Advances in Cancer Research Volume 54

ADVANCESINCANCERRESEARCH VOLUME 54

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ADVANCES IN CANCER RESEARCH Edited by

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

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

Volume 54

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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CONTENTS

CONTRIBUTORS TO VOLUME 51.........................................................................

xi

The Role of DNA Methylation in Cancer

PETERA . JONES AND JONATHAN D . BUCKLEY I. I1. I11. IV. V. VI . VII . VIII . IX. X.

Introduction ................................................................................................. CpG Islands ................................................................................................. Transduction of the Methylation Signal .................................................... DNA Methylation in Tumor Cells ............................................................. Methylation in Uncultured Tumor Tissue ................................................ Effects of Chemical Carcinogens on DNA Methylation........................... Role of DNA Methylation in Tumor Diversification ................................ DNA Methylation during Oogenesis and Spermatogenesis .................... DNA Methylation and Genomic Imprinting ............................................. Conclusion ................................................................................................... References., ..................................................................................................

1 2 4 7 9 11 13 14 16 19 19

Genetic and Epigenetic Losses of Heterozygosity in Cancer Predisposition and Progression

HEIDIJ . SCRABLE.CARMEN SAPIENZA.AND WEBSTERK. CAVENEE I. I1. 111. IV. V. VI .

Introduction ................................................................................................. Genetics and Predisposition ....................................................................... Loss of Heterozygosity and Tumor Progression ....................................... Loss of Heterozygosity in Mixed Cancer................................................... Epigenetic Inactivation of Alleles in Human Cancer............................... Conclusions .................................................................................................. References .................................................................................................... V

25 29

37 42 49

58 59

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CONTENTS

Genetic and Molecular Studies of Cellular Immortalization

JAMES R. SMITHAND OLIVIA M. PEREIKA-SMITH I. 11.

Introduction ..................................................................... Short-Tenn Analysis of Cell Fusion Products ...........................................

\‘.

Discussion .......I......................._........................................

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

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

64 73 76

The Function of RAS Genes in Saccharomyces cerevisiae JAJiES

R. BROACHAND ROBERTJ. DESCHENES

I. Introduction ......................................... 11. Sfodel for Ras Protein Function in Yeast ................................................... 111. Yeast Ras Proteins .........._... I\’, Components of the Ras-cAMP Pathway .............. .... . ...... ...... ........... V. Targets of the cL4MP-DependentProtein Kinase \’I, To What Signals Do R A S Genes Respond?............................................... VII. LVhat Is Ras Doing References...........................

79 82 86 101 111 122 128 132

Retroviral Integration in Murine Myeloid Tumors to Identify Evi-7, a Novel Locus Encoding Zinc-Finger Protein

N. G. COPELAND ASD N. A. JENKINS Introduction ..................... RI Slouse Strains ......................................................................................... Identification o f a New Common \ ’ i d Integration Site, Eci-f , in AKXD Myeloid Tuiiiors .................................................................... 11’. Relationship of Eci-l to Other Zinc Finger Proteins ................................ V. Activation of Transcription ofEl;i-I by Viral Integration in Fim-3 ......... 1’1. Additional Zinc-Finger Proteins Implicated in Neoplastic Disease .......

I, 11. 111.

................................... ............ ........................ ................................... I

141 143 145 151 154 154 154 155

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Metastatic Inefficiency

LEONARD WEISS I. I1. 111 IV V VI VII .

. . . .

Introduction ................................................................................................. Metastatic Inefficiency-Documentation .................................................. The Metastatic Process ............................................................................... Metastatic Inefficiency-Random and Nonrandom Events ..................... The Molecular Biology of Metastatic Inefficiency .................................... Consequences of Metastatic Inefficiency .................................................. Conclusions .................................................................................................. References ....................................................................................................

159 160 161 178 187 199 202 203

Growth Regulatory Factors for Normal. Premalignant. and Malignant Human Cells in Vitro

MEENHARD HERLYN.ROLAND KATH. NOELWILLIAMS. ISTVAN VALYI.NAGY. AND ULRICH RODECK I. I1. I11.

IV . V. VI .

Introduction ................................................................................................. Growth of Normal Human Cells in Vitro .................................................. Human Melanocytic Cells as a Model for Studies on Tumor Progression ................................................................................................... Growth Factor Independence of Human Tumor Cells from Metastatic Lesions ......................................................................................................... Autocrine Growth Stimulation of Human Tumor Cells and Strategies for Growth Inhibition .................................................................................. Summary ...................................................................................................... References ....................................................................................................

213 214 216 224 226 231 232

The Lymphopoietic Microenvironment in Bone Marrow

PAULW. KINCADE I. I1. I11. IV. V. VI .

Introduction ................................................................................................. Evolution of Experimental Approaches ..................................................... Long-Term Bone Marrow Cultures............................................................ Differentiation Steps and Lineages ........................................................... Lymphocytes in Long-Term Cultures ....................................................... Initiation of Long-Term Cultures ...............................................................

235 237 240 241 242 245

...

CONTENTS

Vlll

VII. VIII. IX. X. XI. XI1 XIII. XIV.

Essential Cells of the Microenvironment.................................................. Recognition and Adhesion between Cells Interleukin 7 ...

I

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

xv. References.......

,..................

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

246 250 252 254 256 257 259 261 261 264

Structure and Function of the B-Lymphocyte Epstein-Barr Virus/CSd Receptor

GLENR. NEMEROW, MARGARET D. MOORE,AND NEILR. COOPER I. Introduction ..... .................... 273 11.

111. IV. V. VI. VII.

Identification a Structure of CR2 ................ Structure and Function of the CR2 Ligands .............................................. Cellular and Tissue Distribution o Functional Pr Summary and Future Prospects ...................................... ........................... References.. ....

274 276 284 287 29 1 294 295

The Opportunistic Tumors of Immune Deficiency

HARRYL. IOACHIM Introduction ......................... ............ States of Immune Deficienc .. ... ,....,. 111. Opportunistic Infections ... IV. Opportunistic Tumors ................................. ................................................ V. Distinctive Features of Op VI. Spontaneous Regression of Opportunistic Tumors ................................... VII. Stromal Reaction of Tumors ........................................................ VIII. d Immune Surveillance of Tumors ...................... References. ....... ...................................................... ,........................... I. 11.

.I I..

30 1 302 303 304 31 1 311 312 3 13 315

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A Note on Concomitant Immunity in Host-Parasite Relationships: A Successfully Transplanted Concept from Tumor Immunology

GRAHAM F . MITCHELL I . Introduction ................................................................................................. I1 . Some Examples of Concomitant Immunity in Parasitized Mice ............. I11. Concluding Comments ............................................................................... References ....................................................................................................

319 320 327 330

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INDEX

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CONTRIBUTORS TO VOLUME 54 Numbers in parentheses indicate the pages on which the author’s contributions begin.

JAMES R . BROACH,Department of Biology, Princeton University, Princeton, New Jersey 08544 (79) JONATHAN D. BUCKLEY,Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern Calqornia, Los Angeles, Calqornia 90033 ( 1 ) WEBSTERK. CAVENEE, Ludwig Znstitute for Cancer Research, Royal Victoria Hospital and McGill University of Medicine, Montreal, Quebec H3A 1A1, Canada (25) NEILR . COOPER, Research Institute of Scripps Clinic, Department of Immunology, La Jolla, California 92037 (273) N. G. COPELAND, Mammalian Genetics Laboratory, BRI-Basic Research Program, NCZ-Frederick Cancer Research Facility, Frederick, Maryland 21 701 (141) ROBERTJ . DESCHENES, Department of Biology, Princeton University, Princeton, New Jersey 08544 (79) MEENHARDHERLYN,The Wistar lnstitute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 (213) HARRYL. IOACHIM, Department of Pathology, Lenox Hill Hospital, New York, New York, 10021, and College of Physicians and Surgeons, Columbia University, New York, New York 10032 (301) N . A. JENKINS, Mammalian Genetics Laboratory, BRZ-Basic Research Program, NCI-Frederick Cancer Research Facility, Frederick, Maryland 21 701 (141) PETERA. JONES, Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033 (1) ROLANDKATH, The Wistar Institute ofAnatomy and Biology, Philadelphia, Pennsylvania 19104 (213) PAULW. KINCADE,Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 (235) GRAHAM F. MITCHELL,The Walter and Eliza Hall Znstitute of Medical Research, Melbourne, Victoria 3050, Australia (319) MARGARETD. MOORE,Research Institute of Scripps Clinic, Department of Immunology, La Jolla, California 92037 (273) xi

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CONTRIBUTORS TO VOLUME 54

GLENR. NEMEROW, Research Znstitute of Scripps Clinic, Department of Immunology, LaJolla, Calijornia 92037 (273) ULRICHRODECK, The Wistar Znstitute of Anatomy and Biology, P k l a delphia, Pennsylvania 19104 (213) CALMENSAPIENZA, Ludwig Znstitute for Cancer Research, Royal Victoria Hospital and McGill University Faculty of Medicine, Montreal, Quebec H3A 1A1, Canada (25) HEIDIJ . SCRABLE, Ludwig Znstitute f o r Cancer Research, Royal Victoria Hospital and McGill University Faculty of Medicine, Montreal, Quebec H3A LAI, Canada (25) JAMES R. SMITH,Roy M . and Phyllis Gough Huffington Center on Aging, and Departments of Virology and Epidemiology, of Cell Biology, and of Medicine, Baylor College of Medicine, Houston, Texas 77030 (63) O L I V LM~. PEREIRA-SMITH, Roy M . and Phyllis Gough Huffington Center on Aging, and Departments of Virology and Epidemiology, of Cell Biology, and of Medicine, Baylor College of Medicine, Houston, Texas 77030 (63) ISTVANVALYI-NAGY, The Wistar Znstitute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 (213) LEONARDWEISS, Department of Experimental Pathology, Roswell Park Memorial Znstitute, Buffalo, New York 14263 (159) NOELWILLIA-MS, The Wistar Znstitute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 (213)

THE ROLE OF DNA METHYLATION IN CANCER Peter A. Jones and Jonathan D. Buckley Kenneth Norris Jr. Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033

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

Introduction CpG Islands Transduction of the Methylation Signal DNA Methylation in Tumor Cells Methylation in Uncultured Tumor Tissue Effects of Chemical Carcinogens on DNA Methylation Role of DNA Methylation in Tumor Diversification DNA Methylation during Oogenesis and Spermatogenesis DNA Methylation and Genomic Imprinting Conclusions References

I. Introduction

Interest in a potential role for DNA methylation in the control of eukaryotic gene expression was first stimulated by two papers published in 1975 by Holliday and Pugh (1975) and Riggs (1975). Since that time considerable evidence has accumulated that 5methylcytosine is implicated in gene control. There have been well over 100 studies that have demonstrated an inverse relationship between methylation and expression, and numerous experiments have shown that methylation can inactivate genes. This evidence has led to the suggestion that hypomethylation may be a necessary but not sufficient condition for gene activity and has been summarized in numerous reviews published over the last few years. The reader is referred to these for a detailed description of the biochemistry of DNA methylation and its role in gene control (Doerfler, 1983; Adams and Burdon, 1985; Razin and Szyf, 1984; Cedar, 1988).All of the available evidence supports the idea that methylation plays a role in cellular memory and participates with other gene control mechanisms to stabilize transcriptionally inactive states. DNA methylation is clearly altered in many cancer cells, and the relevance of these alterations to malignancy has also been the subject of many review articles (e.g., Holliday, 1979; Riggs and Jones, 1983; Jones 1985). Some of the reasons for suspecting that the epigenetic 1 ADVANCES IN CANCER RESEARCH, VOL 54

Copyright 0 1990 by Academic Press, Inc All riehts of reoroduction in any form reserved

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PETER A. JOXES AND JONATHAN D. BUCKLEY

control exerted by 5-methylcytosine might be of relevance to carcinogenesis and tumor development were summarized in Advances in Cancer Research by Riggs and Jones (1983).This review will therefore concentrate on developments since that paper was published that have strengthened the hypothesis that aberrations in methylation are a key factor in the development and expression of the malignant state.

II. CpG Islands CpG methylation sites are markedly underrepresented in vertebrates (Josse et al., 1961) and are not scattered randomly throughout the genome but rather are clusterd in specific regions called CpG islands or HTF regions (Tykocinski and Max, 1984; Bird, 1986; Gardiner-Garden and Frommer, 1987). The enrichment for CpG within these islands is due to a lack of the overall suppression of the dinucleotide in eukaryotic genomes and also to their relative G + C richness (65% G + C compared to 40% for bulk DNA) (Bird, 1986). CpG islands have been found to be associated with the 5' ends of many tissue-specific and housekeeping genes as well as with the 3' ends of some tissue-specific genes. Bird et al. (1985)have estimated that there are -30,000 islands per haploid genome in the mouse. They suggested that the islands may be associated with genes and serve to identify sequences that ought to be constantly available in the nucleus for transcription. In support of this hypothesis, Lavia et al. (1987) used two randomly isolated islands as probes and detected multiple transcripts of RNA from several mouse tissues. Cloned cDNAs for the major transcripts of one island were isolated and used to construct a transcritipnal map. The authors found that the island contained the origin of a pair of divergent transcripts that were probably mRNA molecules. The results therefore support the view that CpG islands often mark genes and suggest that bidirectional transcription may be a common feature of island promoters. Many studies have shown that the CpG islands are unmethylated in all tissues tested. Bird et a2. (1987)analyzed both CpG frequency and methylation across part of the human a-globin locus. The CpG frequency was not reduced and none of the tested CpG sites were methylated in the DNA from erythroid or nonerythroid tissues, although flanking CpG sites were methyalted. This observation therefore confirms the results of a large number of other studies, which have shown that CpG islands are not methylated in the germ-line or adult tissues (Bird, 1986; Gardiner-Garden and Frommer, 1987). These kinds of

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experiments have added weight to the hypothesis of Bird (1986) that methylation of the CpG islands does not control gene expression of tissue-specific or housekeeping genes in normal development. Rather, the islands are protected from methylation and remain unmethylated, which has ensured their survival in the face of the strong tendency for 5-methylcytosine to deaminate to thymine. The clear exception to the general rule that CpG islands are not methylated is seen with genes located on inactive X chromosomes in female mammals. Numerous studies have shown that methylation of these areas is associated with transcriptional inactivity. Examples are hypoxanthine phosphoribosyltransferase (HPRT), glucose-6phosphate dehydrogenase (GGPD), and phosphoglycerate kinase (PGK), in which the islands are methylated on inactive X chromsomes but unmethylated on the active chromosome (Wolf and Migeon, 1985; Toniolo et al., 1988; Keith et al., 1986). Experiments with the mouse H P R T gene have also demonstrated that sites within the 5' region of the gene are completely unmethylated when carried on the active X and extensively methylated when carried on the inactivated chromosome. The same sites become demethylated in H P R T genes reactivated either spontaneously or after 5-azacytidine (5-Aza-CR) treatment (Lock et al., 1986). There is therefore considerable evidence that a methylationassociated mechanism participates in the silencing of genes on the inactive X chromosome. However, methylation does not appear to be the initial step by which the gene inactivation event occurs. X inactivation takes place early during embryogenesis and results in the inactivation, at random, of one of the initially active chromosomes. Lock et al. (1987) have demonstrated that methylation of the H P R T gene on the inactive X chromosome occurs after the inactivation event. Therefore, 5-methylcytosine in the CpG island is thought to stabilize the transcriptionally inactive state. If Bird's hypothesis (1986) is correct, then the methylation of CpG islands should never occur in cells that are part of the germ line. Indeed the germ line may be sequestered from the rest of the embryo during embryogenesis and escape X inactivation (Monk et al., 1987). DNA methylation therefore appears to be a mechanism whereby cells can control the expression of genes with similar promoter regions in the presence of ubiquitous transcription factors. As far as dosage compensation is concerned with the X chromosome, the methylation of the CpG island in somatic cells helps to ensure that only one chromosome is active, thus achieving functional hemizygosity for X-linked

4

PETER A. JONES AND JONATHAN D. BUCKLEY

genes. This has been the only clear demonstration of methylation of CpG islands to date; however, several indirect experiments have suggested that methylation may control genes located on autosomes. The metallothionein gene family is present in a broad range of eukaryotic species and is expressed in many different cell types and tissues. All vertebrates synthesize at least two metallothionein isoforms (MT1 and MT2), which have different amino acid sequences. The situation in humans is complicated by the presence of multiple M T l isoforms (Schmidt and Hamer, 1986).The single gene for MT2 is ubiquitously expressed in all human cells in response to cadmium; however, the two genes encoding MT1 isoforms are expressed in a highly specific reciprocal fashion that correlates with the embryonic germ layer origin of the cells. Cells derived from mesoderm and endoderm express predominantly the M T l e gene, whereas cells derived from ectoderm, intermediate mesoderm, or lateral mesoderm express predominantly the M T l f gene in response to a heavy-metal stimulus. Schmidt and Hamer (1986) showed that the ability of cells to express the different isoforms could be altered by prior treatment with S-AzaCR, suggesting that the reciprocity of gene expression in these cells was controlled by DNA methylation. Similar results were obtained for the M T I b gene by Heguy et al. (1986). These authors found that the S’-flankingregion of the gene was highly methylated in HeLa cells that did not express M T l b but unmethylated in hepatoma cells that do express the gene. The HeLa cells could be induced to express M T l b after S-Aza-CR treatment, suggesting a strong influence of methylation in inducing cis-acting suppression. Thus, methylation could play a key role in preferential silencing of a particular subset of genes in the presence of similar promoter regions and common transcription factors.

I l l . Transduction of the Methylation Signal Although we have known for >10 years that methylation and gene expression are inversely correlated, very little is understood of the mechanisms by which the methylation signal is transduced. Early experiments by Vardimon et al. (1982) showed that the expression of a cloned adenovirus gene was substantially inhibited by in vitro modification. Dramatic downregulation of expression of a variety of genes after methylation was also seen in other systems (e.g., Busslinger et al., 1983; Kruczek and Doerfler, 1983). For example, the transcription of the adenovirus 2 E2a gene is reduced below detectable levels by the methylation of three HpaZZ sites within its upstream region (Kruczek

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and Doerfler, 1983; Knebel et al., 1987). In vitro methylation of the cloned Moloney sarcoma virus (McGeady et al., 1983) or the Ha-ras genes (Borrello et aZ., 1987, 1988) limits their transforming activity after transfection. The in vitro methylation of specific sequences in the human P-globin gene has been shown to influence strongly its expression in either fibroblasts or erythroleukemia cells (Yisraeliet al., 1988). Thus, as mentioned earlier, the methylation of CpG sequences within the promoter region of genes can substantially and rapidly alter their expression. Studies on the regulation of eukaryotic gene expression have demonstrated that transcription results from the binding of multiple factors to the promoter region (Wolfee and Brown, 1988). Conceivably, methylation may block the binding of a particular transcription factor to its target sequence, thus preventing the activity of the gene in question. Several studies have shown that some but not all methylation signals are transduced in this way. We used synthetic oligonucleotides (Harrington et al., 1988) to investigate the effect of cytosine methylation on the binding of the transcription factor Spl (Dynan and Tijan, 1983)to its target sequence known as the GC box. The GC box sequence appears frequently in the promoters of genes containing CpG islands and might be an important sequence in which methylation could block factor binding. We failed to detect any effect of 5-methylcytosine in the internal CpG sequence of the GC box on Spl binding using DNase I footprinting or gel retardation analysis. These results did not address the possibility that the activity of Spl was influenced by the presence of a methyl group in its recognition sequence. These findings were confirmed and extended by Holler et al. (1988), who showed that Spl could bind and activate transcription frop a methylated GC box. Hoeveler and Doerfler (1987) found that although methylation of specific CCGG sequences within the E2a promoter of adenovirus DNA-blocked transcription, DNaseI protection analysis showed that the binding of factors to methylated promoter sequences was not influenced. Ben-Hattar and Jiricny (1988) have confirmed and extended the results found with Spl and investigated the effect ofmethylation of a single CpG sequence within the GC box on the activity of the thymidine kinase (TK)gene. The presence of a single 5-methylcytosine in the recognition sequence in the GC box blocked TK transcription but did not affect the affinity of the Spl protein for its respective recognition sequences. In contrast to these findings, cytosine methylation can block the binding of some transcription factors to target sequences. Experiments by Becker et aZ. (1987) have shown that the methylation

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PETER A. JONES AND JONATHAN D. BUCKLEY

of two cytosine residues could prevent the binding of an unknown transcription factor to the promoter region of the tyrosine aminotransferase gene. Watt and Molloy (1988) found that methylation blocked binding of a HeLa cell-transcriptional factor required for optimal expression of the adenovirus major late promoter. Thus, while the binding of some factors may not be affected directly by methylation, the binding of others may be strongly influenced by the presence of the modified base within the recognition sequence. Given the fact that methylation has such strong effects on the transcriptional activity of genes but does not necessarily block factor binding to upstream promoter regions, how then might the methylation signal be recognized in these cases? Some experiments have suggested that methylation may act by a more indirect mechanism to target DNA to inactive chromatin configurations. For example, Keshet et al. (1986)showed that the presence of cytosine methylation could direct transfected DNA to a DNase I-insensitive configuration after integration into chromatin. Detailed experiments by Buschhausen et al. (1987)also suggested that the inethylation signal may be transduced by a mechanism requiring a native chromatin configuration. These authors showed that the inhibitory effect of methylation on the herpes TK gene required that the promoter region become assembled into a chromatin configuration before the effects of methylation on transcription were seen. Naked methylated TK genes were expressed early after injection into TK- cells. However, if the methylated template was combined with histones before injection into the cells, the effect on transcription was immediate. The effects of methylation on gene expression are therefore likely to be complex and to require the presence of a chromatin structure in some cases for the appropriate recognition of the signal. One other mechanism by which methylation signals may be transduced is by the binding of negatively acting factors to methylated regions of DNA. Huang et al. (1984) and Wang et al. (1986a,b) have isolated proteins from human placenta that preferentially bind to certain methylated DNA sequences. Conceivably these sequencespecific and 5-methylcytosine-specific binding proteins might act to silence genes by binding to the promoter regions, thus precluding the access of more specific factors to their target sequences. Alternatively, they may stabilize inactive chromatin conformations. The mechanisms by which methylation signals are recognized therefore remain obscure. The binding of some, but not all, transcription factors may be influenced directly b y DNA modification, but methylation certainly does not directly block the binding of all transcription factors. The most likely explanation in the latter cases is that

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the signal is transduced by more indirect mechanisms such as altered chromatin conformation, which precludes and prevents the activities of the more specific factors. IV. DNA Methylation in Tumor Cells

Several publications since 1983 have expanded the data base that alterations in DNA 5-methylcytosine levels and patterns are very common in cultured tumor cell lines and primary tumors (Tables I and 11). Kuo et al. (1984)examined the methylation of HpaII and Hhal sites in DNA isolated from normal rat livers and from transplantable hepatomas. They found that Hhal sites in the a-fetoprotein gene were less methylated in hepatoma cell DNA than in liver DNA and found a generally decreased methylation of CCGG sequences in rat hepatoma DNA than in normal rat livers. Specific hypomethylation at a site within the c-myc gene was also seen in three of five human tumor cell lines by Cheah et al. (1984). Studies in OUI laboratory have shown that decreased methylation is not a universal feature of all human tumor cell lines, and substantial methylation decreases were not present in all explants of human pediatric tumors (Flatau et al., 1983). Methylation was decreased substantially in a fibrosarcoma, and in some neuroblastomas and rhabdomyosarcomas. However, Wilms’ tumor cell lines or medulloblastoma cell lines had methylation levels that were very similar to those obtained in human fibroblasts. Interestingly, four low-passage retinoblastoma cell strains had levels of methylation of -3.9%, which is a relatively high level of modification for human cells and tissues (Gama-Sosa et al., 1983). We also failed to detect consistently decreased methylation levels in a series of other human tumor cell lines using probes for the Ha-rus, TK, and al-collagen genes (Chandler et al., 1986). However, interpretation of experiments using cultured cell lines is complicated by the problem in obtaining the appropriate normal control cells. Also, there is substantial evidence that methylation levels and patterns can change in culture (Shmookler-Reis and Goldstein, 1982; Wilson and Jones, 1983), which also complicates interpretation of the results. Decreased 5-methylcytosine levels have been observed during metastasis in nude mouse models (Table I). Liteplo and Kerbel (1987) determined the total levels of methylation in a series of related highly metastatic cell lines isolated from a poorly metastatic human melanoma tumor cell line MeWo. The authors interpreted the observations to support the hypothesis that alterations in cytosine methylation may play a part in the generation of tumor cell heterogeneity.

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DNA AM^^^^^^^^^^

IN

TABLE I CULTURED CELLS AND TRANSPLANTABLE TUMORS

Tumor type

Assay method

Transplantable rat hepatomas Human tumor cell lines Human melanoma cells

Gene probe with a-fetoprotein c-myc Probe

Human pediatric tumor explants Human tumor cell lines Human lung and other tumor lines

HPLC

HPLC

Gene probes Calcitonin and other chromosome 11 probes

Methylation level

Reference

Decreased

Kuo et al. (1984)

Decreased in three of five Decreased in metastatic nodules Variable

Cheah et al. (1984)

Variable but heterogeneous Increased

Liteplo and Kerbel (1987) Flatau et al. (1983) Chandler et ~ l . (1986) Baylin et al. (1986); DeBustros et al. (1988)

In contrast to these observations, which have generally found decreased DNA methylation in cell lines associated with transformation and malignancy, Baylin and his colleagues (1986,1987; DeBustros et al., 1988) observed substantial hypermethylation within specific regions of human chromosomes in tumor cells. These authors examined small-cell lung carcinoma and lymphoma for methylation of the calcinonin gene located on chromosome l l p . The unusual hypermethylation patterns were found less frequently in other tumor cell types, suggesting that there was a correlation between abnormal calcitonin gene methylation and differentiation events. These studies have subsequently been extended for other markers on chromosome l l p (DeBustros et aZ., 1988). It appears that there is a hot spot for abnormal methylation on the short arm of chromosome 11, an area known to harbor several putative tumor suppressor genes. Baylin has postulated that the increased regional DNA methylation within these tumors may participate in, or mark, chromosomal changes associated with gene inactivation events central to the development of tumors during multistep carcinogenic changes. In view of the current interests in tumor suppressor genes (Klein, 1987), the concept that increased methylation may be associated with the silencing of suppressor genes within tumors is particularly attractive and provocative.

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V. Methylation in Uncultured Tumor Tissue

In contrast to the situation with cultured tumor cell lines, where there is always the question of potential artifacts being induced by culture conditions and the difficulty in obtaining appropriate normal tissue for comparison, there have been substantial advances made in our knowledge of methylation changes in uncultured tumors (Table 11).Generally decreased levels of DNA methylation were observed in a careful study of a large number of human tumors by Gama-Sosa et al. (1983) using high-performance liquid chromatography (HPLC) techniques. The authors proposed that metastatic neoplasms have significantly lower genomic 5-methylcytosine levels than most benign neoplasms or normal tissues. Similar observations have been published by Bedford and van Helden (1987), who reported decreased levels of methylation in metastatic prostatic carcinoma cells but not in less malignant tumors. A thorough study completed by Feinberg et al. TABLE I1 DNA METHYLATION IN UNCULTURED TUMORS Tumor type Diverse primary and secondary tumors Prostrate carcinoma Colon adenomas and carinomas Colon and lung carcinoma

Rat stomach tumors Human lymphoid and myeloid malignancy

Assay method

Matched control

Methylation change

Reference Gama-Sosa et al. (1983)

HPLC

No

General decrease

HPLC

Yes

HPLC

Yes

Specific genes

Yes

HPLC

Yes

Specific gene (pepsinogen) Specific gene (calcitonin)

Yes

Bedford and van Decrease in metastatic Helden (1987) carcinoma, not in nonmetastatic 8-10% decrease in Feinberg et al. all examined (1988) Feinberg and Decrease in Vogelstein benign and malignant (1983a,b); tumors Goelz et al. (1985) Decrease Ichinose et al. (1988) Heterogeneous Ichinose et al. (1988) pattern Baylin et al. Increased (1987)

Yes

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PETER A. JONES AND JONATHAN D . BUCKLEY

(1988) using human colonic cells has shown an unequivocal hypomethylation of tumor DNA. These studies were aided by the availability of normal tissue in close proximity to the tumors, guaranteeing that the appropriate controls were available. They showed an average reduction in genomic 5-methylcytosine content of 8-10% in all colon adenomas and adenocarcinomas. In contrast to the earlier reports, there was no significant difference between benign and malignant tumors, suggesting that the alteration in methylation occurs very early in the genesis of these particular tumors. The studies by Feinberg et al. (1988) complemented earlier work on changes within specific loci in tumors (Feinberg and Vogelstein, 1983a,b; Goelz et al., 1985). The earlier observations had demonstrated alterations in methylation levels of specific genes including growth hormone, y-globin, a-chorionic gonadotropin, and ycrystallin in all 23 neoplastic growths examined (Goelz et al., 1985). The data suggested that hypomethylation was a consistent biochemical characteristic of human colonic tumors and, since it was also seen in the adenomas, the methylation defect preceded the acquisition of malignancy. Goelz et al. (1985) also made the suggestion that the hypomethylation might inhibit chromosome condensation and lead to chromosome mispairing and nondisjunction. There is evidence that drug-induced hypomethylation can lead to decondensation and chromosome abnormalities (Schmid et al., 1983;Bianchi et al., 1988).Thus, methylation defects may be important in promoting allelic deletions, which are thought to be important in the genesis of tumors (Vogelstein et al., 1988). A study by Ichinose et a2. (1988)demonstrated alterations in methylation of the tissue-specific gene pepsinogen within stomach neoplasms (MNNG). induced in rats by N-methyl-N‘-nitro-N-nitrosoguanidine The methylation patterns of the genes were different from those of the normal tissues, but there was not a simple correlation between methylation and expression of the genes. However, the observation that the patterns were altered is consistent with the other data discussed earlier, which have shown that methylation is changed within tumor cells. Baylin et al. (1987) have also examined the methylation of the calcitonin gene in human lymphoid and acute myeloid malignancies. I n support of their observations with cultured cells (see earlier), these studies showed that there was an increase in the numbers of CCGG sites methylated in the 5’ region of the calcitonin gene in 90% of patients with non-Hodgkin’s lymphoid neoplasms and in 95% of tumor cell DKAs extracted from patients with acute nonlymphocytic leukemia.

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In general the evidence suggesting that methylation changes are associated with the transformed state has accumulated since the early 1980s. The studies with uncultured tissues have been particularly important in establishing a relationship between these two parameters.

VI. Effects of Chemical Carcinogens on DNA Methylation Since the pioneering studies of Drahovsky and Morris (1972) and Drahovsky and Wacker (1975), many studies have demonstrated that chemical carcinogens can interfere with the DNA methylation system (Riggs and Jones, 1983). Several later studies have extended these findings. For example, carcinogens can induce decreases in genomic 5-methylcytosine in normal bronchial epithelial cells (Wilson et al., 1987). Not all cells respond to carcinogens by heritable decreases in DNA methylation. Krawisz and Lieberman (1984) could find no substantial 5-methylcytosine decreases in Raji cells, S49 cells, or human diploid fibroblasts after treatment wich chemical carcinogens or ultraviolet (UV) radiation. However, these latter studies may not have been sensitive enough to detect biologically important methylation changes. There are two corollaries to the hypothesis that chemical carcinogens act in part by inhibiting DNA methylation: (1) Chemical carcinogens should be able to activate nonexpressed genes in suitable selectable systems in a similar manner to that demonstrated for 5-Aza-CR. (2)Agents that inhibit methylation such as 5-Aza-CR should be carcinogenic. Many experiments since 1984 have addressed these issues. MacArthur et al. (1985) showed the treatment of a cadmiumsensitive metallothionein-negative mouse cell line with two directacting carcinogens N-ethylnitrosourea or N-acetoxy-2acetyIaminofluorine, or with UV irradiation, induced a substantial increase in phenotypically stable cadmium-resistant variants. The increased cadmium resistance of the cells was demonstrated to be due to the activation of metalloethionein genes by the carcinogens. Previous studies from this group (Lieberman et al., 1983) had demonstrated that UV radiation could induce MTI gene activation, and that this was associated with extensive DNA demethylation. Thus, it is possible the mechanism of activation of metallothionein inducibility was due to a demethylation event induced by the carcinogen. Further studies by Barr et al. (1986) showed that an inactive but functionally intact hamster TK gene could be activated by MNNG in Chinese hamster cells. The authors proposed that the chemicaI carcinogen activated the TK

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gene by focal demethylation resulting in low TK activity, whereas demethylation throughout the genome resulted in much higher levels of TK activity. Ivarie and Morris (1986) have also shown that MNNG and 5-Aza-CR can induce a 50- to 100-fold increase in the reversion frequency of a mutant HeLa cell line harboring an HPRT gene silenced by methylation. Thus, chemical carcinogens such as MNNG have epigenetic affects in addition to their well-known ability to be mutational agents. Some DNA-alkylating agents can promote enzymatic methylation at CpG sites in certain cases. Ethyl methane sulfonate (EMS) can silence prolactin gene expression in GH3 cells (Ivarie and Morris, 1982), and direct experiments have shown that methylation of poly dC-dG with EMS stimulates the methyl-accepting ability of the DNA by rat DNA methyltransferase (Farrance and Ivarie, 1985).Carcinogens can therefore have multiple effects on DNA methylation, acting either to decrease or to increase methylation depending on the specific lesion induced in DNA. Macnab et al. (1988) have demonstrated that some viruses such as herpes simplex virus can induce the hypomethylation of host cell DNA synthesized after infection. The inhibition of host cell DNA methylation may therefore be an important step in the transformation of cells by herpesviruses. The second corollary of the hypothesis that DNA methylation and carcinogenesis are linked predicts that agents that inhibit DNA methylation should be capable of transforming cells. Our early experiments with 5-Aza-CR (Benedict et al., 1977) showed that the drug was capable of transforming the 10T1/2 cell line in addition to inducing changes in differentiation within the cells (Constantinides et al., 1977). These experiments have been confirmed by Rainier and Feinberg (1988). Studies by Harrison et aZ. (1983)and Gadi et al. (1984) showed that some differentiated cell types induced by 5-Aza-CR were also tumorigenic. Walker and Nettesheim (1986) demonstrated that 5-AzaCR induced the transformation of rat tracheal epithelial cells in primary culture. Carr et al. (1984) showed that the drug could induce tumors in male Fisher rats, and Dendra et al. (1985) reported that the drug could potentiate the initiation of carcinogenesis in rats being fed diets containing various liver carcinogens. Finally, Samid et al. (1987) have shown that 5-Aza-CR can reinduce the transformation of TUS transfected cells that have been reverted to a nontumorigenic phenotype by interferon treatment. This evidence clearly shows that 5-Aza-CR can induce the transformation of cells and also that carcinogens that can inhibit DNA methyl-

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ation are capable of activating the expression of some genes known to be silenced by methylation. Overall the data strongly support the notion that methylation and carcinogenesis are linked. VII. Role of DNA Methylation in Tumor Diversification

Interpretation of experiments relating methylation changes to tumor progression are complicated because it is not certain whether the alterations observed are a cause or a result of the transformed phenotype. Fresh information that methylation plays a role in tumor progression and control of cellular behavior during metastasis has been obtained by a series of experiments using 5-Aza-CR to change DNA methylation patterns and to test the effects of the changes on tumor diversification and metastasis. Frost and Kerbel (1983)and Frost et al. (1984)were the first to show that 5-Aza-CR could strongly influence the tumorigenicity of cells by altering their immunogenicity. These initial findings were expanded by Kerbel et al. (1984),who suggested that the changes in tumorigenic potential were induced by altered methylation patterns. The same kinds of changes could also be seen at similar frequencies by treatment of the cells with agents known to be mutagenic, such as MNNG and EMS. Thus some of the effects of these carcinogenic and mutagenic agents on cellular behavior may be due to epigenetic effects on DNA methylation. Liteplo and Kerbel (1987) also showed that 5-Aza-CR could induce TK activity in a spontaneously enzyme-deficient murine tumor line when its metastatic potential was also altered by drug treatment. This provided additional evidence that methylation might be implicated in the control of the mtastatic phenotype. A series of studies from Olsson’s lab (Olsson and Forchhammer, 1984; Olsson et al., 1985a,b) showed marked effects of methylation changes on tumor cell behavior. They demonstrated that clones of murine Lewis lung carcinoma cells selected for nonmetastatic potential could be converted to a metastatic phenotype by brief exposure to 5-Aza-CR. The ability of human tumor cell lines to grow in semisolid medium, a property often associated with tumorigenicity, was also shown to be markedly responsive to 5-Aza-CR treatment. Olsson et al. (1985b) also tested the response of cloned lines of human squamouscell lung carcinoma and small-cell lung carcinomas to 5-Aza-CR7phorbol esters, and retinoic acid. 5-Aza-CR brought about a shortening of doubling time and increased cloning efficiencies, whereas the other two agents appeared to act in opposite directions. WhiIe the effects of 5-Aza-CR on cellular phenotype were quite variable, the experiments

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suggested strongly that alterations in DNA methylation had profound influence on the behavior of tumor cells as measured by the expression of several different parameters. The role of DIVA methylation in the expression of the metastatic phenotype in B16 melanoma cells was investigated by Trainer et al. (1985). Both 5-Aza-CR and 5-fluoro-2-deoxycytidine caused dosedependent increases in the abilities of B16 cells to form experimental lung metastases. Measurements by HPLC on 5-methylcytosine levels after treatment showed that these were decreased by the drug. The metastatic capacity of human tumor cell lines in nude mice has also been found to be strongly influenced by 5-Aza-CR (Ormerod et al., 1986). The drug induced a 40-fold increase in the number of lung tumor nodules compared to control cell lines. The cells populating the lung tumor nodules retained increased metastatic capacity through several cycles of growth in uitro followed by reinjection into nude mice. Examination of the methylation levels showed that 5-Aza-CR had induced a significant decrease in methylation directly after treatment but that this extensive hypomethylation was not maintained in the cell lines derived from the lung nodule lines. These studies have shown in general that 5-Aza-CR can markedly increase the rate of tumor progression and diversification. However, in certain cases 5-Aza-CR can retard the progression of cells to a more malignant phenotype. Babiss et al. (1985) showed that the rate of progression of adenovirus 5-transformed rat embryo cells was retarded by 5-Aza-CR treatment. Several experiments have shown that other drugs such as hydroxyurea that do not cause DNA hypomethylation and may in fact cause hypermethylation (Nyce et al., 1986) can also substantially alter the metastatic activity of cells (Frost et al., 1987; McMillan et af., 1986; Alvarez et al., 1988).Thus not all of the effects of 5-Aza-CR on tumor metastasis may necessarily be due to changes in DNA methylation. Clearly the drug might act in some cases to change the phenotype in cells by mechanisms more related to its inherent toxicity. However, it remains true that 5-Aza-CR is the most potent agent for altering tumor progression and metastasis in these defined systems. The final proof that 5-Aza-CR acts through a DNA methylation-linked mechanism will require the isolation of the putative genes activated by drug treatment. VIII. DNA Methylation during Oogenesis and Spermatogenesis

Several studies have demonstrated substantial differences in the methylation of DNA sequences in sperm and oocytes. These differences in methylation may be important in controlling the preferential

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expression of genes from either haploid genome during embryogenesis. Groudine and Conklin (1985) examined the chromatin structure of constitutively expressed genes, tissue-specific genes, and inactive genes in chicken sperm chromatin. They found specific sites of hypomethylation in sperm DNA within constitutively expressed genes, but not within globin genes or inactive genes. This corresponded to the location of altered chromatin structure (hypersensitive sites) in somatic tissue and spermatogonial cells. The authors also found that considerable de novo methylation occurred during spermatogenesis, so that regions within and around the genes became methylated but hypersensitive sites did not. This de novo methylation occurred between the spermatogonial stage and the first meiotic prophase. The undermethylated regions were postulated to play important roles in the activation of the paternal genome during embryogenesis. Monk et al. (1987) examined globin methylation during mouse development. The egg genome was strikingly hypomethylated whereas the sperm genome was methylated at a level similar to that found in mature tissues. Monk et al. (1987)also found a loss of genomic methylation during preimplantation development, with the embryonic and extraembryonic lineages becoming progressively and independently methylated to different extents. Sanford et al. (1987) have examined the methylation levels of dispersed, repeated, and low-copy-number gene sequences during gametogenesis and early embryogenesis in the mouse. These sequences were extensively hypomethylated in diplotene oocytes and highly methylated in DNA from sperm. Thus the results indicate that there are genome-wide DNA methylation differences between oogenesis and spermatogenesis. Repeated sequences in DNA from cleavagestage embryos from inner cell masses were methylated at intermediate levels consistent with the idea that the different methylation levels present in sperm and eggs were carried through to the early embryonic stages of development. These findings are in line with the idea that DNA methylation may play a role in genomic imprinting in mammalian development. Since earlier studies by Chapman et al. (1984) and Rossant et al. (1986) had shown that these same sequences were highly methylated in the embryonic portion of the conceptus by 7.5 days after conception, the initial differences between the maternal and paternal genomes may be obliterated at later stages of development. However, not all sequences that are differentially methylated by passage through the paternal or maternal lines behave in this way, since some differences persist into adult stages of development (see later).

