P

E M P G E T P P

L F P I

D M

E s

a

E R

R D A T P P V S P I N M E D Q E R

FIG.11. PEST analysis of Jun proteins. PEST regions are characterized by elevated occurrence ofthe amino acids proline (P),glutamic acid (E), serine (S), and threonine (T) within a short sequence starting and terminating with positively charged residues. Their occurrence in a protein is correlated with rapid turnover. PEST regions are identified and scored by computer using an algorithm developed by Rogers et al. (1986).We thank Dr. Martin Rechsteiner (University of Utah) and Dr. William Boyle (Salk Institute) for their help with the PEST analysis.

jun:

ONCOGENE AND TRANSCRIPTION FACTOR

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PEST sequences are common markers for proteins with a short intracellular half-life (Rogers et al., 1986). Interestingly, the PEST score of v-Jun is less than that of the c-Jun proteins and that of Jun B is significantly lower than that of both viral and cellular Jun proteins. Additional negative control could be exerted at the transcriptional level through Fos. The Jun-Fos dimer has been reported to downregulate the transcription offos (Sassone-Corsi et al., 1988b). Less Fos would mean a reduction in the amount of Jun-Fos complex that can stimulate Jun transcription. Thus Jun transcription appears to be linked through a feedback loop to the transcription of Fos. The Jun protein is posttranslationally modified not only by phosphorylation (Curran et al., 1984, Angel et al., 1988a)but also by glycosylation (Jackson and Tjian, 1988). The carbohydrate side chains in Jun are 0 linked. There appear to exist two populations of Jun in the cell, one glycosylated, the other nonglycosylated. As with phosphorylation, a regulatory effect is suspected of glycosylation as well, but such an effect still remains to be proved. The data on positive and especially on negative regulation ofjun are still very fragmentary. They do not yet allow a comprehensive synthesis of the effects of all signals that control Jun. But it is already clear that the regulation ofjun will turn out to be quite complex and that it will be a key to understanding transcriptional activity in the cell. IX. The Oncogenicity of jun: Increased Dosage or Qualitative Change of the Jun Protein?

The viral j u n gene is responsible for tumor induction in ASV-17infected chickens and for transformation of cultured cells in uitro (Ball et al., 1989). Oncogenic transformation of normal primary chicken embryo fibroblasts by v-jun is a single-hit event (Cavalieri et al., 1985a,b). Expression of viral jun in chicken embryo fibroblasts is sufficient to cause oncogenic transformation. However, although viraljun is an efficient transforming agent, the transformed cells have a limited lifespan in culture: v-jun is not immortalizing (Ball et al., 1989). Tumors in the animal may sustain their more prolonged growth in part by continuously recruiting new cells through infection of adjacent tissue. The tumor cells may also undergo secondary genetic changes. An investigation ofjun oncogenicity must deal with two main questions: (1)What alterations of the cellularjun bring out its oncogenic potential? (2) What is the specific function of transformingjun that is responsible for oncogenicity? The alterations that activate the oncogenic potential of cellularjun may be either quantitative or qualitative. The

26

PETER K. VOGT AND TIMOTHY J. BOS

viral Jun protein is an efficient transcriptional activator in certain cells (Angel et al., 1988a; Imler et aZ., 1988a). It is indistinguishable from its cellular counterpart in two important properties: sequence-specific DNA binding and dimer formation with the Fos protein (Bos et al., 1988, 1989). Yet the viral protein differs structurally from cellular Jun. A comparison of the amino acid sequences of the two genes defines these differences. Starting with the carboxyl-terminal portion of the molecule, there are three nonconservative amino acid substitutions, two of these in the DNA-binding domain. The amino-terminal region of the viral Jun protein has suffered a 27-amino acid deletion; it also differs from cellular Jun by the addition of a Gag tail that is linked to the Jun sequences by 12 cell-coded amino acids (Nishimura and Vogt, 1988).Are any of these differences between viral Jun and cellular Jun essential for transforming activity?jun deleticn mutants and recombinants between cellular and viraljun provide a partial answer (Bos et al., et al., 1990): (1)A v-junlc-jun recombinant, in which the DNAbinding domain is derived from the cellular gene while the rest is viral, still transforms chicken embryo fibroblasts. The amino acid substitutions in the DNA-binding domain of viral j u n are therefore not essential for transformation. This conclusion is also supported b y the fact that no difference in DNA binding to the consensus AP-1 site and to two mutants of the site has been detected between v-Jun and c-Jun (Bos et al., 1988). ( 2 ) As already mentioned, the amino-terminal Gag tail of the v-Jun protein is dispensable for transformation. This includes the 12 cell-derived linker amino acids between Gag and Jun proper (Ball et al., 1989). ( 3 ) From the amino terminus of the v-Jun coding domain at least 20 amino acids can be deleted without abolishig transforming activity. (4)Deletion of the 98 amino-terminal amino acids of the Jun protein, including virtually all of the transcriptional activator domain, almost completely abolishes transforming activity. These results suggest that the amino terminus of v-Jun is essential for transformation, but it is not clear whether a qualitative change in that region of Jun is required. Data on this question come from a construct in which the cellular j u n gene was inserted into the RCAS avian retrovirus expression vector (Hughes and Kosik, 1984; Hughes et al., 1987) and the DNA of this vector was transfected into chick embryo fibroblasts. In the transfected cultures, foci of transformed cells which were morphologically similar to those seen after ASV 17 infection appeared. Therefore, overexpression of the cellular Jun by a retroviral LTR (long terminal repeat) appears to be sufficient to induce transformation. However, the number of foci produced by c-jun per unit DNA was reduced, compared to that found in control cultures transfected

jun: ONCOGENE AND TRANSCRIPTION FACTOR

27

with the same amount of DNA from a viral j u n expression vector. Chicken embryo fibroblasts transformed with the RCAS vector carrying c-jun are poorly tumorigenic in young chickens (Bos et ul., 1990). Since chickens are not inbred, the injected transformed cells are probably rejected and tumors arise from host cells that have become infected by the c-jun-containing retrovirus released from the injected cells. Additional studies will be needed to define the differences between the oncogenic potential of viral and cellularjun. These conclusions are in accord with studies in rat cell culture which have uncovered new features of jun-induced transformation (Schiitte et ul., 1989). In this work a Moloney leukemia virus-based vector carrying the human cellularjun gene was used. Primary rat embryo fibroblasts cannot be transformed by this vector alone. However, they become morphologically transformed and acquire tumorigenic potential if they are cotransfected with an activated Ha-rus gene. Cotransfection with myc is not effective. This result brings to mind the observation that rus is able to induce cellularjun (Imler et ul., 1988b)and raises the question of a possible cooperative role played by the indigenous c-jun gene in this cotransformation by external c-jun and rus vectors. In contrast to primary rat fibroblasts which are not immortal, Rat-1 cells, a continuous fibroblastic cell line, can be transformed by the Moloney virus vector carrying the human c-jun gene alone; cotransfection with activated rus is not necessary. These data support the conclusion that overexpression of cellular jun in normal mammalian cells can complement other constitutive growth signals to bring about oncogenic transformation but that by itself j u n maybe insufficient to induce oncogenesis in the mammalian system. Aside from the aforementioned work with rat cells, it has been difficult to transform mammalian cells with viral or cellular jun (A. Levinson, 1988 personal communication; P. K. Vogt, 1988 unpublished observations). This difficulty may be related to the fact thatjun is a poor transcriptional activator in some cell types. For instance, NIH 3T3 cells, widely used in transfection tests for oncogenicity, fail to show transcriptional activation by transfected jun, either because of some difference in the posttranslational modification of the Jun protein or because of the lack or inactivity of a (still unknown) cellular factor that may be required for the physiological activity of Jun (Imler et al., 1988a). Information on qualitative or quantitative changes that can make cellular j u n oncogenic has been steadily accumulating. However, the mechanism of viral jun oncogenesis remains unknown. A preliminary question here concerns the roles of the indigenous cellularjun andfos

28

PETER K. VOGT AND TIMOTHY J. BOS

genes. Since j u n transcription is positively regulated by Jun itself, alone or acting in conjunction with Fos, any cell that expresses a j u n gene introduced from outside, be it viral or cellular, would be expected also to overexpress the indigenous cellular j u n . Preliminary observations on jun-transformed chicken cells suggests that this autoregulation is not operative and that such transformed cells only express the introduced but do not overexpress the resident gene (Bos et al., 1990). Fos, on the other hand, is downregulated by the Jun-Fos heterodimer (Sassone-Corsi et al., 1988b). This autoregulation of Jun and Fos may result in an excess of Jun protein under some conditions of mitotic stimulation. Jun- Jun homodimers would form with transcriptional regulator properties that are distinct from those of the Jun-Fos heterodimers. Thus, a quantitative change in the level of Jun could lead to a qualitative change in the control of transcription. The fact that the transcriptional activator domain of Jun seems necessary for transformation suggests that Jun induces cancer through its role as transcriptional regulator. Although current work shows that in most instances JUII activates transcription of genes, Jun is also able to exert a negative control, as can be seen in the regulation offos transcription. If aberrant transcriptional control is accepted as the root cause o f j u n oncogenesis, then it becomes important to identify the relevant target genes. Many genes have AP-1 binding sites and are regulated by AP-1. Overexpression of some of these genes could conceivably contribute to the malignant cellular phenotype. Examples are collagenase, which may enhance tumor invasiveness (Angel et d., 1987), growth-related genes such as 11-2 (Fujita et al., 1986), or viral sequences, e.g., VL 30 (Wasylyk et al., 1988). It is not self-evident, however, that the gene targets for j u n oncogenesis will b e found among those that are normally regulated by AP-1. It is equally possible that the changes that makejun oncogenic result in an alteration ofthe target spectrum and that the oncogenically relevant genes are among those that have been either added or deleted. In any even, the number of genes important in transformation can be expected to be much smaller than the total number of genes regulated by jun.

X. The Main Functions of Jun Form a Hierarchical Order: A Hypothesis

The Jun protein has three distinct, separately assayable functions that are located in different domains of the molecule: (1)dimerization, (2) DNA binding, and (3)transcriptional activation. The viral Jun, and to some extent the cellular Jun proteins, have a fourth function, onco-

jUn:

ONCOGENE AND TRANSCRIPTION FACTOR

29

genic transformation. Dimerization is mediated by the leucine zipper domain (Kouzarides and Ziff, 1988; Sassone-Corsi et al., 1988c; Bos et al., 1989; Gentz et al., 1989; Schuermann et al., 1989; Turner and Tjian, 1989). Contact with DNA is probably made by a short basic region that is located adjacent to the amino terminus of the leucine zipper (Kouzarides and Ziff, 1988; Gentz et al., 1989; O'Shea et al., 1989; Turner and Tjian, 1989). Transcriptional activation requires acidic regions in the amino-terminal third of the Jun protein (Struhl, 1988). Dimerization of Jun is a prerequisite for DNA binding. Only homoor heterodimeric forms of Jun have been found to bind to DNA (Halazonetis et al., 1988; Nakabeppu et al., 1988, Gentz et al., 1989; Turner and Tjian, 1989). Mutational analyses of the leucine zippers of Jun, Fos, and of C/EBP also show that dimerization is necessary for DNA binding: all mutants in the zipper region that are defective in dimerization also fail to bind to DNA (Kouzarides and Ziff, 1988; SassoneCorsi et al., 1988c; Gentz et al., 1989; Landschulz et al., 1990; Schuermann et al., 1989; Turner and Tjian, 1989).On the other hand, mutants in the putative DNA contact region of Fos or of C/EBP can be found that do not interfere with dimerization but prevent DNA binding. Such mutants have a trans-dominant lethal phenotype (Kouzarides and Ziff, 1988; Landschulz et al., 1990; Gentz et al., 1989; Turner and Tjian, 1989). The existence of similar mutants can be postulated for Jun. Neither type of mutant-the one preventing dimerization or the other preventing DNA binding-can be expected to allow transcriptional activation. Conversely, deletion of presumptive transcriptional activator domains has no effect on dimerization or DNA binding (Kouzarides and Ziff, 1988; Nakabeppu et al., 1988; Bos et al., 1989; Sassone-Corsi et al., 1988~).In principle activator-negative mutations could also be trans-dominant lethals, but information on this possibility is still lacking. Unlike the DNA binding-negative mutants which are simply nonfunctional, activator negatives may function as specific repressors of transcription. These emerging relationships between the functions of Jun (as well as of Fos, C/EBP, or GCN4) are summarized in Fig. 12. Without dimerization there is neither DNA binding nor transcriptional activation. Without DNA binding there can be dimerization but no transcriptional activation, and without transcriptional activation both DNA binding and dimerization can occur. The domain required for DNA binding of Jun (or Fos, C/EBP, or GCN4) is bipartite: it consists of the dimerization domain proper and the DNA contact domain (Gentz et al., 1989; Landschulz et al., 1990; Turner and Tjian, 1989). Similar arguments about a hierarchical order of functions can be made

30

PETER K. VOGT AND TIMOTHY J. BOS

L I Transcriptional activation L I oncogenic transformation ~

FIG.12. Proposed hierarchial order of Jun functions. In this hypothetical scheme the higher level functions are prerequisite for lower level functions; e.g., dimerization is a precondition for DNA binding, transcriptional activation, and oncogenic transformation. Oncogenic transformation depends on ability to trans-activate,to bind to DNA, and to dimerize.

for Fos. Viral Jun and, under certain conditions, cellular Jun are tumorigenic. Circumstantial evidence suggests that this oncogenic transformation results from an aberration in the transcriptional control function of Jun. It would then follow that transcriptional control, DNA binding, and dimerization must be operative for Jun to induce oncogenesis. The oncogenic potential would be the last one in the hierarchy of Jun functions, dependent on the other three. There is some indirect evidence for this view from mutational studies on the Fos leucine zipper: mutants eliminating dimerization of Fos also abolish its transforming potential (Schuermann et al., 1989). XI. Jun Is a Signal Converter Many growth signals originate outside the cell with a hormone or growth factor. From there they are transduced in centripetal direction by chains of interacting proteins and second messengers. Virtually all components of these signal chains show oncogenic potential. Their genes have appeared as transforming effectors in genomes of retroviruses and, after mutation, amplification, or genetic rearrangement, they have been found as important determinants of the oncogenic cellular phenotype in nonviral tumors. It is likely that all components of cellular growth signals have an inherent oncogenic capability that can be activated by regulatory or structural changes of the corresponding genes. Incoming growth signals of the cell are typically of short duration. They set in motion a long-term cellular response that consists of programmed changes in the patterns of gene transcription. The

jUn: ONCOGENE

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31

program starts with the transcriptional activation of immediate early genes, independent of protein synthesis (Almendral et al., 1988;Ryder et al., 1988).The conversion of incoming signal into response occurs in the cell nucleus and at least some of it is performed by transcription factors. Many of these transcriptional regulators combine both afferent and efferent functions, acting as terminal receptors of the growth signal and as initiators of the programmed response. Jun is such a signal converter: it is activated by external growth signals and in conjunction with Fos and probably other proteins it alters transcription of specific genes, thereby triggering the cellular growth response. Interestingly, the transcriptional activation of the j u n gene itself is part of this response. The Jun-Fos complex is essential to growth-related transcriptional control. It also offers a unique opportunity to analyze oncogenesis at a level that is closest to the genome and to the regulation of gene expression.

ACKNOWLEDGMENTS Work of the authors is supported by U.S.Public Health Service Research Grant C A 42564 and Grant 1951 from the Council for Tobacco Research. We thank Glennis A. Harding for expert editorial assistance and Martha Termaat, Sarah Olivo, Gloria Barreras, and Esther Olivo for their help in producing the manuscript. The authors thank Tom Curran and Robert Tjian for stimulating discussions and for comments and suggestions on this article.

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Nakabeppu, Y., Ryder, K., and Nathans, D. (1988).Cell 55,907-915. Nishimura, T., and Vogt, P. K. (1988).Oncogene 3,659-663. O’Shea, E. K., Rutkowski, R.,and Kim, P. S. (1989).Sctence 243,538-542. Paluh, J. J., Orbach, M. J., Legerton, T. L., and Yanofsky, C. (1988).Proc. Natl. Acad. Sci. U.S.A.85,3728-3732. Perkins, K. K., Dailey, G . M., and Tjian, R. (1988).EMBO]. 7,4265-4273. Pertovaara, L., Sistonen, L., Bos, T., Vogt, P., Keski-Oja, J., and Alitalo, K. (1989).Mol. Cell. Biol. 9, 1255-1262. Piette, J., and Yaniv, M. (1987).EMBO]. 6, 1331-1337. Piette, J., Hirai, %I., and Yaniv, M. (1988).Proc. Natl. Acad. Sci. U.S.A.85,3401-3405. Probst, F., and Vande Woude, G . F. (1985).Nature (London) 315,516-518. Quantin, B., and Breathnach, R. (1988).Nature (London)334,538-539. Rauscher, F. J., 111, Sambucetti, L. C., Curran, T., Distel, R. J., and Spiegelman, B. M. (1988a).Cell 52,471-480. Rauscher, F. J., 111, Cohen, D. R., Currant T., Bos, T. J., Vogt, P. K., Bohmann, D., Tjian, R., and Franza, B. R.,Jr. (1988b). Science 240,1010-1016. Genes Deu. Rauscher, F. J., 111, Voulalas, P. J., Franza, B. R.,Jr., and Curran, T. (1988~). 2,1687-1699. Rogers, S . , Wells, R.,and Rechsteiner, M. (1986).Science 234,364-368. Ryder, K., and Nathans, D. (1988).Proc. Natl. Acad. Sci. U.S.A.85,8464-8467. Ryder, K., Lau, L. F., and Nathans, D. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 14871491. Ryseck, R.-P., Hirai, S. I., Yaniv, M., and Bravo, R. (1988).Nature (London) 334,535537. Sagata, N., Oskarsson, M., Copeland, T., Brumbaugh, J., and Vande Woude, G. F. (1988). Nature (London)335,519-525. Sassone-Corsi,P., Lamph, W. W., Kamps, M., and Verma, I. M. (1988a).Cell 54,553-560. Sassone-Corsi,P., Sisson, J. C., andVerma, I. M. (1988b).Nature(London)334,314-319. Sassone-Corsi, P., Ransone, L. J., Lamph, W. W., and Verman, I. M. (1988~). Nature (London) 336,692-695. Schonthal, A., Herrlich, P., Rahmsdorf, H. J., and Ponta, H. (1988).Cell 54,325-334. Schuermann, M., Neuberg, M., Hunter, J. B., Jenuwein, T., Ryseck, R., Bravo, R.,and Miiller, R. (1989).Cell 56,507-516. Schiitte, J., Minna, J. D., and Birrer, M. J. (1989). Proc. Natl. Acad. Sci. U.S.A. 86, 2257-2261. Setoyama,C., Frunzio, R.,Liau, G., Mudry, J. M., and De Crombrugghe, B. (1986).Proc. Natl. Acad. Sci. U.S.A.83,3213-3217. Sharma, A., Bos, T. J., Pekkala-Flagan, A., Vogt, P. K., and Lee A. S. (1989).Proc. Natl. Acad. Sci. U S A . 86,491-495. Shaw, G . , and Kamen, R. (1986).Cell 46,659-667. Sherr, C. J., Rettenmier, C. W.. Sacca, R., Roussel, M. F., Look A. T., and Stanley, E. R. (1985). Cell 41,665-676. Struhl, K. (1987a).Cell 49,295-297. Struhl, K.(1987b). Cell 50,841-846. Struhl, K. (1988).Nature (London)332,649-650. Turner, R.,and Tjian, R. (1989). Science 243,1689-1694. Vogt, P. K., Bos, T. J., and Doolittle, R. FI (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 3316-3319. Wasylyk, C., Imler, J. L., and Wasylyk, B. (1988).EMBO]. 7,2475-2483. Waterfield, M. D., Scarce, G. T., Whittle, N., Stroobant, P., Johnson, A., Wasteson, A,,

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PROTOONCOGENE C - ~ O SAS A TRANSCRIPTION FACTOR Robert J. Distel and Bruce M. Spiegelman Dana-Farber Cancer Institute and the Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School, Boston. Massachusetts 02115

I. Introduction 11. The Fos Gene and Its Expression 111. Role of Fos in Cell Growth and Differentiation IV. Fos Is a Participant in Sequence-Specific DNA Binding V. The Connection between Fos and Jun VI. The Interaction of c-Fos and c-Jun VII. Fos Can Trans-Activate Gene Promoters via the TRE VJII. Fos Stimulates the Binding of Jun to DNA IX. Fos May Interact with Other Sequences or Protein Complexes X. The TRE Is Subject to Regulation by c-Fos and Other Factors XI. Conclusions References

I. Introduction The phenotypic properties of transformed cells and tumors result from an abnormal program of gene expression that leads to disturbances in the control of cell proliferation and differentiation. To date some 50 oncogenes capable of causing these abnormal cellular functions have been isolated and in many cases their normal cellular counterparts, protooncogenes, have been cloned. The locations and properties of proteins encoded by many of these oncogenes or protooncogenes have implicated them in the processes of intercellular communication and intracellular signal transduction from the cell membrane to the appropriate cytoplasmic or nuclear target. For example, the oncogene v-sis product bears homology to a subunit of the secreted platelet-derived growth factor (Downward et al., 1983; Waterfield et al., 1983) and the erbB andfms oncogene products have striking homologies to growth factor receptors (Sherr et d., 1985; Ullrich et al., 1984). Inner membrane and cytoplasmic oncogenes include src (Courtneidge et al., 1980; Kreuger et al., 1980; Cross et al., 1984), ras (Buss and Sefton, 1986), raf(Morrison et al., 1988; Rapp et al., 1983), and mos (Papkoff et al., 1983). Finally, the nuclear protooncogenes c-erbA (Sap et al., Weinberger et al., 1986), c-fos (Sambucetti and 37 ADVANCES IN CANCER RESEARCH, VOL 55

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Curran, 1986),and c-jun (Angel et al., 1988; Bohmann et al., 1987)have been implicated more directly in gene regulation. The speculation that the nuclear oncogenes and protooncogenes are involved as transacting factors in the regulation of a set of genes is particularly intriguing; it is possible that they might give direct insight into how a mutation in a single gene can affect an entire genetic program. In this way, the oncogene becomes a tool not only for understanding the mechanism of uncontrolled proliferation in neoplastic disease but also may shed light on the complex regulation of normal cell differentiation. With the observation that v-erbA encoded the high-affinity nuclear receptor for thyroid hormone (Sap et al., 1986; Weinberger et al., 1986), it became clear that the nuclear oncogene family includes factors that can function directly in the activation of other genes. Recent discoveries have clarified the function of the nuclear protooncogene c-fos as a transacting factor that acts together with a second protooncogene, c-jun, to mediate transcriptional activity through the consensus sequence TGACTCA. This article does not attempt to give an exhaustive review of the c-fos literature, as several good reviews have recently appeared (Curran, 1988; Verma and Graham, 1987; Muller, 1986; Verma et al., 1984). Rather, we will focus on recent advances in the understanding of cellular gene regulation by c-fos. II. The Fos Gene and Its Expression

c-fos is the cellular homolog of the transforming gene (v-fos) of the murine sarcoma viruses FBJ and FBR (Curran et al., 1983; Finkel et al., 1966; Van Beveren et al., 1983). Characterisitic of these viruses is the causation of osteogenic sarcomas with a short latency after injection into newborn mice (Finkel and Biskis, 1968; Curran and Teich, 1982a,b; Curran and Verma, 1984). Both viruses have been shown to transform fibroblasts in culture (Curran et aZ., 1982; Curran and Verma, 1984; Finkel et al., 1966, 1973; Van Beveren et al., 1983, 1984). The v-fos and c-fos genes are both capable of transforming fibroblasts although the c-fos gene requires structural alterations, as discussed below (Miller et al., 1984). The protooncogene is highly conserved among vertebrates (Curran et al., 1983; Molders et al., 1987; Van Straaten et aZ., 1983); its protein is phosphorylated and located exclusively in the nucleus (Curran et al., 1984). The predicted molecular weight of the 380-amino acid c-fos protein is about 42K, although it often runs in polyacrylamide gel electrophoresis with an apparent molecular weight of between 54K and 62K, probably due to an unusually high proline content and to posttranslational modification

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AS A TRANSCRIPTION FACTOR

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(Curran et al., 1984; Muller et al., 1987; Verma et al., 1984). Phosphorylation on both v-and c-Fos appears to be primarily on serines and threonines (Barber and Verma, 1987). FBJ-v-Fos is less phosphorylated than c-Fos because a large portion of the carboxyterminus is lost by a frame-shift mutation; phosphoamino acid analysis has shown that the carboxyterminal region is the primary site of phosphorylation (Barber and Verma, 1987). The v-fos and c-fos proteins are immunoprecipitated from fibroblasts in tight but noncovalent association with basic cellular phosphoproteins called fos-associated proteins (Faps) (Curran and Teich, 1982b; Curran et al., 1985; Franza et al., 1987). Rat fibroblasts contain a Fap of 39 kDa (p39; Curran and Teich, 1982b; Curran et al., 1985) and rat pheochromacytoma cells (PC12 cells) have a Fap of 40 kDa (Franza et al., 1987). Like c-Fos, the p39 phosphoprotein is also primarily modified on serines and threonines (Barber and Verma, 1987). c-fos protein and mRNA are undetectable in most quiescent cells and require stimulation by hormones, serum, mitogens, or other ligands to reach easily detectable levels (see Curran, 1988, for review). Another means of elevating c-Fos levels is to tranfect cells with an activated c-fos gene. Activated constructs usually replace the 3’ untranslated region of c-Fos with the long terminal repeat (LTR) of FBJMSV (pMMV) (Miller et al., 1984). Interestingly, overexpressed pMMV c-Fos has a shorter half-life than induced endogenous c-Fos (Curran et al., 1984; Curran and Morgan, 1986; Greenberg and Ziff, 1984)and the rates of phophorylation also appear to be different for the two gene products (Curran et al., 1984; Curran and Morgan, 1985, 1986; Morgan and Curran, 1986; Muller et al.,1984b). The c-Fos derived from the transfected c-fos gene requires about 2 hr to be completely modified (Curran et al., 1984), whereas induced c-Fos appears to be fully modified within 15min (Curran and Morgan, 1985; Morgan and Curran, 1986; Muller et al., 1987). The level of modification for c-Fos appears to be dependent on the inducing agent used (Morgan and Curran, 1986). There are a number of immunologically related proteins that have been designated Fos-related antigens (Fras). Polyclonal antibodies made to amino acids 127 through 152 of c-Fos have detected at least three Fras of 30, 35, and 46 kDa in stimulated cells (Curran and Morgan, 1985; Franza et al., 1987; Kruijer et al., 1984; Muller et al., 1984b). The 46-kDa Fra has a peptide map distinct from that of c-Fos (Kruijer et al., 1984). The genes coding for the 35-kDa protein (Fra 1) (Cohen and Curran, 1988) and the 46-kDa protein (fos B) (Zerial et al., 1989) have been cloned and their products have several regions of

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homology to c-Fos. (Collectively we will refer to c-Fos and Fras as Fos.) Fras can be distinguished from Faps by treating cell lysates under strongly denaturing conditions before immunoprecipitation. Under these conditions only c-Fos and Fras but no Faps are precipitated (Curran et al., 1985). Ill. Role of Fos in Cell Growth and Differentiation The observation that protooncogenes might be involved in normal cell cycle regulation (Doolittle et al., 1983; Cochran, 1984; Downward et al., 1983; Waterfield et al., 1983) led to the examination of the transcription of a number of protooncogenes after treating quiescent cells with mitogens. c-fos became a focal point of interest when it was determined that the transcriptional activation of this gene is one of the earliest nuclear events occurring in response to mitogenic agents (Greenberg and Ziff, 1984; Kruijer et al., 1984; Muller et al., 1984a). This transcriptional induction has been shown to be transient in most cells, beginning minutes after stimulation (Greenberg and Ziff, 1984). The level of mature mRNA is at a maximum within 30 min and declines to undetectable levels within 90 min, while protein synthesis follows the accumulation of mRNA with a half-life of 2 hr (Curran and Morgan, 1986; Muller et al., 1984b). In keeping with this tight control of expression, it appears that the principal means of cellular transformation by v-fos is related to overexpression of its protein product (Miller et al., 1984) and derives in part from the fact that v-fos contains deletions in the carboxy-terminus of the protein and 3 ’ untranslated region of the mRNA, which appear to influence RNA stability (Fort et al., 1987; Meijlink et al., 1985; Rahmsdorf et al., 1987). Sequences in the coding region also play some role in mRNA stability (Kabnick and Housman, 1988; Shyu et al., 1989). Deletion of regions of the 3’ untranslated mRNA of c-fos and addition of a strong viral promoter make the c-fos gene capable of transforming cells without changing the primary sequence of the gene product (Miller et al., 1984). However, changes in the coding segment of the v-fos gene do contribute to transformation as transfection of cells with the v-fos protein-coding sequence linked to the 3’ untranslated mRNA from c-fos still results in focus formation (Miller et al., 1984) and a single substitution (Glu to Val at amino acid 138) in the primary amino acid sequence of FBR-MSV v-Fos renders it a more potent immortalizer of cells (Jenuwein and Muller, 1987). The rapid and transient induction of c-jos in response to many biological effectors (Greenberg and Ziff, 1984; Cochran et al., 1984) and other oncogenes (Sassone-Corsi and Borrelli, 1987; Schonthal et al.,

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1988) as well as the nuclear localization and extensive posttranslational modification of its product have suggested that c-fos activity might be a distal intermediate in the process of signal transduction. Functioning as a kind of nuclear switch, c-fos could translate diverse short-term events beginning with receptor binding, or other cell membrane-mediated events, into short- and long-term changes in gene expression (Morgan and Curran, 1986). The function of initiating the expression of long-term changes in gene expression is analogous to the function of viral immediate early genes (Lau and Nathans, 1987). The rapid appearance of c-Fos after treatment of quiescent cells with mitogens implicated c-fos in the early events in proliferation as a “competence” gene. This suggested role of c-fos in cell proliferation is consistent with the fact that c-fos is specifically induced by competence factors and not by the progression factors in platelet-poor plasma (Greenberg and Ziff, 1984; Cochran et al., 1984; Bravo et al., 1985) and many treatments that induce competence also induce c-fos (Verrier et al., 1986). In addition, experiments in which c-fos expression is blocked by antisense RNA in fibroblasts implicate c-fos in the transition from GI to S in quiescent cells (Nishikura and Murray, 1987). Similarly, microinjection of affinity-purified anti-c-Fos antibodies into quiescent cells inhibits cells in the progression from Go to GI or in early GI (Riabowol et al., 1988). It is not yet clear whether c-fos plays a critical role in continually cycling cells as there are reports that antisense mRNA expression does not affect growing cells (Levi and Ozato, 1988; Nishikura and Murray, 1987) although others have reported an inhibitory effect (Holt et al., 1986). The search for expression of protooncogenes in uiuo showed that expression of c-fos at high levels was restricted mostly to certain prenatal tissues such as visceral yolk sac, amnion, midgestation fetal liver (Mason et al., 1985; Muller et al., 1982, 1983) and the nervous system (Caubet, 1989). After birth, only hematopoietic cells, including macrophages (Gonda and Metcalf, 1984; Mitchell et al., 1985; Muller et al., 1984b, 1985), neutrophils (Kreipe et al., 1986), and mast cells (Conscience et al., 1986),display constitutive high levels of c-Fos (Muller et al., 1985; Conscience et al., 1986). In adult tissue, c-fos can be induced in heart by P-adrenergic agonists and pressure overload (Barka et al., 1987; lzumo et al., 1988), in the CNS by pharmacological treatment or direct neural stimulation (Dragunow and Robertson, 1987; Hunt et al., 1987; Morgan et al., 1987; Sagar et al., 1988), and in liver by glycine (Vasudevan et al., 1988). High levels of c-fos expression are associated with the induction of differentiation in several cell systems for the monocytic/macrophage

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lineage (Gonda and Metcalf, 1984; Mitchell et al., 1985; Muller et al., 1984b, 1985) and with the differentiation of certain embryonic tissues; this has led to the speculation that this gene is involved in the process of cellular differentiation. Intriguing as these correlations are, the induction of c-fos apparently does not occur in the differentiation of other monocytic/macrophage cell lines and makes the argument for a general role of c-fos in the differentiation of this cell type inconclusive (Mitchell et al,, 1986; Muller, 1986). It is clear, however, that a variety of differentiated cell types retain the ability to induce c-fos in response to particular biological effectors (Barka et al., 1987; Hunt et al., 1987; Dragunow and Robertson, 1987; Morgan e t al., 1987; Sagar et al., 1988; Vasudevan et al., 1988; Izumo et al., 1988),suggesting strongly that the role of c-fos is not just limited to cell growth but extends to tissue and cell type-specific gene expression. Furthermore, work done in differentiated cell types illustrates that the low levels of Fos present constitutively even without specific ligand stimulation play an important role in the expression of tissuespecific genes. Adipocytes express a lipid-binding protein termed adipocyte P2 (aP2) and the function of the promoter of this gene depends upon the binding of protein complexes containing Fos to its specific DNA-binding site (Distel et al., 1987; Rauscher et al., 1988a; Herrera et al., 1989), despite the fact these cells contain relatively low levels of Fos. Evidence for c-fos involvement in aberrant cellular differentiation is observed in bone tissue. Transgenic animals carrying the c-fos gene driven from a heterologous promoter express this protein in many tissues but show abnormalities only in bone development (Ruther et al., 1987). Infections with FBJ and FBR murine sarcoma viruses cause osteosarcomas exclusively (Curran, 1988). IV. Fos Is a Participant in Sequence-Specific DNA Binding

That c-fos itself might be a trans-acting factor with respect to the activation of other genes has been inferred from several observations: it is rapidly and transiently induced after stimulation of cells with a number of ligands; the protein accumulates rapidly in the nucleus after synthesis; it can be released from chromatin by DNase 1and micrococcal nuclease digestion (Sambucetti and Curran, 1986; Renz et al., 1987); and it has an affinity for double-stranded DNA (Sambucetti and Curran, 1986; Muller et al., 1987; Renz et al., 1987). In an attempt to ask whether v-fos could trans-activate type I11 collagen, one of the gene products known to be activated upon v-fos transformation of

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cells, v-fos constructs were cotransfected with the collagen type I11 promoter linked to a reporter gene (Setoyama et al., 1986). These experiments showed that the v-fos gene was capable of trans-activating the collagen type I11 promoter as well as the Rous sarcoma virus (RSV) LTR, although these experiments could not elucidate whether transactivation directly involved the v-fos protein. Direct involvement of c-fos or c-fos-like proteins (Fos) in sequence-specific DNA-binding complexes was first demonstrated by the observation that the cis-acting fat-specific element 2 (FSE2) involved in the control of the differentiation-linked expression of the adipocyte P2 gene (aP2) bound a nuclear protein complex that included Fos (Distel et al., 1987).The DNA binding of the protein complexes, as determined by gel shift assays, could be completely blocked by treating with anti-c-Fos antibodies prior to adding the DNA fragment (Distel et al., 1987) while preincubating the antibody with its cognate peptide prevented the inhibitory effect of the antibody. A role for Fos in the actual binding of these protein complexes to DNA was suggested by the fact that these antibodies could also specifically disrupt preformed protein/DNA complexes. The direct association of Fos with this DNA was shown by the ability of UV light to cross-link Fos to radiolabelled FSES (Distel et al., 1987).These studies strongly suggested that Fos was directly involved in a transacting complex but initial experiments were not able to distinguish whether this binding was due to c-Fos itself or to Fras. It was subsequently demonstrated by gel shift assays and Western blots that both v- and c-Fos bind to the FSES sequence and that the amount of binding activity was directly related to the amount of these proteins in a given cell extract (Rauscher et al., 1988a). Franza et al. (1988) demonstrated that the FSES sequence could be used to specifically purify c-Fos and Fras by a microscale DNA affinity precipitation assay. V. The Connection between Fos and Jun

The recognition of an association between the protooncogenes c-fos and c-jun came as the result of a number of seemingly unrelated lines of research. In brief, work on the SV40 early promoter had revealed an enhancer sequence designated AP-1 (Lee et al., 1987a) and a similar functional sequence was reported for the polyoma enhancer (designated PEA1; Piette and Yaniv, 1988) and in the activation of cellular genes by the tumor promoter 2-tetradecanoyl-phorbol-13-acetate(target sequence is designated TRE; Angel et aZ., 1987).A factor called activating protein-1 (AP-l), purified by DNA affinity chromatography using the AP-1 sequence, was capable of stimulating transcription from

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constructs containing the AP-1 site in vitro (Lee et al., 1987b) and was shown to contain the human cellular homolog of the avian tumor virus oncogene v-jun (Bohmann et d , 1987; Bos et uZ., 1988; Maki et al., 1987; Vogt et al., 1987). An examination of the 28-bp FSE2 sequence that bound Fos-containing complexes (Distel et al., 1987)revealed that it contained the octomer consensus sequence ATGACTCA (Rauscher et al., 1988a; Franza et al., 1988; Lucibello et aZ., 1988), which was equivalent to the TRE and the AP-l-binding site. (We will use the term TRE when referring to the consensus sequence ATGACTCA to reduce confusion with the trans-acting protein AP-1.) It was demonstrated using gel shift assays that a synthetic oligomer duplex containing the TRE from the human metallothionein IIa gene competed as efficiently for Fos binding to the FSE2 sequence as did the FSE2 sequence itself. The only homology between these two synthetic oligomers was the TRE, strongly suggesting that the Fos binding reported by Distel et al. (1987) was through the TRE. Supporting this idea, antibodies reacting with c-Jun were capable of specifically immunoprecipitating a protein complex photocross-linked to the FSE2 sequence oligomer (Rauscher et al., 1988a). In addition, both the FSE2 sequence and an AP-1 site from the LTR of the gibbon ape leukemia virus (GALV)could be used in DNA affinity precipitation assays to specifically purify C-FOS,as assayed on high-resolution two-dimensional gel electrophoresis (Franza et at., 1988).The affinity purification assays also specifically purified Fras and Faps. Since c-Fos and c-Jun appeared to be simultaneously present in the same extracts and have the same specific binding site, it was suggested that these trans-acting factors were likely to compete for the same binding site or interact via protein-protein interactions to form a single DNA-binding complex (Rauscher et al., 1988a; Franza et ul., 1988). VI. The Interaction of c-Fos and c-Jun

That c-Fos and c-Jun interact directly was shown by a combination of immunological and structural techniques demonstrating that c-Jun was identical or very similar to the previously identified Fap p39 (Rauscher et aZ., 1988b; Chiu et al., 1988; Sassone-Corsi et al., 1988a). Rauscher et al. (198813)demonstrated that p39 isolated by immunoprecipitation with anti-c-Fos antibodies comigrated on high-resolution two-dimensional gel electrophoresis with the protein immunoprecipitated by anti-Jun antibodies. A sequential series of immunoprecipitations with either anti-c-Fos or anti-Jun antibodies showed that the p39 precipitated by anti-c-Fos antibodies was recognized by anti-Jun anti-

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bodies (Rauscher et al., 1988b; Chiu et al., 1988; Sassone-Corsi et al., 1988a). Furthermore, c-jun purified from cells with an anti-Jun antibody and p39 purified with anti-c-Fos antibody showed identical twodimensional tryptic peptide maps (Rauscher et al., 1988b; Chiu et al., 1988; Sassone-Corsi et al., 1988a). If c-Fos and c-Jun are found in a single complex, then transcription factor AP-1 purified by sequence-specific DNA affinity chromatography might be expected to contain c-Fos. Indeed, it was shown that affinity purified AP-1 preparations contained, in addition to c-Jun, substantial amounts of several proteins that were immunologically identified as c-Fos and Fras (Rauscher et al., 1988b). Using radioactively labeled TRE as a probe of blotted proteins from purified AP-1 preparations, three proteins bound the TRE specifically; p39/Jun, a Fra of 36 kDa, and an 80-kDa protein of unknown identity. The bands which corresponded to c-Fos showed no binding to the TRE DNA (Rauscher et al., 1988b).When proteins from anti-c-Fos immunoprecipitations were isolated and renatured from polyacrylamide gels, only those that migrated with p39 showed binding to the TRE in gel shift assays (Sassone-Corsi et al., 1988a). In these experiments the bands corresponding to c-Fos were incapable of binding to the TRE, similar to the results achieved with blotted proteins (Rauscher et al., 1988b). The observation that c-Fos by itself does not effectively bind TRE DNA led to the hypothesis that c-Fos binds to this site indirectly through its interaction with c-Jun. VII. Fos Can Trans-Activate Gene Promoters via the TRE

Since c-Fos binding to the TRE appears to depend on c-Jun, it was of interest to investigate whether c-Fos depends on c-Jun in order to trans-activate gene expression. c-fos and v-fos expression vectors were cotransfected into cells along with reporter plasmids containing either the collagenase promoter (which contains a TRE) or several copies of the TRE and the herpesvirus thymidine kinase promoter linked to a reporter gene (Lucibello et al., 1988; Schonthal et al., 1988). The overexpression of c-Fos was shown to trans-activate the reporter constructs via the TRE, whereas control plasmids containing nonbinding mutations in the TRE were not trans-activated (Lucibello et al., 1988; Schonthal et al., 1988). In these experiments, induction of antisense c-fos mRNA completely blocked activation of the TRE constructions by TPA, suggesting that TPA mediates activation of these genes via c-Fos (Lucibello et al., 1988; Schonthal et al., 1988). The cloning of c-jun allowed similar cotransfection experiments to

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ROBERT J. DISTEL AND BRUCE M. SPLEGELMAN

be carried out to assess the contribution of both c-jun and c-fos separately and together (Bohmann et al., 1987; Angel et al., 1988; Lamph et al., 1988; Ryder and Nathans, 1988; Quantin and Breathnach, 1988; Ryseck et al., 1988; Illmer et al., 1988). F9 embryo carcinoma cells, which have little endogenous TRE-binding material (Piette et al., 1988), were used to demonstrate the activation of the TRE upon transfection with c-jun (Angel et al., 1988; Sassone-Corsi et al., 1988a; Lamph et al., 1988; Ilmer et al., 1988). On the other hand, cotransfection of c-fos and c-jun expression vectors have been reported to cause an increase in trans-activation of the TRE over c-fos or c-jun alone (Sassone-Corsi et al., 1988a). Similarly, F9 cells that express c-fos from an inducible promoter upon hormonal induction trans-activate a TREcontaining promoter only when c-jun is contransfected with the reporter gene (Chiu et al., 1988). Clearly, the addition of both protooncogenes greatly enhances the activity of the TRE, most likely by the formation of a single complex in the cells (Chiu et al., 1988). In addition, c-fos plays a role in the trans-activation of the TRE as antisense RNA to c-fos completely prevents trans-activation via the TRE when cells are stimulated by a number of agents (Lucibello et al., 1988; Schonthal et al., 1988). Whether endogenous c-fos is required for the trans-activation by c-jun alone has not been established. VIII. Fos Stimulates the Binding of Jun to DNA

The role of c-Fos in modulating the DNA-binding activity of c-Jun has been investigated using proteins synthesized in rabbit reticulocyte translation systems from purified mRNA. These defined translation products have been tested for their ability to form homodimers and heterodimers and to bind the TRE in a sequence-specific fashion (Halazonetis et al., 1988; Nakabeppu et al., 1988; Rauscher et al., 1988c; Kouzarides and Ziff, 1988). This simple system for making translation products also allowed for the creation of deletions in the amino and carboxyl ends of the molecules, which were then used to determine the regions required for protein-protein interactions as well as the stoichiometry of the Fos-Jun interaction. c-Fos alone translated in rabbit reticulocyte lysates does not bind the TRE even at high levels of DNA (Halazonetis et al., 1988; Nakabeppu et al., 1988; Rauscher et al., 1988c; Kouzarides and Ziff, 1988) in agreement with earlier results from protein blots (Rauscher et al., 1988b) and renaturation experiments (Sassone-Corsi et al., 1988a). In addition, the cotranslation of truncated 35S-labeled c-Fos and intact c-Fos does not result in the formation of homodimers (Halazonetis et

PROTOONCOGENE C-fOS AS A TRANSCRIPTION FACTOR

47

al., 1988).On the other hand, when c-Fos and c-Jun are cotranslated or mixed after translation, heterodimeric c-Fos/c-Jun complexes are formed and these complexes bind to the TRE DNA in a sequencespecific fashion (Halazonetis et al., 1988; Nakabeppu et d., 1988; Rauscher et al., 1988c; Kouzarides and ZifT, 1988).In contrast to C-FOS, in uitro-translated c-Jun (Jun-A) alone does bind to the TRE DNA, as do Jun-B and Jun-D, which share extensive homology with c-Jun (Hal; et al., 1988). azonetis et al., 1988; Rauscher et al., 1 9 8 8 ~Nakabeppu The affinity of the heterodimeric c-Fos/c-Jun complex for the TRE DNA has been determined to be between 8 and 25 times that of the c-Jun homodimer and the c-Fos/c-Jun affinity is similar to that found for related yeast transcription factor GCN4 (Hope and Struhl, 1986; Halazonetis et al., 1988, Nakabeppu et al., 1988; Rauscher et al., 1 9 8 8 ~Kouzarides ; and Ziff, 1988).To explain the increased affinity of measured the the heterodimer for TRE DNA, Rauscher et al. (1988~) half-life of dissociation of both c-Jun homodimers and c-Fos/c-Jun heterodimers from TRE DNA and found that the half-time of dissociation of the Jun-DNA complex was on the order of seconds whereas the c-Fos/c-Jun-DNA complex decayed with a half-time of approximately 3.5 min. Apparently, the interaction of c-Fos and c-Jun stabilizes the binding of the complex and may account for much of the increased affinity observed. The nature of the Fos-Jun heterodimer interaction with DNA is not clear but may be analogous to that for the yeast transcription factor GCN4. Both c-Jun and c-Fos share homology with GCN4 in regions of about 30 amino acids, rich in basic residues, followed by a region with 4 or 5 leucines spaced at 7-residue intervals. It has been proposed that the basic amino acids may interact with DNA while the leucine region forms long amphipathic a helix (a “leucine zipper”) which may interact with another such helix to form a very stable dimer (Landschultz et al., 1988). For GCN4 the arginine-rich region and the “leucine zipper” are sufficient to give dimerization (Hope and Struhl, 1986,1987).Several groups have begun to ask what regions are necessary for c-Fos/cJun interaction and DNA binding. In uitro translation products containing deletions of large regions of the amino- and carboxy-termini of c-Jun and c-Fos indicated that the ability of heterodimers to bind DNA in a sequence-specific manner resides in the regions of c-Fos and c-Jun containing the basic amino acids and the putative leucine zipper (Halazonetis et al., 1988; Nakabeppu et al., 1988; Kouzarides and Ziff, 1988; Gentz et ~1.~1989; Turner and Tjian, 1989; Neuberg et al., 1989). This also appears to be true for Fral (Cohen et al., 1989) and for Jun-B, Jun-D, and v-Jun (Bos et al., 1989; Ryder et al., 1988). Specific amino

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ROBERT J. DISTEL AND BRUCE M. SPIEGELMAN

acid substitutions of valine or alanine for leucines within the leucine heptad repeat domain of c-Fos demonstrate that they are necessary for the formation of Fos-Jun heterodimers. Substitution of the helixdestabilizing amino acid proline into the leucine repeat domain also causes loss of dimer formation (Gentz et ul., 1989; Turner and Tjian, 1989). Though the leucine-rich dimerization domain is necessary for DNA binding. the region rich in basic amino acids with monology to GCN4 and CREB (CAMPresponse element binding protein) has also been shown by deletion and amino acid substitution to be necessary for DNA binding and is believed to directly interact with the DNA (Gentz et al., 1989; Turner and Tjian, 1989; Schuermann et al., 1989; Neuberg et al., 1989). It was originally suggested that the monomers of the liver enhancerbinding protein C/EBP dimerize via the hydrophobic interaction of the leucine side chains. Such a structure would require an antiparallel arrangement of the protomers (Landschultz et al., 1988, 1989). However, spectrophotometric and hydrodynamic analyses of synthetic peptides made to the GCN4 putative “leucine zipper” suggest that a stable dimer in this protein is formed in a parallel arrangement of protomers (O’Shea et al., 1989).The effects of multiple substitutions of the leucines in this domain of c-Fos and c-Jun in this region are also more consistent with a parallel orientation of the subunits in the heterodimer (Gentz et al., 1989). Given the observation that c-Jun and c-Fos form a heterodimer and each monomer contains a conserved basic domain, each protein may recognize a half-site of the TRE palindrome binding site. Such a structure seems likely given the trans-dominant effect that deletions of the basic region of c-Fos have in preventing the DNA binding of c-Jun (Gentz et al., 1989; Turner and Tjian, 1989). A major question as yet unresolved is how c-Fos acts to increase the trans-activating activity of c-Jun. Two general hypotheses (not mutually exclusive) are that c-Fos regulates the affinity of Jun for DNA or that it specifically regulates the interaction of c-Jun with the transcriptional machinery of the cell. Given the data from in vitro DNA binding, the increased affinity of the c-Fos/c-Jun heterodimer compared to the c-Jun homodimer may be enough to explain the increased transcriptional activity of this complex. However, there is evidence that c-Fos may also increase the affinity of the Jun transcription complex for RNA polymerase by providing a negative domain (Lech et al., 1987) similar to the negative domain found in GCN4 and GAL4 that has been predicted to interact with other transcription factors and/or RNA polymerase I1 (Giniger and Ptashne, 1987; Hope et al., 1988).

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49

IX. Fos May Interact with Other Sequences or Protein Complexes

A key question is whether all of the biological effects of c-Fos occur via its interaction with c-Jun and subsequent binding to the TRE target sequence. It has been reported that c-Fos can act as a repressor of its own gene transcription (Sassone-Corsi and Borrelli, 1987; SassoneCorsi et al., 1988b; Schonthal et al., 1988,1989; Wilson and Treisman, 1988) and this trans-repression occurs at either the TRE or other elements (Sassone-Corsi e t al., 1988b; Schonthal et al., 1989). The other sequences responsible for the downregulation have yet to be determined, although there is some sequence homology between the c-fos gene promoter and the HSP70 gene promoter, which is also downregulated by overexpression of c-Fos (Sassone-Corsi et al., 1988b; Wilson and Treisman, 1988). A unique property of this regulation is that v-Fos is not capable of downregulating the c-fos promoter or the HSP70 promoter, suggesting that unlike the trans-activation function, transrepression may require the intact carboxy terminus of c-fos (SassoneCorsi et al., 1988b; Wilson and Treisman, 1988). It has been suggested that post translational modification of the carboxy terminus of c-Fos may play a role in its ability to act both as a transactivator and transrepressor (Wilson and Treisman, 1988).Others have suggested that the carboxy terminus of c-Fos is capable of forming a zinc finger (Molders et al., 1987). If such a structure forms, it may facilitate interaction with other proteins or with a different sequence of DNA. From these results, it appears possible that c-Fos may aIso work by interacting with other DNA-binding proteins or DNA sequences other than the TRE. X. The TRE Is Subject to Regulation by c-Fos and Other Factors

A complex picture appears in the understanding of gene regulation by c-Fos. The TRE appears to be a site potentially controlled by a number of related trans-acting factors: Jun-A, Jun-Byand Jun-D homodimers, as well as Fos/Jun heterodimers (Nakabeppu et al., 1988; In addition to the FoslJun family, the TRE can Rauscher et aZ., 1988~). bind the trans-acting factors that recognize the cyclic A M P response element, although at a substantially lower affinity (Hai et al., 1988; Hurst and Jones, 1987; Nakabeppu et al., 1988; Rauscher et al., 1988c; Yamamoto, 1988). This protein or proteins called CREB or ATF are biochemically distinct from c-Fos and c-Jun (Hai et al., 1988). Interestingly, the nucleotide sequence of a cloned cDNA of CREB protein from placental JEG cells predicts that, like c-Jun and C-FOS,this molecule contains a putative leucine zipper and a basic domain adjacent to

50

ROBERT J . DISTEL AND BRUCE M. SPIECELMAN

the basic region in c-Jun (Hoeffler et al., 1988). The cyclic AMP response element (CRE) resembles the TRE but has an additional guanidine TGACgTCA. A direct comparison of the ability of phorbol esters and cyclic AMP to stimulate trans-activation through the TRE and the CRE sequence in JEG-3 and Hep-G2 cells demonstrates that both 8-bromocyclic AMP and TPA can trans-activate the TRE while the CRE sequence is only trans-activated by 8-bromocyclic AMP. These observations make it formally possible that the TRE can be activated directly or indirectly by CREB as well as Fos/Jun complexes (Deutsch et al., 1988). Such a c-Fos/Jun-independent pathway is also suggested by the observation that the trans-activation of the TRE-containing transin gene by PDGF is dependent on c-Fos whereas EGF induction ofthis gene is not (Kerr et al., 1988).However, since trans-activation is measured long after stimulation and c-Fos and c-Jun turn over quite rapidly, it is possible that the relevant trans-acting factors here may be Fos and Jun-like molecules with different kinetics of accumulation, such as Fras (Curran and Franza, 1988).

XI. Conclusions

The past few years have seen a rather startling leap in our understanding of how the protooncogene c-fos protein may function as a transcription factor. At this point we can say with some certainty that at least some portion of c-Fos function occurs via its interaction with c-Jun and subsequent binding of this complex to the target sequence, termed TRE. This paradigm, though probably generally relevant to many actions of c-Fos, is almost certainly a great oversimplification when the regulation of particular genes in particular cell types is examined. The problem faced here is one of specificity. How can one protooncogene play an important role in the stimulation of cell growth, nuclear responses to a large number of hormones and ligands, and regulation of gene expression in resting, differentiated cell types? The answer, as implied throughout this article, is undoubtedly in the molecular complexities shown by this molecule and its related family. First, the Fos gene family itself appears to have many members, as does the Jun family. Second, the proteins of both of these families can undergo extensive covalent modifications. Third, it is not unlikely that Fos will be found to interact with other proteins outside the Jun family. Finally, as impressive as the potential number of different Fos protein complexes could be, it is highly unlikely that all Fos binding sites will be equivalent, both with respect to the cis-acting sequences and other

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protein factors that may potentiate or inhibit Fos-complex binding. Thus, a major effort in the next few years must move from the general to the specific: the detailed mechanisms of how the Fos family and its related proteins regulate particular gene activation or repression events.

ACKNOWLEDGMENTS We thank Ms. Adah Levens for assistance in the preparation ofthis manuscript and Dr. Michael Greenberg for helpful comments. R.J.D. is supported by a postdoctoral fellowship from the National Institutes of Health and B.M.S. is an Established Investigator of the American Heart Association.

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Nishikura, K., and Murray, J. M. (1987). Mol. Cell. Biol. 7,639-649. O’Shea, E. K., Rutkowski, R., and Kim, P. S. (1989). Science 243,538-541. Papkoff, J., Nigg, E. A., and Hunter, T. (1983). Cell 33, 161-172. Piette, J., and Yaniv, M. (1988). EMBO]. 6, 1331-1337. Piette, J., Hirai, S., and Yaniv, M. (1988).Proc. Natl. Acad. Sci. U S A . 85,3401-3405. Quantin, B., and Breathnach, R. (1988).Nature (London)334,538-539. Rahmsdorf, H. J., Schonthal, A., Angel, P., LiRin, M., Ruther, U.,and Herrlich, P. (1987). Nucleic Acids Res. 15, 1643-1659. Rapp, U. R., Goldsborough, M. D., Mark, G. E., Banner, T. I., Groffen J., Reynolds, F., and Stephenson, F. R. (1983). Proc. Natl. Acad. Sci. V.S.A.80,4218. Rauscher, F. J., 111, Sambucetti, L. C., Curran T., Distel R. J., and Spiegelman, B. (1988a). Cell 52,471-480. Rauscher, F. J., 111, Cohen, D. R., Curran,T., Bos, T. J.,Vogt, P. K., Bohman, D.,Tijan, R., and Franza, R. (1988b). Science 240,1010-1016. Rauscher, F. J., 111, Voulalas, P. J., Franza, R. J., and Curran T. (1988~).Genes Deo. 2, 1687-1699. Renz, M., Verrier, B., Kurz, C., and Muller, R. (1987). Nucleic Acids Res. 15,277-292. Riabowol, K. T., Vosatka, R. J., Ziff, E. B., Lamb, N. J., and Feramisco, J. R.(1988).M o l . Cell Biol. 8, 1670-1676. Ruther, U., Wagner, E. F., and Muller, R. (1985). EMBO]. 4, 1775-1781. Ruther, U., Garber, C., Komitowski, D., Muller, R.,and Wagner, E. F. (1987). Nature (London)325,412-416. Ryder, K., and Nathans, D. (1988). Proc. Natl. Acad. Sci. U S A . 85,8464-8467. Ryder, K., Lau, L. F., and Nathans, D. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 14871491. Ryseck, R.,Hirai, S. I., Yaniv, M., and Bravo, R.(1988).Nature (London)334,535-537. Sagar, S. M., Sharp, F. R.,and Curran, T. (1988). Science 240,1328-1331. Sambucetti, L. C., and Curran, T. (1986). Science 234,1417-1419. Sap, J., Munoz, A,, Damm, K., Goldberg, Y., Ghysdael, J., Leutz A., Beug, H., and Vennstrom, B. (1986). Nature (London)324,635-640. Sassone-Corsi, P., and Borrelli, E., (1987). Proc. Natl. Acad. Sci. U.S.A.84.6430-6433. Sassone-Corsi, P., Lamph, W. W., Kamps, M. and Verma, I. (1988a). Cell 54,553-560. Sassone-Corsi, P., Sisson, J., and Verma, I. M. (1988b).Nature (London) 334,314-319. Schonthal, A,, Herrlich, P., Rahmsdorf, H. J., and Ponta, H. (1988). Cell 54,325-3334. Schonthal, A., Buscher, M., Angel, P., Rahmsdorf, H. J., Ponta, H., Hatlori, K., Chiu, R., Karin, M., and Herrlich, P. (1989). Oncogene 4,629-636. Schuermann, M, Neuberg, M., Hunter, J. B., Jenuwein, T., and Ryseck, R.-P. (1989).Cell 56,507-516. Setoyama, C., Frunzio, R.,Liau, G., Mudryi, M., and decrombrugghe, (1986).Proc Natl. Acad. Sci. U.S.A.83,3212-3217. Sherr, C. J., Retternmier, C. W., Sacca, R.,Roussel, M. F., Look, A. T., and Stanley, E. R. (1985). Cell 41,665-670. Shyu, A,, Greenberg, M. E., and Belasco, J. G. (1989). Genes Deo. 3,60-72. Strickland, S., and Mahdavi, V. (1978). Cell 15,393-403. Turner, R.,and Tjian, R.(1989). Science 243, 1689-1694. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A. W., Lee, J., Yarden, Y., Libermann, T. A., Schleeinger, J., Downward, J., Mayes, E. L. V., Whittle, N., Waterfield, M. D., and Seeberg, P. H. (1984). Nature (London)309,418-425. Van Beveren, C., van Stratten, F., Curran T., Muller, R,and Verma, I. M. (1983). Cell 32, 124 1- 1255.

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Van Beveren, C., Enami, S., Curran, T., Muller, R., and Verma, I. M. (1984).Virology 135 229-243. Van Straaten, F., Muller, R., Curran, T., Van Beveren, C., and Verma, I. M. (1983). Proc. Natl. Acad. Sci. U S A . 80,3183-3187. Vasudevan, S., Lee, G., Rao, P. M., Rajalakshmi, S., and Sarma, D. S. R. (1988). Biochem. Biophys. Res. Commun. 152,252-256. Verma, I. M., and Graham, W. R. (1987).Adu. Cancer Res. 49,29-52. Verma, I. M., Curran, T., Muller, R., van Straaten, F., MacConnell, W. P., Miller, A. D., and Van Beveren, V. (1984).In “Cancer Cells 2” (G.F. Vande Woude et al.,eds.), Cold Spring Harbor Lab., Cold Spring Harbor, New York. Verrier, B., Muller, D., Bravo, R., and Muller, R. (1986). E M B O J . 5,913-917. Vogt, P. K., Bos, R. J., and Doolittle, R. F. (1987). Proc. Natl Acad. Sci. U.S.A. 84, 33 16-33 19. Waterfield, M. D., Scarce, G. T., Whittle, N., Stroobant, P., Johnsson, A., Wasteson, A., Westermark, B., Heldin, C., Huang, J. S., and Deuel, T. F. (1983). Nature (London) 304,35-39. Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Groul, D., and Evans, R. 1986). Nature (London)324,641-646. Wilson, T., and Treisman, R. (1988). EMBO J . 7,4193-4202. Yamamoto, K. (1988). Nature (London)334,494-498. Zerial, M., Toschi, L., Ryseck, R.-P., Schuermann, M., Muller, R., and Bravo, R. 1989). E M B O J . 8,805-813.

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STUDIES ON THE POLYOMA VIRUS TUMOR-SPECIFIC TRANSPLANTATION ANTIGEN (TSTA) Tina Dalianis Department of Virology, Stockholm City Council, MicrobiologicalLaboratory, 107 28 Stockholm,Sweden, and Departmentof Tumor Biology. Karolinska Institute. 104 01 Stockholm, Sweden

I. Introduction 11. Definition of the Polyoma Tumor-Specific Transplantation Antigen (TSTA) 111. Studies on the Immune Response against Polyoma Virus-Induced Tumor Development and Polyoma Virus-Induced Tumors A. In Vivo Studies B. In Vitro Studies C. Summary IV. Molecular Biology of Polyoma Virus V. Initial Studies on the Nature of the Polyoma Tumor-Specific Transplantation Antigen VI. Recent Studies on the Nature of the Polyoma Tumor-Specific Transplantation Antigen A. Studies on the Relationship between Polyoma TSTA and the Presence of Polyoma Virus-Coded T Antigens B. Studies on the Direct TSTA Activity of T Antigens and Attempts to Define TSTA Epitopes on T Antigens VII. Present View of the Polyoma Tumor-Specific Transplantation Antigen VIII. Future Prospects References

I. Introduction

Polyoma virus was detected independently by Gross (1953) and Stewart (1953) as a contaminant, in extracts from leukemias induced by murine leukemia virus (MLV), that was able to induce neoplasms of the salivary glands and other tissues in mice. The same virus was later also shown to induce a multitude of tumors when used to inoculate newborn mice, hamsters, rabbits, and rats and was therefore designated polyoma virus (Stewartet al., 1958; Eddy etal., l958,1959a,b). It was found to be present in several different laboratory colonies and strains of mice and to be common in wild populations of mice (Law et al., 1959; Rowe et al., l958,1959a,b). The virus was originally isolated and grown in vitro by Stewart et al. (1957) and was found to be able to induce in vitro transformation of untransformed mouse and rat cells (Vogtand Dulbecco, 1960). However, although polyoma virus is com57 ADVANCES IN CANCER RESEARCH, VOL. 55

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

58

TINA DALIANIS

mon in mouse populations, and is definitely potentially oncogenic in newborn mice of susceptible strains, detectable tumors are not normally induced by this virus (Stewart and Eddy, 1958).Hence, a naturally occurring potentially oncogenic virus had been detected that did not induce tumors under natural conditions. Polyoma virus provides a useful laboratory model for the study of tumor viruses. Unlike simian virus 40 (SV40) and adenovirus, experiments using polyoma virus may be performed in the natural host. The research following the discovery of polyoma virus has concentrated on (1)discovering the natural history of polyoma virus infection, including the study of immune responses that prevent tumor development, as well as analysis of the target antigen recognized by the immune system, and (2) defining the molecular biology of the virus in productive infection and during cell transformation. Ultimately the progress in the understanding of the immune responses that prevent tumor development and the molecular biology of the virus has led to the ability to understand the nature of the target structure recognized by the immune system. This article is concerned with the attempts to define the target structure recognized by the immune system.

II. Definition of the Polyoma Tumor-Specific Transplantation Antigen (TSTA) As mentioned above, newborn mice in pol yoma virus-carrying colonies do not develop polyoma tumors. This is due to the presence of maternal neutralizing anti-viral antibodies in the newborn mice, which delay infection for several weeks after birth (Stewart et al., 1958b; Law et ul., 1959). By the time the young are susceptible to polyoma virus infection their immune systems have matured and they are resistant to the oncogenic effects of the virus. Similarly, adult rodents inoculated with polyoma virus do not develop polyoma tumors, either. Studies performed by Habel(l961) and Sjogren et al. (1961)indicated that adult hamsters or mice immunized with polyoma virus had an increased resistance against the outgrowth of a subsequent inoculum of a syngeneic polyoma-induced tumor, as compared to nonimmunized controls. In further studies different polyoma tumors were used for immunization of mice. These mice were subsequently challenged with tumors of either polyoma or nonpolyoma origin, and this resulted in the rejection of polyoma tumors, i.e., cross-immunization between polyoma tumors, but no rejection of nonpolyoma tumors (Sjogren, 1961, 1964a). A common antigen was therefore suggested to be present on all mouse polyoma tumors

STUDIES ON THE POLYOMA VIRUS

TSTA

59

and this antigen was later functionally designated as the polyoma tumor-specific transplantation antigen (TSTA).

Ill. Studies on the Immune Response against Polyoma VirusInduced Tumor Development and Polyoma Virus-Induced Tumors

A. In Vivo STUDIES Studies by Law et al. (1959) demonstrated that mice bearing polyoma tumors had detectable quantities of virus-neutralizing antibodies. This suggested that anti-viral antibodies were not sufficient to cause tumor rejection. Therefore further studies were initiated in order to define the importance of the humoral versus the cellular immune response for prevention and outgrowth of polyoma tumors. Studies of newborn and also older mice and rats from polyomauninfected litters, i.e., with no neutralizing antibodies present, indicated that the animals were susceptible to the tumorigenic effect of polyoma virus only within the first week of life, suggesting the possibility that a mature immune system was necessary for tumor prevention (Law and Ting, 1965). Neonatal thymectomy in rats drastically increased the frequency of tumors that developed and also increased the period of susceptibility to tumor development by polyoma virus from l week to 2-3 weeks (Vandeputte et al., 1963). A similar effect was observed when rats were treated with anti-lymphocytic serum (Vandeputte, 1968), and this effect was emphasized when thymectomy and treatment with anti-lymphocytic serum were combined (Vandeputte, 1968). Allison and Law (1968) demonstrated that it was possible to induce polyoma tumors in adult mice after inoculation with polyoma virus, if the animals had previously been thymectomized and treated with anti-lymphocytic serum. To establish the relative importance of humoral and cell-mediated immunity in controlling oncogenesis, restoration experiments were carried out in such mice (Allison, 1974).Tumor development could be prevented by (1)passive transfer of anti-viral antibodies within 24 hr of viral infection, or (2) passive transfer of immune lymphocytes up to 7 weeks after virus infection. Anti-viral antibodies received 7 days after virus infection could not prevent tumor development, in contrast to passive transfer of immune lymphocytes, as demonstrated in Table I. Furthermore, treatment of immune lymphocytes with anti-8 serum and complement, i.e., depleting the population of immune T cells, abolished protection against tumor development. It was therefore concluded that T cells were crucial for

60

TINA DALIANIS

TABLE I DEVELOPMENT OF TUMORS IN CBA MICE INFECTED AS ADULTSWITH POLYOMA VIRUS' Preliminary treatment

Restoration at 7 weeks

Normal rabbit globulin Thymectomy and antilymphocyte globulin

None None Normal lymphoid cells Sensitized lymphoid cells Sensitized lymphoid cells Sensitized lymphoid cells treated with anti-8 serum and complement Antibody at 24 hr Antibody at 7 days None

Thymectomy and antilymphocyte globulin Thymectomy and antilymphocyte globulin Th ymectomy

Number of animals

Percentage with tumors

24 14 10 11 10 6

0 100

12 10 20

17 90 O

90

O 0 83

" From Allison (1974).

the protection against polyoma tumor development, while the effect of the anti-viral antibodies was due to a reduction of infectious viral particles.

B. In Vitro STUDIES In vitro test systems were also employed in the study of polyomaspecific humoral and cellular immune reactions. This approach could potentially allow detailed characterization of the immune response against polyoma virus-induced tumors. If favorable, it could also allow elucidation of the nature of the target antigen TSTA. Difficulties occurred in correlating the i n vitro findings to the actual events i n vivo during the rejection of a polyoma tumor, and surprisingly few polyoma-specific in vitro reactions have been obtained. 1. Cellular Zmmune Response a. The Colony-Znhibition Assay. In the colony-inhibition (CI) assay tumor cells are incubated in a Petri dish overnight, after which lymphocytes are added. Three or four days later the outgrowing tumor cells are counted (Hellstrom, 1967). This test can also be performed with the addition of anti-serum and complement instead of the addition of lymphocytes (Hellstrom and Sjogren, 1965). Polyoma-specific growth inhibition of polyoma tumor cells has been obtained with lymphocytes from pol yoma tumor-immunized rats (Datta and Vande-

STUDIES ON THE POLYOMA VIRUS

TSTA

61

putte, 1971; Sjogren and Borum, 1971). Such growth inhibition was elicited both by lymphocytes from polyoma tumor-bearing rats and from rats having rejected a living polyoma tumor challenge. It was also shown that CI could be blocked by using serum from rats with growing tumors, but not from animals which had rejected a polyoma tumor. It was concluded from these results that a cellular immunity is also present in tumor-bearing animals, but that the rejection of the tumor is inhibited by components in the serum. b. Cytotoxic Tests. One exceptional report by Greene et al. (1982) demonstrated the possibility of obtaining a strong polyoma-specific cytotoxicity using spleen cells from mice immunized in a 51Cr-release assay; however, no further reports have been published since then. Repeated attempts by others to achieve polyoma-specific cytotoxicity in a 51Cr-release assay have been unsuccessful (Vandeputte, 1982; T. Ramqvist, unpublished observations). c. Macrophage Migration Inhibition Assay (MMZ). In the macrophage migration inhibition test peritoneal exudate cells from polyoma virus-immunized or nonimmunized animals are incubated together with cell or membrane extracts of polyoma or nonpolyoma origin, or together with synthetic peptide antigens in a small droplet in a migration chamber overnight (Szigeti et al., 1982; Ramqvist et al., 1986; Reinholdsson et al., 1989). The areas of migration of the macrophages are then measured, and the migration areas of macrophages exposed to the antigen are divided by the migration areas of the macrophages without antigen. In this way a migration index (MI) is obtained. Polyoma-specific MIS have been obtained with cell and membrane extracts from polyoma tumors, as well as with synthetic peptide antigens derived from the sequence of small and middle T antigens (Szigeti et al., 1982; Ramqvist et al., 1986; Reinholdsson et al., 1989). This test is believed to represent an in uitro model of delayed hypersensitivity and graft rejection responses (Bloom and Bennet, 1966). T helper cells presumably identify an antigen and release lymphokines which attract macrophages and inhibit their migration. This is thought to be a very early step in the activation of lymphocytes. 2. Humoral Immune Responses a. Virus Hemagglutination-InhibitingAntibodies. Virus hemagglutination-inhibiting antibodies are present in mice that have been infected with polyoma virus (Law et al., 1959). They recognize the viral capsid antigens of polyoma virus and have a neutralizing effect against virus infection; however, such antibodies are present in tumor-bearing mice, and do not prevent tumor development, as mentioned above (Law and Ting, 1965; Allison, 1974).