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IV. DNA Methylation and Genomic Imprinting The maternal and paternal genetic complements of mammalian embryos are not functionally equivalent during development (Surani et al., 1987). After fertilization the two haploid gene sets cooperate to direct the development of a complete embryo, and there is substantial evidence that memory of the gametic origin of the two haploid gene sets is maintained during early development. This process, called imprinting, refers to the marking of certain genes for differential utilization by their passage through the maternal or paternal germ lines. Experiments with mice have clearly shown that imprinting is confined to specific chromosomal regions, and it has been proposed that the phenomenon exists in mammals to prevent the formation of homozygotic fetuses (Surani et al., 1987). Imprinting must be established before or during gametogenesis, must persist during early cell division, must be stably inherited during DNA replication, and should potentially be reversible during new gametogenesis (Monk, 1988). As pointed out in several reviews, DNA methylation is a particularly attractive molecular mechanism for the propagation of imprinting (Monk, 1987,1988). Indeed several papers have clearly shown that the methylation status and sometimes the expression of transgenes in mice can be dependent on whether they have been inherited from the mother or father mouse. Reik et al. (1987) used random DNA insertions into transgenic mice to probe the genome for modified regions that might be subjected to methylation changes during transmission from male or female mice in different strains. One of the seven loci studied showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. Importantly, the methylation pattern was faithfully reversed upon each germ-line transmission to the opposite sex. Similar results were obtained b y Sapienza et al. (19871, who also showed that the methylation patterns of exogenous DNA sequences in transgenic mice could be changed by switching their gamete of origin in successive generations. Swain et al.(1987)extended these experiments to study the methylation and expression of an autosomal transgene constructed by fusion of the Rous sarcoma virus long terminal repeat (LTR) and the myc gene. If the transgene was inherited from the male parent it was expressed in the heart and in no other tissue. On the other hand, if the gene was inherited from the female parent, it was never expressed in any tissue tested. The pattern of expression correlated precisely with the imprinted methylation state evident in all tissues. Thus, the meth-

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ylation of the gene acquired by its passage through the female parent was eliminated during gametogenesis in the male, which resulted in the subsequent expression of the gene in the heart. While these experiments do not show that the regions studied are necessarily the ones normally imprinted, and therefore relevant to mammalian development, they nevertheless show conclusively that the methylation status of a gene can be altered by its passage through the male or female germ line. Given the observation that methylation can lead to the suppression of genetic information, it may well be that early mammalian embryos are functionally hemizygous for some genes located on autosomes. This may be a mechanism to ensure that parthenogenic development could not occur. All of these results are consistent with DNA methylation playing a role in marking the paternal and maternal gene sets during early development. However, definitive proof that methylation is involved in impriting will require the isolation of a gene differentially expressed during early embryogenesis depending on its gametic origin. Nevertheless, the fact that there is evidence for differential gene utilization during development, and that methylation differences are substantial, strongly suggests that these differences may have relevance to the development of certain kinds of cancer. Many of the tumors that arise in early childhood are thought to have developmental etiology, and imprinting could conceivably play a role in the development of certain kinds of human cancer. It is not certain that imprinting exists in humans. However, it would appear that both maternal and paternal chromosomes are necessary for normal human embryogenesis, since hydatidiform moles, which contain only paternal genes, do not develop normally (Lawler et ul., 1982). This cannot be due simply to a requirement for heterozygosity, since some hydatidiform moles derive from a double fertilization by two separate sperm and are heterozygous (Lawler et al., 1982). Results from our laboratory on the methylation patterns of individual alleIes of the rus gene have also suggested that individual alleles of genes may be differentially methylated in humans (Chandler et ul., 1987). These experiments showed unequivocally that the two rus alleles present in human cells can bear different methylation patterns. If individual alleles can be differentially methylated, then it might be anticipated that this could alter the inheritance of certain kinds of human cancer. For example, if cells are functionally hemizygous for certain autosomal genes, then the frequency of occurrence of deleterious mutations at these loci would be many orders of magnitude higher than that anticipated for a diploid cell. The differential methyl-

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ation could also result in different mutation frequencies for the two alleles because of the known inherent mutagenic activity of 5methylcytosine (Radman and Wagner, 1986). Cytosine residues undergo spontaneous deamination to uracil, which-not being a normal component of DNA-is readily recognized and repaired by DNA excision repair enzymes. Methylated cytosine residues, on the other hand, deaminate at a higher frequency to produce thymine residues. Since the double-stranded DNA now has a TG pair mismatch, two repair mechanisms are possible: replace the T with C or G with A, with the latter giving rise to a permanent base substitution. Although there is some evidence that the cell differentially excises the T rather than the G in this situation (Brown and Jiricny, 1987), it has been suggested that the methylated cytosine residues are hot spots for mutation (Radman and Wagner, 1986). In fact the majority of restrictionfragment-length polymorphisms in the human genome are associated with C-to-T transitions (Barker et al., 1984). The hvo alleles of a gene in the same cell may therefore have different mutation rates if they bear different DNA modification patterns. Thus, if the genes have been imprinted by their passage through the paternal or maternal germ lines, there is the possibility that the gene inherited from each parent might have different mutation frequencies. There is in fact some evidence that such a situation might occur in Wilms’ tumor, which has been associated with homozygosity for chromosome l l p (Schroeder et al., 1987).The observation that homozygosity for specific chromosomes is found in many cancers (Hansen and Cavenee, 1987) has provided experimental evidence for a two-hit model for carcinogenesis, originally proposed by Knudson (1971).Sporadic tumors were postulated to result from two somatic events such as a mutation of one allele followed by loss of the normal allele, whereas familial tumors might be caused by inheritance of a mutant allele followed by loss of the wild-type gene. In most investigations on the allelic compositions of tumors, genetic markers within the tumors have been compared to normal tissue from the same individual without regard for the parental origin of the markers. However, strikingly unusual results have been achieved with Wilms’ tumor: in all of seven cases studied, the allele lost was that inherited from the mother (Schroder et al., 1987; Reeve et al., 1984). In five of these cases the tumor was unilateral and was not associated with any developmental anomaly, implying that each was due to a new somatic mutation on the paternally derived chromosome. This non-Mendelian behavior of alleles during the formation of this tumor suggests strongly that imprinting might be involved. Thus, the

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differential methylation patterns imposed on genes during their passage through paternal and maternal germ lines may well alter their susceptibility to spontaneously occurring mutations and thus alter the genetics of human cancer. X. Conclusions

The evidence that DNA methylation plays a fundamental role in carcinogenesis and tumor diversification has strengthened considerably. Carcinogens have been shown to inhibit methylation and to activate genes. Agents that inhibit methylation can transform cells and markedly increase the rate of tumor diversification. Many studies have shown that considerable alterations in methylation are evident in naturally occurring human and animal neoplasms at early stages of tumor development. Studies on methylation changes in early embryonic development have raised the interesting possibility that 5-methylcytosine could alter the genetics of develpment of childhood cancers. Overall the evidence is pervasive, but definitive proof will only be available when genes responsible for transformation and malignancy are isolated and characterized within these systems.

ACKNOWLEDGMENT This work was supported by grant R35 CA49758 from the National Cancer Institute.

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Wilson, V. L., Smith, R. A,, Longoria, J., Liotta, M. A., Harper, C. M., and Harris, C. C. (1987).Proc. Natl. Acad. Sci. U.S.A. 84,3298-3301. Wolf, S. F., and Migeon, B. R. (1985). Natzre (London)314,467-469. Wolfee, A. P., and Brown, D. D. (1988). Science 241,1626-1632. Yisraeli, J., Frank, D., Razin, A,, and Cedar, H. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 4368-4642.

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GENETIC AND EPIGENETIC LOSSES OF HETEROZYGOSITY IN CANCER PREDISPOSITION AND PROGRESSION Heidi J. Scrable, Carmen Sapienza, and Webster K. Cavenee Ludwig Institute for Cancer Research, Royal Victoria Hospital and McGill University Faculty of Medicine, Montreal,Quebec H3A 1Al Canada

I. Introduction 11. Genetics and Predisposition A. Cytogenetics of Retinoblastoma B. Molecular Genetics of Predisposition to Retinoblastoma 111. Loss of Heterozygosity and Tumor Progression IV. Loss of Heterozygosity in Mixed Cancer A. Association between Developmental Malformations and Tumors B. Tumors with Phenotypically Distinct Elements V. Epigenetic Inactivation of Alleles in Human Cancer A. Predisposition B. Progression VI. Conclusions References

I. Introduction There is an increasingly large body of evidence that supports the involvement of genetic lesions in the etiology of human cancer. The aggregation of tumors with occurrence incidences much greater than would be expected by chance in families such as that shown in Fig. 1is perhaps the single strongest formal corroboration of this notion. The occurrence of several types of cancer in individual members of a family is indicated in Fig. 1A. An attempt to discern a formal genetic definition of predisposition to the colon cancer trait is futile because its transmission is clouded by a spotty pattern of distribution. When the same family is categorized for a different phenotypic characteristic, stomach cancer, familial aggregation is also apparent; the formal description of a mutation giving rise to this disease is also unclear because transmission appears limited to the first few generations. Furthermore, the frequent occurrence of endometrial cancer in this family provides a phenotype that is not particularly informative given its necessarily sex-limited nature. Consolidation of the preceding phenotypic data makes it clear that cancer is prevalent in this family and that 25 ADVANCES IN CANCER RESEARCH, VOL. 54

Copyright 8 1990 by Academic Press,Inc.

A l l riEhts of reDroduction in any form reserved.

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A

I

T 7

B

FIG.1. Aggregation of cancers of different histogenesis in a family. (A) Individual members ofthe pedigree who developed cancers of the colon (diagonal stripes), stomach (crosshatching), or endometrium (dots). (B) Segregation of cancer in the family. In all cases, filled symbols represent affected individuals; circles represent females and squares represent males. This family was first ascertained by Warthin (1913).

cancers of each type have occurred uniquely in each affected individual. A phenotypic distillation accomplished by removing the qualifying tissue distribution designation and relying only on whether the family members have developed a tumor or not is shown in Fig. 1B. In this simplified display, cancer appears in the family with the formal genetic behavior of an autosomal dominant Mendelian trait. That is, disregarding the site of disease, approximately half the progeny of an affected parent have developed a tumor and no sex bias is obvious.

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This raises two paradoxes: (1) how a single mutation can give rise to any type of cancer given the complex nature of such a phenotype, and (2) how a single mutation can elicit diseases that are quite disparate with respect to the tissue affected. Regarding this latter point, it may be important to note that although this family has developed more than one tumor type, the distribution is actually quite limited in light of the large number of potential cancers that are not apparent. It may also be germane to note that familial aggregation of tumors of mixed histological types is often mirrored by the sequential occurrence in other individuals of second primary lesions after being affected first by cancer of different histogenesis. The final point is that in families such as that shown in Fig. 1, who appear to be transmitting heritable risk, the clinical description of the disease invariably shows focal lesions. This, of course, suggests that the mutation predisposes to, but is not itself sufficient to cause, the disease. On the surface this is entirely consistent with the notion that cancer represents the phenotypic manifestation of the accumulation of a critical load of genetic damage (Foulds, 1957; Nowell, 1976). This proposal is outlined in Fig. 2. The predictions it makes are of two types depending on the entry point into which the pathway is viewed. If this point is at the beginning, the circuitry can be viewed as an initiating genotoxic event with clonal outgrowth, perhaps due to incurred prolifEvents :

tNormaltInitiation+

Promotion-

ProgressionFTumor4

FIG.2. Pathway by which the increasing accumulation of genetic damage leads to malignant progression. N, Normal cell; I, initiated cell; Ti, Tz,TS, T4 refer to progressively more damaged cells in the promotion and progression stages of malignancy.Filled symbols indicate those cells that have undergone lethal mutations. This figure is redrawn from its original proposal by Nowell (1976),and all subsequent figures in this review utilize it in various permutations.

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eration advantages (N + I, Fig. 2). There is, however, a large body of experimental evidence indicating that this initial damage must be genetically fixed and then compounded. A cellular mechanism that causes biallelic fixation of the first event could cause the irreversible conversion to an expanded initiated clone (I 4TI, Fig. 2). Further damage to this clone would then permit (or perhaps even force) it to a stage of higher malignancy (TI + Tz, Fig. 2). Iteration of this process would then culminate in a fully malignant clone (Tz + T3 + Tq, Fig. 2). If the entry point is at the end of the pathway, then one is considering a cellular mass (T4)that is greatly removed from normality and that carries all of the genotoxic damage suffered in each of the previous stage transitions. This hypothesis, then, predicts that tumor cells of the ultimate stage will carry each of the events, cells of the penultimate stage will carry each ofthe events less the last one, and so on. Thus, the dissection of the pathway by which a normal cell becomes fully malignant may be viewed as the unraveling of a nested set of aberrations. Although this model does not specify the nature of the events that cause this malignancy progression, there is much reason to believe that many entail activating or inactivating mutations of genes encoding growth-related molecules. The intimate involvement of chromosomal aberrations in the neoplastic pathway has been postulated since the early part of this century. The major proponent of this idea was Boveri, who noticed that mitotic abnormalities often led to aberrant development of sea urchin embryos. These observations were concordant with the frequent occurrence of nuclear and mitotic abnormalities in carcinoma biopsy samples described during the same period by von Hansenmann. Since these pioneering studies, the technology necessary to elucidate chromosomal structure has become progressively more powerful. Malignant cells of many types have been subjected to cytogenetic analysis, and a massive number of deviations from the normal karyotype have been described, including chromosomal aneuploidy, translocation, deletion, and regional amplification (Helm and Mittelman, 1987).Moreover, many of these somatic alterations occur at high frequency in particular malignancies and so appear to be specific in that sense. Whether these chromosome aberrations are the cause or the result of the neoplastic process is generally unclear, but the studies described here make a strong primafacie case for the former, at least in some cancers. Furthermore, the power of molecular genetics in sorting through this array of cytogenetic abnormalities and in drawing mechanistic inferences regarding submicroscopic genetic and epigenetic defects in the process is illustrated by some of the studies reviewed here.

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II. Genetics and Predisposition

As already discussed, the earliest event in the pathway toward tumorigenesis is termed the initiating event in experimental chemical carcinogenesis. In the human population, such ,initiations may be transmitted as inherited predisposition (Fig. 3). At least 50 different forms of human cancer have been observed to aggregate in families as well as to have corresponding sporadic forms (Mulvihill, 1977). Obviously, these individuals represent a valuable resource in attempts to define the targets of initial genotoxic damage. In many of these cases the aggregation occurs with a pattern consistent with the transmission of an autosomal dominant Mendelian trait reminiscent of the family in Fig. 1. This interpretation is, however, at odds with three lines of evidence. First, if a single mutation were sufficient, in and of itself, to elicit a tumor, then families segregating for autosomal dominant forms of cancer would be expected to have no normal tissue in the diseased organ. This expectation is in direct contrast to the clinical observation of discrete tumor foci amidst normal, functional tissue in such individuals. Second, elegant epidemiological analyses (Knudson, 1986) of sporadic and familial forms of several cancer types have indicated that the conversion of a normal cell to a tumor cell requires multiple events. Finally, there is a substantial body of evidence derived from somatic

FIG.3. Pathway by which increasing genetic damage leads to malignancy: genetic nomenclature. N, Normal; P, predisposed; H, homozygote; H1, H2, H3, homozygotes with increasing levels of progressional damage. Such a pathway must necessarily begin in a single somatic cell in sporadic cases but can initiate in any cell in heritable cases as proposed by Knudson (1971).

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cell hybrids that indicates dominance of the wild-type phenotype in the presence of tumorigenic mutations (Stanbridge, 1987; Klein, 1987). Thus, in the context of the malignancy progression models in Figs. 2 and 3, the transition of cells from one stage to the next could be viewed as an accumulation of genetic lesions.

A. CYTOCENETICS OF RETINOBLASTOMA One important line of evidence in support of a genetic origin for cancer is the frequent finding of constitutional chromosomal abnormalities in patients with specific types of tumors. Perhaps the best characterized of these are deletions involving the chromosome region 13q14, which are found in normal tissues of 3-5% of children with bilateral retinoblastoma, a tumor of embryonic neural retina (Francke, 1976). Several such cases have now been reported and, although these deletions were rare and the extent of deletion varied considerably, the smallest overlapping region of aberration was the band 13q14. Furthermore, analysis of tumor cells from patients with normal constitutional karyotypes indicated that -5% of cases had tumor-specific deletions of chromosome 13, each of which included the q14 band (Balaban et al., 1982; Benedict et al., 1983; Squire et al., 1985).Though striking, the significance of these observations was unclear for several reasons. First, only a small proportion of cases carried visible constitutional or somatic deletions. Second, in those cases in which a 13q14 deletion was apparent in all cells, not all retinal cells were neoplastic; clearly these alterations were insufficient to elicit disease. Finally, tumor (but not constitutional) karyotypes have shown several other chromosomal aberrations in addition to deletion of 13q14, such as triplication of lq23-lqter and an isochromosome 6p (Squire et al., 1985). These complications notwithstanding, deletion cases have been useful in defining the region of the genome likely to contain a locus involved in the genesis of retinoblastoma through inference from the physical map and also genetic-linkage mapping. In the latter, activity levels of esterase D, an enzyme of unknown physiological function, were shown to be reduced in patients with deletions of 13q14 as compared to their karyotypically normal family members (Sparkes et al., 1980).This enzymatic activity also displayed isozymic forms in the human population, so that the cosegregation of a specific allelic variant with retinoblastoma could be determined in families (Sparkes et al., 1983). In fact, no meiotic recombination events could be detected, strongly suggesting that the genetic locus influencing tumor develop-

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ment resided in the 13q14 region. This was particularly important given the aforementioned rarity of cytogenetically detectable deletions of chromsome band 13q14 and because the primary mutation has not been characterized in most familial-form retinoblastoma cases. The involvement of this region has been substantiated by the identification and cytogenetic analysis of a remarkable kindred (Strong et al., 1981). In this family, both affected and unaffected members were described who carried an interstitial 13q14 deletion. However, several of the unaffected members also carried a chromosome 3 that included an interstitial translocation of this 13q14 region. Thus, all family members who were constitutionally monosomic for the region developed disease, whereas all who were either disomic or trisomic were spared. These data strongly suggested that the deletional event predisposed to retinoblastoma, that this region did indeed contain a locus or loci involved with tumor development, and that the initial mutation was unlikely to be acting in a dominant genetic fashion at the level of the individual tumor cell. Nonetheless, in the context of the requirement for multiple events in tumorigenesis (Knudson, 1986), such deletions could act as the first “hit” and, when they are germinal, they could confer the risk of tumor formation in an autosomal dominant manner, depending only on the number of cells at risk for further damage.

B. MOLECULAR GENETICS OF PREDISPOSITION TO RETINOBLASTOMA A model has been proposed (Knudson, 1971; Hethcote and Knudson, 1978)encompassing the aforementioned cytogenetics and the observation that familial cases are generally multifocal and bilateral whereas sporadic cases typically manifest as unilateral, unifocal disease of later diagnosis. According to the model, as few as two stochastic mutational events are required for tumor formation. The hereditary cases would have inherited a germinal mutation that does not in itself cause the tumor but rather predisposes each retinal cell to a further transforming event. In this model, the nonhereditary cases would arise by a similar mechanism but both events would have to occur in the same somatic cell. Thus, the two forms of the disease could be viewed as resulting from the same two-step process at the level of the aberrant retinal cell, the difference being the inheritance or somatic occurrence of the first mutation. Several predictions can be made about the nature of the second tumorigenic event in this model. First, the autosomal dominant hereditary form of retinoblastoma, in the absence of a gross chromosomal deletion, should involve the same genetic locus as that invoIved in

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cases showing large deletions of chromosome 13. Thus, the first step in the pathway toward tumorigenesis in these cases should be a submicroscopic mutational event at the tumor ( T M R ) locus. Second, the same genetic change that has occurred as a germ-line mutation in hereditary retinoblastoma should occur as a somatic genetic alteration of the TMR locus in a retinal cell in nonhereditary retinoblastoma. Third, the second step in tumorigenesis in both heritable and nonhereditary retinoblastoma should involve somatic alteration of the normal allele at the TMR locus in such a way that the mutant allele is unmasked. Thus, the first mutation in this process, although it may be inherited as an autosomal dominant trait at the organismal level, is, in fact, a recessive defect in the individual retinal cell. The model that arises (Cavenee et al., 1983) from these considerations is shown in Fig. 4, which outlines specific chromosomal mechanisms that would allow phenotypic expression of a recessive germinal mutation of the T M R locus. In this model, the heritable form of the disease arises as a germinal mutation of the TMR locus and is inherited by an individual who therefore is an obligate heterozygote (t/+) at the TMR locus in each of his somatic and germ cells. A subsequent event in any of his retinal cells that results in homozygosity for the mutant allele (i.e., mutant at the TMR locus on both chromosome homologs) will ultimately result in a tumor clone. Chromosomal mechanisms that could accomplish this loss of constitutional heterozygosity include (a) mitotic nondisjunction with loss of the wild-Qpe chromsome, which would result in hemizygosity at all loci on the chromosome; (b) mitotic nondisjunction with duplication of the mutant chromosome, which results in homozygosity at all loci on the chromosome; or (c) mitotic recombination between the chromosomal homologs with a breakpoint between the TMR locus and the centromere, which would result in heterozygosity at loci in the proximal region and homozygosity throughout the rest of the chromosome including the T M R locus. Regional events such as gene conversion, deletion, or mutation must also be considered. Heritable and sporadic retinoblastoma could each arise through the appearance of homozygosity at the TMR locus, the difference being two somatic events in the sporadic case as compared to one germinal and one somatic event in the heritable case. The approach that has been taken to examine these hypotheses relies on the variability of DNA sequences among humans. Chromosome-specific, single-copy segments of the human genome, isolated in recombinant DNA form, can be used to recognize polymorphisms at the corresponding chromosomal locus. Sequence variation in restriction endonuclease recognition sites, giving rise to restriction-

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tmr

33

t

Chromosomes :

FIG.4. A model for chromosomal mechanisms that can accomplish the conversion of a normal (N) cell to a cell that is homozygous (H) for inactivation of a tumor (TMR)locus. Predisposition occurs either by inheritance or by somatic occurrence of a mutation that converts a wild-type (+) allele to an inactive allele (TMR).A tumor could then occur by elimination of the remaining wild-type allele by nondisjunction (A), nondisjunction/ duplication (B), mitotic recombination (C), or regional aberration (D), as shown by Cavenee et aE. (1983).

fragment-length polymorphisms (RFLP) of the locus defined by the probe, are revealed as distinct bands on an autoradiogram and represent alleles of the locus (one from the paternally derived and one from the maternally derived chromosomal homolog) and behave as Mendelian codominant alleles in family studies. These RFLP can be used as linkage markers in inherited disorders, including retinoblastoma. If a disease locus is located close to a polymorphic RFLP marker locus, they are likely to segregate together in a family. Therefore, the genotype of DNA markers can be used to infer the genotype at the retinoblastoma locus, and thus to predict if the offspring has inherited the

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predisposition. Chromosome segregation during tumorigenesis can also be determined in each patient by comparing the child’s constitutional and tumor genotypes at each of these marker loci. Recombinant DNA segments have been isolated from human chromosome 13 and used in several such cases to determine somatic changes in the germline genotypes in the manner illustrated in Fig. 4. Detailed analyses of many retinoblastomas (Cavenee et al., 1983, 1985; Dryja et al., 1984) have shown that such events are common and are detected in about three-fourths of all retinoblastoma tumors. These rearrangements fall into four different classes as illustrated in Fig. 4. For example, in 20 of 33 tumors, one constitutional allele was missing at all informative loci along the entire chromosome, and 19 of these tumors contained two intact chromosomes 13 as determined either b y cytogenetic analysis of the tumor cells or densitometric quantitations of the autoradiographic signal of the remaining alleles. Therefore, these losses of alleles must involve two separate events: a nondisjunction resulting in loss of one chromosomal complement and either a duplication of the remaining homolog or an abnormal mitotic segregation of the chromosomes resulting in isodisomy as illustrated by the Retin 409 case in Table I. In one case, data consistent with the sole loss of chromosome 13 were obtained. Evidence for mitotic recombination between the chromosome homologs was provided in 4 of the 33 tumors (one example is Retin 412, Table I). The constitutional genotype was maintained at all informative loci in 9 of the 33 tumors, and therefore, in these cases the mechanism of attainment of homozygosity could not be determined. These studies strongly suggest that the second component in tumor initiation consists of a specific chromosomal rearrangement resulting in physical loss of the balancing wild-type allele. This inference was corroborated by examining cases of heritable retinoblastoma and showing that the chromosome 13 homolog retained in these tumors was derived from the affected parent as would be predicted. Two examples are shown in Table I: Retin KSBH and Retin

462F. It is noteworthy that although the unmasking of predisposing mutations at the RBI locus occurs in mechanstically similar ways in sporadic and heritable retinoblastoma cases, only the latter carry the initial mutation in each of their cells. Heritable cases also seem to be at greatly increased risk for the development of second primary tumors, particularly osteogenic sarcomas (Kitchin and Ellsworth, 1974). This high propensity may not be merely fortuitous but may be genetically determined by the predisposing RBI mutation. This notion of a pathogenetic causality in the clinical association between these two rare

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TABLE I FOR LOCION CHROMOSOME 13q IN RETINOBLASTOMAS Loss OF HETEROZYCOSITY AND OSTEOSARCOMAS Alleles present at locus" Tumor type Retinoblastoma Sporadic Retin LA69 Retin 409 Retin 412 Heritable Retin KS2H Retin 462F Osteosarcoma Sporadic Osteo 03

Tissueb D13S1

D13S7

D13S4

D13S5

013853

Mechanism

N T N T N T

1,s 2 2,2 2,2,2 172 14

1,l 1 172 2,292 2,2 2,2

N T N T

2,2 2,2 1,2" 172

1,l 1,1 1,l 1,1

Mitotic recombination

1,2

Isodisomy

N 132 T 24 Osteo 06 N 192 T 1,s Osteo 09 N 22 T 2,2 Second primary to retinoblastoma Rbl-1 N 1,2 T 171 Rb108 N 19 T 17172

Chromosome loss Isotrisomy Mitotic recombination Isodisomy

191

1,l 1J 172 171 2,2 2,2,2 172 171

Isodisomy Isodisomy Translocation, isodisomy Translocation, isodisomy

a Alleles designated in bold type are combinations that were heterozygous in constitutional tissue. N, Normal; T, tumor. 'D13S6 was examined, not D13S1. Not determined.

'

tumor types was tested by determining the constitutional and osteosarcoma genotypes at RFLP loci on chromosome 13 (Hansen et al., 1985). The data (Table I) indicated that osteosarcomas arising in retinoblastoma patients had become specifically homozygous around the chromosomal region carrying the RBI locus. Furthermore, these same chromosomal mechanisms were observed in sporadic osteosarcomas, suggesting a genetic similarity in pathogenetic causality. These find-

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ings are of obvious relevance to the interpretation of human mixedcancer families such as the one in Fig. 1, as they suggest differential expression of a single pleiotropic mutation in the etiology of clinically associated cancers of different histological types. A likely explanation for the association between retinoblastoma and osteosarcoma is that both tumors arise subsequent to chromosomal mechanisms that unmask recessive mutations in either one common locus that is involved in nornial regulation of differentiation of both tissues, or in separate loci that are located closely within chromosome region 13q14. In either case, germ-line deletions of the retinoblastoma locus may also affect the osteosarcoma locus. Deletions are likely to be an important form of predisposing mutation at the RBI locus because a considerable fraction of bilateral retinoblastoma cases carry visible constitutional chromosome deletions, and submicroscopic deletions have been detected by reduction of esterase D activity and by molecular analyses using a cDNA for a gene that is, in all likelihood, the transcription product of the retinoblastoma locus (Friend et al., 1986; Lee et al., 1987; Fung et al., 1987). The information derived from these studies raises two points relevant to familial predisposition to cancer. Chromosomal mechanisms capable of unmasking predisposing recessive mutations occur in more than one tumor and, at least for chromosome 13, clinically associated tumors share this mechanism of pathogenesis. This latter point suggests that these loci have pleiotropic tissue specificity; however, this pleiotropy appears to be restricted to a small number of tissue types. A more extensive discussion of this point in relation to the model in Fig. 4 follows. The data in this section, however, suggest a general approach to identifying the chromosomal positions of loci the recessive alleles of which predispose to human cancer. The approach takes advantage of specific and frequent somatic chromosomal alterations in tumors and draws its power from the conjoint use of such information and familial genetic analysis. Clearly, one would anticipate that a segregating tumor trait will be genetically linked to the predisposing mutation that elicits it (Hansen and Cavenee, 1987). This single characteristic should provide a means of distinguishing predisposing from progressionally acquired genetic damage, since the latter would be unlikely to be genetically linked to the former in families. In any case, the relevance of this use of genetic analysis to define the first steps of malignant progression, which involve initial monoallelic genotoxic damage and its biallelic fixation (Fig. l),is established by these studies. The more general applicability of the approach has been established as well and is reviewed in Nordenskjold and Cavenee (1988).

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37

111. Loss of Heterozygosity and Tumor Progression

The models shown in Figs. 2 and 3 predict that the ultimate stage of malignancy should encompass all previously occurring aberrations in addition to its own particular event. Clearly, the foregoing section suggests that the attainment of complete defectiveness at a “tumor locus” is one means of initiating the pathway of tumorigenesis. There is no a priori reason that similar mechanisms should be excluded from comprising at least some more distal events as well. In order to test this hypothesis we searched for a disease system characterized by the increasing acquisition of histologically-defined malignant criteria. This seemed a real consideration because other means of clinical progression could comprise, for example, a dominantly acquired increase in drug resistance, invasive capacity, metastatic potential, or growth factor responsiveness, as shown in Fig. 5. We chose the glial tumors for these first efforts at utilizing genotypic

E

Imr

Additional Events : Hemi/ Homozygosiiy- other locus

Chromosomes: +

IQ

i++tmr

it

10

Oncogene Activation Growth Factor Response Growth Foctor Receptor Amplification Drug Resistance Metastatic Potential

FIG.5. Additional genotoxic damage is required in the progression of homozygously defective (H) cells toward frank neoplasia. Potential events in the later steps of the pathway that could contribute, singly or in combination, are listed at right.

38

HEIDI J. SCRABLE E T A L .

analyses to place tumors into various stages of malignant progression. Gliomas, as a class, are the most common primary neoplasms of the central nervous system. Tumors of this type can be subclassified according to their cellular differentiation, displaying either astrocytic, oligodendrocytic, ependymal, or mixed composition, with astrocytic tumors occurring most frequently. Prognoses for individuals having astrocytoma vary according to the histopathologically-assessed malignancy grade of the tumor; however, all adult malignancy grades (grades II-IV) of astrocytoma respond poorly to radiation and/or chemotherapy and the 5-year survival rate for individuals with the most malignant form, glioblastonia (or GB, astrocytoma grade IV) is <5% (Burger et al., 1985).The propensity of low-malignancy-grade astrocytomas to relapse with recurrent tumors that often display a malignant progression accentuates the severity of the disease. Several cytogenetic analyses of high-malignancy-grade tumors have described frequent chromosome aberrations in direct preparations and shortterm cultures of astrocytomas (Rey et al., 1987; Bigner et al., 1988). In contrast, studies involving astrocytic tumors of low malignancy grade have consistently demonstrated cells with normal karyotypes. This could be because the analyzed mitoses are not representative of the tumor cells or, alternatively, genetic information could be lost following chromosomal mechanisms not detectable at the level of cytogenetic analysis. Such mechanisms might include the deletion or rearrangement of relatively small regions of chromosomes, mitotic recombination with balanced interchromosomal exchange, and chroniosomal loss with duplication as described before for retinoblastoma. In order to determine whether astrocytomas and glioblastomas share a progressional lineage, and whether specific losses of heterozygosity were preferentially associated with some of the stages of the pathway, we initially compared constitutional and tumor genotypes at loci on each human chromosome for samples from 39 adult cases of astrocytoma representing each malignancy grade. Data obtained for the loci on chromosome 17p are shown in Table 11. This subset of tumors with astrocytic differentiation lost heterozygosity through the loss and duplication or mitotic recombination mechanisms described in Fig. 4, regardless of malignancy stage (James et al., 1988). With regard to the pathway shown in Fig. 2, we cannot be sure that such events comprise the initial genotoxic damage/fixation steps; they may occur somewhat later in the progression. They do, however indicate shared insults among all the stages and, as such, in all likelihood represent early events that appear to confer selective advantages used in the outgrowth of the clone. Clearly, it would be desirable to uncover events

Astrocvtoma

IV

G 14 G21

N T N

T

TABLE I1 Loss OF HETEROZYGOSITY IN STAGES OF GLIOMA MALIGNANCY Alleles present at locus" Chromosome 17

172 171 192 172

192 272 13 29

1,1 1,1

172 171 172 272 172 191 172 1,1 172 131 172 171

DlOSl

D17S5

Chromosome 10

D10S4 172 172 172 172 132 172 172 122 172 2 14

2,2 2,2 192 172 172 132 172 1 1J 1

1

PLAU

172

172 1,2 172 1,l 1,l 2,2 2 1,1 1

Mechanismc Mitotic recombination; none Mitotic recombination; none Mitotic recombination; none Mitotic recombination; none Isodisomy; monosomy Mitotic recombination; monosomy

a Alleles designated in bold type are combinations that were heterozygous in constitutional tissue. N, Normal; T, tumor. First mechanism given is chromosome 17; second is chromosome 10. Not determined.

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HEIDI J. SCRABLE E T A L .

that occurred at progressively later stages so as to define the aforementioned nested set of aberrations in the pathway. A first step was accomplished by taking advantage of cytogenetic analyses of direct preparations and short-term cultures of malignant astrocytomas that have provided information regarding the gross chromosomal changes taking place in these tumors. For example, monosomy for chromosome 10has been detected in about one-third of grade IV astrocytomas (Bigner et al., 1988). We compared constitutional and tumor genotypes at loci on chromosome 10 for the 39 cases of astrocytoma analyzed for chromosome 17 as already mentioned. Allelic combinations were determined with probes homologous to three different chromosome 10 loci: DlOSl, D10S4 and PLAU. Representative data obtained with samples of various histological grades are shown in Table 11. Each of 28 grade IV GB tumors examined showed loss of constitutional heterozygosity at one or more of the chromosome 10 loci, and these losses appeared to be elicited by nondisjunction resulting in monosomy. In sharp distinction, none of the 11 tumors of lower malignancy grades showed a loss of alleles at any of the chromosome 10 loci examined. The results of these analyses are displayed schematically in Fig. 6. The data have two major implications. The first of these is the demonstration of a clonal origin of the cells composing these tumors. The cellular pleomorphism of malignant astrocytomas and karyotypic heterogeneity of in uitro-derived cell subpopulations arising from primary tumors have complicated attempts to determine the nature of any relationship between the cells that constitute this type of neoplasm. These data show that grade IV astrocytomas arise from the expansion of cells deficient in all or part of chromosomes 10 and 17. The second issue involves the histopathological evidence that astrocytomas progress and become more malignant with time. Because the losses of heterozygosity for chromosome 10 loci were restricted to tumors of the highest malignancy grade, it may be that this aberration is an event of tumor progression, rather than of initiation. Conversely, it is possible that the etiologies and ontogenies of astrocytomas exhibiting low and high degrees of cellular differentiation have no interrelating molecular pathways. However, the postoperative, posttherapeutic recurrence and histological progression of astrocytoma are well documented, providing clinical support for the ontogenetic relationship suggested by the shared chromosome 17p aberrations. The model shown in Fig. 6 predicts that genomic aberration will be cumulative throughout the process of malignancy. Such aberrations can be recessive or dominant mutations, amplification of genes for

LOSS OF HETEROZYCOSITY IN CANCER PREDISPOSITION

E a s:

41

Homozygote 17p

Cells -

:

EGF Remptor Amplification----- - - - -

-

-------_________

FIG.6. Specific genetic alterations in stages of astrocytic malignancy. In these brain tumors, specification can be placed on the mechanisms of genotoxic damage proposed in Fig. 5. These events encompass loss of heterozygosity for loci on chromosome 17p in all stages, epidermal growth factor receptor (EGFR) amplification in the penultimate and ultimate stages, and hemizygosity for chromosome 10 loci in the last stage, as described by James et al. (1988). N, Normal; P, predisposed; A2, astrocytoma grade 11; A3, astrocytoma grade 111; A4, astrocytoma grade IV (glioblastoma multiforme); x on a chromosome indicates an inferred recessive mutation.

growth factors or their receptors, as well as acquired cellular aggression characteristics. Such events appear to occur (or be selected for) in a specific order in brain tumors, but the model does not require this to be so. In fact, a similar accumulation of genetic abberations has been uncovered in the transition of normal colonic epithelium to adenomatous polyps to colon carcinoma (Vogelstein et al., 1988). Thus, although these data are only correlative at present, the identification of these nonrandom events may serve as the beginning of a genotypic, rather than phenotypic, approach to the definition of the molecular underpinnings of human tumor predisposition and progression.

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HEIDI J. SCRABLE E T A L .

IV. Loss of Heterozygosity in Mixed Cancer Our discussion to this point has considered unilinear variation of the original model of Nowell (1976) and the mechanistic involvement of genotoxic damage and chromosomal rearrangement in the process. There is, of course, no reason that more than one progressional event could not occur in different cellular derivatives of an irreversibly committed precursor. In fact, such a relaxation in consideration and the consequent potential for branchpoints in the pathway leads to models capable of addressing other perplexing aspects of cancer biology. In this section, we discuss in this context examples of cancer syndromes that clearly have a Mendelian component, but whose phenotypic variation points to the existence of additional genetic or epigenetic factors. These syndromes may be divided into two classes: the first consists of aggregates of developmental anomalies and a propensity for tumor formation within a single individual, whereas the second is represented by tumors that contain two phenotypically distinct components within a single neoplasm. A. ASSOCIATION BETWEEN DEVELOPMENTAL MALFORMATIONS AND TUMORS

1. Beckwith-Wiedemann Syndrome Beckwith-Wiedemann syndrome (BWS) is a congenital disorder consisting of developmental anomalies (Beckwith et al., 1964; Wiedemann, 1964) with associated neoplastic disease (Sotelo-Avila et at., 1980). The developmental anomalies are characterized by excess growth at the cellular (adrenal cortical cytomegaly), tissue (pancreatic, renal, and pituitary hyperplasia), organ (macroglossia, hepatomegaly), whole-body-segment (hemihypertrophy), or even whole-body (giantism) levels. Other characteristics of the syndrome include omphalocele or umbilical hernia, facial flame nevus, renal medullary dysplasia, and hypoglycemia that may be secondary to pancreatic islet cell hyperplasia. Of particular interest for this discussion is that >lo% of all individuals with BWS will develop rare cancers, including Wilms’ tumor, hepatoblastoma, rhabdomyosarcoma, and adrenal carcinoma, in association with the growth excess disorders that characterize the syndrome. Although most cases of BWS are sporadic, several instances of apparent autosomal dominant inheritance have been described (Sommer et al., 1977; Best and Hoekstra, 1981), albeit with reduced penetrance and variable expressivity. Cytogenetic examina-

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43

tion of constitutional cells from children with BWS has sometimes shown structural abnormalities of chromosome 11 including duplication of the llp13-p15 region (Waziri et al., 1983; Turleau et al., 1984) and duplication of l l p 1 5 only. The significance of these observations lies in their implication of the short arm of chromosome 11as a likely location for at least one of the genes involved in the syndrome. I n fact, genetic-linkage analysis supports this conclusion: familial predisposition to the syndrome segregates with loci in the l l p l 5 . 5 region of the 1989). It is a reasonable assumption, then, that genome (Koufos et d., tumors might arise in BWS patients by fixation of predisposition in somatic cells by attainment of functional homozygous defectiveness for a locus in llp15.5, as proposed in Fig. 3. Furthermore, it could be envisaged that additional progressional events that differed in homozygous kidney, liver, straited muscle, or adrenal cortex could give rise to distinct tumor types in the same individual, which would be concordant with clinical evidence (Sotelo-Avila et d.,1980; Y. Tsunematsu and S. Watanabe, personal communication). The model arising as a result of these considerations is shown in Fig. 7. This model predicts,

Events:

I-NormoltPredisposed

-+

HOmozygote~Progression~,Tumor

HOmozygote+Progression-Tumor+

Tumor A

Tumor 0

FIG.7. Tumors of two different types could arise subsequent to a common predisposing mutation if progressional damage of different types either is tissue-specific in effect or elicits differentiation along two different lineages. Symbols are as in previous figures.

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HEIDI J. SCRABLE E T A L .

at its simplest, that tumors of two different types arise in a bilinear way from a predisposed cell. The experimental expectation is that, whatever the accumulated progressional differences between the tumors, the initial fixation of the predisposition ( P + H) should be held in common. Furthermore, sporadic forms of the tumors should share both the monoallelic (N+ P) and biallelic (P + H) damage, at least in a proportion of cases. In order to test this hypothesis, we examined allelic combinations at loci on chromosome l l p in the tumors associated with the syndrome ofWilms’ tumor (Koufos et al., 1984),as well as rhabdomyosarcoma (Koufos et al., 1985; Scrable et al., 1987)and hepatoblastoma (Koufos et al., 1985). Wilms’ tumor, a neoplasm of embryonal kidney, exhibits several features analogous to those previously discussed for retinoblastoma: constitutional deletions of chromosome region 1lp13 appear to predispose to the disease (Riccardi et aZ., 1978; Slater and de Kraker, 1982), chromosome 11 abnormalities involving the same region are frequent in tumor tissue (Kondo et al., 1984), and sporadic and inherited autosomal dominant forms have been described (Matsunaga, 1981).These cases imply that mutant forms of one or more loci contained within the l l p 1 3 band predispose to tumor development but are not sufficient to elicit the cancer because discrete tumor foci are seen amidst a background of normal kidney, even in cases in which the l l p 1 3 band of one homolog is missing in the germ line. Thus a second, postzygotic event appears to be required as well (Knudson and Strong, 1972). In order to determine whether chromosomal mechanisms similar to those described earlier for retinoblastoma play a role in the development of Wilms’ tumor, DNA samples from normal and tumor tissues were analyzed for their genotypic combinations at loci on chromosome 11 (Koufos et al., 1984). Examples of the data are shown in Table I11 and indicate loss of germ-line alleles, which seemed to arise by chromosomal loss and duplication in the majority of tumors. Similar results were obtained in three other laboratories as well (Orkin et al., 1984; Reeve et aZ., 1984; Fearon et al., 1984). A likely explanation for these results, based on previous work with retinoblastoma, is that mitotic segregation events occurred in each predisposed kidney cell such that one chromosome homolog was lost and the remaining homolog was duplicated during the process of tumorigenesis. We infer that the remaining chromosomes are each defective at the Wilms’ tumor locus on chromosome 1lp. Evidence in support of this idea was provided by the introduction of a normal chromosome 11into Wilms’ tumor cells by microcell transfer (Weissman et al., 1987).The resulting hybrid cells

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45

TABLE I11 Loss OF HETEROZYCOSITY FOR LOCION CHROMOSOME l l p IN THREE CLINICALLY ASSOCIATEDTUMORS Alleles present at locus. HBBC Tumor type

Tissueb

PTH

yC

yA

DllS1.2

INS

HRASl

Wilms’ tumor Wilms 3 Wilms 11 Wilms 16 Rhabdomyosarcoma Rhabd 6 Rhabd 26 Rhabd 31 Hepatoblas toma Hepat 1 Hepat 2 Alleles designated in bold type are combinations that were heterozygous in constitutional tissue. N, Normal; T, tumor. Not determined.

lost the ability to form tumors in nude mice, although other properties of the parental cell line were unaffected. Control experiments with introduction of other chromosomes into the,tumor cells failed to affect their neoplastic properties. Together, all of these data suggest the Wilms’ tumor cells are homozygously defective for a locus that functions as a phenotypic suppressor of tumorigenicity. Furthermore, the data are consistent with the model in Fig. 7, in which cells that suffer a homozygous loss of function of the BWS locus (or a closely linked one) can progress to this associated tumor type. Rhabdomyosarcoma is a soft-tissue malignant tumor of skeletal muscle origin that exists in two principal subtypes that are distinguished on the basis of histological and clinical characteristics. The clinical

46

HEIDI J . SCRABLE E T A L .

association between Wilms’ tumor, the embryonal subtype of rhabdomyosarcoma, and other specific rare tumors in individuals with BWS and the development of more than one rare tumor in the same individual could be simply circumstantial. Alternatively, the clinical associations could reflect a common etiological event and each of the developmental anomalies, including each of the tumor types, could arise after mutation of the same locus. Such mutations could be revealed by mitotic segregation events, similar to those demonstrated for Wilms’ tumor and retinoblastoma, which would serve to produce rhabdomyosarcomas that have lost constitutional heterozygosity. The experimental test of this hypothesis (Table 111) showed that embryonal rhabdomyosarcomas specifically lost constitutional heterozygosity at loci on chromosome l l p (Koufos et al., 1985)and, in more detailed analyses of mitotic recombination events, Ilpl5.5-llpter (Scrable et al., 1987); this was the same region identified by cytogenetic (Waziri et al., 1983; Turleau et al., 1984) and genetic-linkage mapping (Koufas et al., 1989) as containing the BWS lesion. Furthermore, examination of allelic combinations of loci in the l l p genomic region in a smaller number of hepatoblastomas (Table 111) and adrenal cortical carcinomas (our unpublished results) showed identical losses of heterozygosity.

2. Other Growth Excess Disorders Associated with Tumors The high risk for cancer development in individuals who have inherited autosomal dominant mutations causing developmental malformations is not restricted to BWS. In fact, the cosegregation of marker loci with the organismal phenotype and concurrent losses of heterozygosity for the same genomic region has been shown for chromosome 22 in bilateral acoustic neurofibromatosis (Seizinger et al., 1986, 1987), chromosome 3 in von Hippel-Lindau syndrome (Seizinger et al., 1988),chromosome 11 in multiple endocrine neoplasia (MEN) type 1 (Larsson et al., 1988), chromosome 5 in familial adenomatous polyposis (Bodmer et al., 1987; Solomon et al., 1987; Leppert et al., 1987),and chromosome 3 in hereditary renal cell carcinoma (Cohen et al., 1979; Zbar et al., 1987).

B. TUMORS WITH PHENOTYPICALLY DISTINCT ELEMENTS

The unilinear progression model depicted in Fig. 5, as well as its variant, which contains comnion predisposition and early-occurring differential progressive lesions (Fig. 7 ) ,appear to serve well in describing sporadic and familial single cancers as well as sporadic and

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47

familial occurrence of concurrent or sequential cancers of different histogenesis. They do not, however, easily encompass another phenomenon of clinical human oncology: the occurrence of tumors with elements of two phenotypically distinct tissues. These tumors, termed “heterotropic,” are exemplified by the three types discussed next, which have in common the presence of rhabdomyoblasts. The first is the rhabdomyomatous variant of Wilms’ tumor. Although these are rare, the first descriptions made by Wilms (1899) included cases of apparently mixed-element kidney tumors composed of various proportions of cells that were striated muscle in phenotype. Because the kidney and muscle tumors, as homogeneous masses, can occur alternatively in the same families and sequentially or concomitantly in individuals, the question of the mechanism of initiation or genetic predisposition arises. The model shown in Fig. 7 would not likely apply in all such cases, because both elements occur in the same location in heterotropic tumors. A variation of the model that could encompass this tumor class is shown in Fig. 8. In this case, predisposition (N + P), the attainment of homozygosity for the predisposing mutation (P + H), and at least some of the steps of in situ progression (H + H 1 + H2) could then undergo differing damage (H2 + H4; H2 + H3), leading to phenotypically different elements within the same tumor mass (composed of H3 and H4 components). Clearly, one

FIG.8. Heterotropic tumors could arise subsequent to a common predisposing mutation if late-stage progressional damage is acquired differentially within a tumor clone and is expressed as different somatic phenotypes. Symbols are as in previous figures.

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HEIDI J. SCRABLE E T A L ,

prediction of such a model is that early events (such as P + H) would be common to all cells of the tumor. To test this, we determined allelic combinations for loci on chromosome 1l p in rhabdomyomatous Wilms’ tumors; two cases are shown in Table IV. In each case, loss of heterozygosity was complete and isodisomy was attained. Although this is an interesting and provocative result, it may not be particularly surprising in light of the data in Table 111, which show that homogeneous rhabdmyosarcoma and Wilms’ tumors have undergone similar chromosomal changes. More surprising were the results obtained with the other two heterotropic tumor types shown in Table IV. The first is ectomesenchymoma, a tumor that apparently arises from migratory neural crest cells with the potential to form both neuroectoderm and mesenchyme, occurs with an anatomical distribution similar to rhabdomyosarcoma, and can have a phenotype of mixed neuroblasts and rhabdomyoblasts (Kawamot0 et aE., 1987).We have analyzed loci on chromosome l l p in some 75 homogeneous neuroblastomas without obtaining evidence of loss of heterozygosity. Thus, if the phenotype of ectomesenchymoma reflected genotypic distinctions at loci in this region, we would expect

TABLE IV Loss OF HETEHOZYGOSITY FOR LOCION CHROMOSOME llp IN HETEROTROPIC TUMORS Alleles present at locus‘ Tumor type