62

TINA DALIANIS

b. Complement-Fixing Antibodies and Nuclear Zmmunofluorescence Staining. It is possible to detect nuclear staining (staining of T antigens) with antibodies from virus-immunized or tumor-bearing animals (Law and Ting, 1965). However, since such antibodies are present in tumor-bearing animals, they have not been regarded to b e sufficient for tumor rejection. c. Antibodies Directed against the Surjace of Polyoma-Derived Tumors. Attempts have been made to demonstrate polyoma-specific antigens (Malmgren et al., 1968; Witz et al., 1976; Kitahara et al., 1978; Witz and Meyer, 1984). Results of such experiments remain inconclusive. It can be demonstrated that cells which are lytically infected can express viral capsid antigens on their surface (Malmgren et al., 1968;T. Dalianis, unpublished observations). However, other studies have demonstrated that tumor-associated surface antigens on polyoma tumors are not ultimately polyoma specific (Kitahara et al., 1978; Witz and Meyer, 1984). Weak subcellular immunofluorescent staining of polyoma-transformed cells with monoclonal antibodies against POlyoma middle T (MT)and large T (LT)has been described (Dilworth et aZ., 1986).

C. SUMMARY In conclusion, the majority of the combined in vivo and in vitro evidence indicates that a cellular response is responsible for the rejection of polyoma tumors. It is therefore not surprising that early attempts to identify a tumor-specific transplantation antigen of polyoma virus by serological methods were without success. Increasing accumulated knowledge of the molecular biology of pol yoma virus during the last 10-15 years, and a general progress in the field of immunology, has enabled the scientific community to take different approaches. IV. Molecular Biology of Polyoma Virus

Polyoma virus is a papovavirus, and has a circular double-stranded DNA genome, surrounded by its capsid, composed of virus-encoded capsid proteins. Virus particles have an icosahedral symmetry with a diameter of 45 nm. The genome consists of approximately 5290 bp and can be divided into two coding regions, the early and late region, and one noncoding region that separates the two former and within which the enhancers, promoters, and the origin of replication are situated as shown in Fig. 1 (for reviews see Griffin et al., 1980; Ito, 1980). Transcription proceeds bidirectionally from the noncoding region and in

STUDIES ON THE POLYOMA VIRUS TSTA

63

FIG.1. Organization of the polyoma virus genome. Sequence according to Griffin et al. (1980). OR, Origin of replication; LT, large T antigen coding region; MT, middle T antigen coding region; VPl-3, viral protein 1-3 coding regions.

both the early and late regions overlapping reading frames are used that enable the virus to use the genome more efficiently. The early region encodes three proteins, large T (LT), middle T (MT), and small T (ST) antigen, of approximately 100,55, and 22 kDa in size. All three are produced in the transformed cell and during the lytic cycle. Due to RNA splicing and frame shifts the three T antigens share an initial 79-amino acid N-terminal sequence. MT and ST share an additional 113 N-terminal amino acids, while the C-terminal sequences are unique for each T antigen (Ito, 1980). LT has a DNA-binding activity, and is located in the nucleus. A full-length LT is essential for lytic infection, since it will bind to the noncoding region and inhibit

64

TINA DALIANIS

transcription from the early region, but initiate replication of the virus and transcription from the late region (Ito, 1980; Cowie and Kamen, 1984). Furthermore, it has an immortalizing oncogene function (Land et al., 1983; Ruley, 1983). MT is located on the inside of the plasma membrane and in the cytoplasm and has no known function in the lytic cycle. However, it is responsible for the transformed phenotype, and has an oncogene function corresponding to that of ras, which enables it to cause transformation in cooperation with nuclear oncogenes, or to transform already established cell lines (Ito, 1980; Land et al., 1983; Ruley, 1983).A proportion of MT complexes with pp6OSrc.The complex has tyrosine kinase activity and has been suggested to be of importance for the transforming ability of M T (Courtneidge and Smith, 1984). More recent studies have demonstrated that MT also complexes with other cellular proteins, one of which has an approximate size of 81 kDa, and which may be a phophatidylinositol kinase (Courtneidge and Heber, 1987). ST is present in the nucleus and the cytoplasm and potentiates the transforming efficiency of M T (Cheng et al., 1988; Cuzin, 1984) and will also potentiate virus production (Martens, et al., 1989). The late region is transcribed only during the lytic cycle, and encodes the three viral capsid proteins. Since transcription from the late region is not necessary, and is often not present in transformed cells, the products of the late region have not been regarded as important for the induction of the polyoma TSTA, and will not be described here (for review see Ito, 1980).

V. Initial Studies on the Nature of the Polyoma Tumor-Specific Transplantation Antigen Prior to the development of the field of polyoma virus molecular biology, no attempts have been made to correlate the presence of polyoma TSTA to any particular product of the virus, since naturally that would not have been possible. Nonetheless, repeated selection in uiuo of polyoma tumors in polyoma virus-immunized mice had been attempted in order to select variants that had lost the polyoma virus tumor-specific transplantation antigen (Sjogren, 1964b). When this proved to be unsuccessful, it was suggested that TSTA itself was a stable antigen and that its presence was correlated to the transforming properties or products of the virus (Sjogren, 1964b). Later efforts mainly concentrated on finding serological reagents that could identify pol yoma-specific antigens. Although antigens were detected serologically on polyoma tumors, they were frequently found

STUDIES ON THE POLYOMA VIRUS

TSTA

65

to be cross-reactive with, for example, embryonic antigens (Kitahara et al., 1978). A separate report described a serologically determined polyoma tumor-associated antigen (Witz et al., 1976). This 70-kDa antigen was, however, later shown to still be present on tumors of polyoma origin that had lost all their polyoma DNA (L. Lania and R . Kamen, 1979 unpublished observations). It is not unlikely, however, as mentioned above, that the difficulties in defining a tumor-specific transplantation antigen serologically in the polyoma system could in part be due to the fact that the immune response against tumor development and tumor rejection is basically dependent on the cellular immune system rather than the humoral immune system. Therefore it is not surprising if antibodies against the tumor-specific transplantation antigen target epitope(s) do not naturally exist, or are difficult to identify. As the knowledge of the molecular biology of polyoma virus increased, attempts were made to correlate the presence of polyoma TSTA with the presence of products of polyoma virus, and in particular with products of the early region of the genome, since this is the only region which is necessarily transcribed in transformed cells. VI. Recent Studies on the Nature of the Polyoma Tumor-Specific Transplantation Antigen

A. STUDIESON THE RELATIONSHIP BETWEEN POLYOMA TSTA AND THE PRESENCE OF THE POLYOMA VIRUS-CODED T ANTIGENS 1. Polyoma Mutant Viruses w i t h Small Deletions in the Coding Region for MT and LT, and MT and ST, Can Still Induce an Anti-TSTA Response By 1979 a number of polyoma virus mutants with small deletions in the early region had been made and characterized. Some of these mutants had deletions in the coding region common for middle and large T antigen, and were designated dl mutants, while others had deletions in the region encoding the middle and small T antigen and were designated hr-t mutants. The hr-t mutant NG-18 (Benjamin, 1970) and dl mutants such as dl 23 (Griffin et al., 1979; Griffin and Maddock, 1979)and dl 1015 (Magnusson and Berg, 1979; Magnusson et al., 1981) were nontransforming, while other mutants such as dl 8 (Griffin et al., 1979; Griffin and Maddock, 1979) and d11013 (Magnusson and Berg, 1979; Magnusson e t al., (1981) could still transform. The mutants and their deleted regions are shown schematically in Fig. 2. The ability of these small-deletion mutants to immunize against polyoma TSTA in uiuo, i.e., to induce an anti-TSTA response, was

66

TINA DALIANIS 70

80

90

10010

10

20

Map Unlts

Large T-antigen Middle T-antlgen

Small T-antigen

FIG.2. The polyoma virus early region. Map positions of virus mutants are demonstrated. Splicing of RNA is required for the expression of the three T antigens (Ito, 1980). The cross-labeling indicates that the C-terminal sequences of LT, MT, and ST are translated in different reading frames. The sequences deleted in the mutants NG-18, dl 8, dl 23, dl 1013, and dl 1015 are indicated.

investigated. By this method it should be possible to correlate the presence or absence of the deleted regions to the presence or absence of TSTA, and hopefully the relationship of TSTA and the virusencoded products would be elucidated. Furthermore, the outcome of the experiments would also address the question of whether or not there is a correlation between the ability of the viruses to transform and their ability to induce an anti-TSTA response, since some of the mutants were transforming and some were not. These studies were initiated by Dalianis et al. (1982), and it was shown that the anti-TSTA response induced upon in vivo immunization with the mutants dl 18, dl 23, dl 1013, dl 1015, and NG-18 was not totally abolished by any of the small deletions present, and showed no correlation to the transforming ability of the mutant used, since some were transforming and others not. Figure 3 shows that small variations in the ability of the mutant viruses to replicate did not affect their immunizing capacity, since the ability of the viruses to induce hemagglutination-inhibiting antibodies is not correlated to the percentage of tumors developing within the different groups immunized with the different mutants. However, the NG-18 mutant replicated very poorly in oiuo and therefore it was not surprising that the antiTSTA response induced by this mutant was impaired yet remained significant. When the poor replicative activity of the mutant strain was compensated for by an increased quantity of the virus used for immunization, an improved anti-TSTA response resulted (Dalianis et al., 1982).

67

STUDIES ON THE POLYOMA VIRUS TSTA

10240-

A

.

p

...

....4 ..

...

+I

5120I-+

.

2560-

.-5

c, CI

1280640-

I

320-

5

160-

+...I

I--+

Ij k....+

8040-

+-.-+

20-

..

Control NG-18

.

..

46

d

d I8

123 d11013

wt

d11015

FIG.3. Immunity against polyoma TSTA after titration of anti-viral antibodies (A) of sera from mice immunized with polyoma virus mutants or wild-type virus. The animals were challenged with 0.5-1 x lo5of the polyoma tumor SEBB. The formation of palpable tumors was scored (B) and the data pooled. Figures within bars indicate the number of tumor takes per total number of mice within each group.

It was concluded that small deletions in the genome of polyoma virus did not abolish its ability to induce an anti-TSTA response, and that polyoma virus mutants that could not transform could still immunize against polyoma TSTA, indicating that TSTA was not correlated to the transforming capacity of polyoma virus. Furthermore, a mutant NG-18 that induced only a full-length large T antigen and N-terminal unstable fragments of small and middle T antigen could also induce an

68

TINA DALIANIS

anti-TSTA response. This was at the time surprising, since only M T was known to have any connection whatsoever with the cell surface, and it was generally believed that a TSTA had to be situated in the plasma membrane and retain a normal configuration detectable from the outside. As the concept of antigen processing had yet to be discussed (Babbit et al., 1985; Townsend et al., 1984, 1986), it was suggested that polyoma TSTA was a cellular product induced by the presence of one or more of the polyoma tumor antigens (Dalianis et al., 1982). Additional studies were performed in order to assess the relationship of the early region products and polyoma TSTA.

2. Cross-lrnmunization between Pol yoma Virus-Transformed Mouse and Rat Cell Lines It was generally presumed that ifpolyoma TSTA really was a cellular product, it would be likely to differ within different species. Previous studies by Habel (1962), where the TSTA of polyoma mouse and hamster tumors had been studied, indicated that this really may be the case; however, since these results were limited, Ramqvist and Dalianis (1984)proceeded with an investigation to resolve if a common polyoma TSTA was present in rat and mouse polyoma tumors. They found that it was possible to immunize mice against polyoma TSTA with rat polyoma tumors, indicating that whatever the target antigen may be in rats or mice, i.e., T antigen derived or of cellular origin, it was definitely closely related between the two species.

3. Zmmunizution against TSTA with Cells or Vuccinia Virus Vectors Expressing lndividual T Antigens Subsequent investigations considered whether TSTA actually was a T antigen. Were subdetectable amounts of T antigens present on the cell surface? Was TSTA correlated to the induction of a cellular product, and if so, which of the T antigens was responsible for the induction of this cellular product? Attempts were therefore made to immunize against polyoma TSTA with tumors expressing different spectrums of T antigens (Dalianis et al., 1984). Cell lines were now available that expressed only MT, e.g., 2.8, (Triesman et al., 1981),or only full-length large T, with nonfunctional fragments of small and middle T antigen, e.g., 1837 (Lania et al., 1979). The anti-TSTA response after immunization with these cell lines obtained by Dalianis et ul. (1984) is represented in Table I1 for one experiment. Table I1 shows a reduced take incidence of the rat polyoma tumor SEBDAS in rats that were immunized with the 2.8 cell

STUDIES ON THE POLYOMA VIRUS

TSTA

69

TABLE I1 TAKES OF SEBDAS IN SYNCENEIC BDX RATSAFTER IMMUNIZATION WITH DIFFERENT RAT LINESO Rats immunized with Cell dose

Untreated controls

SEBDAS

2.8

59-3-1

1837 114

105 105 1o5

8/10' 919 10113

2/5d 1/3d

2/4d 2/6d

014 4/4d 1/7d

416

Tumor takes: Percentage takes:

27/32 84

3/17 18***

4/10 40*

5/15 30***

5/10 50

019

Rat-1

515

515 100

From Dalianis et al. (1984). Irradiated cells were for immunization. Figures denote the number of rats that died with progressively growing tumors over the total number of rats. Prolonged latency compared to controls. * p < 0.05 compared to untreated controls. *** p < 0.001 compared to untreated controls.

line that expressed only MT, indicating that LT was not necessarily essential for the induction of an anti-TSTA response. Similar results were obtained with the 1837 cell line that expressed only full-length LT, with nonfunctional fragments of MT and ST, indicating that a full-length LT may indeed be sufficient for an anti-TSTA response without the presence of full-length MT and ST. Table I1 also shows that a nonpolyoma line, Rat-1, does not immunize against polyoma TSTA, while a wild-type polyoma tumor, SEBDAS, as well as the cell line 59-3-1, transformed with a polyoma mutant that lacks the C-terminal part of LT (Szigeti et al., 1982) can immunize against polyoma TSTA. It was concluded from these studies that the presence of either MT or LT could be sufficient for the induction of an anti-TSTA response. Furthermore, these findings did not support the model that only one specific cellular product was involved in the anti-TSTA response, since it would be unlikely that the presence of M T in one cell line, and the presence of full-length LT and nonfunctional fragments of MT and ST in the other cell line, would result in the induction of the same cellular product. The results of Dalianis et al. (1984)were later supported by the more recent findings of Lathe et al. (1987), where MT, ST, or LT were introduced separately or together into vaccinia virus vectors and used for the immunization of rats against polyoma tumors. A successful

70

TINA DALIANIS

anti-TSTA response was elicited with vaccina virus vectors introducing M T or LT or all three T antigens intracellularly, while the introduction of only ST in the vaccinia virus vector did not result in an anti-TSTA response. Thus it was demonstrated that the presence of either M T or LT in an intracellular context was sufficient to induce an anti-TSTA response. Taken together the results of Dalianis et al. (1984)and Lathe et al. (1987) suggested that the expression of M T or LT was correlated to the anti-TSTA response and that this was likely to be a direct effect, although an indirect effect could not be entirely excluded. There are three possible explanations as to why the vaccinia construct carrying ST did not immunize against polyoma TSTA. One possibility is that ST did not possess TSTA activity, or alternatively that the vector construction was suboptimal, resulting in, for example, inefficient processing of ST (Lathe e t al., 1987).A third possibility was that this vector preferentially induced a humoral immune response rather than a cellular immune response (R. Lathe, personal communication). 4. Selection against Polyorna TSTA

The accumulated data did suggest that TSTA could be a T antigen (Ramqvist and Dalianis, 1984; Dalianis et al., 1984; Lathe et al., 1987) and therefore a repeated attempt to select a TSTA-negative variant was made (Ramqvist et al., 1987).The aim of this study was to correlate the disappearance of TSTA with changes observed in the expression of polyoma virus products (Ramqvist et al., 1987). A previous attempt by Sjogren (196413)had been unsuccessful, possibly because TSTA was in some way related to the product responsible for transformation. Therefore in this study a mouse polyoma tumor, SEBB, was fused to a mouse Moloney virus lymphoma, YACUT. In this way, even if selection would be directed against a polyoma product responsible for transformation, for example M T or LT, the Moloney lymphoma partner in the somatic hybrid, SEBB/YACUT, would be able to contribute to the transformed phenotype and still enable a successful selection of a polyoma TSTA negative variant. As demonstrated in Table 111, after 10-13 passages of the hybrid in polyoma virus-immunized mice two TSTA-negative variants were obtained, SEBB/YACUT B and SEBBI YACUT C (Ramqvist et al., 1987). These were tested for T antigen expression. SEBB/YACUB B had lost all polyoma DNA, and therefore all T antigen expression (Ramqvist et al., 1987). SEBB/YACUT C had also lost all T antigen expression as judged by immunoprecipitation experiments, but part of the polyoma genome was still present (Ramqvist et al., 1987). SEBB/YACUT C had retained 1.5 copies of the 2.5 integrated copies of the polyoma genome that were found in the origi-

STUDIES ON THE POLYOMA VIRUS TSTA

71

TABLE 111

DECREASE INTSTA EXPRESSION' Take incidence Experiment 1

2 3

4

5

6

SEBBIYACUT subline Parental A B C Parental B C Parental B C Parental

B C Parental B C Parental B C Parental B C C (in uitro)

Selective passage 0 0 0 3 3 3 0 8 8 0 8 8 0 12 12 0 12 12 0 13 13 0 13 13 13

Cell dose lo3

Virus-immunized

(%)

(W

517 (71)

017 (0) 017 (0) 017 (0) 019 (0) 119 (11) 118 (12) 014 (0) 215 (40) 214 (50) 015 (0) 115 (20) 313 (100) 014 (0) 314 (75) 314 (75) 014 (0) 314 (75) 214 (50) 014 (0) 113 (30) 214 (50) 014 (0) 314 (75) 315 (60) 014 (0) 014 (0) 014 (0)

lo4

618 (75)

105 103 104 105 103 103 103 104 104 104 103 103 103 104

717(100) 415 (80) 515(ioo) 515(ioo) 314 (75) 415 (80) 515(100) 414(ioo) 315 (60) 313(100) 314 (75) 314 (75) 414(100) 314 (75) 214 (50) 112 (50) 314 (75) 014 (0) 314 (75) 314 (75) 215 (40) 415 (80) 114 (25) 214 (50) 314 (75)

lo4 104 103 103 103 104 lo4 104

lo4

lo5 lo6

a

Control

From Ramqvist et al. (1987).

nal parental hybrid SEBB/YACUT. The lack of T antigen expression was originally attributed to the loss of the one of the polyoma virus genomes; hwoever, after in uitro culture of the SEBB/YACUT C line for a few months, although no change was observed in the 1.5copy of the polyoma virus genome, T antigens were reexpressed in the same way as for the unselected parental hybrid (Ramqvist et al., 1987).When the in vitro-passaged, T antigen-expressing SEBB/YACUT C was tested for TSTA expression, it was found that TSTA expression was regained (Table 111). Thus, without any selective pressure from the

72

TINA DALIANIS

immune system, both T antigen and TSTA were reexpressed in this mutant. The presence of TSTA could not be correlated with any specific T antigen; however, it was concluded, also in accordance with the results of Dalianis et al. (1984) and Lathe et al. (1987), that it was likely that TSTA could be related to more than one T antigen, and that more than one active TSTA epitope per T antigen could be present (Ramqvist et al., 1987).

B. STUDIES ON THE DIRECTTSTA ACTIVITYOF T ANTIGENS AND ATTEMPTS TO DEFINETSTA EPITOPES ON T ANTIGENS

1. lmrnunization against Polyoma TSTA with Puriied T Antigens: A TSTA Epitope Resides within the 191 N-Terminal Amino Acids Common to MT and ST In order to address the question of whether T antigens were directly responsible for the anti-TSTA response elicited, one would have to immunize with purified T antigens. It had previously been described by Pallas et al. (1986) that it was possible to grow T antigens or parts of T antigens in bacteria, and that these T antigens could be purified and used for immunizing mice in order to obtain monoclonal antibodies directed against T antigens. Immunization of mice with these purified T antigens, illustrated in Fig. 4, was now performed by Ramqvist et al. (1988). Constructs are shown that express ST antigen, the mutant Py 1387T M T antigen that lacks 37 C-terminal amino acids (Carmichael et al., 1982), and a fusion product between P-galactosidase and the 73 N-terminal amino acids common to all three T antigens (Fig. 4). Mice were subcutaneously immunized twice, with a 4 to 6 week interval, initially with Freund’s complete adjuvant and thereafter incomplete adjuvant together with purified proteins at a concentration ranging

pTR1300

I

Promoter

IS.D.IPY Common I

pTRl330

I

I

Promoter

IS.D.1

pTR1340

I

Promoter

1S.D.I

Small T

LAC-Z

J

1I

py-1387T Middle T

J

FIG.4. The 1300 and 1300A series of plasmids: schematic diagram of the region containing promoter, Shine-Dalgarno sequence (S.D.), and gene. The coding sequences common to all three polyoma viruses (PY-Common),for an enzymatically active P-galactosidase fragment (LAC-Z),and for ST and Py 1387T MT are indicated.

STUDIES ON THE POLYOMA VIRUS

TSTA

73

between 3 and 47 pg/immunization. Two weeks after the second immunization the mice were challenged with living cells from polyoma and nonpolyoma tumors. No immune response was elicited against nonpolyoma tumors; however, an immune response was observed against pol yoma tumors after immunization with purified Py 1387T M T or ST proteins, but not with the P-galactosidase fusion product (Ramqvist et al., 1988). Table IV presents details of these data such as the incidence of tumor takes and mean tumor diameter (MTD) of the polyoma tumor SEBB/YACUT after immunization with pol yoma virus, purified T antigens, or bacterial proteins from four separate experiments. Immunization of mice with polyoma virus, purified ST, or Py 1387T M T led to a significant decrease in MTD (days 1214 and days 16-17, with an inoculum of lo5 and lo4 cells/mouse) of the polyoma tumor SEBB/YACUT as compared to controls, whereas this was not the case after immunization with bacterial proteins or the P-galactosidase fusion product (Table IV). It had now been demonstrated that it was possible to induce an anti-TSTA response with purified polyoma T antigens. Although it is a formal possibility that the ST and Py 1387T MT proteins used for immunization were biologically active, either directly or indirectly as processed proteins, and thus could induce new cellular antigens by integrating into cells, this was considered unlikely (Ramqvist et al., 1988).This would have required internalization and transportation of these proteins to their normal intracellular location and exertion of their normal biological functions. It was therefore suggested that antigenic epitopes on the T antigens themselves induced the anti-TSTA response (Ramqvist et al., 1988).If so the T-antigenic epitopes must be expressed in some way on the cell membrane. Since T antigens have previously not been detected on the outside of the cell surface (Ito, 1980)it was suggested by Ramqvist et al. (1988)that T antigens may be expressed on the cell surface in small quantities detectable only by the immune system or, more likely, that they were processed and copresented to the immune system together with major histocompatibility complex (MHC) determinants, as had been described earlier for the influenza nucleoprotein (Townsend et al., 1984, 1986) and for other antigens (Babbitt et al., 1985). Immunization against polyoma TSTA with purified T antigens was clearly not as efficient as immunization with polyoma virus, as also shown in Table IV; however, it was as efficient as immunization with a polyoma tumor cell extract (Ramqvist et al., 1988), indicating that the possibility of polyoma virus replicating in uivo and boosting the immune response could be responsible for the more efficient immuniza-

TABLE IV INCIDENCE OF TUMOR TAKESAND MEANTUMOR DIAMETER (MTD) OF SEBBIYACUT IN IMMUNIZED AND NONIMMUNIZED (CONTROL) A x CBA M I C E ~ Mice immunized with Control Experiment Cell number dose Takes

1 2 3 4

105

414

105

414

104

414 414 414 414

105 105

104

Wild-type virus

ST

MT

MTD

Takes

MTD

Takes

MTD

10 10 9 12 7 7

014 214 114 214 114 1I4

-

214 314

-

2 3

414

4

4

314

5

414

4 3

-

114

Takes

Fusion protein

Bacteria MTD

Takes

MTD

Takes

MTD

414

5

414

10

314

4 -

414 414

6 14

414 314

9 15

8.1 f 1.9 14.2 f 3.4

414

8.7 f 0.6 14.8 f 8.6

5

013

Totals:

lo5 10"

718 4.4 16/16 9.7 f 2.7b 5/16* 3.0 f 2.0** 12/16 4.7 f 1.1* 518 4.2 f 1.3*** 0/3** 218 3.0 818 8.0 f 2.6

f

1.7** 12/12 414

314

~

From Ramqvist et al. (1988). Mean tumor diameter (MTD)of total number of tumor-bearing mice f SD. The MTD was calculated at days 12-14 for the lo5 groups and at days 1 6 1 7 for the 10" groups. * p < 0.001. ** p < 0.01. *** p < .05 as compared to control by Student's t test (MTD) or Fischer's exact test (takes).

STUDIES ON THE POLYOMA VIRUS

TSTA

75

tion observed. The observation that the fusion protein that contains the 73 amino acids common to all T antigens did not immunize must be viewed with considerable caution. The lack of an anti-TSTA response could be because the 73 amino acids involved do not include any antigenic epitopes. It is, however, also possible that the N-terminal73 amino acids do possess TSTA epitopes, but that the fusion product was rapidly degraded, had an undesirable configuration of the polyoma virus sequences, or was subject to antigenic competition (Ramqvist et al.,1988). The reason for discrepancy between the results of Lathe et al.(1987) and Ramqvist et al. (1988) regarding the ability of ST to immunize against polyoma TSTA remains unclear; however, there is an obvious difference between the systems. The vaccinia vector introduces ST within the cell, and it is not known if ST introduced in this context allows correct processing of ST. In the experiments of Ramqvist et al. (1988) ST was introduced externally, which may have enhanced its presentation, and since the processed, externally introduced ST would also share 191 amino acids with MT, this could theoretically result in production of antigenic epitopes also obtained by the presence of intracellular MT. It was concluded from the study of Ramqvist e t al. (1988) that T antigens themselves were directly involved in the TSTA function, and that at least one TSTA epitope was likely to reside within the 191amino acid region common to MT and ST. These findings also suggested once again that antigenic epitopes on more than one T antigen, and perhaps on all T antigens, could be involved in the TSTA function. 2. A TSTA Epitope Resides within the First 113 N-Terminul Amino Acids Common to MT and ST In order to further define regions where T antigen epitopes could be situated, a study was performed by Reinholdsson et al. (1988). The transformed rat cell line 2.8, expressing only MT, was examined for its ability to be rejected in rats immunized with a rat cell line 1837, expressing only full-length LT and nonfunctional 113-amino acid Nterminal fragments of ST and MT. If cross-immunization between the two cell lines 2.8 and 1837 was obtained, it would suggest the presence of a shared epitope. This TSTA epitope would have to be situated within the N-terminal 113 amino acids common to ST and MT, of which the first 79 were also common to LT, since the 2.8 and 1837 cell lines have only these amino acids in common. As positive control immunogens the wild-type polyoma tumor wtRat-lz, and the MTexpressing line 2.8 were chosen, and as negative control immunogens

76

TINA DALIANIS

the nonpolyoma cell lines Rat-1 and SV40-Rat-1, a Rat-1 line transformed by simian virus 40 (SV40),were used. The results are displayed in Table V and show that it is possible to immunize against the MTexpressing transformed 2.8 cell line with itself, wtRat-lz, the wild-type polyoma virus-transformed cell line, as well as with the 1837 line that expresses only full-length LT and 113 N-terminal amino acids of M T and ST. None of the nonpolyoma tumors induced an anti-TSTA response (Table v). These findings indicated that a common TSTA epitope must indeed be present within the 113 N-terminal amino acids of ST and MT. It was also observed, however, that the 2.8 line immunized more efficiently against itself, compared to the immunizing effect of the LT-expressing 1837 and wtRat-lz lines. This was unexpected and may not be relevant, but it is possible that the 2.8 cell line could also express a private antigen. Alternatively, the presence of all three T antigens in wtRat-lz and the presence of the whole of LT in 1837could result in the presentation to the immune system of several polyoma TSTA epitopes. A predominant response to some of these latter epitopes could explain the less favorable response to one/some of the

TABLE V TAKES O F 2.8 IN

SYNCENEIC FISCHER RATS AFTER ~MMUNIZATIONWITH

DIFFERENT RAT

C E L L LINES‘

Rats immunized with

Experiment number

Untreated controls

1 2

415” 313 617 213 515 414

414

24/27

14/19

618

89

74

75

3 4

5 6 Total tumor takes: Percentage takes:

Rat-1

SV40-Rat-1

wtRat-lz

315 213 314

313

013 515

1I3 213 115

1837

2.8

014

115 012

013 014 314

013 013

314

114 1I4

7119

6/19

2/23

37*

32*

9**

014 314

From Reinholdsson et d.(1988). Figures denote the number of rats that died with progressively growing tumors over the total number of rats. * p < 0.05 as compared to the Rat-1 group by Fischer’s exact test. ** p < 0.001. ‘I

77

STUDIES ON THE POLYOMA VIRUS TSTA

polyoma TSTA epitopes present in the MT-transformed 2.8 line (Reinholdsson et al., 1988). 3. Immunization against Polyoma TSTA with Synthetic Peptides Derived from the Sequence of MT: The MT Amino Acids 162-1 76 Constitute a TSTA Epitope To define epitopes of polyoma TSTA in detail, peptides corresponding to different regions of MT were synthesized (Ramqvist et al., 1989). The aim of this study was to see if it was possible to obtain a rejection response against polyoma tumors after immunization of mice with single synthetic T antigen-derived peptides, and if so to use peptides to map polyoma TSTA epitopes. To reduce the number of different peptides produced an algorithm devised by Margalit et al. (1987) that selects for amphipathic a helices was employed for prediction of likely T-helper cell epitopes. Although it is not known if T-helper cells specifically participate in the rejection response against polyomainduced tumors, it has been demonstrated that T cells for this response (see above). Six peptides, all derived from the sequence of MT and with a length of 15-19 amino acids were produced. Their amino acid sequences, their presence on T antigens, and their nomenclature are presented in Table VI. Mice of CBA or (A x CBA)Fl origin were immunized three times TABLE VI THEAMINOACID COMPOSITION OF THE SIX MT-DERIVED PEPTIDES AND THEIR PRESENCE IN THE DIFFERENT T ANTIGENS Present in Designation MT1-lOU MT24-38 MT51-66 MT 162- 176 MT193-207

MT380-305

MT amino acid number

Amino acid sequence

ST M T LT

1-19 24-38 51-66 162-176 193-207 380-395

MDRVLSRADKERLLELLKL + Cb WCDFGRMQQAYKQQS + C LMQELNSLWGTFKTEV + C TRDVLNLYADFIASM RRSEELRRAATVHYT + C RAHSMQRHLRRLGRTL

+ + + + + + + + + + + - + - + -

Peptide synthesis: The protected peptides were assembled by solid-phase synthesis on a cross-linked polystyrene support, using t-Boc-amino acids. The syntheses were performed on a peptide synthesizer (Applied Biosystems 430A) utilizing a standard program. Peptides were cleaved from the resin and deprotected by hydrogen fluoride. Final products were analyzed by reversed-phase HPLC and verified through amino acid analysis. On four of the peptides an extra cystein was added.

78

TINA DALIANIS

with 3-week intervals with Freund’s complete and thereafter incomplete adjuvant alone, or together with approximately 3-10 pg/ immunization of each of the six uncoupled synthetic peptides, or with all six peptides together. Two weeks after the last immunization, mice were challenged with tumor cells of polyoma origin or nonpolyoma origin. No immunization effect was observed in any of the immunized animals against tumors of nonpolyoma origin. Immunization with the uncoupled synthetic peptide, corresponding to amino acids 162-176 of polyoma MT and ST, was able to induce a decrease in tumor progression of polyoma tumors. The mean tumor loads (MTLs) observed for the SEBB/YACUT polyoma tumor (pooled for six different experiments after immunization with the individual peptides, the pooled peptides, wild-type polyoma virus, or with Freund’s adjuvant alone) when the mean tumor loads for controls were 20 mm are illustrated in Fig. 5. N o other peptide other than MT162-176 induced a significant decrease in tumor progression. In mice which received this peptide a 47% decrease of the MTL was obtained (Fig. 5).No reduction in tumor takes was observed in any of the groups with the exception of the polyoma virus-immunized group (Ramqvist et al., 1989). An unexpected result was the fact that immunization with all six pooled peptides did not result in decreased tumor progression. One explanation for this could be that the peptides compete for MHC binding, and it is possible that nonresponsive peptides also bind to MHC, thus competing for binding of the immunizing peptide MT162-176 A similar situation has been shown for other antigen types by Babbitt et al. (1986). In summary, this study demonstrated that immunization of CBA and (A X CBA)F1 mice with a peptide sequence (MT162-176) derived from the protein products of two oncogenes (polyoma MT and ST) can induce a specific tumor graft rejection. The anti-TSTA response induced by this peptide was weaker than the response obtained after polyoma virus immunization, but comparable to that induced by purified M T or ST (Ramqvist et al., 1988). The weaker anti-TSTA response induced by a single peptide as compared to immunization with wild-type virus was possibly due to the fact that several target epitopes can be generated by the virus, and that continuous virus replication in uiuo is likely to result in a more efficient response. Furthermore, although it is possible for the immune system to detect and respond to this epitope in CBA or (A x CBA)F1mice, it is not evident that this epitope is the dominant target epitope during an anti-TSTA response after immunization with polyoma virus. The existence of at least one other TSTA epitope within the first 113 amino acids of M T and ST (Reinholdsson et al., 1988)has already been

STUDIES ON.THE POLYOMA VIRUS

79

TSTA

c

E E

20

Y

W

lu

16

0

-1

b

z

12

E C

8

lu

a,

I

4

0 1

2

3

4

5

6

7

8

9

10

Fic.5. I n oioo growth of polyoma tumor SEBBlYACUT with an inoculum of 2-10 x lo4 cellslmouse in six different experiments (assayed by the combined total MTL) in different groups of immunized mice. The combined total MTL of the different groups of immunized mice are indicated at the time point when the MTL of nonimmunized controls within each experiment were 20 mm. It was calculated as the sum of the mean tumor diameters of the tumors present within each specific type of group, divided by the total number of mice. The different bars correspond to groups of nonimmunized mice (l), Freund's adjuvant (2), polyoma virus (3), peptide MT1-IQ (5), M T U - ~ ~ (6), MT51-66 (71,MTl62-176 (81, MTIY3-207 (91, peptide MT380-395 (lo), and a mixture of these peptides (4). Error bars denote the standard error. The combined total MTL of the virus or MTl62-176 groups were significantly lower than in the control group as evaluated with the Student t test (p < 0.0001). Immunization was performed with the different peptides three times, with 2-week intervals with doses from 3 to 10 pg/inoculation/ mouse together with Freund's adjuvant.

discussed. This epitope would clearly not overlap with MT162-176. These studies were, however, performed in rats and it is possible that different epitopes are recognized in different species, as well as in different strains of rats and mice. The presence of a third TSTA epitope situated in the C-terminal part unique for MT, recognized by Fischer rats, has recently been suggested by R. Lathe, (personal communication). Therefore, although a significant immune response after immunization with the other five peptides was not obtained, it cannot definitely be concluded that they do not constitute TSTA epitopes. There could be other reasons for the lack of response. The MHC of the mouse strains tested may not bind these peptides, or the dose used for immu-

80

TINA DALIANIS

nization, or the immunization procedure, may not have been optimal; such situations have been discussed in a review by Townsend (1987).

4. Polyoma T Antigen-Derived Synthetic Peptides Induce Polyoma Virus-Specqic Macro phage Migration Inhibition ( M M I ) As mentioned earlier (Szigeti et al., 1982; Ramqvist et al., 1986), it is possible to detect polyoma-specific macrophage migration inhibition (MMI) when peritoneal exudate cells (PEC) from polyoma virusimmunized mice, but not from control mice, are exposed to cell or membrane extracts from polyoma tumors. Although it has been shown that it is possible to immunize against polyoma TSTA both with cell and membrane extracts ofpolyoma tumors (Ramqvist et al., 1988)it has still not been possible to examine whether the MMI assay really identifies relevant TSTA epitopes. Since it was possible to immunize against polyoma TSTA with purified T antigens, and a specific peptide MT162-176, an attempt was made by Reinholdsson et al. (1989) to study if the MMI assay does detect TSTA epitopes. The ability of purified T antigens and T antigen-derived synthetic peptides to induce polyoma specific MMI was assayed. It was shown that it was possible to obtain a polyoma-specific M M I with PEC from polyoma virus-immunized mice, but not with PEC from controls, when these were exposed independently to three Sepharose-coupled peptides corresponding to amino acids 1-19 and 51-66, common to MT and ST, and amino acids 380-395, unique for M T (Reinholdsson et aZ., 1989). Nevertheless, purified ST and Py 1387T MT, as well as the Sepharose-coupled peptides corresponding to amino acids 51-66 and amino acids 162-176, common to MT and ST, did not elicit MMI with effector cells from polyoma virusimmunized mice. This was despite the presence of the amino acid sequences 1-19, 51-66, and 380-395 of the three MMI-active peptides described above within MT, and for two peptides (amino acids 1-19 and 51-66) also within ST. However, ST induced MMI with PEC from ST-immunized mice, and M T induced MMI with PEC from MT-immunized mice, but not vice versa, and in addition all peptides could induce MMI with PEC derived from mice immunized with a mixture of all five peptides. Furthermore, membrane extracts from the polyoma tumor SEBB/YACUT could induce MMI with PEC derived from mice immunized with either ST or a mixture of the five MTderived peptides (Reinholdsson et al., 1989). In conclusion, it was not possible to correlate the ability of immunogens such as ST, Py 1387T MT, peptide MT162-176 (corresponding to M T amino acids 162-176), or polyoma virus mutants that are able

STUDIES ON THE POLYOMA VIRUS

TSTA

81

to immunize against polyoma TSTA (Ramqvist et al., 1988, 1989; Dalianis et al., 1982), with their ability to induce an efficient MMI if PEC were derived from polyoma wild-type virus-immunized mice (Reinholdsson et al., 1989; Szigeti et al., 1984).A possible explanation is that the anti-TSTA response obtained in v i m , that ultimately results in tumor rejection, may be due to a combined effect of several immune effector mechanisms, while the MMI response reflects only a limited part of the immune system. Furthermore immunization of mice with one polyoma immunogen, be it peptide, T antigen, virus, or virus mutant, may induce a preferential immune response against one or a few immunodominant epitopes, which may not necessarily correspond to those primarily exposed by a second pol yoma immunogen. Thus, although the MMI assay can detect polyoma specificity, it most probably represents only a limited part of the immune system, and cannot be used alone to assay for polyoma TSTA activity. However, if used cautiously it may be able to further the understanding of preferential immune responses against specific TSTA epitopes, depending on the specific combinations of immunogens and antigens that are used (Reinholdsson et al., 1989). VII. Present View of the Polyoma Tumor-Specific Transplantation Antigen Polyoma virus TSTA has now been shown to be a product of at least one of the polyoma T antigens, MT, since it has been possible to immunize against polyoma TSTA with (1)a cell line that expresses only MT, (2)a vaccinia virus recombinant expressing MT, (3)a purified MT antigen mutant, and (4) a synthetic peptide corresponding to amino acids 162-176 of MT (Dalianis et al., 1984; Lathe et al., 1987; Ramqvist et al., 1988, 1989). It is likely, but not proved, that LT also possesses TSTA activity, since it has been shown by Lathe et al. (1987) that a vaccinia vector expressing only LT will also induce an anti-TSTA response. This result must be viewed with some caution, since it is not evident that the introduction of LT intracellularly by vaccinia virus will result in processing of LT identical to that obtained when LT is introduced in the context of virus infection. However, recent studies by Guizani et al. (1988) suggest that LT expressed from vaccinia virus recombinants retains its specific DNA-binding activity and ATPase and nucleotidebinding activities, and thus appropriate posttranslational processing of LT is not prevented. However, the possibility remains that LT introduced intracellularly by vaccinia virus vectors may be processed in

82

TINA DALIANIS

such a manner that the resulting products are similar to those of MT, as 79 N-terminal amino acids are shared between MT and LT. It is also possible that ST possesses TSTA activity, since it has been demonstrated that it is possible to induce an anti-TSTA response when immunizing with purified ST in an extracellular context (Ramqvist et al., 1988). However, this result must also be viewed with caution. The possibility that extracellular processing of ST results in the presentation of peptide epitopes shared by M T cannot be excluded. Thus, so far, there is no direct evidence that ST constitutes TSTA in its natural context, and furthermore it must be pointed out that immunization of rats with a vaccinia vector expressing ST did not result in an anti-TSTA response (Lathe et al., 1987). In conclusion, separate antigenic epitopes on M T that can constitute polyoma TSTA have been demonstrated (Reinholdsson et al., 1988; Ramqvist et al., 1989; R. Lathe, personal communication). The fact that a peptide 14 amino acids in length is immunogenic does suggest that M T is indeed processed and presented in the context of the MHC to the immune system (Ramqvist et al., 1989). Indirect evidence by Lathe et al. (1987) and Ramqvist et al. (1987, 1988) suggests that antigenic epitopes on both LT and ST can constitute TSTA. Thus, it has now been shown that a T antigen can have direct TSTA activity. However, this does not exclude the fact that virus infection or transformation per se may also induce additional private antigens, or tumor-associated differentiation antigens, that may add to the TSTA activity of these cells. VIII. Future Prospects

Polyoma virus, although oncogenic, does not induce tumors in immunocompetent mice. A detailed study ofthe antigenic epitopes of the tumor-specific transplantation antigen, along with detailed studies of the immune responses against these antigens, will enable us to understand the limitations and abilities of the immune system to respond against tumors induced by this naturally occurring oncogenic virus in its natural host.

ACKNOWLEDGMENTS The author wishes to thank Dr. R. Lathe for discussions of unpublished data, and V. E. Prince for helpful discussion in preparing the manuscript. This work was supported by the Swedish Cancer Society. The author is a recipient of a Travelling Fellowship from Wellcome Trust.

STUDIES ON THE POLYOMA VIRUS

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83

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GROWTH DOMINANCE OF THE METASTATIC CANCER CELL: CELLULAR AND MOLECULAR ASPECTS R. S.Kerbel Mt. Sinai Hospital Research Institute. end Departmentof Medical Genetics, Universityof Toronto, Toronto, Ontario, M5G 1x5 Canada

I. Introduction 11. The Selective Nature of the Metastatic Phenotype

111.

IV. V.

VI. VII.

Dynamic Heterogeneity Model of the Origin and Nature of the Metastatic Cancer Cell Clonal Dominance (Growth Preference) of Metastatically Competent Variant Cells in Primary Tumors: Genetic Analysis Human Malignant Melanoma as a Paradigm for the Growth Dominance of Metastatic Cancer Cells The Role of Growth Factors in the Growth Preference and Dominance of Metastatically Competent Cells A. Growth Stimulatory and Autocrine Growth Factors B. Loss of Response by Metastic Cells to Growth Inhibitory Molecules: Metastatic Cells as Effective Cellular Scavengers C. The Role of Tissue-Specific Growth Factors and the Microenvironment in Metastic Tumor Growth Ectopic Gene Expression and the Pleiotropic Nature of the Growth-Dominant Metastatic Phenotype Conclusions References

I. Introduction

In 1979 Fidler, upon considering all of the properties that he felt were necessary for a cancer cell to successfully metastasize, likened the metastatic cell to a decathlon champion (Fidler and Cifone, 1979). Almost a decade later Hart and his colleagues discussed the notion that the metastatic cell “represents the apotheosis of the neoplastic process” (Hart et al., 1989).These analogies are essentially based on two important considerations: (1)that metastatic cancer cells are the progeny of specialized, genetically mutant subpopulations which emerge during the sequential process of tumor progression (Nowell, 1976, 1989);and (2) that metastasis involves a large number of steps, all of which must be successfully completed in order for a clinically detectable metastasis to form. The list of these steps is well known, and indeed has become virtually obligatory to summarize in almost every research paper-or review-written on the subject. Admittedly the list 87 ADVANCES IN CANCER RESEARCH, VOL. 55

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of steps involved is impressive and for certain carcinomas would include the following: (1)progressive growth at the primary site of tumor growth; (2) penetration of basement membranes separating epithelial tissues from the underlying stroma; (3) vascularization of the tumor; (4) entry into the body’s vasculature, either the lymphatics or blood vessels, the latter of which must include penetration (digestion) of the subendothelial matrix, and adhesion to endothelial cells with their subsequent retraction; (5)migration and survival in the bloodstream or lymphatics, which may be facilitated by the formation of tumor celltumor cell homotypic aggregates (microemboli) or heterotypic aggregates with normal cells such as platelets; (6)arrest in a distant capillary bed; (7) exit from the vasculature or its disruption by mechanical expansion; (8) migration, perhaps of a single clonogenic tumor cell, into the parenchyma of a “foreign” or ectopic organ site; (9) progressive growth from one or a small number of clonogenic cells to a mass of lo9 cells or more. When this list is considered in the light of results suggesting that metastases are clonal growths (Talmadge et ul., 1982; Fidler and Talmadge, 1986; Ootsayama et al., 1987; Talmadge and Zbar, 1987; Korczak et al., 1988, 1989; Chambers and Wilson, 1988; Kerbel et ul., 1989), i.e., that they ultimately arise from the expansion of a single tumor cell, the formidable nature of the successful metastatic cell becomes apparent. Numerous detailed reviews have been written summarizing the many properties thought to be responsible for conferring metastatic competence to tumor cell populations. Most of these reviews are generic in scope in that they cover a very broad range of factors involved in metastasis (e.g., Fidler and Hart, 1982; Schirrmacher, 1985; Hill, 1987; Hart et al., 1989).Others have been dedicated primarily to one aspect associated with the metastatic process or metastatic cancer cell, e.g., organ specificity for secondary sites of tumor growth (Hart, 1982; Nicolson, 1988), the contribution of oncogenes (Nicolson, 1986; Mareel and Van Roy, 1986; Chambers and Tuck, 1988; Greenberg et al., 1989),genetic factors, in general, regulating the metastatic phenotype (e.g., Ling et al., 1985; Hill et ul., 1986; Pickford and Franks, 1988; Collard et al., 1988; Feldman and Eisenbach, 1988), the role of the cell-surface glycoproteins and oligosaccharides (Nicolson, 1984; Raz and Lotan, 1987; Dennis and Lafert6,1987), the role of specific adhesion molecules (Taylor-Sher et ul., 1988) or autocrine motility factors (Liotta and Schiffmann, 1988), the contribution of basement membranes, extracellular matrix, and various proteolytic enzymes (Liotta et al., 1982; Thorgeirsson et ul., 1985; Liotta, 1986), and the impact of growth factors (Herlyn et al., 1989). Readers should also consult an excellent textbook devoted to the principles of

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cancer metastasis (Weiss, 1985) for a rigorous review of all aspects of the subject. In view of these (and other) reviews, it would not seem particularly productive to present yet another one cataloguing all of the known differences between metastatic cancer cells and their nonmetastatic counterparts. I have instead chosen to review, in more detail, and in the light of recent findings, the concept of the apotheotic nature of the metastatic cancer cell from the perspective of its “growth-dominant’’ nature, and how this is manifested as well as regulated at both the phenotypic and genotypic levels. In many types of cancer it is now increasingly clear that the metastatic cell is the result of not one, but probably a series of sequential genetic changes-just as Nowell (1976)predicted. These changes endow metastatically competent cells with what turn out to be rather formidable growth advantages so that even within the primary tumor they come to completely dominate it over time. This new concept is at odds with the prevailing thinking that metastatically competent mutant subpopulations of cells remain as a silent or cryptic minority within the primary tumor and that it is their ability to successfully depart the primary site and establish secondary tumors elsewhere in the body that solely defines their nature. The growth dominance of the metastatic cancer cell at the primary site is a reflection of its powerful ability to successfully survive and grow relentlessly elsewhere in the body, even from a single cell in an ectopic environment. How this may come about will be discussed in some detail. It could, as we shall see, ensue from a heightened sensitivity to stimulatory autocrine or paracrine growth factors, produced by the tumor cells themselves or by their nonmetastatic tumor cell counterparts, or by cells from normal host tissues. Alternatively, or in addition, it could be a reflection of a decreased sensitivity to growth-inhibitory molecules. These changes are also invariably accompanied by many others involving elevated secretion of proteases, altered expression of adhesion molecules, etc., leading to the pleiotropic metastatic phenotype, and this must ultimately be a reflection of fundamental differences between metastatic and nonmetastatic cancer cells with respect to gene regulation and signal transduction. Indeed, the notion will be discussed that many of the phenotypic and genetic changes which are thought to be causative of neoplastic transformation may in fact be related to acquisition of metastatic competence rather than tumorigenic growth per se. This fact may be overlooked when one simply analyzes and compares normal (or immortalized) cells to their neoplastic transformed counterparts, without any regard for the metastatic competence of the latter.

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This article will explore these concepts and hypotheses, and their implications, by attempting to focus on a number of recent key findings and paradigms, rather than by an exhaustive summary of the literature.

II. The Selective Nature of the Metastatic Phenotype Much of the current thinking about the origin and nature of metastatic cancer cells can be traced to the clonal evolutionary nature of tumor growth-a phenomenon often referred to as “tumor progression” (Nowell, 1976, 1989).By now the basic elements of this process are well known, and are supported by a large body of literature. It is assumed that the vast majority of neoplasms, regardless of their origin, arise from the neoplastic conversion of a single altered normal cell (Fialkow, 1976;Woodruff, 1988). This neoplastic stem cell, or progenitor, must possess some kind of growth advantage over its normal cell counterparts and thereby is able to give rise to a neoplastic clone. Within this expanding clone, new mutants continually arise in a sequential manner by various types of genetic mutations or heritable epigenetic alterations. A very small fraction of these mutant cells are assumed to possess some kind of additional growth advantage over the parent tumor clone and as a result their progeny will come to displace (overgrow) the original tumor cell clone over time. This Darwinianlike sequential process can then be repeated again, giving rise to the sequential appearance of subclones which behave in an increasingly aggressive manner. Eventually a subclone will emerge that acquires competence for metastatic spread. A review of the literature reveals that acquisition of metastatic competence b y mutant subclones is usually thought to be associated with an ability to escape from the primary tumor-rather than dominating it-and this is usually attributed to a number of factors, especially to an increased capacity for secretion ofa spectrum ofproteases, e.g., type IV collagenase or plasminogen activator. As shall be discussed, this is a somewhat oversimplified concept in that there is increasing evidence that metastatically competent mutant cells can manifest a strong growth preference over their nonmetastatic counterparts within the primary tumor and so in time come to dominate it (Kerbel et ul., 1988, 1989). This is not meant to imply that such growth preference or dominance is all that is required for tumor cells to acquire metastatic competence. This clearly is not the case, but it will be argued that this growth preference is a major behavioral trait of the metastatic cancer cell and that it is probably necessary-though not sufficient-for expression of metastatic ability. It also helps resolve some of the confu-

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sion in the metastasis literature, which has arisen as a result of the discrepant findings regarding the relative metastatic aggressiveness (or other phenotypic traits) oftumor cells recovered from metastases as opposed to tumor cells recovered from primary tumors. In 1973 Fidler reported the results of studies which for the first time provided convincing evidence for the selective nature of the metastatic phenotype. Using the mouse B16 melanoma cell line, syngeneic mice were intravenously injected with the same number of tumor cells. The lung tumor nodules which arose in the animals were removed, pooled and adapted to tissue culture, and the process repeated. With each successive selection it was found that the tumor cells possessed a greater lung colonization ability. In this way the classic B16F1 and B16F10 paired melanoma cell lines were isolated (Fidler, 1973). The B16F10 cells were shown to stably express a greater organ colonization (“artificial metastatis”) phenotype, even after prolonged serial culture in uitro and have been analyzed in great detail by a large number of investigators for factors which control or influence metastatic potential (see Maslow, 1989, for a recent review). Fidler’s 1973 study became a landmark and was quickly followed by others who attempted to repeat his findings using a variety of other experimental animal (or human) tumor models. It became apparent that the results could not be reproduced in many cases (e.g., Weiss et al., 1983; Alexander, 1984; Milas et al., 1983; Vaage, 1988).It will be noted that in some cases the organ colonization or artifical metastasis assay was employed whereas in others the so-called “spontaneous metastasis” assay was used. In the latter assay, tumor cells are injected in such a way so that a primary solid tumor forms at the site of inoculation, from which metastatic cells are spawned to spread to distant organs. The method of inoculation in this type of assay is variable, and includes subcutaneous, intramuscular, and intraorgan (orthotopic) injections (Fidler, 1986). The colonization assay presumably encompasses only the later stages of hematogenous tumor cell dissemination and metastasis, whereas the spontaneous metastasis assay involves all ofthe steps-both early and late-known to be involved in metastasis. A typical and seemingly straightforward experiment would be to compare the relative metastatic aggressiveness of tumor cells obtained from locally growing primary tumors versus their metastases, upon reinoculation into new animal hosts. This can be done directly, that is, with no intervening cell culture step; alternatively the tumor cells can be adapted to tissue culture first (to establish cell lines) after which they can be tested in uivo. Both types of procedure were undertaken and the results were often contradictory.

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It is instructive to consider in some detail a few of the failures to uncover evidence that tumor cells populating metastatic lesions are more metastatic than their parallel primary tumors and the conclusions drawn from the results. Thus, Alexander (1984)examined a number of transplantable rat sarcomas and found that cells from locally growing primary tumors (obtained after intramuscular injection) were no less metastatic upon retransplantation when compared to cells from either lymph node or lung metastases. Neither were any differences in tuniorigenic and immunogenic properties found. According to Alexander, this experiment appeared to confirm the earlier conclusions that Weiss (1980) had made after a critical review of the clinical literature. Weiss Failed to uncover any evidence for consistent differences lietween the cancer cells in primary cancers and their metastases, either in respect to cytogenetics, immunology, or drug sensitivity (Weiss, 1980). As a result, Alexander concluded “specific selection within metastases of cells with properties which make then) more liable to metastasize is not a major factor in the hiology of cancer. The fate of tumor cells which are released from a tumor and the likelihood ofthcir giving rise to a metastatic lesion seeins to be determined by a series of random pracesses. I t is an interesting question as to why in all the experiments in which direct comparisons were made (ofprimary tumors to metastases) there is so little indication of selection.” (Alexander, 1984)

Indeed it is an interesting question and an answer will be outlined in this article which, in effect, defines the essence ofthe metastatic cell. It shall be argued that the answer does not lie in Alexander’s assertion that the positive selection results obtained by Fidler and many others were the result of an artifact brought about by the introduction of i n oitro culture of the tumor cells to be tested. More recently, Vaage (1988)reported the results ofa series ofcareful experiments designed to see if spontaneous metastases from a recently derived spontaneous mouse mammary adenocarcinoma were any more metastatic than the primary tumor after serial retransplantation. A total of seven C3H/He and C3Hf/He mammary tumors were examined. The tumors were transplanted orthotopically into the mammary fat pads, and were never adapted to tissue culture. In no case was evidence obtained to show that metastases were inore aggressively metastatic that the parallel primary tumor. Increases in metastatic potential did occur in three of the seven tumors during serial passage and did so at approximately the same transplant generation in both the primary tumor and metastases. Vaage concluded by suggesting that the spontaneous metastases were not derived from a subpopulation of cells with inheritable high metastasizing potential, but developed “

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through stochastic events from the average tumor cells that entered the circulation” (Vaage, 1988). Like Alexander (1984), Vaage suggested that some of the data obtained by others showing greater metastatic potential of metastases compared to primary tumors could have been the result of technically inappropriate protocols. These include the use of long-established tumor cell lines and ectopic injection procedures. As a result, the tumors might lose their similarity to the original neoplasm, and this fact “would have downgraded their relevance” (Vaage,

1988). On the face of it, the results of Alexander, Weiss, Vaage, and many others when taken together would appear to pose a serious challenge to the notion that metastases are generated from highly specialized genetically mutant subpopulations of tumor cells which emerge during the course of primary tumor growth (Weiss, 1980, 1983).It is also surprising that similar protocols using different tumor lines have resulted in such divergent results and conclusions. Although the argument that artifacts may result from the use of cultured cell lines, or inappropriate injection procedures might seem reasonable, it is simply not compatible with more recent reports from Fidler’s group and others who have evaluated the metastatic potential of fresh human tumor biopsy material (such as colorectal carcinomas) in athymic nude mice (e.g., Morikawa et al., 1988). The tumor cells were not adapted to culture and in many cases orthotopic injections were also employed; nevertheless evidence was obtained that highly metastatic variants could be selected from the primary tumor specimens and/or that metastases from patients metastasized more aggressively than their primary tumors upon transplantation in nude mice (Giavazzi et al., 1986; Morikawa et al., 1988). How can these discrepant results be resolved? There are, broadly speaking, two solutions. One is to argue that although the metastatic phenotype is a genetically controlled trait, it is inherently dynamic or unstable. This is the so-called “dynamic heterogeneity” model of metastasis put forward by Ling, Hill, Chambers, and Harris (reviewed in detail in Ling et al., Hill et al., 1986; Hill, 1987). Alternatively, it can be argued that once a rare, metastatically competent mutant subclone emerges within a primary tumor, the mutation is stably expressed, and the progeny of that clone not only can metastasize (i.e., leave the primary tumor) but also, over time, come to largely if not completely dominate the primary tumor mass itsev. This is the clonal dominance theory of metastasis (Kerbel et al., 1987; Waghorne et al., 1988; Kerbel et al., 1989). As a result of the marked growth advantage which metastatically competent cells have in primary tumors, the primary tumors, will, given enough time, become increas-

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ingly difficult to genotypically and phenotypically distinguish from their metastases. The evidence for this will b e presented in detail in this article, and it comes from studies in both clinical oncology and basic research. The dynamic heterogeneity model will be discussed first to highlight some conceptual differences and similarities to the clonal dominance model.

DYNAMIC HETEROGENEITY MODELOF THE OHIClN AND

NATUHE OF

THE METASTATIC CANCER CELL

The dynamic heterogeneity model of metastasis had its roots in cloning experiments designed to isolate and study cell variants of varying metastatic potential. Fidler and Kripke (1977) reasoned that if metastatic variants preexisted in cell lines such as the B16 melanona it should be possible to isolate them simply by cell-cloning procedures. If the frequency of metastatic variants was high enough then it might be possible to successfully isolate one or a few clones having high-grade metastatic potential. This was found to be the case. The results of Fidler and Kripke were quickly confirmed by others (e.g., Kerbel, 1979; Chambers et al., 1981). However, in the majority of these studies it was noted that the clones were not phenotypically stable. Thus a clone that was initially found to be highly metastatic (usually after intravenous inoculation) “drifted” so that after a period of several weeks or months in culture its metastatic or organ colonization properties partially or fully returned to the parental wild-type levels. Indeed, it is generally the case that such tumor cell clones, isolated i n uitro, tend to revert rather quickly for whatever property is examined, a behavioral trait sometimes referred to as “phenotypic drift” (Nicolson, 1987; see also Schirrmacher, 1980). Ling, Hill, and co-workers reasoned that these results could be explained by assuming the acquisition of metastatic competence was the consequence of an unstable genetic mutation. Or it could come about as a result ofheritable (but unstable) epigenetic changes such as those brought about by alterations in DNA methylation (Frost and Kerbel, 1983; Kerbel et al., 1984a). With respect to genetic niechanisms, for example, it is well known that genes can be amplified extrachromosomally in the form of minute or double-minute (DM) minichromosomes, as a result of a strong toxic selection pressure, e.g., exposure to a chemotherapeutic drug with the subsequent development of resistance to the drug, and perhaps other drugs as well (Schimke, 1988). In the absence of the drug, however, these episomal-

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like genetic elements, because they lack centromeres, tend to be segregated rather quickly from the cells at division, leading to a commensurate loss of drug-resistance (Schimke, 1988). In contrast, the same gene amplified in the form of intrachromosomal homogenous staining regions (HSRs) is not rapidly lost and the associated degree of drug resistance tends on average to be much more stable. Ling, Hill, and co-workers reasoned a similar process might be at work in respect to metastasis. For example, an invading tumor cell confronted with a seemingly impenetrable barrier such as a basement membrane might amplify a gene coding for a protease, allowing it to locally digest the membrane and thereby breach it. Once this is achieved there would no longer be any selection pressure to maintain the gene in an amplified state. Cells populating metastases or primary tumors would therefore appear similar in that neither might show evidence for any demonstrable amplification of the gene encoding the protease, i.e., the gene was amplified, but only transiently, during a particular stage of the multistep process of metastasis. A series of experiments was undertaken to assess the putative dynamic nature of the metastatic phenotype. This was generally done by cloning several mouse tumor cell lines, e.g., the B16 melanoma, the KHT sarcoma, embryonal carcinoma cells, and the RIF-1 fibrosarcoma (Harris et al., 1982, 1987; Harris and Best, 1988; Hill et al., 1984; Young and Hill, 1986; Korycka and Hill, 1989).The clones were carefully grown to defined population sizes and then tested for their organ colonization capacities after intravenous inoculation. The results obtained indicated that variant cells expressing a specific metastatic (i.e., organ colonization) phenotype could be generated stochastically at a rapid rate during their growth in uitro. It was also found that the phenotype could be lost rather rapidly, i.e., the phenotype was unstable. Using Luria-Delbruck fluctuation analysis the effective rate of generation of metastatic variants was calculated to be in the range of per cell generation (Ling et al., 1985; Hill et al., 1986).It was argued that the acquisition of metastatic competence in the clones would depend on the inherent rate of (forward) mutations, the cell population size of the clones, and the back rate of mutation. The term “dynamic heterogeneity” therefore derives from the fact that a dynamic equilibrium would b e expected to develop in which the frequency of metastatic variants in a given clonal population would be controlled by the rates of generation and loss of the variants (Ling et al., 1985; Hill et al., 1986). The dynamic heterogeneity model, if correct, would provide a

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simple explanation for an inability to detect phenotypic differences between tumor cells from primary tumors and their metastasesincluding relative metastatic ability upon retransplantation. This is because the model predicts that authentic metastatic variants, as a result of their phenotypic instability, would always remain as a numerically silent or cryptic minority subpopulation, riot only within. the primary tumor but etien within metastases themselties. Thus if’ one was attempting to detect evidence for the expression of, say, a particular cell adhesion molecule (or its cognate gene) in metastatic cells, its presence would remain masked or difficult to detect because the proportion of metastatic cells might never be great enough in the tumor samples to permit easy detection. Hill (1987) used this argument to help explain the frequent failure to find consistent differences between metastatic cells and their nonmetastatic counterparts in respect to such properties as membrane proteins or glycoproteins, surface carbohydrates, enzymes, adhesion properties, or surface change. While this might help explain the failures, what can be said of the successes to detect such differences? As will be briefly summarized later in this article (and most recently in Hart et ul., 1989, in greater detail) there are now a large number of compelling examples of molecular structures which are consistently detected (or lost) in metastatic cells, and which in some cases have been shown to have a cause-and-effect relationship with respect to expression of metastatic competence. Some are generic in nature, being associated with many different kinds of tumor, e.g., type IV collagenase or plasminogen activator (PA) production (Axelrod et ul., 1989) and secretion, whereas others are specific to one particular type of tumor (or a small number of tumor types), e.g., expression of the intracellular adhesion molecule called ICAM-1 on human metastatic melanoma cells (Johnson et d., 1989). Such findings force one to consider alternative theories to help explain the existence of genetically mutant metastatic subpopulations-even in the face of ostensibly unequivocal results showing no obvious differences between metastases and primary tumors in respect to metastatic potential. A fairly straightforward explanation is presented and reviewed in the next two sections based on the growth preference or “clonal dominance” of metastatically competent cells in primary tumors. This will be followed b y a discussion of the possible factors that are involved in conferring such growthdominance properties to metastatic tumor cells and how they help define the nature, or essence, of the metastatic cancer cell itself.

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111. Clonal Dominance (Growth Preference) of Metastatically Competent Variant Cells in Primary Tumors: Genetic Analysis

The experimental analysis of the growth characteristics and dynamics of metastatic subpopulations within primary tumors requires genetic, biochemical, or immunologic markers to distinguish the metastatic cells from their nonmetastatic counterparts, and to follow their respective fates over time. In this respect it is well known that there is a variety of genetic and phenotypic markers which have been used to successfully establish the clonal nature of tumors and metastases, and to study in general the clonal evolution of tumor growth (see Woodruff, 1988, for review; see also Fialkow, 1976; Kerbel et ul., 1988). They include chromosome/cytogenetic markers, enzyme polyniorphisms, immunoglobulin markers, and drug-resistance markers. More recently, endogenous molecular genetic markers (Vogelstein et d . ,1989) such as restriction fragment length polymorphisms (RFLPs) have been employed. A few years ago a new method was adopted by two groups (Kerbel et ul., 1987, 1988; Talmadge and Zbar, 1987; Talmadge et al., 1987) that had already been used with considerable success to study cell lineage and cell fate in early and later embryonic development, hematopoiesis, and in the nervous system (see Price, 1987, for review). The method exploits the random integration of transferred foreign DNA molecules into the genomes of recipient cells as a way of generating large numbers of cells containing unique and identifiable genetic markers which are identified by Southern blotting. The principles involved have been explained in detail by Price (1987) and ourselves (Kerbel et d.,1988). In brief, because transfected plasmid DNA, or proviral DNA copied from the RNA of retrovirus vectors, integrates in a more or less random fashion, digestion of genomic DNA with, say, a restriction enzyme which cuts outside the integrated DNA will generate unique-sized fragments of DNA incorporating the foreign DNA and host flanking 5’ and 3’ DNA in any given transfectant or infectant. This is so because the distance of the nearest upstream or downstream relevant restriction sites flanking the inserted DNA recognized by the restriction endonuclease used to digest the DNA will vary from one cell (i.e., transfectant or infectant) to another. Assuming a single copy of the plasmid or proviral DNA is inserted, each clone will contain a unique DNA marker (i.e., restriction fragment of variable length) detectable by Southern blotting using an appropriate hybridization probe. One way in which this has been exploited to study cell lineages in

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tumors and the clonal dominance of primary tumors by metastatically competent cells is schematically summarized in Fig. 1. A plasmid (or retroviral vector) containing a dominant selectable drug-resistance marker (e.g., the neo gene) is used to transfect or infect a mouse or

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Genetic tagging by random integrations of foreign DNA clone

plasmid transfection

<

retroviral vector Southern analysis infection

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Southern Blot Analysis FIG.1. Sc iematic representation of the wocedure used to study tumor cell ineage relationships during it1 a i m growth of primary tumors and metastases. A mouse or human tumor cell line is genetically tagged in aitro by random integrations of foreign D N A so that a large number of individual clones is isolated, each bearing its own unique genetic signature (which can b e visualized by Southern blotting using an appropriate hybridization probe). The foreign D N A is transferred either by plasmid transfection or retrovirus vector infection. A large number ofclones is then pooled, which results in the disappearance of the unique signature associated with any given clone: D N A from such a pooled population will instead present as a faint smear provided a sufficiently large nuniber of clones are pooled. T h e pooled population is used a s an inoculum to inject syngeneic or nude mice. Some time later the primary tumors and metastases are removed and analyzed by Southern blotting for their relative clonal compositions and clonal identities. In the scheme shown above, each clone is associated with a single, unique-sized fragment capable of hybridizing with a tieo-specific hybridization probe. This assunies a single insertion o f a single copy ofthe neo-containing plasmid or proviral D N A , and that a restriction enzyme which does not cut within the foreign integricted D N A is used for digestion of genomic D N A . This method was used to determine the fact that primary tumors can become overgrown by the progeny of a single metastatically 1989, competent clone (Kerbel et ul., 1987; Waghorne e t ul., 1988).(From Kerbel et d., with permission of the puhlisher.)