Tissueb CAT PTH H B B C ( y G ) D11S12 INS

Rhabdomyomatous Wilms’ tumor Wilms 1

N

-<

T

1,l

T

172 1J 192 29

1,2 2,2 1,2 1,l

1,3 3,3 2,3 3,3

N T

1,2 1,l

1,2 1,l

1,2 1J

1,l 1,l

1,2 1,l

N

1,2 2,2

1,l 1,l 1,2 1,l

1J 1,1 1,l 1,1

1,2 2,2 1,2 2,2

2,2 2,2 1,2 2,2

N

Wilms 5 Ectomesench ymoma Rhabd 29 Triton tumor Rhabd 41

T Rhabd 42

N T

~~~

_____~

-

1,l 172 2,2 2,2

12 1,l

~

Alleles designated in bold type are combinations that were heterozygous in constitutional tissue. * N, Normal; T, tumor. ‘ Not determined.

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49

only partial losses to be apparent, at best. Rather, homogeneous loss of alleles at these loci was observed (Table IV), consistent with an early event followed by clonal outgrowth and, at later stages, differential accumulation of progressive damage as proposed in Fig. 8. The final example in support of the model is another tumor of neural crest derivation termed a “Triton tumor.” The neural crest also gives rise to the Schwann cell of the neurilemmal sheath. A tumor composed of malignant Schwannoma and rhabdomyosarcoma is defined as a Triton tumor. We analyzed two such cases for alleles at chromosome l l p loci (Rhabd 41 and Rhabd 42 in Table IV) with the expectation that any alterations would occur in proportion to the relative number of rhabdomyoblasts in the tumors. The data showed complete losses of heterozygosity at all informative loci tested in both cases, consistent with both of the conclusions drawn from the other two heterotropic tumor types and the model in Fig. 8. Four features are common to these three tumor types: (1)The heterotropia is toward rhabdomyosarcoma, misplaced in a tumor of peripheral nerve, neural sheath, or kidney. (2) The heterotropic rhabdomyosarcoma is of the embryonal type. (3)The mixed tumor displays loss of alleles at loci on chromosome l l p as does homogeneous embryonal rhabdomyosarcoma. (4) The nonrhabdomyosarcoma components maintain their individual phenotypes despite uniform molecular genetic characteristics of embryonal rhabdomyosarcoma. Consistent with the model in Fig. 8, the loss of heterozygosity observed in these tumors appears to occur prior to the branchpoint in the pathway. Subsequent genetic events occurring within one or more cells of this particular clone appear to give rise to a single neoplasm that contains cells of more than one phenotypic class. V. Epigenetic Inactivation of Alleles in Human Cancer

The two-mutation model proposed by Knudson (1971; Knudson and Strong, 1972) has provided a strong theoretical framework within which to interpret data from studies on many different forms of human cancer. Support for one or more aspects of this model has come from studies on retinoblastoma (Cavenee et al., 1983, 1985; Dryja et al., 1984), familial adenomatous polyposis (Solomon et al., 1987), MEN type 2 (Mathew et al., 1987a,b; Simpson et al., 1987), and neurofibromatosis (Wertelecki et al., 1988). The strongest case in support of this model is that of retinoblastoma. The existence of this disease in both sporadic and inherited forms (Knudson, 1971),rare instances of constitutional deletions involving chromosome band 13q14 in inherited

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HEIDI J. SCRABLE E T A L .

cases (Strong et al., 1981), and cytogenetic (Francke, 1976) and molecular genetic data demonstrating “loss of heterozygosity” at loci on chromosome 13q in tumor DNA (Cavenee et al., 1983) are all observations that are consistent with the predictions of Knudson’s model. In the preceding sections we have reviewed evidence that supports the general principle that many types of tumors are the result of two or more mutational events: a predisposing mutation that results in the ianctivation of an allele at a tumor suppressor locus, and an additional event that results in the inactivation or loss of the remaining allele (Fig. 4). As presented, the model in Fig. 4 assumes that the predisposing event is an alteration in the nucleotide sequence of a tumor suppressor allele. However, the model has no formal requirement that such sequence changes be the only mechanism of allele inactivation (Moolgavkar and Knudson, 1981). It does not detract from the conceptual validity of the model if the predisposing event is the epigenetic inactivation of a tumor suppresor allele. One process by which otherwise genetically identical alleles may be rendered functionally different is genome imprinting. Genome imprinting is most conveniently described as an epigenetic allele inactivation process that is dependent on the gamete of origin (Sapienza, 1989). In the mouse, the effects of this process are observed as mutant phenotypes dependent on the gamete of origin (Johnson, 1974; McCrath and Solter, 1984a; Cattanach and Kirk, 1385), developmental failure of zygotes containing only maternal or paternal genetic contributions (McGrath and Solter, 1984b; Surani et al., 1984), and differences in the expression or methylation of hemizygous transgenes (Reik et al., 1987; Sapienza et al., 1987; Swain et al., 1987; Hadchouel et al., 1987).In the human, there is less direct evidence that maternally and paternally derived genetic information is differently imprinted, but several phenomena suggest this to be true. Both juvenile-onset Huntington’s disease (Farrer and Conneally, 1985) and neonatal myotonic dystrophy (Glanz and Fraser, 1984) are autosomal dominantly inherited diseases that show pronounced gamete-of-origin effects. In addition, hydatidiform moles are the result of zygotes that contain only paternally derived genetic information (Szulman and Surti, 1984). There are two important differences between alleles inactivated by imprinting and alleles inactivated by nucleotide sequence changes. The first is that the inactivation of an allele by the former process is reversible and epigenetic (McGrath and Solter, 198413; Surani et al., 1984).Thus, imprinted alleles do not carry a “mutation,” in the classical sense, even though such epigenetic changes may have the same effect on phenotype. The second important difference concerns the

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51

specificity of allele inactivation. While most mutations that alter nucleotide sequence are equally likely to be carried by either gamete, genome imprinting affects alleles in a gamete-specific way. The maternal allele at some loci will be affected, while the paternal allele will be affected at other loci.

A. PREDISPOSITION If we invoke the activity of a genome imprinting process as an epigenetic mechanism of tumor suppressor allele inactivation, the model in Fig. 4 may be redrawn as shown in Fig. 9. The predicted consequence of this process is that any diploid cell that contains one imprinted allele and one nonimprinted allele will be functionally hemizygous at this locus. The genotype of such a cell will be homozy-

-

Events :

l-Gometogenesis+Namol+

Predisposed-tHomozygote+Tmwi

FIG.9. Predisposition could take the form of allelic inactivation specific to the gamete of origin, rather than mutational DNA sequence alteration. Following the conversion of an active (+, 0 )allele to an inactive state (i, B),a genetically normal but predisposed cell (P) could attain functional nullizygosity by nondisjunction (A), nondisjunction/ duplication (B), or mitotic recombination (C),as shown to the right of the diagram.

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HEIDI J. SCRABLE E T A L .

gous wild type, but phenotypically it will behave as though it were heterozygous for a nonfunctional allele. This cell will be susceptible to any somatic event that results in the inactivation or loss of the single functional allele. If such an event occurs, a daughter cell will be produced that behaves as though it were homozygous for a recessive (loss-of-function) trait, even though the remaining allele or alleles may be wild type in sequence. Therefore, mutant alleles that show a preference for inheritance from only the female or only the male parent suggest the involvement of genome imprinting. As described in Section IV, Wilms’ tumor is a pediatric nephroblastoma that exists in both sporadic and familial forms (Knudson and Strong, 1972). Cytogenetic analyses of tumor tissue have established an association between Wilms’ tumor and chromosome band l l p 1 3 abnormalities (Riccardi et al., 1978; Slater and de Kraker, 1982). That such abnormalities are unlikely to be due to secondary, progressional events is indicated by the existence of constitutional chromosome l l p deletions and unbalanced translocations (Yunis and Ramsay, 1980) in some familial cases. In addition, molecular genetic studies have provided evidence for the occurrence of somatic events by demonstrating loss of heterozygosity at loci on chromosome l l p (Koufos et al., 1984; Orkin et d.,1984; summarized in Table 111). Familial forms of the disease show an autosomal dominant mode of inheritance (Knudson and Strong, 1972), but at the cellular level the disease appears to be recessive. The observation that Wilms’ tumor cell lines may be rendered nontumorigenic by the addition of a wild-type chromosome 11 (Weissman et al., 1987) supports this contention and is consistent with the existence of a Wilms’ tumor suppressor locus. All of these observations have striking parallels in retinoblastoma, and are therefore consistent with Knudson’s model. However, three additional sets of observations are not consistent with this model. First, although familial Wilms’ tumor appears to be inherited as an autosomal dominant trait, the penetrance of the trait is incomplete (Matsunaga, 1981). The variable penetrance is not easily attributable to the random occurrence of additional somatic events because the penetrance shows a generation effect: the trait is nonpenetrant in some generations, but highly penetrant in subsequent generations. Second, despite cytogenetic and molecular genetic evidence for chromosome l l p involvement in the Wilms’ tumor phenotype, predisposition to the disease does not cosegregate with marker loci on chromosome l l p (Grundy et al., 1988; Huff et al., 1988). Third, there is a marked preference for retention of only paternally derived chromosome 1l p alleles in tumor tissue (Schroeder et at., 1987; Wilkins, 1988). None of

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these observations is predicted by the conventional interpretation of Knudson’s model (Fig. 4). However, if we consider the model in Fig. 9, two wild-type alleles that are identical in nucleotide sequence and lie at the same locus will behave differently. Passage of one of these alleles through gametogenesis of one sex has no effect on the ability of this allele to be expressed. Passage of the same allele through gametogenesis of the opposite sex, however, results in its functional inactivation. We may describe this allele as being “imprinted.” Because the alleles that lie at this locus are identical, the differential establishment of the imprint cannot be a property of the allele itself, but must reflect the gamete-oforigin-dependent activity of another gene or genes. We may describe such a gene as an “imprinting” gene. Because two types of genes are involved in this process, two classes of mutations are predicted. The first class consists of mutations in genes that are imprinted and the second encompasses mutations within genes that imprint. While it is possible that a particular phenotype is the result of mutations in either class, the distinction is important in the analysis of phenomena that involve genome imprinting because the genetic behavior of mutations within each class will differ. If some tumor suppressor loci are imprinted, the model in Fig. 9 predicts that the alleles at loci that become isodisomic in these tumors will generally have come from the same parent; that is, they will have been subject to the same gamete-of-origin-dependent allele inactivation process. In Table V, we have compiled the available data on the parental

PREFERENTIAL Loss Tumor type Sporadic cases Wilms’ tumor Embryonal rhabdomyosarcoma Retinoblastoma Heritable cases“ Wilms’ tumor Pheochromocytoma (MEN2A)

OF

TABLE V MATERNALLY DERIVED ALLELES IN TUMORS

Number of cases

Chromosome on which alleles lost

Parental origin of retained alleles

9 5 2

11 11 13

9/9 Male 515 Male 212 Male

1 1

11 1

Male Male

~~

~

Familial cases in which the mother of the proband was affected. The data are compiIed from Reeve et al. (1984), Schroeder et al. (1987), Wilkins (1988), Dryja et al. (1984), Grundy et al. (1988), Mathew et al. (1987a), and our unpublished data.

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origin of alleles retained in several types of sporadic tumors. A strong and statistically significant bias is found in the parental origin of retained alleles in both Wilms’ tumor and embryonal rhabdomyosarcoma. The isodisomic chromosome l l p alleles found in these tumors, with one possible exception (cited in Wilkins, 1988), are inherited from the father. While both the model in Fig. 4 and that in Fig. 9 require two events to create a tumorigenic cell, they differ in the predictions they make with respect to the inheritance of familial cancers. If both types of first events are possible, the existence of two classes of familial tumors is predicted. In the first class, tumor suppressor alleles that carry alterations in nucleotide sequence will be inherited as the predisposing mutation (Fig. 4).In such families, the disease will be genetically linked to markers on the chromosome that carries the tumor suppressor allele. Examples of this type have been found in retinoblastoma families (Sparkes et al., 1983) and familial adenomatous polyposis (Solomon et al., 1987). In the second class, an epigenetically inactivated tumor suppressor allele will be inherited as the predisposing mutation (Fig. 9). Because the inactivation of this allele need not be dependent on the allele itself, but will reflect the activity of the gene or genes involved in generating or maintaining the genome imprint, the inheritance of the tumor phenotype will not be linked to the tumor suppressor locus. This prediction is consistent with data obtained for two different Wilms’ tumor families (Grundy et al., 1988; Huff et al., 1988). Two additional cases are of interest in this regard (Table V). The first is a case of familial Wilms’ tumor described by Grundy et al. (1988). In this case, an affected mother had a child who was also affected with Wilms’ tumor. The child’s tumor exhibited loss of heterozygosity at loci on chromosome 1l p . However, the alleles retained in the tumor were not derived from the affected mother, but from the unaffected father. A similar unexpected observation was made for loss of alleles on chromosome 1 in a pheochromocytoma from a familial case of multiple endocrine neoplasia type 2 (MENBA) by Mathew et al. (1987a). Both of these studies are consistent with the model in Fig. 9. The simplest interpretation of these results is that, while the locus that gives rise to the tumor phenotype is likely to be located on the chromosome at which allele loss occurs, the predisposing mutation is located elsewhere in the genome. Furthermore, the predisposing mutation must act in trans on a putative tumor suppressor allele in a nonrandom fashion with respect to parental origin. These data also imply that the genome imprints observed in somatic tissue are not finally established until after fertili-

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55

zation, a conclusion that is consistent with data for the mouse (Sanford et al., 1987; Sapienza et al., 1988).There is no a priori reason to expect that any particular type of familial tumor will be restricted to one or the other class. Given a large enough number of families, examples of both types may be found within any particular disease. In the mouse, genome imprinting is a process that affects multiple loci (reviewed by Solter, 1988). Genetic experiments employing translocation chromosomes indicate that some imprinted loci are syntenic, but loci on at Ieast six chromosomes produce visibly aberrant phenotypes (Cattanach, 1986).If imprints in humans specific to the gamete of origin also affect multiple loci, then the inheritance of an allele that influences either the allocation of cells bearing an imprint to the somatic lineage, or the creation of such cells from functionally diploid cells, might result in the cosegregation of more than one type of disease trait. The inheritance patterns of affected traits and the variety of phenotypes are predicted to be complex, but one might predict that such trans-acting modifiers would sometimes give rise to multiple incompletely dominant, syntenic or nonsyntenic mutant phenotypes within an individual or between individuals within the same family. One class of candidate disorders is represented by mixed genetic cancer families. In these families, cancer, as a general phenotype, appears to be segregating as an autosomal dominant trait (Li and Fraumeni, 1969,1982). However, different members of the same family have different types of tumors (see Fig. 1, for example). This inheritance pattern may be explained by the segregation of an imprinting allele that trans-inactivates alleles at tumor suppressor loci on more than one chromosome. Because the formation of a tumor requires a somatic event in addition to the primary event of inactivation of one allele, the probability of the occurrence of the somatic event will determine which tumor phenotype appears (Fig. 7 ) .This model predicts that isodisomic chromosomes within these tumors will have the same parental origin in different individuals (regardless of the affected chromosome) and that the allele that predisposes to cancer in these families will map to the same unlinked locus, independent of tumor phenotype. A formally analogous model may explain the other class of mixeddisease phenotype observed-that of single tumors with heterogeneous elements. Thus, a rhabdomyomatous Wilms’ tumor (Table IV), with its apparent blastemal component arising from a locus in l l p 1 3 and the rhabdomyomatous element arising from a locus in l l p 1 5 could be seen to arise from a single cell in which alleles at both loci have been epigenetically inactivated (Fig. 8).The time during development

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or the location of the cell within which these events occurred might influence whether an individual developed Wilms’ tumor, rhabdomyosarcoma, BWS, or other clinically associated disorders. Similarly, the occurrence of such events in a cell that bears imprinted alleles on more than one chromosome that has not yet committed to either the neural crest or the myogenic lineage might result in the appearance of ectomesenchymoma, which contains elements of both neuroblastoma and rhabdomyosarcoma (Table IV).

B. PROGRESSION In the preceding section, we have reviewed evidence that the inactivation of a tumor suppressor allele may be an epigenetiqrather than a genetic event. Similarly, the progressional changes that occur within some kinds of tumors (Fig. 6) are also treatable within the confines of an epigenetic model (Fig. 10, part 4). In the absence of any epigenetic inactivation events, models that seek to correlate progressional changes with genetic events must invoke a relatively large number of independent mutations, all of which must occur somatically, within the same cell. In the case of advancedstage glioblastoma, for example, loss of heterozygosity is observed at loci on three different chromosomes. The probability that three mutations have occurred independently, each followed by a nondisjunction or mitotic recombination event, would seem very low. However, if an individual cell were inappropriately to express imprinting functions, alleles at multiple loci on several chromosomes are predicted to be affected. The subsequent occurrence of a somatic event that resulted in isodisomy or hemizygosity at a tumor suppressor locus might give rise to a proliferative cell that becomes clonally expanded-a lowgrade astrocytoma (James et al., 1988), for example. Within this expanded, mitotically active population, the occurrence of an additional genetic event might result in a separate clone of cells with different morphological characteristics, that is, a higher grade malignancy. This model predicts that progressional changes are associated with the sequential occurrence of genetic events (Foulds, 1957; Nowell, 1976) and that progressional stages of the same tumor type will show “concerted nonsyntenic allele loss” (Law et al., 1988; Vogelstein et al., 1988). Although the application of a genome imprinting model to tumor progression has broad explanatory power, it is difficult to make novel predictions in such cases. Because imprinting functions must be proposed to be aberrantly expressed in a terminally differentiated diploid

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? :&C

d

FIG.10. Pathway by which loss of heterozygosity can be effected by genetic or epigenetic inactivation during predisposition and progression of cancer. Designations of wild-type, active, mutant, and inactive loci are as in previous figures.

cell, it is difficult to imagine a mechanism that might discriminate between maternally and paternally derived alleles, in the absence of some global primary imprint that has persisted, without phenotypic effect, throughout the life of the organism. It is easier to imagine that

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the parental origin of alleles lost within an individual tumor would be random with respect to parental origin, even though they may still reflect epigenetic inactivation. VI. Conclusions We have attempted to illustrate that a large number of familial and sporadic cancers are mechanistically explainable in terms of Knudson’s original model, or a modification of this model. The modification of Knudson’s model to include heritable epigenetic inactivation of a tumor suppressor allele as the result of genome imprinting does not change the predictions of the model with regard to the sequence of events that lead to tumorigenesis, nor does the modified model exclude the occurrence of “classical” mutations (alterations in nucleotide sequence). Both Knudson’s original model (Knudson, 1971; see also Fig. 4)and the model presented in Fig. 9 require two events. The first event is the inactivation of one allele at a tuinor suppressor locus, and the second event is the inactivation or loss of the remaining functional allele. The model in Fig. 9 differs from that in Fig. 4 in three respects : the prediction of the parental origin of isodisomic chromosomes in tumor cells, the map location of disease predisposition loci, and the nature of the initial allele inactivation event. The model in Fig. 9 also predicts that cells that bear imprinted tumor suppressor alleles have a high probability of becoming tumorigenic. Because cells that bear imprinted chromosomes are thought to be present early in the development of all individuals, we argue that in most individuals, cells that bear imprinted chromosomes do not persist into the somatic lineage, a view that is consistent with data obtained for the mouse (Sanford et al., 1987). However, given the presence of sufficient numbers of such cells, the probability that a tumor is formed becomes high because only a single additional event is required. Affected individuals are presumed to be mosaics of cells that bear imprinted alleles (which are functionally hemizygous at affected loci) and cells that do not (which are functionally diploid) (Sapienza et al., 1988; Sapienza, 1989). The frequency with which such mosaics are created may be influenced by stochastic factors, resulting in sporadic disease, and/or genetic factors, resulting in inherited disease. The combination of genetic and epigenetic events in tumor initiation and progression is predicted to give rise to complex and variable genomic alterations (Fig. 10).However, an understanding of the nature and number of these events is likely to yield significant insight into many biological problems.

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GENETIC AND MOLECULAR STUDIES OF CELLULAR IMMORTALIZATION James R. Smith* and Olivia M. Pereira-Smith Roy M. and Phyllis Gough Huffington Center on Aging, and Departmentsof Virology and Epidemiology, 'of Cell Biology, and of Medicine, Baylor College of Medicine. Houston, Texas 77030

I. Introduction 11. Short-Term Analysis of Cell Fusion Products A. Studies with Heterokaryons B. Studies with Reconstructed Cells 111. Long-Term Proliferation Potential of Hybrids A. Fusions Involving Only Normal Diploid Cells B. Fusions of Normal with Immortal Cells C. Fusions of Various Immortal Cell Lines with Each Other IV. Microinjection Experiments A. Effect of mRNA Isolated from Normal Cells B. Effect of the Oncogenes H-rus, EIA, and SV40 T Antigen on Normal Cells V. Discussion References

I. Introduction

Body homeostasis is maintained by positive and negative regulatory signals in the various cells and organs of an animal. Changes in regulation of these signals could lead to loss of cell proliferative response, as is observed in the aging immune system, or alternatively to uncontrolled cell divisi,on, one step in the progression toward neoplasias, which increase with age. Therefore, changes in regulation of cell proliferation are an extremely important component of not only the general aging phenotype, but also onset of tumorigenicity. The loss of cell proliferative ability of normal animal cells in culture following the accruement of a defined number of population doublings is well documented. This phenomenon has been proposed as a model for aging at the cellular level (Hayflick, 1965). Data in support of the model include the observations that a direct correlation exists between species life span and in vitro life span (Rohme, 1981), but that this correlation is inverse with donor age (Martin et al., 1970; Schneider and Mitsui, 1976; Goldstein et al., 1978; LeGuilly et al., 1973; Bierman, 1978). The frequency of reversal of the senescence phenotype (immortalization) varies greatly with different animal species. The probability of 63 ADVANCES IN CANCER RESEARCH, VOL. 54

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immortalization (spontaneous and induced) in mouse cells is extremely high, whereas in human cells spontaneous immortalization has never been shown to occur and induced immortalization using agents such as virus or chemical carcinogens is very low (probably 2 years (Matsumura et al., 1979; own unpublished observations). The major loss of function is in the ability to synthesize DNA and divide. One set of studies attempted to understand the reason for this loss of growth factor response in senescent cells by short-term studies of DNA synthesis in the nuclei of heterokaryons or by reconstructing cells.

A. STUDIES WITH HETEROKARYONS Norwood et al. (1974) fused normal young with senescent human cells and determined the ability of the nuclei in a heterodikaryon to synthesize DNA, as measured by tritated-thymidine autoradiography. The cytoplasm of the cells was prelabeled with either tritiated methionine or [ '*C]thymidine to allow for identification of heterodikaryons by double-layer autoradiography. They found that senescent cells could inhibit DNA synthesis in the nucleus of the otherwise proliferation-competent young nucleus. The control young homodikaryons were unaffected in DNA-synthetic capability, indicating that the result was not due to the experimental manipulation. Stein's group independently confirmed these observations (Yanishevsky and Stein, 1980). Their studies showed further that the block in senescent cells was at initiation of DNA synthesis, since young

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nuclei that had already entered the S phase of the cell cycle at the time of fusion were not inhibited in progression through the S phase. These results led to the hypothesis that senescent cells produced an inhibitor of DNA synthesis initiation that was diffusible and could affect a young nucleus in the common cytoplasm of the heterodikaryon. Norwood’s group demonstrated that this inhibitory activity could be overcome by fusion of senescent human fibroblasts with the immortal cell line HeLa or with immortal simian virus 40 (SV40)-transformed cells (Nonvood et al., 1975). DNA synthesis was reinitiated in senescent nuclei in a large number of the heterodikaryons resulting from these fusions. However, it is not known if the senescent nucleus carried out a complete round of semiconservative replication. These results showed that, although senescent cells would not synthesize DNA under standard circumstances of mitogen stimulation, they were not structurally incapable of carrying out DNA synthesis. Stein and Yanishevsky (1979), on the other hand, shortly thereafter found that senescent human cells were able to inhibit DNA synthesis in the nuclei of the immortal cells T98G (human glioblastoma cells) or Rk-13 (rabbit kidney cells). Stein and co-workers (1982) have since demonstrated that the ability of an immortal cell to overcome the DNA synthesisinhibitory activity of senescent cells is dependent on the presence of DNA tumor viral genomes (SV40 in the case of immortal SV40transformed cells and papillomavirus DNA in the case of Hela cells), since a carcinogen-derived immortal cell line was unable to override the senescent cell inhibitory activity. These results led to the idea that there are t w o classes of immortal cells, one that can respond to the inhibitor of DNA synthesis present in senescent cells and another that is able to overcome the inhibitory activity. In order to determine whether this inhibitory activity was associated with the cytoplasm or the nucleus of the senescent cell, Nonvood’s group and ours independently proceeded to analyze the DNAsynthetic capability of young cell nuclei in cybrids made from fusion of senescent cytoplasts with whole young cells. We both determined that the senescent cytoplast was as capable of inhibiting DNA synthesis in the young nucleus as was the whole cell (Burmer et al., 1983; Drescher-Lincoln and Smith, 1983).We then asked whether a protein was involved by treating cytoplasts prior to fusion with the protein synthesis inhibitors, cycloheximide and puromycin. We demonstrated that the inhibitory activity was lost following treatment with either agent, with even a short treatment (as little as 2 hr with cycloheximide) resulting in loss of the inhibitory activity. This indicated that the

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inhibitor of DNA synthesis in senescent cells was a protein(s) or that its action was mediated by a protein(s). If the cytoplasts were allowed to recover in the absence of cycloheximide, the inhibitory activity was regained within -3-4 hr, indicating that the message coding for this inhibitory protein(s) was long-lived (Drescher-Lincoln and Smith,

1984). To determine whether the protein(s) was associated with the cytoplasm or the membrane of the cells, we treated the senescent cytoplasts with 0.125%trypsin in the cold for 1 min. During such a treatment, the trypsin would not be likely to enter the cytoplasm of the cells. We determined that the trypsin treatment eliminated the inhibitory activity and that recovery of the cytoplasts following trypsin treatment permitted a return to activity in approximately the same time frame as was seen in the case of cycloheximide treatment. These results indicated to us that the protein(s) inhibitor of DNA synthesis in senescent cells was most likely located on the outside surface of the membranes (Pereira-Smith et aE., 1985).Stein and we have since independently shown that isolated surface membrane preparations from senescent cells and proteins extracted from these membranes are capable of inhibiting DNA synthesis in proliferation-competent young cells when the membranes or proteins are added to the cell culture medium in which the young cells are grown (Pereira-Smith et al., 1985; Stein and Atkins, 1986). Young, logarithmically growing cells do not express a protein(s) inhibitor of DNA synthesis. However, if young cells are made nondividing (quiescent) by growth factor deprivation for at least 2 weeks, or by growth to high density in the presence of growth factors, they express an inhibitor of DNA synthesis (Pereira-Smith et al., 1985; Stein and Atkins, 1986). The inhibitor of quiescent cells has many properties similar to senescent cells (Pereira-Smith et al., 1985).It is a protein(s), is trypsin-sensitive, and is present in surface membraneenriched preparations and in the proteins extracted from these membranes. The major difference is that it is not inactivated by cycloheximide treatment for 524 hr, indicating that it is a very stable protein(s) with a long half-life. We were able to demonstrate that the quiescent cell inhibitory activity did involve a protein by trypsin-treating cytoplasts and allowing them to recover from the treatment in the presence and absence of cycloheximide before fusion. Cytoplasts incubated with cycloheximide never recovered inhibitory activity, whereas those not exposed to the protein synthesis inhibitor did. At this time we do not know if the DNA synthesis inhibitors expressed in senescent and quiescent cells are different or the same protein(s)

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constitutively expressed in senescent cells and mitogen-regulated in quiescent cells. We are presently pursuing purification of the protein(s) from senescent cells in order to answer this question. Our current hypothesis is that expression of this protein(s) is an important component of the mechanisms leading to senescence.

B. STUDIESWITH RECONSTRUCTEDCELLS In an attempt to determine the role of the nucleus and the cytoplasm in the process of senescence, Wright and Hayflick (1975) treated cytoplasms of whole normal young human fibroblasts with iodoacetate and rotenone to induce irreversible cytoplasmic injury. They then fused these injured cells to cytoplasms derived from both young and old donor cells, and determined the proliferation potential of the cybrids. Selection was based on the fact that only the cybrids would survive for longer than a few days, whereas the cytoplasts and injured cells would be nonviable over an extended period of time. They determined that old-donor cytoplasm was as capable as young-donor cytoplasm in rescuing the treated young cells. They concluded that the cytoplasm was not involved in the processes leading to senscence and that these were dictated by the nucleus. Interpretation of these results is complicated by the fact that enucleation is never 100% efficient and that the controls showed that injury to the cytoplasm was not lethal to all cells. It is also possible that the iodoacetate and rotenone treatment might have destroyed some senescent-specific inhibitory activity. Muggleton-Harris and Hayflick (1976)performed experiments using micromanipulation to ask the same question and found, in contrast to Wright and Hayflick (1975), that both the old cytoplasm and the old nucleus had an impact on the division potential of the reconstructed cell. In this case, reconstructed cells included fusions of young cytoplasts with young karyoplasts, old cytoplasts with young karyoplasts, and young cytoplasts with old karyoplasts. They found that whenever an old component was involved, the number of reconstructed cells capable of dividing at least six times decreased dramatically as compared with the young cytoplast-young karyoplast control. These results indicated that both nucleus and cytoplasm played a role in the effect on aging of a cell. However, again there was some difficulty in interpreting the results of this study; namely, the technical difficulties involved in micromanipulation, which affects viability of the reconstructed cells, and the fact that the cells were followed through only a limited number of divisions.

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I l l . Long-Term Proliferation Potential of Hybrids A. FUSIONS INVOLVING ONLYNORMALDIPLOIDCELLS A major problem in attempting long-term studies of the proliferative potential of hybrids from fusions involving normal cells is the generation of mutants that allow for biochemical selection for hybrids. Normal human cell cultures have limited life spans of -50-70 population doubiings (PD). The cultures are heterogenous with respect to the doubling potential of the individual cells in the population (therefore, all cells would not be capable of 50 PD), and 20 PD would be used up in the process of obtaining a million cells of a mutant clone. Littlefield (1973)made an attempt to fuse senescent normal human cells with each other and with young cells. In the latter case he used a hypoxanthine, aminopterin, thymidine (HAT) selection system by fusing young Lesch-Nyhan cells with senescent cells and reported that he was unable to obtain hybrids that could grow to any significant extent. He did obtain small clones that survived the selection system and might have been hybrids, but since he could not karyotype the clones, he had no further confirmation that they were hybrids. One of his conclusions was that the failure of young and aged fibroblasts to form large hybrid clones was “surprising, as if aging were dominant in such a cross.” In fact, his result and conclusion were correct, as later studies showed. Hoehn and co-workers (1978) attempted to circumvent this problem by fusing populations of young and old (but not senescent) diploid human cells having different glucose-6-phosphate dehydrogenase (G6PD) isoenzymes. Hybrids were selected by flow cytometery (sorting for a 2n DNA content) and confirmed by the presence of a CGPDAB heteropolymer band. The conclusion from this study was that the life span of hybrids from short- and long-lived fusions was intermediate between that of the parental cells fused. The problem with this interpretation is that the data were very limited. Analysis of hybrid life spans was restricted to only rapidly proliferating hybrid clones, and the in vitro life span of these hybrid clones was compared with the in vitro life span of the total mass-culture parental cell lines. The clonal life spans of the parent cultures were not examined. It has been documented by Martin et al. (1974) and Smith and Hayflick (1974)that there is tremendous heterogeneity in the division potential of the individual cells in a mass culture of human diploid fibroblasts. Young mass cultures are composed of senescent cells, cells capable of very few doublings, as well as cells capable of extensive division

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potential. Therefore, it is difficult to interpret the data generated by this study. We were fortunate to obtain, through continuously improving cell culture reagents, a human diploid fibroblast line derived from fetal lung that was capable of achieving 5100 PD in vitro (Duthu et al., 1982).The cell line was normal despite this extremely long in vitro life span. With this cell line, we were able to obtain a spontaneous mutant clone resistant to ouabain, which was grown up and used to obtain a hypoxanthine phosphoribosyltransferase-negative (HPRT-) mutation by selection in 6-thioguanine. This cell line had -45 PD remaining to it at this time and could be used as a universal hybridizer, since fusing it with any wild-type cell would allow for selection in medium containing HAT and ouabain. We also obtained a ouabain-resistant mutant clone from a Lesch-Nyhan (HPRT-) cell line that had -28 PD remaining to it after growth to a million cells. Isolation of these cell lines allowed us to proceed with studies of the proliferative potential of the hybrids from fusions of young and old normal cells (Pereira-Smith and Smith, 1982). We decreased the heterogeneity of the parental populations by using in both cases a clone rather than a mass culture of cells. We had previously shown that clonal populations were much more homogeneous with respect to the division potential of the individual cells in the population than were mass-culture populations (Smith and Whitney, 1980). In clonal populations, one obtains two clear modes of cells. In the case of a clone early in its in vitro life span, one observes a low-population-doubling mode with 5 10 PD and a high-populationdoubling mode that usually ranges between -35 and 50 PD. The number of cells in the low-doubling-potential mode increases with increasing in vitro life span until one finally observes only this mode in an old clonal culture. Following fusion of clonal populations early and late in their in vitro life span, we determined the proliferation potential of the hybrids and compared these with the proliferative potentials of the individual cells in each parent population fused. This analysis demonstrated that the division potential of the hybrids was more like that of the old parental cells than the young, indicating that senescence was dominant as had been shown in the case of both the reconstruction and short-term heterokaryon experiments, as well as Littlefield’s fusion results (1973). We were also able to demonstrate that no complementation occurred when we fused clones at the end of their in vitro life span with each other to yield hybrids having life spans greater than either of the

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parents. If, as has been hypothesized, senescence is the result of accumulation of damage through random errors or mutations, one would expect to observe such complementation. The conclusions from fusions involving normal cells are that the senescent phenotype is dominant and that senescence is not the result of random accumulation of damage.

B. FUSIONS OF NORMALWITH IMMORTAL CELLS In early studies in which hybrids from fusion of normal with immortal human cells were used to assay tumorgenicity, the conclusion had been that the phenotype of immortality was dominant, since it was possible to obtain hybrids that could grow to large cell numbers (Stanbridge, 1976; Croce and Koprowski, 1974).The interpretation of this result was that the senescent cells lacked some genes necessary for DNA synthesis and cell division and that these were turned on again in a dominant fashion in the immortal cells. This view was held for a long time until a report by Bunn and Tarrant (1980),in which they showed that some hybrids obtained from the fusion of HeLa cells with normal human diploid fibroblasts yielded hybrids that had limited division potential. This was a very exciting but unexpected result and was questioned on the possibility that the particular HeLa cell line they had used was genetically unstable and that some critical immortalizing Hela DNA had been lost in the hybrids that had limited life spans. Bunn and Tarrant (1980) also observed that if they maintained these nondoubling hybrid populations for varying periods of time in culture, in some cases they would observe foci of dividing cells appearing at a frequency of 1-2 in 10’ cells. These would have regained the immortal phenotype and could grow without limit. Muggleton-Harris and DeSimone (1980) fused normal cells with immortal SV40-transformed cells by micromanipulation, and reported that the majority of the fusion products (98%)had an extremely limited division potential of 5 6 PD. The 2%that resulted in large clones were assumed to be immortal and were not carried to the end of their in vitro life span. We decided to complete this experiment using a biochemical selection system for hybrids and fused immortal SV40-transformed cells with normal human cells (Pereira-Smith and Smith, 1981). What we observed was that the majority of the hybrids (70%)had extremely limited division potential ( 5 7 PD). About 30%of the hybrids proliferated more extensively between 16 and 62 PD, but all eventually

-

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ceased division. At the time that the hybrid populations had ceased doubling, the hybrids continued to express the SV40 T antigen, indicating that the viral genome was present and expressed in these cells. We tested the functional ability of the expressed SV40 T antigen to induce DNA synthesis in senescent normal cell nuclei in heterokaryons and determined that it was intact (Pereira-Smith and Smith, 1987). We also observed the occurrence of rare immortal variants in these nondoubling populations at frequencies similar to those observed by Bunn and Tarrant (1980). Since we had shown that both small hybrid clones (those having <7 PD) as well as the ones that could proliferate as many as 62 PD still expressed SV40 T antigen, there was one other possibility that could explain our result: namely, that integration of the viral genome was different in hybrids that had limited doubling potential. We performed a restriction endonuclease analysis of the immortal SV40transformed parent used in the fusion, hybrids that ceased doubling, and the immortal variants that arose in these hybrid populations. There were no differences in the Southern blot band patterns of the integrated viral genomes in any of the cells. The conclusion from this set of studies was that the phenotype of immortality was recessive and that cellular senescence was dominant. An explanation for the early results is that in those studies the emphasis was on the study of the tumorigenic phenotype and that probably the most viable, rapidly growing hybrids were selected. These studies did not plate the fusion mixtures at clonal densities such as Bunn and Tarrant (1980) and we (Pereira-Smith and Smith, 1981) did; therefore, immortal variants probably arose frequently in the large populations of cells. The other possibility is that limitedlife span hybrids might have been observed in these studies if the actual number of population doublings that the hybrids could achieve before they were used for the tumorigenicity assay had been determined. To determine the generality of the dominance of cellular senescence in hybrids, we then fused normal human cells with a variety of immortal human cells: independently derived SV40-immortalized human cell lines and tumor-derived cell lines, some of which were expressing activated oncogenes. In all fusions the hybrids that we obtained had limited division potential (Pereira-Smith and Smith, 1983). On the basis of these data, we conclude that immortality occurs as a result of recessive changes in the growth control mechanisms of the normal cell.

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C. FUSIONS OF VARIOUSIMMORTAL CELLLINESWITH EACHOTHER Since the phenotype of immortality was recessive, we were able to exploit this fact to determine the number of ways by which cells could become immortal. This allowed us to identify the number of genes or sets of genes or processes that were involved in cellular senescence that could be modified to yield immortal cells. The approach we took was to fuse different immortal human cell lines with each other. If the immortal parents fused had the same recessive change, we would obtain hybrids that were immortal. If, however, the immortal parents fused had immortalized by different events, complementation of these defects would occur to yield hybrids with limited life span. In an initial study, we fused an immortal SV40-transformed cell line with a small number of immortal human cell lines and were able to identify two complementation groups for indefinite division (Pereira-Smith and Smith, 1983).We followed this with a more extensive study involving 26 different immortal human cell lines and to date have identified four complementation groups for indefinite division (Pereira-Smith and Smith, 1988; and unpublished observations). The analysis involved using a cell line (having mutations that would allow for hybrid selection) that we arbitrarily assigned to complementation group A, which was fused to all of the other test cell lines. If immortal hybrids were obtained, the test cell lines were assigned to group A; if limited-lifespan hybrids were obtained, the test cell lines were assigned to not-group A. We then selected a cell line from notgroup A to represent group B and continued fusions with not-group A cell lines. To strengthen the analysis, cell lines that had been assigned to group A were also fused with cell lines representative of groups By C, and D (as they were identified) to demonstrate that immortal hybrids were obtained only in the fusions with the cell line representative of group A and that there was complementation in all other fusions. An additional control was fusion of our double-mutant cell lines representative of each group, with the wild-type cell lines from which they were derived to demonstrate that indeed the hybrids proliferated indefinitely. We have been unable to assign a cell line to more than one complementation group. This result, along with the fact that the mutant cell lines that had been generated following mutagen treatment do not assign to groups other than that of the wild-type parent from which they were derived, indicates that we are working with a limited number of very specific genes that can be modified to yield immortal cells. This is reflected in the fact that the frequency of spontaneous immortalization in human cells is very low. It also indicates that the genes are

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most likely autosomal and that it takes two events to eliminate both alleles. We included a wide variety of immortal human cell lines in this analysis to determine if we could find any parameter that might correlate with complementation group assignment. Our findings were primarily negative. There was no correlation between cell type, embryonal layer of origin, or type of tumor and complementation group assignment. Expression of activated oncogenes did not affect group assignment. The one strong correlation we did see was that 9 in 10 of the immortal SV40-transformed cell lines we examined (derived from skin and lung fibroblasts, keratinocytes, and amnion cells) all were assigned to the same group, indicating that this virus immortalizes different human cells by the same processes (Pereira-Smithand Smith,

1987, 1988). The one exception was a cell line derived from xeroderma pigmentosum (XP) fibroblasts following transfection with an origin-defective SV40 virus. We could not attribute the different assignment of the cell line to the XP nature of the cells or use of origin-defective virus because another independently derived immortalized XP cell line (of the same XP complementation group) and an origin-defective virustransformed cell line were assigned along with the rest of the SV40 cell lines. At this time we do not know why this cell line assigned differently. The important result of assignment of immortal cell lines to specific groups is that we know that we are dealing with a limited number of genes, sets of genes, or processes, and that the possibility of identifying them exists. We can now take a focused approach to determine what common genetic changes have occurred in cell lines within a group that might account for the fact that they have immortalized. We are also applying the technique of microcell fusion, introducing chromosomes from normal cells into the cell lines within each group to determine whether any particular chromosome can restore the senescent phenotype of limited proliferation potential to the immortal cells. IV. Microinjection Experiments

A. EFFECTOF mRNA ISOLATED FROM NORMALCELLS The idea that the phenotype of nonproliferation is dominant over the proliferative phenotype was further reinforced by the results of microinjection experiments in which poly(A)+ RNA from a number of sources was microinjected into young proliferation-competent cells.

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Some of the RNAs inhibited DNA synthesis in these cells. The most potent inhibitor that has been described thus far is the one that has been isolated from senescent human diploid fibroblasts (Lumpkin et al., 1986a). Significant inhibition was obtained when as little as 0.010.03 mg/ml of poly(A)+ RNA was injected into young human fibroblasts. This was in contrast to very little inhibition obtained when as much as 5 mg/ml of RNA isolated from young proliferating cells was injected. Inhibitory activity was also obtained with RNA isolated from quiescent human diploid fibroblasts. However, the RNA had to be used at a concentration > 1 mg/ml in order to obtain significant inhibitory activity upon microinjection. The inhibitory activity found in senescent human fibroblasts has some features in common with normal resting cells found in the body. For example, RNA isolated from resting liver has been found to inhibit normal human fibroblasts, HeLa cells, and NIH 3T3 cells at concentrations of 0.5-1 mg/ml. (Lumpkin et ul., 1985; Pepperkok et aZ., 1988a), and mRNA isolated from resting T lymphocytes will inhibit HeLa cells or normal human diploid fibroblasts IMR-90 when microinjected at concentrations of -0.5 mg/ml (Pepperkok et al., 1988b).

B. EFFECTOF THE ONCOGENES H-rus, ELA, AND SV40 T ANTIGENON NORMALCELLS Since various studies had demonstrated the expression of DNA synthesis-inhibitory activity in both proteins (Pereira-Smith et al., 1985; Stein and Atkins, 1986) and mRNA (Lumpkin et aZ., 1986a) extracted from senscent cells, we thought it would be of interest to determine whether oncogenes could override this activity and allow senescent cells to synthesize DNA (Lumpkin et al., 198613).We began our experiments using both the protooncogene and oncogene forms of c-H-rus DNA isolated from placenta or the bladder carcinoma cell lines EJ and T24. We included quiescent cells (young cells made nondividing by either removal of serum growth factors or growth to high density in the presence of serum growth factors) because DNA synthesis-inhibitory activity had also been observed in these cells. All the DNAs (protooncogene- and oncogene-derived) at concentrations of 1 mg/ml (-500 copies injected per cell) were able to induce DNA synthesis equally efficiently in a large number of the quiescent cells as measured by tritiated-thymidine autoradiography (80%injected cells labeled with tritiated thymidine versus 10% in the uninjected cells). However, senescent cells did not respond by synthesizing DNA. This lack of response was not due to the inability of senescent cells to

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express the DNA, as detected by immunofluorescence using antibodies against the protein product of the DNA. We then injected a combination of oncogenes that had successfully fully transformed primary rodent cells (Ruley, 1983).c-H-ras DNA was microinjected along with DNA coding for the EIA adenovirus early region. This combination also failed to induce DNA synthesis in the senescent cells, despite expression of the protein products as detected by immunofluorescence microscopy. This lack of response of senescent cells to microinjected DNAs was not due to an inability to respond to any stimulus, because SV40 large-T antigen DNA was able to elicit a response in 22%of the injected cells when as few as 35 copies of the DNA were injected per cell. These results further demonstrate the rigorous control of cell division that exists in normal human cells, and provide additional supporting evidence for a role of the senescent-cell inhibitor in the mechanisms involving senscence.

V. Discussion It is apparent from this review that the field of cellular senescence is at the point at which molecular techniques can now be applied to reveal the basic mechanisms involved in the process. It is interesting to compare the sequence of the genetic studies of cellular aging with those in the field of tumorigenicity and see how similar they have been. Initial hybrid studies had indicated that tumorigenicity was a dominant phenotype, but later studies from many laboratories demonstrated that this phenotype was recessive. Attempts have been made to identify complementation groups for tumorigenicity but have not yielded clear results because of the complex nature of the tumorigenic phenotype. However, whole and microcell hybrid studies, combined with cytogenetics, have allowed the identification of chromosomes believed to carry tumor suppressor genes. Sager and co-workers (O’Brien et al., 1986) and Cairns and Logan (1983) have postulated cellular senescence as one mechanism for tumor suppression, and Sager’s data support this idea (O’Brien et al., 1986).As studies in the field of aging progress, the role of senescence in tumor suppression will become more clear. Since the tumorgenic phenotype has been clearly separated from that of immortalization in cells in culture, it will be of interest to determine at the chromosomal and later the gene level how these phenotypes impinge on each other. Studies of cellular senescence should therefore yield insight not only into normal growth control and the role of loss of proliferative potential with age, but also in the areas of tumorigenicity and abnormal development.

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JAMES R. SMITH AND OLIVI.4 M. PEREIRA-SMITH

ACKOWLEDGMENTS This work was supported by grants from the National Institutes of Health AG04749, AG05333, and PO1 AG07123. We thank Theresa Calkins for excellent secretarial assistance.

REFERENCES Bierman, E. L. (1978).In Vitro 14,951-955. Bunn, C. L., and Tarrant, G. M. (1980). E x p . Cell Res. 127,385-396. Burmer, G. C., Moltulsky, H., Zeigler, C. J., and Nonvood, T. H. (1983).E x p . Cell Res. 145,7944. Cairns, J., and Logan, J . (1983). Nature (London)304,582-583. Croce, C. M., and Koprowski, H. (1974).Science 184,1288-1289. Drescher-Lincoln, C. K., and Smith, J. R. (1983).E x p . Cell Res. 144,445-462. Drescher-Lincoln, C. K., and Smith, J. R. (1984). Exp. Cell Res. 153,208-217. Duthu, G . S., Braunschweiger, K. I., Pereira-Smith, 0. M., Norwood, T. H., and Smith, J. R. (1982).Mech. Ageing Dec. 20,243-252. Goldstein, S., Moerman, E. J., Soeldner, J. S., Gleason, R. E., and Barnett, D. M. (1978). Science 199,781-782. Hayflick, L. (1965). E r p . Cell Res. 37,614-636. Hoehn, H., Bryant, E. M., and Martin, G. M. (1978).Cytogenet. Cell Genet.21,282-295. LeGuilly, Y., Simon, M., Lenoir, P., and Bourel, M. (1973). Geroiitologia (Basel) 19, 303-313. Littlefield, J. W. (1973).J.Cell. Physiol. 82, 129-132. Lumpkin, C. K., Jr., McClung, J . K., and Smith, J. R. (1985).E x p . Cell Res. 160,544-549. Lumpkin, C . K., Jr., McClung, J. K., Pereira-Smith, 0. M.,and Smith, J. R. (1986a). Science 232,393-395. Lumpkin, C. K., Knepper, J. E., Butel, J . S., Smith, J. R., and Pereira-Smith, 0. M. (1986b).Mol. Cell. Biol. 6,2990-2993. Martin, G. M., Sprague, C. A,, and Epstein, C. J. (1970).Lab. Znuest. 23,86-92. M a e n , G. M., Sprague, C. A,, Nonvood, T. H., and Pendergrass, W. R. (1974). Am. J . Pathol. 74,137-154. Matsumura, T., Zerrudo, Z., and Hayflick, L. (1979).J.Gerontol. 34,328-334. Muggleton-Harris, A. L., and DeSimone, D. W. (1980). Somatic Cell Genet.6,689-698. Muggleton-Harris, A. L., and Hayflick, L. (1976).E x p . Cell Res. 103,321-330. Nonvood, T. H., Pendergrass, W. R., Sprague, C. A., and Martin, G. M. (1974).Proc. Natl. Acad. Sci. U.S.A. 71,2231-2235. Nonvood, T. H., Pendergrass, W. R., and Martin, G. M.(1975).J.Cell BioZ. 64,551-556. O’Brien, W., Stenman, G., and Sager, R. (1986). Proc. Natl. Acad. Sci. U.S.A.83,86598663. Pepperkok, R., Schneider, C., Philipson, L., and Ansorge, W. (1988a).E x p . Cell Res. 178, 369-376. Pepperkok, R., Zanetti, M.,King, R., Delia, D., Ansorge, W., Philipson, L., and Schneider, C. (1988b). Proc. Natl. Acad. Sci. U S A . 85,6748-6752. Pereira-Smith, 0. M., and Smith, J. R. (1981). Somatic Cell Genet. 7,411-421. Pereira-Smith, 0. M.,and Smith, J. R. (1982). Somatic Cell Genet. 6,731-742. Pereira-Smith, 0. M . , and Smith, J. R. (1983). Science 221,964-966. Pereira-Smith, 0 .M., and Smith, J . R. (1987). MoZ. Cell. Biol. 7, 1541-1544.

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Pereira-Smith, 0. M., and Smith, J. R. (1988). Proc. Natl. Acad. Sci. U . S A . 85,60426046.

Pereira-Smith, 0. M., Fisher, S. F., and Smith, J. R. (1985).E x p . Cell Res. 160,297-306. Rohme, D. (1981). Proc. Natl. Acad. Sci. U.S.A.78,5009-5013. Ruley, H. E. (1983). Nature (London)304,602-606. Schneider, E. L., and Mitsui, Y.(1976). Proc. Natl. Acad. Sci. U.S.A.73,3584-3588. Smith, J. R., and Hayflick, L. (1974).J . Cell Biol. 62,48-53. Smith, J. R., and Whitney, R. G. (1980). Science 207,82-84. Stanbridge, E. J. (1976). Nature (London)260,17-20. Stein, G. H., and Atkins, L. (1986). Proc. Natl. Acad. Sci. U S A . 83,9030-9034. Stein, G . H., and Yanishevsky, R. M. (1979). E x p . Cell Res. 120,155-165. Stein, G . H., Yanishevsky, R. M., Gordon, L., and Beeson, M. (1982). Proc. Natl. Acad. Sci. U S A . 79,5287-5291. Wright, W. E., and Hayflick, L. (1975).E x p . Cell Res. 96,113-121. Yanishevsky, R. M., and Stein, G. H. (1980).E x p . Cell Res. 126,469-472.

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THE FUNCTION OF RAS GENES IN Saccharomyces cerevisiae James R. Broach and Robert J. Deschenesl Department of Biology. Princeton University, Princeton, New Jersey 08544

I. Introduction A. The ras Gene Family and Related GTP-Binding Proteins B. R A S Genes in Saccharomyces cerewisiae 11. Model for Ras Protein Function in Yeast 111. Yeast Ras Proteins A. Do Rasl and Ras2 Proteins Have Distinct Functions? B. Expression of R A S l and RAS2 C. Functional Domains of Yeast Ras Proteins D. Carboxyl-Terminal Modification and Membrane Localization IV. Components of the Ras-CAMP Pathway A. Adenylate Cyclase B. CAMP-Dependent Protein Kinase C. Phosphodiesterases D. Protein Phosphatases E. CDC25 F. Other Components of the Pathway V. Targets of the CAMP-Dependent Protein Kinase A. Proteins Involved in Carbon Metabolism B. Feedback Regulation of the Ras-CAMP Pathway C. Proteins Involved in Growth Control VI. To What Signals Do RAS Genes Respond? A. Carbon Source B. Nutrient Sufficiency C. Other Signals VII. What Is Ras Doing? References

I . Introduction A. THErus GENEFAMILY AND RELATED GTP-BINDINGPROTEINS rus genes constitute a family of highly conserved genes, the prominence of which as objects of investigation arises from their role as etiologic agents in a large percentage of human tumors. rus genes

Present address: Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242. 79 ADVANCES IN CANCER RESEARCH, VOL. 54

Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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JAMES R. BROACH AND ROBERT J. DESCHENES

have been found in every eukaryotic organism in which they have been sought, including all mammalian species, as well as Aplysia (Swanson et al., 1986), Drosophila (Brock, 1987; Mozer et al., 1985; Neuman-Silberberg et al., 1984; Schejter and Shilo, 1985), Dictysotelium (Reymond et al., 1984),Aspergillus (T. Som and J.R. Broach, unpublished observation), Saccharomyces (Powers et al., 1984; DeFeo Jones et al., 1983), and Schizosaccharomyces (Nadin Davis et ul., 1986; Fukui and Kaziro, 1985). The involvement of ras genes in cancer was first suggested by their identification as the transforming agents of Harvey and Kirsten rat sarcomaviruses (Barbacid, 1987).This supposition was reinforced by results from several groups, who used DNA transvection protocols to identify activated versions of cellular rm genes in a variety of human, mouse, and rat tumors (Shih et al., 1981; Krontiris and Cooper, 1981; Perucho et al., 1981; Pulciani et al., 1982; Balmain and Pragnell, 1983; Sukumar et al., 1983; Eva and Aaronson, 1983; Guerrero et al., 1984). The ability of these activated alleles to convert benign primary cell lines into tumorigenic, transformed cells provided compelling evidence that these activated genes compose part of the machinery responsible for the transformed state. Numerous additional experiments have served to confirm this hypothesis (Barbacid, 1987). Substantial effort has been expended to ascertain the normal role of ras in mammalian cells and to explain why activating mutations cause such dramatic alterations of the growth properties of a cell. Scolnick and colleagues demonstrated that the product of mammalian ras genes, designated p21, binds GTP and possesses weak GTPase activity (Temeles et al., 1985; Gibbs et al., 1984; Scolnick et al., 1979). Mutations that activate the transforming potential of the protein either diminish its GTPase activity or enhance its rate of GTP/GDP exchange (Sigal et al., 1986a; Temeles et al., 1985; Scolnick et al., 1979; Gibbs et al., 1984; Tabin et al., 1982; Reddy et al., 1982; Taparwosky et al., 1982; Walter et al., 1986).On the basis of these properties and by analogy to heterotrimeric G proteins that mediate various receptor-induced cellular responses (Fleischman et a!., 1986; Hagag et al., 1986),a number of investigators have proposed that ras gene products function in signal transduction (Lowy and Willumsen, 1986; Bourne and Sullivan, 1986). ras protein could, for example, couple production of an undetermined second messenger to the binding of serum growth factors to cellular receptors. As compelling as this hypothesis is, it has not been unequivocally ratified nor have the components of this postulated signal transduction pathway in mammalian cells been defined (Birchmeier et al., 1985; Bar-Sagi and Feramisco, 1985; Beckner et al., 1985). The biochemical and structural features of ras place it as a member of

ms GENES I N YEAST

81

a large class of small GTP-binding proteins. This class has grown to encompass such functionally diverse proteins as translation elongation factor EF-Tu, components of the secretory machinery Yptl (Schmitt et al., 1988; Segev et al., 1988; Gallwitz et al., 1983) and Sec4 (Salminen and Novick, 1987; Goud et al., 1988), other protooncogenes such as rul and raf, and products of genes of unknown function such as Rho (Anderson and Lacal, 1987; Madaule et al., 1987; Madaule and Axel, 1985).These proteins most likely execute their function through a cycle of GTP/GDP exchange and GTP hydrolysis (Barbacid, 1987). Interaction of the ras-like protein with an activating agent stimulates exchange of bound GDP for free GTP. In this GTP-bound state, the ras-like protein can stimulate activity of a target protein. As a consequence of this interaction or simply as a stochastic process, the bound GTP is hydrolyzed. The GDP-bound protein is then no longer able to stimulate or associate with the target and the protein must be reactivated by recharging with GTP. Clearly, this cycle can be mobilized to serve as an intermediate step in signal transduction, as has been proposed for ras. However, as Bourne (1988)has pointed out, signal transduction is not the only use to which these GTP-binding proteins could be applied. He suggested that such proteins could impart a vectorial component to a complex biochemical process, such as translation or secretion. GTP hydrolysis would ensure a unidirectional flow to the overall process. Another possible function of such a GTP exchangeGTP hydrolysis cycle would be as a proofreading step in a complex pathway. This could ensure that the proper acylated tRNA is positioned on a ribosome or that the appropriate vesicles dock with a particular Golgi stack. Thus, ras is one of many structurally and functionally related proteins, which are likely to participate in distinct ways in a wide variety of cellular processes.

B. R A S GENESIN Saccharomyces cerevisiae

Saccharomyces cerevisiae contains two genes that are highly homologous to human ras genes (Powers et al., 1984; DeFeo Jones et al., 1983). Recognition of their presence initially fostered the prospect of opening a second front in the assault on ras and of applying the awesome armamentarium of yeast molecular genetics to the problem of molecular oncology. This expectation was fortified by the early observation that human and yeast rus genes are functionally interchangeable. Human ras genes can ameliorate the growth defects arising from inactivation of yeast R A S genes (Kataoka et al., 1985a),and minimally tailored versions of a yeast R A S gene can induce proliferative transformation of mouse fibroblast cells (DeFeo Jones et al., 1985).This early

82

JAMES R . BROACH AND ROBERT J . DESCHENES

euphoric expectation has diminished somewhat. Subsequent experiments have documented that, while yeast Ras proteins function predominantly-if not exclusively-as a modulator of adenylate cyclase (Toda et al., 1985), metazoan ras genes appear to exert their influence independent of 3’,5’-cyclic adenosine monophosphate, or CAMP (Bourne, 1985; Beckner et al., 1985; Birchmeier et al., 1985). Despite the apparent disparity of the pathways in which yeast and human rus genes reside, appreciation of the details of the function of yeast Ras protein can provide significant insight into the nature of ras activity in metazoans. For example, since yeast and human ras genes are functionally interchangeable, each protein must be able to interact with a normal target protein(s) in the heterologous host. Precise delineation of the Ras interaction domain of the target protein in yeastmost likely adenylate cyclase-could provide a probe to extract the currently elusive mammalian target of activated ras. In a broader context, yeast Ras proteins appear to influence the cell’s decision between quiescence and mitotic growth. Appreciation of the mechanism by which this decision is effected in a smaller eukaryotic organism should offer a conceptual framework in which to examine the same process in larger eukaryotes. In this review we describe our current understanding of the function of R A S genes in the yeast S. cereuisiae and the components of the pathway in which it acts. Barbacid (1987) has contributed an excellent and thorough review of ras gene structure and function, focusing primarily on mammalian systems but including information on ras genes in other organisms as well. In addition, reviews on R A S genes in yeast have appeared at timely intervals (Tamanoi, 1988; TatchelI, 1986; Matsumoto et al., 1985a). Information on yeast R A S genes continues to accumulate at an accelerating pace. This accumulation provides us with an increasingly detailed conceptualization of the role of R A S genes in yeast and its participation in regulation of cell proliferation. Although several central questions remain unresolved, we feel that a summary of our knowledge at this point, identification of unanswered questions, and speculation on the possible roles of R A S in yeast should prove provocative and valuable. II. Model for Ras Protein Function in Yeast