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human tumor cell population so that a large number of independent clones is obtained. Every clone will have its own unique genetic marker, detectable by Southern blotting using, for example, the neo gene or a fragment of it, as a hybridization probe. If a large number of the clones are pooled, the DNA obtained from such a mixture will present as a faint, broad smear on a Southern blot since no given clone will exist in a high enough proportion to enable its unique “clonotypic” genetic signature to be seen. If such a mixture is then injected into a mouse, the relative clonal composition of primary tumors and any metastases which subsequently emerge in the animal can be determined by Southern blot analysis assuming some clonal selection has occurred. For example, if the primary tumor has been overgrown by the progeny of one or a small number of clones this would be easily detected. Similarly, if metastases are derived from the seeding of organs by single tumorcells, i.e., are clonal growths, as previously shown or suggested by cytogenetic analysis (Talmadge et al., 1982; Fidler and Talmadge, 1986), this too could be easly determined. The lineage relationship of different metastases (located in the same or in different organs) to each other, or to the primary tumor, could also be established (Kerbel et ul., 1987; Talmadge and Zbar, 1987; Korczak et al., 1988)as well as the relative rates of clonal selection. This approach was used to study lineage and clonal evolution in a mouse mammary carcinoma (called SP1) in syngeneic CBA/J mice (Kerbel et al., 1988; Waghorne et al., 1988) and, more recently, in a human malignant melanoma (called MeWo) grown in nude mice (Kerbel et al., 1989). The results in the SP1 tumor studies revealed evidence for clonal dominance of primary tumors by metastically competent tumor cells variants. The SP1 mammary tumor does not normally metastasize from a subcutaneous implantation site (Kerbel et al., 1987). However, the process of calcium phosphate-mediated transfection results in as many as 10-20% of the clones acquiring this phenotype (Kerbel et al., 1987).The metastases are normally found in the lungs after subcutaneous implantation. Thus after the plasmid pSV2neo is used to genetically tag SP1 cells, approximately 1 out of every 10 or 20 clones may be competent for metastasis. When a population of between 50 and 100 pSV2neo-transfected SP1 clones was selected in G418 and pooled, and the DNA extracted from the cells, it was found to present as a faint smear on Southern blotting after being probed with pSVzneo (Kerbel et al., 1987). This cell mixture was injected into syngeneic CBA/J mice and the primary tumors were removed about 6-7 weeks later along with solitary lung metastases from each animal. When these tumors were analyzed by Southern

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blotting all were found to be essentially composed of the progeny of a single clone (Kerbel et ol., 1987); moreover, the identity of the clone was the same from one animal to another, whether it was a primary tumor or a metastasis. Further analysis showed this was not due to an inability of the other injected clones to form tumors. Thus if primary tumors were removed at earlier time points, e.g., 3 weeks after injection, the tumors were found to be populated by a large number of the injected clones (Waghorne et ul., 1988). But, remarkably, by 4-5 weeks after injection;-just a week or two later-dominance of the tumors b y the single clone (called neo 5 ) became readily apparent (Waghorne et al., 1988).This is shown in Fig. 2. The results therefore seemed to indicate that a single clone, initially present in the mixture in a ratio as low as 1 : 50 to 1 : 100 of the cells, came to dominate primary tumors in an exponential-like manner. Furthermore, this clone was metastatically competent. Reconstitution experiments in which a single genetically tagged, metastatically competent clone was mixed with an excess of the parental nonmetastatic (and unmarked) SP1 tumor cells revealed that the degree of enrichment of the metastatic clones was on the order of 5- to 50-fold (Waghorne et al., 1988).This is shown in Fig. 3. Thus, these experiments showed that if a metastatic variant preexisted in a tumor cell population as a “silent” clone its progeny came to overgrow the primary tumor mass over time, indicating a clear growth preference of the metastatic cells over their nonnietastatic counterparts. This raises the question of what would occur if a population of metastatic cells was genetically tagged and similarly injected into a group of animals: would clonal dominance still be observed, and if so, would the same clone always predominate from one primary tumor to another? The answer would appear to depend on the number of such clones that are pooled and injected. For example, Korczak et al., (1988) used retrovirus vectormediated proviral insertions to genetically tag a metastatic variant of the SP1 cell line (obtained by exposure to hydroxyurea). Because this is a high-efficiency gene transfer technique a large number of individual marked clones can be obtained in a single-step selection procedure, e.g., lo4 or los clones (Korczak et ul., 1988, 1989). If as niany as lo4 or lo5 SP1 clones are pooled and injected into syngeneic mice as few as 10 (or less) dominant clones were observed in the primary tumors obtained 6-7 weeks later; moreover the identity of these clones was not predictable from one tumor to another. In this situation there may not be a single metastatic clone which can consistently dominate all others. However, if a fewer number of such clones were to be pooled and injected (say 10 or 20) then the statistical circumstances for

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a single dominant clone to consistently emerge would presumably be more favorable. Frost and co-workers have obtained evidence that this may be the case (P. Frost, unpublished observations): they pooled equal numbers of 11 different chromosomally distinguishable clones of a human breast carcinoma and the pooled population was injected into nude mice. The primary tumors were always found to be domi-

FIG.2. Evolution of “clonal dominance” in primary tumors by metastatically conipetent cell variants. A pooled population of SP1 mouse mammary carcinoma cell clones, each with a separate genetic marker, was injected subcutaneously into syngeneic CBA/J mice or maintained in culture for 77 days in 5% fetal calf serum (i.e., in nonliiniting growth conditions). The genetic markers were generated by calcium phosphatemediated transfection of the plasmid pSVpneo (Waghorne et al., 1988). A time-coiirse analysis of changes in the clonal composition of pSVpneo-transfected and genetically tagged SP1 tumor grown in nitro or in uino was then undertaken. The photograph shows the results of Southern blot hybridization analysis of genomic DNA from (A) a pSV2neotagged SP1 cell population recovered from frozen stock and maintained in tissue culture for 0, 22, or 77 days (lanes 1 to 3); (B) primary tumors (“1”s”) and tumor cells cultured from whole lungs (“mets”) removed at the indicated number of days after Subcutaneous inoculation of the day 0 cell population shown in lane 1 (lanes 4-8); and (C) tumor cells cultured from whole lungs 37 days after intravenous inoculation of the day 0 cell ) digested with BarnHI, separated by electrophoresis population (lane 9). DNA (5~ gwas on 0.6%agarose gels, transferred to nitrocellulose, and hybridized with a 32P-labeled pSVpneo probe. The results indicate effective dominance of the primary tumors by day 28 to day 35.At 21 days (lane 4) the primary tumors are clearly populated by many clones, but by day 28 many of these clones have disappeared. The results shown in lane 9 indicate a large number of the SP1 clones can grow in the lungs after intravenous inoculation compared to subcutaneous inoculation. (From Waghorne et al., 1988, with permission of the publisher.)

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Frt:. 3. (A) Clonal dor~iinaiice/overgrowthof primary tumors by metastatically competent cells as assessed by “mixing” experiments using Southern blotting. A genetically tagged (and spontaneously metastatic) clone of the SP1 niouse mammary carcinoma cell line was obtained by random integration of the plasmid pSVzneo after calcium phosphate-mediated transfection (Kerbel et a [ . , 1987; Waghorne et al., 1988).This clone was designated “neo 5.” T h e neo 5 clone was mixed at various ratios with the parental (unmarked nonmetastatic) SP1 cells s o that the neo 5 cells were always in the minority. Th e various mixtures were then injected subcutaneously into separate CBA/J mice. Primary tumors were removed 6-7 weeks later along with individual lung tumor/ nodules, and evidence for overgrowth of the neo 5 clone was sought hy Southern blotting using pSVpneo as a probe. Lane 1 is the SPl (nontagged) parental line. Lanes 2-6 are 50 : 1, 20 : 1, 10 : 1, 5 : 1, and 2 : 1 ratios of SP1 to n e o 5 cells, respectively. Lane 7 is the neo 5 clone. Lanes 8 and 9 are primary tumors removed from mice 1and 3 after injection of a 1 : 1 S P l / n eo 5 mixture. Lanes 10,11, and 12 are primary tumors from mice 1,2, and 4 after injection of it 10 : 1 S P l / n e o 5 mixture. Lanes 13 and 14 are primary tumors from mice given a 100 : 1 mixture of SP1 to neo 5 cells. Lanes 15-19 are lung metastases from mice given the 10: 1 or 100: 1 mixtures, as indicated. Th e mouse number and lung nodule number is indicated as 1-1, 1-2, etc., on top of lanes 15-19. It is apparent that overgrowth of neo 5 cells can he seen in the primary tumors analyzed in lanes 10 and 11. About a 5- to 10-fold enrichment has occurred in these cases over the 6- to 7-week observation period. N o obvious enrichment is seen in lanes 13 and 14 because the initial ratio o f n eo 5 cells to SP1 cells ( 1 : 100) in the inoculum was too low to result in more than

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nanted by the same single clone. Thus the results point to a process of genetic convergence rather than genetic divergence occurring over time during the course of tumor progression (Hein et al., 1988). However the convergent dominant clones, though homogeneous for the genetic marker used to detect them, may b e heterogeneous in other characteristics such as chromosome numbers and markers (Bell et al., 1989). How can such striking clonal dominance phenomena be explained? The most obvious explanation is by intrinsically shorter population doubling times. Even a slightly shorter population doubling time will endow a cryptic cell subpopulation with what can be a striking exponential growth advantage over time (see, e.g., materials and methods section of Carroll et al., 1988). This explanation does not appear, however, to explain the results of Waghorne et al. (1988), showing dominance of metastically competent variants of SP1 when mixed with an excess of nonmetastatic SP1 variants. This is because the tuniorigenic behavior, lag times, and growth rates of the two types of population are indistinguishable in Giuo when analyzed separately (Waghorne et al., 1988; D. Theodorescu and R. s. Kerbel, unpublished observations). This implies interclonal interactions between the cryptic metastatically competent clones and their nonmetastatic counterparts may facilitate the growth preference and eventual dominance of the former in primary tumors. The release of growth factors from the nonmetastatic cells which preferentially stimulate the growth of the metastatic cells could conceivably bring this about. Evidence for this has been obtained in vitro and in vivo implicating TGFp as the molecular growth factor mediator (D. Theodorescu and R. S. Kerbel, unpublished observations). It is also consistent with recent findings indi10% neo 5 cells in the primary tumors by the time they were removed. It will also be noted that some of the lung metastases lack the presence of the pSVpneo integration. These are SP1 cells which apparently metastasized as a result of being “recruited” by the neo 5 cells (Waghorne et ul., 1988). (B)A similar experiment was undertaken as described in (A) except that a rus-transfected metastatic clone of SP1 was used (called ras 1)in place ofneo 5. When probed with a ras gene fragment a number of insertion sites are apparent (lane 7). Once again growth dominance of the metastatic clone is seen, even after a 100 : 1mixture of SP1 to ras 1 cells are injected (lane 9). The degree ofenrichment of the ras 1 clone is about 20- to 50-fold over the 6- to 7-week observation period. From lanes 10, 11, and 14 it is also apparent that further genetic changes (rearrangements) of the transfected ras genes have occurred in some of the metastases. The DNA in (A) was digested with BamHI and probed with pSV2neo; that in (B) was digested with XbuI and probed with a 2.9-kb SstI fragment of each experimental group as in Fig. 2. Controls are SP1 DNA mixed with known amounts of“tagged” DNA. (From Waghorne et al., 1988, with permission of the publisher.)

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cating that metastatic variants produce and respond to growth factors in a fundamentally different manner from their nonnietastatic counterparts. This literature will be reviewed in Section V, which deals with growth factors. The results are also pertinent to the notion of tumors being cellular “ecosystems” in which the presence of one clone can influence the behavior of another clone (Heppner, 1984, 1989). This has been thoroughly documented by a number of groups, e.g., Heppner, the Millers, and Poste and co-workers (Poste and Greig, 1982; Heppner et al., 1983,Heppner 1984,1989).A striking variety ofphenotypes can be affected by these interclonal interactions, including immunogenicity (Miller et al., 1980),drug resistance (B. E. Miller et al., 1981, 1985; Tofilon et ul., 1984), production of proteases such as type IV collagenase (Lyons et al., 1989), and metastatic aggressiveness (Poste et ul., 1982; Poste and Greig, 1986). To this list we can apparently add clonal dominance of metastatically competent cells in primary tumors, at least in some situations. Other interesting examples of clonal dominance effects have been described recently, e.g., by the Millers and their co-workers (Miller et ul., 1987, 1988). These investigators examined the relative growth characteristics and clonal composition of tumors derived from mice given an injection of a mixture of two distinguishable breast carcinoma variants. The variants were line 4T07, a thioguanine-resistant subline, and line 168, which is thioguanine sensitive. The cell lines have a common genetic background, namely from a spontaneously arising mammary tumor in a BALB/cfC3H mouse (Miller et al., 1987). Mixtures of the two variants, in various ratios, were injected into mice and the resultant tumors were removed 6 weeks later. The tumors were found to be composed almost entirely of 4T07 cells even when the inoculum contained a much higher proportion of 168 cells (Miller et al., 1987).Subsequent studies revealed line 4T07 cells suppressed the growth of line 168 cells by an active mechanism not involving host immunity, but which required close cell contact (Miller et al., 1988). This implicated the release of growth-inhibitory factors by line 4T07 which somehow mediate suppression of line 168 cells. It should b e noted that neither line 168 nor line 4T07 is capable of spontaneous metastasis. Thus, such dominance effects can also clearly manifest themselves in mixtures of small numbers of clones where none is metastatic. It would be of interest therefore, in the system described by the Millers, to assess the relative dominance of metastatically competent lines derived from 4T07 or 168 when mixed with 4T07 and 168 cells. The results in this section provide genetic evidence to show the

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marked growth preference and dominance of metastatically competent cells in primary tumors. Other genetic marker systems, e.g., drugresistance genetic markers showing similar results, have been summarized elsewhere (Kerbel et al., 1988). All of these studies utilized rapidly growing transplantable animal tumors. Do the results of such experimental systems have relevance to human cancer? The answer would appear to be affirmative, the basis for which is summarized in the next section. IV. Human Malignant Melanoma as a Paradigm for the Growth Dominance of Metastatic Cancer Cells Of all the various types of human neoplasms, probably none better illustrates the selective nature of the metastatic phenotype, its growth dominant nature, and, indeed, the principles of tumor progression than malignant melanoma. Melanoma is increasing at a faster rate in North American than any other type of cancer, and it is estimated that about 1 in every 100 children born today will eventually develop superficial spreading type melanoma (Clark, 1988). Although most forms of melanoma are curable in their early stages they tend to be very aggressive in later phases of progression of the disease, and cure rates at these points are low. Before discussing the nature of the human metastatic melanoma cell it is necessary to briefly review some of the more important aspects of the natural history of the disease. There are four common forms of malignant melanoma as well as several uncommon forms (Clark, 1988; Clark et al., 1984). The common forms are (1) superficial spreading type melanoma, (2) nodular type melanoma, (3)lentigo maligna melanoma, and (4)acral lentiginous melanoma. The most prevalent of these is the superficial spreading type and it evolves in a stepwise fashion as do the lentigo maligna and acral lentiginous melanomas. In contrast, nodular type melanoma represents an example of direct tumor progression (Clark, 1988; Clark et al., 1984). Malignant melanomas are tumors of melanocytic cells which are normally nonmitotic cells found in the basal layer of the epidermisthe outermost layer of the skin. They usually are found as single cells surrounded by keratinocytes, with the ratio of keratinocytes to melanocytes being about 30 to 1. The first step in the multistep process of melanocytic neoplasias (with the exception of nodular type melanoma) is the formation of the common acquired melanocytic nevus or the commqn mole (Clark, 1988). Thought to be due primarily to exposure to ultraviolet light, nevi begin to appear between the first and second

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year of life and this continues for approximately 20 years (Clark, 1988). Gradually these moles disappear and become tan skin tags. Their appearance and disappearance is accompanied by precise histologic changes (Clark, 1988). Most important of these is an increase in the number of melanocytes in the basal epidermis and an associated hyperpigmentation. Subsequently the dividing melanocytes begin to form small nests and single melanocytes migrate into the dermis. With time a terminal differentiation-like process ensues and the cells evolve along the lines of Schwann cells, forming structures similar to nerve endings so that the entire lesion may become composed o f a delicate neuromesenchynie. It then flattens and disappears. Depending upon where the common acquired melanocytic nevus is found, it is classified differently (Clark, 1988). Thus, when the cells are limited to the basal layer of the epidermis the lesion is called “lentigo.” Nest forniation in the epidermis denotes a “junctional nevus,” and when melanocytes are found in both the epidermis and dermis the lesion is called a “compound nevus.” Finally, once melanocytic division in the epidermis ceases the lesion is called a “dermal nevus.” The next stage in melanoma progression is the formation of the “dysplastic nevus.” While the vast majority of growing melanocytic nevi eventually differentiate and disappear, as described above, occasionally a few do not follow this pattern; instead, signs ofa combination of an abnormal growth pattern and cytologic abnormality of the melanocytes become apparent. The lesion becomes larger and more irregular but at this stage it is not yet neoplastic. More than half of the melanomas (of the superficial spreading type) have a mole precursor, the majority of which manifest dysplasia (Clark, 1988). The various phases of preneoplastic and neoplastic progression of human melanocytic neoplasias are summarized in Fig. 4. Within a dysplastic nevus additional genetic changes can take place resulting in the emergence of a neoplastic cell (or cells) which gives rise to the first phase of primary melanoma growth. This stage of melanoma progression is termed the radial growth phase (RGP).The tumor cells at this early stage have limited invasive properties but are not yet competent for metastatic spread. Thus they can invade the dermis but are confined to its most superficial layer-the papillary dermis (Clark et ul., 1984; Clark, 1988). The tumor cells tend to be arranged in nests or small nodule-like structures, and the nests do not appear to have a growth preference over the surrounding cells. Although the cells grow in all directions (i.e., upward in the dermis, peripherally in the epidermis, downward from the epidermis into the dermis, and peripherally in the papillary dermis), the net clinical

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NORMAL MELANOCYTE

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"thin"lesion (<0.76rnrn) "thick" lesion (>0.76rnrn)

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FIG.4. T h e stages of progression of melanocytic neoplasias in humans. Cell cultures can be established from normal melanocytes, dysplastic nevi, R G P primary melanomas, V G P primary melanomas, and distant melanoma metastases, which are indicative of growth doniirtance of metastatically competent variant melanoma cells in primary melanoma tumors. As a result, detailed genotypic and phenotypic comparisons can be made between the various stages both in oitro and in oioo (Clark, 1988; Herlyn et ul ., 1987, 1989).As described in the text, such studies have revealed increasing degrees of similarity of advancing V G P primary lesions to distant metastases (e.g., Fig. 5).

growth is horizontal, i.e., at the periphery, along the radii of an imperfect circle (Clark, 1988). Following the radial growth phase is the so-called vertical growth phase (VGP) of primary melanoma growth. It represents the emergence of a sublesion (presumably a subclone) within the primary RGP lesion (a nodule within a nodule) and nicely illustrates the principle of tumor progression and clonal evolution of tumor growth as defined by Nowell (1976, 1989).After a variable period (usually 1-2 years, but as long as 10 years) the character of growth in the dermis of the RGP melanoma cells changes focally. The cells now grow as spheroidal nodules and manifest a clear growth preference/dominance over the rest of the tumor in the papillary dermis. The net direction of growth tends to be perpendicular to that of the radial growth phase. Some of the pathologic features which distinguish the VGP from the RGP include the following: (1)VGP cellular aggregates tend to be larger than

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the mostly intraepidermal RGP aggregates; (2) the dominant site of growth shifts from the epidermis to the dermis and the VGP lesion can extend into the lower half ofthe reticular dermis (which lies below the papillary dermis); ( 3 ) the cells are usually less pigmented in VGP lesions and there is much less ofa cellular immune response present in VGP lesions compared to RGP lesions (Clark, 1988). The prognosis of patients with RGP lesions is outstanding- 100% ciire rates are achieved by surgical excision. With respect to VGP lesions, prognosis depends on the thickness of the lesion and levels of' invasion (Rreslow, 1970; Clark, 1988).It stands a s a remarkable clinical observation that VGP lesions 0.76 mni or less in thickness are associated with an excellent prognosis-about a 95% cure rate can be obtained by surgical excision. In contrast, prognosis becomes more oniinoiis in lesions greater than 0.76-111111thickness; the thicker t h e lesion, the worse the prognosis. Thus, when the VGP produces a lesion of greater than 4.0-mm thickness 7 5 4 0 % of affected patients will die of metastatic disease. Levels of tumor invasion as defined by Clark (1988) can also b e a useful as a prognostic indicator (although not as much as tumor thickness in the vertical growth phase). Level I tumors are in situ, i.e., they lie above the basement membrane which separates the dermis from the epidermis. Level I1 tumors are those in which the invasive cells are present only in the papillary dermis (this generally coincides with the radial growth phase). Level 111 tumors are nodular VGP lesions that impinge the reticular dermis. Level IV invasion indicates invasion hetween the collagen bundles of the reticular dermis. Finally, level V invasion refers to tumors that extend into the subcutaneous fat . Beyond 1.7O-mm t h ickne s s 1e s ions , in format ion about levels of invasion can provide useful additional prognostic information (Clark, 1988). Malignant melanoma is usually thought of as a particularly virulent form of cancer. This reputation is not entirely justified because unlike inany other neoplasms it i s curable when dealt with in its early stages (i.e., during the RGP or thin VGP stages). Its reputation as an aggressive tumor derives largely from its ability to fornm metastases in almost every organ, and also its resistance to therapy. However, even with respect to metastasis, there are some restrictions. Initially, melanoma metastases usually involve the regional lymph nodes, but metastases via the bloodstream can also occur. It is true that such hematogeneous metastases are unusually widespread compared to other types of neoplasm. However, many melanoma patients die with metastases largely confined to the central nervous system (CNS) whereas others manifest widespread visceral (including cutaneous) metastases (Akslen et d.,

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1988). In other words there are cluster patterns, thus implying the metastatic process is not random, even in malignant melanoma. When considering the progression of human malignant melanoma it is important to note that there is one form which does not conform to the sequence ofevents just described, namely the nodular type. It is an example of direct tumor progression in which competence for metastasis is expressed in the initial lesion, i.e., it does not go through a radial growth phase: the tumor is in the vertical growth phase from the outset. It must be said that many other human neoplasms, e.g., lung cancers, renal cell carcinomas, pancreatic tumors, to name a few, behave similarly to nodular type malignant melanomas in this respect because they too do not appear to go through easily detectable sequential stepwise changes associated with superficial spreading type melanoma (or colorectal carcinomas). Whether the nodular type melanoma goes through a pronounced abbreviated form of progression or is metastatic from the initial stem cell stage is not yet clear. The relationship of tumor thickness to patient outcome, i.e., metastasis, in particular the 0.76-mni “breakpoint” figure, is intriguing. What does it signify and what can it teach us about the biology ofmetastasis? Folkman and others have persuasively argued that most carcinomas originate in the avascular epithelial compartment as in situ lesions, and remain separated from the underlying vascular bed until the basement membrane is breached, either by the tumor itself, or by new capillary vessels, or both ( Folkman, 1987).Once breached, the tumor nodule can become vascularized, which is an absolute requirement for it to grow beyond about 1 mm in diameter (Folkman, 1985). Rapid expansion and metastatic dissemination of the tumor can then occur. From the foregoing discussion on the natural history and progression of human malignant melanoma we might therefore ask why radial growth phase or thin vertical growth phase primary melanoma nodules found i n the dermis are not competent for metastasis. Presumably they must have breached the basement membrane separating the epidermis from the dermis. The answer may reside in the fact that dysplastic nevi (Yaar et ul., 1988) and early (mainly epidermal) melanomas (Schmoeckel et al., 1989) are surrounded b y a thin basement membrane, the integrity of which is largely maintained. As VGP tumors thicken, focal lesions in the surrounding basement membrane begin to appear and eventually the membranes are not seen in more advanced dermal lesions (Schmoeckel et uZ., 1989). Presumably this occurs because the metastatically competent melanoma cells are in an environment where there are fewer keratinocytes (the epithelial cell source responsible for making the basement membrane) and because the melanoma cells

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produce basement menibrane-degrading enzymes such as type IV collagenase (Turpeennienii-Hujanen et d.,1986). Folkman has discussed the notion that the increased melanoma thickness beyond 0.76-mm thickness correlates with neovascularization because 0.76 mm is approximately the longest distance permissible between the outer layer of melanoma cells and the nearest open capillaries in the dermis (Folkman, 1987). In any case there is now evidence that the basement membrane becomes disrupted and breached at around the 0.75-mm level of thickness (Kirkham, 1987). Also, it has been determined that melanomas of less than 0.75- to 0.8-mm thickness are not neovascularized, whereas virtually all those greater than 0.8-to 0.9-mni thickness are (Srivastava et al., 1986,1989). These findings, along with the progression stages of melanoma, indicate that cell variants having metastatic competence emerge within early (thin) V G P primary lesions and manifest a clear growth preference over their metastatically incompetent neigh1)ors. If the primary lesion is left intact long enough it should be overgrown by the metastatically competent cells along the lines of the clonal dominance model described in the previous section, and elsewhere (Kerbel et al., 1989). If this were the case then one should detect marked genotypic and phenotypic differences between R G P or thin V G P primary lesions and metastases, whereas strong similarity, or even identity, between advanced V G P lesions arid metastases should b e the norm. A wealth of recent evidence indicates that this is indeed the case; some of this evidence is summarized in Table I, a few examples of which will Ile highlighted here and in the next section on growth factors. Many different groups have attempted to detect and define human melanoma-specific antigens by immunization of mice with human melanoma cells in order to produce monoclonal antibodies (e.g., Brown et al., 1981; Houghton et al., 1982, 1987; Kan-Mitchell et u l . , 1986; Ruiter et d., 1984; Herlyn et al., 1987; Holzmann et al., 1987). Occasionally a hybridoma is found which appears to react preferentially with metastatic melanoma cells. A striking example ofthis is the monoclonal antibody called P3.58 (Johnson et ul., 1989). The P3.58 antigen was subsequently identified by gene cloning and nucleotide sequence analysis to be identical to intracellular adhesion molecule-1 (ICAM-1) by Johnson et a1. (1989). A combination of in situ immunohisto-chemistry and in uitro analyses provided clear evidence that ICAM-1 is not detectable on quiescent melanocytes and is rarely found on benign preneoplastic lesions (dysplastic nevi) or on thin primary melanomas of less than 0.75-mm thickness (Johnson et al., 1989; Natali et al., 1990). An example of this is shown in Fig. 5. In striking contrast,

TABLE I Fcxcrrox OF TUMOR PROGRES5lON ISDICA.TI\.EOF THE GROWTH DOMINANCE OF 1hfETASTATICALLY COMPETENT VARIANTS I N PRlXlARV LE5lONS

PHENOTYPIC CHANGES IN HUMAN MELANOCYTES AS

Property examined

Normal or nevocellular nevi melanocytes

Dysplastic nevus nielanocytes

.4

RGP primary melanomas

VGP primary melanomas; “thin”-earl y lesions

VGP primary melanomas; thick-advanced lesions

hlelanoma metastases

References Halaban et al. (1988); Halaban (1988) Holzmann et al. (1987); Johnson et al. (1989); Natali et al. (1990) Ruiter et al. (1984); Holzmann et al. (1987)

Autocrine production of bFGF Expression of ICAM-1

No

No

No

Probably not

Probably yes, but variable

Yes

NO

No

No

NO

Yes

Yes

Expression of HLA-Dr (class 11) antigens

No

No

Negative or low

Expression generally increases with increasing tumor thickness

Yes

Response to TPA i n ljitro

Growth Growth stimulation stimulation

Growth stimulation

Expression generally increases with increasing tumor thickness Variable

Variable

Expression of “P9/MUC18” antigen Nonrandom changes in chromosomes 1.6, and 7 Growth factor independence in oitro

No

NO

No

No or low degree of expression

Yes

Growth inhibition Yes

Halaban (1988); Halaban et al. (1986) Holzniann et al. (1987)

No

NO

No

NO

Yes

Yes

Herlyn et 01. (1987)

No

No

No

No

Yes

Yes

Kath et 01. (1990)

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H. S . KEHBEL 100

50

Nevi

I $0.75

0.7G1.5

1.51-3.0

23.0

Met

Primary Tumors (thickness in mm)

FIG.5. Changes in the expression of ICAM-1 with progression of hiiiiian tnaligiiatit mrlanoirw ( Johnson r’t l l / . , 1989). ICAM-1 expression was determined b y imnir~noprrosidase staining of frozen tissue sections using monoclonal antibodies P3.58,’and P3.58”, which recognize ICARI-1 tleteriiiinants, Positive lesions contained 2 5% stained cells. Nevi, and “thin” primary lesions were found to h e ICAM-1 nrgative whereas distant metastases arid triore advanced (>0.75 nun thick) primary lesions were both positive. Met, metastases. (After Johnson et NI., 1989, with permission of the authors and pubI i slier).

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the majority VGP primary lesions of greater than 0.76-nini thickness are ICAM-1 positive, as are melanoma metastases (Johnson et ( i l , , 1989). Expression is heterogeneous, ranging from 5 to 90% of the cells and increases somewhat with increasing tumor thickness. The kinetics of appearance of ICAM-l/P3.58 expression in primary human melanomas are remarkably similar to the clonal dominance of primary mouse tumors by metastatically competent cells described in the previous section and elsewhere (Kerbel et a l . , 1988).It illustrates that the results of the genetic tagging experiments are relevant to the progression of at least some types of human neoplasms. This conclusion is also substantiated by one other important and interesting item of information listed in Table I, namely the growth factor requirements of human melanoma cells in tissue culture. Herlyn and colleagues have undertaken an exhaustive comparative analysis of primary melanomas and melanoma metastases using a large tumor cell bank of cell lines established from many independent lesions (Herlyn et al., 1985, 1987).A large number of features were examined, such as nonrandom chromosome abnormalities, expression of melanoma-associated antigens, growth in nude mice, and growth in culture. The results of these studies indicated that cells cultured from the vertical growth phase, but not from the radial growth phase, of primary melanomas were similar to metastatic melanoma cells (Herlyn et ul., 1985). One exception was thought to be growth factor requirements: it was initially concluded that metastatic but not primary melanoma cell lines grew in uitro independently of exogenous growth factors (Rodeck et ul., 1987). Subsequent analysis revealed, however, clear evidence for growth factor independence in uitro of primary melanoma cells obtained from aduunced but not early or intermediate primary lesions (Kath et d . , 1990).The nature ofthese factors will be discussed in more detail in a later section with respect to the mechanisms governing the growth preference and dominance of inetastatically competent cells. Although human malignant melanoma has been used as a paradigm to illustrate the growth preference and dominance of metastatically competent variants within primary tumors, other interesting examples could b e cited as well. Thus human colorectal carcinomas represent another class of tumors which go through a well-defined sequence of preneoplastic lesions and stages or primary tumor growth. Dyplastic colonic epithelial cells can give rise to adenomas--benign proliferative lesions-which progress from type I to type I11 adenomas. The type I11 adenomas can progress to Duke’s A primary neoplasias which in turn can progress to Duke’s B, C, and finally D primary tumors. Distant metastases, normally first detected in the liver, represent the

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final stage in the progression of colorectal carcinoma. The Duke’s staging system is h e d mainly on the level of invasion i n t h e bowel wall and presence of metastases. Unlike melanoma, priniary tumor size has little impact on prognosis (W. Miller et ul., 1985). For example Duke’s B 1 carcinomas are those that have penetrated to muscularis propria without distant metastasis whereas H2 tumors have penetrated through the bowel wall without metastases. Duke’s D carcinomas have niestastasized to distant sites (Astler and Coller, 1954). If metastatically competent colorectal carcinoma cells have a growth advantage in the primary lesion it would be predicted that the proportion of such cells would increase with increasing levels of primary tumor invasion of the bowel wall, and would come to dominate Duke’s D tumors. There is indirect evidence that this is the case. Fidler and co-workers, and others, have published a series of papers describing the metastatic properties of established human colorectal cell lines or fresh biopsy specimens in athymic nude mice (Fidler, 1986; Giavazzi et d., 1986;Cajot et ul., 1986; Morikawa et d., 1988).Although colorectal carcinomas grow well in nude mice they tend not to metastasize i f injected ectopically, e.g., sul)cutaneously (Fidler, 1986). If, however, the tumor cells are injected into an orthotopic site such as the cecum, liver metastases may develop in the mice. It appears that the presence and level of liver metastases depend upon the Duke’s level of the tumor being analyzed: Duke’s I3 tumors tend to be non- or very weakly metastatic whereas Duke’s D tumors are highly metastatic, coniparable, in fact, to the metastatic potential in nude mice ofhepatic metastases obtained from colorectal carcinoma patients (Morikawa et ul., 1988). Moreover, while it is possible to easily select more metastatic sublines from Duke’s B colorectal carcinomas, it is far more difficult to do so from Duke’s D tumors, using progressive selection methods in nude mice (Morikawa et ul., 1988). All of these results are consistent with the idea that the Duke’s D tumors are composed largely of variant metastatically competent cells whereas the earlier stage tumors contain proportionately fewer such cells: the marked growth preference/ dominance of metastatically competent cells brings this change about. The results summarized in this section graphically illustrate the clonal dominance/growth preference of metastatically competent cells in human primary tumors but also highlight the crucial role that time plays in manifestation of the phenomenon. They raise several important questions. First, what are the factors which explain the growth preference of metastatically competent cells in priniary tumors? Second, how important are they with respect to the process of metastatic dissemination and their growth in secondary organ sites? Third, is the

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growth preference/dominance of metastatically competent or metastatic cells ull that is required to express metastatic potential, or is it a necessary but not sufficient property (Liotta, l988)?Considerations of these questions form the hasis of the next section of this review. V. The Role of Growth Factors in the Growth Preference and Dominance of Metastatically Competent Cells

A. GROWTH STIMULATOHY AND AUTOCHINE GROWTH FACTORS It would seem logical to begin any discussion on the possible niechanisms governing the growth preference and dominance of metastatically competent cells from the perspective of polypeptide growth factors. The contribution of various normal or tumor cell-derived stirnulatory or inhibitory growth factors-be they autocrine, paracrine, or endocrine-has assumed a dominant position in the current literature with respect to the developnient and expression of the neoplastic or tumorigenic phenotype (e.g., Goustiii et al., 1986; Heldin et ul., 1987; Sporn and Roberts, 1988). By comparison, relatively little is known about their contribution to the metustatic (malignant) phenotype. However, the viewpoint will be put forward in this section that many of the concepts currently in vogue concerning the role of growth factors in the development and expression ofthe neoplastic phenotype may actually be relevant primarily or only to the metustutic tumor cell variant. These include “growth factor independence” or aritocrine growth of tumor cells, and loss of response to certain inhibitory growth factors by cells when they become neoplastic. To illustrate these points we will turn again to the example of human malignant melanoma. A very large number of long-term cell lines obtained from human melanomas have been established, mostly from lymph node metastases. However, a considerable number of lines from vertical growth phase primary tumors and even some from radial growth phase primary tumors have also been successfully established (Heryln et uZ., 1987, 1989). In addition, it is now possible to grow nornial human melanocytes in culture (Eisinger and Marko, 1982; Eisinger et uZ., 1985; Halaban and Alfano, 1984; Halaban et al., 1987) and melanocytes from dysplastic nevi (Halaban et ul., 1986). Consequently the growth factor requirements and characteristics of metastatically competent melanoma cells-whether they are from primary tumors or metastatic lesions-can be rigorously evaluated and compared to normal nonneoplastic melanocytes or primary melanoma cells which are not metastatically competent. Several

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investigators have contributed to this area and much of this important work was recently summarized b y Herlyn and colleagues (Herlyn et a l . , 1989). The most notal)le finding concerning the comparative growth factor properties and requirements of melanoma cells is that metustntic melanoma cells exhibit a striking growth factor autonomy i n tiitro whereas metastatically incompetent primary melanoma cells (e.g., those obtained from RGP or thin VGP lesions), as well as nonneoplastic melanocytes, exhibit varying degrees of growth factor dependence (Rodeck et ul., 1987; Herlyn et al., 1989).Thus Gilchrist and colleagues found normal tnelatiocytes required choleratoxin (as a stimulator of intracellular CAMP levels) for growth and they were stimulated b y a factor called MGF-melanocyte growth factor (Gordon et al., 1986). I n contrast, offour melanoma cell lines tested (the source of the lines was not indicated) none was stimulated by MGF and choleratoxin had little consistent effect (Gordon et al., 1986). At this point one might conclude that the neoplastic cells have reduced growth factor requirements in comparison to their normal counterparts. However, other studies b y Halaban and colleagues (Halaban et al., 1986, 1987, 1988; Halaban, 1988) and by Herlyn and co-workers (Rodeck et ul., 1987; Herlyn et al., 1989; Kath et d.,1990)show clearly this would be an overly simplistic conclusion. What these two groups did was to compare the growth factor requirements and response of various primary melanomas of various stages in addition to cell lines established from metastases. Halaban's studies showed that the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate) was required for the growth of newborn or adult melanocytes or for melanocytes from dysplastic nevi whereas it actually inhibited the growth of the metastatic melanoma cell lines (Halaban et al., 1986). The inhibitory effects of TPA on metastatically competent melanoma cells have heen noted independently by numerous investigators (e.g., Dooley et al., 1988; Wilson et al., 1989; Herlyn et al., 1989). Halaban observed that the growth factor requirements of primary melanonias tested were in general more stringent than for melanoma cell lines established from metastases, and concluded that the acquisition of independence from mitogenic growth factors is a late event in melanoma progression (Halaban et al., 1986).Subsequent studies revealed evidence that normal or abnormal preneoplastic melanocytes or primary melanomas depend on specific factors such as basic fibroblast growth factor (bFGF) whereas metastatic melanoma cells do not (Halaban, 1988).Halaban et al. (1988) also found that metastatic melanoma cells produce bFGF at the niRNA and protein levels, unlike normal melanocytes. They therefore concluded that b F G F is an autocrine growth factor for melanoma

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cells and this presumably explains why addition of bFGF to metastatic melanoma cells has no stimulatory effect. It would also appear that another metastatic melanoma cell-derived growth factor (MGF) described by Eisinger and colleagues (Eisinger et al., 1985)is similar to bFGF. Richmond and colleagues (Richmond et al., 1982, 1985; Richmond and Thomas, 1986) have also described a different autocrine-like growth factor from human melanoma cells called MGSA (melanoma growth stimulatory activity). Recently cDNA characterization established that the deduced amino acid sequence for human MGSA is identical to the deduced amino acid sequence of the human gro cDNA (Richmond et al., 1988; Anisowicz et al., 1988). No function has been assigned to the human gro cDNA but it is known not to be melanoma specific (Anisowicz et al., 1987; Wen et al., 1989). In fact it can be synthesized by stimulated endothelial cells (Wen et ul., 1989).Whether expression of MGSAlgro protein, like bFGF, is associated with metastatic or metastatically competent cells of melanocytic origin but not with nonmetastatic melanoma cells is not firmly established although preliminary evidence has indicated nevus melanocytes do not produce MGSA (Richmond et al., 1982,1985). A very detailed analysis of the growth requirements of human melanoma cells has also been undertaken by Herlyn and colleagues (summarized in Herlyn et al., 1989).They found that normal melanocytes and melanocytes from common acquired and congenital nevi have very similar growth requirements (with the exception that nevi melanocytes require less bFGF). Thus, stimulatory effects are seen with TPA, bFGF, insulin, and substances which enhance intracellular levels of CAMP such as a melanocyte-stimulating hormone (a-MSH). When some of these factors were omitted from the culture medium, e.g., insulin or insulin-like growth factor-I (IGF-1),cells from primary melanomas were severely inhibited whereas cell lines established from metastases could be quickly adapted to protein-free growth conditions and continued to proliferate for over 6 months under the same conditions (Heryln et al., 1989). As discussed earlier, it was found that TPA inhibited the growth of the metastatic (and primary) melanoma cell lines. Rodeck et al. (1987) found that insulin was the strongest single growth factor for primary and metastatic melanoma cell lines. The metastatic cell lines remained growth responsive to epidermal growth factor (ECF), insulin, and transferrin and responded more vigorously to these exogenously added niitogens than the primary cell lines tested. Rodeck et (11. (1987)concluded that IGF-1 and insulin are major growth factors for melanoma cells and act via the type I I G F receptor. Insulin or IGF-1 do not appear to be melanoma cell autocrine growth factors as is b F G F or MGSA.

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At this point one would reasonably conclude that there is a gradual evolution toward growth factor autonomy in the progression of human malignant melanomas and that primary melanomas are never a s growth f k t o r autonomous or responsive as are melanoma metastases. From the clonal dominance model, however, one would predict otherwise, that is, given enough time, primary melanomas would behave similarly or identically to metastatic melanomas. Evidence for this was obtained by Herlyn’s group (Kath et al., l990), who examined the compar.‘1t’1ve growth factor requirements of early and intermediate versus advmced primary melanoma lesions. “Advanced” lesions were those in which the patient had clear evidence of simultuneous metastases. In contrast, the patients with early or intermediate lesions did not have macroscopic metastases detectable at the time of removal of the primary tumor; these were clinically delayed or absent, i.e., they appeared later (9 months to 7 years) during the course of their disease (intermediate lesions), or not at all (early lesions). These investigators found that the melanoma cells from the early or intermediate lesions were unable to proliferate in a medium depleted of exogenous growth factors and required addition of at least one such factor, such as insdin or IGF-1. On the other hand, t h e cells of advanced primary melanomas were indistinguishable from autologous metastatic cells as demonstrated by their complete independence from the exogenous growth factors. Kath et al. concluded that the cells representing the metastatic lesions overgrew the primary tumors in advanced, but not in early or intermediate, melanoma lesions. These results are complemented by those of S a u vaigo et a1. (lYSCi), who examined the growth factor requirements of a human melanoma cell line called MeWo and a more aggressive metastasizing (in nude mice) variant called LCI that was partially selected in nude mice (Kerbel et id., 1984b). The more aggressive variant was found to exhibit a greater degree of growth factor autonomy in vitro in that it divided persistently in completely serurn-free medium (Sauvaigo et ul., 1986). From the foregoing results it is apparent that acquisition of (complete) growth factor independence is not a property of neoplastic melanocytes unless they are also metustatically competent. This underscores the need to more carefully qualify t h e current and popularly held notion of a direct association of growth factor independence and the development ofcancer (Haas et al., 1984,1986; Sporn and Roberts, 1985; Lang et nl., 1985; Wheeler et nl., 1986; Hays et ul., 1988)and is consistent with the notion put forward by Chadwick and Lagarde (1988)that “the metastatic process involves selection of the most autonomous cells.” Their studies, and those of others, reinforce the asso-

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ciation of complete growth autonomy i n uitro and the metastatic phenotype. Thus a Chinese hamster lung fibroblast called CC139 was injected into nude mice. Tumors were removed, established in culture, and their growth factor requirements were determined. The CC139 cells are not tumorigenic and it is thought that the rare tumors which arose in nude mice were the consequence of additional genetic changes in a subset of cells in uiuo (Chadwick and Lagarde, 1988).The CC139 cells were found to require several growth factors, namely insulin, a-thrombin, and epidermal growth factor (EGF) to proliferate in culture. In contrast, cells from pulmonary metastases proliferated in serum-free medium. Moreover, some CC139 variants selected in uitro for reduced growth factor requirements were found to be metastatic after implantation into nude mice. Chadwick and Lagarde also demonstrated that growth factor-independent metastatically competent variants were present in varying proportions in primary tumors obtained from CC139-injected nude mice although a time-course analysis was not done to determine if the proportion of such cells increased with time and eventually dominated the primary tumors. In any case, the results clearly indicated achievement of growth factor autonomy was associated with a more aggressive malignant phenotype, which also confirmed and extended earlier results from the same laboratory (Renwick et ul., 1986). Greenberg and Wright, and their colleagues, also recently uncovered evidence for an association between growth factor autonomy and the metastatic phenotype (Schwarz et ul., 1988).Earlier studies by this group have demonstrated that transfection of H-rus into the mouse C3H 10T''2 immortalized nontumorigenic fibroblastic cell line resulted in the isolation of tumorigenic clones of varying metastatic potential (Egan et al., 1987).In general, highly metastatic clones expressed higher levels of ras mRNA than clones which were deficient in metastasis. The metastatic clones were found to exhibit a significantly reduced dependence on serum for cell proliferation in uitro (Schwarz et ul., 1988).Finally, Herlyn et al. (1989) have recently summarized evidence showing an association between growth factor independence of human colorectal carcinomas (and other types of carcinomas) and the metastatic phenotype. These results, taken together with the demonstration of clonal dominance of primary tumors by metastatically competent cells, mean that growth factor requirements of spontaneous or experimentally induced animal tumors will vary directly with the extent to which they are overgrown (dominated)by metastatically competent variant cells. This would help explain, for example, the results of Ethier and Cundiff (1987),who found that a subset of primary

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mammary tumors induced in female Lewis rats by chemical carcinogens grew independently in zjitro of growth factors required by normal rat mammary epithelial cells. Cells from 5 of 18 independent primary tumors exhibited requirements for insulin, EGF, and choleratoxin identical to those of growth factor-dependent normal epithelial cells; in contrast, cells from 9 of 18 tumors expressed independence of one, two, or all three of these factors. Not surprisingly, the latter cell lines were tumorigenic in syngeneic rats whereas the five fixtor-dependent cell lines were not (Ethier and Cundiff, 1987). Unfortunately, their metastatic characteristics were not evaluated but it seems reasonable to predict that the cell lines able to grow in the absence of insulin, EGF, and choleratoxin would be metastatic, or the most aggressively so. While growth factor autonomy, autocrine loops, and enhanced responses to stimulatory growth factors such as insulin, IGF-1, and E G F are important in explaining the growth preference and dominance of metastatically competent cells, this may only be part of the story: one also has to consider the idea that metastatic cells may be less responsive to growth inhibitory molecules compared to nonmetastatic tumor cells. This notion is discussed in the next part of this section.

B. Loss OF RESPONSEBY METASTATICCELLSTO GROWTH INHIBITORY MOLECULES: METASTATICCELLSAS EFFECTIVE CELLULAR SCAVENCEHS Part of the proliferative basis for the neoplastic phenotype has been attributed, as discussed above, to the production of stimulatory growth factors which act in an autocrine fashion (Goustin et al., 1986; Roberts and Sporn, 1988).There are also a number of growth factors which act as strong inhibitors of cell growth, and it is thought that loss of responsiveness to such factors by neoplastic cells would also confer increased proliferative potential to them (e.g., Moses et al., 1987).The paradigm here is transforming growth factor /3 (TGFP). There are several members of the TGFP family which are regarded as among the most potent inhibitory growth factors known for epithelial and hemopoietic cell lineages (Keski-Oja et al., 1988a; Roberts and Sporn, 1988).A summary of the properties of T G F p is beyond the scope of this article but can be found in numerous recently published reviews (Moses et al., 1987; Sporn et al., 1987; Keski-Oja et al., 1988b; Roberts and Sporn, 1988). The main point to be made here is that loss of response to the growth inhibitory effects of TGFP by tumor cells may be a function of their metastatic competence. This point has been rarely considered in the

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numerous studies undertaken to evaluate the effects of TGFP on normal versus tumor cell proliferation. Thus the reports that nornial epithelial cells from a certain organ such as the liver can be growth inhibited whereas their neoplastic counterparts cannot (e.g., McMahon et UZ., 1986; Fausto and Mead, 1989)are usually not supplemented by any information about the metastatic properties of the neoplastic cells. Recently, however, this parameter has begun to be examined and the results indeed support the idea that metastatically competent cells are less vulnerable to being growth inhibited by TGFP, or alternatively, are more sensitive to being growth stimulated. Wright, Greenberg, and their colleagues recently examined the effects of TGFP on a number of H-rus-transfected tumorigenic clones obtained from the C3H10T1’2 fibroblast cell line (Schwarz et al., 1988). As summarized above, the clones varied considerably in their metastatic properties and this was found to be correlated with levels of rus expression. Schwarz et aZ. (1988)found that the parent (nontumorigenic) 10T’” cells and a nonmetastatic cell line derived from the 10T”’ cells were growth inhibited by TGFPl whereas the metastatic clones were all stimulated, as much as about sixfold. Brattain’s group has also reported results showing different effects of TGFP as a function of malignant status (Hoosein et d . ,1989).Three groups of human colon carcinoma cell lines were examined for their response to two molecular forms of TGFP (TGFPl or TGFP’). Aggressive, poorly differentiated cells (group I) did not respond to growth inhibitory effects of TGFPl or TGF& while the less aggressive well-differentiated cells of group I11 did so by being growth inhibited. The moderately welldifferentiated cells of group I1 were also growth inhibited in two out of three cases (Hoosein et uZ., 1989). Although the metastatic characteristics of the various cell lines were not documented it is probable that the group 1 cells would be the most aggressively metastatic. The results of Schwarz et d .(1988)and Hoosein et uZ. (1989)point to what is clearly emerging as an important theme in the biology of the metastatic cancer cell: the effect of a given multifunctional growth factor, or group of such factors, on metastatically competent tumor cells will frequently tend to favor their growth over that of their nonmetastatic counterparts. This in turn will facilitate both their growth preference and dominance within primary tumors and their ability to grow into macroscopic metastatic deposits elsewhere. As such, metastatic cells might be thought of as highly effective cellular scavengers. In considering this hypothesis it should be pointed out that most growth factors are known to be multifunctional (Sporn and Roberts, 1988).That is, almost any given growth factor-not just TGFP-can

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stimulate or inhibit cell growth depending upon the circumstances. These include (1) whether the cells are normal or neoplastic, (2) the tissue lineage of the cells, ( 3 ) cellular density and three-dimensional status ofthe cells being tested, and (4)whether they are dividing or not at the time a growth factor is added (Roberts and Sporn, 1988). To this list we can now add the metastatic status (or metastatic potential) of neoplastic cells: it may turn out to be the most important single parameter influencing the response of tumor cells to a number of different growth factors. GROWTH FACTORS AND THE C. THEROLEOF TISSUE-SPECIFIC MICHOENVIHONMENT IN METASTATICTUMOR GROWTH Before concluding any discussion on the role of growth factors in the growth preference of metastatic cells, reference should be made to the involvement of a possible new family of growth factors, namely those made by normal host tissues which facilitate tumor cell growth in a seemingly organ-specific manner. It is of course well known that many tumors metastasize in a predictable manner, giving rise to organspecific patterns of metastasis (Weiss, 1985;Auerbach, 1988; Nicolson, 1988). Many factors may help govern this intriguing phenomenon, including ( 1) preferential adhesion to microvascular endothelial cells in a given organ, (2) preferential adhesion to organ parenchymal cells, ( 3 ) local tumor cell stimulation by growth factors secreted by normal host cells in a given organ, and (4)chemoattraction by factors released by host cells in a given organ. Evidence for all these possibilities has been obtained and was recently summarized in detail by Nicolson (1988). One recent and interesting example of a possible organ-specific growth factor comes from Nicolson’s laboratory (Cavanaugh and Nicolson, 1989).They isolated and purified from mouse lung tissue a lowmolecular-weight polypeptide which was found to stimulate the growth in uitro of a variety of mouse tumor cell lines provided the cell lines were capable of metastasizing to the lungs in uiuo. The existence of such tissue-specific growth factors may be necessary to ensure efficient clonogenic growth of metastatic tumor cells in an ectopic organ site since even when ex-uiuo- transplantable tumors are injected orthotopically into syngeneic animals a minimum of several thousand to lo4 (or lo5)cells is required to achieve a tumor “take.” Considering this fact, the ability of one, or a small number of tumor cells to give rise to a metastasis is an extraordinarily impressive achievement. The heightened sensitivity to stimulatory growth factors, whether they are produced by the tumor cells themselves, or by normal host cells, along

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with a decreased sensitivity to inhibitory growth factors (many of which may also be produced by normal host cells) may explain why this can occur. Ultimately the altered growth factor requirements and responsiveness of metastatically competent tumor cell variants must be reflected in alterations in how they respond to cell signals at the cell surface and transmit these signals to the nucleus to bring about changes in gene expression. Signal transduction and the nature of second signal pathways in metastatic cells (versus their nonmetastatic counterparts) is an area of research that is still in its infancy (see Hart et al., 1989, for details) and will not be reviewed here. Reports are now appearing showing, not surprisingly, that activators of protein kinase C such as pathway of signal transTPA, or of the calcium/calmodulin-dependent duction, can alter metastatic behavior (e.g., Gopalakrishna and Barsky, 1988; Korczak et al., 1989; Lester et al., 1987, 1989). Although the information in this area is limited, it is worth emphasizing (again) that many of the studies designed to uncover differences between “normal” and “neoplastic” cells-in this case with respect to signal transduction-may in fact be relevant only, or primarily, to the metastatic cancer cell. This is because of the ability of metastatically competent cells to clonally dominate primary tumors over time. Thus, analysis of established tumor cell lines obtained from primary tumors may be equivalent to the study of a cell line established from a metastasis. In addition, if a cell line being studied is composed of a mixture of metastatically competent and incompetent cells the signal transduction characteristics may be more complex and variable compared to those obtained with pure populations of either.

VI. Ectopic Gene Expression and the Pleiotropic Nature of the Growth-Dominant Metastatic Phenotype Although the growth dominance of the metastatic cancer cell has been discussed primarily from the perspective of growth factors, this is not meant to leave the impression that this is the sole feature that distinguishes metastatic variant cells from their nonmetastatic counterparts. As if to reinforce this point, Maslow recently summarized the results of a literature search documenting all of the known differences between B16F1 (low metastatic) and its highly metastatic (B16F10) variant (Maslow, 1989): over 61 differences were noted. This certainly emphasizes the massive pleiotropic changes that are associated with the metastatic cancer cell, and make it necessary to exercise restraint in ascribing the acquisition of metastatic competence primarily to one

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particular property (Liotta and Stetler-Stevenson, 1989).The comparative properties of nonmetastatic versus metastatic human melanoma cells summarized in this article highlight this pleiotropy as well (e.g., see Table I). Thus, if an examination of the levels of certain proteases such as plasminogen activator (PA) or type IV collagenase was undertaken in growth factor-independent (and metastatic) tumor cells it would probably reveal the enzyme levels to be increased. Conversely, the growth factor requirements of tumor cell variants selected for increased invasive capacity would b e expected to be less stringent than their noninvasive counterparts. In short, the two phenotypes are somehow linked, expressed, and coregulated in the same cell subpopulation. To illustrate this point attention is drawn to a recent study from Denhardt’s group (Khokha et nl., 1989). Nontumorigenic mouse 3T3 cells were transfected with plasmids capable of making antisense RNA to the messenger RNA encoding TIMP (tissue inhibitor of metalloproteinases). The object of the experiment was to determine the impact that selective upregulation of certain proteases brought about by TIMP inhibition would have on cell behavior. Surprisingly, the transfectants were found to be tumorigenic and metastatic in nude mice and highly invasive in uitro, thereby implicating TIMP as a tumor suppressor gene. Perhaps, however, this is not so surprising considering the intimate and complex interrelationships of growth factors and proteases. As summarized by Laiho and Keski-Oja (198$)), there are numerous examples of growth factors which induce the secretion ofproteases, e.g., bFGF and PA synthesis (e.g., Sato and Rifkin, 1988; Presta et nl., 1986),and conversely, of proteases regulating the activity of growth factors, e.g., plasminogen activator/plasmin activation of latent T G F p (Lyons et nl., 1988). These interrelationships may form the basis for negative and positive feedback loops which regulate growth factors and protease activity in cells (Roberts and Sporn, 1988; Laiho and Keski-Oja, 1989).Such regulatory loops may become uncoupled in cancer cells, especially in their metastatic variants, leading to a highly invasive, motile and growth-dominant phenotype. What might cause such uncouplings is currently a matter of speculation. However, one obvious possibility is the loss of master regulatory genes, e.g., recessive tumor suppressor genes of which the retinoblastoma (Rb) gene is the paradigm (Cavafiee and Hansen, 1986; Marx, 1988; Seemayer and Cavanee, 1989) and which have heen noted in a large variety of human tumors, including maligant melanoma (Dlacopoli et ul., 1989). Such gene (or allelic) losses may be cumulative, involving a number of independent loci over time, a s seems to be the case in human colorectal carcinomas (Baker et ul., 1989; Vogelstein et

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aZ., 1989). Moreover, some of the losses may involve metastasis suppressor genes such as nm23 (Steeg et al., 1988) or WDNMI (Dear et aZ., 1988) rather than tumor suppressor genes. The net result will be acquisition of increasing degrees of growth dominance and ectopic gene expression associated with newly emerging tumor clones (Vogelstein et al., 1989). Ectopic gene expression could provide additional growth or invasive advantages, and do so in a number of ways. Obviously, if the ectopically expressed genes encode growth factors (or their receptors), proteases, or adhesion molecules, etc., it is selfevident how they could directly endow cells with a selective advantage. But this could also come about indirectly. Thus, ectopic expression of the serotonin Ic receptor in mouse 3T3 cells (induced by transfection of the serotonin lc receptor gene) can trigger their neoplastic transformation if the receptor is activated by serotonin binding (Julius et al., 1989).Why this comes about is not known. However, in view of these results we might pause to consider the consequences of the increased number of cases of ectopic gene expression that are often encountered in metastatic cancer cells. For example, neither b F G F nor ICAM-1 is expressed in tumorigenic but metastatically incompetent primary human malignant melanoma cells (see Table I) whereas they are expressed in metastatically competent variant of these cells. How many other examples of such ectopic gene expression in metastatic melanoma cells might there be? And what would be their cumulative effect on tumor cell growth? The possibilities are numerous but acquisition of increased levels of growth dominance seems assured.

VII. Conclusions This article summarizes the evidence for the growth-dominant nature of the metastatic cancer cell-how it contributes to the overgrowth of primary tumors, to metastatic tumor growth in distant organs, and the reasons these may come about. The growth-dominant phenotype appears to be a fundamental property of the metastatic cancer cell in a broad spectrum of animal and human neoplasms. The implications of this concept are broad and extend well beyond the study of metastatis per se. To being with, we need no longer pay homage to the viewpoint that metastatic variants never compose more than a tiny minority of the tumor cells in primary tumors. Given enough timeand in some situations this may b e quite short-a primary tumor may become the equivalent of a distant metastasis. As such, failure to detect genotypic and phenotypic differences between autochthonous primary tumors and their metastases is often to be expected-not be-

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moaned-and does not in any way detract from the hypothesis that metastases are generated from preexisting genetically variant subpopulations. Thus, the finding of an altered gene in a primary tumor which is also present in metastases from the same patient may not be uncommon (Varley et al., 1989) and does not disqualify the gene from being involved in the metastatic process. This concept also effectively transforms the study of metastasis from a somewhat esoteric, albeit important, area of cancer research to one that is fundamental to understanding the cancer cell as a whole. Thus “subversion of growth regulatory pathways” (Heldin et al., 1987) and “growth factor autonomy,” to cite a few examples, are concepts that may largely apply to the rnetastutic cancer cell rather than to the cancer cell per se. It is also worth highlighting some of the clinical implications of the growth-dominance concept of the metastatic or metastatically competent cell. One example which comes to mind is breast cancer. An intensive worldwide search continues for reliable prognostic markers to help predict which women with primary (lymph node negative) breast cancer will develop metastatic recurrence of their disease, about 30%of whom will do so (Hellman and Harris, 1987). It is evident that a prognostic marker will probably be associated with the aggressive, metastatically competent subpopulation within the primary melanoma tumor. Hence, the greater the proportion of such cells, the greater the probability of recurrent disease and of obtaining a positive result with a putative prognostic marker. Is there a sharp breakpoint beyond which the proportion of metastatically competent cells within primary breast tumors will inevitably lead to recurrent disease? This is not a far-fetched idea when the example of human malignant melanoma i s recalled: primary melanoma lesions of less than 0.76-mm thickness rarely metastasize, whereas those greater than 0.76-mm thickness have an increasing tendency to do so (as discussed in Section IV). If reliable, in situ single-cell detection assays for metastatic breast cancer cell can be developed we may eventually encounter a similar situation in breast (and other) cancers. That is, once the proportion of metastatically competent tumor cells in a primary cancer attains a certain value, the probability of recurrence may take a quantum leap. Finally, the inefficiency of the multi-step metastatic process (Weiss, 1986) may b e compensated for by the metastatic cell clonal dominance phenomenon, i.e., the more cells in a primary tumor capable of metastasis, the greater the probability that one (or a few) of them will complete all the steps associated with metastasis. The growth preference or dominance of the metastatic cell has been discussed in this article primarily from the perspective of growth fac-

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tors. However, it must be stressed that the metastatic cell simultaneously acquires and manifests an impressive arsenal of phenotypic characteristics which facilitate cellular motility, migration, implantation, and invasion-all features necessary for metastasis. Along with the acquisition of a high degree of growth factor autonomy (and refractoriness to inhibitory growth factors) these characteristics together endow a cancer cell with formidable powers for progressive growth in the primary tumor site, for dissemination, and finally for progressive growth in a distant organ site. Viewed from this perspective, the notions that the metastatic cell represents “the apotheosis of the neoplastic process” or is the cellular equivalent of a decathlon champion are indeed appropriate. ACKNOWLEDGMENTS

I am most grateful for the excellent secretarial assistance of Lynda Woodcock in preparing this article. The author’s research is supported by grants from the National Cancer Institute of Canada, the Medical Research Council of Canada, and the U.S. Public Health Service, National Institutes of Health (CA 41233). The author is also a Terry Fox Senior Cancer Research Scientist of the National Cancer Institute of Canada.

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Taylor-Scher, B., Bargatze, R., Holzmann, B., Gallatin, W. M., Matthews, D., Wn, N., Picker, L., Butcher, E. C., and Weissman, I. L. (1988).Adu. Cancer Res. 51,361-390. Thorgeirsson, U. P., Turpeenniemi-Hujanen, T., and Liotta, L. (1985). Znt. Rev. E x p . Pathol. 27,203-234. Tofilon, P. J., Buckley, N., and Deen, D. F. (1984). Science 226,862-864. Turpeenniemi-Hujanen, T., Thorgeirsson, U. P., Rao, C. N., and Liotta, L. A. (1986).261, 1883-1889. Vaage, J. (1988).Znt. J . Cancer 41,855-858. Varley, J. M., Armour, J., Swallow, J. E., Jeffreys, A. J., Ponder, B. A. J., T’Ang, A. T., Fung, Y. K. T., Brammar, W. J., and Walker, R. A. (1989). Oncogene 4,725-730. Vogelstein, B., Fearon, E., Kern, S. E., Hamilton, S. R., Preisinger, A. C., Nakamura, Y., and White, R. (1989).Science 244,207-211. Waghorne, C., Thomas, M., Lagarde, A. E., Kerbel, R. S., and Breitman, M. C. (1988). Cancer Res. 48,6109-6114. Weiss, L. (1980). Pathobiol. Ann. 10,51-81. Weiss, L. (1983). Invasion Metastasis 3, 193-207. Weiss, L. (1985). “Principles of Metastasis.” Academic Press, Orlando, Florida. Weiss, L. (1986). Cancer Rev. 3, 1-24. Weiss, L., Holmes, J. C., and Ward, P. M. (1983). B r . J . Cancer 47,81-89. Wen, D., Rowland, A., and Derynck, R. (1989). Emboj. 8, 1761-1766. Wheeler, E. F., Rettenmier, C. W., Cook, A. T., and Sherr, C. J. (1986).Nature (London) 324,377-380. Wilson, R. E., Dooley, T. P., and Hart, I. R. (1989). Cancer Res. 49,711-716. Woodruff, M. F. A. (1988). A d o . Cancer Res. 50,197-229. Yaar, M., Woodley, D. T., and Gilchrest, B. A. (1988). Lab. Invest. 58, 157-162. Young, S. D., and Hill, R. P. (1986).Clin. Exp. Metastasis, 4, 153-176.

THE PATHOGENESIS OF BURKITT’S LYMPHOMA Ian Magrath Lymphoma Biology Section, Pediatric Branch. National Cancer Institute. Bethesda. Maryland 20892

I. Introduction 11. Definition of Burkitt’s Lymphoma A. Historical Perspective B. Histology C. Immunophenotype, Cytogenetics, and Genotype 111. Clinical and Epidemiological Features A. Anatomical Distribution of Tumor B. Role of Environmental Factors in the Pathogenesis of Endemic Burkitt’s Lymphoma C. Burkitt’s Lymphoma in Other Geographic Regions D. Burkitt’s Lymphoma in Immunosuppressed Individuals E. Implications of Clinical Findings for Pathogenesis IV. Phenotype of Burkitt’s Lymphoma Phenotypic Differences between Sporadic and Endemic Burkitt’s Lymphomas V. The Nonrandom Chromosomal Translocations Associated with Burkitt’s Lymphoma Parallels with Animal Tumor Models VI. Structure and Function of c-myc A. Structure of the c-myc Gene and Its Products B. Regulation of Transcription of c-myc C. Functions of the c-myc Protein(s) D. c-myc and Cell Proliferation VII. Timing of the Translocation in Relation to B Cell Differentiation A. Translocations Outside the Switch Region B. Switch Region Translocations C. Translocations Involving Light Chain Genes D. Restriction of the Translocation Time Frame VIII. Mechanism of Translocation A. Possible Mediation of Immunoglobulin Signal Sequences B. Sequence Alterations Adjacent to Breakpoint Locations IX. Structural Changes in c-myc Brought About by the Translocations and Their Possible Functional Consequences A. Breakpoints Far Upstream (5’) of c-myc B. Breakpoints in the 5’ Regulatory Region of c-myc C. Breakpoints within the First c-myc Exon or First Intron D. Breakpoints Downstream of c-myc X. Breakpoints on Chromosomes 14,2, and 22 and Their Functional Significance with Regard to c-myc Expression A. Chromosome 14 Breakpoints B. Chromosome 2 and 22 Breakpoints 133 ADVANCES IN CANCER RESEARCH, VOL. 55

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XI. Correlation of Breakpoint Location with Geography XII. Effect of the Chromosomal Translocations on c-myc Expression A. Evidence that c-myc Is Constitutively Expressed in Burkitt’s Lymphoma B. Stability of c-myc mRNA in Burkitt’s Lymphoma C. Differential Usage of the c-myc Promoters in Burkitt’s Lymphoma D. The c-myc Protein in Burkitt’s Lymphoma E. Absence of Expression of the Normal c-myc Gene in Burkitt’s Lymphoma F. Role of Immunoglobulin Sequences in the Expression of the Translocated c-myc Gene in Burkitt’s Lymphoma XIII. Consequences of Deregulation of c-myc A. Mice Transgenic for c-myc B. Replacement of the Requirement for a Translocation by a Viral PromoterControlled myc Gene C. Chicken Bursa1 Lymphoma XJV. The Role of Other Genetic Abnormalities A. Introduction of Viral Oncogenes into Animal Tumor Models B. Possible Role of Anti-Oncogenes C. Defects in DNA Repair XV. EBV and Burkitt’s Lymphoma A. The Epidemiology and Biology of EBV Infection B. EBV-Induced Lymphocyte Transformation C. Specific T Cell Recognition of EBV-Infected B Cells D. EBV Latent Gene Expression in Burkitt’s Lymphoma E. Reduced Susceptibility to T Cell Cytolysis of Burkitt’s Lymphoma Cells F. Other Evidence for a Pathogenetic Role for EBV in Burkitt’s Lymphoma G. Possible Role for an EBV Transactivator Gene H. Animal Models of EBV-Induced Lymphoproliferation XVI. Synthesis A. Cellular Origins B. Environmental Factors and Immunosuppression C. Significance of Clinical and Biological Differences D. Molecular Consequences of the Chromosomal Translocations E. The Need for Additional Genetic Factors F. Probable Role of EBV in a Subset of Burkitt’s Lymphoma XVII. Coda-Clinical Significance References

. , . riverrun, past Eve and Adam’s, from swerve of shore to bend of bay . . . JAMES JOYCE

I. Introduction

When, in 1958, Burkitt described a “sarcoma” of the jaw in African children (Burkitt, 1958), he could hardly have conceived that this rather exotic African curiosity would contribute so hugely to the science and practice of oncology. Indeed, there are few disciplines within the broad compass of oncology that have not profited, in some way, by information derived from the study of this tumor. The initial focus was,

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of necessity, almost exclusively on treatment and epidemiology. Reports of occasional cures with chemotherapy alone (radiation was not available in Uganda and surgery had proved to be of limited benefit) provided enormous encouragement to pioneering chemotherapists of the era who were faced with the limited benefits of single-agent chemotherapy coupled to a high toxic cost. Today, some 60 to 80% of patients with Burkitt’s lymphoma can be cured with appropriate drug combinations (Magrath et al., 1984; Magrath, 1988). Burkitt and his colleagues also recognized the existence of climatic determinants of the distribution of the “African lymphoma” (Burkitt, 196213; Haddow, 1963) and proposed, accordingly, that the disease is caused by an arthropod-vectored virus (Haddow, 1970). Although evidence to substantiate a role for an arthropod vector was never obtained, the hypothesis motivated a search which led to the discovery of Epstein-Barr virus (EBV), an important human pathogen that is associated with some subtypes of Burkitt’s lymphoma, as well as an increasing list of other lymphoid and epithelial tumors (Epstein et al., 1964; Ablashi and Salahuddin, 1989; Barriga et al., 1988a). In recent years, progress in the fields of immunology and molecular genetics has permitted a series of fundamental observations to be made, which have led to a quite detailed knowledge of the biology and molecular pathogenesis of Burkitt’s lymphoma (Magrath, 1985). During the beginnings of the immunological renaissance of the 1960s and 1970s, the detection of immunoglobulin on the surface of Burkitt’s lymphoma cells (Klein et al., 1968) was one of several observations which crystallized the seminal concept that the non-Hodgkin’s lymphomas share many of the characteristics of their normal counterpart cells in the immune system, and led to modern attempts to utilize immunophenotyping in the classification of the malignant lymphomas (Magrath, 1981; Cohen and Jaffe, 1990). The subsequent demonstration that functional immunoglobulin genes are assembled by a process of recombination of discontinuous genetic subunits during B cell differentiation (Hozumi and Tonegawa, 1976; Early et al., 1980; Leder et al., 1980; Adams and Cory, 1983), and the discovery of the chromosomal translocations associated with Burkitt’s lymphoma (Zech et al., 1976), paved the way for the development of an understanding, at a molecular level, of the pathogenesis of this tumor. Once immunoglobulin genes had been cloned, and their chromosomal locations identified (Croce et al., 1979; Hobart et al., 1981; Kirsch et al., 1982; Erikson et al., 1981; McBride et al., 1982; Malcolm et al., 1982), it became clear that the translocations always involved one of the chromosomal regions containing sequences coding for an immunoglobulin LI

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chain (14q32,22qll, or 2pll-12). This finding in a B cell tumor was provocative, but equally intriguing was the finding that regardless of whether the translocation was the most frequent 8;14, or less frequent 8;22 or 2;8 (the so-called “variant” translocations), the q24 region at the distal end of chromosome 8 was invariably involved. The presumptive conclusion from these observations, that the chromosomal translocations were likely to be of pathogenetic significance, was soon reinforced by the demonstration that band q24 on chromosome 8 was the location of the c-myc gene (Dalla-Favera et al., 1982), the cellular homolog of the transforming element of the avian myelocytomatosis virus, MC29 (Roussel et al., 1979; Sheiness and Bishop, 1979; Sheiness et d . ,1980a,b). With the use of cell lines derived from tumors (Magrath et al., 1980a,c; Lenoir et al., 1985; Kiwanuka et al., 1988), it was rapidly established that c-myc is translocated in the 8;14 translocations from its position on chromosome 8 into the immunoglobulin heavy chain region of chromosome 14 (Dalla-Favera et al., 1982; Taub et al., 1982; Erikson et al., 1982), while in the variant translocations part of the immunoglobulin light chain region is translocated to a position downstream (3’)of c-myc (Croce et al., 1983; Davis et al., 1984; Erikson et al., 1983b; Hollis et al., 1984; Malcolm et al., 1985). These observations provided the foundation for a series of elegant studies which have demonstrated beyond any reasonable doubt that the deregulation of c-myc is a critical component of the pathogenesis of Burkitt’s lymphoma. At the present time there remains much to be learned of the mechanisms whereby the c-myc gene is deregulated, and of the biochemical consequences of this. The precise function of c-myc remains unknown, although its expression has been shown to be necessary for cellular proliferation (Kelly et al., 1983). It also remains to elucidate the presumptive role of EBV in Burkitt’s lymphoma, and to determine what factors are necessary, in addition to deregulation of c-myc, to induce neoplasia (for a considerable amount of evidence suggests that c-myc deregulation alone is insufficient). In spite of the numerous challenges remaining, Burkitt’s lymphoma has provided a paradigm from which to extrapolate to other systems, and promises to remain in the van of research into the identification of the biochemical components of neoplastic transformation, as well as the utilization of molecular pathological information for the definitive diagnosis and subclassification of a tumor-an essential basis for meaningful research into epidemiology and treatment. Furthermore, the molecular genetic changes associated with tumorigenesis, being absent from normal cells, provide a poten-

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tial target for highly selective anticancer therapy. Burkitt’s lymphoma, therefore, also provides a valuable model for the tentative exploration of such novel treatment strategies (Magrath, 1989,1990b). In this article, the overwhelming evidence that deregulation of c-myc is a critical component of the pathogenesis of Burkitt’s lymphoma is summarized and discussed, and consideration is given to the potential pathogenetic role of EBV. Clinical and epidemiological observations pertinent to the pathogenesis of this tumor are presented, and information derived from relevant animal tumors, particularly those in which the homologous chromosomal translocations are present, has been used liberally to provide insights into the structural and functional changes which have been described in Burkitt’s lymphoma.