The Ras-CAMP pathway as we currently understand it is diagrammed in Fig. l, and a list of genes the products of which are components of or impinge on this pathway is provided in Table I. The

83

GENES IN YEAST

P1, P2, P3,

-1

...

P1-P, P2-P, P3-P,

Proteln phosphatase

Go Arrest,

Sporulation

...

1

Energy Activation, Altered Transcription, Cell Growth

FIG.1. The Ras-CAMP pathway in Saccharomyces cereuisiae. The roles of the pathway’s components, each indicated by its gene designation (see Table l),are schematically diagrammed. See text for full description. Dotted line indicates A kinase-catalyzed feedback inhibition. The parallel lines at the top of the figure denote the plasma membrane. AAX designates the C-terminal three amino acids of Ras. P1, P2, and P3 refer to undefined protein targets of A kinase. P1-P, etc., refer to their phosphorylated counterparts.

pathway is depicted in broad storkes in the following paragraphs with the details and caveats elaborated in the next section. Ras 1 and Rase proteins are synthesized as cytoplasmic precursors, which become extensively modified at the carboxyl terminus upon maturation. The mature proteins are acylated, proteolytically cleaved, methyl-esterified, and localized to a membrane fraction, presumed to be the inner surface of the plasma membrane. Some step in this maturation process requires the product of the R A M 1 gene. When charged with GTP, mature Ras protein is capable of stimulating adenylate cyclase, the product of the CYRl locus. This activation is most likely effected through a direct interaction of the two proteins. GTP bound to Ras protein is hydrolyzed, though not released, either as a direct consequence of Ras protein’s interaction with adenylate cyclase or simply as a stochastic event. In the GDP-bound state, Ras protein is not able to stimulate adenylate cyclase. Reactivation of Ras protein requires exchange of bound GDP for free GTP, a process catalyzed by

84

JAMES R. BROACH AND ROBERT J. DESCHENES

TABLE I GENESOF THE Ras-CAMP PATHWAY Gene

Alias

BCYl

SRAl

CDC25

CYR.2

CYRl lRAl PDEl

IACI, SRA4, CDC35 PPDI

PDE2

SRAS

RAM1

DPRl, SGP2, SUPH

R4S1 R4S2

SCH9 SRAG SRAS SRAlO S RV2 TPKl TPK2 TPK3

Y-4K1

sUPC S R A S , PK-25

Product and/or function Regulatory subunit of CAMPdependent protein kinase Ras protein activator; GTP/GDP exchange factor Adenylate cyclase Inhibitor of Ras function Low-affinity (high K,) CAMP phosphodiesterase High-affinity (low K , ) CAMP phosphodiesterase Required for Ras protein and a mating-factor maturation GTP-Binding protein modulating adenylate cyclase activity GTP-Binding protein modulating adenylate cyclase activity Protein kinase that when overexpressed can substitute for protein kinase A Regulator of R A S 1 transcription Suppressor of rasl ras2" alleles Suppressor of rasl r ~ s 2 alleles ~' Suppressor of activated RAS2 alleles One of three catalytic subunits of CAMP-dependent protein kinase One of three catalytic subunits of CAMP-dependent protein kinase One of three catalytic subunits of CAMP-dependent protein kinase Protein kinase the loss of which suppresses the growth defect of t p k l tpk2 tpk3 strains

References.

1-3 4-7 1,7-10 11,12 13,14 15,16 17-19 20-22 20-22 23 10,24 25 25 26 27 27 27 25

' References: 1. Matsumoto et a/., 1982a; 2. Toda et a/., 1987a; 3. Kunisawa et al., 1987; 4. Robinson et a/., 1987;5. Broek et a/., 1987; 6 . Camonis et al., 1986; 7. Pringle and Hartwell, 1981; 8. Matsumoto et a/., 1984; 9. Kataoka et al., 1985b; 10. Cannon et at., 1986; 11. Matsumoto et a/., 1985b; 12. Tanaka et al., 1989; 13. Uno et al., 1983a; 14. Nikawa et al., 198%; 15. Wilson and Tatchell, 1988; 16. Sass et al., 1986; 17. Powers et a/., 1986; 18. Fujiyama et al., 1987; 19. Nakayama et al., 1988; 20. DeFeo Jones et al., 1983; 21. Powers et a/., 1984; 22. Toda et a/., 1985; 23. Toda e f al., 1988; 24. Breviario et a/., 1986; 25. Garrett and J. R. Broach, 1989; 26. FedorChaiken and J. R. Broach, unpublished observation; 27. Toda e t al., 198%.

u s GENES IN YEAST

85

the product of the CDC25 gene. The activity of Cdc25 protein, located in the cytoplasm, appears to be stimulated by glucose through an as yet undetermined mechanism. Unequivocal evidence that other signals, such as nitrogen limitation, impinge on Cdc25 has been elusive, although this possibility has by no means been excluded. Recently, the product of a locus called lRAl has been proposed to stimulate hydrolysis of GTP bound to Ras and thereby regulate Ras activity. Signals that inhibit or enhance the activity of the Iral protein could extend or diminish the lifetime of Ras protein’s activated state. If this conjecture is substantiated, then signals into the Ras-CAMP pathway could enter through either Cdc25 or Iral. The sole function of CAMPin the cell appears to be to activate the CAMP-dependent protein kinase (A kinase). This kinase is most likely a heterotetramer, composed of two regulatory subunits, encoded by the BCYl locus, and two catalytic subunits, redundantly encoded b three separate genes, T P K l , T P K 2 , and T P K 3 . In the absence of CAM$ Bcyl protein restraints the catalytic subunits by serving as a tightly associated, competitive inhibitor of the enzymes. Upon binding CAMP, Bcyl protein releases the catalytic subunits, which are then freed to phosphorylate their numerous cellular targets. Targets of the A kinase include enzymes involved in metabolism of storage carbohydrates, enzymes situated at strategic points in the glycolytic-gluconeogenic pathway, enzymes required for phospholipid metabolism, transcription factors associated with expression of specific genes, proteins involved in production of cAMP itself, and other proteins currently unidentified. In general, high A-kinase activity induces breakdown of stored carbohydrates, activation of the glycolytic pathway, induction of transcription of a large number of growth-specific genes, such as those encoding ribosomal proteins, repression of certain stress proteins, and downmodulation of the RascAMP pathway. This leads, directly or indirectly, to depletion of carbohydrate reserves, sensitivity to nutrient starvation and heat shock, diminished capacity to use nonfermentable carbon sources, and inhibition of sporulation. In the reciprocal situation, low levels of A-kinase activity induce accumulation of carbohydrates, activation of gluconeogenesis, reduced transcription of growth-specific genes, and induction of various stress-related proteins. This leads to diminished capacity for growth on nonfermentable carbon sources, enhanced sporulation in rich media, and hyperaccumulation of carbohydrate reserves. The complete loss of kinase activity results in cessation of growth even in rich media, with cells accumulating at the beginning of the cell cycle.

86

JAMES R. BROACH AND ROBERT J. DESCHENES

At the present time some, but by no means all, of the physiological responses of the cell to high or low cAMP levels can be explained on the basis of differential activities of known targets of the A kinase. Effects on mobilization of carbohydrate reserves are, to a first approximation, accounted for by effects of phosphorylation on activities of enzymes responsible for synthesis versus degradation of these reserve compounds. Carbon source utilization properties may be attributable in part to transcriptional regulation of R A S genes, to effects of phosphorylation on key constriction points in the glycolytic pathway, and to regulation by phosphorylation of sugar transport. Cell cycle effects and sensitivity to starvation and heat shock are less readily explained. However, an increasing body of data suggests that yeast cells can attain a distinct physiological state outside the normal mitotic ceIl cycle when they are nutrient-starved or enter stationary phase. This state is analogous to the Go state described for mammalian cells. Yeast cells in this Go state are more refractory to heat shock and nutrient starvation and are able to persist in this quiescent state for extended periods without significant loss of viability. cAMP levels can influence the cell’s decision to enter this Go state, and this may be the way that cAMP affects the cell’s response to stress. These hypotheses will be elaborated later.

Ill. Yeast Ras Proteins Yeast Ras proteins share considerable homology with the mammalian ras proteins (Dhar et ul., 1984; Papageorge et al., 1984; Powers et al., 1984),although significant differences are evident in the lengths of the respective proteins and the amino acid sequence of certain domains (see Fig. 2). The yeast Rasl and Rase proteins are 309 and 322 amino acids long, whereas Ha-ras protein is 189 (Fig. 2). The difference in length is due to a 7-residue extension at the amino terminus and a large insertion in the C-terminal half of the protein. The homology between RAS family members depends on the portion of the molecules being compared. In the amino terminus, Ha-ras, Rasl, and Ras2 are nearly 90% homologous, whereas in the C terminus there is virtually no sequence similarity (Fig. 2).A short sequence including an invariant cysteine residue is always found at the carboxyl end of all Ras proteins (Deschenes and Broach, 1987; Powers et al., 1986; Willumsen et al., 1984). The extent of sequence homology between either of the yeast R A S genes and mammalian Ha-rus is significantly higher than that of Ha-rus to any of the other currently known small GTP-binding proteins of yeast.

RAs 33.42 01230 64-73

87

GENES IN YEAST

120

143

173

189

0

Ha-ras

no homology

Ha-raslRAS2 L ; I j - 4 5 % *

+80%

RASlIRAS2

7

19 4 0 - 4 9 37 71-80

127

150

4

180

C -A- A- X

322

YEAST RAS E

Ab

guanine

phosphateribose

GTP BINDING

GTP BINDING

VARIABLE

MEMBRANE

ANCHOR

FIG.2. Diagram of the structure of Ras proteins indicating functional domains and regions of homology between members of the family. Positions defining regions important for GTP binding (including phosphate, ribose, and guanine contact points), a putative effector contact region (E),and the monoclonal antibody Y13-259epitope (Ab) are indicated. Details regarding the mutations from which the map is derived can be found in Fig. 3 and in the text. Percentage homologies refer to similarities in protein sequence.

A. DO h

S 1 AND RAS2

PROTEINS HAVEDISTINCT FUNCTIONS?

The predicted amino acid sequences of the two yeast RAS proteins are 86%identical over the first 180 amino acids. Except for the terminal eight residues, which are identical at seven of eight positions, little additional sequence homology is found throughout the remainder of the proteins. The presence ofthis large variable domain might indicate that distinct functions exist for the two proteins. To date, though, no unequivocal demonstration of specialization of Ras 1 and Ras2 proteins has emerged. Deletion of either R A S l and RAS2 alone has no deleterious effect on the growth of yeast strains in rich media, while deletion of both genes is lethal (Kataoka s t al., 1984). This indicates that R A S l and RAS2 are functionally interchangeable for growth in rich medium. The situation is different for growth on a nonfennentable carbon source such ethanol, glycerol, or acetate. In this case, R A S l rus2 strains fail to grow, whereas rasl RAS2 strains are viable (Breviario et ul., 1986; Fraenkel, 1985; Tatchell et al., 1985). This observation initially suggested that the two yeast Ras proteins had different roles under differ-

88

JAMES R. BROACH AND ROBERT J. DESCHENES

ent physiological conditions. Subsequent analysis, though, has indicated that differential requirement for the two R A S genes under these growth conditions is a consequence of differential regulation of the genes, rather than differences in the function of the encoded proteins. Specifically, R A S l is transcribed very poorly in medium containing only nonfermentable energy sources (Breviario et al., 1986).Thus, the growth defect of a R A S l rus2 strain on nonfermentable carbon sources can be attributed to insufficient levels of Ras protein in the cell. In support of this conclusion, Breviario et ul. (1986)found that one class of extragenic suppressors of the growth defect of R A S l rus2 on nonfermentable carbon sources, those mapping to the sru6 locus (Cannon et al., 1986),act by increasing transcription of the R A S l gene. In a complementary set of experiments, Fasano et al. (1988)have observed that Ras2 protein crippled by mutation fails to support growth on nonfermentable carbon sources while maintaining the capability of promoting growth on glucose. Thus, growth on nonfermentable carbon sources correlates strictly with the level of Ras protein expression, rather than with a particular Ras protein expressed. A second phenotypic consequence of ras2 inactivation not exhibited by rasl mutants is induction of sporulation. ras2-homozygous diploids sporulate on rich media, while rasl -homozygous diploids do not (Toda et al., 1985). In contrast to growth on nonfermentable carbon sources, sra6 mutations do not suppress the hypersporulation defect of R A S l ras2 strains (Breviario et aZ., 1986). This may indicate that R A S l and RAS2 play functionally distinct roles in suppressing sporulation. Alternatively, this difference may simply reflect the need for a different threshold level of Ras protein for promoting haploid growth versus suppressing sporulation. This point is currently unresolved. Kaibuchi et ul. (1986)have suggested that Rasl, but not Rase, may affect phosphatidylinositol (PI) turnover, independent of its role in modulating adenylate cyclase activity. These investigators noted that refeeding glucose to glucose-starved cells stimulated the rate of PI turnover and that this stimulation was enhanced in a rasl strain, but unaffected in a rus2 mutant. However, the extent of rasl enhancement in these experiments was minor. In addition, subsequent experiments have not confirmed the role of R A S genes in PI metabolism as it was proposed by these investigators (see Section V). At this point, the role of R A S genes in PI metabolism is unresolved and, accordingly, a distinction between Rasl and Ras2 proteins in promoting PI turnover cannot be considered established.

RAs B. EXPRESSION OF RASl

AND

GENES IN YEAST

89

BAS2

A close examination of the expression of R A S l and RAS2 reveals a complex system of control (Breviario et ul., 1988). The levels of R A S l and R A S 2 mRNA vary depending on the availability of nutrients. As noted before, the levels of R A S l mRNA in cells grown on nonfermentable carbon sources are very low. In addition, as cells enter stationary phase, the levels of both R A S l and RAS2 mRNA become very low, However, when Brevario et ul. (1988) examined Ras proteins under these conditions they found that Ras2 protein levels remained constant even though mRNA levels were markedly reduced. They concluded that the efficiency of translation of the R A S 2 mRNA must increase under these conditions, although they did not formally rule out the possibility that increased protein stability accounts for the persistence of Ras2 protein. From these observations, it appears that the cell uses a variety of mechanisms to ensure that sufficient Ras protein is present in the cell under a wide range of physiological conditions and that these mechanisms are applied nonuniformly to R A S l and RAS2 gene expression. C. FUNCTIONAL DOMAINS OF YEAST RASPROTEINS Identification of the functional domains of Ras proteins has relied on mutational analysis, the three-dimensional (3D) crystal structure recently solved for Ha-rus, and analysis of sequences conserved between members of the Ras family. In the following, we describe our current knowledge of yeast Ras protein structure with respect to GTPbinding and hydrolysis domains, antibody epitopes, putative effector interaction regions, phosphorylation sites, and membrane anchoring sequences. The current assignment of functional domains of Ras proteins from mammals and yeast is presented in Fig. 2. Figure 3 shows the sequence information and a summary of much of the mutational analysis that provides the basis for the assignments presented in Fig. 2. Figure 4, taken from a paper by De Vos et ul. (1988), shows the 3D structure of Ha-rus derived from a Ha-ruslGDP crystal. Using the 3D structure of Ha-rus to analyze mutant yeast RAS proteins is potentially compromised by the fact that the large C-terminal variable domain of the yeast Ras proteins may alter the structure of the Nterminal domain. However, this approach is to some extent validated by the fact that, in almost all cases examined, analogous mutations in yeast R A S genes and mammalian rus oncogenes have the same phenotypic consequences.

RAS.? RASl

H-ras

RAs2 RASl

H-ras

RAsZ RASI

H - ras

L-RSYGI-YI- - - -TRQG--D-- - - -V-EI-QHKLRKLNPPD-SGPGC

I 220

RAS2

RASl

160

230

160

240

290

RAs1 H - ras

260

260

270

280

RKHSNAANG~SS~~ASIESKTGtAGNQATNGKTQTIDNSTGQAGQANAQSANTVNNR SWLDNSLTNAGTG-SSKSA~GETT-RTDEK-YVNQNWNNEGN-KYSSNGNGNRSDISRGNQNNALN

_ _ _ _ - - - - _ _ _ _ _ _ _ _I _X 8_ _ _ _ RAS2

180

170

300

310

*st*

tts a o

VNNNSKAGQVSNAKQARKQQAAPGGNTSEASKSGSGGCCIIS -------SRSKQS-EFQK-SSANARKEY HSCK-VS-

i

szo

FIG.3. Sequences of yeast and mammalian ras proteins and summary of the location and consequences of specific amino acid alterations. The predicted amino acid sequence of yeast Rasl and Ras2 proteins (Powers et al., 1984) and human c-H-ras protein (Barbacid, 1987) are shown. Dashes indicate the same amino acid as that immediately above. Specific amino acid changes in various mutant alleles of yeast R A S genes are shown above the sequence, and those in mutant alleles of human ras genes are shown below the sequence. Mutations are categorized as activating (capital letter designation), inactivating (lowercase letter designation), and neutral (italicized letter designation). * Translation terminator mutation. Elaborating comments and references regarding specific alleles are noted here, designated by superscript numbers 1-20 in the figure. 1. Marshall e t al. (1987); Breviario et 01. (1986). 2. Mutation present in ras2 causes temperature sensihvity for growth on glycerol in a rasl background (Fasano et al., 1988). 3. Mutation causes reduced biological activity that can be suppressed by second-site mutations in CYRl (Marshall et al., 1988).4. Temeles et al. (1985). 5. Marshall et al. (1987). 6. Substitution of any amino acid except proline yields transformation activation

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1. Residues lnvolved in GTP Binding and Hydrolysis

In yeast and mammalian cells, Ras proteins bind and hydrolyze GTP (Gibbs et al., 1984,1987; Scolnick et al., 1979; Colby et al., 1986; Field et al., 1987; De Vendittis et al., 198613; Tamanoi et al., 1984; Temeles et al., 1985).The residues involved in binding and hydrolysis of GTP have been inferred from mutational analysis, from biochemical analysis of mutant proteins, and, in some cases, from inspection of the crystal structure of wild-type and mutant Ha-ras. Two domains of Ha-ras protein-one around GlyI2 the second between AIa5’ and G1u“-are associated with GTP hydrolysis (Yamamoto and Perucho, 1984; Lacal et al., 1986b; Fasano et al., 1984; Bos et al., 1985; Yuasa et al., 1983; Seeburg et al., 1984; Feig et al., 1986).Biochemical analysis of position-12 Ha-ras mutants reveals that GTP binding is normal, but GTPase activity is lowered by -100-fold (McGrath et al., 1984).Mutational analysis of yeast RAS2 gene suggests that the equivalent position (Gly’’) plays an analogous role in GTP hydrolysis (Toda et d . , 1985; Kataoka et al., 1984).In the crystal structure of Hams, Gly” is in a tight loop (loop 1 in Fig. 4A) that straddles the P-y phosphodiester bond of GTP. The presence of gIycine at this position appears to be essential for normal GTPase activity, since the only substitution at position 12 that does not diminish GTPase activity is proline (Seeburg et ul., 1984). Recent solution of the crystal structure of Ha-rasva’ protein has shown that substituting valine for glycine at position 12 (Reddy et al., 1982; Seeburg et al., 1984; Sikumar et al., 1983; Taparowsky et al., 1982). 7 . Transformation activation induced by substitution of V or D, but not S (Bos et el., 1985; Fasano et al., 1984). 8. Mutation causes reduced GDP/GTP binding and the mutant Ha-ras causes dominant temperature sensitivity when introduced into yeast (Sigal et al., 1986a).9. Mutations diminish Ha-ras activity without affecting GTP binding or hydrolysis (Marshall et al., 1988; McCormick, 1989). 10. Fasano et al. (1984). 11. Substitution of any amino acid except P or E yields transformationactivation (Taparowsky et al., 1983;Brown et al., 1984;Yuasaet al., 1983; Bos et al., 1985;Tsuchida et al., 1982; Dhar et al., 1982). 12. Double mutation in ras2 causes temperature sensitivity for growth on glycerol in a rasl background (Fasano et al., 1988). 13. Deletable without affecting Ha-ras activity (Willumsen et al., 1986).14. Walter et d.(1986).15. Dominant suppressor of cdc25 (Camonis and Jacquet, 1988). 16. Mutation causes enhanced GTP/ GDP exchange (Feig et al., 1986). 17. Deletable without affecting Ha-ras activity (Willumsen et al., 1985; Lacal et al., 1986b). 18. Deletion mutant renders yeast cells CDC25-independent (Marshall et al., 1987). 19. Mutations block palmitate addition, prevent localization of protein to the membrane, and diminish R A S function; missense mutation also blocks proteolytic cleavage and carboxyl-terminus methyl esterification (Deschenes and Broach, 1987; Deschenes et al., 1989). 20. Mutation blocks palmitate addition, prevents localization of protein to the membrane, and diminishes ras function (Willumsen et al., 1984).

A

N

FIG.4. Three-dimensional structure of mammalian Ha-ras. Schematic diagrams of two different views of mammalian Ha-ras show the location of different loops (Ll, L2, etc.) on the molecule (A), as well as the position and extent ofvarious domains (B) within the molecule. These include the effector domain (E), region in which most activating alleles are found (A), guanine nucleotide-binding region (G), phosphate-binding region (P), and ribose sugar binding region (S). The amino (N) and carboxyl (C) termini are also indicated. These diagrams are taken from De Vos et al. (1988) and are generously provided by Dr. Sung-Hou Kim.

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causes a significant increase in the size of loop 1 (Fig. 4A) and an attendant loss of two hydrogen bonds (from the backbone amide groups of residues 12 and 13)to the @phosphate of GDP. Tong et al. (1989) have postulated that this change in the conformation of the catalytic site could account for the loss of GTPase in the mutant protein. Mutations yielding substitutions at positions 59 and 61 of mammalian rus (Fasano et al., 1984; Der et al., 1986a) and at the equivalent positions of yeast R A S l (Temeles et al., 1985) induce activation, although the biochemical basis of activation is not entirely clear. For example, the Ala5' to Th?' mutation has been reported to lower GTPase activity by some laboratories (Temeles et al., 1985; Gibbs et al., 1984), increase the GTP/GDP dissociation rate in another study (Lacal and Aaronson, 1986b), and have no effect on GTPase in a third study (Lacal et al., 1986a).Consideration of the location of amino acids 59 and 61 on the Ha-ras crystal structure (loop 4, Fig. 4A) fails to clarify why mutation of these residues resuIts in activation. AIthough Thr5' appears to be in position to accept the y-phosphate during hydrolysis, which would account for autophosphorylation of the viral protein, loop 4 does not seem to be capable of directly affecting binding or catalysis. De Vos et al. (1988) suggest that the effect of Thr5' activating mutations may be mediated through loop 4-loop 1interactions rather than direct contact with GTP. An alternative explanation is that mutations within this domain diminish interaction with cellular GTPase-activating protein (GAP) and thereby extend the average lifetime of GTP-bound ras protein (see later). Mutations that affect GTP binding have aided in defining salient features of the CTP-binding domain. Included in this group are mutations that change Lys16to Asn and Asp"' to Ala in Ha-ras (Sigal et al., 1986a), and to Ile in yeast Rase (Camonis and Jacquet, 1988). These residues were selected by identifying regions of sequence conservation between members of the large family of GTP-binding proteins and then testing proteins specifically mutated at the conserved residues for GTP binding. Reduced GTP binding in these mutants is attributed to an increase in the dissociation rate for bound nucleotide. Additional studies have pointed to a conserved sequence, NKXD at position 116-119, as being important for GTP binding (Der et al., 1986b). Using an in situ-binding assay to identify GTP-bindingdefective mutants, Feig et al. (1986) isolated additional mutant alleles of Ha-ras at positions 83, 119, and 144 that affect GTP binding. The decrease in GTP-binding affinity in each of these mutants was found to be caused by an increase in the dissociation rate constant for guanine

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nucleotide (Sigal et al., 1986a; Camonis and Jacquet, 1988; Der et al., 1986b). Since the intracellular concentrations of GTP are significantly higher than GDP (Proud, 1986), the activity of Ras in the cell is dictated by the nucleotide dissociation rate constant. That is, mutant ras protein with elevated GDP/GTP dissociation rates should be in the activated, GTP-bound configuration more often than is the wild-type protein, since it would more rapidly equilibrate with intracellular guanine nucleotide pools. Consistent with this hypothesis, Feig et al. (1986) found that the GTP-binding mutants at amino acid positions 83, 119, and 143 transform NIH 3T3 cells with the same potency as v-Ha-ras. In addition, Reynolds et al. (1987) have isolated in Ha-ras mutant from a rat hepatoma with a mutation at residue 117. Therefore, two distinct classes of mutations are capable of causing transformation. One class activates Ras by inhibiting the GTPase activity, the second class by increasing the dissociation rate constant for nucleotide. The identification of a protein that stimulates GTPase activity of mammalian ras protein provides an additional complication to the mutational analysis of ras activation (McCormick, 1989). Trahey and McCormick (1987) have shown that extracts of Xenopus contain a protein that can stimulate GTP hydrolysis catalyzed by N-ras p21. This protein, which has been designated GAP (for GTPase-activating protein), is present in mammalian cells as well as Xenopus and has been purified from beefbrain (Gibbs et al., 1988).Sequence analysis of cDNA encoding GAP suggests that the protein shares weak homology with a domain of yeast adenylate cyclase and with the regulatory domains of a number of protein kinases (Vogel et al., 1988; Trahey et al., 1988).It is not yet clear whether GAP represents the normal target of rus in metazoan cells or whether GAP serves to regulate the average duration of the GTP-bound, or activated, form of ras (Cales et al., 1988; Adari et al., 1988; Vogel et al., 1988; McCormick, 1989). In either case, mutations of ras that interfere with ras-GAP interaction would tend to enhance activation of ras. Consistent with this prediction, Trahey and McCormick (1987) have presented evidence that the degree of activation of mutant N-ras proteins with different amino acid substitutions at positions 12,59, and 61 correlates better with the loss of stimulation by GAP than with the reduction in intrinsic GTPase activity. Yeast cells do not contain a GAP-like activity when assayed using Ha-ras protein as a substrate (Adari et al., 1988). However, genetic and sequence analysis of a locus designated I R A 1 (formerly PPDl) suggests that it encodes a protein that acts on yeast Ras proteins in a manner analogous to the action of GAP on Ha-ras (Tanaka et al., 1989).

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Expression in yeast of mutant Ha-ras defective in GTP binding causes dominant, temperature-sensitive lethality (Sigal et al., 1986yL. Lethality can be overcome by the coexpression of yeast RAS2”“’ . From these results Sigal et al. (1986a) proposed that Ha-ras protein defective for GTP binding forms dead-end complexes with cellular effectors, an interaction that can be overcome by expressing a constitutively activated R A S allele. This model accounts for the data but fails to explain why the same mutant Ha-ras causes transformation in mammalian cells. The discrepancy may reflect the inherent differences in the function of RAS in fungi versus metazoans. This highlights the fact that although Ha-ras complements loss of yeast RAS activity, significant differences in yeast and mammalian ras function do exist. 2. Antibody Epitopes Monoclonal antibodies (mAb) have been raised to Ha-ras in an attempt to probe the protein’s structure and function. Epitopes for some of these antibodies lie in well-conserved regions. For example, mAb Y 13-259, Y 13-4, and Y 13-128 all recognize an epitope between residues 63 and 70 of Ha-ras. These mAb react with Ras proteins from other species, including those from yeast. Binding of these antibodies inhibits ras activity, without affecting either GTP binding or hydrolysis (Lacal and Aaronson, 1986a). The hypothesis that this region of ras defines an effector contact zone can be dismissed because deletions in this region do not affect function (Willumsen et al., 1986). Most likely, antibody binding in this region blocks ras activity by steric hindrance of a true effector domain. 3. Effector Domains A11 models of RAS function predict interaction of Ras protein with one or more target proteins. Identification of a domain of Ras through which this interaction is effected has been probed by mutational analysis. A mutation in this hypothetical effector domain would be expected to abolish Ras activity without affecting GTP binding or hydrolysis, protein stability, or the overall conformation of the protein. It should also lie in a conserved region of the protein. Sigal et al. (198613) have suggested that such an effector domain lies between residues 32 and 40 of Ha-ras and at the analogous position in yeast Rase. Mutations in this region of Ha-ras diminish activity without measurably altering any known biochemical property of the protein. In addition, this region corresponds to an open loop that lies on the surface of Ha-ras as judged by analysis of the crystal structure (De Vos et al., 1988). When “effector” mutations are constructed in the yeast RAS2 gene

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(ras2Ser42), a stable protein is made. This mutant protein is inactive even though its biochemical properties are indistinguishable from those of wild-type protein. Consistent with the assumption that this protein is “effector”-defective, Marshall et al. (1988) found that ras2Ser42 mutants could be suppressed by second-site mutations in the gene encoding adenylate cyclase (CYRI), the likely target of yeast Ras. This observation, though, does not constitute irrefutable proof that position 42 lies in the effector domain, since any CYRl mutation that stimulated cyclase activity should suppress a crippled rus2 mutant. Further evidence will be needed to show conclusively that this region of the Rase protein is the site of interaction with adenylate cyclase.

4. Variable Domain The most striking structural difference between different Ras protein structures lies in the carboxyl portion of the molecules, outside the GTP-binding domains. This variable region shows no homology among members of the ras family and can vary in size from 5 to 140 amino acids from protein to protein. Deletion analysis has been performed to assess the function of this region. The 20-amino acid Ha-ras variable domain has been deleted to 5 residues, and also expanded with random sequences up to 50 residues with little significant effect on the ability of the protein to transform NIH 3T3 cells (Willumsen et al., 1985). In yeast Ras protein, in contrast, the variable domain may play a significant role in function. The variable domains of Rasl and Ras2 are 119 and 132 amino acids in length, respectively. Marshall et al. (1987)excised the variable region of the RAS2 gene while retaining the membrane anchoring domain. Expression of this RASB(-V) gene in yeast not only complemented rus2 strains but also rendered the cell independent of CDC25 function. Expression in yeast of Ha-rus, which naturally lacks an extended variable domain, also rendered the cell CDC25 independent. For both RAS2(-V) and Ha-ras expressed in yeast, a higher ratio of GTP to GDP is found bound to the protein following its immunoprecipitation from cell extracts than is found with wild-type Rase protein (Gibbs et ul., 1987). In sum, these results suggest that the variable domain of yeast RAS serves in one of two possible capacities. It could serve to stimulate hydrolysis of bound GTP, functioning essentially as an intramolecular GAP analog. Alternatively, the variable region could restrict GTP/GDP exchange. In this latter model, Cdc25 protein would act either directly or indirectly on the variable region to enhance GTP/GDP exchange. Distinguishing between these models will require further analysis.

-

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5. Phosphorylation Ras2 has been shown to be phosphorylated by A kinase in vitro (Resnick and Racker, 1988)and in vivo (Sreenath et al., 1989; Cobitz et al., 1989).The site or sites ofphosphorylation have not been identified, although circumstantial evidence suggests that a primary site lies in the carboxyl third of the molecule (Sreenath et al., 1989).A reasonable A-kinase consensus phosphorylation site ‘is present at position 214. RasZ protein appears to be phosphorylated in vivo at a second site as well, possibly through an A kinase-independent mechanism (Sreenath et al., 1989; Cobitz et al., 1989). However, by phosphopeptide analysis, Cobitz et al. (1989) have shown that the same tryptic fragments are labeled when purified yeast Rase is phosphorylated in vitro with A kinase as those found in in uivo-phosphorylated Ras2 protein. Rasl protein is also phosphorylated in uiuo, although the site and consequences of this phosphorylation are unknown. The possible role of Ras protein phosphorylation in feedback regulation of CAMPproduction is discussed later (see Section V). D. CARBOXYL-TERMINAL MODIFICATION AND MEMBRANELOCALIZATION Ras proteins must become membrane-localized to function (Deschenes and Broach, 1987; Willumsen et al., 1984), yet all Ras proteins begin as soluble proteins with no significant stretches of hydrophobic residues. Membrane localization requires a complex series of posttranslational modifications culminating in the covalent attachment of a fatty acid to a cysteine residue near the C terminus of the protein. In all Ras proteins, and many related lipidated proteins, the cysteine is found in the conserved-sequence context Cys-A-A-X, where A is any aliphatic amino acid and X is the C-terminal residue (Table 11).Mutating the invariant Cys residue prevents membrane attachment without significantly affecting the other biochemical activities of Ras. Mislocalized Ras protein will not activate adenylate cyclase in yeast (Deschenes and Broach, 1987), nor will v-Ha-ras mutated at this position transform NIH 3T3 cells (Willumsen et al., 1984). Closer examination of the events involved in processing the C terminus has revealed a complex series of steps that include proteolytic cleavage, fatty acylation, and carboxylmethylation. This processing pathway is discussed in the following paragraphs. 1. Fatty Acylation

Localization of Ras protein to the inner surface of the plasma membrane is accompanied with covalent attachment of a fatty acid. If cells

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TABLE I1 C-TERMINAL AMINO ACID SEQUENCES OF ras PROTEINS AND SOME FUNGAL SEX FACTORS" Protein Human Ha-ras K-ras-2A N-rus Drosophila D-ras Dict yostelium D-ras Saccharomyces cerecisiae RASl

RAs2 a-Factor YPTl YPTIE" Trernella Tremerogen A-9291-1 Tremerogen A-10

Amino acid sequence

---Ser-Cys-Lys-Cys-Val-Leu-Ser ---1Ie-Lys-Lys-Cys-Ile-Ile-Met ---GIy-Leu-Pro-Cys-Val-Val-Met ---Arg-Phe-Lys-Cys-Cys-h.let-Leu ---Lys-Cys-Gln-Cys-Leu-Ile-Leu

---Gly-Gly-Cys-Cys-Ile-Ile-Cys ---Gly-Gly-Cys-Cys-Ile-Ile-Ser ---Asp-Pro-Ala-Cys-Val-Ile- Ala ---Gly-Gly-Cy~-Cy~

---Gly-Gly-Cys-Cys-Val-Leu-Ser ---Ser-Gly-Gly-Cys ---Am-Gly-Tyr-Cys

Primary literature for ras sequences are reviewed in Barbacid (1987). Saccharomyces cerecisiae a-factor sequence is from Brake et al. (1985), Tremella sex factor sequences are from Sakagami et al. (1981), and Ishibashi et al. (1984), and the YPTl sequences are from Molenaar et al. (1988).

are labeled with [3H]palmitate, the membrane-bound form of Ras is labeled (Sefton et al., 1982; Shih et al., 1982) and the incorporated label can be released exclusively as palmitate (Buss and Sefton, 1986; Fujiyama and Tamanoi, 1986; F. Tamanoi, personal communication). Since bound lipid is sensitive to hydroxylamine treatment under conditions that would cleave a thioester linkage (Chen et al., 1985), palmitate has been presumed to be linked to the conserved cysteine by a thioester bond. However, failure to isolate the expected modified cysteine residue or tryptic peptide from yeast Rase protein has prompted speculation that linkage of the acyl group to the cysteine thiol may occur through an intermediary group (Deschenes et al., 1989; F. Tamanoi, personal communication). Alternatively, and consistant with recent speculation, the conserved cysteine of Ras2 protein, like that of a-factor (see below), may be modified by a thioether linkage to farnesine, with a separate cysteine serving as the site of acylation by palmitate.

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2. Proteolytic Cleavage Maturation of Ras proteins is associated with a change in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The processed form of the protein migrates as if it were -400 Da smaller than the precursor (Sefton et al., 1982; Shih et al., 1982; Fujiyama and Tamanoi, 1986; Tamanoi et al., 1988; Deschenes and Broach, 1987; Willumsen et al., 1984).Although the mobility shift was initially presumed to result from acylation, new evidence indicates that the protein is proteolytically processed by removal of the terminal three amino acids. First, Tamonoi et al. (1988)have detected a soluble form of Ha-ras p21 expressed in yeast, which is not fattyacylated but which migrates faster by SDS-PAGE than does the initial translation product of Ha-ras. This is consistent with proteolytic cleavage preceding fatty acylation. Second, Deschenes et al. (1989) have presented evidence sugegsting that the C-terminal residue of mature Ras2 protein is cysteine. This would be true only if the terminal three residues of the primary translation product of RAS2 were removed. Although only structural analysis of Ras2 protein purified from yeast will unequivocably confirm that it undergoes proteolytic cleavage during maturation, the current evidence to this effect is reasonably compelling. Recent results demonstrating that ras protein in mammalian cells undergoes a similar processing step further fortifies this conclusion (Gutierrez et al., 1989). 3. C-Terminal Methyl Esterification Ras2 protein from yeast cells and Ha-ras protein from mammalian cells are posttranslationally methyl-esterified in vivo (Clarke et al., 1988; Deschenes et al., 1989). This has been established by assaying Ras proteins, isolated from cells labeled with [methyl-3H] S-adenosylmethionine, for base-specific release of tritiated methanol (Stock et al., 1984). The site of methyl esterification of Rase protein from yeast has been shown biochemically to be a C-terminal cysteine, apparently on a a-carboxyl group. This assignment is supported by the fact that Ras proteins that lack the C-terminal five amino acids (Cys-Cys-A-A-X) or in which Cys319 is changed to Ser are not methylated (Deschenes et al., 1989). 4. Zdentijkation of Genes Involved in R A S Protein Maturation Mutants defective in maturation of Ras protein have been isolated as second-site suppressors in strains carrying activated alleles of RAS2. By selecting heat shock-resistant revertants of such a strain, Powers et

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al. (1986) and Fujiyama et al. (1987)independently isolated alleles of the same gene, designated raml or dprl, that affected processing of RAS proteins (For convenience, the gene is referred to as RAM1 in this discussion, although both designations are still used in the literature). In addition to suppressing the heat shock-sensitivity phenotype of the RAS2va'19 allele, raml mutations cause sterility in a MATa background (Fujiyama et al., 1987; Powers et al., 1986), by restricting biosynthesis of a-factor hormone. A possible explanation for the pleiotropic effect of raml mutations is that Ras proteins and a-factor may share one or more processing steps in common. a-Factor is a 12-amino acid pheromone produced by MATa cells and required for conjugation with MATa cells. Analysis of a cDNA clones of MFAl and MFA2 a-factor genes reveals that it is initially synthesized as 36- and 38-amino acid precursors. The precursor sequences terminate with the Ras proteinprocessing consensus sequence Cys-A-A-X (see Table 11).Comparison of the structure of mature yeast a-factor, determined by fast atom bombardment-mass spectrometry (Anderegg et al., 1988), to the predicted sequence of the primary translation products indicates that they undergo at least two proteolytic cleavages, one of which is immediately distal to the conserved cysteine. In addition, mature a-factor contains a farnesine residue attached via a theioether linkage to the side chain of this cysteine, and a methyl ester group at the C terminus. It is possible that one or more of these steps are shared by the Ras proteins and that RAMl carries out one of these steps. The specific function of the RAMl product is currently unidentified. If, as recent evidence suggests, yeast RAS2 protein is modified by farnesylation of the conserved cysteine residue, then RAMl could possibly encode a protein catalyzing this reaction. On the other hand, evidence consistent with a role of RAMl in proteolysis has been presented. First, RAMl activity appears to be required early in the Ras maturation pathway, since Ras protein accumulates as a slowmigrating, soluble protein in ram1 mutant strains (Tamanoi et al., 1988). Second, although the nucleotide sequence of RAMl does not exhibit any significant stretches of homology with known proteases or methyltransferases, RAMl and thioproteases share a common feature (Goodman et al., 1988).The active site of thioproteases consists of two conserved regions, a Cys-Trp followed by a His-Ala -160 residues away (Ohno et al., 1984). This sequence combination spaced 157 residues apart is also found in the Raml protein (Goodman et al., 1988). Further analysis of Raml, though, suggests that the actual function is more complicated. First, raml alleles have been recovered as suppressors of yeast G-protein mutants (Nakayama et al., 1988) and as suppressors of secretion defects associated with kar2 mutants, the

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yeast homolog of mammalian binding protein, or BIP (Rose and Misra, 1989; M. Scidmore and M. Rose, personal communication). In addition, complete inactivation of R A M 1 results in temperature-sensitive growth rather than unconditional lethality (Nakayama et al., 1988). These results do not rule out the possibility that Raml protein functions as a protease required conditionally for processing a diverse set of proteins, although they make it less likely. An alternative explanation is that Raml plays an as yet undefined role in lipid metabolism, affecting the physical properties of yeast membranes directly and maturation of Ras protein and a-factor only indirectly. This question clearly remains to be resolved.

5. Processing and the Control of R A S Activity In mammalian cells, both palmitoylation and carboxylmethylation appear to be reversible modifications (Clarke, 1985; Chelsky et al., 1985; Staufenbiel, 1988). For instance, palmitate on mammalian N-ras turns over with a half-life of -20 min, raising the possibility that an acylation-deacylation cycle may be involved in the regulation of Ras activity (Magee et al., 1987).Another particularly intriguing example is the cell cycle-dependent carboxylmethylation of lamin B (Chelsky et al., 1987), which apparently is methylated in intact nuclei and demethylated during mitosis. Perhaps Ras proteins are continuously cycled in and out of the membrane by a sequence of reactions including lipidation, methyltransferase, methylesterase, and lipase activities. Any of these steps could play an important role in regulating RAS activity. One can imagine a Ras modification cycle that follows the cell cycle just as the lamin methylation cycle is coordinated with the cycle of nuclear replication. It is interesting in this regard that demethylation of S . cerevisiae a-factor abolishes activity (Anderegg et al., 1988).The possibility that Ras activity in yeast might be modulated by alterations in S-adenosylmethionine levels in the cell or by subtle changes in lipid composition offers another avenue through which Ras could respond to diverse environmental or internal signals. IV. Components of the Ras-CAMP Pathway

A. ADENYLATECYCLASE

1. Structure Saccharomyces cerevisiae contains a GTP-stimulated adenylate cyclase encoded by the gene, CYRl or CDC35 (Casperson et al., 1983; Matsuomoto et al., 1982a, 1984). Mutations in this gene were isolated by Matsumoto et al. (1982a) in a screen designed to identify mutants

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dependent on addition of exogenous cAMP for growth. This screen yielded recessive mutations in two genes, cry1 and cyr2, and dominant mutations in a third gene, CYR3. CYRI was shown to encode the structural gene for adenylate cyclase and to be allelic to the cell cycle gene CDC35 (Matsumoto et al., 1984). CYR2 and CYR3 are allelic to CDC25 and BCYI, respectively (see later). The CYRl gene, cloned by complementation and sequenced, encompasses an open reading frame sufficient to encode a protein of 2026 amino acids (Casperson et al., 1985; Kataoka et al., 1985b). A schematic diagram of the gene is presented in Fig. 5. Several domains can be defined within the gene. Catalytic conversion of ATP or cAMP can be effected by a truncated version of the gene carrying only the carboxyl 20% of the predicted protein. Fragments encompassing this region can complement cyrl mutations, although adenylate cyclase activity produced from this truncated gene is not responsive to modulation by Ras (Kataoka et al., 1985b; Uno et al., 1987). Near the center of the gene is a 590-amino acid stretch that is composed of 26 repeats of a 23-amino acid, leucine-rich sequence (Kataoka et al., 1985b). This sequence may be required for membrane attachment. In addition, it may contribute to RAS protein binding. 2. RAS-Responsive Domains Several approaches have been taken to identify the region of adenylate cyclase that mediates Ras responsiveness (Uno et al., 1985, 1987). Uno et al. (1985, 1987) have attempted to reconstruct a Ras-responsive

LEUCINE REPEATS

CATALYTIC DOMAIN

b-

w\S

-

RESWNSIVE DOMAIN

I .-

FIG.5. Map of the protein-coding region of the adenylate cyclase gene cloned from yeast. The region between 710 and 1300 contains 26 repeats of the sequence: PXX(L,V,I)XXLXXLXXLXLXXNX(L,V,I)XX(L,V,I). Expression of the C terminus (1609-2026) results in active adenylate cyclase, but Ras-responsive cyclase requires a larger region (see text). The positions of two mutations that alter Ras activation of cyclase, residues 1547 (Marshall et al., 1988) and 1651 (De Vendittis et al., 1986a), are indicated. ~

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yeast adenylate cyclase in Escherichia coli mutant strains devoid of endogenous adenylate cyclase activity. Adenylate cyclase activity could be detected in extracts of such strains expressing the C-terminal 434 amino acids of CYRl ,although this activity was not dependent on the presence of Ras proteins or GTP. Extending the portion of the CYRl gene expressed in E . coli to include the C-terminal701 residues yielded cyclase activity that was Ras-dependent. This would localize the Ras-responsive region to the area between the leucine repeats and the catalytic domain. However, attempts to repeat these experiments have not succeeded (Field et al., 1988). An alternative approach to defining Ras-responsive regions of adenylate cyclase has been to construct specific mutations of CYRl in vitro and then examine the activity of the mutant protein in vivo following introduction of the mutant gene into S . cerevisiae. By this assay, almost any lesion between a site 100 amino acids upstream of the leucine-rich domain to the catalytic domain diminishes the Ras-mediated GTP dependence of the protein (M. Wigler, personal communication). Finally, Wigler and colleagues have used an in vivo-competition assay as a means of defining potential Ras interaction sites within adenylate cyclase. They examine the degree to which expression of a specific portion of the CYRl gene can alleviate RAS2vd'g hyperstimulation of wildtype adenylate cyclase in vivo. Expression of any fragment encompassing the leucine repeats appears sufficient to provide competition (M. Wigler, personal communication). Thus, the leucine repeat region may constitute a Ras protein-binding domain, but other portions of the molecule are required as well for Ras-mediated modulation. Mutations in CYRl have been isolated based on their ability to suppress various ras mutants. De Vendittis et al. (1986a) isolated second-site suppressors of a ras2ts strain. One such mutation mapped to the C terminus of CYRl and changed Thr'651 to Ile'651. Several possible mechanisms could account for suppression by this mutation, including elimination of a threonine phosphorylation site, alteration of a protein-protein interaction, or activation of adenylate cyclase basal activity. A second mutation in CYRl was isolated by selecting for suppressors of a ras2 mutant defective in the putative effector domain (see earlier). This suppressor changes Asp'547to a Tyr (Marshall et al., 1988), a site that lies between the leucine repeats and the catalytic domain. In both cases, the multiplicity of mechanisms by which suppression can be explained prevent these CYRl mutations from being invoked to define the Ras interaction domain or even to document that Ras protein physically interacts with adenylate cyclase.

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3. Biochemical Characterization Limited information is available from the biochemical characterization of yeast adenylate cyclase. Heideman et al. (1987)extracted the enzyme from the yeast membranes with detergent and measured its hydrodynamic properties and activation by GTP. From these studies, it was difficult to establish whether additional proteins interact with adenylate cyclase, since activity sedimented as a very large complex of M, 594,000. Varino and Londesborough (1976) similarly found that cyclase activity was associated with a large complex of M , 450,000. Dimers of adenylate cyclase or dimers associated with additional proteins could account for this size. Trypsin treatment of adenylate cyclase stimulates catalytic activity but removes its ability to be stimulated by Ras protein. Heideman et a1. (1987)suggest a model in which cyclase activity is ordinarily inhibited by an unidentified cellular protein and Ras activation occurs by antagonizing this inhibitory factor. The interaction site for this putative negative factor would be outside the catalytic domain and would be removed by trypsin. Field et al. (1988)used an epitope addition method to purify adenylate cyclase from yeast. The CYRI gene was modified by insertion of an oligonucleotide that directs addition of nine amino acids to the amino end of the protein. This nonopeptide functions as an epitope for a previously prepared mAb. Adenylate cyclase containing this epitope was then purified using an antibody affinity column. Silver staining of a protein gel of immunopurified and glycerol gradient-purified adenylate cyclase revealed two bands of M , 200,000 and 70,000. The M , 70,000 protein does not appear to be a breakdown product of adenylate cyclase, but whether it represents a protein that functionally interacts with adenylate cyclase or merely adventitiously associates with the protein must await further analysis,

4. Do Ras Proteins and Adenylate Cyclase Directly Interact? Several lines of evidence suggest that Ras stimulation of adenylate cyclase is not mediated by a third protein, but involves direct physical interaction of the two proteins. However, none of these is conclusive. First, purified adenylate cyclase can be stimulated by purified Ras protein in vitro (Field et al., 1988). However, as noted earlier, this purified cyclase preparation contains at least one other unidentified protein, which might serve to mediate Ras activation. Second, despite extensive mutational analysis of the Ras-CAMP pathway in yeast, no gene has been identified whose product can be invoked to function as an intermediary between Ras proteins and cyclase. Third, coexpres-

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sion of the CYRl and BAS2 genes in E . coli was reported to yield functional cyclase activity (Uno et al., 1985), akhough this observation has not been duplicated (Field et al., 1988).Finally, suppression analysis and in uiuo interference assays are consistent with direct interaction (De Vendittis et al., 1986a; Marshall et al., 1988; M. Wigler, personal communication). In sum, these provide strong circumstantial, but not unequivocal, evidence for direct interaction of RAS protein and cyclase.

B. CAMP-DEPENDENT PROTEIN KINASE The yeast A kinase is a heterotetramer consisting of two identical, CAMP-binding regulatory subunits and two catalytic subunits. This complex was initially identified by examining extracts of yeast for CAMP-bindingproteins (Takai et al., 1974; Kudlicki et al., 1978).Identification of genes encoding these components of the A kinase was accomplished by various approaches (Matsumotoet al., 1982a; Cannon and Tatchell, 1987; Lisziewicz et al., 1987; Toda et al., 1987b). Matsumot0 et al. (1982a) obtained mutations in the gene for the regulatory subunit by isolating second-site revertants of cyrl-2 strains, selecting for CAMP-independent growth. These investigators proposed that mutations in a gene designated BYCl (bypass requirement for cyclic AMP), defined the regulatory subunit oTA kinase. This assignment has been confirmed by sequence analysis of the cloned gene and examination of the protein expressed from it in E . coli (Cannon and Tatchell, 1987; Toda et al., 1987a; Johnson et al., 1987). Genes encoding the catalytic subunit of A kinase have been identified in ras2 suppressor screens and as high-copy suppressors of cdc25 strains (Lisziewicz et al., 1987; Toda et al., 1987b; Cannon and Tatchell, 1987). Lisziewicz et al., (1987) isolated a gene, designated PK-25, on the basis of its ability to suppress a cdc25 mutant when carried on a high-copy vector. Sequence analysis of PK-25 revealed that it was 48% homologous to mammalian A kinase. Toda et al. (1987b) identified three separate genes, TPKl, TPK2, and TPK3, each able to suppress a cdc25 mutation when carried on yeast low-copy plasmid vectors. TPKl and PK-25 are identical genes (Lisziewicz et al., 1987; Toda et al., 198713).Cannon and Tatchell(l987) also isolated a gene for a yeast A kinase by selecting for second-site suppressors of ras2 strains (Cannon et al., 1986).One set of dominant suppressors defined a locus SRA3. On cloning and sequencing the dominant suppressor allele of SBA3, Cannon and Tatchell(l987) found it to be homologous to mammalian A kinase. The sequence of SRA3 is identical to that of

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JAMES R. BROACH AND ROBERT J. DESCHENES

T P K l and PK-25 thoroughout the majority of the coding region. However, the SRA3 sequence diverges from that of the other two genes in its 5’-flanking region and the initial several amino acids of the coding region. It is not clear whether this difference represents a cloning artifact or whether it reflects a genome rearrangement that might explain activation of the locus to yield suppressor activity. Uno et aE. (1984b) proposed that cyr2, isolated in their original screens for mutants in CAMP metabolism, encoded the catalytic subunit of the CAMP-dependent protein kinase. This has subsequently been shown to be incorrect and that cyr2 is allelic with cdc25. The three TPK genes are functionally redundant (Toda et al., 198713). Mutational inactivation of any two of the three genes has little phenotypic consequence to the cell. However, elimination of all three genes is lethal. This indicates that all three genes are expressed in yeast and that each catalytic protein has extensive overlapping substrate specificity with the other two. Mutational inactivation of the regulatory subunit (BCYI gene product) yields unrestricted A-kinase activity, resulting in a number of phenotypic consequences for the cell. These include depletion of carbohydrate reserves, sensitivity to heat shock and nutrient starvation, diminished growth capacity on nonfermentable carbon sources, and suppression of sporulation. Discussion of the rationale for these phenotypes is presented in subsequent sections. The severity of these phenotypes of bcyl strains depends on the particular allele tested, with gene disruptions yielding the most severe phenotypes and the bcyl-I allele yielding a relatively mild phenotype. In addition, low viability of bcyl strains on entry into stationary phase means that they do not store well and often acquire second-site suppressors when recovered from storage. This is a significant caveat in using these strains or in interpreting results of experiments using these strains. The interaction between the regulatory and catalytic subunits of the CAMP-dependent protein kinase has been probed by mutational analysis (Kuret et al., 1988; Levin et al., 1988). Kuret et al. (1988) constructed mutations in the regulatory-subunit gene ( B C Y I )corresponding to a domain termed the “hinge” region, at which the catalytic subunit is proposed to bind in the heterotetrameric holoenzyme. Hinge region mutants in the regulatory subunit either increase or decrease its ability to inhibit the catalytic subunit, depending on the particular residue substituted (Kuret et al., 1988).In these mutants the binding of CAMP is not markedly altered. These results are consistent with a role of hinge region in Bcyl protein in associating with the catalytic subunit. Mutations can be also selected in the catalytic

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subunit ( T P K I ) that alter its interaction with the regulatory subunit. A dominant T P K l suppressor of ra~as2~~ alters T h P 1to Ala. The biochemical consequence of this mutation is a decrease in the interaction of T P K l and BCYl by 100-fold without significant alteration of the catalytic rate (Levin et aZ., 1988). C. PHOSPHODIESTERASES The enzymatic breakdown of cAMP is accomplished by the activity of CAMP phosphodiesterases. Two enzymes carry out this function in yeast. One binds cAMP with high affinity ( K , = 0.2 p M ) and one with lower affinity ( K , = 3 pM) (Londesborough and Suoranta, 1983; Suoranta and Londesborough, 1984). The gene for the high-affinity phosphodiesterase (PDE2) and lower affinity form (PDEI) have been isolated (Nikawa et al., 198713; Wilson and Tatchell, 1988; Sass et al., 1986). Overexpression of the piosphodiesterase genes will suppress defects associated with RAS“”’ . A pdel pde2 double mutation suppresses the lethality normally associated with a rasl ras2 double mutation (Sass et al., 1986), and mutational inactivation of the highaffinity gene pde2 restores the ability of RAS2 ras2 strains to grow on nonfermentable carbon sources (Wilson and Tatchell, 1988).These results are consistent with a role for phosphodiesterases in reducing intracellular levels of CAMP. Despite these observations, cAMP concentrations do not appear to differ greatly in strains lacking PDE genes from those strains containing the genes. In addition, in one study, cAMP levels were slightly higher in pde strains that lack the R A S genes than in pde strains in which the R A S genes are functional (Nikawa et al., 1987a,b). These results can be explained by assuming that cAMP production is regulated by feedback inhibition mediated by the A kinases and that phosphodiesterases may be activated by Akinase phosphorylation. This hypothesis is elaborated in the next section (see Section V).

D. PROTEIN PHOSPHATASES One feature of a signal transduction system based on phosphorylation by protein kinases is that the reactions are reversible by protein phosphatases. In yeast, protein phosphatase activities have been biochemically described in the context of analyzing various reversible metabolic pathways (Horn and Holzer, 1987; Fosset et at., 1971; Huang and Cabib, 1974b; Ortiz et al., 1983; Hemmings, 1981; Wingender-Drissen and Becker, 1983b). Genetic characterization of

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protein phosphatases has not kept pace with biochemical characterization. Matsumoto et al. (1985b)reported isolating a mutant defective in a protein phosphatase in a selection for suppressors of cyr2. Strains with the suppressor mutation p p d l were reported to have reduced protein phosphatase activity. However, the predicted protein product of the cloned PPDl gene is not homologous to any known protein phosphatases, but rather is homologous to mammalian GAP, the GTPase-stimulating factor from mammalian cells (Tanaka et al., 1989). PPDl has been renamed IRA1 to reflect its role in regulating Ras activity, rather than in dephosphorylating proteins.

E. CDC25 Mutations in CDC25 were initially isolated as temperature-sensitive alleles that caused cell cycle arrest as unbudded cells (Hartwell, 1974; Robinson et al., 1987; Broek et al., 1987). cdc25 mutations have significantly reduced intracellular CAMP levels (Broek et al., 1987; Camonis et al., 1986; Martegani et al., 1986).The lethality of cdc25 mutations can be suppressed by RAS2va'19 mutations, which diminish the inherent GTPase activity of the Ras protein. In addition, as noted before, a deletion mutation of RAS2 that removes the C-terminal variable domain of the protein also suppresses cdc25 mutations (Marshall et al., 1987). The protein derived from this mutant RAS2 gene maintains a higher ratio of GTP to GDP bound in viuo than does the wild-type Ras2 protein (Gibbs et al., 1987). Since mutations of R A S that increase the amount of GTP bound to the protein render the cell independent of Cdc25 activity, Cdc25 protein most likely serves to activate wild-type Ras protein or maintain it in an activated state. Cdc25 protein could either promote exchange of GDP bound to Ras protein for GTP in the cytosol or inhibit the intrinsic GTPase activity of Ras. As noted later in the discussion of I R A I , the former hypothesis is more likely to be correct. Regardless of the mode of action of Cdc25, results of Powers et al. (1989) support the hypothesis that Cdc25 and Ras proteins directly interact. They isolated two dominant lethal mutations in RAS2. The effect of these mutations is reversed by overexpression of CDC25, but only in a strain carrying a normal copy of RAS2. These results are interpreted to indicate that the mutant Ras protein forms a nonproductive, irreversible complex with Cdc25 protein. Confirmation of this hypothesis has been obtained by S. Jones and J. R. Broach (unpublished observations), who find that immunoprecipitation of Cdc25 protein in a strain containing the dominant lethal R A S 2 allele yields coprecipitation of U S 2 protein.

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The CDC25 gene has been cloned by complementation and sequenced (Robinson et al., 1987; Broek et al., 1987; Lisziewicz et al., 1987; Camonis et al., 1986). The gene can potentially encode a 1589amino acid protein (Fig. 61, and immunoprecipitation with antipeptide antibody has identified a CDC25-specific 180-kDacytoplasmic protein in yeast extracts (S. Jones and J. R. Broach, unpublished observations). In the course of cloning CDC25, several investigators noted that the C terminus of the gene was sufficient for complementation (Camonis et al., 1986; Broek et al., 1987; Munder et al., 1988). This information coupled with deletion analysis suggests that only the region from nucleotide 2500 (BglII site) to the C terminus is required for CDC25 activity. Munder et al. (1988) have proposed specific functional domains of CDC25, deletion of which affects some phenotypessporulation and growth on nonfermentable carbon sources-while not affecting others, such as growth in rich medium. However, this distinction may reflect different threshold levels of CDC25 activity required for various phenotypes, rather than the existence of specific functional domains. In most cases, testing the viability of deletions agrees with the results of complementation by different pieces of the CDC25 gene. However, Lisziewicz et al. (1987)find that a C-terminal deletion from the BamHI site to the end of the gene is viable, whereas expressing the BglII-Hind111 fragment does not complement a cdc25 mutation (Ca-

CDC25

1

500

1000

1500

2000

2500

3000

3500

4764

4000

+

PLASMID COMPLEMENTATION

-2

-L - L L

MLETlONS I

I

1

V V

3

)

( (

-

3 )-

4

FIG.6. Diagram of the CDC25 gene showing the endpoints of plasmid constructs and deletions that define the functional domain of the protein (crosshatched area). The letters at the left indicate whether that deletion is lethal (L) or viable (V) to the strain. The numbers 1-4 on the right refer to references for the data presented: 1. Robinson et al. (1987);2. Broek et al. (1987); 3. Munder et al. (1988);4. Lisziewicz et al. (1987).

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monis et al., 1986). Most of the other studies of this gene, though, suggest that the carboxyl half of the protein is required for function, although the extreme carboxyl terminus may not be essential.

F. OTHERCOMPONENTS OF THE PATHWAY Several other genes have been implicated as direct or indirect components of the Ras-CAMP pathway. SCHS was isolated for its ability to complement a cdc25 mutation (Toda et al., 1988). DNA encompassing the complementing activity was sequenced and found to encode a protein homologous to A kinase. Overexpression of S C H S suppresses the loss of viability arising from inactivation of R A S , CDC25, CYRl, or all three TPK genes. Inactivation of S C H S yields yeast strains that grow but that have extended G1 periods of the cell cycle. These results have been interpreted to suggest that SCHS encodes a protein kinase that is part of a growth control pathway, which is at least partially redundant with the cAMP pathway. Z R A l , formerly PPDI (Matsumoto et at., 1985b),encodes avery large protein that includes an extended domain in the carboxyl terminus with homology to mammalian GAP (Tanaka et al., 1989; McCormick, 1989). Insertional inactivation of irul can suppress cdc25 mutations but not rasl rus2 or cyrl mutations. In addition, strains with irul insertional mutations are heat shock-sensitive, whereas the same strains carrying cdc25 as well are heat shock-resistant. These results suggest that IRA1 and CDC25 both act on Ras proteins but with opposite effects. This is most easily reconciled with the homology data and the information available on CDC25 function by suggesting that Cdc25 protein promotes CTP/GDP exchange while Ira1 protein stimulates the intrinsic GTPase activity of Ras. YAK1 is a gene identified in a selection for suppressors of T - U S mutations in a rasl backaground. Garrett and Broach (1989) found that null mutations of this locus suppress lethality associated with loss of rasl ruse function as well as with loss of all three tpk genes. The possible role of this protein in the Ras-CAMP pathway is discussed in Section V, “Targets of the CAMP-Dependent Protein Kinase.” CASl was identified as a mutation that allowed a cyrltSto grow at 35°C without cAMP (Boutelet et al., 1985). Biochemical analysis revealed that adenylate cyclase activity is altered in these strains. CASl is not allelic with RASI, RAS2 or CYRl. Its function has yet to be elucidated. The functions of a number of loci that suppress phenotypes associated with loss of ras activity (SRA genes) or hyperactivation of RAS

~ ~ ~

M S GENES IN YEAST

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(SRV or SUP genes) have not yet been clarified. These may define critical protein phosphatases or identify components of the pathway not yet appreciated. V. Targets of the CAMP-Dependent Protein Kinase

A number of yeast proteins have been shown, with varying degrees of rigor, to be in vivo targets of CAMP-dependent A kinase. These are listed in Table 111. The evidence for their assignment as substrates of A kinase and the consequences of phosphorylation on biological activity are described in the following. TABLE I11 POSSfBLE in V i V O SUBSTRATES OF THE YEAST CAMP-DEPENDENT PROTEIN I<JNASE Enzyme Trehalase Trehalose-6-phosphate synthase Glycogen phosphorylase Glycogen synthase Phosphofructokinase-2 Fructose-2,6-bisphosphatephosphatase Fructose-l,6-bisphosphate phosphatase

High-affinity glucose transporter High- and low-affinity galactose transporter Maltose transporter ADRl -Encoded transcription activator Rasl,2 proteins Adenylate cyclase High- and low-affinity CAMP phosphodiesterase