II. Definition of Burkitt’s Lymphoma The definition of any malignant tumor is a function of the tools available for its characterization. In the last century tumors were defined on the basis oftheir clinical features or gross pathological appearance (Magrath, 1990a). Today, histological examination of fixed sections of the tumor is the most widely used diagnostic method, and although subjective histological criteria have served us well throughout most of this century, their shortcomings are readily apparent in the hemopoietic neoplasms (Magrath, 1981). In such tumors, cytogenetics, immunophenotyping, and genotyping have been increasingly used in recent years as adjuncts to histological diagnosis, and all of these methods of characterization can be applied to Burkitt’s lymphoma. However, the application of these more objective diagnostic methods has also created a problem in taxonomy, for the histological entity of Burkitt’s lymphoma does not coincide exactly with the cytogenetic entity. Moreover, it is clear that several genotypic subgroups exist within the group of tumors bearing an 8;14 or variant translocation (Pelicci et al., 1986b; Neri et al., 1988; Barriga et a!., 1988a). These findings are changing our concepts of the definition of Burkitt’s lymphoma, and new nomenclature will need to be developed. In the past, it has been debated as to whether Burkitt’s lymphoma is a clinical syndrome or a pathologic entity (Carbone et al., 1969). Today, discussion must center around whether histologic, cytogenetic, or molecular genetic characteristics should have priority in defining the neoplasm and its subtypes.

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A. HISTORICAL PERSPECTIVE

The: high incidence of jaw tumors and lymphomas had been recorded in equatorial Africa by a number of clinicians and pathologists since the first missionary doctors had arrived at the turn of the century (DeSmet, 1956; Thijs, 1957; Davies et al., 1964a,b) but it was Denis Burkitt, a surgeon, who first recognized on clinical grounds that the same tumor could present as a jaw tumor or an abdominal mass frequently affecting the ovaries (Burkitt, 1958,1962a). These tumors had previously been variably diagnosed as sarcomas and dysgerminomas. O’Conor, a pathologist working in Uganda at the same time as Burkitt, recognized that these tumors were lymphomas (O’Conor and Davies, 1960, 1961), and h e and others subsequently reported the identification of histologically identical tumors in specimens which had been preserved in pathology departments in Europe and the United States (Dorfman, 1965; O’Conor et al., 1965; Wright, 1966). B. HISTOLOGY I n 1969 a panel of distinguished pathologists attempted to provide a histological definition of Burkitt’s lymphoma (Berard et al., 1969), but partly because of the subjective nature of histology, partly because of the large variability in the quality of histopathology in different hospitals, and partly because not all pathologists agreed that the tumor should be defined exclusively on morphological grounds, this definition has not led to a high degree of uniformity among pathologists in making a diagnosis of Burkitt’s lymphoma. When O’Conor, Dorfman, and Wright described histologically identical tumors outside Africa, for example, they excluded patients with bone marrow involvement on the grounds that such patients must have leukemia rather than Burkitt’s lymphoma. As recently as 1969, the merits of a clinical versus a histopathological definition of Burkitt’s lymphoma were still being debated (Carbone et al., 1969). In addition to confusion over whether it is appropriate to define malignant neoplasms on the basis of histology alone, or whether geography, clinical features, or virus associations should also be part of the definition, most pathologists have recognized that each of the features originally included in the histological “definition,” like all biological parameters, is inhomogeneous and subjective. Moreover, since in the United States Burkitt’s lymphoma was originally separated from lymphomas which had previously been referred to as “undifferentiated” lymphomas in the Rappaport histological classification- scheme (Rap-

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paport, 1966; Rappaport and Braylan, 1975), lymphomas which resemble Burkitt’s lymphoma but are considered to have too much pleomorphism, or too many single large nucleoli, are often diagnosed as undifferentiated lymphomas of non-Burkitt’s type, even though the majority are immunologically and cytogenetically identical (Grogan et al., 1982; Miliauskas et al., 1982; Whang-Peng and Lee, 1990). In the recent National Cancer Institute (NC1)-sponsored “Formulation” of non-Hodgkin’s lymphomas (Rosenberg et al., 1982) the undifferentiated Burkitt’s and non-Burkitt’s lymphomas have been categorized together as “lymphomas of small non-cleaved cells,” although such tumors can still be subcategorized as Burkitt’s and non-Burkitt’s types. The term “small noncleaved cell” stems from the proposal of Lukes and Collins (1974) that undifferentiated lymphomas arise in a cell type of similar morphology to one which can be observed in the germinal centers of secondary lymphoid follicles. While there is no direct evidence that morphologically similar cells in a germinal follicle are the normal counterparts of Burkitt’s lymphoma, it is probable that one or more subgroups of tumors with “undifferentiated” histologyparticularly those with 14;18 translocations (Fifth International Workshop on Chromosomes in Leukemia-Lymphoma, 1987)-do arise from germinal center cells. Rare tumors with both 8;14 and 14;18 translocations have been described (Fifth International Workshop, 1987; Gauverky et al., 1988) as has progression from follicular lymphoma to Burkitt’s or Burkitt-like lymphoma (Catovsky et al., 1977; Garvin et al., 1983; Mintzer et al., 1984; Gauverky et al., 1988). Lymphomas of small noncleaved cells should in no way be equated with other lymphomas described as of small cell origin in the NCI formulation, since their clinical and biological behavior is completely different. In addition, small noncleaved lymphoma cells are considerably greater in size than the cells of other small-cell lymphomas. In fact, the size of a Burkitt’s lymphoma cell is closer to that of some large-cell lymphomas from which, needless to say, some Burkitt’s lymphomas are difficult to distinguish. Given the degree of variability of the cell appearances in different parts of the same tumor, it is not surprising that Burkitt’s lymphoma merges morphologically, on the one hand with other histological types of undifferentiated lymphomas, and on the other with large-cell lymphomas. Indeed, in patients with HIV infection, the molecular changes typical of Burkitt’s lymphoma are found in most of the tumors, regardless of whether they are classified as small noncleaved or large-cell lymphomas (Pelicci et al., 1986a). A further difficulty which arises when morphology is used as the sole diagnostic criterion is that it has become clear that a small proportion of

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tumors which would be diagnosed as Burkitt’s lymphoma by the majority of experienced hematopathologists differ from other Burkitt’s lymphomas with regard to either immunophenotype or cytogenetics or both. This is most apparent in the case of patients presenting with a form of acute leukemia referred to as ‘‘L3)’in the French-AmericanBritish classification scheme of the morphology of acute lymphoblastic leukemia: a category which predominantly represents Burkitt’s lymphoma (including characteristic cytogenetic findings) in leukemic phase (Bennett et al., 1976; Mittelman et al., 1979; Magrath and Ziegler, 1980; Roos et al., 1982; Ganick and Finlay, 1980).Some of the L3 leukemias, however, differ phenotypically or genotypically from Burkitt’s lymphoma in that they do not express surface immunoglobulin or they lack the typical translocations of Burkitt’s lymphoma (Mangan et al., 1985; Walle et al., 1987; Gauverky et al., 1988).A small proportion may even possess T cell characteristics (Michiels et al., 1988; Kame1 et al., 1989). These problems have led to difficulty in obtaining accurate incidence rates of Burkitt’s lymphoma, even in countries or regions where reliable population statistics are available. Even now, the incidence of the disease in the United States and most European countries can only be roughly estimated. This problem would be improved if more precise, objective diagnostic criteria were agreed upon and uniformly applied-unfortunately, an unlikely eventuality. AND GENOTYPE C. IMMUNOPHENOTYPE, CYTOGENETICS,

There can be little doubt that the biochemical changes which lead directly to neoplasia, or their genetic antecedents, are likely to provide the best definition of a tumor. While, theoretically, similar biochemical changes could lead to neoplasia in different cell types, information so far available suggests that this is unlikely to be so. This is doubtless a consequence of the biochemical differences which exist in cells of different lineages. Not only do such differences mean that the same genetic lesion in different cells may have different consequences, but they dramatically alter the probability of the occurrence of the genetic lesion. We might therefore redefine Burkitt’s lymphoma as a neoplasm of B lymphoid cells in which there is a chromosomal translocation which causes an immunoglobulin gene to come to lie on the same chromosome as c-myc. This definition would have the immediate advantage of excluding small noncleaved lymphomas in which a 14;18 translocation is present (Fifth International Workshop, 1987). Such tumors occur primarily in individuals over 40 years old and may indeed have an origin from cells of the germinal centers of lymphoid

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tissue, as do other lymphomas with 14;18 translocations (Whang-Peng, and Lee, 1990).Such a definition would also have the potential disadvantage, however, that it would include neoplasms which, on morphological grounds, are presently classified as separate tumors (e.g., small noncleaved lymphomas and large-cell lymphoma) (Fifth International Workshop, 1987; Pelicci et al., 1986a; Chenevix-Trench et al., 1986). Whether such morphological differences are of biological significance remains to be seen. In addition, as will become clear from a more detailed consideration of the variety of molecular abnormalities which can be associated with the same histological appearance, this definition would also include neoplasms with differences in the structural genetic changes which result from the chromosomal translocation. Since such structural differences correlate with epidemiological and clinical differences, they probably delineate separate pathologic entities with different etiologies. Because of this, it seems likely that the subgroup of lymphomas defined, as above, on the basis of the cell lineage and the principle features of the associated genetic changes, will need to be subdivided into several subcategories according to the specific structural changes identified (Table I). The latter, in turn, may reflect the state of differentiation of the cell in which the chromosomal translocation occurred, since gene expression and chromatin structure probably influence the location of chromosomal breaks. It is, perhaps, also appropriate to mention a caveat; that it is possible that in rare tumors the same functional changes occasioned by the chromosomal translocation could rarely be brought about in the absence of a translocations. This has, in fact, been described in a small number of mouse plasmacytomas with an interstitial deletion in chromosome 15 (Wiener et al., 1984), and also in human cell lines (Ohno et al., 1989). It is apparent that the newer tools of cytogenetics and genotype already permit a more precise, objective classification of the lymphomas of the B cell lineage but it is likely to be some time before general agreement on their usage is reached. It is important to recognize that each of these diagnostic tools defines a slightly different group of tumors and that pathogenetic definitions will certainly require modification as new findings are made. In this article, the term Burkitt’s lymphoma will be applied to small non-cleaved lymphomas with either an 8;14,8;22, or 2;8 chromosomal translocation. Ill. Clinical and Epidemiological Features Just as the epidemiological features of a malignant tumor reflect the external milieu of the population at risk as well as the internal milieu (genetic factors) of each individual patient, so the clinical features of

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TABLE I DEFINITION AND CLASSIFICATION OF “BURKITT’S LYMPHOMA” A tumor of B cell lineage (expresses B-specific markers such as CD19 and CD20 and usually surface IgM) which has the morphology of an immature or activated B lymphocyte and is associated with genetic changes which cause immunoglobulin gene regions to lie on the same chromosome as c-myc, induce structural changes in the gene, and result in its deregulation

Genotypic subtypes include the following:

1. There is a break in chromosome 8 upstream of c-myc outside the EcoRI fragment that encompasses c-myc a. The heavy chain enhancer element is juxtaposed to the second and third exons of c-myc b. The heavy chain enhancer element is not juxtaposed to the second and third exons of c-myc 2. There is a break within the EcoRI fragment that encompasses c-myc, upstream of the c-myc promoter, PI a. The heavy chain enhancer element is juxtaposed to the second and third exons of c-myc b. The heavy chain enhancer element is not juxtaposed to the second and third exons of c-myc 3. There is a break within the first exon of c-myc a. The heavy chain enhancer element is juxtaposed to the second and third exons of c-myc b. The heavy chain enhancer element is not juxtaposed to the second and third exons of c-myc 4. There is a break within the first intron of c-myc a. The heavy chain enhancer element is juxtaposed to the second and third exons of c-myc b. The heavy chain enhancer element is not juxtaposed to the second and third exons of c-myc 5. There is a break downstream of c-myc with juxtaposition of K constant sequences, possibly including the kappa enhancer element 6. There is a break downstream of c-myc with juxtaposition of A constant sequences Type 1 is nearly always associated with EBV, types 2-6 may or may not be associated with EBV Type 1 is the predominant African or endemic form, type 4 the predominant sporadic and AIDS-related form. Types 5 and 6 may be equally divided between sporadic and endemic forms

the disease are a consequence of the biology and biochemistry of the tumor cells. This is obvious when cancer of a specific organ is considered, but while not so obvious, is equally true for diseases of tissues which are spread throughout the body (e.g., neoplasms of the immune system). Thus the distribution patterns of a disease, both at a popula-

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tion level as well as at an anatomical level in the individual patient, provide information of critical importance to an overall understanding of pathogenesis.

A. ANATOMICALDISTRIBUTION OF TUMOR The most striking feature of the clinical syndrome that Burkitt originally described in Uganda (now commonly referred to as endemic Burkitt’s lymphoma) was the very high frequency of jaw involvement. Some 60% of children with Burkitt’s lymphoma have one or more tumors of the mandible or maxilla, while orbital tumors, some of which may arise in the superior aspect of the maxilla, are also prominent (Burkitt and O’Conor, 1961; Burkitt, 1966, Wright, 1970; Magrath, 1982,1983a). This was a particularly striking finding, since jaw tumors of any histology are infrequent in children. A particularly curious, and perhaps, ultimately, informative feature of jaw tumors in African children is their age association (Burkitt, 1966; Magrath, 1985). This may be explained by the close physical relationship between the tumor cells and the developing molar tooth buds (Adatia, 1970), a situation which suggests that such tumors may have a requirement, at least initially, for one or more growth factors which are present at this location. A possibly parallel situation is the occurrence of breast involvement in pubertal/adolescent girls or lactating women (Shepherd and Wright, 1967). Breast involvement has not been described in young, prepubertal girls, and jaw involvement is much less frequent in adults. Other characteristic features of endemic Burkitt’s lymphoma include the high frequency of central nervous system disease and the low frequency of bone marrow involvement. Both the endemic and sporadic forms of Burkitt’s lymphoma are associated with a high frequency of abdominal tumor (Magrath and Sariban, 1985), although in the sporadic disease, unlike the endemic disease, tumor occurs particularly often in the right iliac fossa; in the appendix, terminal ileum, and ascending colon. Jaw tumors are unusual in North American patients, as are orbital tumors. This difference could arise from differences in the requirement of the tumor cells for growth factors. Even when jaw tumors are present, their clinical features (size, involvement of multiple jaw quadrants, etc.) differ from those in African children (Sariban et al., 1984), although young children with typical African-type jaw tumors have occasionally been reported from European countries (Philip et al., 1982). Bone marrow involvement is common in patients with the sporadic form of the disease (Magrath and Ziegler, 1980). In fact, this may be the commonest site of involvement since 20% of patients who present with

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Burkitt’s lymphoma have overt marrow infiltration, another perhaps 20% of patients have occult bone marrow disease (Benjamin et al., 1983; Magrath, 1988),while some 2-5% of patients who present with acute lymphoblastic leukemia are subsequently determined to have L3 leukemia with the typical Burkitt’s translocations and c-myc rearrangements, i.e. (predominantly), Burkitt’s lymphoma in leukemic phase (Mittelman et al., 1979; Magrath and Ziegler, 1980; Berger et uZ., 1979; Blick et al., 1986, Mathieu-Mahul et al., 1985). Since acute lymphoblastic leukemia is approximately 12 times as common as Burkitt’s lymphoma in Europe and the United States, the L3 leukemia is numerically approximately half as frequent as all forms of sporadic Burkitt’s lymphoma (Magrath, 1988). Thus, one might argue that some twothirds of all patients with sporadic “Burkitt’s lymphoma” have disease in the bone marrow. This is in striking contrast to the disease in equatorial Africa, and, coupled with the other clinical differences, strongly suggests that there are significant differences in the biology of endemic as compared to sporadic Burkitt’s lymphoma-a suggestion that gains strength from the pattern of EBV association in Burkitt’s lymphoma. Almost all endemic tumors, but perhaps only a quarter of sporadic tumors, contain EBV DNA, in spite of the fact that more than 75% of patients with sporadic Burkitt’s lymphoma are EBV seropositive (P. H. Levine et al., 1982). Not only does the endemic form of the disease differ with regard to clinical characteristics and EBV association, but its incidence is considerably higher than that of the sporadic variety. Some 2 or 3 children (less than 16 years old) per million develop Burkitt’s lymphoma in Europe and North America, while the incidence of endemic Burkitt’s lymphoma is between 50 to 100 per million. While genetic factors should not be underestimated, their importance is presently unknown, and they must, in any event, be closely interwoven with the environment, since black children in the United States have a lower incidence of Burkitt’s lymphoma than whites (NCI SEER program). The translocations, however, occur in both endemic and sporadic varieties of Burkitt’s lymphoma (Douglas et al., 1980).

B. ROLEOF ENVIRONMENTAL FACTORS IN THE PATHOGENESIS OF ENDEMIC BURKITT’S LYMPHOMA The importance of environment in endemic Burkitt’s lymphoma is clearly demonstrated by the distribution of this disease, which appears to be dependent upon temperature and rainfall (Burkitt, 1962b, 1970b). The climatic conditions which predispose to Burkitt’s lymphoma correspond to those required for the breeding of the mosquito vectors of

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malaria, and it has been proposed, on this basis, that malaria predisposes to the development of Burkitt’s lymphoma (Haddow, 1970; Burkitt, 1970a). However, all evidence relating those two diseases is indirect. For example, malarial infection has been reported to increase the incidence of Moloney virus-induced lymphomas in mice (Wedderburn, 1970)and to depress the immune response to this virus (Bomford and Wedderburn, 1973). More recently, it was shown that acute malaria, which increases B cell proliferation and induces marked elevation of serum IgM and IgG levels (Jayawardena, 1981),also impairs EBV-specific T cell responses (Whittle et al., 1984). The latter could result in a larger pool of EBV-infected cells, as well as increasing the number of replication cycles which each EBV-infected cell undergoes. Since the risk of development of Burkitt’s lymphoma is a function of the likelihood of the occurrence of a chromosomal translocation, an increase in the size and growth fraction of the target cell population for lymphomagenesis would significantly increase the incidence of Burkitt’s lymphoma. The finding of the West Nile prospective seroepidemiological study-that children with higher titers of antibodies against the EBV capsid antigen are at increased risk to develop Burkitt’s lymphoma (de Th6 et al., 1978)-could well reflect a greater size in the pool of EBV-infected cells in such children compared to children with a lower risk of developing Burkitt’s lymphoma. This is entirely consistent with the hypothesis of Klein (1983; Klein and Klein, 1985), that EBV-infected cells are indeed the target cell population for lymphomagenesis; but Lenoir and Bornkamm, who have proposed that Burkitt’s lymphoma results when EBV infects a cell in which a translocation has already occurred (Lenoir and Bornkamm, 1988), would presumably argue that the high anti-VCA titer reflects a greater number of cells producing EBV, thus providing more virus particles to infect other cells. However, there i s no evidence that EBV infection of B cells in vivo occurs at a significant rate after primary infection (see below). This is probably mainly due to the presence of circulating neutralizing antibodies (directed against EBV membrane antigens) which prevent EBV infection of other cells (Rickinson et al., 1977). Thus, the probability that a rare cell which develops a chromosomal translocation has a significant likelihood of being infected by EBV would appear to be extremely low. C. BURKITT’S LYMPHOMA IN OTHERGEOGRAPHIC REGIONS A survey of the scant information from other geographic regions suggests that there would be much to be gained from repeating Burkitt’s original African safari on a world scale in order to determine

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whether correlations exist between clinical and molecular characteristics and EBV association. In equatorial Brazil, for example, unlike in temperate South American countries, a high proportion of the rather few patients with Burkitt’s lymphoma that have been described have jaw tumors (Chaves, 1977; Dalldorf et al., 1969). A review of 47 patients reported from South America gave an average of 48% of patients with jaw tumors, while in 45 patients from Asia 51% had jaw tumors (Lenoir et al., 1984). Similarly, in the lower socioeconomic groups in Turkey, jaw tumors occur in 50% of patients with Burkitt’s lymphoma (A. 0. Cavdar, personal communication), while the frequency appears to be lower in the more affluent sector of the population. In the Middle East and North Africa, the incidence ofjaw tumors from published data appears to be similar to that in the United States and Europe (Lenoir et al., 1984). While these data must be interpreted with caution, since they are usually selected (individual institutes, different diagnostic criteria, etc.), they do suggest strongly that there are likely to be clinical differences (and probably, therefore, biological differences) in Burkitt’s lymphoma in different geographic regions. It seems likely that at least two forms of Burkitt’s lymphoma, broadly characterized as endemic and sporadic, frequently coexist in the same country, the predominant form depending upon the average social status of the population, or possibly upon specific, but as yet uncharacterized, environmental factors. Insufficient data exist to be able to correlate EBV association with the clinical features of the disease. There is not, however, an obvious association between a high incidence of jaw tumors and the presence of EBV in the tumor cells. In North Africa, for example, in contrast to equatorial Africa, approximately 85% of Burkitt’s lymphomas are EBV associated (Lenoir et al., 1984),butjaw tumors are the primary disease site in only 22% of patients-a similar figure to that observed in the United States: (Sariban et al., 1984). Very few tumors in other parts of the world have been examined for the presence of EBV.

D. BURKITT’SLYMPHOMA IN IMMUNOSUPPRESSED INDIVIDUALS The high incidence of non-Hodgkin’s lymphomas in patients with inherited or acquired immunodeficiency syndromes has long been recognized (Magrath, 1983b; Filipovich et al., 1984, 1987, 1990; Ziegler et al., 1984a,b).Unfortunately, most ofthese tumors have been characterized on the basis of histology alone, and although many have been diagnosed as Burkitt’s lymphoma, the majority have been referred to as immunoblastic lymphomas (or in earlier classification

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schemes, reticulum cell sarcomas). Recently, it has become clear that many of the lymphoproliferative syndromes arising in immunosuppressed individual are polyclonal proliferations of EBV-containing B cells, ranging from clear-cut fatal infectious mononucleosis to localized tumor masses (Magrath, 1983b; Cleary et al., 1986; Hanto et al., 1981, 1985). Such processes are almost certainly the human counterpart of EBV induced lymphoproliferation/lymphomas in primates (Magrath, 1983b, 1989b; Young et al., 1989).There can be little doubt that such processes arise because of failure of the patient’s immune system to control the proliferation of EBV-infected cells. Indeed, in iatrogenically immunosuppressed patients, withdrawal of immunosuppressive drugs often results in resolution of the process (Starzl et al., 1984). However, it has been observed that some of these polyclonal lymphoproliferative processes may evolve into a true monoclonal lymphoma (Hanto et al., 1982), often bearing the chromosomal translocations characteristic of Burkitt’s lymphoma (Chaganti et al., 1983; Magrath et al., 1983b; Whang-Peng et al., 1984), while in other cases a true (i.e., cytogenetically defined) Burkitt’s lymphoma may arise apparently de nouo, or preceded only by chronic lymphadenopathy (Magrath, 198313; Filipovich et al., 1987; Purtilo, 1987). This has been best documented in patients with HIV infection, in whom it has been shown that small numbers of expanded B cell clones (defined by immunoglobulin gene rearrangements) coexist with the monoclonal neoplasm (defined by a c-myc rearrangement) in some 60% of cases (Pelicci et al., 1986a). The presence of additional clones of B cells in malignant lymphomas which are not preceded by a recognized immunodeficiency syndrome has been documented, but at a much lower rate-10% or less (Pelicci et al., 1986a), while there has been debate over several years as to the meaning of multiple immunoglobulin gene rearrangements detected in the lymphomas associated with cardiac transplant recipients (Cleary et al., 1984; Cleary and Sklar, 1984). Recently, using the terminal repeat region of EBV as a clonal marker (see below), it appears that such tumors probably evolve from polyclonality to monoclonality (Sklar and Weiss, 1988; Cleary et al., 1988). In patients with an HIV-associated lymphadenopathy syndrome oligoclonal B cell proliferation has also been observed in some 20% of lymph node biopsies (Pelicci et al., 1986a),consistent with the finding of oligoclonal immunoglobulin bands on serum electrophoresis in similar groups of patients (Papadopoulos et al., 1985), suggesting that malignant transformation occurred in an expanded, but nonmalignant clonal population of B cells as a consequence of a chromosomal translocation. It has been shown that HIV is not present in the lymphoma

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cells of patients with HIV-associated Burkitt’s lymphomas (Pelicci e t al., 1986a).

EBV- and HIV-Associated Lymphomas In patients with HIV infection there is considerable evidence for a deficiency in the regulation of EBV-infected B cells, including elevated anti-EBV antibody titers, higher numbers of circulating cells capable of giving rise to spontaneous, EBV-containing continuous cell lines, and impaired ability to suppress immunoglobulin secretion in uitro by EBV-infected B cells (Birx et al., 1986; Ragona and Sirianni, 1987; Ernberg, 1989). Nevertheless, only approximately 40% of the high-grade lymphomas that arise (containing a c-myc rearrangement) contain EBV (Subar et al., 1988; Ernberg, 1989), suggesting that, whereas EBV-containing clones may be more likely to undergo malignant transformation, the process is not confined to EBV-infected cells. In addition, this raises the possibility that EBV does not have a role in the actual process of neoplastic transformation in any of the tumors, but that it provides, in addition to the HIV-induced immunosuppression, an effective drive to proliferation of the target cell population. This is reminiscent of the twin factors of malaria and early EBV infection in Burkitt’s lymphoma. There are, however, a number of important differences between HIV-associated lymphomas containing c-myclimmunoglobulin gene translocations and endemic Burkitt’s lymphoma. The clinical spectrum of disease, for example, is significantly different (i-e.,jaw tumors are rare and bone marrow and central nervous system involvement is common in HIV-associated lymphomas) (Ziegler et al., 1984a,b). It is possible that this largely relates to differences in age and other host factors related to the spread of tumor in the two high-susceptibility groups. It is unlikely, however, that an age difference, if it is relevant at all, is the only explanation, since there are also molecular differences between endemic Burkitt’s lymphoma and the “Burkitt’s lymphoma” of patients with HIV infection in the United States, namely that the pattern of rearrangements of c-myc is very similar to that of sporadic Burkitt’s lymphoma (see below) rather than endemic Burkitt’s lymphoma (Pelicci et al., 1986a; Neri et al., 1988). The extremely high incidence of HIV infection in equatorial Africa is likely to alter the pattern of Burkitt’s lymphoma, although no reports of this have been published so far. It will be important to determine whether HIV-associated African Burkitt’s lymphoma is associated with a sporadic or endemic type of molecular rearrangement. In spite of the differences between endemic and HIV-associated Burkitt’s lymphoma, there is also at least one interesting similarity

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between them-the likelihood of late recurrence. In African patients late recurrences, although uncommon, have been documented up to 6 years after presentation with the original tumor (Biggar et al., 1981). It has often been speculated that such late recurrences actually represent the reinduction of new tumors, and GGPD isoenzyme data in heterozygous females appear to confirm at least the possibility that this is so (Fialkow et al., 1972). In contrast, in sporadic tumors, such late relapses have not so far been described. Recently, we described a patient with HIV-associated Burkitt’s lymphoma in whom a second clonally discrete Burkitt’s lymphoma developed after some 2.5 years of diseasefree survival (Barriga et al., 1988b). Since most patients are not cured of their first tumor or die from infectious complications, it is rare that the opportunity to document such a phenomenon arises, although it is possible that it might otherwise be common. It seems likely that the occurrence of late relapses in Burkitt’s lymphoma is indeed due to the reinduction of a new tumor, and the likelihood of this must reflect the magnitude of the overall risk for the development of Burkitt’s lymphoma-greatest in HIV infection (2-5% of patients will develop a malignant non-Hodgkin’s lymphoma), intermediate in endemic Burkitt’s lymphoma, and least in sporadic Burkitt’s lymphoma.

E. IMPLICATIONS OF CLINICAL FINDINGS FOR PATHOGENESIS A number of insights relevant to the pathogenesis of Burkitt’s lymphoma are to be gained from its clinical features. As is clear from the above descriptions, both endemic and sporadic forms of Burkitt’s lymphoma are primarily extranodal diseases. Tumors occur predominantly in the bowel, the jaw (associated with developing teeth), other bones, the bone marrow, the central nervous system, the pleurae, the testes, and the liver (Wright, 1970; Magrath and Sariban, 1985).Lymph nodes in the mesentery or even peripheral lymph nodes can be involved, but these are not infrequently in the regional drainage area of a tumor arising from extranodal tissue. Presentation with involvement of multiple peripheral lymph node groups is rarely seen in Burkitt’s lymphoma. This fact alone makes it unlikely that the normal counterpart cell of Burkitt’s lymphoma arises in the germinal center of peripheral lymph nodes. This statement, however, must be qualified. First, in individuals over 40 years in whom lymphomas with the morphology of small noncleaved cells, but containing 14;18 chromosomal translocation (Fifth International Workshop, 1987), it is likely that such tumors (which ought to be clearly differentiated from Burkitt’s lymphoma) do indeed arise in germinal center cells. Second, a number of

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tumors containing both 14;18 and 8;14 translocations have been described. This suggests that the genetic changes associated with Burkitt’s lymphoma may occur in existing neoplasms which have originated in germinal center cells. Further study at a molecular level of these dual 14;18/8;14 tumors is likely to be revealing. IV. Phenotype of Burkitt’s Lymphoma

The phenotype of a lymphoid neoplasm is clearly a valuable aid to determining the cell type from which it originates, although it cannot be assumed that the predominant cell type in the tumor cell population is necessarily the same as that in which tumorigenesis occurred. Differentiation of the malignant clone could well have occurred in uiuo. In addition, caution must be exercised in extrapolating directly from the phenotype of cell lines to that of in uiuo tumors. However, with these caveats in mind, the examination of the phenotype of Burkitt’s lymphoma can provide much useful information. Burkitt’s lymphoma was shown many years ago to express surface immunoglobulin, and therefore to be of B cell origin (Klein et al., 1968). As expected, therefore, all Burkitt’s lymphomas express the B cell-restricted antigens CD19 (B4), CD20 (Bl),and CD22 as well as B cell-associated antigens including BA-1 (CD24) and HLA-DR (Favrot et al., 1984; Sandlund et al., 1986; Rowe et al., 1985; Cohen et al., 1987). The vast majority of tumors express surface IgM; only a small percentage lack IgM and express other heavy chain classes (Gunven et al., 1980). In some cell lines there is evidence of expression of more than one heavy chain class, due either to differential splicing of a large transcript encompassing much of the heavy chain locus, or to transcription of unrearranged constant region immunoglobulin genes (Magrath et al., 1980b). IgD is uncommonly observed on the surface of Burkitt’s lymphoma cells, although it has been described in L3 leukemia (Preud’homme et al., 1981) and can frequently be detected in the cytoplasm of cell lines (Kiwanuka et al., 1988). Only rarely have cell lines been observed in which class switching with deletion of p genes has been documented (Hamlyn and Rabbitts, 1983). A single light chain type is expressed by the fresh tumor cells, reflecting the monoclonal origin (Gunven et al., 1980). Some freshly isolated tumors, usually presenting as leukemias, have a pre-B phenotype in which p chains are present in the cytoplasm but are not expressed on the surface (Cohen et al., 1987),and rare cases of Burkitt cell (L3) leukemia bearing 8;14 translocations and expressing terminal deoxyribonucleotide transferase (TdT) have been reported (Secker-Walker et al., 1987; Drexler et al., 1986). These find-

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ings indicate that at least some Burkitt’s lymphomas have an immature, precursor B phenotype. TdT has a role in the production of immunoglobulin (and T cell receptor) diversity by inserting random nucleotides close to the junctions of the D immunoglobulin (or T cell receptor) gene segment during the process of VDJ joining and is expressed in lymphoid neoplasms arising from precursor cells of the B and T lineage. TdT expression has not otherwise been described in Burkitt’s lymphomas. In contrast to the occasional pre-B phenotype, many cell lines, as well as tumors in zjiuo, secrete IgM (Benjamin et al., 1982) which in large-volume sporadic tumors can be detected as a discrete monoclonal band by a sensitive method of serum electrophoresis (Magrath et al., 1983a). This characteristic, which is usually associated with a relatively mature phenotype, appears to be an uncommon attribute of cell lines derived from endemic tumors. Thus, it would appear that phenotypically Burkitt’s lymphoma is as homogeneous as it is clinically and histologically, but that these phenotypic differences correlate, in general, with the geographic origin and/or the EBV association of the tumor. PHENOTYPIC DIFFERENCES BETWEEN SPORADIC AND ENDEMIC BURKITT’S LYMPHOMAS The difference in EBV association between endemic and sporadic Burkitt’s lymphomas might be expected to be reflected in phenotypic differences-either because of differing susceptibility of the cells to EBV, or as a consequence of the presence of EBV itself in the tumor cells. Differences in the expression of complement receptors, one of which (that for C3d, now known as CD21) is known to serve as a receptor for EBV (Magrath et al., 1980a; Fingeroth et al., 1984; Inghirami et al., 1988; Bare1 et al., 1988), have been correlated with Epstein-Barr nuclear antigen (EBNA) positivity (Freeman et al., 1982; Benjamin et al., 1983; Gaither et al., 1983). It is possible that the CD21 molecule is orientated differently in the cell membrane in EBVpositive versus negative tumors, since monoclonal antibodies binding to different epitopes of this protein (e.g., B2 and HB5) have different degrees of reactivity with the same cell lines (Sandlund et al., 1986; Favrot et al., 1986). Interestingly, EBV-negative cell lines can be infected with EBV-expression of the CD21 molecule is low, but not necessarily absent, and can be induced with methylated xanthines (Magrath et al., 1980a, 1981). However, the relatively small number of cells which are converted to EBVpositivity are frequently lost from the

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population, either because of loss of virus, or because EBV-infected cells are at a growth disadvantage in such cultures. In contrast, stably infected BJAB and Ramos cells have decreased serum requirements (Klein et al., 1974, 1975; Steinitz and Klein, 1975, 1976, 1977), and amplification of c-myc has been described after EBV infection of BJAB (Lacy et al., 1987). The relevance of latter findings to the pathogenesis of Burkitt’s lymphoma remains unclear, particularly since the BJAB line, derived from an African patient originally diagnosed as having Burkitt’s lymphoma, lacks a translocation involving c-myc. A number of other differences between endemic and sporadic tumors have been described, almost exclusively based on studies of derived cell lines. Almost all sporadic tumors, but a minority of endemic tumors, secrete IgM (Benjamin et al., 1982)and this characteristic appears to correlate with the chromosome 8 breakpoint location even better than does geographic origin (Pelicci et al., 1986b). The common ALL antigen (CDlO), which is expressed on B cell precursors and a minority of germinal center cells, is also expressed on almost all Burkitt’s lymphomas in uiuo, but this antigen is frequently lost when EBV-positive tumors are grown in uitro as cell lines (M. Rowe et al., 1985,1986,1987). Because of this, only about 50% of EBV-positive cell lines express CD10. Loss of CDlO from EBV-positive Burkitt’s lymphoma cell lines is associated with variable degrees of expression of activation antigens such as Ki-1, Ki-24, CD22, and CD39 (Rowe et al., 1987), and MacLennon et al. (1988) have described a similarity between germinal center cells and ll EBV-positive Burkitt’s lymphoma cell lines with regard to the level of expression of 14 different antigens. Interestingly, EBV-transformed lymphoblastoid cell lines did not show the same pattern of antigen expression. The interpretation of this finding is complicated by the observation that alterations in the expression of activation antigens occur during early passages of EBV-positive Burkitt’s lymphoma cell lines. Consistent with the findings that loss of CDlO is paralleled by an increase in the expression of activation antigens in uitro, an inverse correlation between the expression of the activation antigen LB-1 and that of CDlO has been reported in both endemic and sporadic cell lines which have been in culture for some time (Ehlin-Hendriksson and Klein, 1984; Ehlin-Hendriksson et al., 1987). EBV-negative cell lines appear to be much more stable phenotypically than EBV-positive cell lines, and almost invariably retain CDlO while failing to express activation antigens. An inverse correlation has also been shown between the expression of mantle zone markers, e.g., BA-1 (CD24) or Tu-1, and CDlO (Favrot et al., 1984), while a positive correlation was observed between 38.13

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and CDlO (Ehlin-Hendriksson et al., 1987). In Favrot’s study, most CD10-positive lines were of sporadic origin and most CD10-negative lines of endemic origin. CDM is expressed on B cell precursors, immature and circulating B cells, as well as mantle zone cells and reacts poorly with germinal center cells, while 38.13 is expressed on germinal center cells as well as a subset of chronic lymphocytic leukemia cells. As expected, CD24 and 35 IC5, another marker of mantle zone cells, inversely correlated with the expression of the germinal center cell marker 38.13 in EBV-negative Burkitt’s lymphoma cell lines (Ehlin-Hendriksson et al., 1987). This correlation was disrupted in EBV-negative lines which were converted to EBV positivity. CD5, an antigen present on some 18% of circulating peripheral B cells and on chronic lymphocytic leukemia cells, is not expressed on Burkitt’s lymphoma cells (Cohen et al., 1987). It is not clear that cell lines bearing variant translocations differ in their surface markers from cell lines with 8;14 translocations, although they tend to be more often EBV positive and more often lack CDlO and express activation antigens (Ehlin-Hendriksson et al., 1987; Cohen et al., 1987). An association between the type of variant translocation and the light chain produced-8;22-bearing lines more often synthesize A and 2;8 lines K-has been reported (Lenoir et al., 1982) although there are exceptions to this (Magrath et al., 198313; Hollis et al., 1984; Denny et al., 1985). Similar concordance has been noted between heavy chain isotype and breakpoint location in mouse plasmacytomas as well as rat immunocytomas (Pear et al., 1988). This supports the idea that the translocations occur temporally close to the point in cell differentiation associated with rearrangement of immunoglobulin gene regions, when such regions are likely to be more “fragile” and therefore more susceptible to translocation. The progressive changes that EBV-containing Burkitt’s lymphoma cell lines frequently undergo in uitro, becoming closer to EBVtransformed lymhoblastoid cell lines (Rowe et al., 1987),must be taken into account when attempting to evaluate differences in phenotype between endemic and sporadic tumors. In fact, these findings suggest that the phenotype of sporadic and endemic, or EBV-negative and EBV-positive Burkitt’s lymphomas, may differ very little in uivo,with the possible exception of the secretion of immunoglobulins. It would also appear that the phenotype of Burkitt’s lymphoma cells in viuo does not resemble that of activated B cells, a conclusion which is supported by the recent observation of a very low level of insulin receptor expression in Burkitt’s lymphoma cell lines (Newman and Harrison, 1989). Rather, the implied lack of activation antigens on the

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in uiuo tumor suggests that the cells are closer phenotypically to resting B cells, although the fact that the cells are actively proliferating may result in some phenotypic differences from true resting cells. For example, it has recently been shown that Burkitt’s lymphoma cell lines usually express receptors for transferrin and for a B cell growth factor, even though these receptors are induced by polyclonal activation of normal B cells (Favrot et al., 1986).Two recognized types of resting B cells are cells which have recently completed antigen-independent differentiation, i.e., virgin B cells, and memory cells derived from cell clones activated by antigen, and it is probable that Burkitt’s lymphoma is the malignant counterpart of one of these cell types, or that different subsets of Burkitt’s lymphoma correspond to each of these normal cell types. V. The Nonrandom Chromosomal Translocations Associated with Bu rkitt ’sLymphoma

Four years after Manolov and Manolova’s observation (1972), that in Burkitt’s lymphoma additional chromatin attached to the long arm (9) of chromosome 14 can usually be observed, Zech et al. (1976) demonstrated that this was due to a translocation involving chromosomes 8 and 14. The localization of the immunoglobulin heavy chain genes to the same band (14q32) involved in the chromosomal translocations (Croce et al., 1979; Hobart et al., 1981; Kirsch et al., 1982) provided probes to further dissect the molecular consequences of the chromosomal translocations and to demonstrate that the c-myc gene, or a part of it, is translocated from its normal position on chromosome 8 (q24) into the heavy chain region (Dalla-Favera et al., 1982, 1983; Taub et al., 1982; Erikson et al., 1982; Adams et al., 1983).The discovery ofthe variant translocations in which the A and K immunoglobulin light chain genes on chromosomes 22 or 2, respectively, are involved in reciprocal translocations with chromosome 8 (Bernheim et al., 1981; Croce et al., 1983; de la Chapelle et al., 1983; Erikson et al., 1983b; Davis et al., 1984; Hollis et al., 1984; Emmanuel et al., 1985) reinforced what appears to be a principle: that the gross structural changes in Burkitt’s lymphoma involve the juxtaposition of c-myc, or at least its proteincoding sequences, with sequences derived from either a heavy or a light chain immunoglobulin gene. In situ hybridization using a c-myc probe and molecular cloning have clearly demonstrated that whereas in the 8;14 translocations c-myc is translocated from its normal position on chromosome 8 to the heavy chain locus on chromosome 14 (Dalla-

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Favera et al., 1982, 1983; Taub et al., 1982; Erikson et al., 1983a; Adams et al., 1983), in the variant translocations c-myc remains on chromosome 8. In this case some part of the light chain locus from chromosomes 2 or 22 is translocated to a position distal to c-myc on chromosome 8 (Croce et al., 1983; Erikson et al., 1983b; de la Chapelle et al., 1983; Davis et al., 1984; Hollis et al., 1984). Since the transcriptional orientation of c-myc is centromere to telomere, and that of the light chain genes is the same, in the variant translocations c-myc and the translocated light chain sequences are in the same transcriptional orientation. In 8;14 translocations, however, since the heavy chain locus is transcribed in the direction telomere-centromere, the transcriptional orientation of the relevant genes differs. This observation has focused attention on the possibility that enhancer regions associated with the immunoglobulin loci may have a critical role in bringing about the functional end result of the translocation. While in some cases recognized enhancer regions such as the heavy chain enhancer situated between the J and switch p regions (Gillies et al., 1983) may be involved in c-myc deregulation (Hayday et al., 1984), in other tumors the reciprocal translocation has caused this enhancer element to lie on chromosome 8 and thus to be unable to influence the expression of the protein-coding portion of c-myc which has been translocated to chromosome 14. In such cases, it is likely that other regions within the immunoglobulin loci provide a positive transcriptional impetus to c-myc. The normal promoter of the immunoglobulin gene involved in the translocation cannot be involved in c-myc transcription since molecular analyses have demonstrated that it is never on the same chromosome as c-myc. It is also often the case (when the chromosomal breakpoint is within the gene) that the normal c-myc promoters are separated from the coding region if c-myc. It is important to note that in spite of the large variation in their positions, the breakpoints on chromosome 8 never disrupt the coding regions for the c-myc protein, nor do the breakpoints on chromosomes 14, 22, or 2 ever disrupt the constant regions of the immunoglobulin chains. The first finding stems from the absolute need of a Burkitt’s lymphoma cell for a functional c-myc protein (the normal c-myc allele which is not involved in the translocation is silent in the vast majority of Burkitt’s lymphomas). The second finding could relate either to the degree of fragility, and therefore the likelihood of breaking, of different regions of the immunoglobulin genes, or to the need for enhancer regions present in the immunoglobulin loci to remain able to influence the expression of c-myc.

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PARALLELS WITH ANIMAL TUMOR MODELS The probability that the chromosomal translocations associated with Burkitt’s lymphoma are of pathogenetic significance is greatly enhanced by the observation that homologous translocations which result in the juxtaposition of c-myc and immunoglobulin sequences occur in mouse plasmacytomas and rat immunocytomas (Marcu et al., 1983; Adams et al., 1983; Erikson et al., 1985; Pear et al., 1986). Plasmacytomas are readily induced in susceptible mouse strains by intraperitoneal injection of pris tane (tetramethylpentadecane), which causes an inflammatory peritonitis (with, quite likely, free radical formation which may give rise to chromosomal damage) containing large numbers of pre-B cells as well as other proliferating B cells at various stages of maturity (Potter and Wax, 1983; Potter, 1982, 1986a,b;Potter et al., 1985). T h e incidence of plasmacytomas varies from 20 to 60% according to the dose of pristane. The latent period can be shortened by simultaneous injection of Abelson murine leukemia virus (along with Moloney helper virus) into the peritoneal cavity (Potter et ul., 1973; Ohno et al., 1984)but the latter is not an obligate requirement for plasmacytoma induction, and probably acts by increasing the proportion or number of cycles of the particular subset of pre-B or B cells susceptible to the transformation event. Murine plasmacytomas contain translocations in more than 95% of cases, the commonest being 12;15 translocations in which the c-myc gene is translocated from its location on chromosome 15 into the immunoglobulin locus on chromosome 12 (Marcu et al., 1983; Adams et al., 1983). A small proportion of the tumors have 6;15 translocations, in which K light chain sequences are relocated from chromosome 6 to a point more than 94 kb 3’ of c-myc on chromosome 15, usually within a 4.5-kb span of a region designated as put-1, which is also a frequent site of integration of proviruses in T cell lymphomas in AKR mice (Graham et al., 1985)and rats (Villeneuve et al., 1986).A second relevant animal tumor model is the spontaneous rat immunocytoma (Bazin et al., 1972, 1974, 1988; Bazin, 1974) in which the chromosomal translocation, in this case 6;7 causes c-myc, located on chromosome 7 in the rat, to be juxtaposed to heavy chain sequences present in chromosome 6 (Siimegi et al., 1983; Bazin et al., 1987; Pear et al., 1986). The similarity of the chromosomal translocations in these animal tumors to those of Burkitt’s lymphoma provides compelling evidence that the juxtaposition of c-myc and immunoglobulin sequences is a critical component of pathogenesis in all three. Further, the analysis of murine plasmacytomas and rat immunocytomas provides information

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of crucial importance to the development of an understanding of the pathogenesis of Burkitt’s lymphoma, since these systems can be experimentally manipulated in order to investigate factors which protect against the tumors, or factors which increase the likelihood of their development. The role of the translocations in deregulating the expression of c-myc and the potential participation of other oncogenes in the induction of these tumors can and have been studied in these systems (see below) and may well have direct application to Burkitt’s lymphoma. A large amount of evidence derived from all three systems implicates the deregulation of the c-myc oncogene as the common pathogenetic feature of these tumors. There is no longer any reasonable doubt that this is the direct result of the chromosomal translocations. Numerous questions remain, to which a greater or lesser degree of enlightenment has been obtained to date, and which are the subject of continued intensive research. These include the mechanism of chromosomal translocation and its timing in relationship to B cell differentiation, the nature of the molecular consequences of the translocations which bring about c-myc deregulation, the biochemical consequences of c-myc deregulation (even the function of c-myc protein has not yet been elucidated), and the participation of other factors in bringing about a truly neoplastic state. These topics will be addressed in the remainder of this article. VI. Structure and Function of c-myc

While the precise biochemical effects of the c-myc protein in the cell remain unknown, there is little doubt that it is involved in the control of cellular proliferation (reviewed in Kelly and Siebenlist, 1986; Marcu, 1987). It is likely that the gene functions as a transactivator, able to bind to the regulatory regions of other genes. The existing evidence favors the possibility that c-myc is necessary for the reception of signals derived from growth factors, presumably through influencing either the expression of growth factor receptors or the proteins concerned in the transduction of the signals from occupied receptors. In interpreting the evidence for this, it is well to remember that cellular proliferation is the result of a cascade of events involving several sets of growth factors and their receptors. Thus, occupancy of one receptor may result in the expression of another, enabling the cell to respond to a growth factor which may already be present in its environs [e.g., antigen triggers the expression of activation antigens including interleukin-2 (IL-2) receptors, which in turn, when occupied, cause the expression of transferrin receptors].

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A. STRUCTURE OF THE c-myc GENEAND ITSPRODUCTS The c-myc gene in mammals and birds is highly conserved (Persson et al., 1984) and consists of three exons which contain a single open reading frame (Watt et al., 1983; Battey et al., 1983; Hamlyn and Rabbitts, 1983).All three exons are present in the messenger RNA, but only the second exon and approximately two-thirds of the third exon encode the major protein product. Thus the messenger RNA carries a long untranslated leader sequence derived from the first exon. Approximately 50% of the c-myc messenger RNA is polyadenylated, and this RNA appears to have a very short half-life of some 30 min, although examination of the half-life of c-myc obtained from total RNA has been estimated to be longer-approximately 1 hr (Nishikura and Murray, 1988). There are three primary promoters for the c-myc gene, PI and P2, which are at and near the 5' border of exon 1 (Battey et al., 1983; Bernard et al., 1983) separated by some 160 nucleotides, and Po over 500 nucleotides upstream of PI (Bentley and Groudine, 1986a). A fourth promoter, Ps, situated in the first c-myc intron has been reported to be operative, albeit weakly, in some rodent and human cell lines (Ray et al., 1987; Ray and Robert-LBzBnGs, 1989). This promoter is also used in some Burkitt's lymphomas. In normal cells some four times as many transcripts are initiated at P2 as at PI, while POtranscripts account for only a small percentage of the c-myc messenger RNA. Protein products of transcripts initiating at the POpromoter have not been identified, and the function of such putative proteins remains speculative. The c-myc gene encodes a protein consisting of 439 amino acids and a predicted molecular weight of 49,000 (Hann and Eisenman, 1984; Ramsey et al., 1984). In sodium dodecyl sulfate gels, however, the protein runs at a molecular weght of 64 kDa. The construction of deletion mutants has suggested that the region of the protein responsible for the inappropriately slow migration is between amino acids 145 and 262 from the amino terminal (Stone et al., 1987). Interestingly, antibodies made against myc peptides precipitate a second protein some 3 kDa larger than the major protein product (Hann and Eisenman, 1984; Ramsey et al., 1984), and recently this was shown to be initiated 14 codons upstream of the ATG codon at which translation of the 64-kDa protein at the 5' end of the second exon is initiated (Hann et al., 1988). The 67-kDa protein initiates at a CTG rather than an ATG codon, but it appears that both proteins can be translated from a single mRNA, and that PI and PZtranscripts do not code for separate protein products (Hann et al., 1988).The importance of both protein products

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is emphasized by the existence of two c-myc proteins in widely differing species and the continuous expression of both proteins throughout the cell cycle (Hann et al., 1985). €3. REGULATION OF TRANSCRIPTION OF c-myc

There is considerable evidence that regulatory elements for c-myc transcription are scattered throughout the 5’ flanking sequences as well as within the first exon and intron. As might be expected, the high degree of homology between the human and murine c-myc genes extends some 2 kb upstream of PI. Five DNase I-hypersensitive sites have been consistently identified in human c-myc (Siebenlist et al., 1984). Such sites are regions in which the chromatin structure differs in order to accommodate the binding of nonhistone proteins putatively engaged in regulation of transcription. Such sites have been observed immediately upstream of the TATA boxes associated with the promoters PI and P2, at a site some 2 kb upstream of PI at a region known to contain a positive regulatory element (enhancer) for c-myc, as well as the recently described autonomously replicating sequences (see below). An additional DNase-hypersensitive site has been described in the region immediately upstream of Po and another between this and the site 2 kb 5’ of PI. Both of the latter sites are close to binding sites for the transcriptional factor N F 1, and are within regions containing positive regulatory elements (Fig. 1).In translocated c-myc genes, new DNase I-hypersensitivity sites have been described in relationship to the adjacent immunoglobulin gene (Dyson and Rabbitts, 1985). Both positive and negative regulatory elements for transcription from P1 and PZ have been described in the 5’ flanking sequences as well as within the gene itself in both murine and human c-myc (Remmers et al., 1986; Bentley and Groudine, 198613; Nepveu et al., 1987a,b; Hay et al., 1987; Marcu, 1987; Marcu et al., 1988). These are described in more detail below in the context of the chromosomal breakpoint locations. One of the negative elements serves to prevent the passage of RNA polymerase I1 beyond the first exon (Bentley and Groudine, 198613; Nepveu and Marcu, 1986; Eick and Bornkamm, 1986), and thus to prevent elongation of the nascent RNA chain. This appears to be an important regulatory mechanism in some cell types (Blanchard et al., 1985), and one which may be deranged in Burkitt’s lymphoma. In the mouse, sequences 5’ of P2 appear necessary not only to P2 utilization, but also to the efficient functioning of the transcriptional block (Marcu et al., 1988; Wright and Bishop, 1989). A positive regulatory element required for transcription from P2 has also been identified at the 3’ end of the first exon (Yang et al., 1986),and negative

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IAN MACRATH n

0o m

0

I

bp -3000

I -2OW

I

-low

I I

1 - 1

0

1M)O

I

I

l

l

I

I

I

I

2000

3000

uxx)

5000

WOO

7000

Boo0

WOO

FIG.1. Diagrammatic depiction of the structure of the human c-myc gene, indicating known regulatory regions and some restriction enzyme cleavage sites. Exons are indicated by the boxes, and distances are indicated in base pairs from PI. Shaded areas depict the regions encoding the smaller (64kDa) protein. The promoters Po, PI, and P2 are shown. DNase 1 hypersensitivity sites are indicated by wedges. The positions of recognized positive and negative regulatory elements are indicated approximately, although many more regions probably exist which have not been precisely mapped. Restriction endonuclease sites are as follow: B. BgZII; Ps, PstI; Pv, PouII; R, EcoRI; C, ClaI; H, HindIII. Binding sites for the nuclear factor NFl are indicated, as are the consensus sequence present in many enhancer elements, the site of the block to transcript elongation, and the binding site in the intron of a putative regulatory protein (PBS). The sequences encoding functional regions of the protein relating to nuclear translocation (MI at residues 320 to 328 and MZ at residues 364 to 274) are shown, as which includes leucines at residues 413,420,427, is the "leucine zipper" region (LZ), and 434.

elements have been described at -923 to -1060 and -424 to -615 from the P1 promoter in the murine c-myc gene (Marcu et al., 1988). Gel retardation assays have demonstrated that nuclear proteins bind to both of these negative regulatory regions. Finally, it is likely that regulatory sequences are also present in the first intron of c-myc, as evidenced by the presence of a binding site for a putative regulatory protein (Zajac-Kaye et al., 1988a,b). C. FUNCTIONS OF THE c-myc PROTEIN($

Both c-myc and v-myc, its viral homolog which lacks the first exon present in the cellular gene, are phosphorylated, located in the cell nucleus, and can bind nonspecifically to nucleic acids, (both DNA and RNA) with high affinity (Donner et al., 1982,1983; Alitalo et al., 1983; Persson and Leder, 1984; Watt et al., 1985; Beimling et al., 1985). Immunofluorescent studies have shown that these proteins localize in a nuclear region which is enriched for small nuclear ribonucleoprotein particles, although the functional significance of this is unknown (Spector et aZ., 1987). The half-life of the c-myc protein is short, 15-30

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min or less (Hann and Eisenman, 1984; Rabbitts et al., 1985b),as might be expected for a molecule with a regulatory function.

1. An Autonomously Replicating Sequence Upstream of c-myc Recently, a region of DNA able to replicate autonomously as an episome in mouse and human cells was cloned from mouse cells (Ariga et al., 1987).The c-myc protein was shown to bind to this sequence and to promote its replication in transfected cells (Iguchi-Ariga et al., 1987a). Moreover, sequences upstream of the human c-myc gene itself have recently been shown to be able to function as autonomously replicating sequences, to be able to bind c-myc protein (or a complex containing c-myc protein), and to have homology with the sequences previously cloned from mouse cellular DNA (Iguchi-Ariga et al., 1988). These sequences are situated between nucleotides 2215 and 2204 upstream of the PI promoter of c-myc (Fig. 1).This is also a region of regulatory activity for the transcription of c-myc (see below) and the sequences with autonomously replicating activity are in the same region as a positive regulatory element for c-myc transcription, which also appears to be functionally dependent upon binding of the c-myc protein (Iguchi-Ariga et al., 1988). The effect of c-myc on transcription may be dependent upon the context, i.e., the presence or absence of other regulatory proteins occupying binding sites which influence c-myc expression. Both cloned human and mouse autonomously replicating sequences and the region approximately 2 kb upstream of c-myc to which the c-myc protein binds contain sequences homologous to TGAATAGTCA, which may be the recognition site for the c-myc protein, or for proteins complexed to c-myc. Thus the c-myc protein is putatively able to promote replication of cellular DNA by binding to replication origins, and it also appears able to influence its own transcription. These findings, which have been partially reproduced in other laboratories (McWhinney and Leffak, 1988),suggest that the c-myc protein has similar functions to the SV40 T antigen, which bears a structural similarity to c-myc and which binds to the origin of replication for SV40 (or pol yomavirus) and promotes DNA replication (DePamphilis and Bradley, 1986). The T antigens also regulate their own transcription, and polyoma replication has been shown to be dependent upon an adjacent enhancer region (de Villiers et al., 1984). The similarities of function are supported by experimental evidence, for c-myc can substitute for SV40 T antigen in SV40 DNA replication (Iguchi-Ariga et al., 1987b) and, like SV40 T antigen and the adenovirus protein ElA, to which c-myc also bears some structural similarity (Ralston and Bishop,

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1983),can cooperate with ras genes in the in vitro transformation of embryonal rat fibroblasts (Land et al., 1983a,b; Parada et al., 1984) and kidney cells (Ruley, 1983). Finally, the functional resemblance between c-mgc protein and the EBNA 1 protein of EBV, which binds to the EBV origin of replication (see below), is intriguing and provides a potential link between EBV and c-myc in the pathogenesis of Burkitt's lymphoma. The possibility that EBNA 1can bind to the autonomously replicating sequences in the upstream regulatory region of c-mgc, or can substitute for it in other transactivating functions, is worthy of investigation. 2. Functional Domains of c-myc Considerable progress toward the localization of the functional domains within the c-myc protein has been accomplished by the construction of a series of in-frame deletion and insertion mutants in a plasmid or retrovirus containing the human c-mgc gene coupled to a Moloney virus promotertenhancer and assessing the effects of the mutations in several functional assays. Both types of mutation in the region extending from amino acid 320 to near the carboxy terminus markedly inhibited or totally abrogated the ability of myc to cooperate with a mutant c-Ha-ras-1 gene in the transformation of primary rat embryo cells (Stone et al., 1987).Several deletions between codons 41 and 201 also abolished cotransformation, as did removal of a portion of the amino terminal between codons 7 and 91, but deletions of the central third of the gene (i.e., between codons 144 and 320) lessened, but did not prevent, cotransforming activity. Interestingly, the ability of retrovirus constructs containing mutant c-mgc genes to transform an established cell line, Rat-la, did not necessarily correlate with the level of activity of these same mutants in cotransformation assays (Stone et al., 1987). While mutations in the carboxy-terminal region led to reduced activity in both assays, other mutations had differing effects in the two assays. These differences were not explicable on the basis of quantitative differences in the expression of the mutant c-mgc proteins. The ability of the proteins with mutations in exon 3 to localize in the cell nucleus was unimpaired except when deletions 3' of codon 320 were present. Immunofluorescence and fractionation studies suggested that regions 106-143 and 371-412 are important for nuclear retention of the protein, while region 320-368 probably directs the protein to the nucleus. Loss of this property did not totally abrogate cotransformation, presumably because such mutant proteins were equally distributed between nucleus and cytoplasm and were not

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exclusively cytoplasmic (Stone et al., 1987). Further studies in which portions of c-myc were fused to chicken pyruvate kinase (Dang and Lee, 1988) permitted the more precise localization of the regions responsible for nuclear translocation. Residues 320-328, designated peptide M1, were shown to induce complete nuclear localization. Their deletion from c-myc resulted in distribution of the protein between the nucleus and cytoplasm, although such proteins retained activity in cotransformation assays. Another region encompassing residues 364-374 (peptide Mz) induced partial nuclear localization of the fusion protein. Deletion mutants of c-myc in this region resulted in mutant proteins that were still able to localize in the nucleus, but were unable to cooperate with ras in cotransformation assays or to transform Rat-la cells. This peptide is probably the reason that partial nuclear localization of M1 deletion mutants occurs, but although highly conserved and doubtless a critically important region of the gene, it is unlikely that the nuclear localizing properties of Mz are essential to normal c-myc function. As might be expected, regions homologous to M1 are present in c-myc and N-myc genes of many different species. The nuclear localization signals of the large T antigens of polyoma and SV40 virus are structurally similar to M1.

3. Oligomerization of c-myc uia Its Leucine Zipper

The recent identification of the periodic repetition (every seventh amino acid) of leucine residues in the DNA-binding region of a newly identified protein (CIEBP) that binds to many viral enhancers (Landschulz et al., 1988) as well as to a sequence (CCAAT) present in many promoters, led to the discovery of similar stretches of leucine heptad periodicity in regions predicted to be a helical in several nuclear oncogenes, includingjun, fos, and myc. In c-myc this leucine repeat region begins 32 residues upstream of the carboxy terminus and extends for 8 turns of a hypothetical a helix. It has been proposed that the long, hydrophobic leucine side chains, which are bulky at their tips, may permit dimerization of proteins through a kind of “zipper” action when present in a-helical regions (Landschulz et al., 1988). Dimerization is likely to be necessary to the conformation of a particular class of DNA-binding proteins to the DNA target site, and the leucine zipper may also permit the formation of heterodimers between molecules which possess similar zipper regions. This has recently been shown to be the case for fos and j u n (Sassone-Corsi et al., 1988a) (see also Chapters 1 and 2, this volume). More recently still, it was shown that highly purified c-myc exists in vitro as tetrads, and that oligomerization is mediated through the carboxy-terminal leucine zipper. De-

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letion of residues 414 to 433 inhibited oligomerization, while the construction of fusion proteins between amino acid residues 372-439 of c-myc, containing the leucine zipper (which includes the leucine residues 413,420,427, and 434), and staphylococcal protein A permitted dimerization and oligomerization whereas other c-myc sequences did not (Dang c-myc 1989). The formation of oligomers in vivo, either with itself, or with other proteins containing the leucine zipper motif, is likely to be of considerable importance to c-myc function. D. c-myc AND CELLPROLIFERATION c-myc is expressed in a wide variety of cell types, and in somatic cells, though not embryonic cells; its level of expression appears to correspond to the rate of cellular proliferation (Pfeifer-Ohlsson et al., 1984, 1985; Stewart et al., 1984a). Transient expression of c-myc follows the stimulation of quiescent, serum-deprived fibroblasts with platelet-derived growth factor (Kelly et al., 1983), or of lymphocytes with mitogen, anti-p, anti-8, anti-rc, IL-4, phorbol ester, or calcium ionophore (Kelly et al., 1983; Smeland et al., 1985; Reed et al., 1985; Klemsz et al., 1989). Regenerating liver is also associated with increased c-myc expression (Makino et al., 1984; Goyette et ul., 1984). c-myc expression is not sufficient to cause DNA synthesis, however, as shown by transfection experiments (Armelin et al., 1984) or microinjection of c-myc protein (Kaczmarek et aZ., 1985),and its expression has been considered to render cells “competent” to undergo cell division. This presumably implies that stimulation by at least one additional growth factor is required for DNA synthesis to commence. Perhaps c-myc is necessary for reception or transduction of the signal received from this growth factor. In some circumstances, myc expression can render cells independent of a growth factor, e.g., a murine T cell line has been rendered independent of IL-2 by transfection with a recombinant retrovirus containing v-myc and an immature myeloid cell line was similarly rendered independent of IL-3 (Rapp et al., 1985; Cleveland et al., 1986). These findings suggest that in these cell lines, c-myc functions after IL-2 or IL-3 in the cascade of events leading to proliferation. As might be expected, during differentiation c-myc is normally down regulated. This has been clearly shown for normal human burstforming units (Umemura et al., 1986) as well as for the promyelocytic cell line, HL60 (Watanabe et al., 1985; McCachren et al., 1986) and murine erythroleukemia cells (Lachman and Skoultchi, 1984; Lachman et al., 1985), to quote just three of many possible examples. Indeed, in the Friend erythroleukemia, constitutive expression of c-myc,

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transfected into the cells in a plasmid construct, has been shown to inhibit differentiation (Coppola and Cole, 1986; Prochownik and Kukowska, 1986; Dmitrovsky et al., 1986). Interestingly, in Burkitt’s lymphoma cells, differentiation can be induced with several different agents, but c-myc expression is not inhibited (Benjamin et al., 1984; Sandlund et al., 1990). The difference in these two systems could be due to differences in the cell types, or to differences in the level of c-myc expression. In the Burkitt’s lymphoma cells the lack of down regulation of c-myc is consistent with the hypothesis that this gene is regulated by sequences which normally regulate the expression of immunoglobulin genes. VII. Timing of the Translocation in Relation to 6 Cell Differentiation Malignant non-Hodgkin’s lymphomas, for the most part, phenotypically resemble normal lymphoid cells which have been “frozen” at specific stages of differentiation (Magrath, 1981).This suggests that the genetic lesions that cause lymphoid neoplasia are also responsible for inhibiting differentiation to a greater or lesser extent. A high proportion of the translocations which are associated with lymphoid neoplasms involve either the T cell antigen receptor or immunoglobulin loci. Both sets of genes undergo rearrangements of their component parts, which are separated in the genome, during the process of generating antigen receptor diversity (Hozumi and Tonegawa, 1976; Leder et al., 1980; Adams and Cory, 1983; Seidman and Leder, 1978; Waldman, 1987). Since this process involves breaking the DNA strand, removing intervening sequences, and approximating the relevant sequences by religation, it is possible that cells in which such recombinational events occur are predisposed to translocations at or close to the sites of genetic rearrangement. Consistent with this possibility is the finding that in Burkitt’s lymphoma chromosomal breakpoints are located within or close to the immunoglobulin gene regions at which physiological rearrangements occur, namely V (variable), D (diversity), J (joining), or S (switch) regions. This suggests that the translocations occur either close to the time of VHDJ H or VJL joining, when the variable region of the immunoglobulin heavy or light chain, respectively, is assembled and approximated to the immunoglobulin constant region, or close to the time of heavy chain class switching. If this is so, the possibility that translocations are mediated by the recombinases which catalyze immunoglobulin gene rearrangements must also be considered, although alternative mechanisms of translocation can be proposed. It is possible, for example, that the regions of physiological recombination are more susceptible to chromosomal breaks

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because of their more open chromatin structure, necessitated by the need for enzyme access to the DNA strands during immunoglobulin gene rearrangement. The ligation of the DNA strands of different chromosomes could involve repair enzymes or topoisomerases rather than site-specific recombinases. This issue is further discussed in a later section. Regardless of the mechanism of chromosomal translocation, since immunoglobulin gene rearrangement occurs in an orderly sequence and normal cells are prevented from rearranging the second p heavy chain allele once the first has successfully rearranged, or from rearranging a A gene if a functional K chain has been produced, the events taking place during B cell differentiation provide a framework on which it may be possible to locate the timing of the translocations occurring in cells destined to become Burkitt’s lymphoma cells. If the translocations are temporally related to physiological recombinational events, then Burkitt’s lymphoma could, theoretically, consist of one or more of three possible subtypes: (1) tumors in which the translocation is temporally associated with VHDJH joining (i.e., heavy chain rearrangement), (2)tumors in which the translocation is temporally associated with VJL joining (i.e,, light chain rearrangement), and (3)tumors associated with heavy chain class switching. While the translocations in types (1) and (2) must take place in immature cells, type (3) could involve either immature or more mature (e.g., activated) cells, since class switching takes place at several stages of B cell differentiation, including the pre-B cell stage (Akira et al., 1983;Alt et al., 1982;Vogler et al., 1981; Burrows et al., 1983). The variability of phenotype and genotype, as well as the occurrence of translocations involving either heavy or light chain genes, suggests that Burkitt’s lymphoma is not homogeneous with regard to the timing of the translocation.

A. TRANSLOCATIONS OUTSIDETHE SWITCHREGION Some further observations regarding the timing of the 8;14 translocation are possible. According to the currently accepted hierarchy of immunoglobulin gene rearrangements, a cell undergoing VHDJ H joining would not yet have rearranged its light chain genes. Since the majority of Burkitt’s lymphomas express light chains, if a translocation occurs during initial VHDJHjoining, then differentiation must have occurred after the translocation. Moreover, such light chain rearrangement within the clone of cells bearing a translocation ought to result in many different light chain types, i.e., there would be polyclonality at the light chain locus. Burkitt’s lymphoma, however, is monoclonal at

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the light chain locus as judged by the expression of either K or A light chains, but not both (Gunven et al., 1980). Since pre-B cells are actively proliferating, it is not likely that light chain rearrangement occurs so close to heavy chain joining that there is no time for cell division, leading to the conclusion that in most Burkitt’s lymphomas the translocation occurs in a cell that has already successfully rearranged a p gene and a light chain gene. The rarity of Burkitt’s lymphomas which fail to express p is also of interest. This suggests that either translocation occurs generally in the nonexpressed allele, such that the functional p gene is left intact, or that rearrangement of the second allele following translocationinduced disruption of the functionally rearranged gene is possible. In fact, a murine B cell lymphoma (NFS-1)which synthesizes PK molecules has been shown to be able to rearrange both A and heavy chain molecules subsequent to the expression of surface IgM (Kleinfield et aZ., 1986). Such a possibility appears unlikely because only about one-third of attempted immunoglobulin gene rearrangements are successful, such that a high proportion of Burkitt’s lymphomas would, in this circumstance, lack immunoglobulin expression. Such tumors are rarely observed. Of course, it remains possible that p chain expression is essential to the neoplastic cell, and that any potential tumor cell clone which does not express p does not develop into a neoplasm and is presumably eliminated. Alternatively, salvage of unsuccessfully rearranged p genes by V H to VH recombination (Reth et al., 1986; Kleinfield et al., 1986),which is a highly efficient process, could occur, Whether or not translocations involve successfully or unsuccessfully rearranged p genes, it would appear that the earliest point at which translocation could occur in monoclonal Burkitt’s lymphomas expressing both heavy and light chains is during or shortly after light chain recombination, i.e., in a cell that has already become a B cell. A small number of Burkitt’s lymphomas or L3 leukemias have been reported in which the tumor cells did not express any light chain (Cohen et aZ., 1987; Lenoir et al., 1982),indicating that in these tumors translocation occurred prior to light chain rearrangement and presumably prevented VJL joining, or that light chain gene rearrangement did not result in a functional immunoglobulin molecule.