Comments Activated by A kinase Inhibited 2- to 4-fold by A kinase Phosphorylated form active; either direct or indirect substrate of A kinase Phosphorylated form inactive; either direct or indirect substrate of A kinase Activated by A kinase-catalyzed phosphorylation Low-K, enzyme inhibited by A kinase-catalyzed phosphorylation Phosphorylation by A kinase increases its sensitivity to inhibition by fructose 2,6-bisphosphate. Subject to “catabolite inactivation” as a direct or indirect effect of A-kinase activity Subject to “catabolite inactivation” as a direct or indirect effect of A-kinase activity Subject to “catabolite inactivation” as a direct or indirect effect of A-kinase activity Inhibited by A kinase-catalyzed phosphorylation Phosphorylated by A kinase; phosphorylation may inhibit activity Inhibited either directly or indirectly by A kinase-catalyzed phosphorylation May be stimulated by A-kinase phosphorylation

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Rigorous demonstration that a portein is an in uiuo substrate of the A kinase is not straightforward. Showing that a protein can be phosphorylated by ATP in the presence of CAMP in an in uitro assay is not sufficient. Such phosphorylation could be an indirect effect or the protein could serve as an adventitious substrate of the kinase in vitro even though it is not naturally an in uiuo substrate. This analysis can be further confused by the fact that some proteins are normally phosphorylated by more than one kinase. Similar caveats apply to genetic analysis. Demonstration that a protein is phosphorylated or altered in its activity in mutant strains or under conditions promoting high A-kinase activity is consistent with, but does not prove, that the protein is a substrate of the kianse. Such effects again could be indirect, as a result of substantial alterations of the physiology of the strain under the conditions examined. Ideally, assignment of kinase targets should rest on the results from a combination of genetic, physiological, and biochemical analyses. In only a few cases to date has this happy convergence been achieved. A. PROTEINS INVOLVED IN CARBON METABOLISM 1. Trehalose S ynthase and Trehelase

Trehalose, a glucose disaccharide used by yeast as a storage carbohydrate, is synthesized from glucose 6-phosphate (G6P) by trehalose-6phosphate synthase and degraded by trehalase. These two enzymes provide a fairly unequivocal example of reciprocal regulation by A kinase-catalyzed phosphorylation. Trehalase can be isolated from yeast in an inactive form, which can be activated in the presence of ATP and CAMPby a soluble yeast protein fraction containing A kinase (van Solingen and van der Plaat, 1975; Uno et al., 1983b; DellamoraOrtiz et al., 1986).These conditions yield phosphorylation of trehalase (Uno et al., 1983b; Dellamora-Ortiz et aZ., 1986).In addition, trehalase activity can be significantly stimulated in t h o by addition of glucose to acetate-grown cells (Thevelein and Beullens, 1985)-a condition that yields a rapid increase in intracellular CAMP level (Thevelein and Beullens, 1985, and later). Finally, the pattern of trehalase activity in strains altered in their expression of A kinase is consistent with the enzyme being activated by A-kinase phosphorylation. Trehelase levels are high in strains containing mutations, such as bcyl, IAC, or p d e l , that elevate A-kinase activity and low in strains containing mutations, cyrl-2 or ras2, that diminish kinase activity (Uno et al., 1983b). In a reciprocal fashion, trehalose-6-phosphate synthase is inhibited

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by A-kinase phosphorylation. Panek et al. (1987) have shown that trehalose synthase activity could be reduced in vitro under conditions promoting A-kinase activity and could be reactivated by treatment with phosphoprotein phosphatase. In addition, they showed that glucose addition to acetate-grown yeast cells yielded a rapid diminution of synthase activity and that a bcyl strain had lower synthase activity than wild-type strains, while a ras2 or a cyrl-2 strain had higher levels of activity. In sum, enhanced A-kinase activity yields degradation of stored trehalose by activating trehalase and suppressing synthase activity, while low levels of A kinase promote trehalose synthesis through inactivation of trehalase and stimulation of synthase.

2. Glycogen Synthase and Phosphorylase Glycogen in yeast is synthesized from UDP-glucose by glycogen synthase and is degraded to glucose 1-phosphate (GlP) by glycogen phosphorylase (Fig. 7). As in the situation with trehalose, the control of glycogen metabolism in yeast is effected through protein phosphorylation, in which both glycogen synthase and phosphorylase are interconverted in a reciprocal fashion between two forms, one active and one inactive, under conditions prevailing in the cell. However, the role of CAMP-dependent protein kinase in this process is still unclear. Glycogen synthase can exist in either an active, G6P-independent form, variously designated as a or I, or a relatively inactive form, termed b or D (FranCois and Hers, 1988; Rothman-Denes and Cabib, 1970). The b form can be allosterically stimulated by millimolar levels of G6P (Franqois and Hers, 1988; Huangand Cabib, 1974a).The b form Ca2+

CAW?

C W ?

AiP

FIG.7. Model for regulation of glycogen metabolism in Saccharornyces cereuisiae. Both glycogen synthase (synthase) and gIycogen phosphorylase (PLase) are interconverted between an active (a) and less active (b) form by phosphorylation and dephosphorylation. The role of glucose 6-phosphate (G6P),calcium, and CAMPin stimulating these interconversions is indicated by the dotted lines. See text for further discussion.

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JAMES R. BROACH AhTD ROBERT J . DESCHENES

is correlated with the presence of covalently bound phosphate, and a-form enzyme can be converted to b-form enzyme by incubation in yeast extracts in the presence of ATP. This in vitro conversion is not significantly stimulated by cAMP or calcium (Franqois and Hers, 1988). Conversion of b form to a form can also be catalyzed in vitro, a process that is stimulated by G6P, apparently through its allosteric effects on glycogen synthase rather than through a direct interaction with a protein phosphatase (Franqois and Hers, 1988). Glycogen phosphorylase also exists in two forms: an active (a) form and a completely inactive ( b )form (Fosset et al., 1971; Becker et al., 1983; Franqois and Hers, 1988). Activation of phosphorylase accompanies incorporation of bound phosphate (Fosset et al., 1971; Becker et aZ., 1983).Phosphorylase can be phosphorylated by CAMPdependent protein kinase from yeast as well as by a 29-kDa “phosphorylase kinase” from yeast, which is distinct from A kinase and is not stimulated by cAMP or calcium (Wingender-Drissen and Becker, 1983a; Pohlig et al., 1983). Franqois and Hers (1988)have shown that conversion of phosphorylase b to phosphorylase a can occur in yeast extracts in the presence of ATP and that conversion is stimulated $0-fold by calcium but only minimally by CAMP.Inactivation of glycogen phosphorylase can also be catalyzed in vitro and, as is the case with glycogen synthase, this process is stimulated b y G6P through an allosteric interaction (Franqois and Hers, 1988; Hwang and Fletterick, 1986), Genetic and physiological analysis of glycogen accumulation is neither straightforward nor entirely consistent with the foregoing biochemical analysis. bcgl and RAS2 activated alleles, which yield high kinase activity, promote glycogenolysis, while cgrl or ras2 mutations lead to hyperaccumulation of glycogen (Fraenkel, 1985;Tatchell et al., 1985). This would suggest that A kinase activates phosphorylase and inactivates synthase. Consistent with this, Franqois and Hers (1988) noted that inactivation of a-form glycogen synthase and activation of b-form glycogen phosphorylase occurs during harvesting of wild-type cells but that these effects are blocked in cdc35 cells. However, in direct contrast to the prediction of the foregoing genetic analysis, Franqois et al. (1988b)observed that glucose addition to starved cells, while transiently inducing cAMP accumulation, stimulated synthase activity and inhibited phosphorylase activity. These effects were completely reversed by subsequent addition of a nitrogen source or a metabolic uncoupler such as dinitrophenol (DNP). The former additive had no effect on cAMP levels and the latter, like glucose, substantially stimulated cAMP synthesis. Thus, in these experiments cAMP levels and synthase-phosphorylase activities bore no relationship.

u s GENES IN YEAST

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Obviously, metabolic regulation of glycogen synthasephosphorylase in yeast is not yet resolved. These two enzymes are clearly regulated by phosphorylation-induced interconversion. However, it is not clear whether A kinase directly acts on these enzymes in wiwo or whether the A kinase impinges on the system indirectly, by stimulating Ca2+mobilization and attendant phosphorylation by a Ca2+-dependentkinase, for example. Alternatively, as is the case in mammalian glycogen synthase, yeast synthase and phosphorylase may be regulated by more than one kinase, either independently or in a dependent order of phosphorylation. These enzymes may also be sensitive to several, potentially competing metabolic signals. For example, feeding glucose to starved cells may provoke phosphorylation through CAMP induction while concurrently provoking dephosphorylation through accumulation of G6P. Whether the outcome is glycogen synthesis or glycogenolysis would depend on the precise balance of these competing reactions. 3. Phosphofructokinase-2 and Bisphosphatase The critical step in regulating flux through the glycolytic pathway is interconversion of fructose 6-phosphate (F6P) and fructose-1,6bisphosphate (F-lY6-P2).The forward, or glycolytic, reaction is catalyzed by phosphofructokinase-1 (PFKl), and the reverse, gluconeogenic, reaction is catalyzed by fructose-1,6-bisphosphatase (F-1,6BPTase). The activities of these two enzymes are stringently and reciprocally regulated by allosteric effectors, the most relevant being fructose 2,6-bisphosphate (F-2,6-P2), which synthesized from F6P by phosphofructokinase-2 (PFK2) and is degraded back to F6P by one or more specific bisphosphatases. Fructose 2,6-bisphosphate stimulates PFKl and inhibits the activity of F-lY6-BPTase.Thus, high levels of F-2,6-P2 stimulate glycolysis and low levels promote gluconeogenesis. These reactions are diagrammed in Fig. 8. CAMP-dependent protein kinase influences the equilibrium between F6P and F-1,6-P2 by stimulating synthesis of F-2,6-P2 and enhancing the sensitivity of F-lY6-BPTaseto inhibition by F-2,6-P2. Conditions that increase cellular CAMPlevels yield activation of PFK2 and a concurrent increase in the cellular concentration of F-2,6-P2 (Noshiro et al., 1987; Franqois et al., 1984,1986).Franqois et al. (1988a) have extensively purified PFK2 from yeast and have shown that it can be phosphorylated in witro by CAMP-dependent protein kinase from beef heart. This phosphorylation stimulates activity -8fold. Two different bisphosphatases that specifically remove phosphate from the 2-position of F-2,6-P2 have been isolated from yeast (Franqois et al., 1988a; Kretschmer et al., 1987).The one with the lower K, (-0.1 pM)

116

JA,MES R. BROACH AND ROBERT J. DESCHENES

[ ............

PFK2

A.

F6P

''.

-

F 2 ,6 - B i a s e

F-&3-

pFK1

BPTase

~

F-2,B-PZ ,.<

....:."

'

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

I.--........................................

.,..'.

F-1 ,6+2

L

I GLYCOLYSIS I FIG.8. Regulation of interconversion of fructose 6-phosphate and fructose 1,6bisphosphate by fructose 2,6-bisphosphate and CAMPin yeast. See text for abbreviations and discussion of the model.

can be inactivated in uitro by phosphorylation by A kinase (Kretschmer et ul., 1987). Fructose-1,6-bisphosphatase is regulated in part by A-kinase phosphorylation. This enzyme can be phosphorylated in vitro by CAMPdependent protein kinase on a serine at position 11(Pohlig and Holzer, 1985; Rittenhouse et al., 1986). In uiuo-labeled protein contains phosphate predominantly on the same residue, supporting the conclusion that A kinase is responsible €or in uivo phosphorylation. Initial studies suggested that phosphorylated bisphosphatase was 2-fold less active than nonphosphoryiated enzyme (Pohlig and Holzer, 1985). However, subsequent studies indicate that the phosphorylated enzyme is unchanged in its maximal activity but that it is several fold more sensitive to inhibition by F-2,6-P2 than the nonphosphorylated form (Rittenhouse et al., 1987). This conclusion is confirmed by analysis of the kinetic parameters of mutant enzymes in which Ser" has been converted to either Ala or Asp. In sum, A-kinase activity affects intracellular levels of F-2,6-P2 and, accordingly, the relative activities of PFKl and BPTase. The degree to which A-kinase regulation of this step per se affects flow through the glycolytic pathway awaits analysis of the in cico consequences of mutations in PFK2 and the various BPTases that render them nonresponsive to A-kinase phosphorylation.

4. Glutamate Dehydrogenase The NAD-dependent glutamate dehydrogenase catalyzes conversion of glutamate to a-ketoglutarate and ammonia. In yeast, the reverse reaction is catalyzed by a separate, NADP-dependent enzyme. In vivo

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activity of the NAD-dependent enzyme is highest when cells are grown on glutamate. This activity is rapidly, but reversibly, lost when glutamate-grown cells are starved for glutamate (Hemmings, 1984). Studies with partially purified enzyme have shown that inactivation can be stimulated in vitro in the presence of ATP. This inactivation correlates with phosphorylation of the enzyme and is reversible by treatment with protein phosphatase (Uno et al., 1984a). Although in vitro inactivation of glutamate dehydrogenase can be stimulated by CAMP,no additional evidence is available to confirm or exclude a role for A kinase in the in vivo regulation of the enzyme. 5. Sugar Transport Systems A number of sugar transport systems, including the high-affinity glucose transporter, the high- and low-affinity galactose transporter, and the maltose transporter, exhibit a gradual loss of activity over several hours following addition of high levels of glucose to the growth medium (Gorts, 1969; Matern and Holzer, 1977; Ramos et al., 1988, 1989). For soluble enzymes that exhibit similar “catabolite inactivation,” such as F-l,g-BPTase, this gradual loss of activity results from proteolysis (Holzer, 1984). Holzer (1984) has proposed that catabolitesensitive enzymes are marked for proteolytic inactivation by protein phosphorylation, although this hypothesis has not yet been rigorously tested. Ramos and Cirillo (1989) have shown that CAMP-dependent protein kinase mediates catabolite inactivation of the galactose transporters. Catabolite inactivation of both transporters is blocked in mutants with constitutive A-kinase activity. Although these resuIts provide compelling evidence that catabolite inactivation is mediated by A kinase, it does not pinpoint the level at which that regulation occurs. It is possible that the transporters are targets of the A kinase and that phosphorylation induces eventual proteolytic degradation in a manner consistent with Holzer’s model. Alternatively, A kinase could activate a specific protease that recognizes a number of different catabolitesensitive enzymes. Another possibility is that A-kinase activity yields specific repression of transporter synthesis (see later). If the transporter proteins are normally turned over, then diminished synthesis would lead to decay in the steady-state level of the proteins. Further study is required to address the precise role of the kinase in catabolite inactivation of sugar transporters.

6. ADRl The product of the ADRl locus mediates glucose-induced repression of transcription of the ADH2 gene, which encodes one of the alcohol dehydrogenases of yeast. Evidence suggests that the product of

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the ADRl gene is a transcriptional activator whose activity, but not synthesis, is regulated in response to the presence or absence of glucose (Denis and Gallo, 1986). The possibility that this regulation is effected through A-kinase phosphorylation of the A d r l protein is suggested by examination of the nature of a mutation, ADRl-SC, that renders the protein partially refractory to inactivation by glucose. This mutation yields a single amino acid change that alters a consensus A-kinase recognition sequence (Denis and Gallo, 1986), suggesting that loss of ability to phosphorylate the protein protects it from glucose inhibition. Consistent with this hypothesis, Cherry et al. (1989) have shown that the Adrl protein can be phosphorylated in uitro by either yeast or mammalian CAMP-dependent protein kinase at the site of the ADRl-5C mutation. In addition, bcyl mutations yield reduced expression of ADH2, an effect that can be interdicted by activated alleles of the ADRl locus, such as ADRl-5C. These results serve to confirm the hypothesis that phosphorylation of Adrl protein by A kinase diminishes its ability to stimulate transcription. This provides at least one avenue for glucose repression of ADH2 expression, although other kinases may also act on ADRl and glucose may affectADH2 expression through other pathways as well (C. L. Denis, personal communication).

B. FEEDBACK REGULATION OF THE Ras-CAMP PATHWAY cAMP synthesis is subject to stringent feedback regulation by the A kinase. Recognition of this fact has significantly clarified the pattern of responses of intercellular cAMP levels to external stimuli or to mutations of various components of the pathway. Nikawa et al. (1987a) most clearly documented the existence of this feedback control by using mutant strains containing low-level, CAMP-independent A-kinase activity. These mutant strains carry insertional disruptions of both bcyl, the single gene for the regulatory subunit, and two of the three redundant t p k genes, encoding the catalytic subunits. In addition, the remaining t p k gene carries a mutation that diminishes, without completely eliminating, activity of the residual catalytic subunit. This allele is designated tpkw. Since the regulatory subunit is completely inactive in this strain, the residual attenuated A kinase is completely refractory to modulation by CAMP. The steady-state level of cAMP in these strains is 1000-to 10,000-fold higher than in the corresponding bcyl TPKi strains. Thus, loss of in uieo A-kinase activity yields a dramatic increase in intracellular cAMP levels. These results provide compelling evidence that synthesis of CAMP is vigorously suppressed

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by A-kinase activity. This conclusion is supported by additional observations reported by Nikawa et al. (1987a). The component or components of the Ras-CAMP pathway that are sensitive to inhibition by phosphorylation by A kinase have not been identified. Although both Cdc25 protein and adenylate cyclase contain consensus A-kinase phosphorylation sites (Arg-Arg-X-Ser),neither protein has yet been shown to be phosphorylated by the A kinase, either in vivo or in vitro. Nonetheless, De Venditiss et al. (1986a)have obtained evidence suggesting that phosphorylation of adenylate cyclase may play a role in feedback regulation. These investigators isolated a second-site suppressor of a r ~ s 2 allele. ~' This suppressor mutation mapped to CYRl and, by sequence analysis, altered a consensus A-kinase phosphorylation site. This result is consistent with the hypothesis that A kinase-catalyzed phosphorylation of adenylate cyclase diminishes its activity. The activity of the Ras proteins themselves may be regulated by phosphorylation. Resnick and Racker (1988)have shown that purified Ras2 protein can be phosphorylated in vitro by mammalian A kinase and that phosphorylated Ras protein is -2-fold less active in stimulating adenylate cyclase in vitro than is dephosphorylated protein. In addition, as noted in an earlier section, both Rasl and Ras2 proteins are phosphorylated in vivo (Sreenath et al., 1989; Cobitz et al., 1989). Rase protein appears to be phosphorylated at two sites, at least one of which may be catalyzed by A kinase. Despite these observations, the extent to which phosphorylation of Ras mediates all or part of A kinase-promoted feedback of CAMPproduction still remains an open question. Regardless of the precise mechanism of feedback regulation, activated alleles of RAS2 appear to be able to ove%ide this regulatory process to some extent. Strains containing RAS2"' alleles are capable of yielding as high an intercellular level of CAMP,at least in a pdel pde2 background, as are the bcyl tpkWstrains already described (Nikawa et al., 1987a). Unclear as yet is whether this suppression of feedback inhibition results from diminished phosphorylation of Ras2 protein or from the ability of the activated protein to circumvent phosphorylation-induced inactivation of Cdc25 or adenylate cyclase. The extent to which inhibition of CAMP turnover contributes to feedback control of CAMPlevels has not been seriously addressed to date. In mammalian cells, CAMPphosphodiesterase activity is stimulated substantially by A kinase (Grant et al., 1988). Indirect evidence suggests that turnover of CAMPin yeast is also stimulated by A-kinase activity. As noted before, Nikawa et al. (1987a)found that CAMPlevels

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were extremely high in strains with very low-level constitutive Akinase activity, even though the strains were PDEl PDE2. Similar levels of CAMP were attained in RAS2va'1" strains that carried wild-type A-kinase genes, but only if the strains were also pdef pde2. Thus, high levels of cAMP can be achieved when A-kinase levels are low, regardless of the presence of intact phosphodiesterase genes. When kinase levels are high, high levels of cAMP accumulate only when the phosphodiesterases are genetically inactivated. In conflict with these observations, the single direct experiment to address the ef'fects of A kinase on phosphodiesterase activity failed to document any alteration of phosphodiesterase activity as a function of A-kinase levels (Nikawa et al., 1987a).To explain this discrepancy, Nikawa et al. (1987a) suggested that the rate of production of cAMP in a bcyl tpkW strain could be qualitatively more rapid than in RAS2va1Lg strains. An alternative explanation is that yeast phosphodiesterases do, in fact, require A-kinase phosphorylation to be activated, but that the extraction or assay procedures used in the single experiment to test this hypothesis masked this requirement. Finally, it is possible that yeast cells possess other mechanisms to reduce cAMP levels, such as specific export, and that these other decay processes are stimulated by A kinase. Clearly, this issue requires further investigation.

C . PROTEINS INVOLVED IN GROWTH CONTROL 1. Transcription Factors

The transcription pattern of yeast cells is dramaticalIy influenced by the level of A-kinase activity. First, as noted before, the activity of the transcription factor encoded by ADRl appears to be inhibited by A kinase-catalyzed phosphorylation, resulting in reduced synthesis of alcohol dehydrogenase encoded byADH2. Second, a number of stressrelated proteins, such as polyubiquitin, whose synthesis is enhanced by heat shock or nutrient starvation, are produced at high levels in cyrl-2 strains and are not inducible in bcyl strains (Tanaka et al., 1988; Shin et al., 1987b). For the UBZ4 gene, which encodes polyubiquitin, this effect appears to be exerted at least in part at the level of transcription through a mechanism independent of heat shock induction. Finally, transcription of a number of growth-specific genes-including a number of genes encoding ribosomal proteins-is substantially repressed under conditions that deplete intracellular cAMP levels (S. Silberberg and J. R. Broach, unpublished observations).

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The mechanisms by which cAMP and A-kinase levels affect transcription patterns in yeast cells has not been elucidated. The bacterial paradigm, which would posit a CAMP-binding protein-such as Bcyl protein-as a CAMP-dependent transcription factor, most likely does not apply in this case. In mammalian cells, cAMP effects on transcription are mediated through sites, designated CRE, in enhancer domains of CAMP-regulated genes. These elements apparently serve as binding sites for transcription factor(s) whose activity is modulated, directly or indirectly, by A kinase-induced phosphorylation (Yamamoto et al., 1988; Montminy et al., 1986). Whether this same situation attends in yeast awaits further dissection of CAMP-responsive yeast promoters and identification and characterization of the factors that interact with them. 2. lnositol Phospholipid Synthesis The role of the Ras-CAMP pathway in stimulating inositol phospholipid turnover in yeast is unclear. Kaibuchi et al. (1986) reported that refeeding glucose to glucose-depleted cells causes a rapid and marked stimulation of phosphate incorporation into phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), and phosphatidylinositol bisphosphate (PIP2). These investigators19notedthat in strains activated for A kinase, such as bcyl and RAS2""' strains, as well as in those diminished for A kinase, such as rasl and ras2 strains, this glucose stimulation of phosphate incorporation into inositol phospholipids was enhanced. On this basis, they concluded that glucose stimulation of PI metabolism was mediated in part by Ras through a mechanism independent of cAMP production. This hypothesis was proposed even though none of the mutations examined eliminated glucose stimulation of PI turnover, nor were the enhancements resulting from mutation of the Ras-CAMP pathway particularly dramatic. In conflict with the conclusions of this study, Uno et al. (1988; Ishikawa et al., 1989) reported that PI kinase and PIP kinase were stimulated by A kinase. They found that activity of these two enzymes was significantly higher in a bcyl strain than in a wild-type strain and was significantly lower in a ras2 strain. Finally, in contrast to both these reports, Holland et al. (1988) noted that the activity of PI kinase isolated from glucosegrown cells was essentially unaffected by treatment with either protein kinase A or by protein phosphatase. Phosphatidylinositol kinase isolated from cells growing exponentially on glycerol as carbon source could be stimulated somewhat by treatment with phosphatase and inhibited 2-fold by treatment with A kinase. This effect is the

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opposite of that suggested by the results reported by Uno et al. (1988). What is obvious from these observations is that the role, if any, of Ras-CAMP in modulating PI turnover is currently unresolved.

3. YAM Garrett and Broach (1989) have identified a gene, YAKl, whose inactivation suppresses growth arrest resulting from complete loss of A-kinase activity. As noted earlier, strains containing insertional mutations in all three T P K genes are unable to grow. However, such strains that also carry an insertional mutation within YAKl are restored for growth. This epistasis places Y A M activity either downstream from A kinase in the Ras-CAMP pathway for growth control or on an alternate pathway that can compensate for loss of A-kinase activity. Sequence analysis of YAKl strongly suggests that it encodes a protein kinase, in the CDC28 family (Garrett and Broach, 1989). However, unlike SCHS, the overexpression of which suppresses loss of T P K function (Toda et al., 1988),loss of the Yakl kinase suppresses loss of T P K function. One explanation for these results is that Yakl kinase is itself a target of A-kinase phosphorylation and that such phosphorylation functionally inactivates the Yakl kinase. Yakl kinase in this scenario would serve as a negative regulator for growth. A second possibility is that Yakl kinase has overlapping specificity with A kinase for a number of target proteins involved in mitotic growth. In this model, Yakl and A kinases would have opposite effects on the activity of the target proteins. Thus, inactivating either T P K - or YAK-encoded kinase alone would, respectively, diminish or enhance the activity of the target proteins, while inactivating both would be compensatory and have an essentially neutral effect on the activity of the proteins. An example of such a scheme is the regulation of cdc2 protein in Schizosaccharomyces pombe, the activity of which is inhibited by a protein kinase encoded by the wee1 gene and stimulated by the product of the cdc25 gene (Russell and Nurse, 1987). In either case, further analysis of the roIe of YAK1 in growth control should serve to pinpoint the relevant targets of the A kinase that affect the cell’s decision to enter the mitotic cell cycle. VI. To What Signals Do RAS Genes Respond? A. CARBON SOURCE Addition of glucose or any related fermentable sugar to yeast cells grown on a nonfermentable carbon source, starved for glucose, or arrested in stationary phase yields a dramatic spike in intracellular

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cAMP concentration (van der Plaat and van Solingen, 1974; Mazon et al., 1982; Franqois et al., 1988b; Mbonyi et al., 1988). Peak cAMP levels, which are generally 10- to 50-fold above preaddition concentrations, are attained within 0.5-2 min after addition. cAMP concentrations then precipitously decline to a basal value only -2-fold higher than preaddition level. As described later, the glucose-induced rise in cAMP levels is, most likely, a consequence of Ras-mediated stimulation of cAMP synthesis. The rapid decline in concentration can be attributed to A kinase-mediated feedback on cAMP production, effected through inhibition of synthesis and probably through stimulation of breakdown as well (Nikawa et al., 1987a). Initial studies on glucose induction of cAMP levels noted that glucose addition to yeast cells results in rapid membrane depolarization and a decrease in intracellular pH from -7.2 to 6.5 (Purwin et al., 1986; Valle et al., 1986). An even more pronounced stimulation of cAMP accumulation could be obtained by treatment of yeast cells with metabolic uncouplers, such as DNP and carbonyl cyanide rnchlorophenylhydrazine (CCCP), which also yield depolarization and rapid reduction of intracellular pH (Purwin et al., 1986; Franqois et al., 1988b). From these observations several investigators proposed that glucose induction of CAMP accumulation was a direct consequence of the differential pH optima of adenylate cyclase (pH 6) and cAMP phosphodiesterase (pH 8) (Purwin et al., 1986; Valle et al., 1986). Lowering the intercellular pH would tend to shift the balance of CAMP production/decay toward synthesis. Subsequent experiments, though, have shown that reduction of intracellular pH and induction of cAMP levels are not strictly correlated and that glucose can induce cAMP accumulation without necessarily lowering intracellular pH (Eraso et al., 1987; Thevelein et al., 198713). Similarly, investigators have found that neither changes in membrane potential nor increases in ATP levels in the cell correlate with cAMP induction (Thevelein et al., 1987a). Recent experiments are consistent with the hypothesis that the Cdc25-Ras pathway mediates glucose stimulation of cAMP accumulation. Mbonyi et al. (1988) reported that genetic inactivation of rasl and rag2 abolished glucose stimulation of CAM: levels. Similarly, a number of investigators have noted that RAS2""' strains, while maintaining a higher basal cAMP level than RAS2 strains, exhibit a substantially reduced spike in cAMP concentration on addition of glucose (Mbonyi et al., 1988; M. Fedor-Chaiken and J. R. Broach, unpublished observations; S. Cameron and M. Wigler, personal communication). These results can be explained by assuming that addition of glucose

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stimulates Ras activity, either by increasing CDC25-catalyzed GTP/ GDP exchange or by inhibiting Ras protein’s inherent or I R A I stimulated GTPase activity. One report suggesting that glucoseinduced stimulation of CAMP accumulation is blocked in various cdc25 strains provides confirmation of this general model, although it does not distinguish between the two possible modes of action (Van Aelst et al., 1988). The precise mechanism by which glucose addition influences Ras activity is currently unknown. One can envision models in which addition of glucose yields increased concentrations of intermediary metabolites, such as G6P, that could serve as allosteric effectors of Cdc25 or Ira1 protein. In this light, Beullens et al. (1988) has shown that glucose-stimulated cAMP accumulation requires at least one functional hexokinase, either HXK1, HXK2, or GLK1, while fructosestimulated cAMP accumulation requires either HXKl or HXK2. This requirement of sugar phosphorylation for sugar stimulation of cAMP production appears not be related simply to sugar uptake (Mbonyi et nl., 1988). In alternative models, cAMP stimulation could involve alterations in intracellular pH or changes in membrane potential. Situation of adenylate cyclase and Ras protein on the plasma membrane could render them or their interactions sensitive to alterations in membrane potential. In addition, interaction of either of these proteins with each other or with other protein modulators of the pathway could readily be influenced by changes in intracellular pH. These two possibilities certainly do not exhaust the various models that can currently be proposed to explain the glucose effect on CAMP. Also, it appears that addition of DNP and glucose may act synergistically on accumulation of CAMP (Franqois et al., 1988b). Accordingly, more than one mechanism may be involved in stimulating CAMP production, so that attempts to identify a single causal pathway may prove futile. Decline in cAMP levels following the initial boost in concentration upon glucose or DNP addition can be attributed, at least in part, to feedback inhibition of cAMP production catalyzed by A kinase (Nikawa et al., 1987a). This is most dramatically documented by following cAMP production upon glucose stimulation of a bcyl tpkW strain (see Section V). In this case, cAMP levels begin to accumulate as in a wild-type strain, but, instead of declining after a short interval, they continue to increase essentially indefinitely, attaining concentrations several thousand times that of the preinduction levels (Nikawa et al., 1987a; S. Cameron and M. Wigler, personal communication). As noted before, whether this indefinite increase is due to loss of induction of breakdown of CAMP as well as to loss of repression of synthesis is not yet clear.

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Finally, it should be noted that although glucose-mediated stimulation of CAMP and A kinase induces diverse and extensive alterations in the metabolic and transcriptional state of the cell, this pathway is not responsible for catabolite repression of most glucose-sensitive genes. Matsumoto et al. (1982b) showed that glucose repression of GALl/ GAL10 gene expression was unimpaired in a cyrl background. Rather, except for ADRl-mediated glucose repression of ADH2 as described earlier, most of the transcriptional effects of glucose are mediated by the Snf 1 kinase pathway.

B. NUTRIENT SUFFICIENCY Initial examination of the various phenotypes associated with loss or activation of Ras function in yeast suggested that the Ras-CAMP pathway mediated the cell's response to nutrient availability. However, subsequent studies have failed to confirm these initial interpretations and current evidence indicates that the Ras-CAMP system may not be involved in nutrient response. Yeast cells that enter stationary phase as a result of depletion of nutrients in the media or exponentially growing cells that are shifted to media depleted of either a nitrogen, sulfate, or phosphate source eventually arrest as unbudded cells (Pringle and Hartwell, 1981). In this arrested state, cells are more resistant to heat shock and can retain viability for extended periods. In addition, diploid cells can sporulate, but only when transferred to media lacking both nitrogen and a fer~ ~ , or cdc2SSstrains similarly mentable carbon source. rasl r u ~ 2cyrlts, arrest as unbudded cells when shifted to their nonpennissive temperature, even in media replete with all required nutrients (Toda et al., 1985; De Vendittis et al., 1986a; Pringle and Hartwell, 1981). Also, homozygous diploid mutants sporulate even on medium containing excess nutrientsJToda et al., 1985; Matsumoto et al., 1983).In contrast, bcyl or RAS2'"' strains do not arrest as unbudded cells, even when their growth medium is depleted of a nutrient. Rather, the distribution of budded and unbudded cells remains essentially unaltered following depletion and cells begin to lose viability with prolonged incubation under these starvation conditions (Toda et al., 1985; Matsumoto et al., 1983).Also, diploid bcyl or R A S ~ " " ' ~mutant ~ strains fail to sporulate in response to nutrient limitation. These epistasis relationships between nutrient availability and mutations in components of the Ras-CAMP pathway suggested a causal connection. Nutrient sufficiency was presumed to induce high CAMPlevels, which in turn prompted cell cycle initiation, while nutrient starvation caused reduced CAMP levels, which imposed arrest at the beginning of the cell cycle.

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Little evidence in support of this model has appeared. Nutrient availability does not correlate particularly well with intracellular cAMP levels. Although refeeding sulfate to sulfate-starved cells has been reported to yield an increase in intracellular cAMP pools (Shin et al., 1987a), other investigators have found that refeeding nitrogenous compounds to nitrogen-starved cells yields almost no detectable alterations in cAMP levels (FranGois et al., 1988b). This is true even though such refeeding schemes yield significant changes in biochemical and transcriptional patterns of the cell in a manner similar to that found following addition of glucose to carbon-starved cells. Freese and colleagues have examined the signals associated with induction of sporulation. They have observed that initiation of sporulation under a variety of conditions most closely correlates with diminution of intracellular GTP pools and not with reduction in cAMP levels (Varrna et al., 1985; Olempska-Beer and Freese, 1987). In addition, sporulation can be efficiently induced by depletion of intracellular guanine nucleotide pools even though cAMP levels remain high under the particular conditions used (Olempska-Beer and Freese, 1987). These results suggest that reduction of CAMP is not a prerequisite for sporulation and that sporulation can occur even when CAMP levels are elevated. Closer inspection of the behavior of bcyl strains upon nutrient depletion suggests that its effect can be explained without postulating a direct role of the gene in growth control. The failure of bcyl cells to accumulate as unbudded cells upon nutrient limitation has been tacitly assumed to indicate that bcyl cells ignore cell cycle arrest signals and continue mitotic growth even after nutrient depletion. This is assumed to lead to eventual depletion of internal reserves and cessation of growth at random points in the cell cycle. In contrast to this expectation, C. Mann (personal communication) found that, rather than continuing to cycle following nutrient depletion, bcyl cells stop growing immediately following a shift to nutrient-free medium. Thus, the random-arrest phenotype can be explained by the failure of a bcyl cell to complete its current cell cycle rather than by a failure to arrest at GI. This observation is not unreasonable when we consider that bcyl cells do not maintain normal levels of storage carbohydrates. Unlike wild-type cells, bcyl cells may not have the metabolic reserves that must be mobilized to complete a cell cycle following nutrient deprivation. Regardless of the explanation, this observation precludes invoking the behavior of bcyl strains as confirmation of a role for the Ras-CAMP system in mediating nutrient control of cell cycle arrest. Also counter to the postulated role of Ras-CAMP in mediating

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nutrient response, Cameron et ul. (1988) observed that strains expressing low-level, constitutive A-kinase activity ( b c y l typk" strains) respond normally to nutrient limitation, even in the absence of both R A S gene, C Y R l , or CDC25. These b c y l tpk" strains grow with only a slightly diminished growth rate. They acquire heat shock resistance but, like wild-type cells, only upon entry into stationary phase. Similarly, homozygous b c y l tpk" strains form spores only when transferred to medium lacking both nitrogen and a fermentable carbon source. Also, these cells change their glycogen content to reflect growth conditions in the same pattern as wild-type cells, Finally, as suggested earlier, all of these growth-coupled changes in physiology occur even if RAS, C Y R l , or CDC25 genes are mutationally inactivated. Thus, yeast cells are capable of responding to nutrient limitation even in the absence of a regulated Ras-CAMP signaling pathway. This would suggest that if the Ras-CAMP pathways plays a role in mediating nutrient starvation response, an efficient alternate pathway also exists. How can these observations be reconciled with the epistasis results described before? One possibility is that the cell's decision to enter or not enter the cell cycle is based on input from several different sensing pathways. The cell would use some integrated average of these various input signals to judge whether to initiate a cell cycle. If the input signal from the Ras-CAMP pathway were at one extreme or the other, as a result of its mutational inactivation or hyperactivation, then this decision-making process could be rendered oblivious to any other signaling pathways. That is, M S - cells would have such a low signal from the cAMP pathway that signals from other pathways indicating normal nutrient levels would go unhee4:d and the cell would arrest. In the reverse situation, a b c y l or RASVa' mutation would induce such a strong, positive signal from the cAMP pathway that signals from other pathways indicating insufficient nutrients would be ignored and the cell would fail to arrest. Finally, if the cAMP pathway sends a neutral signal, as would be the case in a b c y l tpk" background, then the cell would arrest or grow on the basis of input from the other sensing pathways. C. OTHERSIGNALS The possibility that other metabolic or extracellular signals impinge on the Ras-CAMP pathway has not been explored. As noted, metabolic uncouplers that lower intracellular pH and depolarize the plasma membrane can induce cAMP levels. This route to cAMP activation could well be exploited by other, more natural agents. Another possi-

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bility is that Ras-CAMP activity could be sensitive to the level of S-adenosylmethionine. Should the methyl ester group on Ras protein be labile and should the demethylated protein be inactive, then Ras activity could be modulated by the rate of methylation. Similar arguments could be made concerning acylation and the lipid content of the cell. No evidence exists at the moment for any of these pathways. However, since we do not fully understand the overarching context of the Ras-CAMP pathway, we should remain open to these possibilities.

VII. What is Ras Doing? Despite the abundance of information that has accumulated pertaining to the role of Ras and cAMP in yeast, we still do not have a clear appreciation of their function in the cell. As we have observed, Ras appears to modulate the level of CAMP at least in response to the availability of fermentable carbon sources. Changes in cAMP levels in turn regulate the activity of CAMP-dependent protein kinase. This kinase affects the activities of a broad spectrum of proteins involved in carbohydrate storage, control of glycolysis, transport of nutrients, and transcription of numerous genes. The sum total of this kinase activity appears to be to enhance the metabolic activity devoted to energy production and mass accumulation of the cell. Thus, to a first approximation, the Ras-CAMP pathway appears to coordinate the disparate processes that are required for expansion of mass (sugar transport, glycolysis, production of components for protein synthesis, etc.) in response to the particular carbon source available. Can this model for Ras-CAMP function account for the various phenotypes associated with loss and with hyperactivation of the pathway? As noted earlier, low Ras-CAMP activity leads to diminished capacity for growth on nonfermentable carbon sources, enhanced sporulation in rich media, and hyperaccumulation of carbohydrate reserves. The complete loss of kinase activity yields cessation of growth even in rich media, with cells accumulating at the beginning of the cell cycle. Hyperactivation of the pathway induces depletion of carbohydrate reserves, sensitivity to nutrient starvation and heat shock, diminished capacity to use nonfermentable carbon sources, and inhibition of sporulation. As noted earlier, these effects on carbohydrate reserves can be accounted for, to a greater or lesser degree, by A-kinase regulation of the enzymes responsible for carbohydrate reserve metabolsim. More difficult to explain are the effects of Ras-CAMP on growth control and sensitivity to stress.

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The effects of Ras activity on growth control could be an indirect consequence of its effects on energy metabolism and transcription. Diminished Ras activity tends to constrict glycolysis and promote deposition of carbohydrates in reserve compounds. In addition, these conditions also restrict expression of genes required for growth, such as those encoding ribosomal proteins. This crippled ability for energy metabolism and expression of critical growth-related genes could simply reduce the ability of the cell to increase its mass. Attainment of a critical “size” appears to be an essential component of executing the first step in the cell cycle, designated “start” (Pringle and Hartwell, 1981). Thus, the reduced metabolic capacity of the cell resulting from reduce Ras activity could account for its arrest at the beginning of the cell cycle. Consistent with this hypothesis is the fact that other restrictions in glycolysis or protein synthesis yield the same terminal phenotype as TUS- cells. CDCIS, mutations of which yield arrest at “start,” encodes pyruvate kinase, and CDC33, which is also required for completion of “start,” encodes a translation initiation factor (Pringle and Hartwell, 1981; Brenner et al., 1988). Similarly, cells treated with low levels of cycloheximide or grown on very low levels of glucose arrest as unbudded cells (Pringle and Hartwell, 1981; Kaibuchi et al., 1986). These observations suggest that cell cycle control by Ras could be an indirect effect of Ras regulation of metabolism. This metabolic model can account for the apparent altered growth control arising from overexpression of the Ras-CAMP pathway as well. As noted before, the failure of bcyl strains to arrest following depletion of nutrients may well be due to a failure of these cells to accumulate nutrient reserves rather than an inherent obliviousness to signals for cell cycle arrest. In addition, high unmodulated levels of cAMP could lead to metabolic imbalance. That is, increased A-kinase activity promotes high transcription rates of ribosomal protein genes, generating a high demand for energy and nutrients to accommodate this enhanced level of metabolic activity. This demand might only be met by growth on glucose, rendering the cell sensitive to growth on other, less readily metabolized carbon sources. Superimposed on this metabolic role of Ras-CAMP in yeast, RascAMP may also be involved in regulating transition between mitotic growth and a quiescent Go state. Evidence has accumulated to suggest that quiescent yeast cells attain a physiologica1 state that is distinct from any stage of the mitotic cycle. Quiescent cells synthesize a subset of proteins that are not made during mitotic growth and, conversely, turn off synthesis of certain growth-specific proteins, the synthesis of which does not cycle during mitotic growth (Iida and Yahara, 1984a,b;

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Shin et ul., 1987a).Thus, the transcription pattern of quiescent cells is quite distinct from that of cells at any stage of the mitotic cycle. In addition, quiescent cells exhibit physiological properties-such as tolerance to stresses like heat shock-that are distinct from those of cycling cells (Cannon and Tatchell, 1987; Francois et al., 1988a). Finally, Derbot et ul. (1987) isolated a mutant that grows normally but fails to emerge from quiescence. Thus, consistent with the view of Go as a distinct state, emergence from quiescence requires genetically defined components that are not required for mitotic growth. How might US-CAMP be involved in the transition between Go and GI? Populations of cells growing slowly have a higher level of resistance to heat shock than do rapidly growing cells, and the percentage of survivors of heat shock correlates well with the percentage of unbudded cells in a population. One way to account for this is to suggest that the thermoprotective Go state can be attained only by cells in GI, but that access to this state is available to the cell at any point in GI between cytokinesis and “start.” We would suggest that a cell traversing GI continues to do so unless it is “bumped” into the Go state by any one of a number of different insults. These would include heat shock, ethanol exposure, nutrient depletion, carbon starvation, and diminished protein synthesis. Consistent with this view, Johnston and Singer (1980) noted that heat shock induced accumulation of unbudded cells. We would further suggest that reduced CAMP levels also serve as one of the incentives to disembark from the mitotic cycle and enter Go. This would account for the arrest phenotype andJhermotolerance of rus- strains. The sensitivity of bcyl and ZUS2’”’ strains to nutrient deprivation or heat shock can be explained in this model by suggesting that they are denied access to Go as a consequence of the high CAMP levels they maintain. Just as described in the previous section, this extremely high signal input from the CAMP pathway would “swamp out” any signals from other pathways prompting entry into Go. Accordingly, even bcyl cells traversing GI at the time of heat shock would not be able to gain entry to Go, rendering them sensitive to the treatment. The sporulation phenotype of mutants in the Ras-CAMP pathway can also be explained in this context. If we assume that sporuiztion can be initiated only from the Go state, then bcyl and RAS’”’ strains, which cannot enter Go, would be incapable of sporulating. On the other hand, rus- strains that precociously enter Go would be in a position to initiate sporulation even in the presence of excess nutrients. As several investigators have noted, the relatively low efficiency of sporulation of rus2 or cyrl diploids in rich media and the abundance of two-spored asci upon sporulation resembles sporulation of

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wild-type cells depleted for carbon source (Matsumoto et al., 1985a). This is consistent with the idea that Ras-CAMP mediates a signal for carbon source availability. This model does not address the mechanism by which the decision to enter Go is effected. One could envision the existence of a central processor that integrates input signals and regulates the battery of genes and proteins whose changes yield the distinct Go phenotype. Alternatively, the transition to Go could occur simply as the accumulated changes in a number of different metabolic, physiological, and transcriptional processes, each sensitive to regulation by CAMP,heat shock, nutrient availability, and other influences. At least some evidence is consistent with this latter view (Tanaka et al., 1988). Does RAS have a role in the cell aside and apart from its ability to activate adenylate cyclase? As noted earlier, Kaibuchi et a2. (1986) have suggested that Ras proteins affect phosphoinositide metabolism independent of their role in CAMPmetabolism, although a consistent story has yet to emerge from analysis of this system. Wigler and colleagues have noted that cyrl strains are capable of growth, albeit extremely slowly, while rasl rus2 strains never grow. This might suggest that R A S genes are required for viability in some process not mediated by CAMP. Consistent with this hypothesis, these investigators have noted that in particular bcyl tpk" strains, the presence or absence of R A S genes can affect the residual heat shock phenotype of the cell (M. Wigler, personal communication). Thus, the possibility still exists that Ras proteins function in some capacity in addition to their role as modulators of adenyalte cyclase. In sum, activation of RAS proteins appears to be promoted by the availability of fermentable carbon sources in the medium. In response to this activation, a panoply of metabolic and transcriptional changes occur, mediated by phosphorylations catalyzed by CAMP-dependent protein kinase. These changes accelerate the metabolic activity of the cell and stimulate mass accumulation. Thus, Ras protein activity serves to coordinate a plethora of disparate processes to yield the balanced pattern of growth essential for viability. As noted in this review, though, substantial research is required to test critically this speculative model of RAS function and to clarify many of the steps associated with this pathway.

ACKNOWLEDGMENTS We are very gratefuI to our numerous colleagues who generously supplied us with information prior to publication. We are also indebted to S. Garrett, S. Jones, S. Silberberg, and M. Fedor-Chaiken for stimulating discussions and critical comments on the

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manuscript. We would like to thank Dr. Sung-Hou Kim for providing us with original figures from his publication and permitting us to use them, and Dr. Sara Jones for creating several of the figures presented in this review. Work from this laboratory described in this review was supported by grant CA41086 from the National Institutes of Health.

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Tabin, C. J., Bradley, S. M., Bargrnann, C. I., Weinberg, R. A., Papageorge, A. G., Scolnick, E. M., Dhar, R., Lowy, D. R., and Chang, E. H. (1982).Nature (London)300, 143- 149. Takai, Y., Yarnamura, H., and Nishizuka, Y. (1974).J. Biol. Chem. 249,530-535. Tamanoi, F. (1988b).Biochim. Biophys. A d a 948, 1-15. Tamanoi, F., Walsh, M.,Kataoka, T., and Wigler, M. (1984).Proc. Nutl. Acad. Sci. U.S.A. 81,6924-6928. Tamanoi, F., Hsueh, E. C., Goodman, L. E., Cobitz, A. R., Detrick, R. J., Brown, W. R., and Fujiyama, A. (1988).J. Cell. Biochem. 36,261-273. Tanaka, K., Matsumoto, K., and Toh-e, A. (1988). EMBOJ. 7,495-502. Tanaka, K., Matsumoto, K., and Toh-e, A. (1989). Mol. Cell. Biol. 9,757-768. Taparowsky, E., Suard, Y., Fasano, O., Shirnizu, K., Goldfarb, M., and Wigler, M. (1982). Nature (London)300,761-765. Taparowsky, E., Shimizu, K., Goldfarb, M., and Wigler, M. (1983). Cell (Cambridge, Mass.) 34,581-586. Tatchell, K. (1986).J . Bucteriol. 166,364-367. Tatchell, K., Robinson, L. C., and Brietenbach, M.(1985). Proc. Natl. Acad. Sci. U.S.A. 82,3785-3789. Temeles, G . L., Gibbs, J. B., D’Alonzo, J. S., Sigal, I. S., and Scolnick, E. M. (1985). Nature (London)313,700-703. Thevelein, J. M., and Beullens, M. (1985).J. Gen. Microbiol. 131,3199-3209. Thevelein, J. M.,Beullens, M.,Honshoven, F., Hoebeeck, G., Detremerie, K., Den Hollander, J. A., and Jans, A. W. H. (1987a).J. Gen. Microbiol. 133,2191-2196. Thevelein, j. M., Beullens, M., Honshoven, F., Hoebeeck, G., Detremerie, K., Griewel, B., Den Hollander, J. A., and Jans, A. W. H. (1987b).J . Gen. Microbiol. 133, 21972205. Toda, T., Uno, I., Ishikawa, T., Powers, S., Kataoka, T., Broek, D., Cameron, S., Broach, J., Matsumoto, K., and Wigler, M.(1985). Cell (Cambridge, Mass.) 40,27-36. Toda,T., Cameron, S., Sass, P., Zoller, M., scott, J. D., McMullen, B., Hurwitz, M., Krebs, E. G., and Wigler, M.(1987a).Mol. Cell. Biol. 7, 1371-1377. Toda, T., Cameron, S., Sass, P., Zoller, M.,and Wigler, M. (1987b). Cell (Cambridge, Mass.) 50,277-287. Toda, T., Cameron, S., Sass, P., and Wigler, M.(1988). Genes Dev. 2,517-527. Tong, L., De Vos, A. M., Milbum, M. V., Jancarik, J., Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E., and Kim, S.-H. (1989).Nature (London)337,90-93. Trahey, M., and McCormick, F. (1987). Science 238,542-545. Trahey, M., Wong, G., Halenbeck, R., Rubinfeld, B., Martin, G. A., Ladner, M., Long, C., crosier, W. J., Watt, K., Koths, K., and McCormick, F. (1988).Science 242,1697-1700. Tsuchida, M., Ohtsubo, E., and Ryder, T. (1982). Science 217,937-939. Uno, I., Matsumoto, K., and Ishikawa, T. (1983a).J . Biol. Chem. 258,3539-3542. Uno, I., Matsumoto, K., Adachi, K., and Ishikawa, T. (1983b).J. Biol. Chem. 258,1086710872. Uno, I., Matsumoto, K., Adachi, K., and Ishikawa, T. (1984a).J. Biol. Chem. 259, 12881294. Uno, I., Matsumoto, K., Adachi, K., and Ishikawa, T. (1984b).]. B i d . Chem. 259,1250812513. IJno, I., Mitsuzawa, H., Matsumoto, K., Tanaka, K., Oshima, T., and Ishikawa, T. (1985). Proc. Natl. Acad. Sci. U S A . 82,7855-7859. Uno, I., Mitsuzawa, H., Tanaka, K., Oshima, T., and Ishikawa, T. (1987). Mol. Gen. Genet. 210, 187-194.

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RETROVIRAL INTEGRATION IN MURINE MYELOID TUMORS TO IDENTIFY EVI-7, A NOVEL LOCUS ENCODING A ZINC-FINGER PROTEIN N. G. Copeland and N. A. Jenkins Mammalian Genetics LaboratoryBRI-Basic Research Program.NCI-Frederick Cancer Research Facility,Frederick,Maryland 21701

I. Introduction 11. RI Mouse Strains

111.

IV. V. VI. VII.

A. Models for Studying the Molecular Genetic Basis of Neoplastic Disease B. AKXD RI Strains Identification of a New Common Viral Integration Site, Eui-1, in AKXD Myeloid Tumors A. Mapping of Evi-1 to Mouse Chromosome 3 B. Detection of Eui-l Rearrangements in Primary NFS/N Myeloid Tumors and Cell Lines C. Location and Orientation of Proviruses Integrated in the Eui-l Locus D. Eui-I Encodes a Zinc-Finger Protein that is Evolutionarily Well-Conserved Relationship of Evi-1 to Other Zinc-Finger Proteins Activation of Transcription of Eui-l by Viral Integration in Fim-3 Additional Zinc-Finger Proteins Implicated in Neoplastic Disease Conclusions References

I. Introduction Retroviruses that do not carry oncogenes induce disease with long latency periods in susceptible hosts. They do not readily transform cells in culture. Over the last few years it has become apparent that these viruses induce disease by the insertional activation of cellular protooncogenes (reviewed by Nusse, 1986). The genes activated by viral integration may represent either known or novel cellular protooncogenes. Thus, these retroviruses serve as useful “retrotransposon tags” for identifying and cloning genes involved in the disease process. Probes to known genes can be used in Southern blot analysis to determine whether they are rearranged by viral integration in tumors. If rearrangements are detected, this provides evidence that the gene is causally associated with tumorigenesis. Novel protooncogenes can be identified by searching for proviral integration sites that are shared in common among two or more independent tumors. Since retroviruses 141 ADVANCES IN CANCER RESEARCH, VOL. 54

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

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N. G . COPELAND AND N. A. JENKINS

integrate a large number of sites in the genome, the probability of finding even two independent tumors containing proviruses integrated into the same chromosomal domain by random chance is exceedingly low. This finding is therefore taken as evidence to suggest that this locus encodes a gene the activation of which by viral integration predisposes cells to tumorigenesis. These loci have been termed common sites of viral integration. More than 15 common sites of viral integration sites have been identified in murine hematopoietic tumors (Table I). These common integration sites have been found to encode cell surface receptors (e.g., Fim-2), serine protein kinases ( e g , Pim-I), and tyrosine protein kinases (e.g., Lck). Recently, a new common viral integration site, Eui-l (ecotropic viral integration site l), was identified in myeloid tumors of AKXD-23 recombinant inbred (RI) mice (Mucenski et al., 1988b).Eui-l identifies a novel locus that encodes a zinc-finger protein (Morishita et aZ., 1988). Zinc fingers were first identified in the Xenopus transcription factor TFIIIA (Brown et al., 1985; Miller et al., 1985). Subsequently, zincfinger proteins have been identified and characterized in a number of species including mouse and human. Zinc-finger proteins are thought TABLE I COMMON SITESOF VIRALINTEGRATION IN MURINE HEMATOPOIETIC TUMORS Locus

Disease

Pim-l

T - c e l l lymphoma R-Cell lymphoma

Pim-2 Put-1 Fis-1

T - c e l l lymphoma T - c e l l lymphoma T - c e l l lymphoma Myeloid leukemia T - c e l l lymphoma T - c e l l lymphoma T - c e l l lymphoma T - c e l l lymphoma Myeloblastic leukemia Myeloblastic leukemia Myeloblastic leukemia Myeloid leukemia Myeloid leukemia Erythroleukemia B-Cell lymphoma

Lck Gin-1 Mlui-1 Mlvi-2 Fim-1 Fiin-2 Fim-8 Eui-1 Eci-2 Spi-1 Ahi-I

Reference Cuypers et al. (1984) Selten e t a / . (1985, 1986);Wirschubsky et al. (1986);Warren et al. (1987); Mucenski et d. (1987a); Hanecak et aZ. (1988) A. Berns (personal communication) Graham et al. (1985);Mucenski et d. (1987a) Silver and Kozak (1986) Mucenski et ~ l(1987a) . Marth et al. (1985);Voronova a n d Sefton (1986) Villemur et al. (1987) Wirschubsky et al. (1986);Mucenski et QZ. (1987a) Mucenski et at. (1987a) Sola et a!. (1986) Sola et al. (1986);Gisselbrecht et al. (1987) Bordereaux et d. (1987) Mucenski et QZ. (1988b) Buchberg et oZ. (1988);Copeland et al. (1989) Moreau-Gachelin et al. (1988) Poirier et ~ f .(1988)

RETROVIRAL INTEGRATION IN MURINE MYELOID TUMORS

143

to represent a family of DNA-binding proteins that regulate gene expression (reviewed by Johnson and McKnight, 1989).Evi-1 represents the first zinc-finger protein identified by retroviral integration and the first implication for a member of this gene family in the transformation of hematopoietic cells. In this review, we briefly describe the status of RI strains as model systems for identifying novel genes involved in hematopoietic disease, such as Evi-1. We also describe Eui-1 and discuss its relationship to other members of the zinc-finger protein family.

It. RI Mouse Strains A. MODELS FOR STUDYING THE MOLECULAR GENETIC BASISOF NEOPLASTIC DISEASE Recombinant inbred strains, derived by brother-sister mating from F1 cross of two preexisting inbred mouse strains (Taylor, 1978), represent stable segregant populations resulting from the chance reassortment of the two parental genotypes. During inbreeding, linked genes tend to remain linked and unlinked genes are randomized in the recombinant phases, so that RI strains derived from mice that differ in disease incidence or disease type provide useful models for identifying genes that affect the disease process. If the disease is retrovirally induced, the retroviruses also serve as useful retrotransposon tags for identifying novel cellular protooncogenes involved in the disease process.

the

B. A M D RI STRAINS The AKXD R I strains represent a valuable RI strain family for identifying and studying genes that affect susceptibility to lymphomas. These strains were derived by crossing mice from two inbred strains that differed significantly in lymphoma incidence: AKR/J and DBABJ. AKR/J is a prototypic highly lymphomatous mouse strain; nearly all of these mice develop T-cell lymphomas by 7-16 months of age. In contrast, the lymphoma incidence in DBAEJ mice is low. The high incidence of lymphomas in AKR/J mice is associated with the expression of two endogenous ecotropic murine leukemia virus (MuLV) loci, Emv-11 and Emv-14 (Jenkins et al., 1982). Although DBA/2J mice also carry an endogenous ecotropic provirus, Emu-3, this provirus carries a small mutation in the gag gene that inhibits its expression (Copeland et al., 1988).The low level of virus expression in

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N. C . COPELAND AND N. A. JENKINS

II)BA/2Jmice is likely to be an important factor contributing to the low tumor incidence in this strain.

1. Lymphoma Susceptibility and Disease Type Among the 23 AKXD R I strains aged to determine their lymphoma incidence, 21 strains developed tumors at appreciable frequency (Mucenski et al., 1986; et al., 1989). The two strains with low tumor incidence did not inherit either of the two expressed endogenous ecotropic MuLV loci from the AKR/J parent, which likely accounts for their low susceptibility to tumors. In contrast, the 21 high tumor incidence strains all carried one or more highly expressed endogenous ecotropic proviral loci. As expected, most of the hematopoietic tumors characterized in these strains were monoclonal and contained somatically acquired proviruses consistent with their induction by viral integration and the subsequent activation of cellular protooncogenes (Mucenski et al., 1987b; Gilbert et al., 1989). Unexpectedly, only six strains developed predominantly T-cell lymphomas like the AKR/ J parent. Eight strains developed predominantly B-cell lymphomas, whereas six strains developed both T-cell and Bcell lymphomas. One strain, AKXD-23, died predominantly of myeloid tumors. Very few myeloid tumors were identified in strains other than AKXD-23. Thus, the AKXD strains have segregated for a number of genes that affect susceptibility to lymphoma. 2. Variation in the Repertoire of Cellular Protooncogenes Activated by Viral lntegration in AKXD Lymphomas with Respect to Cell Type Eight loci previously shown to serve as common sites of viral integration in murine or rat hematopoietic tumors were screened by Southem blot analysis for evidence of virally induced rearrangement in A M D tumors (Mucenski et al., 1987a; data not shown) (Table 11). With the exception of M y b , virally induced rearrangements were detected in all loci examined. The vast majority of rearrangements were observed in T-cell lymphomas. These results suggest that the number of loci that are activated by viral integration in T-cell lymphomas is large and that the repertoire of cellular protooncogenes activated by viral integration in AKXD lymphomas varies with respect to hematopoietic cell type. The large number of AKXD tumors representing many diverse histological types should provide ideal reagents for isolating new protooncogenes involved in hematopoietic disease.

RETROVIRAL INTEGRATION IN MURINE MYELOID TUMORS

145

TABLE I1 VIRALLYINDUCED REARRANGEMENTS IN AKXD LYMPHOMAS

Tumor type"

Number of tumors analyzed

Stem Pre-B B T Myeloid Mixed

6 29 90 111 g 13

Locus Myc

l

-

Pot-1

-

-

-

5 -

1"

-

21

Fis-1

-

-

4 -

Pim-1

Pim-26

Myb

1 1 3 14

-

-

-

-

3 -

-

-

-

-

-

2 -

-

Mloi-1

5 -

Mloi-2

'Stem cell lymphomas did not contain rearrangements in the immunoglobulin heavy (IgH), immunoglobulin K light (Igrc), or T-cell receptor P chain (TP) genes and were classified in the lymphoid lineage by histopathological analysis. Pre-B-, B-, and T-cell lymphomas were classified according to their rearrangements in IgH, IgK, and TP genes (Mucenski et aZ., 1986). Myeloid tumors were classified solely by histopathological analysis. Mixed tumors displayed phenotypes of two different cell lineages. Approximately one-half of the AKXD lymphomas were scored for Pim-2 rearrangements. Displayed B-cell and T-cell characteristics.

H I . Identification of a New Common Viral Integration Site, Evi-I, in AKXD Myeloid Tumors Because the majority of cellular protooncogenes previously shown to be activated by viral integration in murine or rat lymphomas were not rearranged by viral integration in AKXD B-cell or myeloid tumors, the AKXD tumors were analyzed further with the hope of identifying novel cellular protooncogenes associated with B-cell and myeloid disease. Southern blot analysis of AKXD-23 myeloid tumor DNAs identified a common integration site that appeared to be shared by all AKXD-23 myeloid tumor DNAs (Mucenski et al., 1988b) (Fig. 1; data not shown). This common ecotropic viral integration site was designated Eui-l , Cellular sequences flanking this common integration site were subsequently cloned and unique-sequence cellular probes isolated. All AKXD lymphoma DNAs were subsequently screened for Eui-l rearrangements, using these unique-sequence probes (Table 111). Eui-l rearrangements were detected in all seven AKXD-23 myeloid tumor DNAs analyzed. Eui-l rearrangements were also detected in two other myeloid tumor DNAs, one from the AKXD-9 and one from

146

N. G . COPELAND AND N. A. JENKINS

FIG.1. Identification of the Eoi-1 common integration site in AKXD-23 myeloid tumor DNAs. High molecular weight DNA (5p g per lane) extracted from lymphomatous spleen (S) and control brain (B)was digested to completion with EcoRI and analyzed by Southern blot hybridization analysis with an ecotropic virus-specific probe (Mucenski ui al., 1988b).

the ADD-15 strain. Thus, Eui-1 rearrangements were detected in all AKXD myeloid tumors analyzed. Evi-1 rearrangements were also detected at low frequency in nonmyeloid lymphomas (Table 111). The distribution of Eui-l rearrangements among tumor types was different from that described previously for Myc, Put-1, Fis-1, Pim-1, Pim-2, TABLE 111 Eoi-l REARRANGEMENTS IN AKXD LYMPHOMAS ~