REGIONTRANSLOCATIONS B. SWITCH Switch region translocations pose fewer questions regarding the status of the immunoglobulin genes at the time that chromosomal recombination takes place. Switching normally occurs after heavy and

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light chain rearrangement has been completed (Kraal et al., 1982), although there are exceptions to this reported in Abelson virustransformed mouse pre-B cells, which have a remarkable propensity to switch from p to y2b heavy chains (Burrows et al., 1983; Shizuo et al., 1983), and human pre-B cell leukemia cells, which switch preferentially to y l (Vogler et al., 1981; Kubagawa et al., 1983).Recently, Altiok et al. (1989) described the frequent occurrence of translocations involving 14q32 in a pro-B cell line, FLEB-14, kept in continuous culture for 4-36 months. The immunoglobulin genes of FLEB-14 were originally germline. In 5 of 7 independently maintained sublines, translocations, each of which involved a different part of the S, region, were observed. Although 8;14 translocations were not observed, these findings confirm the possibility that translocations involving the S, region can occur in very immature B cells, and indeed, may be more likely to occur than in more mature B cells in which such translocations are rarely seen. Interestingly, the majority of Burkitt’s lymphomas with switch breakpoints have translocations which involve the p switch region. Burkitt’s lymphoma also nearly always expresses IgM. In contrast, mouse plasmacytomas usually express IgA and most have translocations into the S, region (Cory, 1986), while rat immunocytomas predominantly secrete E and have translocations which involve S, (Pear et al., 1988). It has been shown that transcription of the relevant heavy chain constant region occurs immediately prior to switching, suggesting that the heavy chain into which switching will occur has been determined prior to the process of switching (StavnezerNordgren and Sirlin, 1986). The high frequency of y2b switches in Abelson pre-B lymphoma cell lines also supports this notion (Burrows et al., 1983). Predetermination of the heavy chain class involved in switching suggests that a change in the chromatin pattern of the target heavy chain occurs prior to switching, presumably to permit access to switch recombinase. It is highly likely that such regions of more open chromatin also have increased susceptibility to translocation, thus explaining the concordance of translocation and heavy chain class. Translocation into regions “prepared for switching” may be due either to actual participation of switch recombinases in the translocational event, or to increased fragility of the chromosomal region consequent upon the altered chromatin pattern. It is worth noting that it is probable that the excluded allele also becomes accessible to recombinases at the same time as the functional allele. This may be a necessary prerequisite for translocation to occur in the excluded allele, as has been clearly demonstrated in some tumors (Tian and Faust, 1987).

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In Burkitt’s lymphoma, several cell lines have been described in which the translocation involves a switch region other than S, (Hamlyn and Rabbitts, 1983; Showe et al., 1985; Haluska et al., 1988).In such tumors c-myc could have first been translocated into the p gene and then, by a switch deletion process, relocated as if it were a variable region into another switch region. Alternatively, c-myc could be translocated directly into one of the other switch regions, which may or may not have already undergone normal physiological switching. The mechanism which applies may depend upon whether class switching is imminent or has already occurred at the time of translocation. In the Raji and EW36 cell lines, true switch deletion of C, seems to have occurred (Hamlyn and Rabbitts, 1983; Haluska et at., 1988), while in the CA46 line the presence of most of the immunoglobulin locus on chromosome 8 without deletion of p suggests that c-myc was translocated directly into the S,1 region (Showe et al., 1985).

C. TRANSLOCATIONS INVOLVING LIGHTCHAINGENES As is the case with translocations involving switch regions in different species, the variant translocations are usually concordant with regard to the chromosome involved and the light chain expressed. Thus 8;22 translocations are usually associated with X gene expression, and 2;8 translocations with K expression (Lenoir et al., 1982). This suggests that the variant translocations usually occur close to the time of light chain rearrangement when the chromatin structure is more susceptible to DNA breakage. Several exceptions to this concordance have been described, however, in two of which the accepted hierarchical order of light chain rearrangement attempts has been violated (Magrath et al., 1983b; Hollis et al., 1984; Denny et al., 1985). Since the translocation prevents expression of the involved allele, it is probable, using the same argument as for translocations into the heavy chain locus, that one light chain has already been successfully rearranged prior to the occurrence of the translocation, and that the translocation occurs into the nonfunctional or excluded allele.

D. RESTRICTION OF THE TRANSLOCATION TIMEFRAME There seems no doubt that there is a specific window (or windows if there are several subtypes of Burkitt’s lymphoma) which occurs in the course of B cell differentiation during which transformation of a normal cell into a Burkitt’s lymphoma cell can occur. The concordance of

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translocation site and immunoglobulin gene expression strongly suggests that translocation occurs close to the time that a normal recombinational event, be it VHDJHjoining, VLJL joining or heavy chain class switching, would occur. As is discussed below, it appears that the regulation of immunoglobulin gene rearrangement is determined by accessibility of the relevant DNA to recombinase, rather than alterations in the level of the enzyme itself. As access of the continuously present enzyme to each gene region is permitted, so too ensues a period during which translocation at this location is possible. Experimental support for this notion of translocation-permissive differentiation windows comes from animal models. For example, mice doubly transgenic (described below) for a c-myc gene driven by an immunoglobulin enhancer (EnMyc) and a p gene encoding a membrane p molecule (pm) had a markedly lower incidence of pre-B neoplasms than mice transgenic for EnMyc alone (Nussenzweig et al., 1988). In addition, such mice were resistant to tumorigenesis by Abelson leukemia virus. The authors surmised that the presence of the rearranged pm gene, which caused allelic exclusion, hastened passage of the pre-B cells through a specific differentiation phase-presumably that associated with VHDJHjoining. In avian leukosis virus (ALV)-induced bursal lymphomas in chickens, tumors occur only if ALV infection occurs during bursal embryogenesis or within a few days of hatching, probably corresponding to the period during which bursal stem cells are present (Maas et al., 1982). These considerations would appear to indicate that translocations in Burkitt's lymphoma can occur at various different points during the process of physiological rearrangement of immunoglobulin genes. The particular window in which the translocation occurred is reflected in the breakpoint location-D or J if during or close to VHDJHjoining, in K or A if close to the time of light chain rearrangement, and in S, if close to the time that switching would occur (although the latter is able to occur throughout B cell differentiation). Only if translocation occurs after heavy chain class switching would it be expected that an immunoglobulin other than IgM would be synthesized by the tumor cells. Clearly, this occurs rarely in Burkitt's lymphoma, but one or two examples, e.g., Raji, which synthesizes IgG, have been described (Hamlyn and Rabbitts, 1983; Kiwanuka et al., 1988). These differences in the level of differentiation of the cells in which the translocations are occurring could be reflected in somewhat different clinical manifestations of the tumor, but to date there is no information on this.

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VIII. Mechanism of Translocation

A. POSSIBLE MEDIATIONOF IMMUNOGLOBULIN SIGNAL SEQUENCES The mechanism, or mechanisms, whereby chromosomal recombination occurs is still largely unknown. Extensive homology between c-myc and immunoglobulin genes is not present, so that homologous recombination is essentially ruled out. While random breaks and religation via DNA repair enzymes or topoisomerases could occur, the occurrence of breakpoints in regions normally involved in immunoglobulin gene recombinations and the correlation between breakpoint location and immunoglobulin isotype or light chain type in rodent and human tumors associated with homologous translocations raises the question as to whether the recombinases which mediate immunoglobulin gene rearrangements are also involved in the process of chromosomal translocations. One might suppose that if such recombinases are involved, there would be a strong tendency for breakpoints in and around c-myc to be localized to specific regions, for such regions to bear recognizable signal sequences, and for the breakpoints on the immunoglobulin bearing genes to be immediately adjacent to heptamer sequences in the J or D regions, or to fall within a switch iegion. While there are some regions in and around c-myc which tend to be frequent sites of breakpoints, it is possible that these are selected for because of the resultant functional disturbance to c-myc rather than because of the presence of particular signal sequences involved with the genesis of the translocation. Sequences which resemble the V, D, and J heptamerhonamer signal sequences have been described (Morse et al., 1989) in the 3‘ end of the c-myc first exon, at the hypermutable PvuII site (see below), while sequences which bear a close resemblance to switch recombination sites have been described at the breakpoints of some mouse plasmacytomas (Dunnick et al., 1983, 1984; Gerondakis et al., 1984). Switch regions are commonly involved in both human and animal tumors. A tetranucleotide, GGAG, in particular, has been identified at the breakpoints of several murine plasmacytomas as well as in Burkitt’s lymphomas (Picoli et al., 1984; Showe et al., 1985). Interestingly, among rat immunocytomas, the majority of which have breakpoints within the S, region of chromosome 6 (and secrete IgE), two tumors were recently described with breakpoints outside switch regions. One of these, a y2a-secreting tumor, had a breakpoint in a DNA repetitive element-a LINE element located within the immunoglobulin heavy chain region-which shares signifi-

172

IAN MAGRATH

cant homology with Sue sequences, while the other had a breakpoint in the first intron of the L locus, in a region of homology with S, (Pear et al., 1988). The GGAG tetranucleotide was found in both c-myc and LINE at the breakpoint region. These findings support the possibility that many of the translocations occur at sequences which resemble switch recognition sequences and are mediated by switch recombinases. Such sequences have been described as “scattered throughout the genome” (Kirsch et al., 1981), but most are probably inaccessible to enzymes. Only a few Burkitt’s lymphomas with a breakpoint far 5’ of c-myc on chromosome 8 have been subjected to molecular analysis. Using DNA regions immediately adjacent to cloned breakpoints as probes in Southern blotting analysis of more than 20 African tumors with breakpoints in this region, no evidence of significant clustering of the breakpoints was obtained (Neri et al., 1988). However, this by no means excludes the participation of recombinases acting on heptamer/ nonamer signal sequences and it is possible that the enzymes can occasionally catalyze breaks and religations in regions which resemble signal sequences, but are not identical. Indeed, the poor affinity of the enzymes and associated protein factors for such regions is consistent with the rarity of translocational events in both cell and human populations. The most suggestive information that recombinases can catalyze chromosomal translocation in cell lines with 8;14 translocations has been derived from three cell lines, Daudi, P3HR1, and a pre-B acute lymphoblastic leukemia (380).All three cell lines have 8;14 translocations which happen to have breakpoints within a small distance of each other (Haluska et al., 1986,1987). Sequences which bear a resemblance to the heptamer and nonamer signal sequences known to mediate immunoglobulin gene rearrangements have been described on clones derived from the normal chromosome 14 in a region adjacent to the breakpoint in PSHR1. The putative “heptamer” sequence is CAATGCC and the putative “nonamer” sequence GTTCTCTCG. However, these sequences lie upstream of what has been called a pseudo-J region, itself upstream of the heptamer and nonamer sequences 5‘ of the J5 region which lies some 90 bp 3’ of the breakpoint. Thus, the breakpoint, like several others that have been cloned (Taub et al., 1984; Denny et al., 1985),has not occurred at a “physiological” region, i.e., one at which immunoglobulin gene recombination normally occurs, although the breakpoint region bears similarities to a physiological recombination region. The sequences of the signal heptamer and nonamer sequences 5’ of 55 are CAATGTG and

THE PATHOGENESIS OF BURKITT’S LYMPHOMA

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GTTCTTTGT. These differ by two and three nucleotides, respectively, from the putative heptamednonamer signals close to the breakpoint location. While it must remain unknown, for the present, whether these sequences are nevertheless able to bind the recombinase, there is evidence that quite wide sequence variation within the heptamer is tolerated (Yancopoulos et al., 1986a).In the case of the 380 cell line, the breakpoint on chromosome 14 does appear to be at a heptamer border, immediately adjacent to a putative N region of additional nucleotides inserted at the 5’ end of J6. In the Daudi cell line, the breakpoint on chromosome 8 is located within a heptamer/ nonamer signal sequence with a 23-bp spacer, as can be seen by an examination of the same region on the normal chromosome 8 (Haluska et al., 1987). Apparent N regions are present immediately adjacent to the breakpoint on both chromosomes. In addition, the region of chromosome 14 immediately adjacent to the breakpoint harbors a D region. Thus, it seems likely that the translocation in this cell line was mediated by recombinases normally mediating VDJ recombination. A nonproductive D-JH rearrangement (involving a truncated J4 region) is present on chromosome 14, with evidence (i.e., altered restriction map of this D region versus the D region involved in the translocation) that a D-D rearrangement also occurred. The 23-bp spacer between the heptamer and nonamer on chromosome 8 is the same size as the spacer normally observed next to variable regions. Moreover, a VHDjunction is present on chromosome 8q- immediately upstream of the breakpoint. Because of these findings, it has been postulated that the translocation occurred as a mistake during attempted VH-D rearrangement. Alternatively, the VDJ recombinase simply used available, accessible signals to bring about the chromosomal recombination. In cell lines with variant translocations, there is evidence that signal sequences may, at least on occasion, mediate chromosomal translocations. Two examples have been analyzed in detail to date. In the BL64 cell line the breakpoint on chromosome 2 has been localized to a region between the conserved heptamer and nonamer sequences immediately 5’ to JS (Hart1 and Lipp, 1987). The breakpoint on chromosome 8 lies within a repetitive (Alu) sequence, but both nonamer and heptamer sequences were present as perfect inverted repeats of the JK signal sequences, although the distance between heptamer and nonamer was not the expected 12 b, but rather 155 b. Nevertheless, these findings strongly suggest that recombinases normally responsible for V-J joining in the K locus were involved in the chromosomal translocation of BL64. In the J 1 cell line the breakpoint on chromosome 2 has been localized to within an aberrantly rearranged and truncated V, I 08

174

IAN MAGRATH

variable gene. Heptamer and nonamer signal sequences were again observed on chromosome 8, although on chromosome 2 only five of the seven heptanucleotide sequences were present and there was no nonamer. A further observation of relevance to the theme of chromosomal recombination is a report of a LINE-1 element replacing an initially expressed variable region, specifically, the VHD region of the immunoglobulin molecule, and combining site-specifically with the remaining J H segment (Yancopoulos et al., 1986b).Sequences very similar to the heptamer/nonamer recognition sequences which normally mediate VHDJHjoining appear to have mediated the LINE-1 to J H join. This observation demonstrates that presumptively recombinase-mediated recombination can occur in an already functioning immunoglobulin gene, a finding that implies that recombinases remain active even after the production of an IgM molecule, and supports the concept that allelic exclusion is mediated by preventing access of the recombinase to the signal sequences rather than through cessation of production or inactivation of the enzyme. Enzyme accessibility is maintained by transcription, a finding which accounts for the transcription of individual segments of immunoglobulin genes shortly before rearrangement occurs. It has also been shown that the most 3' unrearranged VH regions can be transcribed under the influence of the heavy chain enhancer (Wang and Calame, 1985), thus retaining accessibility to enzyme, while more distant VH regions remain inaccessible. LINE sequences are known to be transcribed and so presumably also permit enzyme access to embedded signal sequences. The persistent presence of recombinase after immunoglobulin rearrangement is important both to the generation of immunoglobulin diversity as well as to the conservation of cells which have unsuccessfully rearranged their immunoglobulin genes (which occurs in some two of three attempts). Moreover, it may have relevance to the possible mechanism of translocation in Burkitt's lymphoma. With regard to the generation of immunoglobulin diversity, it has been shown that already productive genes, or genes which are nonfunctional because of a shift in the reading frame occasioned by the addition of nucleotides (N) by terminal transferase, can undergo VH to VHDJ" rearrangement. I n this process, whereby the major part of the already rearranged V region is replaced by an upstream VH region, either an out-of-frame variable region is returned to an in-frame, functional molecule, or there is simply substitution of one variable region for another (Kleinfield et al., 1986;Reth et al., 1986).I n either event, an isolated heptamer sequence embedded in the variable region appears to be responsible for site-

THE PATHOGENESIS OF BURKITT’S LYMPHOMA

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directed recombination of this kind. The majority of VH regions (but not light chain V regions) in mice and humans contain internal, conserved heptamer sequences presumably able to mediate such events. Whether this mechanism is ever utilized in the genesis of chromosomal translocations is not certain, but there can be little doubt that the enzymatic machinery necessary to accomplish it persists in the cell for some time after immunoglobulin gene rearrangement has occurred. Moreover, since there is a precedent for the utilization of isolated heptamers instead of hepatmednonamer combinations (which also applies in the K locus), the likelihood of recombinase-mediated chromosomal translocation is increased, since the probability that a sequence bearing sufficient resemblance to a signal heptamer motif to bind recombinase will be found in a relevant chromosomal region is also increased. These data suggest that heptamer/nonamer-like signal sequences are at least occasionally, and possibly frequently, involved in chromosomal recombinations which occur in or close to c-myc. Moreover, repeat sequences (e.g., LINE-1, A h ) which are scattered widely through the mammalian genome have also now been implicated. It remains possible that translocations utilize recombinases which can bind to poorly matched signal sequences, or signal sequences without the correct spacers. Clearly, if well-matched signal sequences were present in c-myc, chromosomal translocations might be much more frequent than they are. B. SEQUENCE ALTERATIONSADJACENTTO BREAKPOINT LOCATIONS The translocation breakpoints which have been cloned indicate that a precise exchange of cleanly broken DNA rarely occurs. Deletions and/or insertions as well as duplications occur close to the breakpoints (Hollis et al., 1984; Denny et al., 1985). For example, in the translocation in BL37,21 bp of DNA from the A 1 locus have been deleted at the cross-over point. In KK124, a cell line with an 8;22 translocation, a 39-bp sequence found on the normal chromosome 8 was present at the breakpoint on both derivative chromosomes. On chromosome 22 an internal 8-bp duplication had occurred within this 39-bp sequence, while on chromosome 8, a second copy of nucleotides 14 to 39 was present very close to the first (5bp away). A 32-bp sequence had been deleted from the breakpoint region on chromosome 22. In the cell line J1, eight nucleotides have been inserted between the variable region in which the chromosome 2 breakpoint occurred and the immediately adjacent J region (Klobeck et al., 1987).This is reminiscent of an N

176

IAN MAGRATH

region (ie., inserted by terminal transferase), although such regions have not been described in K genes. In this same cell line, several point mutations, consistent with somatic mutations induced in immunoglobulin genes, were also present close to the breakpoint in the immediately adjacent regions of both chromosomes. In the cell line JBLB, the entire first exon of c-myc has been duplicated, giving rise to an abnormal large c-myc message (Taub et al., 1984). In Daudi, 9 nucleotides were lost from chromosome 8 and 15 from chromosome 14 during the translocation (Haluska et al., 1987). The presence of deletions and/or duplications have suggested that DNA breakage occurs at a different site on each strand (Gerondakis et al., 1984).The overlapping strand is then either degraded by an exonuclease activity, giving rise to a deletion, or functions as a template on which the opposite strand can be resynthesized. In the latter circumstance the same sequence will be present on both chromosomes involved in the translocation. Often a small number of nucleotides are inserted at the chromosomal junctions, as in the 380 and J1 cell lines described above. The occasional insertion of such nucleotides, similar to N regions which are added on either side of the D region of the immunoglobulin heavy chain gene during its rearrangement, may be mediated by terminal deoxyribonucleotide transferase (TdT). This would support the probability that some translocations occur temporally close to the time of VHDJHjoining, when TdT is present in the cell. An alternative explanation to the participation of immunoglobulin gene recombinases in the translocational event is that the regions involved in immunoglobulin gene rearrangement are relatively fragile because of the open chromatin structure needed for recombination to be able to occur. In this case breaks may occur at random within the fragile region, and only those which produce deregulation of c-myc will permit the cells to become neoplastic. The breakpoints in and around c-myc may also occur at relatively fragile sites, in this case a presumptive consequence of the expression of this gene in a proliferating cell. IX. Structural Changes in c-myc Brought about by the Translocations and Their Possible Functional Consequences The juxtaposition of c-myc with an immunoglobulin constant region is not the only consequence of the nonrandom chromosomal translocations associated with Burkitt’s lymphoma. It appears that in all tumors, regardless of whether the breakpoint is far upstream of c-myc, within the gene, or far downstream, there are also structural changes in

THE PATHOGENESIS OF BURKITT’S LYMPHOMA

177

c-myc. Up to the present, not one tumor has been generally accepted to contain two absolutely normal c-myc genes, even among the endemic tumors in which the majority of Burkitt’s lymphomas bear chromosome 8 breakpoints which are at a considerable distance from c-myc. A structural change in c-myc can therefore be considered to be the second general feature of Burkitt’s lymphoma. The structural changes in c-myc vary from a single point mutation to separation of the second and third exons of the gene from its first exon. Such diverse differences must clearly have different immediate consequences, even though the final end result of the translocations may be, in all cases, the inappropriate expression of c-myc. In addition, the various types of structural change correlate to a remarkable degree with the differences in clinical features and epidemiology noted above.

A. BREAKPOINTS FARUPSTREAM(5’)OF c-myc In the majority of endemic Burkitt’s lymphomas, the breakpoint on chromosome 8 lies far upstream of the c-myc gene. In several cell lines derived from African Burkitt’s lymphomas, the chromosome 8 breakpoint has been molecularly cloned, and regions adjacent to the breakpoint used as probes to determine whether other tumors have breakpoints in a similar location (Haluska et al., 1986; Neri et al., 1988). To date, this strategy has not revealed rearrangement of the DNA detected by such probes in the majority of tumors, indicating that there is considerable variability in the breakpoint location in the upstream region of c-myc. Thus, it appears unlikely that the upstream breakpoint has any significance beyond causing translocation, and directly or indirectly, structural changes in c-m yc. In particular, alteration in the function of a gene upstream of c-myc consequent upon the breakpoint location appears to be unlikely, although this possibility cannot yet be completely excluded.

Functional Significance of Mutations in the First c-myc Exon In all tumors with a far 5’ chromosome 8 breakpoint that have been examined in sufficient detail, mutations have been detected in the first exon of c-myc-usually in the 3’ region in proximity to a PvuII restriction enzyme site (Pelicci et al., 1986b). The mechanism whereby these mutations are induced is quite unknown although it has been speculated that juxtaposition to immunoglobulin heavy chain sequences could be relevant insofar that immunoglobulin variable regions accumulate somatic mutations as part of the process of the generation of

178

IAN MAGRATH

antibody diversity. Translocation brings c-myc into a location which approximates that of the normal immunoglobulin variable region in a rearranged immunoglobulin gene, such that it may be subject to mutation induction via the same mechanism as an immunoglobulin variable region (Rabbitts et al., 1984). In a small number of cell lines, it has been shown that there is a correlation between the presence of exon mutations and abrogation of the normal block to elongation of the RNA transcript that is recognized as an important means of regulating the expression of c-myc (Eick and Bornkamm; 1986, Bentley and Groudine, 1986b). This block, situated at the first exonhntron junction, prevents the production of transcripts derived from the protein-coding region of c-myc and can be readily detected by nuclear run-off experiments in which the relative proportion of nascent nuclear transcripts derived from exon 1 and exon 2 can be determined. In quiescent cells, exon 1 transcripts are far in excess of transcripts containing exon 2, indicating failure of the polymerase to read through the second (and third) exons, whereas when such cells are activated, the relative proportion of transcripts containing exon 1 and those containing exon 2 becomes more equal. Abrogation of the elongation block-i.e., a failure to inhibit chain elongation (Cesarman et aZ., 1987)-could be an important mechanism whereby c-myc is deregulated in Burkitt’s lymphoma. However, abrogation of the elongation block does not appear to be the only consequence of first exon mutations. These same mutations may result in failure to produce the 67-kDa protein initiated near the 3’ end of the first exon. Thus, in 5 of 11cell lines examined in which the breakpoint on chromosome 8 is not within the c-myc gene, only the 64K protein could be detected (Hann et aZ., 1988). This is entirely consistent with the demonstration that the introduction of mutations in the 5’ region of the first exon can abolish the production of the 67K protein in uitro. Whether this is significant to the development of Burkitt’s lymphoma, or is a secondary effect, is a matter for speculation at present.

B. BREAKPOINTS IN THE 5’ REGULATORY REGIONOF c-myc The presence of regulatory elements immediately upstream of c-myc has been documented in several different laboratories and by a variety of techniques. Among the most direct of these are experiments in which various lengths (containing putative regulatory elements) of the c-myc 5’ region are linked via the c-myc promoters PI and Pz to a reporter gene (i.e., one whose expression is readily detected) in a

THE PATHOGENESIS OF BURKITT’S LYMPHOMA

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plasmid construct which is then transfected into various cell types and the degree of transcription observed (Hay et al., 1987). These approaches can be embellished by inducing site-specific mutations or deleting parts of the 5’ c-myc region in the plasmid, and observing the effect on reporter gene expression. Complementary information is obtained by measuring the binding of proteins to specific sequences in this 5’ region, e.g., by observing a reduction in the electrophoretic mobility of fragments to which proteins are bound, or by exonuclease assays in which digestion of a segment of DNA is protected at sites where proteins are bound (Levens and Howley, 1985; Hay et al., 1987). Regulatory elements identified in this way usually correspond to DNase-hypersensitive sites, presumably because of the need for increased accessibility (of protein factors) to the DNA at these locations (Siebenlist et al., 1984; Dyson and Rabbitts, 1985). In Fig. 1, the present state of knowledge regarding the 5‘ regulatory elements of c-myc is summarized. Several points become apparent. First, there are both positive and negative regulatory elements for transcription initiation from PI and PZ(Hay et al., 1987). Two major positive elements have been identified, one between 353 and 1257 bp upstream of PI and the other between 1257 and 2329 bp upstream. Both elements contain hypersensitive sites for DNase I hydrolysis and sequence motifs suggestive of binding sites for the transcriptional enhancer protein N F l . The region between -353 and -1257 bp also contains a sequence (GTGGAAGG)similar to the consensus sequence found in many transcriptional enhancers. Disappearance of a protein factor which binds to the element between - 1257 and -2329 is associated with a decrease in c-myc transcription and the onset of differentiation in HL60 cells (Siebenlist et al., 1988).The region between -2119 (a PstI site and -2329 (the Hind111 site), which contains the autonomously replicating sequence described above, has recently been analyzed in more detail, with some surprising results (Iguchi-Ariga et al., 1988). It appears to contain more than one enhancer element (sequences -2329 to -2286 and -2215 to -2204) and probably a negative regulatory element (sequences -2286 to -2232). The c-myc protein binds to the region between sequences -2225 and -2141, at the autonomously replicating sequence, and plasmids containing these sequences, but not plasmids containing adjacent sequences, were able to inhibit c-myc expression in HL60 cells, presumably due to competition with the cellular c-myc gene for c-myc protein (Iguchi-Ariga c-myc 1988). These results suggest that c-myc itself may have a positive regulatory effect, at least in HL60 cells. A negative element has also been identified in the region between

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IAN MAGRATH

293 to 353 bp upstream of P1 (Hay et al., 1987).This region bears some resemblance to a transcription suppressor element for p-interferon, and like the latter displays dyad symmetry (Goodbourn et al., 1985, 1986). The negative effect of this element can be overridden by the upstream positive elements, but its removal, in the presence of the latter, permits some enhancement of transcription. Thus the second point to be made is that the activity of elements close to the first exon of c-myc may only become apparent in the absence of the upstream region. For example, in the absence of the upstream positive regulatory elements, transcription from the P1 promoter appears to be totally dependent upon a third positive element situated between 101 and 293 bp 5' of PI. This element is not required in the presence of the upstream transcriptional enhancer regions. In one cell line, MCll6, derived in this laboratory, the breakpoint on chromosome 8 lies very close to the latter element, thus separating c-myc from its upstream positive regulatory elements. In addition, the -293/- 101 element appears to have been rendered nonfunctional by the translocation such that in MC116 c-myc is transcribed only from the PZ promoter-as demonstrated by S1 protection assays (Nishikura and Murray, 1988; Barriga et aZ., 1988a). It is apparent that breakpoints within the 5' region of c-myc could have a variety of effects depending upon which regions remain attached to the translocated c-myc gene, and whether additional adjacent mutations are associated with the translocation. For example, a breakpoint immediately upstream of the negative element would result in suppression of c-myc expression in the absence of an enhancer function derived from another source, while mutations of the suppressor element could contribute to enhanced transcription of c-myc. Breakpoints within 353 bp upstream of P1 (i.e,, between the CZaI and SmaI sites) have the effect of removing the normal c-myc enhancer. If the breakpoint on chromosome 14 is upstream of the heavy chain enhancer (situated between the J and S regions), the latter, presumably, simply replaces the normal c-myc enhancer. In effect, the c-myc gene is likely to be regulated as if it were an immunoglobulin gene. This appears to be the case in the murine plasmacytoma fusion gene used to generate EH-myc transgenic mice (see below). When the breakpoint in chromosome 14 is downstream of the enhancer element, e.g., within the switch region, the heavy chain enhancer is translocated to chromosome 8, but other enhancer elements almost certainly exist within the immunoglobulin region and presumably influence c-myc transcription in this circumstance. It seems highly probable that when the chromosome 8 breakpoint is in the immediate upstream region of

THE PATHOGENESIS OF BURKITT’S LYMPHOMA

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c-myc, deregulation occurs as a consequence of the net effect of the residual regulatory elements attached to the translocated c-myc gene and the regulatory elements residing in the remaining immunoglobulin gene components on chromosome 14. In effect, the c-myc gene enhancers (and in some cases, promoters) are lost and replaced by immunoglobulin gene enhancers. In some circumstances, depending upon the precise breakpoint locations on chromosomes 8 and 14, additional positive regulatory elements may be required. These could be provided, directly or indirectly, by EBV (see below). WITHIN C. BREAKPOINTS

THE

FIRST c-myc EXONOR FIRST INTRON

Breakpoints downstream of PI and PZremove both the regulatory elements and the normal promoters from c-myc. In this circumstance there is evidence that transcripts are initiated at a “cryptic” promoter (P3) present in the first intron (ar-Rushdi et al., 1983; Cory, 1986). It is pertinent that breakpoints within the intron appear never to occur downstream of this region-presumably such breakpoints are not conducive to c-myc expression. Nearly all of the normal regulatory mechanisms relevant to c-myc transcription, including transcriptional controls and the block to transcript elongation, are abrogated by exon/intron breakpoints, the exception being positive and negative transcriptional regulatory elements which are situated in the exon or remaining part of the intron itself. Although it has been suggested that truncation of the gene in this way could result in its constitutive expression, available information does not suggest that this is the case. Truncated transgenes unattached to an enhancer do not induce neoplasia in mice (Adams et al., 1985). Moreover, expression of truncated c-myc genes in plasmacytoma cells depends upon the immunoglobulin sequences to which they are linked. In one series of experiments a truncated gene linked to the heavy chain immunoglobulin enhancer, but not one linked to a switch region, was expressed when transfected into a mouse plasmacytoma line (Feo et al., 1986). Similarly, truncated genes attached to switch regions are not expressed in somatic cell hybrids between lymphoblas toid cell lines and Burkitt’s lymphoma cell lines, in which the lymphoblastoid phenotype predominates (Croce et al., 1984), although they are expressed when the same Burkitt’s lymphoma cell lines are fused to plasmacytoma cells. These results suggest that truncated c-myc genes are not active without a positive drive provided by a transactivating factor which must bind to enhancer elements associated with the gene. The protein factors which bind to such elements are presumably not provided by the

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IAN MACRATH

lymphoblastoid milieu. Since most of the normal c-myc regulatory region is missing from the translocated, truncated gene, alternative enhancers must be provided. These are likely to be derived from immunoglobulin gene sequences juxtaposed to c-myc, but in some circumstances an additional exogenous positive transcriptional drive may be necessary; this could be provided by an EBV gene.

D. BREAKPOINTS DOWNSTREAM OF c-myc Chromosomal breakpoints downstream (telomeric) of c-myc occur in the variant translocations. Manolov and colleagues (1986) reported, on the basis of cytogenetic data from high-resolution G banding in a small number of cell lines, that the breakpoint on chromosome 8 in tumors with an 8;22 translocation is more distant from c-myc than the breakpoint in 2;8 tumors. However, this information conflicts with molecular data, since some of the cell lines identified as having a breakpoint in the more distant, so-called “divergent” region of band 8q24.22 actually have a breakpoint close to c-myc (e.g., BL2). In addition, molecular analysis places several 2;8 breakpoints further away from c-myc than some 8;22 breakpoints (Sun et al., 1986; Henglein et al., 1988). Clearly, predicting breakpoint locations on the basis of cytogenetic data can be hazardous. The large spread of the variant translocation breakpoints on chromosome 8-ranging from approximately 7 kbp downstream of the normal c-myc promoters in the cell line BL37 (Hollis et al., 1984)to more than 300 kbp downstream (Henglein et al., 1988, 1989; Shtivelman et al., 1989)-does not immediately suggest a unifying theme to account for the functional changes consequent upon the translocations. It has been pointed out that the first approximately 38 kb of DNA downstream of c-myc contains many repetitive sequences (Alu), and that such sequences are often associated with deletions, rearrangements, and amplifications (Sun et al., 1986). A number of nonlymphoid tumors have, in fact, been associated with amplifications of c-myc which extend a considerable distance into this 3‘ region (Sun et al., 1986; Graham and Adams, 1986; Mengle-Gaw and Rabbitts, 1987). Thus, it is possible that this whole, rather extensive region is particularly susceptible to DNA strand breakage, and hence to chromosomal translocation, but whether elements in this region contribute directly to the deregulation of c-myc, or contribute to transformation in another way, is unknown. There are no clear differences that have emerged to date between the regions where breakpoints occur in 2;8 versus 8;22 translocations. In fact, among the chromosome breakpoints which have been iden-

183

THE PATHOGENESIS OF BUFUCITT'S LYMPHOMA

tified so far, 8;22 and 2;8 translocations appear to be interspersed among each other (Henglein et aZ.,1988, 1989) (Table 11).A common feature of the variant translocations is that both are frequently, and perhaps always, associated with mutations within c-myc. In one study of 15 cell lines with 8;22 translocations, 9 had mutations detectable by restriction endonuclease analysis, with many of them clustered around the PvuII enzyme site close to the 3' end of the first exon (Szajnert et al., 1987)-a similar finding to that which has been observed in tumors with a far 5' breakpoint. Of the 26 c-myc mutations found in the 15 cell lines, 18were located in a 600-bp region comprising the 3' half of exon 1 and the 5' half of the intron. It is highly likely that more detailed analysis would reveal mutations in the remaining six lines.

TABLE I1 VARIANT TRANSLOCATION BREAKPOINTS ON CHROMOSOME 8

Cell line

Translocation

Breakpoint location"

<10

BL37 KK124 J1 BL2 LY47 LY67 PA682 BL64 BL21 LY91 JBL2 LY66

8;22 8;22 2;8 8:22 8;22 8;22 8;22 2;a 2;8 2;8 2;8 2;8

25-35 10-50 >40 >40 >47 140 140 140 >260 >300

BL33 BL60 BL47 BL50 BL84 BUS BL90 BL99 BL104 MWIKA L660

8;22 8:22 8;22 8:22 8;22 8;22 8;22 8;22 8;22 8;22 8;22

NK NK NK NK NK NK NK NK NK NK NK

<10

-

EBV status

+ + + + + + + + + + +

Light chain K K K

A

none h K

K K

none none A A

none A A

h A A K

none

a Measured in kilobase pairs in a downstream (3') direction from the c-myc promoters. NK, not known.

184

IAN MACRATH

The origin of these mutations is unknown, as is the case of those associated with 8;14 translocations. It seems less likely than in the case of far 5’ breakpoints that they are a consequence of juxtaposition to immunoglobulin sequences and consequent exposure to the same process that normally causes somatic mutation in V regions: in the variant translocations the breakpoints are often very far from the c-myc gene, and it is uncertain as to whether the somatic mutation mechanism can operate over such distance. Moreover, somatic mutation is not a prominent mechanism for diversifying light chain V regions. This, of course, leads to less confidence that the similar mutations associated with 8;14 translocations are caused by the same mechanism that leads to V H somatic mutation. Whatever the mechanism, it seems probable that the same consequences ensue. As mentioned, the mutation at the 3’ end of the first exon may cause abrogation of the block to chain elongation and, in many cases, failure to synthesize the 67-kDa c-myc protein (Cesarman et al., 1987; Hann et al., 1988). 1. Point Mutations within the First Intron Some of the mutations that have been described in the intron may also be of importance to c-myc regulation. In particularly, a point mutation identified within a 20-bp region of the intron of a cell line bearing an 8;22 translocation (PA682) has been shown to prevent the binding of a nuclear protein-quite probably a regulatory protein (Zajac-Kaye et al., 1988a,b). This 20-bp region appears to encompass the recognition sequence for the restriction enzyme MaeIII (GTAAC). Mutations in this 20-bp region, including mutations within the MaeIII site, were found in four of the six additional lines for which sequence data were available. These included three lines with 8;22 translocations and one (Raji) with an 8;14 translocation. Two of the lines (KK124 and BL2) each had only one mutation in a 300-bp region at the 5‘ end of the intron, and both were within the MueIII recognition sequence. Daudi (8;14)and BL37 (8;22) did not have mutations in this region. The mutations do not appear to attenuate the block to c-myc message elongation at the 5‘ exon border, since a significant excess of nascent exon 1 message compared to exon 2 message was demonstrated in the PA682 cell line. The likeliest explanation for the function of this protein binding region is that it is a negative regulatory element. Indeed, Chung et al. (1986) have suggested that such elements exist within the c-myc intron. It is possible that the element interacts with other c-myc regulatory elements; if so, its importance as a regulator of transcription of c-myc in Burkitt’s lymphoma would vary according to the presence or absence of other structural alteration in the gene.

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2. The Human Equivalent of the Murine pvt-1 Locus The murine put-1 locus was originally isolated in association with an aberrantly rearranged K gene in a murine plasmacytoma, ABPC4, and was subsequently shown to be the most frequent site of breakage on chromosome 15 in murine plasmacytomas bearing a 6; 15chromosomal translocation (Webb et al., 1984; Banerjee et al., 1985; Cory et al., 1985)and to be a frequent site of insertion of retroviral DNA in mice and rats (Graham et al., 1985; Villeneuve et al., 1986). DNA cloned from the breakpoint of a Burkitt’s lymphoma cell line (JBL2) bearing a 2;8 translocation has been shown to be homologous with put-1 (Graham and Adams, 1986).Recently, put-1 was also identified as a site of interstitial deletion in a cell line derived from human adult T cell lymphoma (Mengle-Gaw and Rabbitts, 1987), while the amplicon in several tumor cell lines bearing amplified myc genes of both murine and human origin contains sequences derived from this region (Graham and Adams, 1986; Mengle-Gaw and Rabbitts, 1987). One of these cell lines (COL0320 HSR) has an amplicon of approximately 300 kbp which has been shown to terminate within 5 kbp ofthe breakpoint of JLB2, indicating that the put-1 like region on human chromosome 8 must be approximately 300 kbp from c-myc (Mengle-Gaw and Rabbitts, 1987). More precise mapping has localized put-1 to approximately 260 kbp downstream of c-myc. Interestingly, Shtivelman et al. (1989) have recently identified a human transcription unit which encompasses put-1 and which begins 57 kbp downstream of c-myc and extends for a minimum of 200 kbp, the transcriptional direction being the same as that of c-myc. This putative gene has been called PVT. It is co-amplified or partly co-amplified (exon 1only) with c-myc in several tumor cell lines, including COL0320, small-cell lung carcimoma lines (H82 and N4517) and a neuroepitheloma cell line (SK-N-MC). Polyadenylated transcripts which hybridize with a probe representing the 5’ domain of PVT have been detected in a variety of human cell lines, including many derived from Burkitt’s lymphoma. In 6 BL cell lines with either 2;8 or 8;22 translocations, abundant, anomalous chimeric transcripts of 0.8 to 1 kbp were detected, which contained a small part of the 5‘ end of PVT and the constant region of the translocated light chain gene (K for 2;8 and A for 8;22translocations). In all lines, however, normal PVT transcripts were also detected-presumably derived from the normal allele (Shtivelman et al., 1989).At present, the functional significance of these findings remains unknown, but the fact that a large number of Burkitt’s lymphomas with variant translocations have breakpoints within PVT could be of relevance to their pathogenesis.

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X. Breakpoints on Chromosomes 14, 2, and 22 and Their Functional Significance with Regard to c-myc Expression Even though our knowledge of the regulation of transcription of c-myc is still quite rudimentary, the presence of positive and negative regulatory elements for the initiation of transcription, as well as the identification of sites of transcriptional pause and block (leading to termination of mRNA elongation prior to reading through the proteincoding regions of the gene), provide powerful testimony to the tight regulation to which c-myc expression is subject. This is no more than would be expected for a gene involved in cellular proliferation, for the consequences of deregulation of such genes are likely to be profound. Because of the complexity of the interactions of the regulatory elements, however, the end result on gene expression of any single lesion within a regulatory element is difficult to deduce. For example, Hay et al. (1987) have shown that the negative regulatory element in the c-myc locus can be overcome not only by the upstream positive elements, but also, at least in experimental systems, by viral enhancers. Since the c-myc enhancer region is governed by the protein factors which bind to it, the absence of these transcriptional factors is likely to lead to the predominance of the influence of the negative element. Alternatively, translocations or exogenous (e.g., viral) influences which bring additional positive elements to bear on the gene could overcome the negative element when the latter is exerting control b y virtue of inactivation of the c-myc enhancer region. It seems highly probable, therefore, that the juxtaposition of immunoglobulin sequences to c-myc in Burkitt's lymphoma is an invariable consequence of the chromosomal translocations because it leads to the provision of additional positive regulatory elements. This is supported by the occasional occurrence of complex translocations in animal tumors in which, in spite of multiple chromosomal rearrangements, immunoglobulin sequences still remain juxtaposed to c-myc. In a plasmacytoma (ABPC45) considered cytogenetically negative for a translocation, for example, a multiple-step process resulted in the heavy chain enhancer coming to lie some 2.5 kb 5' of c-myc from which it is separated by a small piece of the 5' S, region and part of the S, region in the same transcriptional orientation as c-myc (Fahrlander et al., 198513). The immunoglobulin enhancer presumably substitutes, in this rearrangement, for normal c-myc enhancer elements lost as a result of the chromosomal translocation-the breakpoint on chromosome 15 lies only 365 bp 5' of c-myc. A second plasmacytoma, ABPC17, also translocation negative, in which a 2.3-kb region bearing the immunoglobulin

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heavy chain enhancer is inserted in the same orientation as c-myc in the immediate 5‘ flanking region of c-myc, has also been described (Corcoran et al., 1985). Similarly, in a rat immunocytoma which had undergone multiple chromosomal rearrangements, c-myc and S, sequences were juxtaposed, the chromosome 7 breakpoint being 850 bp upstream of c-myc. S, and c-myc sequences were orientated head to head, but in addition, sequences from the S,1 region were placed distal to the S, sequences in a tail-to-tail orientation, necessitating an inversion (Pear et al., 1986). The alternative view to the positive regulation hypothesis is that chromosomal breaks within the immunoglobulin regions are simply more likely in B cells because these regions are particularly fragile. Deregulation of c-myc in this case is presumed to be caused exclusively by the structural changes which occur in the gene, perhaps in some cases aided by the biochemical changes brought about by the presence of EBV. Such a view seems eminently reasonable when translocations bring the immunoglobulin enhancers (either E H or E,) to lie on the same chromosome as c-myc, but separated by a considerable distance such that there is doubt as to whether the sphere of influence of the enhancer could encompass the c-myc promoters. However, if the immunoglobulin region plays no direct role in the functional disturbances which lead to neoplasia, it is difficult to conceive that these regions would be invariably involved in the translocations. The tendency to assume that enhancers have relatively limited spheres of influence is also changing as more information becomes available. Such distances, in any event, should not be calculated simply in terms of the length of the intervening DNA strand, since the tertiary structure of the nucleoprotein could bring regions apparently far removed on a linear scale into close proximity. Moreover, since truncated c-myc genes-or even normal c-myc genes-are incapable of inducing tumors in transgenic mice (Adams et al., 1985; Cory, 1986) and are not expressed in Burkitt’s lymphoma/lymphoblastoid cell line hybrids (Croce et al., 1984; Feo et al., 1986), there seems little doubt that the structural damage to c-myc is an insufficient explanation per se for its pathological deregulation and that juxtaposition (i.e., relocation to the same chromosome as c-myc) of some part of the immunoglobulin locus (not necessarily the recognized enhancers) is essential to tumorigenesis. As is clear from a consideration of the variability of the breakpoint on chromosome 8, several different mechanisms may lead to the deregulation of c-myc in Burkitt’s lymphoma. If we accept the notion that immunoglobulin juxtaposition is essential to oncogenesis, it would

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seem very likely that the breakpoint on the immunoglobulin chainbearing chromosome would correlate with the breakpoint on chromosome 8. The invariable association of breakpoints 5' or within c-myc with breakpoints on chromosome 14, and breakpoints 3' of c-myc with breakpoints on chromosomes 2 or 22, provides the beginnings of just such a correlation. Preliminary data obtained from Southern blotting have extended this observation to a molecular level within the 8;14 translocation group. In general, breakpoints far 5' of c-myc are associated with breakpoints upstream of the S, region of chromosome 14 (i.e,, in the genetic components of the immunoglobulin variabIe region) while breakpoints within or immediately 5' of c-myc are associated predominantly with breakpoints within the S, region (Neri et al., 1988).This suggests that the position of the breakpoint on chromosome 8 determines the kind of influence required from the immunoglobulin region for pathological deregulation of c-myc. The results of ongoing analyses of breakpoints on chromosomes 8 and 14 are likely to be more revealing in this regard.

A. CHROMOSOME 14 BREAKPOINTS Even though there remains much to be learned of the regulatory input from the immunoglobulin gene-bearing chromosomes, some general considerations are instructive.

1. Breakpoints Upstream of the Immunoglobulin Enhancer The heavy chain enhancer region (EH)situated between J and S, (Queen and Baltimore, 1983)is an obvious candidate for a pathological role in Burkitt's lymphoma with 8;14 translocations, although in some tumors a breakpoint downstream of EH (generally in the switch region) results in its translocation to chromosome 8, where it could only influence the remaining portion of c-myc (if any), and not the translocated protein-coding portion of c-myc which is now situated on chromosome 14. However, EHlies on the same chromosome as c-myc in all tumors in which the chromosome 14 breakpoint is upstream of Eft, i.e.,when the breakpoint is in either the J, D, or V regions (Fig. 2). A number of experiments using plasmid constructs or transgenic mice have clearly demonstrated that EH is capable of driving the transcription of the c-myc gene. The probable role of EH in the pathogenesis of at least some tumors bearing c-myc/immunoglobulin translocations is strongly supported by the fact that some, at least, of the EH-c-myc fusion genes used were actually derived from tumors, such as the EH-myc construct used by Adams et al. (1985).By inference, this almost certainly applies

I11

I

I1

I

c

J

O

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111

c

6

O

t

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8

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8

C

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I/-=

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I FIG.2. Diagrammatic depiction of the range of structural consequences of an 8;14 translocation. c-myc exons are indicated by the roman numerals I, 11, and 111. The p joining, switch, and constant regions are indicated as J, S, and C, respectively. (A) Chromosome 14 breakpoints within J; (B) chromosome 14 breakpoints within S.

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to Burkitt’s lymphoma, and in one Burkitt’s lymphoma cell line (Manca), the influence of EH on c-myc expression has been clearly demonstrated (Hayday et al., 1984). While it is debatable as to whether EH can influence c-myc expression in far 5’ breakpoints in which c-myc may lie more than 50 kb upstream of EH, this possibility should certainly not be arbitrarily excluded. It has been shown, for example, that retroviral long terminal repeat regions, which contain enhancers, can influence the expression of oncogenes as much as 100 kb distant. Moreover, the very large size of the T, locus suggests that any enhancers present in the J-C intron of T, must be effective over a distance of up to 40 kb. Whether or not EHis essential to the expression of c-myc in tumors with far 5’ breakpoints may only be definitively answered by devising a method of abrogating its function, e.g., by introducing DNA which competes for the proteins which bind to EH,or inducing site-specific mutations in the cell-still a tall order.

2. Breakpoints Downstream of E H In tumors with a breakpoint in the S , region, E H cannot influence c-myc expression (Fig. 2). In such tumors the location of the relevant immunoglobulin positive element has not been identified in Burkitt’s lymphomas. Recently, however, a novel mechanism whereby c-myc expression can be influenced was described in mouse plasmacytomas (Julius et al., 1988). The most common chromosomal translocations in these tumors, which are cognate with those of Burkitt’s lymphoma, are 12;15 translocations in which the breakpoint sites on chromosome 15 are in the first exon or intron of c-myc, while on chromosome 12 the breakpoint is nearly always in a switch region. Thus, in mouse plasmacytomas EH is usually translocated to chromosome 15 and therefore unable to participate in the cis regulation of c-myc. Julius and colleagues (1988) observed that most c-myc transcripts in such tumors are between 1.8 and 2.0 kb in size, consistent with an origin in the first c-myc intron, but some transcripts of up to 2.8 kb are observed. In the BALB/c plasmacytoma, MPCl1, transcripts larger than the normal c-myc message are observed even though this tumor has a breakpoint within the first c-myc exon. cDNA cloning and sequencing of c-myc messenger RNA (mRNA) from MPC demonstrated that these large transcripts, which can account for up to 50% of the steady state c-myc message, initiate in the antisense strand of the heavy chain, predominantly in the switch region (S,za in MPC ll),but infrequently in the first intron of the Cyearegion. Such transcripts read through (5‘ to 3’) the translocated c-myc gene, giving rise to hybrid or chimeric mRNAs containing switch and c-myc sequences. Both c-myc proteins (65Kand

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68K) were detected in this cell line, indicating that initiation of translation in the c-myc first exon CUG codon, which gives rise to a 68K protein, the homolog of the human 67K c-myc protein, occurred in those transcripts which contained the 3’ end of the first c-myc exon. A predicted chimeric protein (67K) initiating in an in-frame AUG codon in the first intron of the Cyearegion was not detected, but such proteins, which would have a different amino terminal, might be present in low quantity or in higher amounts in other tumors. S1 protection experiments performed on RNA from another plasmacytoma (J558L) with a breakpoint in c-myc exon 1 also indicated that some of the transcripts in this tumor must originate within the immunoglobulin region (Julius et d.,1988). Thus the finding in M P C l l is not unique. Moreover, an octanucleotide sequence (ATTTGCAT) which has been described in association with immunoglobulin V genes was observed upstream of the 5‘ terminus of one of the cDNA clones derived from MPC11. This octamer is believed to have enhancer activity as well as being able to function as a promoter, and in MPCll it was situated immediately adjacent to a sequence, ACCTGGG, which is also found immediately adjacent to the E H octamer and which is known to bind nuclear proteins. These findings strongly support the probability that other enhancer/promoter sequences are present in the immunoglobulin heavy chain locus, and that these regions play an important role in the transcription of c-myc in tumors with c-myclimmunoglobulin translocations, either as positive elements for initiating transcription from c-myc itself, or as promoters for transcripts originating in immunoglobulin sequences adjacent to the breakpoint in tumors in which c-myc is translocated into the immunoglobulin switch region.

B. CHROMOSOME 2 AND 22 BREAKPOINTS Switch regions are not present in the genes coding for immunoglobulin light chains, and most breakpoints on chromosomes 2 and 22 lie within the intron between the J and light chain constant regions, or within the J region itself. However, too few tumors have been analyzed for any consistent pattern to have emerged. The rather exceptional BL37 line, in which the breakpoint on chromosome 8 is only some 7 kb from the c-myc promoters [about 400 b 3’ of the poly(A) addition site of c-myc], has a breakpoint on chromosome 22 approximately 5 kb 5‘ of the JA1region on chromosome 22. In KK124, the chromosome 22 breakpoint was shown to be approximately 5 kb 5’ of the variable region of a nonfunctionally rearranged but intact A gene. These two tumors are, however, unusual, for whereas in both of them myc/A

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fusion genes were created, in most other tumors bearing 8;22 translocations that have been analyzed the breakpoints are too far downstream of c-myc for this to have occurred (Taub et al., 1984; Rabbitts et al., 1984; Szajnert et al., 1987; Henglein et al., 1988).Whether or not A region enhancer sequences are relevant to the deregulation of c-myc is a matter for conjecture, which must be based more upon extrapolation from breakpoints on other immunoglobulin gene-bearing chromosomes than on data derived from analysis of the A gene or the 8;22 breakpoints. Unfortunately, there is no translocation in murine plasmacytomas homologous to the 8;22 translocation in Burkitt's lymphoma. Several 2;8 translocations have been studied at the molecular level. Breakpoints have been identified immediately 5' of the J, region in the JBL2 and BL64 cell lines (Taub et al., 1984; Hameister and Adolph, 1986), and further upstream of C, in several other lines, including J1, BL21, LY66, and LY91. In J1, the breakpoint may be within an aberrantly rearranged V, gene (Erikson et al., 198313; Emmanuel et al., 1984; Malcolm et al., 1985; Hameister and Adolph, 1986) and in BL21 and LY66 the breakpoint lies somewhere between J, and C, since the J, region is translocated to chromosome 8 (Rappold et al., 1984). In LY91 a major part of the JKregion remains on chromosome 2 (Rappold et al., 1984). In most of these lines the K chain enhancer, situated between the J and C, regions, is translocated to chromosome 8, but the distance from c-myc varies. In J1 the K chain enhancer element is close enough to c-myc to provide a positive transcriptional stimulus (Henglein et al., 1988), but in the other cell lines reported so far with 2;8 translocations c-myc is approximately 300 kb upstream of this region, a distance which would appear to be too great for the influence of the enhancer to overcome. As with the 8;22 translocations, whether or not the light chain gene has a functional role in deregulation of c-myc in addition to a role in mediating the translocation remains speculative. However, even if the recognized enhancer cannot be utilized, other enhancer elements within the immunoglobulin gene may be. The light chain gene may maintain an open chromatin structure and could, at the same time, be responsible for the generation of mutations within c-myc. The distance over which mutagenesis is active, in this context, is quite unknown. XI. Correlation of Breakpoint Location with Geography

It is clear from the above discussion that the mechanism whereby the c-myc gene is deregulated, which is primarily a consequence of the locations of the breakpoints on the chromosomes participating in the

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THE PATHOGENESIS OF BURKITT’S LYMPHOMA

translocations, may differ greatly in different tumors. The breakpoint locations do not vary in a random fashion, however, but correlate with the geographic origin of the tumor. Thus, the most common chromosome 8 breakpoint location in endemic Burkitt’s lymphoma is far 5’ of the c-myc gene. In contrast, the most common chromosome 8 breakpoint location in sporadic Burkitt’s lymphoma is within the first c-myc intron (Pelicci et al., 1986b; Barriga et al., 1988a). As shown in Fig. 3, there is overlap between endemic and sporadic tumors with regard to the chromosome 8 breakpoint locations, but to date we have not observed an endemic tumor with an intron breakpoint, while far 5’

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SPORADIC

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FIG.3. Illustration of the differences between endemic and sporadic Burkitt’s lymphoma in breakpoint locations on chromosome 8. The five breakpoint regions in and around c-myc are depicted on the cartoon (lower part of figure) of the first and second exons of the c-myc gene (left and right solid rectangles, respectively). The number of tumors with a breakpoint in each region is shown in the histogram. (From Barriga eta!., 1988.)

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breakpoints in sporadic tumors are very uncommon. Another way of viewing these data is that a quite sharp watershed exists at the first exon border with regard to endemic and sporadic tumors. In endemic tumors, in which breakpoints are nearly always 5’ of Pi (21 of 24), and in most cases Po as well, the gene is transcribed from its own promoters. In contrast, in the majority of sporadic tumors, in which breakpoints are generally within the first exon or intron (18 of 27), the transcription start site is at a cryptic promoter within the first intron. While preliminary data suggest that endemic tumors are also associated with chromosomal breakpoints upstream of the switch region on chromosome 14 and sporadic tumors with switch region breakpoints (Neri et al., 1988) (Fig. 4) more tumors need to be examined before the validity of this apparent association can be said to be definitive. The tumors occurring in patients with HIV infection appear to be similar to sporadic tumors with regard to the distribution of breakpoint locations (Pelicci et al., 1986a; Barriga et al., 1988a). Thus, hypotheses that Burkitt’s lymphoma in patients with HIV infection is likely to resemble the endemic form of the disease with regard to breakpoint locations (Haluska et al., 1988a) are not supported. The differences in chromosomal breakpoint locations in sporadic versus endemic Burkitt’s lymphoma correlate with the described differences in EBV association and clinical features, and suggest strongly that the etiology, and probably the cell of origin of these two forms of the disease, differ. Moreover, these data rejuvenate the possibility that EBV may have a direct role in tumorigenesis, for they indicate quite clearly that Burkitt’s lymphoma, as defined histologically, is not homogeneous. Thus, even though EBV cannot be of pathogenetic importance to Burkitt’s lymphoma in general, it may well be an essential factor in a subset of tumors. These issues are discussed further below. At the present time, much remains to be learned of the pattern of breakpoint distribution in Burkitt’s lymphoma in various poorly studied world regions. In North Africa there is a high rate of EBV association, but a largely sporadic pattern with regard to clinical characteristics (Ladjadj et al., 1984). In South America there appears to be a high incidence ofjaw tumors occurring in the tropical zones of the North. In Brazil, for example, the few cases reported from the equatorial region appear to conform to an African pattern (Chaves, 1977; Dalldorf et al., 1969), whereas in southern South America, i.e., in the temperate region, the tumor appears to clinically resemble the sporadic form of the disease (Cebrian-Bonesana et al., 1978; Drut et al., 1982). Clearly, it would be important to determine whether or not these tumors are EBV positive and to carry out molecular analyses of chromosomal breakpoint locations. In a small number ofArgentinian tumors, three of

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v)

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FIG.4. Illustration of the differences between endemic and sporadic Burkitt's lymphoma and Burkitt's lymphoma associated with AIDS in breakpoint locations on chromosome 14.These are divided into those 5' of the HindIII site (see cartoon) between the S,and J regions (many of which will be in the J region) and those 3' of this HindIII site (most of which will be in the switch region). (From Neri et al., 1988.)

four had rearranged c-myc genes (Guttierez et d.,1990), supporting the probability that in temperate South America the tumor conforms to the sporadic type. XII. Effect of the Chromosomal Translocations on c-myc Expression

A consideration of the structural changes in c-myc provides strong, though indirect evidence that a major element in the pathogenesis of Burkitt's lymphoma is the deregulation of c-myc. This possibility can

0

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IAN MAGRATH

only be seriously considered if the c-myc allele involved in the translocation is the expressed allele in Burkitt’s lymphoma. Evidence for this comes from two sources. First, in tumor cell lines in which there is a truncated c-myc gene, i.e., one in which the breakpoint on chromosome 8 is within the first intron, first exon sequences cannot be detected by Northern blot in the messenger RNA although c-myc transcripts are readily detected by a third exon probe (Taub et al., 1984; Magrath et al., 1988). Second, somatic cell hybrid experiments have been performed in which Burkitt’s lymphoma cell lines were fused to mouse plasmacytoma cells and clones which contained either the derivative chromosomes or their normal alleles were isolated (Nishikura et al., 1983). In the case of hybrids in which one of the parents was a cell line bearing an 8;14 translocation four types of clone, each containing only one of the chromosomes 8, 14, derivative 8, or derivative 14, were isolated, Human c-myc expression was detectable only in clones bearing the derivative chromosome 14, and not in those containing a normal chromosome 8, while immunoglobulin expression was detected only in those clones possessing a normal chromosome 14. There are rare exceptions to the latter (Versnel et al., 1986). Similar experiments were performed with cell lines bearing 2;8 and 8;22 translocations. In each case, c-myc expression was detected only in clones carrying the derivative chromosome 8, and not in clones carrying the normal chromosome 8 (Erikson et al., 1983b; Croce et al., 1983).Thus, with rare exceptions, in Burkitt’s lymphoma the normal c-myc allele is not expressed. The significance of this observation is further discussed below. The juxtaposition, as a result of the translocations, of c-myc to immunoglobulin genes, coupled to the findings in mouse plasmacytomas described above, strongly supports the notion that c-myc is regulated in Burkitt’s lymphoma as if it were an immunoglobulin gene. However, examination of the steady state level of c-myc mRNA in Burkitt’s lymphoma cell lines does not reveal a marked elevation compared to other (inuitro) proliferating B cells (Maguire et al., 1983). In fact the levels overlap with those of EBV-transformed B lymphoblastoid cell lines and many other malignant neoplasms which have no intrinsic defect, as far as is known, in the c-myc gene. Thus, simple measurement of the steady state level of c-myc RNA in Burkitt’s lymphoma cells, as in mouse plasmacytoma (Keath et al., 1984b),provides no explanation for the neoplastic behavior, and would not lead to the conclusion that the genetic lesion of Burkitt’s lymphoma involves the c-rnyc gene. Further, sequence analysis of c-myc genes from Burkitt’s lymphoma indicates that in the majority of tumors the protein-coding

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sequences of c-myc are entirely normal (Battey et al., 1983; Stanton et al., 1984a,b), excluding a functional abnormality of the c-myc protein as relevant to pathogenesis (there are occasional exceptions to this, as in the cell lines Raji, CA46, and LY65) (Rabbitts et al., 1983b; Showe et al., 1985; Murphy et al., 1986). This seeming paradox is really no paradox at all. The level of c-myc expression in Burkitt’s lymphoma is clearly sufficient to provide a powerful drive to proliferation. But whereas the proliferation of antigen-stimulated or EBV-infected B cells is regulated by exogenous factors, this is not the case in Burkitt’s lymphoma. In other words, the basic pathological lesion must reside in the inability of the cell to shut off cellular proliferation and while other theoretical possibilities may be considered possible, the structural changes described above strongly support the probability that the defect lies in the gene itself. Either it cannot respond to exogenous factors which normally lead to its down regulation, or, as may be the case for cells in the antigen-independent differentiation pathway, a “ preprogrammed” or internal system for inhibiting c-myc expression is ineffective. Either of these two possibilities is consistent with the absence of expression of the normal c-myc allele in Burkitt’s lymphoma.

A. EVIDENCE THAT c-myc Is CONSTITUTIVELY EXPRESSED IN BURKITT’S LYMPHOMA At a cellular level, the demonstration of constitutive expression of a gene requires that it continue to be expressed under circumstances in which a normal gene would be down regulated. Since the normal counterpart cell of Burkitt’s lymphoma has not been definitively identified, such circumstances are not easy to contrive. One approach has been to construct somatic cell hybrids with Burkitt’s lymphoma cells in which the partner cell would not normally permit expression of c-myc. In hybrids between a normal human lymphoblastoid cell line (GM1056) and murine plasmacytoma cells (NP3), the human c-myc gene is not expressed in clones which retain the normal human chromosome 8 (Nishikura et al., 1983). Similarly, hybrids between HL60 (a promyelocytic leukemia cell line) and another murine plasmacytoma cell line, P3Bu4, failed to express human c-myc, even when the amplified c-myc gene carried by HL60 was present in the hybrid cell clone (Nishikura et al., 1983). The failure of the plasma cell internal milieu to support expression of the normal human c-myc gene is consistent with the finding that c-myc is down regulated (usually biphasically) in a number of in uitro differentiation systems, including HL60 cells, and

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Friend erythroleukemia (F-MEL) cells, and also that in the latter system transfection with a constitutively expressed c-myc gene (controlled by the SV40 early promoter) prevents differentiation (Coppola and Cole, 1986; Prochownik and Kukowska, 1986; Dmitrovsky et al., 1986). In contrast to the results with GM1056/NP3 hybrids, human c-myc was expressed in hybrids between several Burkitt’s lymphoma cell lines (P3HR1, JD38, ST486, Daudi, and J1) and plasmacytoma cells which retained the chromosome bearing the c-myc gene involved in the translocation (14q+ for P3HR1, JD38, and Daudi, 8q+ for J1) (Nishikura et aZ., 1983; Croce et al., 1983, 1984, 1985; Erikson et al., 1983b). An additional hybrid between P3HRl and the murine fibroblast cell line LM-TK- which retained the 14q+ chromosome expressed very low levels of human c-myc and also failed to support expression of human immunoglobulin genes (Nishikura et al., 1983). These results indicate that the c-myc gene of Burkitt’s lymphoma is expressed in conditions which lead to suppression of the normal c-myc gene. Additional and perhaps more direct confirmation of the constitutive expression of the c-myc gene of Burkitt’s lymphoma has been obtained by the demonstration that the induction of differentiation (confirmed b y morphology and the demonstration of an increase in IgM expression) in several Burkitt’s lymphoma cell lines did not result in down regulation of c-myc (Benjamin et al., 1984; Sandlund et al., 1990). In fact, in some cases expression of c-myc may even have been increased. Such a result would be expected if c-myc expression is coupled to that of immunoglobulin genes. Confirmation that down regulation of the normal c-myc gene would have been expected under these circumstances was provided by examining the expression of the isolated first exon of c-myc in cell lines in which the gene is broken, i.e., there is a first intron breakpoint, in which case the first exon is often expressed as an approximately 900-bp transcript. In such lines the first exon and its associated regulatory sequences remain intact on chromosome 8 and differentiation-inducing agents caused down regulation of first exon transcripts.

B. STABILITY OF c-myc mRNA IN BURKITT’SLYMPHOMA It has been demonstrated that c-myc RNA has a short half-life in Burkitt’s lymphoma-in the region of 10-15 min when poly(A) RNA levels are measured after actinomycin D transcriptional blockade, and closer to 1 hr when total RNA is measured (Nishikura and Murray, 1988). In cell lines in which the breakpoint lies within the first c-myc

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exon or intron, c-myc transcripts are abnormal and could therefore be processed differently in the nucleus or cytoplasm. Indeed, when transcripts are initiated in the intron, the absence of the 5’consensus splice sequence results in failure to splice out the remains of the first intron, so that the mature c-myc mRNA in this circumstance not only lacks a first exon, but also contains intron sequences (ar-Rushdi et al., 1983). Similar observations have been made in mouse plasmacytomas (Prehn et al., 1984; Keath et al., 1984b; Calabi and Neuberger, 1985; Cory, 1986). It has been shown that the half-life of such messages is prolonged by a factor of approximately twofold (from a half life of 16 to 30 min in plasmacytomas and Burkitt’s lymphomas with an intact c-myc gene to a half life of an hour or more in plasmacytomas and Burkitt’s lymphomas with truncated genes), leading to similar fold increases in steady state levels (Rabbitts et al., 1985a; Piechaczyk et al., 1985; Eick et al., 1985). This is believed to be due to the presence of the intron sequences in the abnormal message, rather than the absence of first exon sequences (Bonnieu et al., 1988), but cellular factors may also be important, as illustrated by the recent isolation of a cytosolic factor from erythroleukemia cells which accelerates c-myc RNA degradation (Brewer and ROSS,1989). It seems unlikely that the alteration in message stability is of major importance to pathogenesis, since increased stability gives rise to a higher level of message but steady state levels in Burkitt’s lymphoma do not appear to be generally increased compared to EBV-transformed lymphoblastoid cell lines. Even in plasmacytomas, stability of c-myc mRNA in tumors with intact genes is not increased, so that at best, increased levels in tumors with truncated genes might be associated with differences in behavior, such as an increased rate of cell proliferation, rather than being an essential element in cell transformation.

C. DIFFERENTIAL USAGEOF THE c-myc PROMOTERS IN BURKITT’S LYMPHOMA An abnormality of transcription in Burkitt’s lymphoma that remains unexplained is the difference in promoter utilization compared to normal proliferating cells. In the latter, some three to five times as many transcripts are initiated at P2 than at PI (Leder et al., 1983). In Burkitt’s lymphomas in which the breakpoint lies 5’ of PI, there are roughly equal levels of transcripts arising from P1 and P2 (except in those cell lines in which a breakpoint or mutations in the 5’ regulatory region results in suppression of transcription from one or other promoter) (Leder et d.,1983; Barriga et d.,1988a; Nishikura and Murray,

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1988).The functional significance of this finding remains unknown. In particular, the 64K and 67K proteins are both transcribed from the same transcripts, and do not appear to relate to the PI and P2 mRNA species (Hann et al., 1988). In Burkitt’s lymphoma the 67K protein, when detectable, is present in much lower quantities than the 64K protein in spite of the approximate equality of P1 and P2 transcripts. There is no evidence that the rates of degradation of P1 and P2 transcripts differ (Dani et al., 1984). Even if they did, since the level of steady state c-myc mRNA is not markedly elevated in Burkitt’s lymphoma, it is unlikely that a difference in the ratio of P1:P2 transcripts is of mechanistic importance to pathogenesis. It seems probable that the altered P1:P2 ratio, rather than being of functional significance in its own right, is a reflection of the altered regulation of c-myc-i.e., that the regulatory region of the gene involved in the translocation is abnormal, and that at least part of the drive to transcription is provided by immunoglobulin enhancers. Some of the c-myc enhancers themselves have a differential effect on P1 versus PZ(e.g., the positive regulatory element for P1 between position -293 and - 101) (Hay et al., 1987). That disruption of the regulatory region can influence P1:P2 ratios is supported by the finding that in murine plasmacytomas there is marked reduction in P2 usage in tumors with breakpoints within 350500 bp of c-myc. Alterations in P1:P2 ratios of this magnitude are not seen in plasmacytomas with other breakpoint locations on chromosome 15. This may reflect the loss of an upstream, cis-acting, positive element and its replacement by immunoglobulin sequences. Also supporting the possibility that the altered P1:P2 ratio occurs as a result of the influence of an immunoglobulin enhancer element is the finding of an abnormal P1:P2 ratio in isolated c-myc first exon transcripts from the Burkitt’s lymphoma cell lines ST486 and JD38, which have breakpoints in the first intron (Nishikura et al., 1985). Such transcripts originate from the normal first exon with an intact regulatory region remaining on chromosome 8, which is closely approximated to the translocated heavy chain enhancer. While both EBV positive and negative cell lines have altered P1:P2 ratios, the recent demonstration that transcription from P2 can be increased by the adenovirus protein E la, which probably acts via a nuclear factor, E2F, known to bind to a DNA element within P2 (Lipp et al., 1989; Thalmeier et al., 1989) suggests an alternative, or contributing explanation to altered PI:P2 ratios in EBV containing cells-namely, modulation by viral genes. In such cells the immunoglobulin enhancer element is more distant from c-myc, since the breakpoint on chromosome 8 is usually far upstream. The recent description of an additional promoter, Po, more than

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500 bp upstream of PI and associated with a DNase I-hypersensitive site, provides an additional potential difference between Burkitt’s lymphoma and normal cells with regard to c-myc products. Po transcripts of 3.1 kb have been observed in most cell types, but represent less than 5% of normal c-myc mRNA (Bentley and Groudine, 1986a). An approximately 12.5K protein is predicted on the basis of the presence of an open reading frame, but this putative protein has no homology with any known protein. Interestingly, the 5’ end of Po lies approximately 50 bases downstream of a sequence, seven bases of which match the 8-base consensus enhancer core sequence GTGGAAAG. It also overlaps with one ofthe N F 1 binding sites (Bentley and Groudine, 1986a). Transcripts with similar 5’ heterogeneity have been described arising in proximity to the immunoglobulin heavy chain enhancer. Clearly, Burkitt’s lymphoma with breakpoints 3’ of the Po site would not be able to transcribe the 3.1-kb mRNA, but the significance of this remains unknown.

D. THEc-myc PROTEIN IN BURKITT’S LYMPHOMA While a small number of Burkitt’s lymphomas have been described in which there are mutations in the protein-coding region of c-myc (Rabbitts et al., 198313,1984;Murphy et al., 1986)the majority of c-myc genes that have been sequenced in Burkitt’s lymphoma have completely normal second and third exons. Moreover, the half-life ofc-myc protein does not differ in Burkitt’s lymphoma from that in cells without a translocation. However, whereas normal cells synthesize both a 64K and a 67K c-myc protein, the latter initiated from the 3’ end of the first exon, many Burkitt’s lymphomas fail to synthesize the 67K protein. Thus, 5 of 10 Burkitt’s lymphoma cell lines with a breakpoint outside the first exon, but containing exon 1mutations, failed to synthesize the 67K protein (Hann et al., 1988). All cell lines with a break in the first intron are incapable of synthesizing the 67K protein. Unfortunately, until the function of the c-myc protein has been determined, and the functional relevance of the difference in the amino terminal of the 64K versus the 67K proteins identified, the significance of this observation will remain unknown.

E. ABSENCEOF EXPRESSION OF THE NORMALc-myc GENEIN BURKITT’S LYMPHOMA It is likely that the lack of expression of the normal c-myc allele in Burkitt’s lymphoma provides a clue to the nature of the cell type in which the translocations occur. While the expression of the c-myc

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allele involved in the translocation fulfills a necessary criterion for the chromosomal translocations to be of significance, there is no discernable a priori reason for the silence of the normal allele. It has been postulated that there is a negative feedback mechanism involved in the control of c-myc, and that the constitutive expression of one gene therefore results in the negative regulation of the other. Such reasoning presupposes that c-myc would be expressed in the normal counterpart cell of Burkitt’s lymphoma-i.e., that this cell type is in a proliferating phase. In such a circumstance, one might seriously wonder what the relevance of deregulated c-myc expression is, particularly in the absence of excessively high c-myc levels. An alternative and more appealing view is that the normal counterpart cell is a nonproliferating cell, i.e., one which does not express c-myc. In such a cell, expression of c-myc would lead to proliferation, and probably prevention of differentiation beyond a point incompatible with proliferation. Three experimental situations provide the opportunity to examine the effect of a constitutively expressed myc gene on the endogenous c-myc gene(s). These are (a) transfection of a c-myc construct into proliferating cell lines, (b) the construction of somatic cell hybrids in which one of the parent lines contains a deregulated c-myc gene, and (c) the examination of c-myc expression in mice transgenic for c-myc. The results have been somewhat conflicting. Evidence generally favoring the existence of a negative regulatory mechanism has been provided by the transfection experiments. In human lymphoblastoid cell lines, avian and rodent fibroblasts, and hemopoietic cells, c-myc constructs controlled by an SV40 promoter or immunoglobulin heavy chain enhancer (in the case of the human cells) or a murine retroviral LTR (in the case of the rodent and avian fibroblasts) did indeed cause inhibition of the normal c-myc allele (Lombardi et al., 1987; Keath et al., 1984a; Rapp et al., 1985).Further, inhibition mediated by high levels of v-myc in mouse 3T3 cells required participation of the v-myc protein, since inhibition was abolished by frameshift mutations and deletions in v-myc (Cleveland et al., 1988). In the lymphoblastoid cell lines there appeared to be a correlation between the level of expression of the transfected, exogenous c-myc gene and the endogenous gene. The latter was completely inhibited or minimally expressed in lines transfected with truncated genes coupled to the (strong) SV40 promoter, but only slightly inhibited in cell lines transfected with a truncated c-myc gene coupled to the immunoglobulin heavy chain enhancer (Lombadi et al., 1987). The latter c-myc construct was poorly expressed. The results of somatic cell hybrid experiments have been less consistent. In cell hybrids between normal mouse spleen cells and Bur-

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kitt’s lymphoma cells (SK-DHL2B or Manca), the normal murine c-myc gene is suppressed (Feo et al., 1985). However, in hybrids between several different Burkitt’s lymphoma cell lines and a human EBV-transformed lymphoblastoid cell line the normal c-myc gene continues to be expressed. In four different hybrid clones in which the Burkitt’s lymphoma parental cell was Daudi the translocated c-myc gene was expressed at variable levels while a normal c-myc gene (presumed but not demonstrated to be from the lymphoblastoid parent) was expressed at similar levels in each of the hybrids, although this level was somewhat lower than in the lymphoblastoid parent (Croce et al., 1985).As discussed further below, in several other hybrid clones constructed with the Burkitt’s lymphoma cell lines Raji, CA46, and ST486, in which the 14q+ chromosome was demonstrated to be present, all the c-myc transcripts were of normal origin. In these clones the deregulated gene provided by the Burkitt’s lymphoma cell line was suppressed (Croce et al., 1984; Nishikura et al., 1985). These data argue against the possibility that there is a negative feedback mechanism which acts on c-myc expression in proliferating cells. In mice transgenic for a c-myc gene coupled to an immunoglobulin heavy chain enhancer, pre-B and B cell lines derived from the lymphoid tumors which develop in such mice express only the transgenic and not the normal c-myc gene (Adams et al., 1985). This is similar to the situation in Burkitt’s lymphoma, but does not necessarily indicate that the normal allele is switched off because of a negative feedback loop. It is probable that these cells would be resting cells in the absence of the deregulated c-myc transgene; in such cells the mechanism of transcriptional inhibition probably differs in that it involves changes in chromatin structure (see below). The issue of a negative feedback loop being involved in the regulation of c-myc expression must also be reconciled with the recent observation that the c-myc protein has been shown to bind to a region of DNA some 2 kbp upstream of the c-myc gene itself and appears to have a positive effect on c-myc transcription, at least in HL60 cells (IguchiAriga et al., 1988). It is possible that the effect of the c-myc protein (whether via a direct or indirect mechanism) varies according to whether the level of c-myc in the cell is high or low. The most efficient means of regulating the rate of c-myc transcription, and hence the intracellular level, would be for the c-myc protein to have an inhibitory effect when present at high levels, but a stimulatory effect when present at low levels. If there were more than one regulatory region to which the c-myc protein binds, each having a different affinity for the protein and a different effect on transcription, the intracellular level of

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c-myc could be kept within a relatively narrow range. A mechanism of this kind has been proposed for the EBV regulatory protein EBNA 1 (see below). Even if we accept the involvement of a negative feedback loop in which the c-myc protein participates in the control of c-myc transcription, there is no reason to assume that this is the mechanism whereby the normal allele in Burkitt’s lymphoma is suppressed. Nishikura and Murray (1988) have shown that in the cell lines Daudi, PSHR1, and Raji, inhibition of protein synthesis by means of cycloheximide does not result in the reexpression of the silent normal allele (the two alleles in these lines can be distinguished by S1 mapping) as would be expected if the latter were suppressed by a protein mediator. These findings are consistent with observations in the Burkitt’s lymphoma cell line, Manca, in which nuclear run-off experiments (a means of examining nascent nuclear transcripts) have shown that the normal allele is, as expected, transcriptionally silent. Similarly, during differentiation of HL60 cells with DMSO, c-myc is down regulated at first via increased blockage to elongation of nascent c-myc messages, but subsequently via altered chromatin structure. Prior to, but not after the alteration in chromatin structure had occurred, down regulation of c-myc was rapidly reversible by withdrawal of DMSO (Siebenlist et al., 1988).In several Burkitt’s lymphoma cell lines as well as in mouse plasmacytoma, the location of DNase I-hypersensitive sites (Siebenlist et al., 1984; Dyson and Rabbitts, 1985; Fahrlander et al., 1985a) and the pattern of methylation (Dunnick et al., 1984) differ in the translocated and untranslocated c-myc genes. Taken together, these observations suggest that the lack of transcription of the normal allele in Burkitt’s lymphoma is not d u e to negative feedback inhibition, but rather that the normal counterpart cells do not express c-myc because of altered chromatin structure and DNA methylation which prevents access of both positive and negative regulatory proteins to the DNA. In proliferative cells, such as lymphoblastoid cell lines, positive and negative regulation mediated by protein factors is likely to be necessary to keep the level of c-myc within acceptable limits. Such cells have a pattern of DNase I-hypersensitive sites identical to that of the transcribed c-myc gene in Burkitt’s lymphoma (Dyson and Rabbitts, 1985). The altered chromatin pattern of the silent c-myc allele strongly suggests that Burkitt’s lymphoma is the malignant counterpart of a cell type in which c-myc would not normally be expressed (unlike lymphoblastoid cell lines). The likely candidate normal counterparts, based on this evidence alone, are virgin B cells or memory B cells, both of which are resting cells, rather than activated B cells expressing c-myc. Such a

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conclusion has an additional corollary. It is most unlikely that chromosomal translocations occur in resting cells. Such events are much more likely to occur during DNA and chromosomal replication. As discussed above, particularly vulnerable genes are those which are being actively transcribed or rearranged. The sequence of events which leads to the development of Burkitt’s lymphoma is likely, therefore, to be initiated in an actively dividing cell, and particularly one which is in the process of, is about to undergo, or has just undergone immunoglobulin gene rearrangement (whether VDJ joining or switch recombination) since these genes, or specific regions within them, have a more open chromatin structure. The translocation leads to an inability of the cell to switch off the transcription of one of its c-myc genes (which would normally be accomplished by altering the chromatin pattern and limiting enzyme access). In addition, other alterations in the regulatory region may further impair the ability of the cell to “fine tune” the level of c-myc, although this may be a secondary problem which has more to do with the actual rate of cell growth or degree of dependence on exogenous growth factors than the fact of being neoplastic. Since in a dividing cell c-myc should be expressed, the inappropriate expression of c-myc brought about by a translocation will only become apparent as differentiation proceeds to the point at which the cell should normally become a resting cell (Fig. 5).

F. ROLE OF IMMUNOGLOBULIN SEQUENCES IN THE EXPRESSION OF THE TRANSLOCATED c-myc GENEIN BURKITT’S LYMPHOMA The series of experiments with somatic cell hybrids mentioned above have also shed light on the regulation of the translocated c-myc gene in Burkitt’s lymphoma (Croce et al., 1984,1985; Nishikura et al., 1985; Feo et al., 1986). Croce and colleagues selected Burkitt’s lymphoma cell lines containing different breakpoint locations on chromosome 8 and 14 and fused them to either a plasmacytoma cell line or to an EBV-transformed lymphoblastoid cell line. Their results are summarized in Table 111, and suggest that the breakpoint on chromosome 14, rather than that on chromosome 8, is the determining factor which governs the expression of translocated c-myc genes in the hybrids between Burkitt’s lymphoma and lymphoblastoid cell lines. It is probably pertinent that the hybrids phenotypically resembled the lymphoblastoid cell line parent rather than the Burkitt’s lymphoma cell line parent, although immunoglobulin from the Burkitt’s component were detectable. When the Burkitt’s lymphoma parent had a chromosomal breakpoint in the switch region, the translocated c-myc gene was not

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IAN MAGHATH

A

Normal VDJ joining

Q

Translocation

resting pre-B and B cells

@

Q

@

deregulation of c-myc

Malignant clone

B

Normal activation

-

Translocation

class switching

-

-

memory cell

deregulation of c-myc

Malignant clone

FIG.5. Illustration of the probable consequence of deregulation of c-myc by a chromosomal translocation: (A) In cells undergoing primary (antigen-independent) differentiation and (B)in cells undergoing secondary (antigen-dependent differentiation). In each case, a cell type is prevented from entering a resting phase by the deregulation of c-myc consequent upon a chromosomal translocation.

expressed. When the breakpoint was upstream of switch, the translocated c-myc gene was expressed. This conclusion must be considered as tentative since only one hybrid with a breakpoint outside the switch region (Daudi) was examined. It contrasts with the situation in Burkitt’s lymphoma/plasmacytoma hybrids in which the translocated c-myc is expressed regardless of the chromosome 8 or 14 breakpoint location. Interestingly, in Daudi, the only cell line in which the lymphoblastoid environment was permissive to the expression of the translocated c-myc gene, the breakpoint location on chromosome 8 brings the immunoglobulin heavy chain enhancer onto the same chromosome as the translocated c-myc gene, but at least 30 kb away. In the

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TABLE 111 EXPRESSION OF THE TRANSLOCATED c-myc ONCOGENE IN SOMATIC CELLHYBRIDS BETWEEN B U R K I LYMPHOMA ~S AND LYMPHOBLASTOID CELLLINESOR PLASMA CELLS Burkitt’s cell line

Breakpoint location on chromosome 14

Breakpoint location on chromosome 8

Expression of Burkitt c-myc

Expression of first c-myc exon“

Hybrids with lymphoblastoid lines ST486 CA46 Raji Daudi

s, S,

s, D region

First intron First intron Flanking region Far 5’ of c-myc

Inhibited Inhibited Inhibited Expressed

Expressed b

Hybrids with plasmacytoma cells ST486 P3HRl

s, JtI

First intron Far 5’ of c-myc

Expressed Expressed

Inhibited

a Relevant in cell lines with a break in the first c-myc intron, when the first exon remains on chromosome 8 and can be expressed separately from the translocated second and third exons. * Not expressed in the parent CA46 cell line.

remaining cell lines this enhancer element is translocated to chromosome 8 and thus is on a separate chromosome from the translocated c-myc gene. It seems probable that the particular regulatory element that is providing a positive drive to the expression of c-myc in Burkitt’s lymphoma is differentiation specific. Thus, the positive elements operative in switch breakpoints may not be active, or are overridden in the hybrids with lymphoblastoid cell lines, whereas the elements operative in breakpoints upstream of switch remain effective. Significantly, the expression of genomic clones containing truncated c-myc genes derived from two different Burkitt’s lymphoma cell lines and juxtaposed to different immunoglobulin sequences differed when transfected into the plasmacytoma cell line NP3 (Feo et al., 1986). A clone derived from the cell line CA46 containing a truncated c-myc gene attached to the S , region into which it was translocated (and therefore lacking the immunoglobulin heavy chain enhancer region) was not transcribed, whereas a clone derived from the cell line SK-DHL2a (Manca) containing a similarly truncated gene attached to the immunoglobulin heavy chain enhancer was expressed, although at a lower level than in the parent cell line. This is entirely consistent with other

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evidence already presented that indicates that cis-regulatory elements are necessary for the expression of the truncated gene derived from CA46. Although this gene is expressed in a CA46/NP3 hybrid cell, in which protein factors are provided by the Burkitt’s lymphoma parent, the plasmacytoma line apparently lacks the necessary proteins to drive the c-myc construct. The NP3 line, however, can apparently provide protein factors able to bind to the immunoglobulin heavy chain enhancer and thus to drive c-myc expression from the similarly truncated gene derived from Manca. XIII. Consequences of Deregulation of c-myc

While there is ample evidence that in Burkitt’s lymphoma the abnormal c-myc gene cannot be switched off, since little is known of the functions of the c-myc protein, there are few insights into the chain of biochemical events whereby this results in continued cell proliferation. The fact that c-myc participates in the proliferation cascade, however, make it an excellent candidate gene for the induction of neoplasia when aberrantly expressed, and there is considerable evidence from experimental systems that its abnormal expression can indeed lead to neoplasia. A. MICE TRANSGENIC FOR c-myc When a cloned gene is inserted into a fertilized mouse ovum it integrates into a chromosome and is introduced during embryogenesis into all the tissues of the mouse. This experimental system provides a direct means of testing the oncogenicity of cellular genes. Transgenes may exhibit tissue specificity depending upon the regulatory elements to which they are coupled, and when governed by an inducible promoter such as that of the metallothionein gene, their expression can also be regulated by exposure of the animal to exogenous agents (Hofmann et al., 1988).c-myc transgenes have been shown to be oncogenic in several murine systems, including one in which the gene is attached to the mouse mammary tumor virus (MMTV) LTR, which is steroid responsive, and another in which it is attached to the immunoglobulin heavy chain enhancer ( E H ) , which is active in lymphoid cells. c-myc attached to MMTV induces mammary carcinomas (Stewart et al., 1984b), while EH-myc (using either human or murine c-myc genes) causes lymphoid neoplasms within a few months of birth (Adams et al., 1985; Schmidt et al., 1988). These findings clearly implicate constitutive c-myc expression as a critical factor in tumorigenesis in these

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systems, and strongly support the probability that deregulation of c-myc is a critical element in the pathogenesis of Burkitt’s lymphoma. A numbc r of additional insights into the mechanisms of tumorigenesis can be gained by a closer examination of transgenic mouse strains susceptible to lymphoid neoplasms.

1. B Cell Neoplusia in EH-mvc Transgenic Mice The ability of a nun . 2r of different c-myc constructs to induce lymphoid tumors has been explored (Adams et al., 1985). Among the primary transgenic animals the entire normal murine c-myc gene, a truncated gene (i.e., lacking exon l),and a truncated gene attached to a metallothionein promoter failed to induce tumors (tumor incidence 0/5, O/ll, and 0/23, respectively), and normal or truncated genes coupled to a viral LTR or the SV40 promoter/enhancer, respectively, were weakly oncogenic (1/13 and 3/21 tumors, respectively, two of the latter tumors being nonlymphoid). A construct derived from the plasmocytoma ABPC 17, in which 2.3 kb of immunoglobulin Db!A including EHand S, sequences have been joined to an intact c-myc gene 361 bp 5’ to exon 1 (referred to as E,-myc) induced tumors in 13/16 mice, while a construct consisting of a truncated c-myc gene coupled to an SV40 promoter in which the enhancer element was replaced by E, induced tumors in 6/17 mice. The tumor susceptibility of the E,-myc mice persisted in 3 generations of 8 independent lineages, 59/63 (94%)mice dying of lymphoid neoplasia within 4 months. These tumors could b e readily transplanted into syngeneic mice. The neoplasms were manifested as multicentric lymph node enlargement with splenomegaly, marrow and peripheral blood involvement, and thymic swelling in some mice. All tumors were of monoclonal or occasionally oligoclonal pre-B or B lymphoid origin (one tumor progressed from pre-B to B) and expressed p and K transcripts. Most tumors arising at different sites were derived from the same cell clone, but occasionally different sites had different clonal origins as assessed by J H rearrangements. Because marker DNA from 4x174 had been introduced into the 3’ end of the c-myc constructs, it could be clearly established that the high levels of c-myc transcripts in all the tumors originated from the transgene, and none from the normal c-myc gene. These transcripts were initiated either at the normal promoters of c-myc, or upstream of these, probably in the S, region as judged by hybridization to an S, probe. The latter transcripts appeared to include only exons 2 and 3 of c-myc. While many of the observations made in the transgenic mice are applicable to Burkitt’s lymphoma, parallels should be applied with

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caution. Of particular relevance is the finding that a polyclonal prelymphomatous state, consisting of marked hyperplasia of abnormally large early and late pre-B cells in the bone marrow (accounting for some 60% of marrow cells) and spleen (approximately 20% of splenocytes), exists prior to the development of clonal malignancy (Langdon et al., 1986). These pre-R ci.lls are not tumorigenic in syngeneic mice under conditions in which more than 90%of recipients of E,-myc lymphoma cells would develop tumors. In addition, there was a paucity of IgD-bearing B cells in the spleen and lymph nodes, suggesting a retardation of B cell maturation. The lymph nodes, however, did not contain pre-B cells. In fetal liver derived from E,myc mice at 18 days of gestation there was an increase in large pre-B cells, indicating that hyperplasia occurs before birth. Moreover, serial studies starting at birth showed a progressive increase in the percentages of pre-B cells in marrow and spleen, more immature pre-B cells predominating in younger mice, gradually giving way to more mature pre-B cells and B cells in older mice, as evidenced by the proportion of cells expressing the late pre-B/B cell marker, THB. The paucity (less than half the absolute values of normal mice of the same age) of B cells in bone marrow and lymph nodes persisted at least to the age of 5 to 7 weeks, but a much greater proportion of B cells were in the S/G2/M phase of the cell cycle in E,-myc mice (23%)than in their normal litter mates (8%). No small lymphocytes were present in the E,-myc mice, although lymphocytes of this size predominate in the normal animal. Interestingly, the 20%of E,-myc mice that had not developed tumors by 14 weeks of age had lower percentages of pre-B cells in their bone marrows and higher percentages of mature B cells in their spleens than their younger E,-myc littermates, suggesting that the latent period for tumor induction correlates with the proliferative drive imparted upon the pre-B cells by the E,-myc transgene. It can be concluded that constitutive expression of c-myc in immature B cells, presumably commencing as soon as protein factors responsible for activating EH are present, leads to hyperplasia of the pre-B compartment, and retardation, but not prevention of differentiation, in the B cell lineage. The lack of small lymphocytes indicates that in E,-myc mice differentiation is not accompanied by entry into a resting phase. The degree of pre-B cell hyperplasia and retardation of B cell differentiation appears likely to depend upon the level of c-myc expression. It is interesting that the degree of hyperplasia appears to stabilize some weeks after birth, and does not itself lead to death of the mice. This indicates that a considerable proportion of the proliferating cells must die. It is also interesting that the resultant tumors are mono-, or rarely oligoclonal, suggesting that a rare second genetic event is

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needed to induce neoplasia. This event is presumably increased in likelihood by the increase in size of the pre-B cell pool (by a factor of 4 or 5 ) and the increased number of cell divisions (at least 10-fold).When the tumors emerge, they do so in lymph nodes and occasionally in thymus-both are sites which are not infiltrated by pre-B cells. Approximately half the tumors are of pre-B phenotype, 19% express surface immunoglobulin, and the remainder are a mixture of pre-B and B cells (Harris et al., 1988).The observation of mixtures of pre-B and B cell tumor cells suggests that tumor cells can undergo differentiation in d u o . This is supported by the observation that many of the permanent cell lines derived from pre-B tumors express surface immunoglobulin. These findings are remarkably consistent with hypotheses, described above, regarding the development of Burkitt’s lymphoma. First, the need for a positive drive to expression of c-myc, provided by immunoglobulin sequences, appears to be established-even gross structural damage to the gene leading to separation of the proteincoding portion from the bulk of the regulatory region does not, by itself, lead to deregulation. Second, the constitutive expression of c-myc in pre-B cells appears to prevent the cell from entering a resting phase (although differentiation from pre-B cell to B cell is not prevented, simply slowed) and sets the scene for the development of neoplasia (see Fig. 5), although at least some of the neoplasms express a more differentiated phenotype. Constitutive expression of c-myc appears to be insufficient for lymphoid tumorigenesis, at least in mice, since clonal neoplasms develop from polyclonal hyperplastic pre-B cells. These findings do not necessarily indicate that constitutive expression of c-myc in Burkitt’s lymphoma is also a prior event to tumorigenesis. It is possible that in the human tumor some other element causes hyperplasia (early EBV infection and malaria, for example, in endemic tumors) which is subsequently complemented by constitutive c-myc expression. In any event, the preneoplastic hyperplasia of pre-B cells in transgenic mice is consistent with the idea that environmental factors may influence the incidence of Burkitt’s lymphoma by causing hyperplasia of the target cell population. It is probable that multiple factors are relevant to the precise timing of outgrowth of the malignant neoplasms in transgenic animals, since back crossing of the original strain with three inbred strains markedly alters the characteristics of the neoplasms (see below).

2. B Cell Neoplasia in EnMyc Transgenic Mice A second mouse model transgenic for a human c-myc gene, containing a murine immunoglobulin heavy chain enhancer inserted into the first c-myc intron (Schmidt et al., 1988), provides some interesting

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similarities and contrasts to the E,-myc model. The c-myc transgene (EnMyc) was expressed only in pre-B cells present in thymus, spleen, and bone marrow of l-month-old mice, but in only the bone marrow of 2- and 8-month-old mice. Similarly to E,-myc mice, the pre-B cells were polyclonal and nontumorigenic in nude mice. The EnMyc construct, unlike E,-myc, appeared not to inhibit B cell differentiation, although its expression was inversely proportional to the expression of K chains (present in 95% of murine B but not pre-B cells). At 2 months of age, mice transgenic for EnMyc developed diffuse lymphadenopathy due to the appearance of malignant pre-B cell tumors capable of being transplanted into nude mice. By the age of 114 days, half of the transgenic mice had developed tumors, and by 400 days, 100% had tumors. Rearrangement of the J H segment demonstrated that the tumors were clonal or oligoclonal, but only 1 of 12 tumors tested had K chain rearrangement and none had A rearrangements. In this transgenic model system it has been shown that the breeding of an additional immunoglobulin p gene, mIgp (encoding the membrane form of the molecule), into the transgenic mice markedly reduced the incidence of pre-B tumors (Nussenzweiget al., 1988). While 50% of EnMyc animals had developed lymphomas by 117 days, none of the mice also bearing the transgenic mIgp gene had developed tumors during this period. After a year, more than 90% of EnMyc mice, but only 40% of the EnMyclmIgp mice, had developed tumors. Interestingly, the tumors in the EnMyc/mIgw mice also differed phenotypically in that 29% were pro-B (prior to immunoglobulin gene arrangement), 24% pre-B, and 24% were of mature B phenotype. This difference in the frequency of various B lineage phenotypes appeared to be due to a reduction in the incidence of pre-B tumors rather than an absolute increase in tumors of other phenotypes. Mice doubly transgenic for EnMyc and the secreted form of the immunoglobulin p molecule developed tumors at a similar rate and within a similar time frame to the EnMyc mice. These tumors were also phenotypically similar to the tumors in EnMyc mice, indicating a lack of effect of the secretory p molecule on tumorigenesis. I n EnMyc/mIgp mice, both EnMyc and mIgp were expressed in the pre-B cells, EnMyc to the same extent as in the EnMyc transgenic mice. In addition, in 40 separate samples, no difference in the proportion of pre-B cells was observed between the bone marrows of EnMyc mice and EnMyc/mIgp mice. However, pre-B cells derived from EnMydmIgp mice were some 70 to 80% less susceptible to transformation by Abelson virus. The authors surmised that the reduc-

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tion in the incidence of tumors in EnMyc/mIgp mice was a consequence of the acceleration of cells through specific stages of the B cell differentiation pathway, and that because of this, the number of cells susceptible to tumorigenesis by EnMyc or by v-abl was reduced. This acceleration was presumed to be a consequence of the inhibition of immunoglobulin gene rearrangement by the membrane form of the p chain. Thus, this model too is consistent with the idea that the incidence of a given tumor is dependent upon the absolute number of cells present which are susceptible to the transforming influence relevant to its pathogenesis. It is interesting that a higher proportion of B cell or mixed pre-B/B cell tumors are present in E,-myc mice. E,-myc, but not EnMyc, inhibited normal B cell differentiation. Variables in the two systems that might account for these differences include the mouse strains(C57BL X SJL)F2 in the case of E,-myc and C57BL/6J x CD1 in the case of EnMyc mice-and the constructs used to create the transgenic strains-the E,-myc construct included a larger immunoglobulin fragment (which contained S, sequences) linked 5’ to the murine c-myc gene, while the EnMyc construct consisted purely of immunoglobulin enhancer sequences inserted into the first intron of the human c-myc gene. It is possible that the level of expression of the two transgenes or that the relative proportions of the two major c-myc proteins varied. Alternatively, variations in the cellular milieu may have resulted in different effects on the differentiation of B cells. Whatever the reason, the observed differences, occurring in lymphomas which are both generated by deregulated c-myc expression in pre-B cells, provide a parallel to the differences in breakpoint locations and clinical features between endemic and sporadic Burkitt’s lymphomas. The latter may well be due to differences in the structural consequences of the translocations at a molecular level, or perhaps to differences in the presumed additional genetic changes that are necessary for the development of the fully neoplastic phenotype * Recently, two separate groups of investigators have shown that similar overgrowth of pre-B cells followed by the development of clonal lymphoid tumors occurs in transgenic mice that carry an N-myc gene coupled to the immunoglobulin heavy chain enhancer (Dildrop et al., 1989; Rosebaum et al., 1989). In the pre-B and B cell tumors, the endogenous c-myc and N-myc genes were silent. Thus, in some circumstances, c-myc and N-myc may perform interchangeable functions, although N-myc has not been implicated in naturally occurring lymphoid neoplasia.

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B. REPLACEMENT OF THE REQUIREMENT FOR A TRANSLOCATION BY A VIRALPROMOTER-CONTROLLED myc GENE The experiments with transgenic mice are admirably complemented by experiments with pristane-induced murine plasmacytomas, which have provided an excellent experimental system in which to examine the role of the translocations involving c-myc in the pathogenesis of these tumors. The approach adopted was to examine the influence of an exogenously provided, deregulated c-myc gene on plasmacytoma induction. In these elegant experiments, pristane-treated mice were injected with Moloney helper virus and defective murine retroviral constructs (J-2 or 5-3 viruses) containing an avian v-myc oncogene and a hybrid murine/avian v-ruf oncogene under the control of the viral LTR. The v-rafoncogene is expressed in J-2 but initially thought not to be in 5-3, because of a 256-bp deletion at the guglraf border which creates a frameshift and failure to synthesize the v-rufprotein. Sensitive mouse strains inoculated with 5-3 following pristane developed plasmacytomas at approximately the same frequency (22%)as they did after pristane alone, but the mean latent period was approximately half as long. Even mice belonging to a strain resistant to the development of plasmacytomas after pristane alone developed a small number of plasmacytomas when also inoculated with J-3. The main point of interest, however, was that among the nine plasmacytomas in J-3-treated mice subjected to cytogenetic analysis, translocations involving c-myc were present in only two of the tumors-both of which failed to express the v-myc gene. The remaining seven, which expressed v-myc (under the control of the viral long terminal repeat), did not contain translocations (Potter et al., 1987; Clynes et al., 1988).These data clearly indicate that the role of the translocation is to provide deregulated myc expression, and that the translocation is not necessary if this requirement for tumorigenesis has already been met. However, these experiments, like those with mice transgenic for c-myc, also suggest that c-myc deregulation is not, by itself, sufficient for tumor induction since the plasmacytomas induced with v-myc (there was one exception) were monoclonal in spite of the fact that many B cells must have been infected by the 5-3 virus. Interestingly, mice injected with J-2 virus, in which the v-rufgene was expressed in addition to v-myc, developed lethal myeloid tumors with an even shorter latent period than the plasmacytomas induced by J-3, but no mice injected with J-2 developed plasmacytomas. Similar myeloid tumors have been induced in pristane-primed mice by retroviruses containing c-myc (Wolff et al., 1986).

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The differences in the biological effects of 5-2 and 5-3almost certainly arise from differences in the functional changes which are required in addition to deregulated myc expression for tumor production in any given cell lineage. The elucidation of the complexities of these additional requirements has been begun by constructing a variety of myc-containing recombinant retroviruses and examining their biological effects (see below). C. CHICKEN BURSAL LYMPHOMA Another model of relevance to the consequences of deregulation of c-myc is the virus-induced lymphoma of the bursa of Fabricius (the site of differentiation of B cell precursors in birds) of chickens. Infection of susceptible birds with avian leukosis virus (ALV)(infection must occur during the embryo stage when the bursa is developing, or immediately posthatching) results ultimately in the development of metastasizing lymphomas in which c-myc has been deregulated (and vastly overexpressed) because of the nearby insertion of an ALV provirus. Only one of the terminal repeat regions is necessary for tumorigenesis, and the virus can be inserted both upstream and downstream of the c-myc gene, since deregulation is a consequence of c-myc being driven by the LTR enhancer element (Hayward et al., 1981).It was, in fact, this model which provided incentive to examine the identity of the chromosome 15-derived rearranged DNA in mouse plasmacytomas (Sheng-Ong et al., 1982). In the chicken bursal lymphoma, the production of true neoplasia is a stepwise process beginning with the proliferation of large, nonneoplastic, basophilic cells in the bursal follicles, referred to now as transformed follicles (Baba and Humphries, 1985), followed by the development of discrete, monoclonal bursal nodules which, ultimately, are able to metastasize (Neiman et al., 1980). The role of myc was examined in this system by transfecting bursal lymphocytes from 18-day-old embryos with a defective retrovirus containing v-myc (HB1) and transfusing them into recipient embryos which had been treated with cyclophosphamide to ablate their bursae, such that the infused cells would reconstitute them (Neiman et al., 1988).At 4 weeks posthatching some of the reconstituted follicles were morphologically indistinguishable from ALV-induced transformed nodules, and expressed high levels of HB1 v-myc (Thompson et al., 1987).These transformed follicular cells, but not normal bursal cells (or even proliferating bursal cells) from chicks of this age, could reconstitute secondary cyclophosphamideablated recipients, in whom all the reconstituted follicles were trans-

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formed and expressed high levels of HB1 v-myc (Thompson et al., 1987).These transformed cells from secondary recipients could again be transplanted, but were markedly less efficient at reconstituting follicles. Interestingly, the transformed follicle cells were shown to be capable of diversifying their immunoglobulin light chain genes (Thompson et al., 1987). These data suggest that HB1 infection of embryonal bursal lymphocytes causes the persistence of cells which function as bursal stem cells. Such cells have disappeared from normal bursae by 4 weeks posthatching, at which time the follicles are normally largely composed of small lymphocytes. Even the proliferating cells present at this time are activated bursal cells which are not able to reconstitute the bursae of cyclophosphamide-ablated chicks. The deregulated v-myc gene, in other words, appears to have prevented the presumptive stem cells in which it is expressed from becoming small resting lymphocytes-a situation remarkably similar to that in transgenic mouse models. That these cells are not themselves neoplastic but readily undergo neoplastic change is demonstrated by the low incidence of metastasizing bursal lymphomas (5%) seen in the primary transplant recipients, and the increased number of lymphomas (approximately 20%) which occur in secondary and tertiary transplant recipients (Nieman et al., 1988).Once again, the preneoplastic state of the HB1-infected cells is paralleled by the preneoplastic pre-B cells of transgenic mice. Moreover, it seems likely that the cell type susceptible to transformation by HB1 infection, as in the case of ALV infection, is the bursal stem cell population. XIV. The Role of Other Genetic Abnormalities An understanding of the totality of the genetic changes which contributes to the genesis of Burkitt’s lymphoma is likely to be more readily gained by first exploring the requirements beyond deregulated myc expression for the induction of tumors of the B cell lineage in the available animal models. In fact, it is from these model systems that the necessity for additional genetic lesions is most clearly perceived. In the transgenic mouse models there is a preneoplastic phase of pre-B cell hyperplasia upon which is superimposed a monoclonal neoplasm-a situation remarkably reminiscent of the development of monoclonal lymphomas in immunosuppressed individuals with B cell hyperplasia, which is often EBV associated. Similarly, in bursal lymphomas in chickens, lymphocytes infected with the HB 1virus bearing v-myc are not themselves neoplastic, although they have a high

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propensity to neoplastic change. Moreover, in both the murine and chicken systems, transformation appears to be remarkably differentiation-stage specific, indicating that the deregulated c-myc gene is only of pathological significance when superimposed upon an appropriate phenotypic background. This has been further confirmed by backcrossing the original E,-myc transgenic strain against inbred mice (Harris et al., 1988; Sidman et al., 1988).The original animal, a male Fz [C57BL/6(B6) X SJL/J], had been bred against (B6 X SJL)Fl females to generate the primary E,-myc strain. This strain was now bred against inbred SJL, B6, or BALB/cAN mice. The offspring of E,-myc mice bred against the SJL strain developed tumors very rapidly (36% per week), while those bred against B6 developed tumors more slowly than the original strain. Interestingly, the transgene converted resistant mouse strains sensitive to pristane-primed plasmacytoma development-apparently having a very similar effect to the v-myccontaining retrovirus, 5-3described above. These findings, while confirming the importance of the genetic background, do not provide any information as to what the changes might be that convert hyperplastic pre-B cells to neoplastic cells.

A. INTRODUCTION OF VIRALONCOGENES INTO ANIMALTUMOR MODELS One experimental approach that has been adopted to explore this question is to infect transgenic mice with retroviruses bearing additional oncogenes. Such experiments have indicated that both v-H-rus and v-ruf oncogenes, but not v-abl, rapidly induce lymphomas in E,-myc mice by 3 weeks of age (Harris et al., 1988). Normal animals or uninfected transgenic animals had not developed lymphomas by this age. A similar approach to that used in transgenic mice has also been used in pristane-primed mice. Pristane-primed mice were infected with a virus designated as “RIM.” RIM contains the coding exons of a normal c-myc gene cDNA coupled to an immunoglobulin heavy chain enhancer and an immunoglobulin V H promoter. It also contains a v-Ha- M S gene controlled by the viral LTR. RIM virus induced a high incidence of monoclonal IgM-secreting plasmacytomas (84% of 94 tumors) with a considerably shorter latent period than pristane alone, and induced plasmacytomas more readily in 3-week-old mice (83% of animals) than in adult mice (28% of animals). In addition, this virus induced plasmacytomas in CDFl mice, which are normally resistant to pristane-induced plasmacytomas. Another virus similar to RIM except for the absence of the v-Ha-ras gene (designated IM) failed to induce plasmacytomas (Clynes et al., 1988), while a virus incorporating the

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identical c-myc coding segments under the control of a Moloney virus LTR induced myeloid tumors in pristane-conditioned BALB/c mice (Wolff et ul., 1986), as did the 5-2 virus expressing v-myc and v-ruf. Finally, infection with the LTR-myc virus of cloned cell lines of pre-B or B phenotype, whether already overtly neoplastic or not, derived from E,-myc mice, resulted in their conversion to neoplastic macrophages producing the myeloid growth factor GM-CSF. These experiments further confirm that deregulated myc expression is insufficient per se; this genetic lesion must occur in an appropriate cellular environment if plasmacytomas are to be induced. Expression of c-myc in the RIM virus, in contrast to the 5-3 virus, is likely to be largely limited to cells of the B lineage, since it is controlled by an immunoglobulin VH promoter and the immunoglobulin heavy chain enhancer. Hence the poor ability of this virus (or its fellow lacking the v-Ha-rus gene) to induce myeloid tumors, as compared to retroviral constructs containing similar c-myc sequences controlled by a viral LTR, is readily explained. However, the v-Ha-rus gene appeared to play an important role in tumorigenesis in this system, since when this gene was absent in infecting viruses they failed to induce plasmacytomas. Even so, these two genes operating together still appear to be insufficient to induce tumors, because the induced plasmacytomas were monoclonal. Nevertheless, the ability of myc and rus to cooperate is well known from experiments in which both are required for the neoplastic conversion of primary rat embryo cells (Land et ul., 1983a,b; Lee et ul., 1985). Similarly, myc and ruf have also been shown to cooperate in in vitro systems, such as in the transformation of bone marrow cells (Cleveland et ul., 1986). Interestingly, transfection of human EBV-transformed lymphoblastoid cell lines with a recombinant retrovirus containing a v-Ha-rus gene induced a more differentiated phenotype as well as inducing tumorigenicity in nude mice (Seremetic et ul., 1988). This could be of relevance to the role of v-Ha-rus in plasmacytomagenesis. The much higher incidence of plasmacytomas in 3-week-old mice is intriguing. One possible explanation is that there may be different proportions of pre-B versus B cells in young mice compared to adults, and pristane may therefore induce higher proportions of target cells in 3-week-old animals. This is reminiscent of the age-related incidence of Burkitt’s lymphoma. As with the 5-3 virus, chromosomal translocations involving c-myc are not required for tumorigenesis by RIM virus, since the resulting plasmacytomas had germ-line endogenous c-myc genes which were not transcribed. This, coupled to the ability of RIM and 5-3 to induce plasmacytomas in resistant mouse strains, suggests that ge-

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netic resistance is related to the likelihood of a translocation occurring rather than a difference in the intracellular milieu in which myc must operate. Eighteen of 24 RIM-induced plasmacytomas in adult mice lacked Moloney proviruses (used as helper virus), indicating that integration of this virus with induction of adjacent cellular oncogenes via LTR enhancement (insertional mutagenesis) does not play a role in the pathogenesis of these tumors. Further evidence of oncogene cooperation in plasmacytomagenesis has been obtained from experiments with retroviruses of the J series (Troppmair et ul., 1988).J5, a virus containing only the MC29-derived v-myc gene, like J-2, which contains an MH2/MC29 hybrid v-myc gene, failed to induce plasmacytomas but was able to induce myeloid tumors. Y7, containing both the MC29 v-myc and a v-ruf gene which had been activated by truncation, caused a similar acceleration of plasmacytoma induction to 5-3. A virus containing only an activated v-ru. gene (Y5) failed to induce plasmacytomas. These data suggested that ruf and myc cooperate in plasmacytoma induction, but failed to explain why 5-3, in which the ruf protein was believed not to be expressed, accelerated plasmacytoma induction. This issue appears to have been resolved, since virus rescued from the ascites of three J-3-induced plasmacytomas induced foci in NIH 3T3 cells with a morphology quite typical for those induced by v-ruf-containing viruses. Thus it appeared likely that the 5-3 virus can undergo a second frameshift mutation which restores v-ruf expression, and that such viruses are selected for in the course of plasmacytoma induction. These data, then, suggest strongly that r u . and myc cooperate in plasmacytoma induction. The retrovirus-mediated introduction of additional oncogenes into both transgenic mice and pristane-primed mice confirms the generally held belief that the rebdiness with which tumorigenesis occurs depends upon the genetic background, and that a single genetic change is not sufficient. Moreover, it also raises the possibility that either multiple additional changes are necessary, or that several different genetic lesions can cooperate with deregulated c-myc in tumorigenesis. Quite possibly both of these apply. Similarly, in Burkitt’s lymphoma, it is possible that several different genetic lesions may be able to cooperate with c-myc deregulation in the process of tumorigenesis. The resultant tumors may, of course, have subtle biological differences. Seen from this perspective, the presence of EBV in only a proportion of Burkitt’s lymphomas is not at all surprising, although whether its putative role in pathogenesis lies at the level of increasing the pool of target cells (similar, perhaps, to the Abelson virus with

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respect to plasmacytoma induction) or contributing directly to the process of tumorigenesis, or both, remains unknown. To date, no other persistent genetic abnormalities apart from the specific chromosomal translocations have been described in Burkitt’s lymphoma, although changes in chromosome 1 occur with a high frequency in sporadic tumors (Douglass et al., 1980; Berger and Bernheim, 1984). In one tumor cell line (Ramos) a mutated N-ras gene was identified (Murray et al., 1983), and in 1of 14 tumors and cell lines, an activated ki-ras gene was detected (Lenoir et ul., 1984), but this is clearly not the rule in Burkitt’s lymphoma. The B-lym gene, originally believed to have been derived from Burkitt’s lymphoma DNA by transfection and transformation of mouse NIH 3T3 cells, having been shown to be of mouse origin (Rogers, 1986; Cooper et al., 1986), is no longer relevant to such considerations. It would appear that continued exploration of the possibilities raised by the experiments with animal models is likely to be the most lucrative source of leads to an understanding of the participation of other oncogenes with c-myc in Burkitt’s lymphomagenesis. B. POSSIBLE ROLE OF ANTI-ONCOGENES

An additional possibility worthy of further exploration is that the deletion or mutation of a recessive gene (anti-oncogene), such that its function is totally lost from the cell, may have a role in the pathogenesis of Burkitt’s lymphoma. Loss of function of a gene likely to be involved in suppression of proliferation rather than increased activity of a gene involved in the induction of proliferation, is an oncogenetic mechanism that has been implicated in retinoblastoma, Wilms’ tumor, rabdomyosarcoma, and an increasingly long list of solid tumors (see recent reviews in Klein, 1987, 1988; Weinberg, 1988; Friend et al., 1988). This possibility is raised in Burkitt’s lymphoma by a number of intriguing observations, notably the structural similarity of c-myc to one of the three conserved regions (region 2) of the adenovirus protein, E l a (Moran and Matthews, 1987; Figge et al., 1988), a region which also bears considerable homology to other virus nuclear proteins including the large T antigens of SV40, polyomavirus, and lymphotropic papovavirus (Stabel et al., 1985).Region 2 of E l a , along with region 1, is known to be essential to several functions of E l a , including its ability to immortalize primary cells, to cooperate with ras, like myc, in in vitro transformation assays, and to induce cellular DNA synthesis (Lillie et al., 1986,1987).Moreover, this region, in all the viral proteins mentioned above, has been shown to participate in the binding of the product of the retinoblastoma gene, a protein with a molecular weight

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of 105kDa, now usually referred to as p105-RB. pl05-RB is believed to suppress cellular proliferation, probably through the transactivation of other genes, and it has been proposed that the normal function of these proteins bearing homologous domains is to bind pl05-RB and inactivate it in order to permit the onset of S phase in infected cells-a necessary prerequisite for viral DNA synthesis (Timmers et al., 1988; Green, 1989). Transformation only occurs in cells which are nonpermissive for viral replication. It is not yet known whether c-myc can bind p105-RB7 but its homology with E l a and the large T antigens suggests that it may be able to. Thus, cells in which there is impaired production or function of an anti-oncogene such as p105-RB7possibly as a consequence of virus infection, may be more susceptible to transformation by a deregulated c-myc gene. EBV could possibly exert an effect through a mechanism of this kind; the demonstration that a latent EBV protein binds an antioncogene would provide support for this. Pertinent to this issue is the recent demonstration that E l a is capable of transactivating c-myc. E l a caused initiation of transcription at the authentic cap site of the P2 promoter, the presence of which was an absolute requirement in the constructs containing part of the c-myc gene which were used to demonstrate this effect (Lipp et al., 1989). A region within Pz, conserved between mouse and man, has considerable homology with the E la-inducible promoter, E2, of adenovirus type 5. This region binds a nuclear transcription factor, E2F, known to be essential to the activation of adenovirus early promoters and enhancers by Ela. An intact E2F binding site is required for both myc expression via P2, and modulation by E l a (Thalmeier et al., 1989). These findings suggest that DNA virus genes may contribute to malignant transformation through these biochemical pathways, and also suggest that the modulation of E2F by cellular factors may be involved in the normal regulation of c-myc transcription. Several other proteins are known to bind to E l a (Whyte et al., 1988) and, under normal circumstances, transformation by adenovirus also requires the participation of another protein, Elb. E l b binds to a cellular protein, p53, which also binds to the large T antigen of SV40 and, like large T, Ela, and c-myc, can cooperate with rus in cell transformation (Land et al., 1983a,b; Lee et al., 1985). p53 is now believed to be another example of an anti-oncogene whose inactivation by binding to viral proteins may, as in the case of pl05-RB, be pertinent to viral transformation (Levine, 1982; A. J. Levine et al., 1982; Green, 1989). It is therefore of considerable interest that an EBNA protein may bind to p53 (Luka et al., 1980; Luka and Jornvall, 1982). This may be pertinent both to transformation by EBV and to the possi-

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bility that c-myc and EBNA may cooperate in the pathogenesis of Burkitt’s lymphoma. Recently, evidence was presented that oligomerization of the c-myc protein, a process dependent upon the leucine zipper domain in a carboxy-terminal peptide (Dang et al., 1989), is necessary for its transforming activity (Stone et al., 1987).Moreover, an inactive c-myc mutant which retained its ability to oligomerize inhibited the ability of wild-type c-myc to cooperate with ras in the transformation of primary rat embryo cells (Dang et al., 1989). This is consistent with the necessity of the c-myc protein to bind to itself or to other proteins as a part of its transforming capability, but also suggests that simple binding of other proteins to c-myc with and resultant inactivation is not a sufficient explanation for the role of c-myc in cooperative transformation with ras, since “activity” of the c-myc protein is required. It is possible that the complex of proteins has an important effect, e.g., in transcriptional regulation. In this respect it is possible that c-myc binds tojun andfos. Both are also nuclear oncogenes, and have been shown to form a heterodimer mediated by the leucine zipper domain offos (SassoneCorsi et al., 1988a). The association offos andjun proteins has been observed in some transcription complexes (Sassone-Corsi et al., 1988b; Franza et al., 1988), and such complexes probably bind to the c-myc regulatory region itself. One probable binding site which has been identified is the negative regulatory element upstream of c-myc (Hay et al., 1989). At present the potential ramifications of these observations cannot be further elucidated. The possible interaction of c-myc with anti-oncogenes and putative transcription factors remains an exciting area for future studies. IN DNA REPAIR C. DEFECTS

A final possible genetic interaction concerns defects in DNA repair enzymes. Mouse strains susceptible to plasmacytoma induction have been shown to have impaired ability to repair DNA compared to normal mice (Sanford et al., 1986; Potter et al., 1988a,b), a defect that presumably increases the likelihood of a translocation developing, since resistant strains are equally susceptible to the induction of translocation-negative plasmacytomas with viral oncogenes (see above). The enzyme poly(ADP) ribose polymerase, which may be involved in DNA repair, has been implicated in resistance to plasmacytomas, and it is possible that increased susceptibility to Burkitt’s lymphoma occurs in individuals with relatively poor ability to repair DNA. This could be relevant to lymphoma induction in Bloom’s syn-

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drome, xeroderma pigmentosum, and ataxia telangiectasia, all of which are associated with chromosomal instability and defects in DNA repair (Paterson et al., 1976; Willis and Lindahl, 1987; Lindahl, 1987; Hecht and McCaw, 1977).In ataxia telangiectasia there is an increased incidence of Burkitt’s lymphoma bearing the characteristic nonrandom chromosomal translocations (Filipovich et al., 1987, 1990). Interestingly, the search for a possible pathogenetic relationship in Burkitt’s lymphoma to a defect of a DNA repair enzyme led to the discovery of an association with a genetic polymorphism for a poly(ADP) ribose polymerase-like gene on chromosome 13. Detailed study of this polymorphism suggests that it is caused by a deletion, raising the possibility that this region of chromosome 13 is close to the site of an antioncogene (Bhatia et al., 1990). In fact, allelic loss at loci on chromosome 13has also been observed in primary gastric cancer (Motomura et al., 1988). XV. EBV and Burkitt’s Lymphoma

The climatic determinants of the distribution of Burkitt’s lymphoma in Africa led to the hypothesis that the tumor is caused by an arthropodvectored virus and to a search for virus particles in the tumor cells. Although virions could not be found in fresh tumor cells, Epstein, Achong, and Barr were able to identify herpesvirus particles by electron microscopy in cell lines derived from African Burkitt’s lymphomas (Epstein et al., 1964). This led to the development of a variety of serological tests for EBV infection (G. Henle and Henle, 1966; W. Henle and Henle, 1979; Henle et aZ., 1970) and the recognition that African patients had a markedly higher geometric mean titer of antibodies against the virus capsid antigen (VCA) and a greater frequency of antibodies against the early antigens (EA),particularly the restricted early antigen (R) (Henle and Henle, 1979), although the rapidly recognized ubiquity of the virus prevented the formulation of a simple hypothesis designating EBV as the primary etiological agent of Burkitt’s lymphoma. It subsequently became clear that the viral genome of this new (then) herpesvirus is present in the tumor cells ofthe majority of endemic Burkitt’s lymphomas (some 96%), but in a minority of sporadic tumors-some 15 to 30% (Lindahl et al., 1974; Anderson et al., 1976; Barriga et al., 1988a). Interestingly, approximately 85% of tumors from North Africa contain EBV DNA, although the clinical characteristics of Burkitt’s lymphomas in North African countries are more similar to sporadic tumors than endemic (Ladjadj et al., 1984). These findings have provided the basis for much research and specula-

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tion on the pathogenetic significance of the association of EBV with Burkitt’s lymphoma. EBV cannot be causally implicated in the majority of sporadic tumors, although some attempts have been made to do this. Theories that the virus could induce neoplasia and then be lost from the tumor cells are untenable because of the significant proportion (some 25%) of seronegative individuals with sporadic Burkitt’s lymphoma, and the occasional occurrence of infectious mononucleosis caused by primary EBV infection occurring after the onset of Burkitt’s lymphoma (Magrath, 198313).It remains possible, however, that the majority of endemic tumors, and possibly EBV-associated sporadic tumors, may have an absolute requirement for EBV. The observations that EBV can transform normal human B lymphocytes and render them immortal in uitro, and can induce fatal lymphoproliferative syndromes and lymphomas in primates as well as similar syndromes in immunodeficient or immunosuppressed patients (Weisenberger and Purtilo, 1986; Cleary et al., 1986; Magrath, 1983b), support the possibility that the virus is of pathogenetic significance in EBV-associated Burkitt’s lymphoma. However, the lymphoproliferative processes occurring in previously immunodeficient or immunosuppressed patients usually differ clinically and pathologically (for example, they are often polyclonal) from Burkitt’s lymphoma. Exceptions include Burkitt’s lymphoma occurring in patients with X-linked lymphoproliferative syndrome and Burkitt’s lymphoma in HIVinfected individuals. It therefore seems likely, as is further discussed below, that the probable mechanisms whereby EBV contributes to the pathogenesis of Burkitt’s lymphoma differ from those involved in the transformation of normal lymphocytes into continuous cell lines, and in the failure to regulate EBV-infected cells in some immunodeficient patients. With this in mind, it is necessary to provide an outline of the basic biology and relevant molecular biology of EBV in order to discuss further the association of this virus with Burkitt’s lymphoma, EBV is a large, enveloped virus whose DNA contains approximately 173,000 bp of linear duplex DNA containing multiple single-stranded breaks (Pritchett et uZ., 1975) and a coding capacity in excess of 80 polypeptides (Baer et al., 1984). Its genome consists of two unique and a longer domain of domains, a short domain of about 15,000 bp (US) about 150,000 bp separated by tandem repeats of a 3071-bp sequence (U,) (Dambaugh et d., 1986). Additional internal repeat regions are present within the larger unique domain, and each end of the viral DNA includes 4 to 12 copies of a 500-bp terminal repeat unit (Kintner and Sugden, 1979). The viral genome contains multiple restriction enzyme sites for BamHI and EcoRI (Given and Kieff, 1978; Dambaugh

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et al., 1980; Bornkamm et al., 1984; Baer et al., 1984), and its resultant polypeptides are often referred to by the BamHI fragments and open reading frames which code for them, the latter having been derived from the complete nucleotide sequence of the B95-8 strain of EBV (Baer et ul., 1984). Thus, BKRFl refers to the first rightward reading frame of the BamHI K fragment. A. THEEPIDEMIOLOGY AND BIOLOGYOF EBV INFECTION EBV gains entry into the human body via secretions, usually saliva, derived from other individuals, which contain virus particles. Infected saliva is usually transmitted to children and young adults such that by adulthood more than 90% of individuals worldwide have antibodies to EBV (Henle and Henle, 1979). Infection occurs earlier in less economically privileged populations. In equatorial Africa, for example, 100% of children are infected by age 3 years (de ThC et al., 1975). Infectious mononucleosis occurs in perhaps 50% of adolescents and young adults as a consequence of primary EBV infection, but at other ages infection is not usually accompanied by a clinical syndrome (Henle and Henle, 1979). At the time of primary infection, EBV establishes a lytic (i-e.,virus productive) infection in epithelial cells of the pharynx, including the ductal cells of the salivary glands (Sixby et ul., 1983, 1984). The virus has also been isolated from uterine cervix secretions and urethral secretions in men seen in clinics for sexually transmitted diseases (Sixby et al., 1986),and can presumably also be transmitted by this route. Undifferentiated cervical epithelial cells have been shown to possess viral receptors and to express EBV nuclear antigens (EBNAs), while more differentiated, i.e., keratinized epithelial cells express antigens associated with virus replication, i.e., early antigens (EA) and virus capsid antigen (VCA)(Sixby et al., 1987). In normal individuals it appears that the lytic infection in epithelial cells persists throughout life, although recently, eradication of pharyngeal EBV was reported after bone marrow transplantation (Gratama et al., 1988). If lytic infection were prevented following intensive preparative therapy, EBVcontaining cells could be lost without infecting others. This would presumably never occur under normal circumstances. Virus released from epithelial cells can infect lymphocytes in adjacent lymphoid tissue, or in capillaries passing through the mucosal tissue. It is also possible that lymphocytes can be infected directly at the time of primary exposure in mucosal associated lymphoid tissue such as the tonsils. EBV has been considered for many years to be B

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lymphotropic, but recently the description of EBV-associated T cell lymphomas (Joneset al., 1988)has raised the possibility that certain T cells in the body may also harbor EBV. However, the extent to which lymphocytes are infected from virus released from epithelial cells after the development of neutralizing antibodies is not clear, and it is possible that the maintenance of infected lymphocytes, which also persist throughout life, occurs independently of the epithelial lytic infection. This is supported by the finding that the frequency of EBV-containing circulating B lymphocytes is not altered even after several months of acyclovir treatment, which prevents virus replication (and therefore infection of circulating lymphocytes from epithelial cells) (Rickinson, 1988; G. Tosato, personal communication). Thus, the concept that EBV-infected B cells are continuously destroyed by T cells in the immune host, but also continuously replenished by infection of B cells circulating through the pharynx (Yao et al., 1985a,b; Rickinson, 1986), seems no longer to be tenable. EBV has been shown to be capable of infecting immature B cells (Hansson et al., 1983; Katamine et al., 1984),and possibly even multipotential stem cells. It has also recently been demonstrated in immature erythrocytes grown in uitro by infecting bone marrow cells depleted of B cells (Baranski et al., 1988). It is therefore possible that EBV-containing B cells are derived by a process of differentiation from a separate reservoir of EBV-containing cells in the precursors of B cells. This would be consistent with the finding that EBV can be eradicated from lymphoid cells as well as from the pharynx after bone marrow transplantation (Gratama et al., 1988). It is also worth noting that if EBV is present in B cell clones reactive with commonly encountered antigens, such that the clones persist as memory cells, EBV could be permanently maintained in such clones. Either of these circumstances are consistent with the observed eradication of EBV following bone marrow transplantation. In this respect, it has recently been demonstrated that although the majority of E BV-carrying B cells are low-density blast cells which are capable, in vitro, both of direct (spontaneous) outgrowth and also of release of EBV with subsequent transformation of other lymphocytes, occasional high-density small lymphocytes capable of direct outgrowth in uitro can be observed (Lewin et al., 1987). Nevertheless, it must be stated that at the present time it is not known how the reservoir of EBV-infected B cells in maintained. EBV gains entry into B lymphocytes via the CD21 molecule (p35) which is expressed on resting B lymphocytes, but not activated B lymphocytes, which are therefore resistant to EBV infection (Iida et al., 1983; Fingeroth et al., 1984; Aman et al., 1984; Inghirami et al.,

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1988).In vitro infection of B lymphocytes results in lymphocyte transformation, immunoglobulin production (Kirchner et aZ., 1979),and the permanent propagation of the EBV-infected clones (Henle et al., 1967; Pope et al., 1968; Katsuki et al., 1977; Aman et al., 1985). EBV-infected B cells in vivo are well controlled by the immune system in normal individuals (see Rickinson, 1986; Tosato, 1987, for reviews). Nonspecific cytotoxicity, e.g., via natural killer cells and HLA-unrestricted T cells (Tomkison et al., 1987), provides the major components of the initial immune response in the naive host. These mechanisms, in addition to lymphocyte mediated antibody-dependent cytotoxicity, may also continue to be active against cells which have entered a virus productive cycle (Kurakata et al., 1989).After primary infection immunoregulation appears to be effected via HLA class I-restricted cytotoxic T cells, and/or suppressor T cells reacting against EBV-specific antigens expressed by EBV-containing cells. Such HLA-restricted, EBVspecific, cytotoxic T cells have also recently been shown to be present in small numbers in the blood of patients with infectious mononucleosis (Strang and Rickinson, 1987). Recently, earlier suggestions that there may be a subpopulation of B cells which harbors EBV, but which is not transformed, and which does not express all of the EBV genes usually associated with latent EBV infection-i.e., infection which does not result in virus proliferation-have been revived (Crawford et al., 1978; Tosato and Blaese, 1985). If such a B cell population exists, some of the presently puzzling features regarding the association of EBV with Burkitt’s lymphoma might become more comprehensible. TRANSFORMATION B. EBV-INDUCED LYMPHOCYTE B cells are transformed by EBV after the establishment of a latent infection within the cell and the expression of a subset of viral genes, referred to here as latent genes (reviewed in Dambaugh et al., 1986). Once inside a cell and uncoated, the viral DNA is circularized and becomes associated with the chromosomes in the form of plasmid or episomal molecules (Lindahl et al., 1976). The failure of phosphonoacetic acid and acyclovir, which inhibit viral DNA polymerase, to prevent the replication of latent EBV genomes in B cells indicates that the viral plasmids are replicated with the cell DNA by cellular DNA polymerases (Summers and Klein, 1976; Colby et al., 1980). During the process of circularization a variable number of the 500-bp terminal repeat regions present at each end of the linear molecule are included in the plasmid (Raab-Traub and Flynn, 1986). In any transformed cell clone derived from the same originally infected cell there are multiple

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plasmid copies, each of which contains the same number of terminal repeats. This has provided a valuable means of determining whether all the cells in a given population have originated from a single EBVinfected cell (Raab-Traub and Flynn, 1986). In so-called lymphoblastoid cell lines derived by transformation of peripheral blood lymphocytes, few of the cells produce virus. In fact, since virion production is associated with cell death, i.e., is lytic, virus particles are absent from the majority of cells in the population. The remainder of the cells are latently infected and express genes which are necessary for the maintenance of the EBV plasmids (Dambaugh et al., 1986; Dillner and Kallin, 1988) and the suppression of genes involved in virion replication such as the viral polymerase and genes which code for structural components of the virus particles. It is the latent genes that are of relevance to the potential role of EBV in Burkitt’s lymphoma since lytic infection, being associated with cell death, is inconsistent with neoplasia. This is somewhat ironic, since the serological observations which provided the initial impetus to the study of the role of EBV in the pathogenesis of Burkitt’s lymphoma were reflecting a response against lytic cycle antigens-VCA and EA (Henle and Henle, 1979). This is presumably explained by the generation of such antigens in dying tumor cells, by the very high body burden of EBV-containing cells, and by the immunosuppression which accompanies a large tumor volume (Magrath, 1974; Magrath and Simon, 1976).

EBV Genes Expressed in Latently Infected B Lymphocytes Most of the genes expressed during latent EBV infection are nuclear antigens (EBNAs),i.e., they are present in the cell nuclei prior to or in the absence of a lytic viral cycle. They do not share antigenic determinants, although some of the messenger RNAs contain identical noncoding sequences since individual transcripts are spliced from a large polycistronic messenger RNA which encompasses almost the entire rightward strand of the viral DNA (Bodescot et al., 1987; Spring et al., 1989). There are about three copies per cell of the five EBNA RNAs which are spliced from this large message (Fig. 6 ) ,at least one of which begins with a long open reading frame encoding a sixth nuclear protein (EBNA 5 or EBNA-leader protein) (Petti et al., 1988; Wang et al., 1987). There are about 60 copies per cell of an RNA transcribed from the leftward EBV strand which encodes the latent membrane protein (LMP) (Spring et al., 1989). The commonest transcripts in latently infected cells are the two small nonpolyadenylated RNAs known as EBERs, which are transcribed by RNA polymerase I11 from the

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K)

k b

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P

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FIG.6. (A) Diagrammatic depiction of the general structure of EBV showing unique

(U)and internal repeat (IR) regions as well as the terminal repeats (TR)and oriP region.

(B) Map of thk EBV BamHI restriction endonuclease sites and resultant fragments. (C) Coding regions of the EBV latent gene products.

BamHI J region. These RNAs are structurally similar and can substitute for the adenovirus VA RNAs (Arrand and Rymo, 1982;Bhat and Thimmappaya, 1983;Glickman et al., 1988)which facilitate the translation of adenovirus mRNAs. They are probably involved in a similar function in latently EBV-infected cells, and may also participate in RNA processing (Glickman et al., 1988). EBNA 1 is a polypeptide whose molecular weight ranges from 65K to 73K in different cell lines (Stmad et al., 1981;Dillner and Kallin, 1988).This is mainly a function of the number of internal repeat units (IR3)contained within the EBV BamHI K fragment which encompasses the reading frame (BKRF1) for EBNA 1. Antibodies against EBNA 1 are primarily responsible for the anti-EBNA reactivity of seropositive individuals and the standard anticomplementary test for EBNA (Reedman and Klein, 1973;Hearing et al., 1984).The predominantly immunoreactive region of the molecule is a 20-to 45-kDa glycine-alanine copolymer of unknown function which is encoded by the IR3 region (Dillner et aZ., 1987;Dillner and Kallin, 1988).The carboxyterminal domain of the EBNA 1polypeptide has been shown to bind to the BamHI C region of the EBV genome, a region known as oriP, which contains the origin of EBV plasmid replication (Yates et al., 1984;Rawlins et al., 1985;Wysokanski and Yates, 1989).The binding of EBNA 1 to oriP is necessary for the stable replication of episomal EBV DNA (Yates et al., 1984,1985;Mecsas and Sugden, 1987;Sugden

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and Warren, 1989),and appears to confer an enhancer activity upon a 1.6-kb inverted repeat region of oriP (Lupton and Levine, 1985; Reisman and Sugden, 1986). EBNA 1binds with the highest affinity to the region containing 20 30-bp repeats in oriP, with somewhat lower affinity to the dyad symmetry region of oriP, and with least affinity to another region of the EBV molecule of unknown function, namely the BamHI Q fragment (Jones et al., 1989). It has been suggested that these characteristics permit EBNA 1to tightly regulate both the number of plasmid copies per cell, as well as its own expression. The selfregulatory characteristics of EBNA 1are similar to those of the SV40 T antigen and of c-myc itself. While binding to the dyad symmetry region may actually initiate replication, the availability of EBNA 1to bind to this region is likely to be modulated by the adjacent high-affinity inverted repeat region and probably requires a host factor for replication to occur (Wysokenski and Yates, 1989). Binding to the inverted repeat region of oriP enhances transcription of EBNA 1as well as that of the other latent proteins (EBNAs 2 to 6) through the two major latency promoters in the BamHI C and BamHI W regions (Sample et al., 1986; Bodescot et al., 1984). In the presence of excess EBNA 1 levels, oriP will be saturated and binding to the lower affinity BamHI Q region will be increased. This region, being one through which the primary EBNA 1transcript passes, could possibly act as a chain terminator for EBNA 1 transcripts, thus providing an element of negative control. Similar mechanisms could pertain to the regulation of the c-myc protein, which also binds to a region some 2 kb 5' of the c-myc gene, as described above. It is also possible that EBNA 1, by binding to oriP in episomal plasmids, enables them to bind diffusely and randomly to chromosomal DNA (Dillner and Kallin, 1988), which is a property of EBNA 1but not the other EBNA proteins. Such binding to chromosomes could be important to the even distribution of EBV episomes between daughter cells at mitosis. Finally, a transgenic mouse model in which EBNA 1 is under the control of EH and the polyoma virus promoter has recently been described in which the mice develop a monoclonal lymphocytic lymphoma that appears to arise in immature B cells (Spring et aZ., 1989). EBNA 2 is a polypeptide with a molecular weight of approximately 82K, which has been shown to be encoded by the BYRFl reading frame of the BamHI WY and H fragments of the EBV genome. This was shown by the demonstration that antisera raised against synthetic peptides deduced from the BYRFl reading frame reacted with EBNA 2 (Dillner et al., 1985). In addition, induction of EBNA 2 has been demonstrated by transfection of cell lines with the BamHI WYH re-

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gion of EBV (Rymo et al., 1985; Mueller-Lantzsch et al., 1985). However, this reading frame differs in different EBV strains, the prototype EBNA 2 (referred to as type A) being present in the B95-8 virus and a second, smaller molecule, type B (molecular weight of 75K) being present in the AG876 virus (Pizzo et al., 1978; Polack et al., 1984; Adldinger et al., 1985). These virus strains have also recently been shown to differ at other loci [i.e., the coding region for the small RNAs and EBNA 3b and 3c (Arrand et al., 1989; Rowe et al., 1989)] and whereas both EBNA 2 and EBNA 1vary markedly in molecular weight in type A strains, both EBNA molecules are of similar size regardless of geographic origin in type B virus (Young et al., 1987). Some virus strains obtained from Burkitt’s lymphoma-derived cell lines, including P3HRl and Daudi, have deletions of approximately 6 kb in their BamHI WYH fragments (and a corresponding lack of EBNA 2) (RaabTraub et al., 1978; Bornkamm et al., 1982; Dambaugh et al., 1984; Jones et al., 1984), but such deletions are never present in EBVtransformed lymphoblastoid cell lines (Dillner et al., 1985, Ernberg et al., 1986). Viruses which contain deletions in BamHI WYH are nontransforming, and it is believed, therefore, that EBNA 2 is necessary for lymphocyte transformation (Dambaugh et al., 1986; Volsky et al., 1984; Dillner and Kallin, 1988). However, when B lymphocytes are infected with P3HR1, EBNAs 1 and 3 as well as the RNA species known as EBER 2 are not expressed even though they are intact in P3HR1, and can be expressed in EBV negative cell lines. Thus EBNA 2 may enhance its effects by influencing the expression of other latent genes (Rooney et al., 1989). The EBNA 2 protein binds very tightly to DNA, high salt concentrations being required to dissociate EBNA 2IDNA complexes. EBNA 2 has been shown by retrovirus-mediated transfection to decrease serum dependence (Dambaugh et al., 1986)in primary fibroblasts (Rat-1 cells) and to induce the activation antigen, CD23 (also known as Blast-2), on the surface of EBV-negative Burkitt’s lymphoma cell lines (Wang et al., 1987). This antigen, which is also induced by IL-4, y-interferon, and LMP (Rousset et al., 1988; Bonnefoy et aZ., 1988; Spring et al., 1989), is a receptor for IgE and is related to the receptor for a B cell growth factor (Gordon et al., 1986a,b), thus providing a possible ’mechanism whereby EBV may transform B lymphocytes. It has even been suggested that CD23, when shed from the cell surface [as occurs after IL-4 stimulation, for example (Cairns et al., 1988)],acts as an autocrine growth factor for B cells (Swendeman and Thorley-Lawson, 1987, 1988). The B95-8 virus strain, but not P3HR1, has been shown to be able to induce the CD21 antigen (C3d and EBV receptor) and Bac 1, a potential B cell growth factor receptor (Calender

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et al., 1987).The difference in the virus strains in this regard presumably relates to the deletion in the BamHI WYH region coding for EBNA 2. Other EBNAs, including 3,4,6 (also known as 3a, 3b, and 3c) encoded by the BamHI E fragment, and 5 (leader protein), have been described (Dillner and Kallin, 1988; Petti and Kieff, 1988; Shimizu et al., 1988; Allday et al., 1988), but their functions to date remain unknown and they will not be discussed further here. LMP is a protein of 58 kDa with six strongly hydrophobic regions, each of which is believed to span the cell membrane of EBV-transformed cells (Fennewald et al., 1984; Dillner and Kallin, 1988; Dambaugh et al., 1986). Both amino and carboxy terminals are on the cytoplasmic side of the cell membrane (Liebowtiz et al., 1986). LMP transfected via a retroviral vector can reduce the serum dependence of mouse fibroblasts (3T3 cells) and induces loss of contact inhibition, anchorage independence, and tumorigenicity in nude mice of Rat-1 cells (Wang et al., 1985). This, however, applies only to the form of LMP expressed during latent infection, and not the truncated form (with a deletion at the amino terminal end) expressed in cells permissive for EBV replication (Wang and Liebowitz, 1988). LMP induces vimentin, CD23, LFA-1, LFA-3 (lymphocyte functional antigens), and ICAM-1 (intercellular adhesion molecule) expression in Burkitt’s lyrnphoma cells and colocalizes with vimentin in B lymphocytes (Spring et al., 1989; Birkenbach e t al., 1989; Liebowitz et al., 1987). It also appears able to function as a target for EBV-specific cytotoxic T cells (Murray et al., 1988). Mice bearing an LMP transgene controlled by the polyoma enhancer/promoter develop hyperkeratosis of the skin (Spring et al., 1989). There can be little doubt that EBV induces lymphocyte transformation via one or more of the genes expressed in latently infected cells. Primary candidates are EBNA 2 and LMP, but it is quite likely that other proteins also participate. One probable mechanism involves the transactivation of cellular genes which code for growth factor receptors, and possibly others which code for growth factors. This is entirely consistent with data supporting the presence of an autocrine growth factor loop in B lymphoblastoid cell lines (Gordon et al., 1984a,b). The expression of latent genes, however, not only leads to cell transformation, but also provides antigenic targets for specifically immune T cells. Antigenicity is further heightened in lymphoblastoid cell lines by the increased expression of the lymphocyte functional antigen, LFA-3, which interacts with CD2 on the surface of T cells (Shaw et al., 1986). Such interactions are believed to be necessary for effective T cell killing.