~~~~

~~

Lymphoma type" Tumor origin

AKXD"

Stem

Pre-B

B

T

Myeloid

Mixedb

116

3129

6/90

21111

919

3/13

Lymphomas were classified according to cell type as described in Table 11. One lymphoma displaying myeloid and B-cell phenotypes, one displaying myeloid and T-cell phenotypes, and one displaying T-cell and B-cell phenotypes contained Eui-1 rearrangements. ' Number of lymphomas containing rearrangementsltotal number lymphomas of each type analyzed. a

RETROVIRAL

INTEGRATION IN MURINE MYELOID TUMORS

147

Mlvi-I, and Mlvi-2 (Table II), and is consistent with the hypothesis that the repertoire of cellular protooncogenes activated by viral integration in AKXD lymphoma-leukemia varies according to tumor histogenesis.

A. MAPPINGOF Evi-1

TO

MOUSECHROMOSOME 3

To determine if Eui-l was identical to any other locus already mapped in the mouse, its chromosomal location was determined (Mucenski et al., 1988a; Buchberg et al., 1989). Evi-1 mapped 18 centimorgans (cM) distal to the carbonic anhydrase (Car) locus on chromosome 3 (Fig. 2). The chromosomal locations of several genes that might be involved in oncogenesis including the protooncogene Nrus and two growth factors, epidermal growth factor (Egf)and the 8 subunit of nerve growth factor ( N g f b ) , which had previously been assigned to chromosome 3 by somatic cell hybrid analysis, were also determined (Fig. 2). The location of Eui-1 is distinct from these loci. Eui-l is also distinct from the macrophage colony-stimulating factor (Csfm) locus (Fig. 2), which has been mapped to chromosome 3 (Buchberg et al., 1989). These results suggest that Evi-1 represents a novel locus involved in myeloid disease.

OF E v i - l REARRANGEMENTS IN PRIMARY NFS/N B. DETECTION TUMORS AND CELLLINES MYELOID

DNAs from cell lines established from myeIoid tumors that arose in virus-infected NFS/N or NFS/N hybrid mice were also screened for rearrangements in the Eui-1 locus (Mucenski et al., 1988b; Morishita et al., 1988).Among 25 interleukin-3 (IL-3)-dependent myeloid cell lines examined, five (NFS-48, NFS-58, NFS-60, NFS-78, DA-1) contained rearrangements of the Eui-l locus. Interestingly, the NFS-60 cell line also had a viral integration in the M y b locus (Weinstein et ul., 1986). This suggests that M y b and Eui-1 gene products cooperate in inducing myeloid disease. E v i - l rearrangements were not detected in two stem cell, two pre-Bcell, two B-cell, and four T-cell lines. E v i - l rearrangements were detected in DNAs of 4 of 18 primary myelogenous leukemias induced in NFS/N or NFS/N hybrid mice. These results indicate that E v i - l rearrangements are not restricted to AKXD tumor cells. The myeloid tumor cell lines provided a convenient source of mRNA for identifying the Eui-1 gene product (see later).

13 12

Es-16 SUl

Evi- I Odc-3 (Car-.Z Amy-2)

34

Es-26

39

my

43

Es-27

54 55 57

i: Fgg, Xmmv-22

Ngf& Nras- I Csfm

FIG.2. Eui-l maps to mouse chromosome 3. The chromosome represents a composite linkage map of mouse chromosome 3. The loci in small print are taken from the June 1988 linkage map of mouse chromosome 3 compiled by T. H. Roderick, M. T. Davisson, A. L. Hillyard, and D. P. Doolittle, the Jackson Laboratory (personal communication). The loci mapped, as described in the text, are shown in large type to the right of the chromosome.

RETROVIRAL INTEGRATION I N MURINE MYELOID TUMORS

149

AND ORIENTATION OF PROVIRUSES INTEGRATED IN THE C. LOCATION Eoi-l LOCUS

The location of proviral integration sites within the Eui-1 locus was determined by Southern blot analysis (Mucenski et al., 198813; Morishita et al., 1988). In AKXD tumors, all Eui-1 rearrangements were caused by ecotropic viral integration. The viral integrations occurred within a 600-base pair (bp) region. Most viral integrations were clustered within a 300-bp region. All proviruses were integrated in the same transcriptional orientation. Among the four myeloid tumor cell lines analyzed in detail, three (NFS-58, NFS-78, DA-1) contained proviruses integrated in the same transcriptional orientation as those in the AKXD tumors. In the NFS-60 myeloid tumor cell line, the provirus was integrated in the opposite transcriptional orientation (Morishita et al., 1988). D. Eui-1 ENCODES A ZINC-FINGER PROTEIN THAT IS EVOLUTIONARILY WELL-CONSERVED Sequences from the Eui-1 locus are evolutionarily well conserved and are present in species as distantly related as chickens and humans (M. L. Mucenski, N. A. Jenkins, and N. G. Copeland, unpublished results). Evolutionarily well-conserved genomic probes from the Evi-l locus were used to screen mRNAs from the NFS-78 myeloid tumor cell line by Northern blot analysis (Morishita et al., 1988) to determine whether viral integration in the Eui-l locus affects transcription within this region. Weak diffuse hybridization in the range of 1-7 kilobases (kb) was detected. Four Evi-1 cDNA clones were isolated: two from an NFS-78 cDNA library (2.2 and 2.5 kb) and two from an NFS-58 cDNA library (3.0 and 5.1 kb). The structure of these cDNAs was determined by restriction enzyme analysis, DNA sequencing, and comparison of the cDNA sequences with the genomic sequences at the viral integration site. Three cDNA clones contained viral sequences at their 5’ ends, which suggests that transcripts can arise by transcription through the 3’ viral long terminal repeat as well as by splicing out of viral sequences into flanking Evi-1 sequences. The diffuse nature of Evi-1 transcripts identified in Northern analysis may result from this differential splicing of viral RNA to Eui-l sequences. In the NFS-60 cell line, the virus is integrated in the opposite transcriptional orientation relative to Eui-1. Viral activation of Evi-1 in this cell line may represent an enhancement mechanism or may result from transcription initiated at a cryptic promoter.

150

N. G . COPELAND AND N. A. JENKINS

The fourth cDNA (5.l-kb cDNA) contained only cellular sequences and was therefore useful in Northern analysis to study transcription from the Eui-l locus. Eui-l transcripts were not detected in a number of niyeloid tumor cell lines lacking viral integrations in E v i - I . They were also not detected in T-cell or B-cell lines. In NFS-60 and DA-1 cells a major transcript of -5 kb was found. In NFS-58 cells transcripts of 5 and 4 kb were detected. In NFS-78 cells transcripts that varied from 1 to 7 kb were observed, similar to what was observed with the initial genomic probes. Taken together, the data indicate that viral integration in the Eui-l locus activates transcription of a gene that is not normally expressed in hematopoietic cells. Virus integration takes place either within or just upstream of two exons that both appear to represent 5’-untranslated exons. The 5.1-kbcDNA identified a single open reading frame 3126 nucleotides in length. The predicted translation product from the first methionine was 1042 amino acids, which would encode a protein of 120 kd with six potential N-linked glycosylation sites. The 3’-untranslated region contained at least five copies of the sequence ATTA. These sequences are associated with mRNA instability presumably mediated by selective RNA degradation (Shaw and Kamen, 1986). While the nucleotide sequence did not show any significant homology with sequences in GenBank, the predicted amino acid sequence showed striking homology to TFIIIA, a Xenopus RNA polymerase I11 transcription factor that regulates the expression of 5s RNA genes during development (Pelham and Brown, 1980; Honda and Roeder, 1980). The region of homology was limited to the zinc-finger domains that are involved in DNA binding (Miller et aZ., 1985; Diakun et al., 1986; Berg, 1986). Zinc-finger domains have been identified in a number of other transcriptional regulatory proteins (reviewed by Johnson and McKnight, 1989), suggesting that Evi-l encodes a transcriptional regulatory protein. The predicted Evi-1 protein contains 10 zinc-finger domains of 27-28 amino acids (Fig. 3). The repeats share the classic histidinecysteine zinc-finger motif (Fig. 3). Seven repeats are located in the amino-terminal region of the protein. The first of these repeats is separated from the other six by a gap of 25 amino acids. The other six repeats are tandemly repeated. Three additional repeats are located in the carboxy-terminal region ofthe protein. Located just distal to these repeats (Asp877 to Glu928) is an acidic domain. Acidic domains have been identified in other transcriptional regulatory proteins and are thought to be involved in bind-

RETROVIRAL INTEGRATION IN MURINE MYELOID TUMORS

151

Domain 1

Domain 2

Consensus X X ~ X X ~ X X X ~ X X X X X ~ X X p i J X X X X ( H / C J X X X X X

F/Y

K/R

S N

R

FIG.3. Amino acid sequence of the zinc-finger repeats in Evi-I. The consensus sequence for the zinc-finger regions of Eoi-1 is shown at the bottom.

ing other cellular proteins involved in gene activation, such as RNA polymerase I1 (Hope and Struhl, 1986; Ma and Ptashne, 1987). IV. Relationship of Evi-1 to Other Zinc-Finger Proteins

A number of zinc-finger proteins have been identified by virtue of their homology with the zinc-finger domain of TFIIIA (Ruppert et al., 1988; Johnson and McKnight, 1989)(Table IV). Based on early experiments with TFIIIA, it has been proposed that each zinc-finger repeat complexes with a single zinc ion via tetrahedral coordination with the spatially conserved cysteine and histidine residues. It has been further hypothesized that the 12-14 amino acids intervening between the cysteine and histidine residues loop out to form a three dimensional structure capable of interacting with DNA and that each zinc-finger repeat interacts with -5 bp of DNA (Rhodes and Hug, 1986). The specificity of DNA binding would then be determined by a combination of the amino acid sequence separating the cysteine and histidine residues, the number of zinc repeats within the protein, and the spatial order of repeats with respect to each other. While Evi-1 as well as a number of the other zinc-finger proteins have not been shown directly

'TABLE IV S u e c l . ~ s s r ~ r c : ~OF~ rZINC-FINCEH o~ PI
C2H2-GLI

CzH2-XJ

Consensus finger sequence

(Y/F)XCX2CX:jFX~W(2fiXRXHTGEKP

Gene

Kruppel ADHl KR-H xfin NCFI-A MKrl,2 Krox4,6,H,Y ,20 Spl HKRI -4 Egr-1 (YIF)XCX3GCX:~(FIY)X5LX~HX:,,~H(TIS)GEKP ULl Mgll GLI2,3 (YJF)XCX~,~CX~FXSLXZ.~HX~.~HXS TFIIIA Serendipity Snail Hunchback pUP1007 Eoi-1

Drosophila Yeast Ilrosophila Xenopus Rat Mouse Mouse tiuman flunlan Mouse Human Mouse Human Xenopus Drosophila Drosophila Drosophila Human Mouse Yeast

Rosenberg et crl. (1986) Hartshorne et ctl. (1986) Schuh et (11. (1986) Ruiz i A1tal)a et al. (1987) Milbrandt (1987) Chowdhury et a1. (1987) Chavrier et (11. (1988) Kadonaga et ul. (1987) Ruppert et al. (1988) Sukhatn~eet al. (1988) Kinzler et a1. (1988) Kinzler et al. (1988) Ruppert et al. (1988) Brown et al. (1985); Miller et al. (1985) Vincent et a1. (1985) BouIay et al. (1987) Tautz et al. (1987) Page et al. (1987) Mucenski e t al. (1988b); Morishita et al. (1988) Stillman et al. (1988)

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to bind to DNA, it is expected that further study will confirm that these proteins represent DNA-binding proteins involved in regulating gene expression. Zinc-finger proteins have been subclassified on the basis of common conserved elements (Ruppert et al., 1988) (Table IV). The GLZKruppel gene family contains the conserved amino acid sequence HTGEKP(Y/F)XC connecting most zinc fingers. The GLZ-Kruppel genes can be further divided into Kruppel (CzHz-Kr) or GLZ (C2H2GLZ) subclasses, depending on spacing within the fingers and other conserved-sequence features (Table IV). In addition, exons of the GLZ subgroup contain only one complete zinc finger, whereas exons of the Kruppel subgroup contains several fingers. The second family, C2H2-&, lacks any consensus sequence connecting the zinc fingers. This family contains Eui-l as well as the prototype zinc-finger protein TFIIIA; pDP1007, a gene that is thought to regulate human testis determination; two Drosophila genes, hunchback and snail, involved in pattern formation in the early embryo; serendipity, a Drosophila blastoderm-specific gene that is thought to perform important but as yet unknown functions in embryonic development and; SW15, a gene that regulates mating-type switching in yeast. The identification of zinc-finger motifs in proteins involved in human and mouse development raises the interesting possibility that Eui-l may function during mouse development. This hypothesis is supported by the pattern of expression of Eui-l. Analysis of a large number of adult tissues for Eui-1 transcripts has identified only two tissues, kidney and ovary, that express Eui-l (J.N. Ihle, personal communication). This lack of expression in most normal adult tissues suggests that Evi-1 functions during embryonic development. In situhybridization studies of mouse embryos will provide important information regarding the developmental and tissue-specific expression of Eui-1 during embryogenesis and may help elucidate its function in normal mouse development. The role of Eui-l in the transformation of hematopoietic cells is also unknown. Myeloid tumor cell lines containing viral insertions in Eui-l are still IL-3-dependent, which suggests that Eui-l acts differently from transforming genes that abrogate the growth factor requirements of myeloid tumor cells. The cell lines are also altered in their ability to terminally differentiate, which may be due to the action of Evi-1 protein. Additional experiments will be required to elucidate the role of Eui-1 in neoplastic transformation.

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V. Activation of Transcription of Evi-7 by Viral Integration in Fim-3 Infection of newborn mice with Friend MuLV (F-MuLV) induces a wide variety of leukemias including T-cell and B-cell lymphomas, erythroleukemias, and myeloblastic leukemias (Wendling et al., 1983). Three common sites of viral integration, Fim-1, Fim-2, and Fim-3, have been identified in F-MuLV-induced myeloid leukemias (Sola et al., 1986; Bordereaux et a!., 1987). Interestingly, the Fim-3 common integration site maps to mouse chromosome 3 and is tightly linked to Eui-1 (Sola et aZ., 1988; Ihle et al., 1989; Bartholomew et al., 1989). No recombinants between Evi-1 and Fim-3 were observed in 241 interspecific backcross mice analyzed. Restriction mapping and hybridization studies of -50 kb surrounding the Fim-3 locus have failed to demonstrate overlapping regions with Evi-1 (Bartholomew et al., 1989).Eui-1 transcripts of -5 kb were detected in two cell lines, DA-3 and DA-34, that have viral integrations in Fim-3 but not in Evi-1 (Bartholomew et al., 1989).Fim-3 transcripts have not yet been identified. This suggests that Fim-3 represents another common integration site within or near the Eui-1 locus and that the primary effect of viral integration in Fim-3 is the activation of Evi-1. Additional physical mapping studies are required to determine the exact physical relationship between Eui-1 and Fim-3. VI. Additional Zinc-Finger Proteins Implicated in Neoplastic Disease The only other zinc-finger protein that has been implicated in neoplastic disease is GLI (Kinzler et al., 1988). GLZ was originally identified as an amplified gene in several human glioblastomas. Cloning and sequence analysis ofthe GLZ cDNA indicated that it contained five zinc-finger repeats. This protein is the prototype of one family of zinc-finger proteins described in Table IV. GLZ is highly conserved among species and is expressed in only a few normal adult tissues. Like Evi-1, the role of GLZ in neoplastic disease remains to be established. VII. Conclusions

Over the last 10 years it has become increasingly clear that retroviruses that do not carry transforming genes provide important retrotransposon tags for identifying new protooncogenes involved in neoplastic disease. The development of RI mouse strains derived from crosses of high and low tumor incidence strains has provided mouse geneticists

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with a number of new model systems for the study of neoplastic disease and for identifying host cellular genes that modulate the disease process. The identification of a new common viral integration site, Evi-1, in AKXD myeloid leukemias illustrates the power of this approach. The finding that Eui-1 encodes a zinc-finger protein suggests a role for this class of transcription factors in neoplastic disease and supports the hypothesis that nuclear protooncogenes represent cellular genes involved in regulating normal eukaryotic gene expression.

ACKNOWLEDGMENTS This research was supported by the National Cancer Institute, DHHS, under contract N01-CO-74101 with Bionetics Research, Inc. We thank Linda Brubaker and Robin Handley for the preparation of this manuscript, and Archibald Perkins, Arthur Buchberg, and Monica Justice for critical review of its content.

REFERENCES Bartholomew, C., Morishita, K., Buchberg, A., Jenkins, N. A,, Copeland, N. G., and Ihle, J. N. (1989).Oncogene 4,529-534. Berg, J. M. (1986).Science 232,485-487. Bordereaux, D., Fichelson, S., Sola, B., Tambourin, P. E., and Gisselbrecht, S. (1987). J . Virol. 61,4043-4045. Boulay, J. L., Dennefeld, C., and Alberga, A. (1987).Nature. 330,395-398. Brown, R. S., Sander, C., and Argos, P. (1985).FEBS Lett. 186,271-274. Buchberg, A. M., Bedigian, H. G., Taylor, B. A., Brownell, E., Ihle, J. N., Nagata, S., Jenkins, N. A., and Copeland, N. G. (1988).Oncogene Res. 2,149-165. Buchberg, A.M., Jenkins, N. A., and Copeland, N. G. (1989).Genomics (in press). Chavrier, P. Lemaire, P., Revelant, O., Bravo, R., and Charnay, P. (1988).Mol. Cell. Biol. 8,1319-1326. Chowdhury, K., Deutsch, U., and Gruss, P. (1987). Cell. (Cambridge, Mass.) 48, 771-778. Copeland, N. G., Jenkins, N. A., Nexe, B., Schultz, A. M., Rein, A., Mikkelsen, T., and JZrgensen, P. (1988).J . Virol. 62,479-487. Copeland, N. G., Buchberg, A. M., Gilbert, D. J., and Jenkins, N. A. (1989).Cum. Top. Microbiol. lmmunol. 149,45-57. Cuypers, H. T., Selten G., Quint, W., Zijlstra, M., Maandag, E. R., Boelens, W., van Wezenbeek, P., Melief, C., and Berns, A. (1984). Cell (Cambridge, Mass.) 37, 141-150. Diakun, G. P., Fairall, L., and Klug, A. (1986).Nature (London)324,698-699. Gilbert, D. J., Taylor, B. A,, Jenkins, N. A, and Copeland, N. G. (1989).In preparation. Gisselbrecht, S., Fichelson, S., Sola, B., Bordereaux, D., Hampe, A., Andre, C., Galibert, F., and Tambourin, P. (1987).Nature (London) 329,259-261. Graham, M., Adams, J. M., and Cory, S. (1985).Nature (London)314,740-743. Hanecak, R., Pattengale, P. K., and Fan, H. (1988).J. Virol. 62,2427-2436.

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Hartshome, T. A., Blumberg, H., and Young, E. T. (1986). Nature (London) 320, 283-287. Honda, B. M., and Roeder, R. G. (1980).Cell (Cambridge, Mass.) 22,119-126. Hope, 1. A., and Struhl, K. (1986).Cell (Cambridge, Mass.) 46,885-894. Ihle, J. N., Morishita, K., Parker, D. S., Bartholomew, C., Askew, D., Buchberg, A., Jenkins, N. A., Copeland, N. G., and Weinstein, Y. (1989). Curr. Top. Microbiol. Immunol. 149,59-69. Jenkins, N. A,, Copeland, N. G., Taylor, B. A., and Lee, B. K. (1982).]. Virol.43,26-36. Johnson, P. F., and McKnight, S. L. (1989).Annu. Reu. Biochem. (in press). Kadonaga, J. T., Camer, K. R., Masiarz, F. R., and Tijian, R. (1987). Cell (Cambridge, Mass.) 51, 1079-1090. Kinder, K. W., Ruppert, J. M., Bigner, S. H., and Vogelstein, B. (1988).Nature (London) 332,371-374. Ma, J., and Ptashne, M. (1987).Cell (Cambridge, Mass.) 48,847-853. Marth, J. D., Peet, R., Krebs, E. G., and Perlmutter, R. M. (1985).Cell (Cambridge, Mass.) 43,393-404. Milbrandt, J . (1987).Science 238,797-799. Miller, J., McLachlan, A. D., and Klug, A. (1985).EMBOJ. 4, 1609-1614. hloreau-Gachelin, F., Tavitian, A., and Tambourin, P. (1988). Nature (London) 331, 277-280. Morishita, K., Parker, D. S., Mucenski, M. L., Jenkins, N. A., Copeland, N. G., and Ihle, J. N. (1988).Cell (Cambridge, Mass.) 54,831-840. Mucenski, M. L., Taylor, B. A., Jenkins, N. A., and Copeland, N. G. (1986).Mol. Cell. Riol. 6,4236-4243. Mucenski, M. L., Gilbert, D. J., Taylor, B. A., Jenkins, N. A., and Copeland, N. G. (1987a).Oncogene Res. 2,33-48. Mucenski, M. L., Taylor, B. A., Copeland, N. G., and Jenkins, N. A. (1987b).J.Virol. 61, 2929-2933. Mucenski, M. L., Taylor, B. A., Copeland, N. G., and Jenkins, N. A. (1988a).Oncogene Res. 2,219-233. Mucenski, M. L., Taylor, B.A., Ihle, J. N., Hartley, J. W., Morse, H. C., 111, Jenkins,N. A., and Copeland, N. G. (198813).M o l . Cell. Biol. 8,301-308. Nusse, R. (1986).Trends Genet. 2,244-247. Page, D. C., Mosher, R., Simpson, E. M., Fisher, E. M. C., Mardon, G., Pollack, J., McGillivray, B., de la Chapelle, A., and Brown, L. G. (1987).Cell (Cambridge, Mass.) 51,1W1-1104. Pelham, H. R., and Brown, D. D. (1980).Proc. Natl. Acad. Sci. U.S.A.77,4170-4174. Poirier, Y ., Kozak, C., and Jolicoeur, P. (1988).J. Virol. 62,3985-3992. Rhodes, D., and Klug, A. (1986).Cell (Cambridge, Mass.)46,123-132. Rosenberg, U. B., Schroder, C., Preiss, A., Kienlin, A., Cote, S., Riede, I., and Jackle, H. (1986).Nature (London)319,336-339. Ruiz i Altaba, A., Perry-O’Keefe, H., and Melton, D. A. (1987).EMBOJ. 6,3065-3070. Huppert, J. M., Kinder, K. W., Wong, A. J., Bigner, S. H., Kao, F.-T., Law, M. L., Seuanez, H. N., O’Rrien, S. J., and Vogelstein, B. (1988).Mol. Cell. B i d . 8,3104-3113. Schuh, R., Aicher, W., Gaul, U., Cote, S,, Preiss, A., Maier, D., Siefert, E., Nauber, U., Schroder, C., Kemler, R., and Jackle, H. (1986). Cell (Cambridge, Mass.) 47, 10251032. Selten, G., Cuypers, H. T., and Berns, A. (1985).E M B O J .4,1793-1798.

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Selten, G., Cuypers, H. T., and Boelens, W., Robanus-Maandag, E., Verbeek, J., Domen, J., van Beveren, C., and Berns, A. (1986).Cell (Cambridge, Mass.) 46,603-611. Shaw, G., and Kamen, R. (1986).Cell (Cambridge, Mass.)46,659-667. Silver, J., and Kozak, C. (1986).J . Virol. 57,526-533. Sola, B., Fichelson, S., Bordereaux, D., Tambourin, P. E., and Gisselbrecht, S. (1986). J . Virol. 60,718-725. Sola, B., Simon, D., Mattei, M-G., Fichelson, S., Borderaux, D., Tambourin, P. E., Guenet, J.-L., and Gisselbrecht, S. (1988).J . Virol. 62,3973-3978. Stillman, D. J., Bankier, A. T., Seddon, A., Groenhout, E. G., and Nasmyth, K. A. (1988). EMBO J . 7,485-494. Sukhatme, V. P., Cao, X., Chang, L. C., Tsai-Morris, C.-H., Stamenkovich, D., Ferrira, P. C. P., and Cohen, D. R. (1988).Cell (Cambridge,Muss.) 53,37-43. Tautz, D., Lehmann, R., Schnurch, H., Schuh, R., Seifert, E., Kienlin, A., Jones, K., and Jackle, H. (1987).Nature (London)327,383-389. Taylor, B. A. (1978). In “Origins of Inbred Mice” (H. C., Morse, 111, ed.), Academic Press, New York. Villemur, R., Monczak, Y.,Rassart E., Kozak, C., and Jolicoeur, P. (1987).Mol. Cell. Biol. 7,512-522. Vincent, A., Colot, H. V., and Rosbash, M. (1985).J . Mol. Biol. 186,149-166. Voronova, A. F., and Sefton, B. M. (1986).Nature (London) 319,682-685. Warren, W., Lawley, P. D., Gardner, E., Harris, G., Ball, J. K., and Cooper, C. S. (1987). Carcinogenesis (London)8,163-172. Weinstein, Y., Ihle, J. N., Law, S., and Reddy, E. P. (1986).Proc. Natl. Acad. Sci. U.S.A. 83,5010-5014. Wendling, F., Fichelson, S., Heard, J. M., Gisselbrecht, S. Varet, B., and Tambourin, P. (1983).In “Tumor Viruses and Differentiation” (E. Scolnick and E. Levine, eds.), pp. 357-362. Liss, New York. Wirschubsky, Z., Tschilis, P., Klein, G., and Sumegi, J. (1986). Int. J . Cancer 38, 739-745.

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METASTATIC INEFFICIENCY Leonard Weiss Department of Experimental Pathology, Roswell Park Memorial Institute,Buffalo, New York 14263

I. Introduction 11. Metastatic Inefficiency-Documentation 111. The Metastatic Process A. Invasion B. Intravasation C. Arrest of Cancer Cells D. Extravasation E. Neovascularization IV. Metastatic Inefficiency-Random and Nonrandom Events Metastatic Subpopulations V. The Molecular Biology of Metastatic Inefficiency A. Overview B. Metastasis C. Metastatic Inefficiency D . Perspective VI. Consequences of Metastatic Inefficiency VII. Conclusions References

I. Introduction

The natural history of metastatic cancer usually culminates in the death of the patient. At first sight, it may therefore appear illogical to consider metastasis as an inefficient process. However, even an inefficient disease process, if repeated often enough, may in time culminate in the death of the patient. In this review, the inefficiency of different steps of metastasis will first be documented against both numerical and temporal backgrounds. On the one hand, inefficiency may be expressed as the death of large numbers, or even the majority of cancer cells entering each step of the metastatic cascade. On the other hand, inefficiency may be manifest as the length of time required to complete one or another step of the metastatic process. Depending on how the question is phrased, much discussion has focused on whether metastasis is a random or nonrandom event, or a combination of both. This often-heated discussion has served a useful 159 ADVANCES IN CANCER RESEARCH, VOL. 54

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

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purpose in generating many useful data and concepts, which are relevant to metastatic inefficiency and will form the basis for the next part of this review. Finally, this review will deal with the consequences of metastatic inefficiency on the patterns of metastasis development and therapy.

I I . Metastatic Inefficiency-Documentation

The existence of metastatic inefficiency is well illustrated by the survival of patients diagnosed as having the nine most common primary cancers of Caucasians, in the localized stage; these were subsequently treated, mostly by resection. The majority of these primary lesions are reasonably expected to fall in the T1 stage (TNM classification) and, where appropriate, to have diameters between 1and 4 cm. In Table I, which is based on studies involving thousands of patients, the decrease in percentage of survivors over time can be explained by assuming that initially the patients fitted into two broad groups. The first were correctly diagnosed as having localized cancer and were cured b y removal of their primary lesions. The second group had undiagnosed metastases. Ignoring recurrence at the primary site and the development of new cancers, the proportions of long-term survivors approximately indicate the group correctly diagnosed as having localized primary lesions. On the one hand, the survival for 220 years of large proportions of

TABLE I RELATIVESURVIVAL RATESFOR LOCALIZED CANCER, 1950-1954” Survival rate Ranked distribution Primary site Female breast Lung and bronchus Colon Prostate Rectum Bladder Corpus uteri Stomach Cervix uteri

(70) 12.9 12.2 9.0 6.6 4.8 4.8 3.8 3.6 3.6

(%)

I-Year

5-Year

10-Year

15-Year

20-Year

98 42 86 86 86 83 95 63 93

83 21 68 60 66 67 84 42 75

72 17

67 17 61 33 59 56 80 38 66

62 60 30 56 53 74 39 65

~

Data from Axtell et a [ . (1976).

64 45 60 60 81 39 69 ~~

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patients with a history of diagnosable primary lesions indicates that, for these cancers, one or more of the various steps in the metastatic process was inefficient. On the other hand, the degree of metastatic inefficiency, which correlates with cases in which seeding of target sites had occurred prior to (or during) resection, but that were incorrectly diagnosed, may be gauged by the decreases between 1- and 5-year survival rates. On the basis of incidence and the temporal course of disease, metastasis therefore appears to be an inefficient, slow process. Ill. The Metastatic Process In order to look further at metastatic inefficiency, the metastatic process will be broken down into its components: invasion, intravasation, arrest of cancer cells, extravasation, and neovascularization.

A. INVASION Invasion culminates in cancer cells occupying space formerly taken by host tissues, and requires that the latter be displaced, degraded, and/or destroyed. Unless cancer cells can enter various disseminative routes, metastasis will not occur, and entry is usually dependent on invasion. Invasive inefficiency will therefore be examined as an early regulatory step in the metastatic process. In this section, specific mechanisms of invasion will first be briefly reviewed. Invasive inefficiency will then be identified with respect to extent and rate, and finally, some of the possible underlying mechanisms of invasive inefficiency will be discussed.

1. Mechanisms of Znvasion There appear to be two separate, but often associated basic mechanisms involved in penetration of tissues by cancer. The first is related to proliferation, in which an actively growing cancer expands along the pathways of least resistance; the second is dependent on the active locomotion of cancer cells through tissues. In the case of human invasive cutaneous melanomas, it appears that invasion toward the subcutaneous tissues is accomplished by repetitive cycles of active locomotion of melanoma cells followed by cessation of movement and proliferation (Suh and Weiss, 1984).In addition to the two basic mechanisms, invasion will be promoted by degradation of the tissues through which it occurs, including breaching of basement membranes, which tend to compartmentalize tumors from connective tissues. In

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the context of metastasis, invasion includes intravasation and extravasation, in which the vascular subendothelium-basement membrane is also breached. Intravasation and extravasation are regarded as similar processes in many respects. The basement membrane is therefore regarded as a major barrier to the invasion of normal tissues by cancer (Burtin et al., 1982; Liotta et d,, 19831, and in many publications (e.g., Daher et al., 1987),fragmentation of the basement membrane is correlated with invasion, metastasis, and prognosis. Following seminal work of Sylven (Sylven and Bois, 1960), there have been numerous attempts to relate invasive capacity to enzyme production and release by cancer cells. According to this approach, a major mechanism in invasion is histiolysis due to a balance between the release of active collagenases (Liotta et al., 1980) and other enzymes (Nakajima et aZ., 1986; Niedbala et al., 1987), as well as enzyme inhibitors (Cawston et al., 1983; Halaka et al., 1983). While in some situations invasive capacity has been shown to increase with cancer cell collagenase activity, in other situations it has not (Eisenbach et al., 1985; Warburton et al., 1987). Expression of an invasive phenotype may involve mutations of cellular genes (protooncogenes) or of control genes (Bishop, 1987). The role of oncogenes in the transcriptional activation of collagenase has been clarified by Schonthal et aZ., (1988) by studies on the phorbol ester-responsive element (TRE) corresponding to the sequence of human collagenase gene. The collagen promoter responds to a number of oncogenes, mediated through the TRE, with an absolute requirement for fos gene expression. A major feature “of c-fos gene activation by various inducers is the transient appearance of fos RNA and fos protein . . caused by the short half-life of mRNA, rapid degradation of fos protein and fast repression of c-fos promoter activity.” On account of the numbers of different oncogenes that may be involved, and the transient nature of the response, it appears unlikely that simplistic correlations will be found between oncogene expression, cancer cell collagenase ievels, and invasion. It would be unwise to confine the search for the underlying mechanisms of invasion to cancer cell-produced enzymes and their inhibitors, with respect to basement membrane degradation. Thus, maintenance of basement membrane integrity depends on both synthesis and degradation. It is often assumed that the penetrated basement membranes are normal; however, reduced synthesis of connective-tissue components may be associated with malignancy (Adams et al., 1982) on the evidence of decreased mRNA levels (Sobel et al., 1981) and transcription rates (Tyagi et al., 1983).Expansion of a tumor may itself I

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cause pressure atrophy of surrounding tissues (Gabbert et al., 1987; Vaupel and Muller-Klieser, 1983); rapid, expansive growth is often associated with invasive capacity. Neutrophil polymorphs are traditionally considered to be part of the host defense system, and appear to be involved in the destruction of cancer cells (Glaves, 1983). However, in fulfilling this protective role, neutrophils often cause damage to tissues, by mechanisms that include the release of matrix-degrading proteases, thereby enhancing invasion; the invasion-promoting effects of dead and dying tissues have been described in relation to stromal degradation (Weiss, 1977a, 1978) and increased cancer cell migration (Turner and Weiss, 1980).Cellular damage to both cancer cells (Glaves, 1986a) and host cells appears to be associated with the generation and release of oxygen-derived free radicals from neutrophils (Ward et al., 1986; Halliwell, 1987; Imlay and Linn, 1988), and the inflammatory response has been shown to promote lung colonization by circulating cancer cells (Orr et d.,1986; Orr and Warner, 1987). In the situation often seen in some sarcomas, where vascular clefts are lined with cancer cells, intravasation occurs by exfoliation rather than invasion. The neovasculature of solid tumors in general tends to leak on account of fenestrations, which facilitate intravasation by cancer cells, with minimal requirements for prior degradation of matrix. Although it is traditionally taught that arteries are resistant to invasion on account of their elastic laminae, as many as 87% of a series of lung cancers were found to invade the pulmonary arteries and their branches, compared with -4% in renal and bowel carcinomas (Kolin and Koutoulakis, 1987). Two different extravasation mechanisms have been observed in cancer cells arrested and surviving in the microvasculature. The first involves active migration of the cancer cells, sometimes through interepithelial cell junctions and sometimes following the migration pathways of leukocytes (Wood, 1958; Kinjo, 1978; DeBruyn and Cho, 1982).The second involves intravascular proliferation of arrested cancer cells, with compression damage to the surrounding vessel (Warren and Gates, 1936; Fonck-Cussac et al., 1969; Locker et al., 1970; Baserga et al., 1970; Machado et al., 1982). After attaining a critical size, the intravascular tumors “burst” out ofthe blood vessels (Chew et al., 1976; Crissman et al., 1985).In the former mechanism, cell locomotion appears to play a primary role, influenced by the physical properties of invaded matrix (Folger et al., 1978); in the latter, cell proliferation plays a primary role, limited by the mechanical properties of the subendothelium-basement membrane. In both cases extravasation

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would be facilitated by basement membrane-degrading enzymes released from the cancer cells themselves, the vascular endothelium and other damaged cells, and leukocytes (Weiss et al., 1988a,b). Cancer cell locomotion is enhanced by released collagenases (Maslow, 1987), interaction with basement membrane-subendothelium constituents, and directed by various degradation products of these structures (Varani and Orr, 1982).