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C. SPECIFIC T CELLRECOGNITION OF EBV-INFECTED B CELLS Following primary infection with EBV, the acute phase of which is largely controlled by NK cells and nonspecific, cytotoxic T cells, normal individuals develop specific T cell-mediated immunological memory which is restricted to cells bearing the same HLA class I antigens (Rickinson 1986; Tosato, 1987). Thus, outgrowth of EBVcontaining B lymphoblastoid lines in vitro is inhibited by the presence of autologous T cells, and EBV-induced immunoglobulin production is likewise rapidly abrogated in seropositive individuals (Tosato et al., 1982; Tosato and Blaese, 1985; Tosato, 1987). The antigens against which the T cell response is directed are incompletely defined, and have long been referred to as “lymphocyte defined membrane antigen” (LYDMA), a term which does not connote a specific antigen, but alludes to the assumption that EBV-encoded antigens must be present in the membrane of EBV-infected cells for a T cell cytolytic response to be elicited (Moss et al., 1981; Rickinson et ul., 1981).An obvious candidate for LYDMA is LMP, since this is known to be membrane associated, and it is likely that this protein is at least responsible for some component of T cell recognition. There is evidence, for example, that LMP is the antigen responsible for the phenomenon of leukocyte migration inhibition in peripheral blood T cells of seropositive individuals (Szigeti et al., 1984,1986; Sulitzeanu et al., 1986).Recently, it was shown that T cells from seropositive individuals but not from seronegative individuals would proliferate in response to a synthetic peptide fragment of LMP (Thorley-Lawson and Israelsohn, 1987). Moreover, some of the clones derived by stimulation with the LMP peptide could specifically lyse an autologous EBVinfected lymphoblastoid cell line, but not autologous EBV-negative lymphoblasts or allogeneic EBV-infected lymphoblasts. Similar clones were derived at a much lower frequency without prior LMP stimulation. Thus, LMP may be one component of the natural EBV-specific T cell response. There is evidence that the EBNAs may also be immunoreactive with T cells. Precedents for an immune response directed against nuclear proteins exist in a number of systems, including influenza. Fragments of such proteins are expressed at the cell surface in the cleft of HLA class I antigens after intracellular processing. In fact, there is no doubt that the EBNAs are antigenic because of the well-defined serological responses against them, which have been described, indicating that the EBNAs can be presented to antigen-responsive cells. The epitopes specific for EBNA 2 types A and B have been shown to reside in the carboxy terminal of the molecule (Rowe and Clarke, 1989). Recently,

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cytotoxic T cell clones were obtained from seropositive individuals which specifically reacted with lymphoblasts infected with either type A or type B EBV, but not with the other (Moss et al., 1988). It seems highly likely that such clones were reacting with EBNAs-either EBNA 2, or possibly epitopes present on other EBNAs which also differ in the A and B strains (e.g., EBNA 3). A somewhat similar observation was made with EBV-negative cell lines that had been infected with EBV. Recognition by EBV-specific, HLA-restricted cytotoxic T lymphocyte preparations was apparent when the lines were converted with B95-8 virus, but not when P3HR1 was used to infect the cell lines. In the latter case, neither EBNA 2 nor LMP was expressed, although there was no difference in the expression of HLA determinants (Murray et al., 1988).Recently, cell mediated immune responses against EBNA antigens were demonstrated by using a leucocyte migration inhibition assay (Szigeti et al., 1989). These findings are critically important to an understanding of the association of EBV with Burkitt’s lymphoma, since ifthe same proteins responsible for lymphocyte transformation also elicit a specific immune response against the EBV-transformed B cell, then the net result is a self-limited lymphoproliferation-exactly the situation in infectious mononucleosis and subclinical EBV infection in normal individuals. The potential for mortality caused by the proliferation of EBVcontaining B lymphoblasts (or immunoblasts) in immunosuppressed individuals emphasizes the critical importance and efficiency of the normal immune response against EBV-infected cells, especially in light of the fact that EBV probably infects in excess of 90% of the human race. It would seem, therefore, essentially impossible that the oncogenic potential of EBV in Burkitt’s lymphoma is exerted via the same mechanisms utilized in the transformation of B lymphocytes, unless there exist parallel mechanisms whereby the neoplastic cells can escape from T cell surveillance against EBV antigens In fact, the presence of EBV in a Burkitt’s lymphoma, should it express all its latent genes, represents a disadvantage to the tumor cell. This very fact makes it probable that EBV does indeed have an important role in the genesis of at least a subset of Burkitt’s lymphomas, for there seems little reason otherwise for EBV-infected cells to be preferential targets for tumorigenesis in endemic Burkitt’s 1ympho:na-in seropositive individuals, EBV-infected B cells are in the minority: less than 1% of the total B cell pool (Moss et al., 1983a; Tosato, 1987). Even infection of the cells after tumorigenesis is rendered most unlikely by these observations, unless EBNA 2 is lacking (certainly not the case in most

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endemic Burkitt’s lymphomas) (Rowe et al., 1987) or down-regulated. In exploring this issue further, the focus of our attention is appropriately directed toward an examination of latent gene expression in Burkitt’s lymphoma and the ability of Burkitt’s lymphoma cells infected with EBV to avoid immunosurveillance.

D. EBV LATENT GENEEXPRESSION IN BURKITT’S LYMPHOMA In recent years it has become clear that expression of the EBV latent genes, especially LMP and EBNA 2, differs in Burkitt’s lymphoma compared to EBV-transformed lymphoblastoid cell lines. Modrow and Wolf synthesized oligopeptides corresponding to the hydrophobic amino-terminal region of LMP and to the repeat unit which is part of the large carboxy-terminal hydrophilic region and raised antibodies against them (Modrow and Wolf, 1986). Interestingly, two polypeptides can theoretically be expressed from the BNLFl reading frame which codes for LMP (Baer et al., 1984), one of which lacks 138 amino acids from the amino terminus (which includes 4 of the transmembrane regions of LMP). The first peptide made by Modrow and Wolf was from this region. By immunoprecipitation experiments, they were able to show that this smaller protein was not expressed in several Burkitt’s lymphoma cell lines but was present in EBV-transformed lymphoblastoid cell lines. Hatzubai et al. (1987) made an antiserum against an Escherichia coli fusion protein containing the carboxy half of LMP and showed that it precipitated a protein from EBV-infected cells and was able to block leukocyte migration inhibition induced by the membrane protein (although interestingly, antibodies directed against LMP do not appear to be a part of the normal humoral response to EBV infection). Using this antibody in a radioimmunoassay, these investigators also measured LMP in a number of cell lines. Eight EBV-negative Burkitt’s lymphoma cell lines were negative, and 18 of 21 EBV-carrying Burkitt’s lymphoma lines were positive with a range from C0.3 to 6.4 units/mg protein in the cell membrane. In contrast, lymphoblastoid cell lines contained levels ranging from 1.5 to 14.8 unitslmg protein. LMP mRNA levels were also uniformly higher in lymphoblastoid cell lines than in Burkitt’s lymphoma cell lines, Masucci et al. (1987) found that levels of LMP in EBV-positive Burkitt’s lymphoma cell lines were between 10 and 20% of the levels in EBV-transformed cell lines derived from the same individuals. The levels of LMP were as low in EBV-converted Burkitt’s lymphoma cell lines as in cell lines which were EBV positive from the outset. For

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reasons discussed below, it is likely that LMP is expressed at even lower levels, or may be absent from tumors in uiuo. In addition to a reduction of LMP expression, it has also been shown that EBNA 2 expression may be absent in EBV-positive Burkitt’s lymphoma. Ernberg et al. (1986), using immunoblotting and anticomplement immunofluorescence, were unable to detect EBNA 2 in 17 of 27 such cell lines, while 26 of 27 EBV-transformed lymphoblastoid cell lines expressed EBNA 2. D. T. Rowe et al. (1986) found that LMP and/or EBNA 2 expression was undetectable in four of eight newly derived EBV-positive Burkitt’s lymphoma cell lines, although they were unable to demonstrate any correlation between the expression of these antigens and susceptibility to T cell killing. Viruses obtained from the cell lines which failed to express EBNA 2 were, however, able to express EBNA 2 in lymphoblastoid cell lines. EBNAs 1and 3 were invariably expressed in these lines. These investigators subsequently extended these data by examining 24 newly derived EBV-positive Burkitt’s lymphoma cell lines (Rowe et al., 1987). They found that some lines retained the biopsy cell phenotype and failed to express activation antigens, EBNA 2, or LMP, while others were, to a greater or lesser degree, phenotypically similar to lymphoblastoid cell lines (i.e., with regard to the expression of activation antigens such as Ki-24, CD23, CD39, and Ki-1). Seven lines which expressed no activation antigens, or Ki-24 only, failed to express either EBNA 2 or LMP. One cell line expressing Ki-24 expressed only EBNA 2 and no LMP, while the remaining line in this group expressed both EBNA 2 and LMP. All remaining 15cell lines, which expressed more activation antigens, also expressed, in all cases, EBNA 2 and LMP. In three biopsies, EBNA 2 and LMP were undetectable. In two lines which were followed during serial passages, there was a change from lack of expression of EBNA 2 and LMP in both to LMP expression in one and low levels of both EBNA 2 and LMP in the other. In all cases there was available an EBV-transformed cell line from the same patient, and in each case EBNA 2 and LMP were expressed. In all 24 cell lines and the biopsy specimens, EBNA 1 was present. Interestingly, EBV-negative Burkitt’s lymphoma cell lines failed to express activation antigens even after several years in culture (M. Rowe et al., 1986) but, when converted to EBV positivity in uitro, began to express such antigens. In the case of one BL-41 converted line, in which there was a striking shift towards a lymphoblastoid phenotype, with high class I HLA and LMP expression, reversion to minimal tumorgenicity in immunosuppressed mice, and low agarose clonability was observed (Torsteinsdottir et al., 1989). This finding is consistent with the expression of CD23 in EBV-

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negative cell lines into which the EBNA 2 gene has been introduced by means of a retroviral vector (Wang et al., 1987). EBNA 5 was first detected by raising antisera against peptides synthesized on the basis of the amino acid sequences deduced from the transcript encoded by the BamHI W, Y, and H fragments (Bodescot et aZ., 1984).These antibodies precipitated a nuclear polypeptide from lymphoblastoid cells whose molecular weight ranged from 22K to 70K [depending upon the number of repeat units (BamHI W) incorporated into the protein]. Using these antisera, EBNA 5 was not detectable in 10 of 11 EBV-containing Burkitt’s lymphomas (Dillner et al., 1986), although it was shown to be present in all 10 lymphoblastoid cell lines tested. Moreover, infection of the EBV-negative Burkitt’s lymphoma cell line, Ramos, failed to induce EBNA 5. Antibodies against EBNA 5 (or rather, against one of the synthetic peptides used in the above experiments) are occasionally present (4 of 42) in EBV-seropositive individuals, These findings, although predominantly obtained from cell lines, indicate that LMP, EBNA 2, and EBNA 5 may be low or absent in EBV-positive Burkitt’s lymphoma cells. There is evidence, based on up regulation of EBNAs 2 to 6 and LMP by 5-azacytidine in a cell line which normally expresses only EBNA 1, that DNA methylation is involved in the suppression of EBNA expression (Masucci et aZ., 1989). While the data clearly need to be extended to include more fresh biopsies in order to obtain a clearer picture of the frequency of expression of these proteins in the tumor cells in uiuo, it is possible to conclude already that LMP, EBNA 2, and EBNA 5 are not essential for the proliferation of EBV-containing Burkitt’s lymphoma cells; i.e., that these proteins, which appear to be essential to EBV-induced lymphocyte transformation, and are expressed in EBV-associated posttransplant lymphoproliforative syndromes as well as the “lymphomas” induced by EBV in cottontop tamarins (Young et al., 1989a,b), do not mediate any role that EBV might have in the pathogenesis of Burkitt’s lymphoma. This is not as surprising as it may seem at first sight, for Burkitt’s lymphoma cells contain a deregulated c-myc gene and would not, therefore, be expected to require the same proliferative drive from EBV genes as lyrnphoblastoid cell lines or EBV-associated lymphoproliferative syndromes arising in immunodeficient hosts. Further, the latent EBV genes also carry a liability for any potential tumor cell, since they also provide antigenic targets for cytotoxic T cells. Indeed, it has been shown that EBV-positive Burkitt’s lymphoma cell lines which express more activation antigens are more susceptible to EBVspecific T cell cytolysis (Rooney et al., 1986). Attention should there-

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fore be focused upon EBNA 1 and perhaps ENBA 3 as genes which may be critical to the pathogenesis of EBV-positive Burkitt’s lymphoma. The high frequency of lymphomas which have been observed in mice transgenic for EBNA 1(Spring et al., 1989)serves to emphasize this point. E. REDUCEDSUSCEPTIBILITY TO T CELLCYTOLYSIS OF BURKITT’S LYMPHOMA CELLS While a powerful drive to proliferation is an important component of the pathogenesis of Burkitt’s lymphoma, unless the tumor cells can escape both specific and nonspecific regulatory mechanisms, neoplastic growth cannot occur. Immune killing of potential tumor cells is particularly likely to occur when foreign antigens, specifically EBV antigens, are present in the tumor cells. Clearly, potentially neoplastic cells must be able to circumvent EBV-directed killing if they are to be manifested as a tumor, particularly since there is good evidence that EBV infection can occur up to 18 months prior to the development of Burkitt’s lymphoma (de The et al., 1978). In other words, only cells which are able to escape the T cell response directed toward EBVinfected cells can proliferate as a neoplasm. The altered expression of latent genes in EBV positive Burkitt’s lymphoma compared to lymphoblastoid cell lines transformed by EBV suggests one mechanism whereby tumor cells can avoid the EBVspecific immune response-suppression of the expression of antigens which can be recognized by T cells (LYDMA). It has, in fact, been clearly demonstrated that EBV-containing Burkitt’s lymphoma cells are less susceptible to EBV-specific T cell-mediated immunoregulation-both allospecific and EBV specific-than EBV-transformed B cells derived from the same patient, although susceptibility of cell lines differed quite markedly (Rooney et al., 1984,1986; Torsteinsdottir et al., 1984, 1986a,b; Klein et al., 1986). The variable susceptibility was shown to correlate with cell surface markers. Cell lines which retain, in uitro, the phenotype of the original tumors from which they were derived (expressing only the common ALL antigen and BLA) were less susceptible to allospecific and natural killer-like (nonspecific) T cell cytotoxicity (i-e., a cytotoxic response primarily directed toward HLA class I antigens) than cell lines which progress in uitro toward a lymphoblastoid phenotype and develop activation antigens such as Ki-24, Ki-1, CD23, and CD39 (Rooney et al., 1986). This correlated with a reduced quantity (or, at least, reduced reactivity with appropriate monoclonal antibodies) of HLA class I and I1 framework

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antigens on the surface of the cells which retained the phenotype of the original biopsy compared to lymphoblastoid cell lines. EBV-negative Burkitt’s lymphoma cell lines are also very poor stimulators in mixed lymphocyte cultures (Lea,systems in which the T cell target is the HLA class I antigen) (Avila-Carifio et al., 1987). Conversion of such lines to EBV positivity was shown to increase their ability to stimulate in mixed lymphocyte cultures, but the explanation for this was not clear and did not seem to be due to increased expression of HLA or activation antigens. Nor was there a difference in the ability of such EBV-converted cells to stimulate EBV-seropositive versus EBVseronegative donors.

1. Reduced Expression of H L A Class 1 Antigens on Burkitt’s Lymphoma Cells Other investigators have observed a lower level of HLA antigen expression on Burkitt’s lymphoma cell lines (both EBV positive and negative) than on lymphoblastoid cell lines (Masucci et aZ., 1987; Torsteinsdottir et al., 1988).Torsteinsdottir and colleagues (1988)have shown that the low expression of HLA (compared to autologous lymphoblastoid lines) applies primarily to the polymorphic determinants, not the framework antigens, and can be up regulated by EBV infection of the cell lines, although the degree to which this occurs appeared to be dependent upon the degree to which the phenotype of the cells shifts in a “lymphoblastoid” direction (Torsteinsdottir et al., 1988). Treatment with y-interferon and tumor necrosis factor have also been reported to result in increased expression of HLA in some cell lines, which was associated, in one case, with increased susceptibility to T cell cytotoxicity (Avila-Carifioet al., 1988).This differential expression of polymorphic versus monomorphic (framework) HLA class I antigens on Burkitt’s lymphoma cell lines is also observed on resting B and T lymphocytes, which is consistent with the possibility that the normal counterpart cell of Burkitt’s lymphoma is a resting B cell (Torsteinsdottir et al., 1988). It is presumably due to the orientation of the HLA molecule in the cell membrane, based on the finding that different polymorphic epitopes on the same HLA molecule may also be differentially expressed.

2. Reduced Expression of Lymphocyte Functional Antigens (LFA) and lntercellular Adhesion Molecules (ICAM)in Burkitt’s Lymphoma In addition to reduced expression of HLA antigens, Burkitt’s lymphoma cell lines have been shown to express lower levels of receptors

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belonging to the integrin family, LFA-1, LFA-3, and ICAM-1, than EBV-transformed lymphoblastoid cell lines (Billaud et al., 1987). EBV-negative Burkitt’s lymphoma cell lines have very poor expression of all of these molecules, while EBV-positive lines have an intermediate expression between EBV-negative cell lines and lymphoblastoid lines (Billaud et al., 1987; Patarroyo et al., 1988). Recently it was shown that LFA-1, ICAM-1, and LFA-3, like activation antigens, are expressed at very low levels, or not at all, in EBV-positive Burkitt’s lymphoma cell lines which show a restricted pattern of latent EBV genes-the usual pattern of biopsied tumors and of cell lines immediately after explantation (Gregory et al., 1988).Cell lines which progressively became more lymphoblastoid in character, and which tended to grow in large cell aggregates, expressed higher levels of LFA-1 and ICAM-1, the molecules which mediate intercellular adhesion. All of these molecules are important to T cell binding to target cells, but the interaction between LFA-3 and CD2 is probably essential for target cell lysis by cytotoxic T cells (Shaw et al., 1986, Shaw and Luce, 1987; Hynes, 1987; Makgoba et al., 1988a,b).The low or absent expression of these molecules thus provides an additional reason for the resistance of Burkitt’s lymphoma cells to T cell immunoregulation (Gregory et al., 1988). Interestingly, LFA-1 also appears able to promote the binding of lymphocytes to the high endothelial venules of lymph nodes and Peyer’s patches, which serve as entry portals for cells circulating in the peripheral blood (Hamman et al., 1988; Pals et al., 1988).Thus, the low expression of this antigen on Burkitt’s lymphoma cells could in part account for the relative infrequency of involvement of lymph nodes. As is the case for HLA molecules, lymphocyte functional antigens are expressed at low levels in resting lymphocytes, and are up regulated in activated cells. This is also consistent with evidence derived from other sources that the phenotype of Burkitt’s lymphoma is much more akin to that of a resting B cell than an activated B cell. Against this explanation is the finding that in EBV-transformed cell lines transfected with c-myc genes under the influence of heterologous enhancers, increased c-myc expression was associated with a decrease in expression of LFA-1 (Seremetis et al., 1988).

F. OTHEREVIDENCE FOR A PATHOGENETIC ROLEFOR EBV IN BURKITT’SLYMPHOMA The demonstration that sporadic and endemic Burkitt’s lymphomas differ with regard to the predominant structural changes in and around the c-myc gene, coupled to their different rates of EBV association,

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makes it highly probable that EBV association in Burkitt’s lymphoma correlates with the breakpoint locations on chromosome 8. We recently demonstrated that such a correlation does exist (Barriga et al., 1988a). Twenty-two of 23 tumors, including 19 endemic and 3 sporadic cases, in which the c-myc gene was not rearranged (i.e., in which there was a far 5’ breakpoint) were EBV positive. The remaining EBV-negative tumor was an unusual sporadic tumor which did not express any immunoglobulin heavy chain. Tumors in which c-myc was rearranged were either EBV positive or negative, but among the sporadic tumors only 1 of 15 tumors with a chromosome 8 breakpoint immediately 5’ of c-myc was EBV positive. These data are suggestive, but must be considered as preliminary. They raise the possibility that certain structural changes brought about by the chromosomal translocation, but not others, have an obligate requirement for the participation of an EBV gene in the process of neoplastic transformation. This, in turn, suggests that EBV is likely to participate in the process of deregulation of c-myc rather than simply cooperating with c-myc, since if all breakpoints on chromosome 8 resulted in c-myc deregulation without the need for the participation of EBV, then there would be no reason to expect an association of EBV with specific breakpoint locations. However, the possibility that different structural changes result in different levels of c-myc expression has not yet been explored, and it remains possible that EBV is necessary when c-myc expression is below a certain level in a given intracellular milieu. An alternative explanation for the correlation between EBV and breakpoint location is that each type of structural change in c-myc occurs in a different target cell, and that EBV can influence the size of some target cell populations but not others, thus influencing the likelihood of the occurrence of a translocation in these populations. These questions will only be resolved by a much more extensive analysis of EBV association and breakpoint location-an investigation that will be considerably aided by utilizing the differences in EBV association of Burkitt’s lymphomas in different parts of the world.

G. POSSIBLE ROLE FOR

AN

EBV TRANSACTIVATOR GENE

If EBV does have a direct role in tumorigenesis, it is likely that this will be via a transactivating gene which influences the expression of another cellular gene. Candidate genes for this include c-ras and c-raf for reasons discussed in the next section. The association of c-fgr expression with EBV infection is also worthy of mention in this context (Cheah et al., 1986). It is also possible, however, that an EBV gene could provide a protein capable of binding to an enhancer region of

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c-myc and inappropriately increasing its expression. Increasing knowledge of the complexities of transcriptional regulation indicate that occupancy of a given cis-regulatory element by a protein may vary in its effect, depending upon the occupancy of adjacent regulatory elements. In some Burkitt's lymphomas, predicting the effects of a given regulatory element is further complicated by the removal of some cis-acting elements by the translocation. This is at its most extreme in the case of intron breakpoints when most, if not all, of the regulatory elements are removed. Such a truncated gene, as discussed above, appears unable to function unless provided with an alternative (heterologous) enhancer element-presumptively part of the immunoglobulin locus, In tumors with intron breakpoints, a role for EBV could depend upon the precise location within the intron of the breakpoint, whether or not there are mutations in remaining intron regulatory elements, and the nature of the juxtaposed immunoglobulin sequences. Naturally, if an EBV gene is to influence c-myc expression, its product must be able to bind to a regulatory element on the remaining part of the c-myc gene, or it must become integrated close enough to the gene to be able to influence its expression. The latter is an unlikely possibility, particularly since the bulk of EBV genomes are episomally situated, but it has not been rigorously excluded except in the case of the cell line Namalwa, in which a single EBV genome is integrated on chromosome 1 (Matsuo et al., 1984; Dambaugh et al., 1986). In tumors in which part or all of the c-myc regulatory region remains intact, an EBV protein could either block the binding of a suppressor protein or bind to a positive element and increase transcription. It seems unlikely that EBV suppresses the block to chain elongation at the end of exon 1,since some EBV-positive cell lines still possess such a block, e.g., PA682 (Zajac-Kayeet a1.,1988b). The intriguing similarity between the region approximately 2 kbp upstream of c-myc, which possesses both enhancer function and can act as an autonomously replicating sequence, and the oriP region to which EBNA 1 binds, suggests that it would be worthwhile exploring the possibility that EBNA 1 can bind to the HindIIIIPstI fragment 5' of c-myc-either directly, or through the intervention of other protein factors. There are, however, no obvious sequence homologies between this region and oriP. Interestingly, tumors with a breakpoint such that this enhancer element is removed from its possible influence on the PI and PZpromoters are almost always EBV negative.

H. ANIMALMODELSOF EBV-INDUCED LYMPHOPHOLIFERATION

A number of animal models have been used to study the induction, by EBV, of fatal lymphoproliferation. These include primates such as

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owl monkeys and cottontop tamarins, which may develop fatal lymphoproliferation when inoculated with transforming strains of EBV (for overview, see Magrath, 1983b) and, more recently developed, SCID mice in which EBV associated B cell “lymphomas” develop when transplanted with peripheral blood Ieucocytes from EBV seropositive, but not EBV seronegative donors (Mosely et id.,1989). These experimental models, which lack characteristic karyotypic abnormalities, more closely parallel the EBV-associated lymphoproliferations and lymphomas associated with immunodeficient or immunosuppressed patients than Burkitt’s lymphoma arising in otherwise normal individuals. Nonetheless, Burkitt’s lymphoma associated with characteristic chromosomal translocations can arise in the setting of immunological insufficiency, so that such models may provide useful experimental systems for the elucidation of the molecular and biological events involved in its pathogenesis.

XVI. Synthesis While many gaps remain and fine detail is often dimly perceived, the last decade has witnessed the emergence of an overall conception of the molecular events which lead to Burkitt’s lymphoma. Based on the molecular abnormalities, it would appear that each tumor is unique in some respect; but at the same time, subgroups with the same fundamental pathogenetic changes can be discerned. Much more information will be required before it can be deduced whether different subgroups arise from different normal cells, or whether different molecular lesions are indicative of different etiological events occurring in relationship to the same normal target cell. However, the following summary comments should be considered as applying to at least a major portion of the disease currently identified as Burkitt’s lymphoma.

A. CELLULAR ORIGINS Two main sources of evidence derived from studies of Burkitt’s lymphoma point toward the probability that the normal counterpart cell of Burkitt’s lymphoma is a resting B cell. First is the lack of expression of the normal c-myc allele. The best interpretation of available evidence suggests that this is primarily a result of a more closed chromatin pattern rather that a suppressor protein (although the latter is probably one mechanism of regulating the c-myc level in cells which express this gene), suggesting that c-myc is not expressed in the normal counterpart cell. Second is the paucity ofactivation antigens and cellular adhesion molecules on the surface of EBV-negative Burkitt’s lym-

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phoma cell lines, freshly biopsied EBV-positive tumor cells, and very recently derived cell lines from EBV-associated Burkitt’s lymphoma, as compared to antigen- or EBV-transformed lymphoblastoid cells. It has become clear that a number of EBV-containing Burkitt’s lymphomas undergo phenotypic progression in vitro such that activation antigens which were not present on the original in vivo tumor cells are expressed on the cultured cell line. Such antigens appear to be induced by EBV itself, although why they are suppressed in vivo is not known. The evidence from Burkitt’s lymphoma cells is supported by the transgenic mouse systems, in which there is more clear-cut evidence that premalignant pre-B cells are unable to enter a resting phase because of the expression of the deregulated c-myc transgene. A distinction should be made between the normal counterpart of the predominant cell type of Burkitt’s lymphoma and the cell type in which the chromosomal translocation occur. The latter is almost certainly a proliferating cell undergoing either primary (antigenindependent) or secondary (antigen-dependent) differentiation, since resting cells are either virgin B cells or memory cells. There is no presently available method of deciding which of these two possibilities applies. Indeed, both may apply, giving rise to two major subtypes of Burkitt’s lymphoma. These subtypes are likely to be further characterized on the basis of other features such as the breakpoint locations; one might surmise that J breakpoints are more likely to arise during primary differentiation (close to the time of VDJ joining), while S breakpoints are more likely to arise during secondary difyerentiation (close to a major time for heavy chain class switching after antigen stimulation), although this is by no means necessarily the case, since heavy chain class switching can occur in immature cells. Thus, the two proposed subtypes could correspond either to endemic and sporadic tumors, or to tumors with breakpoints outside or within an immunoglobulin switch region. It appears likely that recombinases normally catalyzing immunoglobulin gene rearrangements mediate some or all of the translocations, the DNA sequences at the breakpoint being either normal signal sequences, sequences that resemble normal signal sequences, or repetitive elements such as LINE or A h . The primary result of the translocation is to cause persistant (deregulated) expression of c-myc when this gene should have normally been shut off, such that the cell continues to proliferate. Activation antigens, other than those that may be directly or indirectly influenced by c-myc expression itself, are not expressed and HLA class I and lymphocyte adhesion molecules are expressed at lower levels than in normal proliferating B cells. If the cell contains EBV, its latent gene expression

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will also, presumably, conform to the pattern expected in a normal resting B cell. At the present time there is no direct information as to whether there is a pattern of latent gene expression specific to resting cells. However, since circulating cells which contain EBV and are capable of undergoing spontaneous transformation in uitro are probably not destroyed by T cell in uiuo,as evidenced by maintenance of the level of such cells in acyclovir-treated patients, they are unlikely to express those EBV genes operationally referred to as LYDMA. These probably include LMP and EBNA 2. Thus, Burkitt’s lymphoma cells do not express those antigens which would result either in their nonspecific suppression by T cells, i.e., activation antigens, or in their specific lysis, by virtue of their expression of EBV coded antigens. €3. ENVIRONMENTAL FACTORS AND IMMUNOSUPPRESSION

The markedly increased incidence of endemic Burkitt’s lymphoma compared to the sporadic form of the disease clearly implicates environmental factors in pathogenesis. The probability that such factors (primarily holoendemic malaria and early infection with EBV) increase the size of the target cell population because of impaired immunoregulation of B cells is supported by the increased incidence of Burkitt’s lymphoma (and histologically classified immunoblastic lymphomas bearing 8;14 translocations) in patients with immunodeficiency syndromes. This notion is further supported by the observation of progression of polyclonal lymphoproliferative processes (unassociated with a chromosomal translocation) in HIV-infected patients to monoclonal Burkitt’s lymphoma, and is entirely consistent with the murine models of lymphomagenesis; mice harboring a transgenic c-myc gene have a marked pre-B hyperplasia prior to the onset of true malignant tumors while in pristane-treated mice the incidence of plasmacytomas can be increased by infection with Abelson leukemia virus. However, the incidence of EBV-containing lymphomas in individuals with HIV infection is somewhat less than 50%, so that in this case EBV-negative cells appear equally good targets for the transformational event. In predominantly EBV-positive endemic Burkitt’s lymphoma holoendemic malaria may be responsible for lessening the control of EBV-infected cells, which provide the targets for lymphomagenesis in equatorial Africa. It would not, however, be appropriate to compare the degree of immunosuppression in an African child with that of an individual with a congenital immunodeficiency disease or HIV infection, although the incidence of Burkitt’s lymphoma in these two groups may be roughly proportional to the degree of immunosup-

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pression. Some 2-5% of adults with HIV infection develop lymphomas as opposed to 0.08-0.15% of African children up to the age of fifteen. Thus, the incidence of EBV-positive Burkitt’s lymphoma is far higher in patients with HIV infection. The reason for the difference in breakpoint location between these two groups is not clear. It could relate to differences in the target cell populations that are expanded, or differences in the age of the patient. For example, it is possible that the target cell is a pre-B cell in an African child and an activated B cell in a patient with HIV infection. Further studies in lymphomas in children with HIV infection or older individuals with endemic Burkitt’s lymphoma may clarify this. There can be little doubt that the risk of developing a lymphoma is a function of the size of the target cell population and that environmental factors which predispose to Burkitt’s lymphoma almost certainly do so by causing an increase in the number of target cells in individuals at risk. Unlike the transgenic mouse model, however, there can be little doubt that hyperplasia of the target cell occurs prior to chromosomal translocation and c-myc deregulation in the pathogenesis of Burkitt’s lymphoma.

C. SIGNIFICANCE OF CLINICAL AND BIOLOGICAL DIFFERENCES

The clear differences in clinical features and EBV association of Burkitt’s lymphoma in different geographic regions, coupled to the correlation between geography and chromosomal breakpoint location, indicates beyond any reasonable doubt that endemic and sporadic Burkitt’s lymphoma are separate, although closely related, diseases. In other words, it is likely that the cell of origin differs, and that etiological factors and pathogenetic mechanisms differ. It would not be unreasonable to reserve the term Burkitt’s lymphoma for the equatorial African disease, and possibly for a biologically identical disease in other world regions, if biological identity can be established. More information is required before precise definitions can be devised, but a reasonable starting point would be EBV association and a far 5‘ chromosomal breakpoint on chromosome 8. More information needs to be collected before inclusion of the chromosome 14 breakpoint in the definition. In a population of such patients there would be expected to be a high frequency ofjaw tumors, particularly in younger children. It should be borne in mind that the geographic separation of these diseases is not absolute, and that sporadic cases almost certainly occur at low frequency in endemic areas and vice versa. Burkitt’s lymphoma has been little studied in most world regions, and it is likely that much information of relevence to an understanding of pathogenesis will be

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gained by studying clinical features, EBV association, and chromosomal breakpoint locations throughout the world. OF D. MOLECULAR CONSEQUENCES THE CHROMOSOMAL TRANSLOCATIONS

The primary consequence of each of the three chromosomal translocations associated with Burkitt’s lymphoma is deregulation of the c-myc gene. This appears to result from at least two components: a structural change in the c-myc gene (which is superimposed upon the persistence of an open chromatin structure such that the gene is able to be expressed) and a positive regulatory influence provided by the juxtaposed immunoglobulin gene region. The influence of the latter in Burkitt’s lymphoma with a far 5‘ breakpoint is less clear because of the large linear distance between c-myc and immunoglobulin sequences; perhaps this is a reason for the EBV association. The structural changes are readily divided into several categories, perhaps the most obvious of which are those which deprive the gene of its normal promoters, and those which permit transcription via PI and Pz. The structural changes can be small in magnitude, for example, as minimal as single-base substitutions in the first exon or first intron. Single-base changes in critical regions such as a regulatory element may result in failure of a protein factor to bind, and consequent loss of function of the element. In the case of mutations in the first exon, the result may be abrogation of the block to transcript elongation, which is a normal component of c-myc regulation, or lack of expression of the 67-kDa c-myc protein, which is initiated in the terminal region of the first exon (or, of course, both). The significance of the absence of the 67-kDa protein in some Burkitt’s lymphomas remains a matter for speculation. The possibility that it is normally involved in the regulation of c-myc is worthy of consideration. Minimal structural changes are associated with chromosome 8 breakpoints far 5’ of c-myc or 3’ of c-myc. More flagrant (but not necessarily more consequential) structural changes occur when the breakpoint on chromosome 8 is close to or within c-myc. In such cases part or even all of the regulatory region of the gene is separated in the genome from the protein-coding region. Frequently, synthesis of the 67-kDa protein is also prevented because the breakpoint is distal to its initiation codon in the first exon. Even these gross structural changes appear to be insufficient in themselves to activate c-myc and, whatever the nature of the structural change in c-myc, it appears that a positive transcriptional impetus is required from the juxtaposed immunoglobulin region. Although it is entirely possible that the juxtaposition of immunoglobulin sequences

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to c-myc in a B cell maintains an open chromatin structure of the latter gene, this is almost certainly insufficient per se to result in deregulation of c-myc. Several potential regulatory elements in the immunoglobulin loci may be relevant to the pathogenesis of Burkitt’s lymphoma, the most conspicuous being the heavy chain enhancer situated between the S, and J regions and the similar K enhancer element between and C,. The distance over which the heavy chain or K chain enhancers can act is unknown, but there are precedents for viral enhancer elements influencing promoters as much as 100 kb distant, so that the immunoglobulin enhancers may well be critical components of deregulation in all breakpoints resulting in their placement on the same chromosome as c-myc. However, the involvement of a gene associated with the human equivalent of pot-1 at the breakpoint of mouse plasmacytomas with 6;15 translocations cannot be excluded in Burkitt’s lymphomas bearing a variant translocation. In many Burkitt’s lymphomas, the breakpoint on chromosome 14 is 3’of the heavy chain enhancer such that it is translocated to chromosome 8 and thus is not on the same chromosome as the translocated c-myc gene. Other enhancer elements must be operative in this case. A second probable site of enhancer function and possibly even aberrant promoter activity (transcribing in an antisense direction) is the S, region. Transcripts reading through c-myc which initiate on the antisense strand of juxtaposed S , regions in Burkitt’s lymphoma have not yet been described, although such transcripts have been reported to account for as much as 50%of c-myc mRNA in some mouse plasmacytomas. Other regions of the immunoglobulin gene loci could provide enhancer functions in some Burkitt’s lymphoma, but there is currently no data to support or refute this possibility. It seems probable that the shift in c-myc promoter utilization (i.e., roughly equivalent amounts of PI and P2 transcripts) in Burkitt’s lymphoma is a consequence of the influence of the immunoglobulin enhancer regions on c-myc expression. Whether this has any pathological consequences is unknown. Similarly, an alteration in the half-life of truncated c-myc transcripts would appear to be of minor consequence to the neoplastic transformational event itself, particularly since c-myc mRNA is not uniformly elevated in Burkitt’s lymphoma compared to other proliferating B cells. Deregulation of c-myc is the immediate consequence of the chromosomal translocations, which leads to the failure to permit the cell in which the translocation occurred to enter a resting phase. This is likely to be a consequence of a functional alteration in a growth factor receptor or growth factor receptor transduction pathway, probably resultant upon the binding of c-myc (possibly in concert with other transactivat-

J.

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ing genes such asfos andjun) to the regulatory region of another gene or genes. Increased growth in the presence of exogenous growth factors could still occur, and might be more evident in early tumors which have undergone little tumor progression. This could account for preferred anatomical sites of tumor cell proliferation, e.g., the jaw in African children, breast in pubertal or lactating females, both doubtless sites of high concentrations of growth factors.

E. THENEED FOR ADDITIONALGENETIC FACTORS The evidence from animal tumors suggests strongly that c-myc deregulation is, in itself, insufficient to induce Burkitt’s lymphoma. To date, however, with the exception of EBV infection, there are few indicators as to what additional factors may be involved. In addition, it is important to differentiate between factors which influence the size of the target cell population or the ability to repair damaged DNA, which increase the risk of development of a chromosomal translocation, and factors which cooperate with the deregulated c-myc gene to induce a state of true neoplasia. The possibility that c-myc interacts with other oncogenes such as ras or rafis raised by experiments with animal models, but there is no evidence that structural or functional abnormalities of such genes are a necessary component of pathogenesis. An intriguing possibility is raised by the sequence homology between c-myc, the adenovirus protein Ela, and the large T antigens of polyoma and SV40, perhaps these genes utilize the same cellular pathway.

F. PROBABLE ROLEOF EBV IN A SUBSET OF BURKITT’S LYMPHOMA EBV may well have two roles in Burkitt’s lymphoma. First, early infection with EBV, which occurs in all African children and probably to some degree in all lesser developed countries and deprived populations, may increase the size of the EBV-infected target cell population. By itself, however, this is unlikely to greatly increase the incidence of Burkitt’s lymphoma, since otherwise one would expect a higher incidence in all less developed countries in addition to equatorial Africa (where an additionaI factor is holoendemic malaria). In addition to this effect on the target cell population, EBV may also have a direct role to play in the pathogenesis of Burkitt’s lymphoma. Its association with specific chromosomal breakpoint locations strongly suggests that the expression of one or more EBV genes may be an essential component of pathogenesis in the presence of some, but not other, structural abnormalities of c-myc. Since the net result of the chromosomal translocations (deregulated c-myc expression) is the same regardless of the

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details as to how this is accomplished, a likely explanation of the association of EBV with specific chromosome 8 breakpoint locations is that EBV itself may participate in the deregulation of c-myc. One possibility is that EBV could provide a transactivating protein which binds to the c-myc regulatory region and enhances transcription. It is not difficult to envisage that such an effect might be essential in the presence of some structural changes in c-myc but not others. Similarly, the nature of the juxtaposition of c-myc with immunoglobulin sequences may also be of importance in determining whether or not there is an absolute requirement for EBV gene products for malignant transformation. It would seem, intuitively, that if EBV does provide an element in the deregulation of c-myc, it is likely to be already present in the cells in which a chromosomal translocation occurs, thus favoring Klein’s hypothesis over Lenoir’s and Bornkamm’s. However, if EBV acts separately but cooperatively with c-myc, then neither hypothesis is favored. The necessity for any EBV gene which is of pathogenetic importance in Burkitt’s lymphoma to be expressed in latently infected cells immediately narrows the possible number of candidates. Moreover, the quite strong evidence that in Burkitt’s lymphoma in uiuo the expression of several of the genes relevant to transformation of lymphoblastoid cell lines (EBNA 2, EBNA 5, and LMP) is low or absent, narrows the field even further. Of the remaining genes known to be expressed in latently infected cells and also in Burkitt’s lymphoma, EBNA 1 must be considered a strong candidate gene for a role in tumorigenesis, since it is known to be a transactivator (and therefore potentially able to bind to the regulatory regions of cellular genes) and is responsible for EBV plasmid maintenance. Rather enticingly, its functional properties are very similar to c-myc, since both proteins are able to bind to sequences which are then capable of autonomously replicating. Moreover, mice transgenic for this gene develop lymphoid malignancies, indicating that EBNA 1can, in some circumstances, be tumorigenic. Clearly, EBNA 1is worthy of intense scrutiny as a potential contributor to the pathogenesis of Burkitt’s lymphoma. XVII. Coda-Clinical

Significance

The quite detailed knowledge of the mechanisms of tumorigenesis in Burkitt’s lymphoma brings with it new problems of classification and nomenclature ranging from the relatively obvious to the more sophisticated. We might question, for example, whether sporadic and endemic, or EBV-negative and EBV-positive, Burkitt’s lymphomas are separate diseases. Or whether lymphomas with the same cytogenetic abnormalities but differing histologies should be considered the same

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disease. And are tumors with chromosome breakpoints within the c-myc gene appropriately classified as pathologically identical to those with breakpoints tens of kilobases away from c-myc-i.e., should the mechanism of deregulation of c-myc be considered a valid taxonomic criterion? Answers to these questions can only be formulated by the establishment of new codifications of pathological criteria, and will, in any event, be of merely temporary value until the next level of comprehension is attained. Nevertheless, Burkitt’s lymphoma, along with a small number of tumors of largely hemopoietic origin, is clearly pointing toward molecular genetics as an important, perhaps even primary, means of classification of malignant neoplasms. The impact of this (quite aside from its academic interest) is not limited to diagnosis, for the molecular events leading to neoplastic growth permit the use of extraordinarily sensitive techniques, such as the polymerase chain reaction, to be applied to the detection of tumor cells (Shiramizu and Magrath, 1990). This should lead to a considerable refinement in the ability to detect residual tumor cells after therapy, and thus permit improved therapeutic decision making. Moreover, the molecular abnormalities of tumor cells provide a truly unique target for therapeutic endeavors, which, if it can be exploited, should for the first time provide a solid foundation for therapeutic selectivity, resulting in minimally toxic but highly effective treatment (Magrath, 1989,1990b).The presence of abnormal transcripts, for example those which contain c-myc intron sequences as a consequence of truncation of the gene and consequent abnormal splicing, provides one such therapeutic target, the utility of which is presently being explored. It seems probable that Burkitt’s lymphoma, rare tumor though it may be, will remain in the vanguard of pathological and therapeutic advances for the foreseeable future.

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MECHANISMS OF SIGNAL TRANSDUCTION TO THE CELL NUCLEUS Erich A. Nigg Swiss Institute for Experimental Cancer Research (ISREC). CH-1066 Epalinges. Switzerland

I. Introduction 11. Early Consequences of Plasma Membrane Receptor Stimulation

A. Production of Second Messengers and Activation of Serine-ThreonineSpecific Protein Kinases B. Activation of Tyrosine-Specific Protein Kinases C. Cross-Talk between Different Signaling Pathways 111. The Nuclear Envelope and Pore Complexes A. The Structures and Properties of Nuclear Pores B. Mechanisms for Nuclear Accumulation of Proteins IV. Mechanisms of Signal Transduction to the Nucleus A. Steroid Hormone Receptors: Prototypes for Ligand-Responsive Transcription Factors B. A Role for Protein Kinase Translocations in Signal Transduction? C. Shuttling Proteins in Signal Transduction: A Hypothesis D. The Anchorage-Release Model: A Unifying Concept for Signal Transduction between Cytoplasm and Nucleus V. The Role of Signal Transduction in Oncogenesis: Concluding Remarks References

I. Introduction

The coordination of cell proliferation and differentiation requires extensive communication between different types of cells. Accordingly, individual cells must be able to respond to a large variety of external stimuli. These are provided by a multitude of soluble agents such as hormones, growth and differentiation factors, and neurotransmitters, as well as by direct cell-cell contact and interactions between cells and extracellular matrix proteins. Irrespective of the chemical nature of the primary regulatory stimulus, signals controlling DNA replication and differential gene expression need to be transduced from the cell surface to the nucleus. As a consequence, signaling across the plasma membrane to the cytoplasm and thence to the nucleus plays a pivotal role in normal development and life of multicellular organisms (for reviews see Gurdon, 1986; McGrath and Solter, 1986; Brachet, 1987; Metcalf, 1989). Moreover, as indicated by the fact that many presently known protooncogene products function in signal 271 ADVANCES I N CANCER RESEARCH, VOLUME 55

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transduction, there is no doubt that deregulation of intracellular signaling pathways contributes in a major way to the development of neoplastic disorders (Weinberg, 1985; Kahn and Graf, 1986; Reddy et al., 1988). In order to reach the nucleus, signals arriving at the cell surface must be transmitted across two distinct physical barriers, the plasma membrane and the nuclear envelope. Based on differences in the mechanisms used for conveying information across these barriers, two fundamentally different signaling pathways can b e distinguished (Fig. 1). Some agents, exemplified here by steroid hormones (Fig. lA), traverse the plasma membrane and execute their regulatory functions by binding to intracellular receptors. It remains uncertain how these hormones traverse the plasma membrane, but there is agreement that, in general, they need to enter the cell in order to execute their functions. Conversely, most peptide hormones, growth factors, and neurotransmitters act upon receptors located in the plasma membrane (Fig. 1B). Although ligand-receptor complexes are rapidly internalized, the significance of this internalization for signal transduction remains uncertain (for references see Johnsson et al., 1985; Keating and Williams, 1988;Hannink and Donoghue, 1988).Likewise, evidence suggesting a direct nuclear action of peptide hormones and growth factors remains difficult to interpret (for discussion see Burwen and Jones, 1987;Evans and Bergeron, 1988; Maher et al., 1989), but it may be premature to dismiss categorically the possibility of a direct nuclear function for all such agents (e.g., Podlecki et al., 1987; Bouche et al., 1987). The present article is written on the premise that most physiological effects of peptide hormones and growth factors result from the occupation of cell surface receptors, and that these effects are mediated by intracellular signaling molecules that are chemically distinct from the primary regulatory factors. An impressive amount of information describes early consequences of ligand binding to plasma membrane receptors, i.e., the production of different types of second messengers and the activation of various protein kinases (e.g., Kikkawa and Nishizuka, 1986; Gilman, 1987; Carpenter, 1987; Berridge, 1987; Yarden and Ullrich, 1988; Rozengurt and Sinnett-Smith, 1988; Pouyssegur et al., 1988; Williams, 1989). Similarly, much progress has been made with respect to the characterization of cis-acting gene regulatory sequence elements and transacting protein factors controlling gene expression (McKnight and Tjian, 1986; Maniatis et al., 1987; Atchison, 1988; Ptashne, 1988; Struhl, 1989; Mitchell and Tjian, 1989). In contrast, little definitive information is available about intermediate stages in signal transduc-

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FIG.1. Representation of signaling pathways utilized by agents acting upon either intracellular receptors [e.g., steroid hormones (A)] or plasma membrane receptors [e.g., peptide hormones (B)]. As illustrated in this scheme, it remains uncertain how steroid hormones and related agents enter the cell, but their nuclear action is comparatively well understood. Conversely, much information is available about the early consequences of plasma membrane receptor occupation by peptide hormones and growth factors, but the mechanisms involved in propagation of signals to the nucleus remain to be unraveled.

tion, that is the actual mechanisms involved in transmission of signals from the cytoplasm to the nucleus. In this article concepts are developed that may contribute to the elucidation of this central part of intracellular signaling. For this purpose, a brief outline of commonly

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observed early consequences of surface receptor stimulation is presented, followed by a summary of what is known about the structure of nuclear pores and the transport of macromolecules across the nuclear envelope. With this information at hand, speculations are then made on possible mechanisms for communication between cytoplasm and nucleus. Hopefully these ideas may stimulate further research on the interactions between cytoplasm and nucleus and the importance of these interactions for the regulation of cell growth and differentiation.

II. Early Consequences of Plasma Membrane Receptor Stimulation It is beyond the scope of this article to review the multitude of physiological consequences of ligand-receptor interactions at the plasma membrane. Instead, the following discussion is meant to illustrate a few common principles that have emerged from the study of many different processes. The inevitable oversimplification inherent in this brief presentation should not divert attention from the fact that the biochemistry involved in transmembrane signaling and the generation of second messengers is very complex (see, e.g., Berridge, 1987; Neer and Clapham, 1988; Berridge and Irvine, 1989),and that different types of cells may respond differently to a given stimulus (e.g., Sporn and Roberts, 1988; Tigges et al., 1989). The first general point to be emphasized below is that protein phosphorylation-dephosphorylation reactions play a pivotal role in amplifying and disseminating incoming signals throughout the cell (see Fig. 2 for illustration). The second point is that signal transduction through the cell does not generally proceed in a linear “domino-type” fashion, but invariably involves multiple lateral “network-type” interactions between different signaling pathways (see Fig. 3 for illustration).

A.

PRODUCTION OF SECOND MESSENGERSAND ACTIVATIONOF SERINE-THHEONINE-SPECIFIC PROTEIN KINASES

Many receptors for neurotransmitters and hormones are coupled, via guanine-nucleotide regulatory (G) proteins, to enzymatic effector systems regulating the production of second messengers (Gilman, 1987; Neer and Clapham, 1988). A particularly well-studied example is provided by the coupling of receptors to adenylate cyclase, a process resulting in the production of CAMP (Benovic et al., 1988) and the subsequent activation of CAMP-dependent protein kinases (PK-A; Flockhart and Corbin, 1982; Edelman et al., 1987; Taylor, 1989). Another major pathway is based on receptor-effector systems regulating

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FIG.2. Highly simplified representation of a few basic aspects of transmembrane signaling and activation ofprotein kinases. For the sake of simplicity, the figure does not represent any of the complexities inherent in both stimulatory and inhibitory G protein functions (Gilman, 1987; Neer and Clapham, 1988; Milligan, 1989), nor does it include the multiple ramifications that arise from the interconversions of various inositol polyphosphates (Berridge, 1987; Michell, 1989; Berridge and Irvine, 1989). Likewise, it exclusively depicts the pathways leading to the activation of protein kinase activities; other processes such as the regulation of phosphatase activities (Cohen, 1989; Hunter, 1989) or changes in ion fluxes (Moolenaar, 1986; Carafoli, 1987; Grinstein et al.,1989; Ganz et ol., 1989)are not included. Based on structural criteria, two different types ofcell surface receptors may b e distinguished: a first family (see A and C) includes receptors for a large variety of hormones and neurotransmitters; these receptors (exemplified by the P-adrenergic receptor) consist of single polypeptide chains with seven hydrophobic membrane-spanning domains (Sibley et al., 1987; Caron, 1989). Generally, such receptors are coupled to effector systems through guanine nucleotide regulatory (G) proteins. Their occupation elicits the production of various types of second messengers which in turn act through regulating intracellular Ca2+ levels and protein kinase activities. A second family [exemplified by the epidermal growth factor (EGF) receptor; see B] comprises receptors with intrinsic tyrosine kinase activity. These receptors contain only a single transmembrane-spanning domain (Carpenter, 1987; Yarden and Ullrich, 1988; Panayotou and Waterfield, 1989; Westermark and Heldin, 1989); in this case, it is likely that receptor oligomerization plays an important role in signal transduction across the plasma membrane (Schlessinger, 1988). Both EGF and platelet-derived growth factor (PDGF) were shown to rapidly induce tyrosine phosphorylation of phospholipase C, pointing to a biochemical link between receptors with tyrosine kinase activities and inositol phospholipid nretabolism (Wahl et uf., 1989; Margolis et af., 1989; Meisenhelder et al., 1989). Further substrates of membrane-associated tyrosine kinases include GTPase-activating protein (Molloy et al., 1989), phosphatidylinositol-3-kinase (Kaplan et al., 1987; Varticovski et al., 1980) and the c-raf gene product (Morrison et al., 1989). AC, Adenylate cyclase; PK, protein kinase; PI, phosphatidyl inositol, PIP2, phosphatidyl-4,5-bisphosphate;IPS, inositol-1,4,5-trisphosphate;PL-C, phospholipase C; DAG, diacylglycerol; CaM, calmodulin.

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FIG.3. Diagram exemplifying major levels of interactions between different signaling pathways. Arrows are meant to illustrate stimulatory interactions, T-shaped endings indicate inhibitory types of interactions. This scheme depicts an idealized and highly oversimplified type of network. First, it depicts only one protein kinase (PK)and one key substrate (S) per signalingpathway, although examples of bona fide kinase cascades have been described, and most protein kinases phosphorylate multiple physiological substrates. Second, it does not include any regulatory interactions based on phosphatase activities. Third, it omits additional levels of regulation, particularly phosphorylation events affecting the enzymes involved in the production or degradation of second messengers, and modulatory interactions involving second messenger-derived metabolites.

the hydrolysis of inositol phospholipids (Hokin, 1985; Berridge, 1987; Michell, 1989).In this case, cleavage ofthe minor membrane phospho(PIP2) by C-type phospholipases lipid pho~phatidyl-4~5-bisphosphate (PL-C) generates two major second messengers, i.e., 1,2-sn-diacylglycerol (DAG) and inositol-1,4,5-trisphosphate(IPS).Both molecules are important in signal transduction in that diacylglycerol is required for stimulation of protein kinase C (Kikkawa and Nishizuka, 1986), whereas inositol trisphosphate triggers release of calcium ions from intracellular stores (Berridge, 1987); calcium ions, in turn, contribute to the regulation of several different types of protein kinases (Kikkawa and Nishizuka, 1986; Edelman et d.,1987; Schulman and Lou, 1989), although they clearly affect the functions of many other proteins as well (Carafoli, 1987).

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B. ACTIVATIONOF TYROSINE-SPECIFIC PROTEIN KINASES Several growth factors and polypeptide hormones activate tyrosinespecific kinase activities. In most cases these activities are intrinsic to the cytoplasmic domains of the respective receptor polypeptides (Carpenter, 1987; Yarden and Ullrich, 1988),but there is also evidence for tight coupling between membrane-spanning receptors (the T cell antigens CD4 and CD8) and nonreceptor-type tyrosine kinases ( ~ 5 6 ’ “ ~ ; Rudd et al., 1988; Veillette et al., 1988, 1989; Barber et al., 1989).This finding may provide a paradigm for explaining the function of other nonreceptor (i.e., “src-type”) tyrosine kinases (Hunter and Cooper, 1985).Also, it should be noted that the family of receptors with associated tyrosine kinase activities includes members that appear to be regulated by a cell surface-associated ligand rather than a soluble factor (Hafen et al., 1987). Recently, phospholipase C (Wahl et al. 1989; Margolis et al. 1989; Meisenhelder et al. 1989) as well as GTPase-activating protein (Molloy et al. 1989) were identified as physiological substrates of receptor-type tyrosine kinases. These intriguing findings establish a link between tyrosine kinase activation and signal transduction pathways utilizing second messengers (see Fig. 2). C. CROSS-TALK BETWEEN DIFFERENT SIGNALING PATHWAYS Pharmacological and biochemical evidence concur to demonstrate that different signal transduction pathways interact extensively with each other. Prominent examples for cross-talk between different signaling pathways include interactions between second messengerdependent systems operating through calcium and those acting through CAMP (e.g., Whitfield et al., 1987), interactions between the pathways using protein kinases A and C (Kikkawa and Nishizuka, 1986; Worley et al., 1986; Yoshimasa et al., 1987; Woodgett et al., 1987), and the regulation of inositol phospholipid turnover by tyrosine phosphorylation (Wahl et al., 1989; Margolis et al., 1989; Meisenhelder et al., 1989). As illustrated in Fig. 3, interactions between different effector systems can occur at multiple levels: a first level concerns regulation of the biological activity and subcellular distribution of membrane receptors by phosphorylation (Panayotou and Waterfield, 1989). Agonistinduced phosphorylation may target the homologous receptor, or receptors specific for other ligands, and frequently leads to “downregulation” or “desensitization” of the receptor concerned (Sibley et al., 1987; Benovic et al., 1988). A second level of cross-talk is provided by

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the fact that the activities of many kinases and phosphatases are themselves subject to regulation by phosphorylation. For instance, several tyrosine-specific protein kinases are phosphorylated in uico at multiple sites, including serine and threonine as well as tyrosine residues, and it appears that serine-threonine-specific kinases contribute to the regulation of tyrosine kinase activity (for references see Hunter and Cooper, 1985; Hunter, 1987; Kazlauskas and Cooper, 1988; Chackalaparampil and Shalloway, 1988; Yaciuk et al., 1989). Conversely, recent evidence suggests that a major function of tyrosine-specific kinases is the regulation of serine-threonine-specific enzymes (Ray and Sturgill, 1988; Czech et al., 1988; Morrison et al., 1988; Draetta et al., 1988; Rossomando et al., 1989). A third major level of integration between different effector systems arises from the convergence of different protein kinases and phosphatases onto a limited number of key substrates. Phosphorylation of individual proteins on multiple sites provides a myriad of possibilities for regulation, as is well illustrated by the complex regulation of glycogen synthase (Cohen, 1986; Roach, 1986; Edelman et al., 1987; Hardie, 1989). In the context of signal transduction, it will be interesting to search for targets of multiple phosphorylation among trans-acting factors controlling gene expression or DNA replication. An important consequence of interactions between different signaling pathways is that any information arriving at the cell surface is not only amplified but also disseminated laterally. Thus, the final physiological consequences of a given signal will generally depend on the status of many other effector systems present in a given target cell. This is true not only for different agents acting through plasma membrane receptors, but also applies to hormones acting through intracellular receptors. For instance, there is increasing evidence to suggest that the activity of steroid hormone receptors may be modulated by protein phosphorylation (for references see Tienrungroj et al., 1987; Auricchio et al., 1987; Orti et al., 1989a,b; Sheridan et al., 1989). Ill. The Nuclear Envelope and Pore Complexes

Before addressing the question of how incoming signals may be propagated from cytoplasm to nucleus, it may be helpful to review briefly what is known about the properties of the nuclear envelope and its function as a permeability barrier for macromolecules.

A. THESTRUCTURE AND PROPERTIES OF NUCLEAR PORES The nuclear envelope consists of two membranes (enclosing a 10- to 30-nm-wide perinuclear space) that are frequently joined and pene-

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trated by nuclear pores complexes (for reviews see Franke et al., 1981; Gerace and Burke, 1988; Nigg, 1988). Depending on the physiological state of a cell, the number of nuclear pores may vary considerably, i.e., from 2 to 4 pores/pm2 in the case of avian erythrocytes to over 60 pores/pm2 for mature amphibian oocytes; in most higher eukaryotic cells there are about 10-20 pores/pm2 or approximately 2000-4000 pores/nucleus (Maul, 1977). Nuclear pores have been studied extensively by different electron microscopic techniques (for reviews see Franke, 1974; Harris and Marshall, 1981; Scheer et al., 1988). These studies demonstrate that nuclear pores consist of several prominent substructures arranged in a complex with eightfold radial symmetry (Unwin and Milligan, 1982;

FIG.4. Transmission electron micrograph of “native” nuclear pore complexes of Xenopus laeois, as visualized by negative staining of nuclear envelopes with uranyl for-

mate. Bar represents 0.25 Fm. Inset: Image reconstruction produced by computer averaging of density maps of 50 pores; note the striking eightfold symmetry apparent from this representation. (Micrographs courtesy of Dr. U. Aebi, Basel, Switzerland.)

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Gerace and Burke, 1988; Reichelt et al., 1990).To illustrate this, Fig. 4 shows an electron microscopic view of nuclear pores of unfertilized eggs ofXenopus laevis, and Fig. 5 represents a schematic side view, as derived from image analysis and three-dimensional reconstruction (Unwin and Milligan, 1982; Reichelt et aZ., 1990). Mass determination studies using scanning transmission electron microscopy indicate that the nuclear pore complex has a mass of approximately 125 X lo6 Da (Reichelt et al., 1990). Although it is well established that most of this mass consists of protein, it is only recently that immunochemical approaches have begun to yield insight into the macromolecular composition of nuclear pores (reviewed in Gerace and Burke, 1988; Nigg, 1989). Interestingly, several of the identified pore proteins were found to carry multiple 0-linked N-acetylglucosamine (0-GlcNAc) residues (Snow et al., 1987; Holt et al., 1987; Davis and Blobel, 1986, 1987; Park et al., 1987). This particular type of glycosylation is remarkable because it results in modifications that are accessi-

FIG.5. Schematic side view of nuclear pore complexes [redrawn in modified form from Unwin and Milligan (1982) and Reichelt et al. (19901. Major constituents of nuclear pores are the outer (R,) and inner (RJ rings, spokes (S), and a central plug (P). Recent mass determination studies based on scanning transmission electron microscopy confirm that the cytoplasmic and nucleoplasmic aspects of the nuclear pore differ from each other, consistent with the existence of distinct vectorial transport mechanisms for nuclear import and export, respectively (Reichelt et al., 1990). Note the continuity of the lipid bilayer of the outer (NM,) and inner (NM,)nuclear membranes at the edges of the nuclear pore; the two membranes enclose a perinuclear space (PNS), a compartment that is continuous with the endoplasmic reticulum. The nuclear lamina, a karyoskeletal structure lining the nucleoplasmic surface of the inner nuclear membrane (Aebi et aZ., 1986; Cerace and Burke, 1988; Nigg, 1989), is supposed to interact with nuclear pores, but no detailed information is presently available on the nature of this interaction. Lamin proteins are depicted here as being anchored to the nuclear membrane through an integral membrane protein receptor (Worman et al., 1988; Senior and Cerace, 1988), as well as by virtue of a lipophilic posttranslational modification, i.e., isoprenylation (Beck et al., 1988; Wolda and Clomset 1988; Krohne et al., 1989; Vorburger et al., 1989). Finally, it should be noted that transmission electron microscopy suggests the existence of filamentous structures emanating from the nuclear pores into both the cytoplasmic and the nucleoplasmic compartment (e.g., Scheer et al., 1988; Richardson et al., 1988). Although not depicted here as being part of the pore complex per se, these structures may well play an important role in targeting appropriate substrates for nuclear translocation to the pores.

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28 1

ble from the cytoplasmic and nucleoplasmic compartments (Hanover et al., 1987; for review see Hart et al., 1988), in contrast to the lumenal disposition of most intracellular carbohydrate (Kornfeld and Kornfeld, 1985; Hirschberg and Snider, 1987).The O-GlcNAc modification is not confined to pore proteins (e.g., Holt and Hart, 1986; Jackson and Tjian, 1988; Kelly and Hart, 1989) and its functional significance remains to be clarified. However, it is likely that the presence of these carbohydrates on pore proteins accounts for the observation that the lectin wheat germ agglutinin inhibits nuclear transport both in vivo (Yoneda et al., 1987; Dabauvalle et al., 1988b; Wolff et al., 1988) and in vitro (Finlay et al., 1987; Newmeyer and Forbes, 1988). The nuclear pore has long been assumed to be the major passageway for nucleocytoplasmic transport. Although the possible existence of alternative mechanisms should not be excluded (for references see Scheer et al., 1988),there is now convincing direct evidence indicating that both import of proteins into nuclei (Feldherr et al., 1984; Richardson et al., 1988; Newmeyer and Forbes, 1988; Featherstone et al., 1988; Dabauvalle et al., 1988a) as well as export of ribonucleoprotein particles from nuclei (Stevens and Swift, 1966; Skoglund et al., 1983; Dworetzky and Feldherr, 1988) occurs through nuclear pores. Moreover, individual pores were shown to function in both import of proteins as well as export of RNA, implying the existence of bidirectional transport mechanisms (Dworetzky and Feldherr, 1988).

B. MECHANISMS FOR NUCLEAR ACCUMULATION OF PROTEINS Nuclear pores determine the rate of nucleocytoplasmic transport of macromolecules at two different levels. On the one hand, they function as molecular sieves allowing free exchange of ions and small molecules. As determined by microinjection of various types of tracers, nuclear pores provide aqueous channels with a functional open diameter of approximately 10 nm (reviewed in Paine and Horowitz, 1980; Peters, 1986; Dingwall and Laskey, 1986). This channel permits diffusion of ions, metabolites, and small proteins (up to approximately 40 kDa). On the other hand, much larger proteins, ribonucleoprotein complexes, and even macromolecular assemblies such as preribosoma1 particles also pass through nuclear pores, implying the existence of active or facilitated transport mechanisms. Although signals specifying nuclear export of ribonucleoproteins remain poorly understood (Zasloff, 1983; Agutter, 1984; Tobian et al., 1985; Schroder et al., 1987), major progress has been made toward understanding nuclear import of proteins. There is now convincing evidence that import of large proteins into nuclei involves a signal- and

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energy-dependent mechanism (Dingwall et al., 1982; Kalderon et al., 1984a,b; Newmeyer et al., 1986; for reviews see Smith et al., 1985; Dingwall and Laskey, 1986; Newport and Forbes, 1987; Goldfarb, 1989; Roberts, 1989),and a number of different signal sequences have been identified within the primary structures of nuclear proteins of both viral and cellular origin (Table I). Identification of these sequences as nuclear location signals is based on several lines of evidence (for references see Table I). First, they were shown to be required for correct nuclear location by deletion analyses and sitedirected mutagenesis. Second, by gene fusion experiments (i.e., experimental transfer of nuclear location signals to proteins with a normally cytoplasmic location) many of the sequences listed in Table I were also shown to be sufficient for nuclear location. Finally, the efficiency of nuclear location signals was demonstrated in microinjection experiments using synthetic signal peptides coupled to different carrier proteins (Goldfarb et al., 1986; Lanford et al., 1986; Chelsky et al,, 1989). Most strikingly, in response to nuclear location signals, nuclear pores were shown to allow translocation of (nondeformable) colloidal gold tracers of up to 28 nm in diameter (Feldherr et al., 1984; Dworetzky and Feldherr, 1988; Dworetzky et al., 1988). Despite this progress toward understanding nuclear import of proteins, several important questions remain unresolved. In particular, the nature and subcellular location of the receptor(s) recognizing nuclear location signals remain to be explored further (Adam et al., 1989; Yamasaki et al., 1989), and it remains to be clarified how the pore opens up in response to such signals. In this context, it is interesting that the efficiency of nuclear import was found to depend on the number of nuclear location signals present in a particular protein (Lanford et d . ,1986; Dworetzky et d.,1988; Fischer-Fantuzzi and Vesco, 1988). Moreover, although a single, comparatively short sequence is sufficient for nuclear localization of SV40 large T antigen (Kalderon et al., 1984a,b;Lanford and Butel, 1984; Colledge et al., 1986), variations in the sequences surrounding the primary signal were shown to contribute to this process (Welsh et al., 1986; Roberts et al., 1987). Similarly, several recent studies indicate that maximal activity of nuclear location signals may depend on extended or multiple sequence motifs (Richardson et al., 1986; Picard and Yamamoto, 1987; Dingwall et al., 1988; Kleinschmidt and Seiter, 1988; Silver and Hall, 1988; see also Rihs and Peters, 1989). Finally, it will be important to clarify to what extent the rate or specificity of nucleocytoplasmic transport is subject to regulation (Jiang and Schindler, 1988; Schroder et al., 1988). One intriguing possibility is that nuclear import of proteins might be modu-

TABLE I AMINOACID SEQUENCES ACTINGAS NUCLEAR LOCATION SIGNALS

Nuclear protein (source)

Deduced signal sequence

Reference

A. Vertebrate proteins c-Myc (human) Lamin AIC (human) Glucocorticoid receptor (rat)" Karyophilic protein N 1 (Xenopus) Nucleoplasmin (Xenopus)

PAAKRVKLD SVTKKRKLE YRKCLQAGMNLEARKTKKKIKGIQQATA VRKKRKT and AKKSKQE (AVK)RPAATKKAGQAKKK(KLD)

Dang and Lee (1988) Loewinger and McKeon (1988); Chelsky et al. (1989) Picard and Yamamoto (1987) Kleinschmidt and Seiter (1988) Biirglin and De Robertis (1987);Dingwall et al. (1988)

KIPIK PRKR GKKRSKA

Hall et al. (1984) Moreland et al. (1985) Moreland et al. (1987)

PKKKRKV APTKRKGS VSRKRPRP and PPKKARED AAPE DLRVLS KRPRP MPKTRRRPRRSQRKRPPTP

Kalderon et al. (1984b) Wychowski et al. (1986) Richardson et al. (1986) Davey et al. (1985) Lyons et al. (1987) Siomi et al. (1988)

B. Yeast proteins ( S . cereoisiae) Ribosomal protein L3 Histone H2B C. Viral proteins Large T antigen (SV40) Capsid protein VPl (SV40)b Large T antigen (polyoma virus) Nucleoprotein (influenzavirus) E l a gene product (adenovirus) ~27""' (HTLV-l)c

Only one of two purported nuclear location signals is indicated here. These sequences, when assayed as synthetic peptides, failed to confer nuclear location to carrier proteins in mammalian cells (Chelsky et al., 1989). 'The HTLV-1 (human T cell leukemia virus type 1)-derived sequence was reported to target proteins to the nucleolus. This sequence may include determinants involved in binding nucleolar constituents.

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lated by phosphorylation of sequences adjacent to nuclear location signals (Rihs and Peters, 1989). In conclusion, the notion of nuclear accumulation through signalmediated active import has now found widespread acceptance. Although this mechanism is well supported by experimental evidence, it is important to emphasize that an alternative model, based on selective binding of nuclear proteins, should not be dismissed (for further discussion see also Zimmer et al., 1988;Paine, 1988).This model invokes nuclear accumulation of certain proteins as a consequence of passive diffusion followed by selective retention (due to the presence of intranuclear binding sites provided by nondiffusible macromolecules, e.g., DNA; Bonner, 1978).Clearly, the two mechanisms are not mutually exclusive, and, depending on the size and properties of a particular protein, either one or the other may be operating.

IV. Mechanisms of Signal Transduction to the Nucleus

The following discussion is based on the assumption that signal transduction between cytoplasm and nucleus involves primarily the translocation of proteins between the two compartments. There is no doubt that in some instances the nuclear availability of cytoplasmically produced metabolites contributes to the modulation of nuclear activities, but this possibility is not considered here. This section presents, first, a brief review of signaling by steroid hormones and other agents acting upon intracellular receptors. Then evidence suggesting that nuclear translocation of protein kinases may play a role in signal transduction will be discussed followed by a speculation that protein shuttling between nucleus and cytoplasm plays a role in integrating nuclear and cytoplasmic activities. Although various signal transduction mechanisms are certain to differ with respect to biochemical aspects, they may resemble each other in conceptual terms. Thus, a tentative model (the “anchorage-release model”) for signal transduction from cytoplasm to nucleus will be presented. This unifying model postulates, first, that signal transduction between the two compartments is accomplished primarily by controlling the nucleocytoplasmic compartmentation of regulatory macromolecules, and, second, that shuttling proteins may play a major role in communication between cytoplasm and nucleus. A. STEROIDHORMONE RECEPTORS: PROTOTYPES FOR LIGAND-RESPONSIVE TRANSCRIPTION FACTORS Much progress has been accomplished with respect to both the molecular characterization of steroid hormone receptors, as well as the

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delineation of cis-acting regulatory DNA sequences conferring hormone responsiveness (for reviews see Yamamoto, 1985; Evans, 1988; Beato, 1989). In brief, hormone-responsive genes were shown to be flanked by short (15-20 bp) palindromic sequences, called hormoneresponsive elements (HRE), and consensus sequences have been delineated for both steroid and thyroid hormones. In general, HREs behave like transcriptional enhancer elements in that they function independently of their precise position or orientation (reviewed in Yamamoto, 1985; Beato, 1989). Over the last few years, steroid hormone receptors were found to belong to a much larger family of receptors that function as ligandresponsive transcription factors. This superfamily includes receptors for both steroid (K. R. Yamamoto et al., 1988) and thyroid hormones (Sap et al., 1986; Weinberger et al., 1986; Benbrook and Pfahl, 1987), vitamine D3 (McDonnell et al., 1988), the morphogen retinoic acid (Petkovich et al., 1987; Gigukre et al., 1987),and receptors for several as yet unidentified ligands (e.g., Thompson et al., 1987; Gigukre et al., 1988; Brand et al., 1988). Sequence comparisons and mutational analyses demonstrate that all of these receptors have a similar modular domain structure, and presumably function in very similar ways (Green and Chambon, 1986; Evans, 1988; K. R. Yamamoto et al., 1988). The major structural features of these receptors include variable N-termini, short but well-conserved central domains, and structurally complex C-terminal regions. The central domains contain cysteinerich sequence elements in a configuration allowing the formation of two “zinc fingers” (Freedman et al., 1988; Green et al., 1988; Severne et al., 1988),a structure common to many nucleic acid-binding proteins (for reviews see Berg, 1986; H u g and Rhodes, 1987; Evans and Hollenberg, 1988). As deduced from mutational analyses, the conserved C-terminal halves of receptor molecules are required not only for hormone binding, but also for receptor dimerization, and nuclear localization (Rusconi and Yamamoto, 1987; Picard and Yamamoto, 1987; Kumar et al., 1987; Kumar and Chambon, 1988). N-termini have been implicated in interactions with the transcriptional machinery, but a detailed functional characterization of those domains requires additional study (Giguhe et al., 1986; Hollenberg et al., 1987; Hollenberg and Evans, 1988). Upon hormone binding, receptors undergo an allosteric structural “ alteration (“activation” or transformation”; Gorski and Gannon, 1976). Although this process remains poorly understood, it is remarkable that deletion of hormone-binding domains resulted in constitutive activation of receptors. This indicates that, in the absence of ligand, the hormone-binding domain inhibits receptor function, and

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that this inhibition is relieved by hormone (Hollenberg et al., 1987; Godowski et al., 1987). According to the classic two-step model of steroid hormone action, receptor activation is accompanied by translocation of ligand-receptor complexes from cytoplasm to nucleus (Gorski and Gannon, 1976). However, in recent years considerable controversy has arisen with respect to the subcellular localization of unoccupied steroid receptors (King and Greene, 1984; Gasc et al., 1984; Welshons et al., 1984; Fuxe et al., 1985; Perrot-Applanat et al., 1985;Wikstrom et al., 1987; Gasc et al., 1989), and whether or not unoccupied receptors can bind HREs (Becker et al., 1986; Bailly et al., 1986; Willmann and Beato, 1986; Denis et al., 1988). Some of these discrepancies might be resolved if unoccupied receptors in uiuo were complexed with other proteins which restricted their intracellular distribution. Specifically, there is evidence that steroid-free receptors form macromolecular complexes through association with a 90-kDa heat shock protein (Catelli et al., 1985; Sanchez et al., 1985; Schuh et at., 1985; Denis et al., 1987), and that hormone-dependent release from this complex may be required for receptor function (Beaulieu, 1987; Groyer et al., 1987; Denis et al., 1988; Bresnick et al., 1989). Major unresolved issues to be addressed in the future concern the activation of receptors by ligands, and the targeting of' the activated receptors to responsive genes. Moreover, it remains to be determined how DNA-bound ligand-receptor complexes interact with the transcriptional machinery, and why some genes are activated by steroid hormones while others are repressed. Finally, it will be of the utmost importance to determine at what levels the regulation of intracellular hormone receptor activity is integrated with other intracellular signaling pathways. B. A ROLE FOR PROTEIN KINASE TRANSLOCATIONS IN SIGNALTRANSDUCTION? Most nuclear proteins are phosphorylated (Olson, 1983; Mitchell and Kleinsmith, 1983), but, until recently, little definitive information was available on the molecular identity of the kinases acting upon nuclear substrates in uiuo (Matthews and Huebner, 1984). Over the last few years, it became increasingly apparent that protein phosphorylation may represent a major mechanism for controlling nuclear activities, and, in particular, gene transcription (e.g., Cadena and Dahmus, 1987; Montminy and Bilezikjian, 1987; Imagawa et al., 1987; Sorger and Pelham, 1988; Krause and Gehring, 1989; Gonzalez and Mont-

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miny, 1989; Gonzalez et al., 1989; Cherry et al., 1989).With respect to mechanisms of signal transduction, it is imperative to identify the responsible protein kinases, and to determine in which cellular compartment the relevant phosphorylation reactions occur. A priori, one may postulate that, in response to appropriate physiological stimuli, protein kinases may translocate from cytoplasm to nucleus. On the other hand, it is conceivable that substrates for cytoplasmic kinases may migrate between cytoplasm and nucleus. As outlined below, available evidence suggests that both of these mechanisms may cooperate in signal transduction. 1. The CAMP-Mediated Pathway It is generally accepted that in eukaryotes most, if not all, physiological effects of CAMP are mediated by CAMP-dependent protein kinases (PK-A). These enzymes are composed of two regulatory (R) and two catalytic (C) subunits that together constitute an inactive holoenzyme of the form R2C2. Binding of cAMP to the regulatory subunit dimer results in release and concomitant activation of the catalytic subunits (Flockhart and Corbin, 1982; Edelman et al., 1987; Taylor, 1989). Gene regulatory effects of cAMP are well established in eukaryotes (Roesler et al., 1988; Karin, 1989) as well as in prokaryotes (de Crombrugghe et al., 1984). However, whereas the role of cAMP in the transcriptional activation of prokaryotic genes has long been understood (de Crombrugghe et al., 1984), its mode of action in eukaryotes constitutes the subject of a long-standing controversy (for references see Lohmann and Walter, 1984). By analogy to prokaryotes, where cAMP acts as an allosteric effector of CAMP-receptor protein (de Crombrugghe et al., 1984), the hypothesis has been advanced that eukaryotic R subunits, following binding of CAMP, might directly function as transcriptional regulators (e.g., Nagamine and Reich, 1985; Constantinou et al., 1985). On the other hand, it has been speculated that transcriptional regulation of eukaryotic CAMP-responsive genes may depend on the phosphorylation of trans-acting gene-regulatory proteins by the C subunit of the kinase (for references see Jungmann and Kranias, 1977; Johnson, 1982). Immunocytochemical studies strongly suggest that nuclear effects of cAMP are mediated by the C subunit (Nigg et al., 1985a,b, 1988). In response to cAMP elevation, the catalytic subunit of PK-A translocates from a perinuclear location to the nucleus, whereas no redistribution of the regulatory subunits can be observed (Fig. 6). Although other workers have reported nuclear translocations of R subunits of PK-A (e.g., Kuettel et al., 1985; Sikorska et al., 1988; Kwast-Welfeld and

FIG.6. Effect of CAMP elevation on the subcellular distribution of PK-A (type 11) subunits in cultured bovine kidney epithelial (MDBK) cells. MDBK cells were incubated for 1hr in the presence (b, d, and f ) or absence (a, a', c, and e) of forskolin ( M), an activator of adenylate cyclase. Then they were fixed and permeabilized using fonnaldehydelTriton X-100, and stained with antibodies specific for either the regulatory (R11) or the catalytic (C)subunit of PK-A, followed by rhodamine-conjugated goat anti-rabbit IgC (for details see Nigg et ul., 1985a,b). (a) Distribution of R I1 subunits in untreated cells. As described in detail elsewhere (Nigg et ul., 1985a; De Camilli et al., 1986). R I1 subunits are concentrated in a perinuclear area corresponding, at the light microscopic

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Jungmann, 1988), we never obtained any evidence to support the idea ofa nuclear function of R subunits (Nigg et al., 1985b; E. A. Nigg, G. S. McKnight, B. A. Hemmings, unpublished observations; see also Fig. 6). Of course, it is difficult to rigorously exclude a nuclear function for all R subunit isoforms, but several recent lines of evidence now strongly implicate the C subunit of PK-A in gene regulation: Stimulation of a CAMP-responsive gene has been accomplished by the addition of purified C subunits to a cell-free transcription system (Nakagawa et al., 1988), and similar results were obtained when microinjecting purified C subunits into living cells (Riabowol et ul., 1988).Likewise, transfection of cultured cells with cDNAs encoding C subunits conferred cAMP regulation to a reporter gene cotransfected under the control of a CAMP-responsive element (Maurer, 1989; see also McKnight et al., 1988). Conversely, expression of a cDNA encoding a specific inhibitor of the C subunit of PK-A abolished cAMP responsiveness of a cotransfected reporter gene (Grove et al., 1987; Day et al., 1989), and mutant cell lines expressing C subunit proteins with reduced kinase activities showed reduced inducibility of CAMPresponsive genes (Jans and Hemmings, 1988; Handler et ul., 1988). Finally, at least two nuclear transcription factors were recently shown to be affected in their biological activities by phosphorylation via PK-A (Montminy and Bilezikjian, 1987; K. K. Yamamoto et al., 1988; Cherry et al., 1989; Gonzalez and Montminy, 1989).On the basis of these data, it appears reasonable to conclude that gene regulation by cAMP involves the C rather than the R subunits of PK-A (Fig. 7). The data shown in Fig. 6 (see also Nigg et al., 1985b, 1988) strongly suggest that nuclear translocation of the C subunit represents an important step in the signal transduction pathway (Fig. 7). However, it would be premature to exclude the possibility that in certain instances the C subunit might act by phosphorylating a cytoplasmic mediator. Specifically, it is conceivable that phosphorylation might regulate the

level, to the location of the Golgi complex; moreover, they are associated with spindle poles in mitotic metaphase cells (a'). (b) Distribution of R I1 subunits in forskolin-treated cells; note that elevations of CAMP levels do not induce any detectable redistribution of R I1 subunits. (c and e): Distribution ofC subunits in untreated cells after fixation at 23°C (c) or 37°C (e), respectively. In the absence of CAMP, C subunits codistribute to a large extent with R I1 subunits, as expected for the inactive holoenzyme. (d and f ) Distribution of C subunits in forskolin-treated cells after fixation at 23°C (d) or 37°C (f); note that, irrespective of fixation conditions, cAMP elevation induces a striking redistribution of C subunits from the perinuclear Golgi area (c and e) to the nucleus (d and f). Bar in (f) represents 20 pm.

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FIG.7. Simplified scheme illustrating likely mechanisms for intracellular transmission of CAMP-mediated gene regulatory signals. A first mechanism is suggested by the results shown in Fig. 6 (see also Nigg et al., 1985b, 1988);it invokes nuclear translocation of activated catalytic subunits of PK-A, followed by intranuclear phosphorylation of trans-acting gene-regulatory factors. A second possible mechanism postulates that cytoplasmic phosphorylation may regulate the compartmentation of regulatory factors. Note that these two mechanisms are not mutually exclusive. TF, Transcription factor; R, regulatory subunit of PK-A; C, catalytic subunit of PK-A.

compartmentation of trans-acting gene-regulatory factors between cytoplasm and nucleus (Fig. 7; see also Shirakawa and Mizel, 1989). Figure 8 shows that the nuclear translocation of the PK-A C subunit is readily reversible in response to lowering of cAMP levels. This reversibility is very rapid in comparison to the turnover rates of PK-A subunits (Hemmings, 1986; Weber and Hilz, 1986); therefore, the results shown in Fig. 8 suggest that activated C subunits are capable of leaving nuclei. This conclusion raises the question of how C subunits, present in the nucleus, “sense” the presence or absence of cAMP on the (cytoplasmically located) R subunits. One possible explanation would be that activated C subunits, once dissociated from the holoenzyme complex, may constantly shuttle back and forth between nucleus and cytoplasm. Considering the molecular mass of C subunits (40 kDa), it appears possible that this protein may cross the nuclear

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29 1

FIG.8. Reversibility of nuclear translocation of C subunits. Immunofluorescent staining of untreated or forskolin-treated MDBK cells was carried out using antibodies specific for C subunits (see Nigg et al., 1985a,b; see also legend to Fig. 6). (a) Untreated M);(c) cells after treatment as cells; (b) cells after treatment for 1 hr with forskolin in (b),followed by a 5-min incubation in drug-free culture medium at 37°C; (d) cells after treatment as in (b), followed by a I-hr incubation in drug-free culture medium at 37°C. Bar in (d) represents 20 p m .

pores by diffusion. If this interpretation is correct, the equilibrium distribution of the protein would be determined by the relative numbers and affinities of cytoplasmic and intranuclear binding sites. Although the apparent accumulation of C subunits within nuclei (Fig. 6) might be interpreted as evidence for an active import process, it is equally possible that such an accumulation might be due to a “phaseaffinity” phenomenon, in that it might reflect the existence of a large number of intranuclear binding sites for the C subunit.

2. Nuclear Translocation of Other Protein Kinases? Activation of protein kinase C (PK-C) rapidly leads to altered transcription of a considerable number of genes, and the possibility has been raised that PK-C might translocate to the nucleus and phosphorylate trans-acting factors (for references see Kikkawa and Nishizuka,

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1986; Woodgett et al., 1987; Cambier et al., 1987; Kiss et al., 1988; Fields et al., 1988).However, at present, evidence supporting the idea of a direct nuclear function of activated PK-C should be viewed with caution. Reported biochemical evidence may be flawed by artifactual protein redistribution during cell fractionation, and most of the available immunocytochemical data do not distinguish between an “intranuclear” and a “perinuclear” location for activated PK-C. Subsequent to activation, plasma membrane-associated PK-C is cleaved by proteolysis to yield a soluble (calcium/lipid-independent)enzyme (Kishimot0 et al., 1989),and it has been speculated that such a soluble form of PK-C might be responsible for the phosphorylation of intranuclear substrates (e.g., Hornbeck et al., 1988). The idea is attractive, but it remains to be determined whether or not under physiological circumstances a truncated form of PK-C accumulates to sufficient levels to be functionally relevant. In addition to PK-A (and perhaps PK-C),other “cytoplasmic” protein kinases may qualify as candidates for a nuclear function. These include the kinases encoded by the protooncogenes vlc-abl (Van Etten et al., 1989) and c-raflc-mil(Morrison et al., 1988; Rapp et al., 1988). In particular, available evidence is consistent with the idea that the rafl mil kinase may play a pivotal role in conveying mitogenic signals to the nucleus (Morrison et aZ., 1988; 1989).Another kinase that may possibly play a similar role is casein-kinase 11. This ubiquitous type of enzyme (Hathaway and Traugh, 1982; Edelman et al., 1987) is activated by mitogenic stimulation (Sommercorn and Krebs, 1987; Klarlund and Czech, 1988; Carroll and Marshak, 1989), and, in addition to acting upon cytoplasmic substrates, clearly phosphorylates several proteins with predominantly nuclear location, including cellular and viral transforming proteins (e.g., Figge et al., 1988; Grasser et al., 1988; Luscher et al., 1989). C. SHUTTLING PROTEINS IN SIGNAL TRANSDUCTION: A HYPOTHESIS

1. Established Examples of Shuttling Proteins The existence of nuclear proteins that shuttle back and forth between nucleus and cytoplasm was postulated some 30 years ago based on the results of nuclear transplantation studies carried out with amoebas (Goldstein, 1958; for review see Goldstein and KO, 1981). Unfortunately, the shuttling proteins in amoebas could not be functionally characterized, nor was it possible, for technical reasons, to explore the generality of the phenomenon in higher eukaryotes. Recently, we have used two independent approaches to ask whether or

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not shuttling of individual nuclear proteins between nucleus and cytoplasm occurs in vertebrate cells. The assays we used consisted of monitoring the equilibration of proteins between nuclei present in interspecies heterokaryons, and in observing the antigen-mediated nuclear accumulation of cytoplasmically injected antibodies (for details see Borer et al., 1989). The results lead us to conclude that two major nonribosomal nucleolar proteins migrate constantly back and forth between nucleus and cytoplasm. At steady state, no significant cytoplasmic pools of these proteins could be detected by subcellular fractionation or immunocytochemistry, and, therefore, the dynamic behavior of these proteins escaped detection by conventional localization studies. This raises the possibility that other proteins which are considered to be stably associated with nuclei may in reality transit through the cytoplasmic compartment. 2. Possible Functions of Shuttling Proteins What is the physiological significance, if any, of protein shuttling between nucleus and cytoplasm? At present, the answer to this question must remain speculative, but it is possible to make two specific proposals: first, shuttling proteins may perform carrier functions and may thus play a role in nucleocytoplasmic transport (Fig. 9A). In particular, active transport of a limited number of carrier proteins into and out of the nucleus might provide the basis for nucleocytoplasmic translocation of a large variety of proteins by a “piggy-back” mechanism. Studies on the nuclear migration of adenovirus DNA polymerase lend some credence to this concept (Zhao and Padmanabhan, 1988). Second, the transient exposure of shuttling nuclear proteins to the cytoplasm may provide a mechanism for cytoplasmic regulation of nuclear activities (Fig. 9B). Specifically, cytoplasmic modification (e.g., by phosphorylation) of shuttling nuclear proteins may regulate either enzymatic activities of such proteins, or their interactions with other proteins. If the population of shuttling proteins were to include transacting factors involved in the regulation of transcription and DNA replication, cytoplasmic modification of such factors could represent an important mechanism for propagating information to the nucleus.

3. Generality of Protein Shuttling How widespread is the phenomenon of protein shuttling in living cells? As discussed above (see also Borer et al., 1989), cytoplasmic pools of shuttling proteins may occasionally be small and difficult to detect by conventional analyses of steady state protein distributions. Nevertheless, many proteins are readily detectable in both nucleus and cytoplasm (e.g., Peterson and McConkey, 1976; Bonner, 1978),

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4. Carrler Function 3

8. Signal Transduction ?

FIG.9. Possible functions of shuttling proteins. Two speculative proposals are illustrated. (A) Shuttling proteins may function as carriers. It is proposed that proteins andlor ribonucleoprotein particles might be transported across the nuclear envelope by virtue ofbinding specifically to any one of a limited number of shuttling carrier proteins. These carriers would be expected to interact transiently with a “translocator complex” located at the nuclear pore. Although the carrier is represented here as being capable of functioning in bidirectional transport, it is equally possible that import and export processes may be mediated by different carriers. (B)It is proposed that cytoplasmic modification of shuttling nuclear proteins may represent a general mechanism for signal transduction between cytoplasm and nucleus. Assuming that even a limited number of modified proteins appearing in the nucleus might be sufficient to elicit a biological response, signal transduction might be rapid. However, if one assumes that cytoplasmically modified proteins would have to reach threshold levels in the nucleus in order to produce physiological responses, such a signalling mechanism would exhibit a comparatively slow response time, and would appear to be ideally suited for transmitting only signals that persisted for a sufficient length of time.

and it is likely, although not proved, that many of those proteins may shuttle back and forth between the two compartments. Moreover, protein shuttling may occur in those instances where shifts in equilibrium distributions can be observed in response to either environmental or developmental cues. Examples include intracellular hormone receptors and protein kinases that migrate to the nucleus in response to appropriate stimulation (see above), small ribonucleoprotein particles

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(Bachmann et al., 1988), and heat shock response proteins (Velazquez and Lindquist, 1984; Welch and Feramisco, 1984; Lewis and Pelham, 1985; Collier and Schlesinger, 1986). Moreover, several proteins were described as redistributing between cytoplasm and nucleus at precise stages of embryonic development, both in invertebrates (Dequin et al., 1984; Servetnick and Wilt, 1987) and in vertebrates (Dreyer et al., 1982; Dreyer and Hausen, 1983; Zeller et al., 1983; Mattaj and De Robertis, 1985). The physiological significance of timed protein translocations to the nucleus during development remains to be explored, but it is an intriguing possibility that this process might relate to the regulation of differential gene expression. Strong support for this notion stems from recent studies on the nuclear distribution of the product of the maternal effect gene dorsal in Drosophila (Rushlow et al. 1989; Steward 1989; Roth et al. 1989). 4. I s the Transcription Factor NFKBa Shuttling Protein? In the light of the hypothesis put forward above, it is interesting to briefly consider the transcription factor NFKB(for review see Lenardo and Baltimore, 1989).This factor was originally identified in the course of investigations on the expression of immunoglobulin K light chain genes in B lymphocytes (Sen and Baltimore, 1986a; Atchison and Perry, 1987). Subsequently, NFKB activity was found to be inducible in a wide variety of cell types, and several viral and cellular genes were shown to be responsive to this factor (for references see Sen and Baltimore, 198613; Nabel and Baltimore, 1987; Bohnlein et al., 1988; Osborne et al., 1989; Lenardo et al., 1989; Dale et al., 1989). In the context of signal transduction between cytoplasm and nucleus, NFKB provides another excellent example that underscores the importance of compartmentation in the regulation of nuclear activities (Baeuerle and Baltimore, 1988a,b). In unstimulated cells NFKB occurs in the cytoplasm in the form of a complex with an inhibitor called IFKB.In response to appropriate stimuli (e.g., activation of PK-C), NFKB is activated and translocates to the nucleus (Baeuerle and Baltimore, 1988a,b). In all likelihood, regulation of the subcellular distribution of NFKB involves modification (presumably via phosphorylation) of IFKB(Sen and Baltimore, 1986b; Baeuerle and Baltimore, 1988b; Shirakawa and Mizel, 1989). Similar to the situation discussed above for the case of the catalytic subunit of PK-A, available evidence suggests that the nuclear translocation of NFKB is reversible (Baeuerle and Baltimore, 1988b), implying that NFKBmay shuttle between nucleus and cytoplasm.

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D. THEANCHORAGE-RELEASE MODEL: A UNIFYINGCONCEPT FOR SIGNAL TRANSDUCTION BETWEEN CYTOPLASM AND NUCLEUS Despite obvious differences in the molecules involved in the various signaling pathways discussed above, comparative analysis of the various situations reveals that signal transduction between cytoplasm and nucleus involves regulated compartmentation of information-carrying proteins (Fig. 10). To illustrate this point, it may be helpful to briefly reconsider glucocorticoid hormone action, the cAMP pathway, and gene regulation by induction of NFKB. In all three cases, regulation of the proteins responsible for signaling to the nucleus involves an anchorage-release type of mechanism: in the absence of stimuli, the signaling proteins exist in macromolecular complexes occurring predominantly in the cytoplasm; in response to appropriate stimuli, however, the signaling proteins are released from these complexes and translocate to the nucleus (Fig. 10D). Interestingly, release from anchorage is accomplished by different means. In the case of the cAMP pathway, the molecule triggering dissociation of the complex (CAMP) binds to the subunit remaining in the cytoplasm (Fig. lOA),whereas, in the case of glucocorticoid hormones, the ligand associates with the migrating receptor (Fig. 1OC). Finally, in the case of NFKB, complex dissociation is presumably mediated by phosphorylation of IFKB,the anchoring moiety in the complex (Fig. 10B). In none of the above cases has it been demonstrated directly that shifts in equilibrium distributions are accompanied by constant shuttling of individual proteins, but available evidence appears to be consistent with this view. If correct for the case of steroid hormone receptors, this notion might contribute to resolve the controversies about the subcellular distributions of unoccupied receptors (see above). On balance, the available evidence indicates that there may be real differences in the steady state distributions of various unoccupied receptors, but it is difficult to believe that these differences reflect fundamental differences in signal transduction mechanisms. Instead, if steroid hormone receptors were shuttling proteins, the nucleocytoplasmic partitioning of a given receptor at steady state would simply depend on its relative affinities for binding to cytoplasmic versus nuclear constituents, and, given the dynamics of the system, would be of comparatively little functional consequence. Consistent with this interpretation, recent studies suggest a dynamic nucleocytoplasmic distribution of the progesterone receptor (Guiochon-Mantel et al., 1989).

@ cAMP-dependent Protein Kinase : Inact Kina

@ NF

- KB:

@Steroid Hormone Receptors:

REC

0

/

@Unifying Concept :

Inactlva Regulator

Regulator

CytopIasm

~ I G 10. . The anchorage-release model: Schematic representation of conceptual similarities among different signal transduction pathways. Despite obvious differences in detailed mechanistic aspects (A-C), available evidence points to regulated compartmentation of signaling proteins as one ofthe fundamental aspects in signal transduction between cytoplasm and nucleus (D). Regulated compartmentation may involve not only formation of macromolecular complexes between essentially soluble proteins, but it may also be based on interactions between regulatory proteins and endomembranes or elements of the cytoskeleton. In the case of regulatory proteins with short halflives, constant turnover may be sufficient to prevent excessive depletion or accumulation of such proteins in any particular compartment. However, in all other cases, protein shuttling (i.e. reversible translocation between the cytoplasm and the nucleus) would be expected to be essential for maintaining a flexible, rapidly responsive signaling system. (For further explanation, see text.) REG, Regulatory subunit, CAT, catalytic subunit; INH, inhibitory subunit; TF, transcription factor; REC, receptor; HSP 90, (constitutively expressed) 90 kDa heat shock protein.

TABLE I1 EVIDENCE FOR INVOLVEMENT OF PROTOONCOGENES IN SIGNALING PATHWAYS ~

Protooncogene

Related proteins and purported biological function

Reference

A. Protooncogenes related to growth or differentiation factors

c-sis

Platelet-derived growth factor (@chain)

int-1 int-2Ihst

Drosophila wingless gene product Fibroblast growth factor

Doolittle et al. (1983);Waterfield et al. (1983) Rijsewijk et al. (1987) Delli Bovi et al. (1987);Yoshida et ol. (1987)

M (9

m

B. Protooncogenes related to plasma membrane receptorsa c-erbB C-fVLS

Receptor for epidermal growth factor Colony-stimulating factor 1

Downward et al. (1984) Sherr et al. (1985)

C. Protooncogenes related to intracellular receptors c-erbA

Thyroid hormone receptor

Sap et 01. (1986);Weinberger et al. (1986)

D. Protooncogenes functioning as protein kinases Tyrosine kinases" c-src family c-raflnil c-nos

Early signal transduction after membrane receptor occupation?

Hunter and Cooper (1985)

Serine/threonine kinases involved in signal propagation? Serine/threonine kinase involved in cell cycle regulation (meiosis)

Rapp et al. (1988) Sagata et a / . (1989)

E. Protooncogenes related to guanine nucleotide regulatory proteins c-ras family

Coupling of membrane receptors to secondary messenger effector systems?

Barbacid (1987)

F. Protooncogenes related to nuclear factors c-jun

AP-1 transcription factor

c-fos

Transcription factor (complex with c-jun)

c-myc c-myb

Transcription factor? Transcription factor?

Vogt et al. (1987); Bohmann et al. (1987);Angel et al. (1988) Curran and Franza (1988);Vogt and Bos (1989) Cole (1986) Biedenkapp et al. (1988)

G. Protooncogene related to angiogenesis ~

c-mas

Jackson et al. (1988)

Angiotensin receptor

W

(D

H. “Tumor suppressor” genes” RB (retinoblastoma gene) Krev-1 P53 Lethal (2) giant Laroue a

Transcription factor? ras-related repressor Nuclear Function Regulator of cell growth and differentiation?

et al. (1988) Whyte Kitayama et al. (1989) Finlay et al. (1989) Jacob et al. (1987)

For a listing of receptor-type and “src-type” tyrosine kinase protooncogenes see Hunter and Cooper (1985)and Hunter et al. (1988). For reviews on tumor suppressor genes in Drosophila, see Gateff (1978,1979).

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V. The Role of Signal Transduction in Oncogenesis: Concluding Remarks

Consistent with the notion that hyperplasia arises from loss of control over normal programs of cell division or differentiation, most of the currently known protooncogene products appear to function in signal transduction pathways controlling these processes (Table 11; see also Land et al., 1983; Weinberg, 1985; Kahn and Graf, 1986; Rassoulzadegan and Cuzin, 1987; Bishop, 1987; Reddy et al., 1988). Likewise, although the molecular analysis of cellular genes capable of suppressing the transformed cell phenotype is at an early stage (Gateff, 1978, 1982; Knudson, 1971, 1985; Sager, 1985, 1989; Klein, 1987; Stanbridge, 1987; Hansen and Cavenee, 1988),the products of the first few characterized “tumor suppressor genes” also appear to function in signal transduction (Table 11). Thus, there is now ample evidence to demonstrate that deregulation of intracellular signaling pathways plays a major role in the etiology of neoplastic disorders. It is to be hoped that an increased molecular understanding of these processes will eventually lead the way to novel diagnostic or therapeutic approaches. ACKNOWLEDGMENTS Because of the breadth of the subject covered in this article, it was inevitable to limit citations to the most recent literature (up to May, 1989) and cite review articles wherever possible. My sincere apologies go to all workers whose contributions received less explicit credit than they deserve. I would like to thank Drs. H. Hilz (University of Hamburg, West Germany) and B. Hemmings (Friedrich-Miescher Institute, Basel, Switzerland) for generous gifts of anti-PK-A antibodies, and Dr. U. Aebi (M. E. Miiller Institute for high-resolution electron microscopy at the Biocenter, Basel, Switzerland) for kindly providing Fig. 4. I also thank Drs. S. Gasser, V. Simanis, and T. Young for helpful discussions and comments on the manuscript. Work in the author’s laboratory was supported by grants from the Swiss National Science Foundation (3.316 and 3.431) the Swiss Cancer League (371.871), and the Cancer League of the Kanton Zug.

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INDEX

Abelson leukemia virus, Burkitt’s lymphoma and, 245 Adenocarcinoma, metastatic cancer cells and, 92 Adenomas, metastatic cancer cells and, 113 Adenovirus, polyoma virus TSTA and, 40 Adenylate cyclase, signal transduction and, 274 Adipocytes, c-fos protooncogene and, 42,43 Amino acids Burkitt’s lymphoma and c-myc, 158, 162,164 EBV, 235,237 c-fas protooncogene and, 38-40,47,48 jun oncogene and, 1 DNA binding, 4-6 family of related genes, 9 leucine zipper, 17-21 oncogenicity, 26 signals, 12, 13 metastatic cancer cells and, 117 polyoma virus TSTA and, 63,72, 75-78,80-82 signal transduction and, 283 Antibodies Burkitt’s lymphoma and c-myc structural changes, 178 EBV, 223,225,227,229,235,237 features, 145, 148 c-fos protooncogene and, 39,41,43-45 jun oncogene and, 14,19 polyoma virus TSTA and, 58-62, 65,66 signal transduction and, 293 Antigens Burkitt’s lymphoma and B cell differentiation, 165 C - ~ Y C ,157, 161, 163 chromosomal translocations, 197 EBV, 223,225-240 features, 145

genetic abnormalities, 220, 221 phenotype, 150,152-154 synthesis, 243,244,249 c-fos protooncogene and, 39 jun oncogene and, 7, 15 metastatic cancer cells and, 111, 113 polyoma virus, see Polyoma virus TSTA signal transduction and, 282,283,293 AP-1 c-fos protooncogene and, 43-45 jun oncogene and, 2,6-8 dimerization, 14-17 oncogenicity, 26,28 regulation, 21,22 signals, 13 ATPase, polyoma virus TSTA and, 81 Autocrine growth factors, metastatic cancer cells and, 89, 111, 115-120 Avian leukosis virus, Burkitt’s lymphoma and, 170,215,216 Avian sarcoma virus 17,jun oncogene and, 2-4,25

B B cells, Burkitt’s lymphoma and, 135, 136,141, 142 c-myc expression, 187 chromosomal translocations, 203,204 deregulation, 209-216 differentiation, 165-170 EBV, 226,227,230-234,238-240 features, 145, 147, 148 genetic abnormalities, 216,218 nonrandom chromosomal translocations, 156,157 phenotype, 150,153,154 synthesis, 243-246,248,250 Bacteria jun oncogene and, 6-8 polyoma virus TSTA and, 72,73

311

3 12

INDEX

Basement membranes, metastatic cancer cells and, 88,95, 108-1 10 Basic fibroblast growth factor, metastatic cancer cells and, 116, 117,124, 125 Bone Burkitt’s lymphoma and, 149 c-fos protooncogene and, 42 Bone marrow, Burkitt’s lymphoma and, 138 deregulation, 210,212 EBV, 225,226 features, 143, 144, 149 genetic abnormalities, 218 Burkitt’s lymphoma, 135-137 c-myc, 157 cell proliferation, 164, 165 gene structure, 158, 159 protein functions, 160-164 regulation of transcription, 159, 160 c-myc expression, 186-192 c-myc structural changes, 176-185 chromosomal translocations, 195-197 absence of normal c-myc expression, 201-205 c-myc expression, 197, 198 c-myc mRNA, 198,199 c-myc promoters, 199-201 immunoglobulin sequences, 205-208 clinical significance, 250,251 definition, 137-141 deregulation of c-myc, 208 chicken bursa1 lymphoma, 215,216 transgenic mice, 208-213 viral promoter, 214,215 EBV and, 223-225 epidemiology, 225-227 latent gene expression, 235-238 lymphocyte transformation, 227-232 pathogenesis, 240,241 T cell cytolysis, 238-240 T cell recognition, 233-235 transactivator gene, 241,242 features, 141-143 clinical findings, 149, 150 distribution of tumor, 143, 144 environment, 144, 145 geography, 145, 146 immunosyppression, 146-149

genetic abnormalities, 216,217 anti-oncogenes, 220-222 defects in DNA repair, 222,223 viral oncogenes, 217-220 geography, 192-195 non-random chromosomal translocations, 154-157 phenotype, 150-154 synthesis, 242 additional genetic factors, 248,249 animal models, 250 cellular origins, 243,244 clinical differences, 246-248 environment, 244,245 molecular consequences, 246 subset, 249,250 translocation mechanism, 171-176 timing, 165-170

C c-fos protooncogene, transcription and, 37,38,50,51 cell growth, 40-42 DNA binding, 42,43,46-48 gene expression, 38-40 jun, 43-45 protein complexes, 49 TRE, 45,46,49,50 c j u n protooncogene, 43-46,48-50 c-myc, Burkitt’s lymphoma and, 136, 137, 140, 142 B cell differentiation, 169, 170 cell proliferation, 164, 165 chromosomal translocations, 195-208 clinical significance, 251 deregulation, 208-216 EBV, 230,240-242 expression, 186-192 features, 148 genetic abnormalities, 216-218, 220-222 geography, 192,193, 195 nonrandom chromosomal translocations, 154- 157 phenotype, 152 proteins, 160-164

INDEX

structure, 157-159,175-185 synthesis, 242,245-249 transcription, 159, 160 translocation mechanism, 171,175,176 Calcium Burkitt’s lymphoma and, 164 jun oncogene and, 7 metastatic cancer cells and, 123 signal transduction and, 276,277,292 Calmodulin, metastatic cancer cells and, 123 Carbohydrate jun oncogene and, 25 signal transduction and, 280,281 cDNA clfos protooncogene and, 49 jun oncogene and, 9 , 1 1 metastatic cancer cells and, 117 signal transduction and, 289 Central nervous system Burkitt’s lymphoma and, 143, 149 clfos protooncogene and, 41 metastatic cancer cells and, 108 Centromeres, metastatic cancer cells and, 95 Chicken bursa1 lymphoma, Burkitt’s lymphoma and, 215,216 Choleratoxin, metastatic cancer cells and, 116,120 Chromatin Burkitt’s lymphoma and, 141 B cell differentiation, 166, 168 c-myc, 159 c-myc expression, 192 chromosomal translocations, 154, 203-205 synthesis, 243,246,247 translocation mechanism, 176 clfos protooncogene and, 42 Chromosomes Burkitt’s lymphoma and, 135-137, 140-142 B cell differentiation, 165-168 c-myc expression, 186-192 c-myc structural changes, 176, 177, 180-183, 185 clinical significance, 251 deregulation, 208,215 EBV, 230,241,242

313

features, 145, 147, 149 genetic abnormalities, 218,220 geography, 192-194 nonrandom translocations, 154- 157 phenotype, 152 synthesis, 242,245-250 translocation, 171-176, 195-208 jun oncogene and, 8 , 9 metastatic cancer cells and, 97, 103, 111, 113 Clonal dominance, metastatic cancer cells and, 96-105 Clones Burkitt’s lymphoma and, 135, 147, 148 B cell differentiation, 166, 167 c-myc, 161,177, 185,190, 191 chromosomal translocations, 196, 197,203 deregulation, 209,210,212,213 EBV, 226,227,233,234,236 genetic abnormalities, 218 nonrandom chromosomal translocations, 154 phenotype, 150,154 translocation mechanism, 172,175 c-fos protooncogene and, 37,49 jun oncogene and, 3 , 4 , 7 , 1 1 metastatic cancer cells and, 88, 121, 125 human malignant melanoma, 107, 110,113 phenotype, 90,95 Collagen c-fos protooncogene and, 42,43 metastatic cancer cells and, 108 Collagenase clfos protooncogene and, 45 jun oncogene and, 7 metastatic cancer cells and, 90,96, 104,110,124 Colony-inhibition assay, polyoma virus TSTA and, 60,61 Colorectal carcinomas, metastatic cancer cells and, 113, 114, 124 Competence factors, clfos protooncogene and, 41 Complement-fixing antibodies, pol yoma virus TSTA and, 62

3 14

INDEX

Cyclic AMP c-fos protooncogene and, 49,50 jun oncogene and, 9,23 signal transduction and mechanisms, 287-291,296,297 plasma membrane receptor, 274,277 Cytoplasm Burkitt’s lymphoma and, 150 c-myc, 162,163 chromosomal translocations, 199 EBV, 232 c-fos protooncogene and, 37 jun oncogene and, 1,2, 15 polyoma virus TSTA and, 64 signal transduction and, 273,274 mechanisms, 284,286,287,289-293, 295-297 nuclear pores, 278,280,282 plasma membrane receptor, 277

D Deletion, polyoma virus TSTA and, 65-67 Deregulation, Burkitt’s lymphoma and, 136, 137 c-myc, 208-216 c-myc expression, 186, 187, 192 c-myc structural changes, 181, 182 chromosomal translocations, 195, 202 EBV, 237,241 genetic abnormalities, 216, 217, 219 nonrandom chromosomal translocations, 155, 157 synthesis, 243,245,247-249 translocation mechanism, 176 Differentiation Burkitt’s lymphoma and, 135 B cell, 165-170 c-myc, 165, 179 chromosomal translocations, 197, 198,204,205,207 deregulation, 210,212,213 EBV, 225 genetic abnormalities, 217,218 phenotype, 150,154 synthesis, 243,244

c-fos protooncogene and, 37,38, 41-43,50 jun oncogene and, 2 metastatic cancer cells and, 106, 121 signal transduction and, 271,298 Dimerization Burkitt’s lymphoma and, 163, 164 c-fos protooncogene and, 47,48 jun oncogene and, 15-21,26,28-30 signal transduction and, 285 DNA Burkitt’s lymphoma and B cell differentiation, 165, 166, 169, 170 c-myc, 160,161,163,164 c-myc expression, 187, 190 c-myc structural changes, 177, 179, 182, 185 chromosomal translocations, 200, 203-205 deregulation, 209, 215 EBV, 223,224,227-231,237 features, 144 genetic abnormalities, 220-223 synthesis, 244,248 translocation mechanism, 171, 172, 175, 176 c-fos protooncogene and, 42-49 jun oncogene and, 2 binding, 4-6 dimerization, 14, 15 family of related genes, 8 , 9 hierarchical order of functions, 28-30 leucine zipper, 17-21 oncogenicity, 27 regulation, 23 signals, 12, 13 transcription factor AP-1, 7, 8 metastatic cancer cells and, 94, 97,99 polyoma virus TSTA and, 62,63,65, 70,81 signal transduction and, 271,278,284, 286,293 Drosophila, jun oncogene and, 9 Drug resistance, metastatic cancer cells and, 95,97,98, 104, 105 Duke’s staining system, metastatic cancer cells and, 114

INDEX

Dynamic heterogeneity, metastatic cancer cells and, 94-96

Early antigens, Burkitt’s lymphoma and, 223,225,228 EBP, see Enhancer-binding protein Ectopic environment, metastatic cancer cells and, 89,93, 114 Ectopic gene expression, metastatic cancer cells and, 125 Electron microscopy Burkitt’s lymphoma and, 223 signal transduction and, 279 Embryo Burkitt’s lymphoma and, 208,215,218, 222 c-fos protooncogene and, 42,46 junoncogeneand,24-27 metastatic cancer cells and, 97 polyoma virus TSTA and, 65 signal transduction and, 295 Embryonal carcinoma cells, metastatic cancer cells and, 95 Endothelium Burkitt’s lymphoma and, 240 metastatic cancer cells and, 88, 117, 122 Enhancer-binding protein Burkitt’s lymphoma and, 163 c-fos protooncogene and, 48 j u n oncogene and, 19,21,29 Environment, Burkitt’s lymphoma and, 244,245 Enzymes Burkitt’s lymphoma and B cell differentiation, 166, 170 c-myc structural changes, 183, 184 EBV, 224 genetic abnormalities, 222,223 translocation mechanism, 171, 172, 174,175 j u n oncogene and, 5 metastatic cancer cells and, 88,97, 110, 123 signal transduction and, 274,278,292, 293

315

Epidermal growth factor c-fos protooncogene and, 50 metastatic cancer cells and, 117, 119, 120 Epithelium Burkitt’s lymphoma and, 135,225,226 metastatic cancer cells and, 88, 109, 113, 120, 121 Epitopes Burkitt’s lymphoma and, 151,234,239 j u n oncogene and, 7,14 polyoma virus TSTA and, 65,73, 75-82 Epstein-Ban nuclear antigen, Burkitt’s lymphoma and c-myc, 162 chromosomal translocations, 204 genetic abnormalities, 221,222 phenotype, 151 synthesis, 244,250 transactivator gene, 242 Epstein-Barr virus Burkitt’s lymphoma and, 135-137, 142, 223-225 c-myc expression, 187 c-myc structural changes, 181-183 chromosomal translocations, 197, 199,200,203,204 clinical significance, 250 deregulation, 21 1 epidemiology, 225-227 features, 144-148 genetic abnormalities, 216,218,219, 22 1 geography, 194 latent gene expression, 235-238 lymphocyte transformation, 227-232 pathogenesis, 240,241 phenotype, 151-153 synthesis, 243-246,248-250 T cell cytolysis, 238-240 T cell recognition, 233-235 transactivator gene, 241,242 j u n oncogene and, 9 , 2 1 Escherichia coli, Burkitt’s lymphoma and, 235 Extracellular matrix metastatic cancer cells and, 88 signal transduction and, 271

316

INDEX

Fat, metastatic cancer cells and, 108 Fibroblasts Burkitt’s lymphoma and, 162, 164 chromosomal translocations, 198,202 EBV, 231,232 c-fos protooncogene and, 38.39,41 j u n oncogene and, 3,23,25-27 metastatic cancer cells and, 119 fos oncogene, j u n oncogene and, 2,22 Fos protein, j u n oncogene and dimerization, 14-17 hierarchical order of functions, 29 leucine zipper, 17-21 oncogenicity, 28 regulation, 23 signals, 31

G G proteins, signal transduction and, 274 p-Galactosidase, polyoma virus TSTA and, 73 GCN4 c-fos protooncogene and, 47,48 j u n oncogene and, 4-7,9 dimerization, 16 hierarchical order of functions, 29 leucine zipper, 17, 19,21 Genetic markers, metastatic cancer cells and, 97 Geography, Burkitt’s lymphoma and, 192-195,246 Glucocorticoids, signal transduction and, 296 Glycoprotein, metastatic cancer cells and, 88,96 Glycosylation, j u n oncogene and, 25 Growth factors, see also specific growth factor Burkitt’s lymphoma and, 143, 154 B cell differentiation, 157, 164 chromosomal translocations, 205 EBV, 231,232 synthesis, 248 c-fos protooncogene and, 37 j u n oncogene and, 1,2,24

metastatic cancer cells and autocrine growth factors, 115- 120 clonal dominance, 103, 104 growth inhibitory molecules, 120- 122 human malignant melanoma, 88, 110,111,113,124-127 tissue specificity, 122, 123 signal transduction and, 272,277,298 Growth-inhibitory molecules, metastatic cancer cells and, 89, 104, 120-122

HeLa cells, j u n oncogene and, 6-8, 13 Hemagglutination, polyoma virus TSTA and, 61,66 Hematopoiesis c-fos protooncogene and, 41 metastatic cancer cells and, 97, 120 Hemopoiesis, Burkitt’s lymphoma and, 137,202,250 Heterodimers Burkitt’s lymphoma and, 163, 222 c-fos protooncogene and, 46-49 j u n oncogene and, 15,16,21,23,28, 29 Heterogeneity, metastatic cancer cells and, 94-96 HIV, Burkitt’s lymphoma and, 139, 147-149,194 HLA, Burkitt’s lymphoma and EBV, 227,233,234,238,239 synthesis, 244, 245 Homodimers c-fos protooncogene and, 46,49 j u n oncogene and, 15,21,29 Homogenous staining regions, metastatic cancer cells and, 95 Homology Burkitt’s lymphoma and, 137 C - ~ Y C ,159-161,163,185 chromosomal translocations, 156,201 EBV, 242 genetic abnormalities, 221 synthesis, 249 translocation mechanism, 171, 172

INDEX

c-fos protooncogene and, 37,40,44, 47,49 jun oncogene and, 1,3-6,9 signal transduction and, 277 Hormone-responsive elements, signal transduction and, 285,286 Hormones c-fos protooncogene and, 38,39,50 jun oncogene and, 1,30 signal transduction and, 271,272 mechanisms, 284-286,294,297 plasma membrane receptor, 274,278 Hybridization Burkitt’s lymphoma and c-myc, 181,185,187,190 chromosomal translocations, 196-198,202,203,205-208 genetic abnormalities, 219 nonrandom chromosomal translocations, 154 jun oncogene and, 8 metastatic cancer cells and, 97,99 polyoma virus TSTA and, 70,71 Hybridomas, metastatic cancer cells and, 110 Hyperplasia Burkitt’s lymphoma and, 210,211,216, 217,245 signal transduction and, 296

I Immune response, polyoma virus TSTA and, 59-62,70,82 Immunization metastatic cancer cells and, 110 polyoma virus TSTA and, 58,65, 68-70,72-82 Immunofluorescence Burkitt’s lymphoma and, 162,236 polyoma virus TSTA and, 62 Immunoglobulin Burkitt’s lymphoma and, 135, 136 B cell differentiation, 165-170 c-myc, 159, 165 c-myc expression, 186-188, 190-192

317

c-myc structural changes, 176-178, 180-182,184 chromosomal translocations, 196, 198,200-208 deregulation, 209,211-213,216,217 EBV, 227,231,233,241,242 features, 145, 147, 148 genetic abnormalities, 217, 218 nonrandom chromosomal translocations, 154- 156 phenotype, 150-153 synthesis, 244-249 translocation mechanism, 171-176 metastatic cancer cells and, 97 Immunosuppression, Burkitt’s lymphoma and, 146-149 EBV, 224,225,234,236 synthesis, 244,245, 250 Influenza, polyoma virus TSTA and, 73 Inhibition Burkitt’s lymphoma and, 231,232,234 B cell differentiation, 165 c-myc, 162, 163, 165 c-myc structural changes, 178,179 chromosomal translocations, 197, 202-204 deregulation, 212,213 genetic abnormalities, 222 c-fos protooncogene and, 41,43,51 jun oncogene and, 9,22 metastatic cancer cells and, 89, 104, 115-117,120-122,124,127 polyoma virus TSTA and, 60,61, 63,66 signal transduction and, 281, 285,286, 289 Insulin Burkitt’s lymphoma and, 153 metastatic cancer cells and, 117-120 Insulin-like growth factors, metastatic cancer cells and, 117,118,120 Integrin, Burkitt’s lymphoma and, 240 Intercellular adhesion molecules, Burkitt’s lymphoma and, 240 Interferon, Burkitt’s lymphoma and, 180, 231,239 Interleukin-2, Burkitt’s lymphoma and, 157, 164 Interleukin-3, Burkitt’s lymphoma and, 164

318

INDEX

Interleukin-4, Burkitt’s lymphoma and, 23 1 Intracellular adhesion molecule, metastatic cancer cells and, 96, 110-113,125

J jun oncogene, 1-4

dimerization, 14-17 DNA binding, 4-6 family of related genes, 8-11 hierarchical order of functions, 28-30 leucine zipper, 17-21 oncogenicity, 25-28 regulation, 21-25 signal conversion, 30,31 signals, 11-14 transcription factor AP-1.6-8

K Keratin Burkitt’s lymphoma and, 225 jun oncogene and, 19 Keratinocytes, metastatic cancer cells and, 105,109 Kidney, Burkitt’s lymphoma and, 162

L Latent membrane protein, Burkitt’s lymphoma and, 228,231-237,244, 250 Lectin, signal transduction and, 281 Leucine c-fos protooncogene and, 47,50 jun oncogene and, 12,13,30 Leucine zipper Burkitt’s lymphoma and, 163, 164,222 jun oncogene and, 17-21

Leukemia, Burkitt’s lymphoma and, 140, 144 B cell differentiation, 167, 168 phenotype, 150, 153 Ligands c-fos protooncogene and, 42,50 jun oncogene and, 1 signal transduction and, 272, 277,285, 286,296 Lipid c-fos protooncogene and, 42 signal transduction and, 292 Liver Burkitt’s lymphoma and, 164,210 c-fos protooncogene and, 41,48 metastatic cancer cells and, 113, 121 Long terminal repeats Burkitt’s lymphoma and chromosomal translocations, 202 deregulation, 208,209,214,215 genetic abnormalities, 217-219 c-fos protooncogene and, 39,43 Lymph nodes Burkitt’s lymphoma and, 149 deregulation, 209-211 EBV, 240 metastatic cancer cells and, 92, 108,115 Lymphocyte-defined membrane antigen, Burkitt’s lymphoma and, 233,238, 244 Lymphocyte functional antigens, Burkitt’s lymphoma and, 240 Lymphocytes Burkitt’s lymphoma and c-myc, 164 deregulation, 210, 215,216 EBV, 224-232,234,237,239,240 synthesis, 244 polyoma virus TSTA and, 59-61 signal transduction and, 295 Lymphoid cells, Burkitt’s lymphoma and deregulation, 208 differentiation, 165 EBV, 226 Lymphokines, polyoma virus TSTA and, 61 Lymphomas, polyoma virus TSTA and, 70 Lymphoproliferation, Burkitt’s lymphoma and, 250

INDEX

M Macrophage migration inhibition assay, polyoma virus TSTA and, 61,80,81 Macrophages Burkitt’s lymphoma and, 218 c-fos protooncogene and, 41,42 Major histocompatibility complex, polyoma virus TSTA and, 73,78, 79,82 Malaria, Burkitt’s lymphoma and, 145, 148,211,245 Mast cells, c-fos protooncogene and, 41 Mean tumor diameter, polyoma virus TSTA and, 73,74 Mean tumor loads, polyoma virus TSTA and, 78 Melanocyte growth factor, metastatic cancer cells and, 116 a-Melanocyte-stimulating hormone, metastatic cancer cells and, 117 Melanoma cell-derived growth factor, metastatic cancer cells and, 117 Melanoma cells, metastatic cancer cells and, 124-126 clonal dominance, 99 growth factors, 116-118 human malignancy, 105-115 phenotype, 91,95,96 Metallothionein Burkitt’s lymphoma and, 208,209 c-fos protooncogene and, 44 jun oncogene and, 7 Metastatic cancer cells, 87-90, 125-127 clonal dominance, 97-105 ectopic gene expression, 125 growth factors autocrine growth factors, 115-120 growth inhibition molecules, 120-122 tissue specificity, 122,123 human malignant melanoma, 105-1 15 phenotype, 90-96 pleiotropy, 123, 124 Methionine, jun oncogene and, 12 Migration Burkitt’s lymphoma and, 158,234 metastatic cancer cells and, 88, 106, 126 signal transduction and, 293,294

319

Migration index, polyoma virus TSTA and, 61 Minichromosomes, metastatic cancer cells and. 94 M itogens Burkitt’s lymphoma and, 164 c-fos protooncogene and, 39-41 metastatic cancer cells and, 116, 117 signal transduction and, 292 Mitosis Burkitt’s lymphoma and, 230 jun oncogene and, 28 Monoclonal antibodies Burkitt’s lymphoma and, 151,238 metastatic cancer cells and, 110 polyoma virus TSTA and, 62,72 Monocytes, c-fos protooncogene and, 41,42 mRNA Burkitt’s lymphoma and c-myc, 158,186,190 chromosomal translocations, 196, 198-201 EBV, 228,229,235 synthesis, 248 c-fos protooncogene and, 39-41,45, 46 jun oncogene and, 14,22,24 metastatic cancer cells and, 116, 119, 124 Murine leukemia virus, polyoma virus TSTA and, 57 Mutagenesis Burkitt’s lymphoma and, 192 jun oncogene and, 19 signal transduction and, 282 Mutation Burkitt’s lymphoma and, 219,220,222 c-myc, 162,163 c-myc expression, 192 c-myc structural changes, 177-180, 183,184 chromosomal translocations, 199, 201,202 EBV, 192,242 synthesis, 247 translocation mechanism, 176 c-fos protooncogene and, 38,39 metastatic cancer cells and, 87,90, 93-96

320

INDEX

polyoma virus TSTA and, 65-67,69, 72,80, 81 signal transduction and, 285,289 Myoblasts, jun oncogene and, 3 Myotubes, jun oncogene and, 3

N Natural killer cells, Burkitt’s lymphoma and, 233,239 Neoplasm Burkitt’s lymphoma and, 137, 140,141 B cell differentiation, 165, 170 c-myc, 181, 187 chromosomal translocations, 157, 196,205 deregulation, 208-213.216 EBV, 224,228,234,238,241 features, 142, 147, 148, 150 genetic abnormalities, 216-218 phenotype, 150,151 translocation mechanism, 176 metastatic cancer cells and, 89, 125, 127 growth factors, 115, 118, 120-123 human malignant melanoma, 105, 106,108,109,113 phenotype, 90 signal transduction and, 272,300 Neoplasms, Burkitt’s lymphoma and clinical significance, 251 synthesis, 248 Neovascularization, metastatic cancer cells and, 110 Neurospora,jun oncogene and, 9 , 2 1 Neutrophils, c-fos protooncogene and, 41 Nuclear immunofluorescence staining, polyoma virus TSTA and, 62 Nucleotides Burkitt’s lymphoma and c-myc, 158, 161 EBV, 225 phenotype, 151 translocation mechanism, 173-176 c-fos protooncogene and, 49 jun oncogene and, 1,4,7,11, 12,14 metastatic cancer cells and, 110 polyoma virus TSTA and, 81

Nucleus, signal transduction to, see Signal transduction to cell nucleus

Oligomerization, Burkitt’s lymphoma and, 163, 164 Oligosaccharides, metastatic cancer cells and, 88 Oncogenes, see also specific oncogene Burkitt’s lymphoma and c-myc expression, 187, 190 chromosomal translocations, 157, 163,207 deregulation, 208,214 EBV, 234 genetic abnormalities, 217-222 synthesis, 248 c-fos protooncogene and, 37,38,40 polyoma virus TSTA and, 58,59,64, 78,82 Oncogenesis, signal transduction and, 296-300

P39 c-fos protooncogene and, 39,45 jun oncogene and, 14 PDGF, see Platelet-derived growth factor Peptides Burkitt’s lymphoma and, 230, 233,235, 237 c-fos protooncogene and, 43,45,48 jun oncogene and, 7,14,19 polyoma virus TSTA and, 61,77-82 signal transduction and, 272 Peritoneal exudate cells, polyoma virus TSTA and, 80,81 PEST sequence, jun oncogene and, 24,25 Phenotype Burkitt’s lymphoma and, 140, 150-154 B cell differentiation, 165, 166 chromosomal translocations, 205

INDEX

deregulation, 211-213 EBV, 236,238-240 genetic abnormalities, 217, 218 c-fos protooncogene and, 37 jun oncogene and, 28-30 metastatic cancer cells and, 88,89, 123-126 clonal dominance, 97,99, 104 growth factors, 115, 119, 120 human malignant melanoma, 105, 110,111 selectivity, 90-96 polyoma virus TSTA and, 64,70 signal transduction and, 300 Phorbol esters c-fos protooncogene and, 50 jun oncogene and, 7,22 Phospholipase, signal transduction and, 276 Phosphorylation c-fos protooncogene and, 38,39 fun oncogene and, 22,23,25 signal transduction and mechanisms, 286,287,289,290,292, 295,296 nuclear pores, 282 plasma membrane receptor, 274, 277,278 Plasma, c-fos protooncogene and, 41 Plasma cells, Burkitt’s lymphoma and, 207 Plasma membrane polyoma virus TSTA and, 64,68 signal transduction and, 272 oncogenesis, 298 receptor, 274-278 Plasmacytoma, Burkitt’s lymphoma and, 141 B cell differentiation, 168 c-myc expression, 186, 190-192 c-myc structural changes, 180, 185 chromosomal translocations, 196-200, 205-208 deregulation, 209,214,215 genetic abnormalities, 217-220,222 nonrandom chromosomal translocations, 156 phenotype, 153 synthesis, 245,247,248 translocation mechanism, 171

32 1

Plasmids Burkitt’s lymphoma and c-myc, 165, 179 EBV, 227-230 synthesis, 250 c-fos protooncogene and, 45 metastatic cancer cells and, 97-99, 124 Plasminogen activator, metastatic cancer cells and, 90,96, 124 Platelet-derived growth factor Burkitt’s lymphoma and, 164 c-fos protooncogene and, 50 jun oncogene and, 1 Pleiotropy, metastatic cancer cells and, 123 Polyoma virus TSTA, 57,58 definition, 58,59 future prospects, 82 immune response studies, 59-62 molecular biology, 62-64 present view, 81,82 studies, 64,65 epitopes, 72-81 T antigens, 65-72 Polypeptides Burkitt’s lymphoma and, 224,225,229, 230,235,237 jun oncogene and, 6 , 7 metastatic cancer cells and, 115, 122 signal transduction and, 277 Pristane, Burkitt’s lymphoma and, 156, 214,217,218 Progression factors, c-fos protooncogene and, 41 Proliferation Burkitt’s lymphoma and B cell differentiation, 167 c-myc, 157,186 chromosomal translocations, 196, 197,199,202,203 deregulation, 208,210,215,216 EBV, 227,233,234,237,238 genetic abnormalities, 220,221 mechanism, 145, 147, 148 nonrandom chromosomal translocations, 156 phenotype, 154 synthesis, 243,244,248 translocation mechanism, 176 c-fos protooncogene and, 37,38,41

322

INDEX

metastatic cancer cells and, 113, 117 signal transduction and, 271 Proline, jun oncogene and, 13, 17 Proteases, metastatic cancer cells and, 95,104,124,125 Protein Burkitt’s lymphoma and c-myc,157-164 c-myc expression, 186, 188, 190, 191 c-myc structural changes, 178, 179, 184 chromosomal translocations, 196, 197,200-204,208 deregulation, 213,214 EBV, 228,230-235,237,241,242 genetic abnormalities, 219-222 nonrandom chromosomal translocations, 154, 155 phenotype, 151 synthesis, 243,247,249,250 jun oncogene and, 1-3 dimerization, 14-17 DNA binding, 4-6 family of related genes, 9 hierarchical order of functions, 28,29 leucine zipper, 17, 19-21 oncogenicity, 25-28 regulation, 22,24,25 signals, 12,30, 31 transcription factor AP-1,7,8 metastatic cancer cells and, 96, 116, 117 polyoma virus TSTA and, 62-64, 72, 73,75 signal transduction and, 271,272 accumulation of proteins, 281-284 mechanisms, 284,286,289-296 plasma membrane receptor, 274, 276,278 Protein kinase, signal transduction and, 272 mechanisms, 284,286-292,297,298 plasma membrane receptor, 274-278 Protein kinase C jun oncogene and, 1,7,22,23 metastatic cancer cells and, 123 Proteolysis metastatic cancer cells and, 88 signal transduction and, 292

Protooncogenes, see ulso specific protooncogene signal transduction and, 271, 292,296, 298,299

Radial growth phase, metastatic cancer cells and, 106-111, 116 Radioimmunoassay, Burkitt’s lymphoma and, 235 Replication Burkitt’s lymphoma and c-myc, 161, 162,179 chromosomal translocations, 205 EBV, 225-230,232 features, 145 genetic abnormalities, 221 jun oncogene and, 3 polyoma virus TSTA and, 62, 64,66, 73,78 signal transduction and, 271,278, 293 Restricted early antigen, Burkitt’s lymphoma and, 223 Restriction fragment length polymorphism, metastatic cancer cells and, 97 Retinoic acid, signal transduction and, 285 Retrovirus Burkitt’s lymphoma and c-myc,162, 164, 185, 190 chromosomal translocations, 202 deregulation, 214, 215 EBV, 231,232,237 genetic abnormalities, 217-219 jun oncogene and, 1,3,4,26,27,30 metastatic cancer cells and, 97-99 Ribonucleoproteins, signal transduction and, 281,294 RIM, Burkitt’s lymphoma and, 217-219 RNA Burkitt’s lymphoma and c-myc, 158-160, 178 chromosomal translocations, 198, 199 EBV, 228,229,231 c-fos protooncogene and, 40,41 jun oncogene and, 22

323

INDEX

metastatic cancer cells and, 97,124 polyoma virus TSTA and, 63 signal transduction and, 281 RNA polymerase, c-fos protooncogene and, 48 Rous sarcoma virus, c-fos protooncogene and, 43

S Second messengers jun oncogene and, 30 signal transduction and, 272,274-277 Serine, signal transduction and, 277,278 Serotonin, metastatic cancer cells and, 125 Shuttling proteins, signal transduction and, 292-296 Signal kansduction c-fos protooncogene and, 37,41 metastatic cancer cells and, 89,123 Signal transduction to cell nucleus, 271-274 accumulation of proteins, 281-284 mechanisms, 284 anchorage-release model, 295,296 oncogenesis, 296-300 protein kinase translocations, 286-292 shuttling proteins, 292-295 steroid hormone receptors, 284-286 nuclear pores, 278-281 plasma membrane receptor, 274-278 Simian virus 40 Burkitt’s lymphoma and C-~YC 161, , 163 chromosomal translocations, 198,202 deregulation, 209 EBV, 230 genetic abnormalities, 220 synthesis, 249 polyoma virus TSTA and, 58,76 signal transduction and, 282 Steroids Burkitt’s lymphoma and, 208 signal transduction and, 272 mechanisms, 284-286,296,297 plasma membrane receptor, 278

Subclones, metastatic cancer cells and, 90,93,107

T T cells Burkitt’s lymphoma and, 140 B cell differentiation, 165 c-myc, 164 EBV, 226,227,232-240 features, 145 nonrandom chromosomal translocations, 156 phenotype, 151 synthesis, 244 polyoma virus TSTA and, 59,61,77 TdT, see Terminal deoxyribonucleotide Teeth, Burkitt’s lymphoma and, 143 Terminal deoxyribonucleotide, Burkitt’s lymphoma and, 150, 151, 176 Thioguanine, metastatic cancer cells and, 104 Threonine, signal transduction and, 277, 278 a-Thrombin, metastatic cancer cells and, 119 Thymus, jun oncogene and, 24 Tissue inhibitor of metalloproteinases, metastatic cancer cells and, 124 Tissue specificity, metastatic cancer cells and, 122,123 Topoisomerases, Burkitt’s lymphoma and, 166,171 TPA c-fos protooncogene and, 45,50 jun oncogene and, 7,22-24 metastatic cancer cells and, 111, 116, 117, 123 Transactivation, Burkitt’s lymphoma and, 232,241,242,249,250 Transcription Burkitt’s lymphoma and B cell differentiation, 169 C - ~ V C ,158, 159, 161 c-mycexpression, 186, 188, 190-192 c-myc structural changes, 178-182, 184,185

324

INDEX

chromosomal translocations, 198-201,203,205 clinical significance, 251 deregulation, 209 EBV, 228,230,237,242 genetic abnonnalities, 218,221,222 geography, 194 nonrandom chromosomal translocations, 155 phenotype, 150 synthesis, 246-249 c-fos protooncogene and, see c-fos protooncogene jun oncogene and AP-1,6-8 dimerization, 15-17 DNA binding, 4-6 family of related genes, 9 hierarchical order of functions, 2, 28-30 oncogenicity, 26-28 regulation, 21-25 signals, 12-14,30,31 polyoma virus TSTA and, 64,65 signal transduction and, 285-288,291, 293,295,299 Transferrin, Burkitt’s lymphoma and, 154,157 Transforming growth factor-/3, metastatic cancer cells and, 103, 120, 121, 124 Transin, c-fos protooncogene and, 50 Translocation Burkitt’s lymphoma and, 135-137, 139-141 B cell differentiation, 165-170 c-myc, 159 c-myc expression, 186-188,190-193 c-myc structural changes, 176-185 EBV, 242 effect on c-myc expression, 195-208 features, 144, 145, 147-150 genetic abnormalities, 218-220,222, 223 mechanism, 171-176 nonrandom, 154-157 phenotype, 150,152,153 synthesis, 243-250 signal transduction and, 282, 284-295 Transplantation antigen, tumor-specific, see Polyoma virus TSTA TRE, c-fos protooncogene and, 43-50

TSTA, polyoma virus, see Polyoma virus TSTA Tumor, Burkitt’s lymphoma and clinical significance, 251 synthesis, 243-246,248 Tumor necrosis factor Burkitt’s lymphoma and, 239 jun oncogene and, 23 Tumor-specific transplantation antigen, polyoma virus, see Polyoma virus TSTA Tumorigenesis Burkitt’s lymphoma and, 136, 150 B cell differentiation, 170 c-myc expression, 187 clinical significance, 250 deregulation, 208-215 EBV, 232,234,236,241 genetic abnormalities, 218-220 geography, 194 metastatic cancer cells and, 89, 124, 125 clonal dominance, 103 growth factors, 115, 117, 121 phenotype, 92 polyoma virus TSTA and, 59 Tumors Burkitt’s lymphoma and, 134-141 B cell differentiation, 166-170 c-myc expression, 186-188, 190, 191 c-myc structural changes, 177,182 chromosomal translocations, 196, 199,203 deregulation, 209-215 EBV, 223,224,228,234,236-238, 240-242 features, 141-150 genetic abnormalities, 216-220 geography, 193-195 nonrandom chromosomal translocations, 156, 157 phenotype, 150-152 translocation mechanism, 171,172 c-fos protooncogene and, 37,43,44 jun oncogene and, 3,22,25,27,28,30 metastatic cancer cells and, 88,89, 124-127 clonal dominance, 97-105 growth factors, 115, 117, 119-123

325

INDEX

human malignant melanoma, 106-109,111-113 phenotype, 90-94,96

Virus-hemagglutination-inhibiting antibodies, polyoma virus TSTA and, 61,66

signal transduction and, 300 Tyrosine kinase polyoma virus TSTA and, 64 signal transduction and, 277,278 Wheat germ agglutinin, signal transduction and. 281

X

v Vaccinia virus vectors, polyoma virus TSTA and.. 69.70.75.81.82 . . . . Vascularization, metastatic cancer cells and, 88 Vertical growth phase, metastatic cancer cells and, 107-111, 113, 116 Virus capsid antigen, Burkitt’s lymphoma and, 223,225,228

Xenopus, signal transduction and, 280

Y Yeast c-fos protooncogene and, 47 jun oncogene and, 4-6,16

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