2. Inuasiue Inefficiency: In Situ Cancers

The progression of in situ to invasive carcinomas provides good examples of temporal inefficiency in the invasive process. a . Uterine Ceruix. Some, but not all benign atypia, progress through different degrees of dysplasia to carcinoma in situ (CIS). This continiium is reflected in the newer terminology for abnormalities of the cervical epithelim associated with an increased risk for invasive carcinoma; these are all contained in the single diagnostic category of cervical intraepithelial neoplasia (CIN). CIN I and I1 correspond to mild and moderate dysplasia, respectively; CIN I11 includes both severe dysplasia and CIS (Buckley et ul., 1982). It is beyond the scope of the present review to discuss the merits of including severe dysplasia in the same category as CIS. Carcinoma in situ is an anaplastic change in squamous epithelial cells or, in 5% of all cervical carcinomas (adenocarcinoma in situ), anaplasia of the endocervix. These lesions neither penetrate basement membranes nor invade the cervical stroma. That CIS is not metastatic is demonstrated by long-term follow-up studies on patients treated conservatively by therapeutic conization (Kolstad and Klem. 1976). However, untreated CIS ultimately progresses to invasive carcinoma, which breaches basement membranes and begins to be associated with vascular invasion and/or lymph node metastasis when the depth of stromal invasion exceeds 3 mm (Roche and Norris, 1975; Perez et aZ., 1985). As reviewed elsewhere (Weiss, 1985b),given an established causal relationship between CIS and invasive carcinoma, some estimate of progression times may be obtained from the peak ages of onset of the two conditions in the large numbers of women subject to regular screening by the Papanicolaou test. The consensus is that progression takes between 8 and 30 years, with a mean of 10 years. h. Breast. Although CIS may be ductal or lobular, only the more common ductal CIS (intraductal carcinoma) will be discussed here. ‘This disease entity is restricted to cancers in which there is no evidence of invasion through the basetnent membrane, and includes le-

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sions detected by mammography, or present in breast tissue removed because of another abnormality (Schnitt et al., 1988). Direct observations on the times taken for in situ lesions to become invasive are limited to those in which CIS was treated by biopsy alone. Not all cases of in situ lesions studies progressed to become invasive carcinoma; however, in two separate studies, those that did so required an average of 6.1 (Page et al., 1982) and 9.7 (Rosen et al., 1980) years after initial biopsy. On the basis of retrospective histologic studies, invasion was found to occur in 45% of previous in situ lesions after an average of 15 years (Toker, 1974); epidemioIogic studies revealed a peak age incidence of in situ lesions 10 years earlier than that for invasive cancers (Lewison, 1976), and calculations based on growth kinetics indicate that it could take -15 years to progress from a single cancer cell to a metastasizing (invasive) lesion (Weiss, 1985b). c. Bladder. Carcinoma in situ is defined as anaplastic cancer cells confined to the urothelium (Richie et al., 1985). Two staging systems are in common use, namely the Jewett-Strong-Marshall system (Marshall, 1952)and the TNM system. Present discussion is limited to the time taken for stage 0, in situ (Tis)carcinomas, which are limited to mucosa, to progress to stage A (TI)or stage B(T2) lesions, which have invaded the lamina propria or the muscularis, respectively. In analyzing data, it should be noted that although stage 0 includes both in situ and papillary carcinomas (TJ, the latter are generally less invasive and the majority can be controlled by conservative local therapy. In contrast, in a series of in situ carcinomas treated locally and in retrospect, inadequately, 73% of patients developed invasive lesions (Utz et al., 1970). In another series, a subset of patients with CIS was identified, some of which showed no signs of progression to invasive lesions after 10 years follow-up (Utz et al., 1980). d . Colorectum. The data on colorectal carcinoma (Sugarbaker et al., 1985) are deficient in the present context. Thus, it is well documented that a small proportion (-1%) of hyperplastic polyps progress to clinically significant, large (>1.5cm diameter) polyps over an average of 8 years (Arthur, 1968). When these polyps are subdivided into villous, intermediate, and adenomatous types, 40, 22, and 5%, respectively, progress into invasive carcinomas (Muto et al., 1975). In the case of adenomatous polyps, progression to an invasive lesion is thought to take an average of 5 years; however, ages of peak incidence give no clue of progression times for villous and intermediate types. From the clinical viewpoint, it is important to discriminate between in situ and invasive lesions (Sugarbaker et al., 1985). If a carcinoma penetrates no deeper than superficial to the muscularis mucosa, it is considered to be

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an in situ (nonmetastatic) lesion (Lane and Kaye, 1967).This appears to be associated with the absence of lymphatics superficial to the muscularis mucosa, and the close association of a lymphatic plexus with the bundles, which constitutes a disseminative route (Fenoglio et ul., 1973). Although delays occur in progression for in situ to invasive carcinomas of the colon, the complexity of the clinicopathologic situation combined with a paucity of relevant data prevent their enumeration.

3. Mechanisms of lnuasive Delay A major manifestation of metastatic inefficiency appears to be the long time taken for progression of in situ carcinomas to invasive lesions, or invasive delay. Where these delays can be documented, consistent estimates for a number of analogous situations in humans indicate progression times of 520 years. Although these observations reveal inefficiency expressed as an extended time base, they d o not in themselves point to underlying mechanisms. In attempting to account for the long delay in the transition from in situ to invasive carcinomas, a number of nonexclusive mechanisms are possible. a. CeZ2 Loss. In situ lesions tend to be small and slow-growing, and mechanical considerations dictate that expansion of these lesions will be superficial, along the path of least resistance. Therefore, one possible cause of delay is that cancer cells are lost by exfoliation into a body cavity communicating with the outside world, at a rate that matches their growth rate. b. Cancer Cell Motility. Cancer cell motility is generally considered to be an important part of the invasive process (Strauli and Weiss, 1977; Suh and Weiss, 1984; Thorgeirsson et al., 1982; Volk et al., 1984), although it is by no means the only factor involved (Weiss, 1985a). Cancer cells may be experimentally selected on the basis of motility (Varani et al., 1985; Tulberg and Burger, 1985; Grimstad, 1988) and invasiveness (Hart, 1979; Poste et al., 1980; Kalebic et al., 1988). Regardless of an absolute correlation between in vitro motility and in 2;itt-oand in vivo invasiveness, and difficulties in the interpretation of selection procedures discussed later, it is possible that part of the invasive delay depends on the emergence of motile and/or invasive subpopulations of cancer cells. As penetration of host tissues by an i i i situ lesion would appear to have survival value for cancer cells, this could constitute a selection pressure for this type of progression. c. Repair Processes. In the cervix and vulva, immunohistochemical studies (Ehrmann et al., 1988) revealed that whereas CIS exhibited

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intact laminin and type IV collagen, in invasive foci defects were seen in these two basement membrane components. Their absence around totally keratinized or necrotic cancer cell nests implied a dependency on living cells. Less laminin was observed around new invasive buds than parent nests, suggesting a cyclic process with laminin loss during active invasion and re-formation during quiescence. The cyclic nature of invasion, as also suggested by Suh and Weiss (1984), together with evidence of increased proliferation of cancer cells in invasive lesions compared with in CIS (Mushika et al., 1988), might also account for delay. The clinical behavior of in situ and invasive lesions in the breast, is distinct, and the former may be cured by local therapy (Azzopardi, 1979);however, it is difficult to exclude invasive lesions by the appearance of an intact basement membrane, because it may be intact in some sections (Carter et al., 1969; Goldenberg et al., 1969), while gaps may be observed in other sections of the same tumor (Ozzello and Sanpitak, 1970; Ahmed, 1978). A sensitive parameter of invasion is the presence of myofibroblasts (Tamini and Ahmed, 1986),which may represent an attempt to repair a damaged basal lamina (Majno, 1979). Repair processes result initially in the formation of granulation tissue followed by that of scar tissue (Vracko, 1974). It has been suggested that synthesis (Ryan et al., 1974) of periductal collagen and elastin by myofibroblasts serves to localize the cancer and account for regressive CIS (Muir and Aitkenhead, 1934; Linell and Ljungberg, 1984). Repair-encapsulation processes of this type could retard stromal invasion, particularly by small, in situ cancers. The cancer cells in basal cell carcinomas (BCC) produce cytokines, which may stimulate collagenase production by fibroblasts and hence promote invasion. However, increased collagen synthesis, indicated by increases in types I and 111 procollagen mRNA, may result in the deposition of a dense and extensive extracellular matrix (Moy et al., 1988), which may inhibit invasion. An exaggerated form of stromal deposition, indicating an inbalance between matrix formation and degradation, is seen in the sclerosing or morphea-like variants of BCC. This may indicate a general delay mechanism in invasion, and may be related to the general failure of BCC to metastasize. d . Subpopulations. The possibility that invasive delay is dependent on the emergence of invasive subpopulations of cancer cells within in situ carcinomas could in theory be explored by use of probes for specific genetic markers of invasiveness; unfortunately, these are not currently available. However, evidence of difference between cancer

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cells in in situ and invasive lesions could well be a useful starting point for such studies, even in the absence of established causal relationships between individual markers and invasive capacity. The low molecular weight cytokeratins are one such group of potential markers, which appear to be expressed in squamous-cell carcinomas but not in normal stratified squamous epithelium (Leader et ul., 1986). The human cytokeratins constitute a group of at least 19 polypeptides, one or more of which are expressed by all epithelial cells (Moll et al., 1982,

1983). Using immunohistochemical reactions with monoclonal antibodies

(mAb) against specific cytokeratins, which appear to be heterogeneously expressed (Teglbjaerg et al., 1985), the progression from dysplasia to invasive carcinoma has been studied in the uterine cervix (Gigi-Leitner et al., 1986; Bobrow et ul., 1986a; Angus et al., 1988). With respect to the (Ab CAM 5.2, the indirect immunoperoxidase technique is negative in the majority of cases (<95%) of normal or rnetaplastic stratified squamous epithelium. In CIN 111, approximately one-quarter show positive staining and, in invasive carcinoma, between 88%(15to 17)(Bobrow et al., 1986a)and 69%(9 of 13)(Angus et aZ., 1988) are positive. Thus, the expression of low molecular weight cytokeratin as revealed by mAb CAM 5.2, might be used to study both progression (Puts et al., 1985)and invasive delay. However, it is difficult in the present context to relate the reported changes to transition from CIS to invasive lesions, because it could be argued that only one-quarter of the cases reported as CIN I11 were in fact CIS and possibly only these have positive staining reactions. It might be expected that this question could be resolved by determining whether foci of microinvasive carcinoma and adjacent CIN react with CAM 5.2. However, at present the results of such studies are contradictory; thus, Wells et ul. (1986)report a weak reaction in only one of six cases; Bobrow et at!. (1986b) report positive staining in two of two cases and both negative and weakly positive areas in adjacent CIN, and positive staining was also reported in two of two cases of microinvasive lesions by Angus et al., (1988). This controversy will doubtless be resolved by examination of more specimens of authenticated microinvasive lesions. For the moment, the evidence appears to indicate that cytokeratin expression changes “late in the sequence of events leading to malignancy with invasive potential” (Bobrow et ul., 19881)). However, neither the relationship of change from CIS to invasive cancer nor the mechanisms are known. Differential expression of cytokeratins may prove useful in detecting differences between cancer cell populations in invasive and nonin-

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vasive cancers of the breast (Bartek eta!., 1985; Altmannsberger et al., 1986). In human breast tissue, reaction with antikeratin mAb AE1 and AE3 indicates more homogeneity in specimens containing intraductal and invasive ductal carcinomas compared with that in infiltrating ductal carcionomas (Sorenson et al., 1987).No differences were detected between in situ and invasive lesions with respect to cytokeratins 18 and 19, where all cancer cells were positive, and complexes of cytokeratins 14 and 5, where all preinvasive and most invasive carcinomas were negative (Jarasch et al., 1988). Thus, in contrast to analogous studies on the uterine cervix, these studies provide no evidence that invasive delay in breast carcinoma is dependent on the appearance of novel, minor subpopulations of cancer cells. e . Conclusions. There seems little doubt that a considerable time is required for an in situ cancer to become invasive. Thus, invasive delay may be considered as contributing to overall metastatic inefficiency. Any process inhibiting normal invasive mechanisms, or the specific mechanisms just outlined, can contribute to invasive delay; however, the rather limited relevant experimental evidence suggests that by themselves none of these various factors could account for delays extending into decades. However, the cumulative effects of a number of the factors could conceivably be responsible for conferring some of the characteristics of a chronic, superficial disease on this early part of the metastatic process. Whatever the underlying mechanisms, invasive inefficiency expressed as temporal delay makes a major contribution to the success of the surgical treatment of early cancer by inhibiting metastasis during the period before diagnosis.

B. INTRAVASATION Before hematogenous metastasis can occur, cancer cells must enter the bloodstream by some sort of invasive process. An attempt was made using mice to assess the relative efficiencies of intravasation of different lines of B16 melanoma cells (Fidler, 1973),by comparing the incidence of pulmonary colonies following their intravenous injection, with that of pulmonary metastases from intramuscular B16 tumors (Weiss et al., 1982).As summarized in Table II., with BL6 cells, which were selected for invasiveness in vitro (Hart, 1979), there was correspondence between high colonization and metastatic potentials. In the case of the FlOFA line, which were selected on the basis of short-term survival in the bloodstream (Weiss et al., 1982), a 100% incidence of pulmonary colonies corresponded to a 50% incidence of pulmonary

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TABLE I1

RELATIVEINTRAVASATION EFFICIENCY OF B16 MELANOMA CELLLINES“ Pulmonary colonies 21 days after lo5 cells iv

B16 Line BL6 F10 FlOFA FlO(lr6) Wild

Animals with tumors

Median (range) tumors per animal

12112 45/45 12/12 3414 1 28/30

240 (77-352) 103 (5-400) 27 (7-95) 3 (0-152) 4 (0-69)

Pulmonary metastases 22 days after 105 cells im Animals with metastases (%)

25/25 23/64 (36) 48/96 (50) 34/68 (50) 12/30 (40)

Median (range) metastases per animal 6 (1-51)

0 (0-19) l(0-242) l(0-10) 0 (0-4)

Mean

SE

8.1 t 2.0 4.0 1.0 14.0 5.5 2.8 +- 0.4 1.8 2 0.3

*

“ Data from Weiss et al. (1982). metastases. In the case of the F10 and FlO(lr6) lines, which were selected on the basis of their respective high and low colonization potentials (Fidler, 1973), no correlation was observed between colonization potential and either the incidence of animals with “natural” metastases or the average numbers of metastases per animal (Weiss et

al., 1982). One possible explanation for this discord between colonization and metastasis, which was also reported by Stackpole (1981)and Trainer et al. (1985),is that the events culminating in intravasation are different from the subsequent events culminating in metastasis, and that intravasation itself is a potent rate regulator for the metastatic process. Because massage often enhances metastasis (Tyzzer, 1913; Hoover and Ketcham, 1975), intramuscular tumors were massaged in an attempt to increase intravasation and induce correspondence with pulmonary colonization following intravenous injection of cancer cells. In the BL6 and wild-type lines metastasis was enhanced by massage, but no enhancement was detected with FIO and FlO(lr6) lines. The results therefore suggest that within the time frame of the experiments, the failure of the B16 BL6 and wild-type tumors to express their colonization potential fully during metastasis is d u e to limitations in intravasation. In the case of the F10 and FlO(lr6) lines, the absence of massage enhancement suggests that at time of massage the cancer cells could not be mechanically displaced into blood vessels. The extent to which this is due to the properties of the cancer cells and/or the vasculature is not known at present.

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The use and limitations of direct counts of circulating tumor cells in studies of metastasis have been reviewed by Glaves (1986b, 1987). In a series of studies on tumor-bearing mice, right ventricular blood was layered onto discontinuous Percoll gradients, passed through 2-pm Nucleopore filters, and the cancer cells on the filters stained and enumerated (Glaves, 1983). From the experimental data (Table 111), estimates may be made of the range of numbers of cancer cells intravasated from intramuscular MC1 and MC2 fibrosarcomas (Glaves and Mayhew, 1984) and B16 melanomas and 3LL (Lewis lung) carcinomas (Glaves, 1983);in Table I11 only one mean volume is selected for each type of primary tumor, although ranges of volumes are given in the original publications. In contrast to MC2, MC1 fibrosarcomas rarely metastasize. It is therefore of interest that
TABLE I11 CANCER CELLSDETECTED IN RIGHT VENTRICULAR BLOODOF MICE BEARINGSPECIFIED INTRAMUSCULAR TUMORS Type of im tumor

Tumor volume (cm3-+ SD)

Number of cancer cells per 2 ml blood

References

MC1 MC2 3LL B16 (Wild)

4.6 f 0.9 4.7 0.6 4.2 3.1

0-8 x 103 0-2.8 x 104 66-700 42-2084

Glaves and Mayhew (1984) Glaves and Mayhew (1984) Glaves (1983) Glaves (1983)

*

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C. ARRESTOF CANCER CELLS During hematogenous metastasis, cancer cells released into the bloodstream are arrested in the vasculature of target organs. Unless cancer cell arrest occurs, metastasis cannot take place; however, because of the inefficiency of this phase, arrest of cancer cells does not guarantee that metastasis will occur. It has been known for many years (Goldmann, 1907; Schmidt, 1903) that “although the blood-stream may teem with cancer cells, there may be no evidence of metastatic tumour formation” (Wilson, 1908).Much of the early evidence has been reviewed elsewhere (Weiss, 1985c), and only two examples will be discussed here. Studies on humans provide an impression of the complexities of the whole metastatic process and the limitations in considering invasive inefficiency in isolation. Estimates were made of the rates at which cancer cells were released directly into the renal vein in patients undergoing radical nephrectomy for primary renal cell carcinoma (Glaves et d., 1988). Data from 3 of 10 reported cases are summarized in Table IV: Patient KT-6 had a 10-cm-diameter primary tumor, which released cancer cells into the renal vein blood at a rate of 3.7 x lo7 per 24 hr; the patient died with multiple metastases 7 months after nephrectomy. However, patients KT-2 and KT-10 had 10- and 6-cmdiameter primary lesions, respectively, which released cancer cells into their renal veins at rates of -5 x lo9and 2 x lo8per 24 hr; yet they TABLE I\’ RELEASEOF CANCER CELLSINTO RENAL VEINSOF PATIENTS WITII RENAL CELLCARCINOMAS‘’

i’atient

Tiirnor diameter (cm)

Number of cancer cells/ml blood

Prorated cancer cell release per 24 hr

IiT-6

10

51

3.7 x 10’

KT-2

10

7309

5.3 x loy

6

315

2.3 x lox

ET- 10

‘*Data from Glaves et ul. (1988).

Patient status Dead 7 months after surgery; multiple inetastases Alive at 66 months; no detectable metastases Alive at 31 months; no detectable metastases

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were both still alive 66 and 31 months after surgery, with no detectable metastases. On the one hand, these two apparently discordant observations may reflect the inherent problem of extrapolating (prorating)from counts made on single blood specimens (Glaves, 1983; Mayhew and Glaves, 1984; Glaves and Mayhew, 1984). On the other hand, the observations indicate that in these cases many cells were released, probably for some time before surgery, but the overriding influence in patients KT-2 and KT-10 was inefficiency in the post intravasation phases of metastasis, and in patient KT-6, a relative efficiency. Studies were made of cell traffic between the lungs and liver (Weiss, 1980a) and liver and lungs (Weiss et al., 1983a) in animals following direct injection of cancer cells into either the tail vein or portal veins. Sampling at various access points indicated that the arrest and major cancer cell destruction occurred in the microvasculature. Accurate numerical estimates of the post intravasation phases of metastasis come from data of the type shown in Table 11, in which known numbers of cancer cells were injected into the tail veins of mice, and the pulmonary colonies were counted 21 days later. Given the clonal origin of these colonies, the observation that lo5 injected B16 cells of types BL6 and FlO(lr6) gave rise to medians of 240 and 3 pulmonary colonies, indicates that their respective efficiencies were only 0.2%and 0.003%. These low levels of efficiency with respect to lung colony formation refer only to the postintravasation phases of metastasis and would probably be even lower if allowance could be made for the preintravasation phases occurring in natural metastasis. Much information on cancer cell traffic has been generated by the use of cancer cells radiolabeled with iododeoxyuridine ( [25Z] UdR) and making y counts on various tissues (Hofer, 1970; Fidler et al., 1977). After tail vein injections of most types of “solid” cancer cells, almost all are immediately arrested in the lungs, but there is slow release of radioactivity over the next 24 hr, to approximately background levels. Alcohol extraction procedures, which in some circumstances leave label behind in cells that were intact at the time of alcohol immersion, indicate a slow loss of cancer cell viability in a number of organs (Fig. 1).However, when the results of alcohol extraction are compared with bioassays of the lungs (Fig. 2), the results of the former are misleading in the present context, and 5 min after injection only 15% of cancer cells arrested in the lungs are viable. Thus, on the basis of animal experiments, postintravasation metastatic inefficiency can be considered as biphasic. There is a rapid phase of intramicrovascular cell death, completed in <5 min, which accounts for 85%of arrested cancer

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Time after injection FIG.1. Pulmonary retention at different times after tail vein injections of [1251]UdRlabeled cancer cells of four types: . . . B16; C - H , Lewis; A- . -A, MC2; t - - O ,

MCL.

+ +,

cells; this is followed by a slow phase, which accounts for the vast majority of the remainder (Weiss et al., 1988, 1989).

1. Rapid Phase of Postintruuasation Cancer Cell Death The rapid phase of cancer cell death within the microvasculature has been associated with shape transition, when cancer cells enter, move along, and are arrested in microvessels having smaller diameters than their own. It has been suggested that shape transitions from the equilibrium spherical configuration, which at constant cell volume require increases in cell surface area, are accomplished by two sequential mechanisms. The first is an increase in apparent surface area, accomplished by unfolding of the normal convoluted cell surface; this is a reversible, nonlethal change that corresponds to cell “deformability,” which depends on the rheologic properties of the whole cell, the cytoskeleton. If adequate cell shape transitions are associated with

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Time After Injection FIG.2. Comparisons between (0) lung y counts after tail vein injection of [1251JUdRlabeled 3LL cells and ethanol extraction, and (0)bioassays of lungs after injection of nonlabeled cells. Reproduced from Glaves (1986b), with permission.

surface unfolding alone, then cancer cells will survive the rapid phase of postintravasation destruction. However, if shape transitions require a true increase in cell surface area, dependent on stretching the surface membrane, then increases in surface area greater than -4% result in increases in membrane tension above a critical level, resulting in membrane rupture and cell death (Weiss and Schmid-Schonbein, 1989). Although the ultimate factors in shape transition-associated death are the mechanical properties of the cancer cell surface membranes, major contributory factors include ( 1) the relative deformabilities of the cancer cells and the microvessels in which they are trapped (Weiss and Dimitrov, 1986), which are partially dependent on the mechanical properties of the surrounding tissues: (2)the pressures in surrounding tissues transmitted through the microvasculature to the trapped cancer cells, particularly in contractile organs including the myocardium

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(Weiss, 1988) and skeletal muscle (Weiss, 1989); (3) physiologic changes in the mechanical properties of tissues-for example, the increases in alveolar capillary tension during inspiration (Weiss and Dimitrov, 1986), and blood pressure differentials between the ends of trapped cancer cells (Weiss and Dimitrov, 1984; Weiss et ul., 1985). It may be mentioned here that shape transitions may also be associated with another facet of metastatic inefficiency, namely dormancy (Gabor and Weiss, 1985).

2. S l o w Phase of Postintruvasation Cancer Cell Death The slow phase of cancer cell death within the microvasculature occurs after arrest. It involves the host defense systems, and interac-

tions between the cellular and humoral components of the inflammatory, immune, and coagulation systems. These factors have been reviewed with an extensive bibliography (Weiss et al., 1989), and limitations of space preclude any useful review here. They are mentioned only to identify the slow phase of postintravasation death of cancer cells.

3. Effect of Postintrauasation Inefficiency On the one hand, in cases where only small numbers of cancer cells are released into the bloodstream, because of either the small size of the tumor (e.g., early cancers) or inherent intravasation defects (e.g., in situ lesions, and possibly BCC), the high level of metastatic inefficiency may result in no cancer cells surviving arrest in the microvasculature. On the other hand, when large numbers of cancer cells are released, even the small fraction surviving will be enough to generate micronietastases, and the effect of the inefficiency will be to retard the rate of metastasis development.

D.

EXTKAVASAI'ION

Cancer cells surviving trauma associated with arrest can extravasate h y migrating in a manner analogous to leukocytes, or alternatively,

following intravascular growth they may burst out of vessels (Wallace

et al., 1978; Crissman et al., 1985). Extravasation is aided by microvascular trauma, including that consequent upon the inflammatory responses to arrest. Relevant trauma includes endothelial retraction and degradation of basement membrane and perivascular tissues, by enzymes released from cancer and noncancer cells. It should be noted that treatment modalities, in addition to postembolic inflammation, may promote the extravasation phase of metastasis (Weiss et al., 1989).

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Thus, the same factors contributing to the slow component of cancer cell damage and death-and hence metastatic inefficiency-during the immediate post intravasation steps of metastasis may also promote the efficiency of the extravasation phase of metastasis. E. NEOVASCULARIZATION Regardless of the inefficiency of the metastatic process, metastases do develop. Cancer cell emboli grow into micrometastases, which may be operationally defined as lesions small enough (maximum diameter 0.1-0.2 cm) to satisfy their nutritional requirements by diffusion (Carlsson et al., 1979). Subsequent growth of micrometastases to overt metastases depends on neovascularization of these lesions (Folkman, 1985; Furcht, 1986). The complexity of the vascularization process is dramatically shown in Dingemans’ reconstruction (1988) of the vascular system of 0.1-cm-diameter B16 melanoma metastasis in mouse liver. Irregular, flattened vascular spaces were seen branching from the portal vessels covering a large proportion of the metastasis surfaces; the hepatic arteries did not penetrate the lesions, which contained central fluid-filled spaces. Non-cancer-specific interactions with pericancerous venules lead to tumor neovascularization, without which further development of micrometastases will not occur. The presence of micrometastases showing no increase in volume constitutes a dormant state. However, it is not at all clear whether this is true dormancy, in which the cancer cells are in Go, or pseudodonnancy, in which cancer cell proliferation is matched by cancer cell loss. Nonetheless, in the present context, failure of neovascularization may act to promote metastatic inefficiency. D’Amore (1988), among others, has discussed the use of angiostatic cancer-specific agents are one basis for antimetastatic therapy. However, because angiogenesis represents a general response to a number of types of noncancerous tissue trauma, effective angiostatic therapy would likely also block vascular repair in normal tissues. Even well vascularized metastases do not grow indefinitely at an exponential rate. While part of the explanation for growth retardation is related to vascular compression and subsequent vascular insufficiency within solid tumors, this is unlikely to be the whole story. However, it appears likely that the two major retardation “points” in the Gompertz equation for turmor growth kinetics are associated with vascular insufficiency both in transition from micrometastasis to overt lesions and in prolongation of volume-doubling times in overt metastases with diameters greater than several centimeters.

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IV. Metastatic Inefficiency: Random and Nonrandom Events

The vast majority of the cancer cells entering the metastatic process do not form metastases. However, it is the behavior of the surviving, active minority of cancer cells, that ultimately determines the metastasis-related fate of the patient. The question then arises whether this minority of metastasis-generating cells is determined on a random o r nonrandom basis. When the metastatic efficiency of a cancer cell population is 1%, has only this 1% a preexisting metastatic potential, rind do the remaining 99% have a preexisting incapacity to metastasize? An individual cancer cell can either generate a metastasis or not. In populations of cancer cells this sharp all-or-none distinction is lost, since some populations of cancer cells produce more or fewer metastases or colonies than others within a given time frame. The relative metastatic behavior of the cancer cell populations of tumors could therefore depend on the numbers of cells present at appropriate times, with a “metastatic phenotype” that could be either a transient or stable property of these cells. Assessment ofthe numbers of cells expressing a metastatic phenotype may be complicated by synergism between then and nonexpressing cells (Miller, 1983). If metastasis were the endpoint of an orderly sequence of events in isolated, noninteractive systems, in which the only variables were static properties of the involved cancer cells themselves, it would be possible to define the metastatic process as random or nonrandom. However, the systems are extremely complex and interactive, and in many respects disorderly. In addition, the properties of the many different types of cancers and cancer cells are different from one another and dynamic. It is therefore unreasonable to expect simple, definitive answers to the overtly simplistic question of random or nonrandom. In the circumstances, within the context of this communication it appears reasonable to rephrase the question in less global terms, and to ask the relative extents to which metastatic inefficiency may be accounted for by random and/or nonrandom processes focused on cancer cell properties.

METASTATICSUBPOPULATIONS On the basis of a series of ingenious experiments in which mouse B16 melanoma cells were exposed to a series of sequentiaI in vivo and in vitro selections, Fidler (1973) established a number of B16 lines. When injected into the tail veins of mice, the cells consistently gave

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rise to different numbers of pulmonary colonies, which sometimes (Weiss et aZ., 1982; Suzuki, 1983) correlated with the occurrence of natural” pulmonary metastases generated from subcutaneous or intramuscular tumors (Table 11).It was suggested that the cancer cells in solid tumors were genetically heterogeneous with respect to metastatic potential, and the metastases arose exclusively from subpopulations with a preexisting metastatic phenotype (Fidler and Kripke, 1977). As shown in Table 11, when lo5 cancer cells of the “highly” metastatic B16 F10 subline were injected into the tail veins of mice, they gave rise to a median of only 103pulmonary colonies, which indicates that even in lines selected for high metastatic or colonization potential, the level of efficiency was only 0.1%. This example of metastatic inefficiency can be interpreted in two ways. One alternative is that only 0.1% of the selected line has the capacity to give rise to pulmonary colonies by a nonrandom process, which in turn implies that within the selected line this subpopulation could potentially be expanded by progression to account for the remaining 99.9% cells. The other possibility is that all of the cells in the subline could have the same colonization potential, but only 0.1% of them survived by apparently random processes. In addition, various combinations of random and nonrandom processes are possible. Using the same basic approach as that used to isolate metastatic variants, cells were isolated from both pulmonary metastases and the primary” tumors generating them, and injected into similar sites in fresh mice. On the one hand, if metastases arose from a small genetically determined subpopulation by a nonrandom process, then the tumors derived from these metastases might be expected to give rise to more metastases than those derived from the “primary” lesion. On the other hand, if the metastatic inefficiency were due to a random process, then no differences would be expected between cells from the metastases and the “primary” lesions. In some experiments, cells from metastases were indeed more metastatic then those from primary tumors (Barut and Klaunig, 1986; Maguida et aZ., 1980; Pal et al., 1983; Talmadge and Fidler, 1982); in other cases they were less metastatic (Milas et al., 1983). In experiments made with B16 melanomas, KHT osteosarcomas, the 3LL and T241 carcinomas, which were carefully controlled with respect to time and tumor mass, cells from metastases were neither consistently nor markedly more metastatic than those derived from primary tumors (Weiss et al., 1983a),in accord with other work (Eccles, 1980; Giavazzi et al., 1980; Mantovani et al., 1981; Eccles et al., 1980; Alexander, “

“

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1982). In a tabulated review (Chambers et al., 1984) of 40 separate experiments of this type made in different laboratories on 23 different murine tumors, 20 experiments showed that the metastases were more metastatic then cells from the corresponding primary lesions, but 20 did not. A similar wide spectrum of results was reported by Volpe and Milas (1988).However, even in experiments in which the metastases were more metastatic, the comparatively small differences did not account for the general level of metastatic inefficiency. At least some of the controversy in interpreting these results stems from criticism of the use of “antique” cell lines even though Fidler’s original selection of successive cell lines for metastasis (1973) was made on the “antique” B16 melanoma. However, it should be noted that cyclic transplantation may exert a selective growth pressure, associated with increased metastatic ability in cells derived from both ‘. primary” tumors and their metastases (Risely and Sherbet, 1987). -Another interpretive complication relates to the use of cancer cells with a history of cultivation i n ljitro (Woodruff and Hodson, 1985; Ossowski and Reich, 1980, 1983; Hand et al., 1983; Rubin and Chu, 1984) in experiments of this general type (Weiss, 1984; Hoon et d., 1986). Some of the aforementioned problems are avoided in Vaage’s study (1988) of spontaneous mammary carcinomas in C3H/He and C3HF/ He mice, which metastasize to the lungs. When implanted into the mammary fat pads of fresh animals, tissues from autochthonous metastases and primary tumors had similar potentials for spontaneous metastasis. In addition, primary tumors and metastases, transplanted as parallel lines through consecutive generations, also maintained similar average metastatic potentials. In an analogous manner to that reported by Kisely and Sherbet (1987), changes in metastatic potential occurred in some tumors during serial passage, but the changes were similar in tumors derived from primary lesions and metastases. The failure to demonstrate consistent, major differences in metastatic potential between cancer from metastases and their primary tumors, parallels attempts to demonstrate other consistent differences between them, over and above those observed in cells growing in different regions of the same tumor, or differences associated with age-related volume (Weiss, 1980, 1985d). In this context, regional differences include those associated with hypoxia, accumulation of lactic acid with regional variation in pH, and abnormal levels of metabolities or other elements. In addition, partly because of limitation in blood supply, necrotic regions are common, and necrosis-associated products may af‘fect local invasion and cancer cell detachment from solid tumors (Weiss, 1977a).

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As emphasized elsewhere, this failure to demonstrate differenceswhile not providing support for a major role of dedicated, minor subpopulations of cancer cells in the genesis of metastasis-neither necessarily disproves this hypothesis nor excludes a comparatively minor role for it. However, these negative observations cannot be dismissed, and their consideration has revealed additional degrees of complexity in the interpretation of this important element of the metastatic process, in relationship to metastatic inefficiency. It is hardly necessary to note that the concept of heterogeneity of cancer cells within tumors (Heppner, 1984) has not been in question since first described in morphologic terms by Virchow (1858).As postulated by Foulds (1949, 1969) and others, cancers appear to progress from “bad to worse,” and the rarity of regression in authenticated cases of cancer (Lewison, 1976) supports Fould’s view that, in general, progression is an irreversible process, although at cancer cell level it may be reversed or accelerated, for example by demethylation (Babiss et al., 1985). Nowell (1976,1982)has argued that progression results from an acquired genetic instability in cancer cells with the “sequential selection of variant subpopulations,” and that selection for invasion and metastatic capacity is part of this process. Cifone and Fidler (1981) demonstrated that the mutation rates, with respect to 6-thiopurine or ouabain resistance, were 3- to 7-fold higher in clones of the UV-2237 fibrosarcoma with “high” pulmonary colonization potential, than clones with “low” potential. These results were interpreted to support the concept that mutational events could at least partially account for tumor progression with respect to metastasis. However, Kendal and Frost (1986)showed that in the series of cells studied by them, ouabain resistance was unrelated to the acquisition of the metastatic phenotype. Furthermore, Yamashina and Heppner (1985) and Kendal and Frost (1986)failed to correlate spontaneous mutation rates with metastasis. The hypothesis of genetic instability has been questioned (Frost and Kerbel, 1983)on the basis of observations that the changes relate to the expression of preexisting genes as distinct from new, mutationdependent products. Emphasis has been placed on the genetic instability of metastatic subpopulations of cells (Cifone and Fidler, 1981; Nowell, 1986)and its possible contributions to tumor progression and colonal evolution. However, as emphasized in the concept of clonal dominance (Kerbel et al., 1988), favorable genotypes may be stable (Kendal and Frost, 1986). If progression were a unidirectional process, going remorselessly from “bad to worse,” then transplantable tumors of the type referred to earlier-where 99.9% of cells injected into the bloodstream do not

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form tumors-should also become progressively more metastatic upon serial transplantation. In fact this is not the general case (Stackpole, 1983;Chambers et al., 1984;Volpe and Milas, 1988),although it sometimes occurs to a limited extent (Risely and Sherbet, 1987; Vaage,

1988). Histologic evidence of the genetic basis of progression comes from Broders’s observations (1925, 1958) on “grading” and malignancy; karyotypic “progression” (Wolman, 1983) and studies of a number of tumors, using the powerful techniques of flow cytometry and static cytophotometry, have confirmed a relationship between aneuploidy and poor prognosis (e.g., Ljungberg et aZ., 1986). In accord with Nowell’s concepts, aneuploidy is associated with some sort of selective advantage on cancer cells that eventually tend to dominate the tumor. However, multiple examinations of cancer cells taken from human tumors do not show reversion from aneuploidy to lesser degrees of ploidy (Sandberg and Yamada, 1966; Sandberg et al., 1967).Therefore, if aneuploidy confers selective advantage, and is causally related to metastasis, it is expected that metastases would contain a greater proportion of aneuploid cells than the heteroploid primary tumors from which they arose. This has not been observed. For example, a flowcytometric analysis of DNA aneuploidy in 365 primary and 291 metastatic human solid tumors revealed no major differences in frequency and degree of aneuploidy between the two groups (Frankfurt et aZ.,

1984). In reviewing the concept that metastases arise from subpopulations of stable metastatic variants, Ormerod and Hart (1987)conclude that as yet no exclusive properties have been identified that unequivocally characterize metastatic subpopulations, and that “the view of metastasis as a selective or non-random event remains to be proven.” Three hypotheses relating to randomness and selection are outlined in Fig. 3, and will be briefly discussed.

1. The Transient Metastatic Compartment One explanation for the failure to detect differences of the required magnitude between primary lesions and their metastases to account for metastatic inefficiency, is that if there is a significant nonrandom element to metastasis, by which a minority of cancer cells have enhanced metastatic capacity, then this enhancement is temporary. The term, transient metastatic compartment, was introduced (Weiss, 1979a) to indicate the view that although all of the viable cancer cells in a tumor have metastatic potential, not all of them can realize this potential at any one time. However, by virtue of location in a tumor (e.g., accessi-

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TRANSIENT METASTATIC COMPARTMENT W

Y

TUMORS

ALL CELLS POTENTIALLY METASTATIC

CLONAL DOMINANCE

DYNAMIC HETEROGENEITY

NOCELLS POTENTIALLY METASTATIC

HETEROGENEOUS

I

1

EPIGENETIC

METASTASES

W

m T

......

METASTASES

GENETICALLY DIFFERENT FROM PRIMARY TUMOR, SIMILAR TO EACH OTHER

i

Y

SOME CELLS POTENTIALLY METASTATIC HETEROGENEOUS

GENETIC (< 10-5/CELLIGENERATION

~

GENETICALLY SIMILAR TO INDIVIDUAL SUBPOPULATIONS IN PRIMARY TUMOR; DIFFERENT FROM EACH OTHER

X

CLONAL OOMINANCE

000000 GENETICALLY SIMILAR TO LATE PRIMARY TUMOR A I M EkCH OTHER

REVERSION

I < lO-’ICELLIGENERATIONl

METASTASES NOW SIMILAR TO PRIMARY TUMOR

FIG.3. The salient differences among three hypotheses relating random or nonrandom events to metastasis.

bility of disseminative routes) and/or random epigenetic events, at any one time the chances of individual cancer cells entering and surviving the metastatic process could be enhanced. In a new location these temporary attributes would be lost, and entry of individual cancer cells into a transient metastatic compartment would again depend on random circumstances. This hypothesis advanced a dynamic view of cancer cell populations in metastasis and accounted for the failure to observe significant enrichment of metastases, with cancer cells expression the metastatic phenotype. In postulating the transient metastatic compartment, consideration was given to modulation of metastasis related properties of cancer cells during different metabolic states and in relation to cell cycle. These are of particular importance because at any one time many tumors show topographic heterogeneity with respect to cancer cells, ranging from death and dormancy to active metabolism and proliferation, and this type of “physiologic” variation is often overlooked by those with subpopulation myopia. Metabolism-associated changes were observed in the metastasis-

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related properties of detachment (Weiss, l977b), adhesion (Weiss and Chang, 1973),and cell surfaces (Weiss, 197713).Cycle-related genes are variably expressed during the cell cycle and may be involved in replication (Denhardt et ul., 1986). Examples of cycle dependency include (1) increases in cancer cell surface protease levels observed just before and during mitosis (Hatcher et d . , 1976), which could enhance invasion (2) heterogeneity in the expression of some surface antigens (Czerniak e t al., 1984), which may be associated with proliferation (Leong et al., 1987) or host resistance (Sarkar et al., 1980); (3) cycle-dependent differences, which have also been observed in adhesion-mediated cell interactions (Elvin and Evans, 1983), as perhaps exemplified by cycle-dependent macrophage-mediated cytostasis (Hamilton et al., 1982). Other factors determining the probability of individual cancer cells entering the bloodstreani are the density, location, and susceptibility to invasion of blood vessels; vascularity, in turn, affects proliferation. In addition to “physiologic” (i.e., metabolic and cycle-associated) modulation and topologic factors, epigenetic changes must be considered that, while not altering the base sequence of cancer cell DNA, produce changes in its secondary structures; among these are DNA hypomethylation (Frost and Kerbel, 1983), gene amplification (Sager et ul., 1985), and gene rearrangement (Feinberg and Coffey, 1982; Sager, 1982).Epigenetic changes may not only modulate gene expression with respect to metastasis, but also impose some degree of heritability on this process. The concept of the transient compartment is supported by the demonstration of modulation of metastatic behavior by epigenetic stimuli (Frost and Kerbel, 1983; Takenage, 1984; Kerbel et al., 1984; Olsson and Forchhammer, 1984; Trainer et ul., 1985; Ishikawa et ul., 1987),which would account for a high rate (Chow and Greenberg, 1980; Harris, 1986) of random mobilization of cancer cells into a metastatic compartment, in the absence of structural genetic change.

2. Dynumic Heterogeizeity This hypothesis proposes that the majority of cancer cells within a tumor are genetically incapable of generating metastases, and that secondary lesions are derived from cancer cell variants, arising by a inutationlike process at a rate of lW5 per cell per generation (Harris et d.,1982; Hill et d.,1988). This high rate is apparently similar for the KHT niurine fibrosarcoma (Harris et al., 1982), the B16 melanoma (Hill c,t ul., 1988),and the MDAY-D2 tumor (Lagarde, 1983).Experiments of this type are subject to several criticisms: (1) Metastatic potential was

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determined by enumeration of pulmonary colonies after tail vein injection of cancer cells; by neglecting invasive inefficiency, this method overestimates metastatic potential. (2) In order to complete the experiments, clonal expansion in vitro had to be employed, and this increases the possibility of artifact (Elmore et al., 1983; Li et al., 1983; Morrow, 1948; also see earlier. (3)The question has been raised of the accuracy of the Luria and Delbruck (1943) technique of fluctuation analysis in this situation (Kendal and Frost, 1986). Mutation rates for NIH 3T3 and CBA SP-1 cells and their metastatic variants were of the order of lop8 per cell per generation, and no significant rate differences were detected between the cells and their metastatic variants. Taking the dubious view that the generation of ouabain resistance corresponds to overall genomic stability, the absence of differences indicates the other genetic or epigenetic events were responsible for (metastatic) progresssion (Kendal and Frost, 1986). The mutation like rates reported by Harris et al. (1982)are therefore possibly too high for the in vivo situation. According to the hypothesis of dynamic heterogeneity, metastases arise from dedicated metastatic subpopulations of cancer cells, which are genetically different from the majority of cancer cells in the primary tumor. The failure to detect such genotypic differences is attributed to a surprisingly high reversion rate (10-1-10-2 per cell per generation) in the metastases. Partial support for this hypothesis comes from the observation that 5-7 days after intravenous injection, KHT cells isolated from microscopic colonies in the lungs, exhibited a 50- to 100-fold increase in colonization efficiency compared with the parent population. Unfortunately, the “considerable variability in the data” (noted by Young and Hill, 1986), together with the small number of data points prevents critical comparison of metastatic efficiencies at subsequent times. Analogous experiments were made with 3LL tumors growing subcutaneously in mice (Weiss et al., 1983a). Small tumor cylinders were taken after 3,7,9, 15, 19, and 21 days and implanted into fresh recipients, which were killed 15 or 21 days later and their pulmonary metastases counted. At 15 days after implantation, significantly more metastases (3-fold increase) were present in recipients of 7-day than 19-day tumors. However, by 21 days significantly more metastases (2-fold increase) had developed in the 19-daythen the 5-day recipients. These observations, which overall show little or no difference in metastatic potential with tumor age, appear more in accord with the concept of the transient metastatic compartment than that of dynamic heterogeneity.

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3. Clonal Dominance Experimental evidence indicates that at the time of examination, both primary tumors (Esumi et al., 1986; Fearon et al., 1987) and metastases (Poste et al., 1980;Talmadge and Fidler 1982) are clonal in origin. Additional support for clonal origin comes from Kerbel and colleagues (Kerbel et al., 1988; Waghome et al., 1988; Korczak et al., 1988)using a spontaneous, CBA/J mouse mammary carcinoma (SP-l), which, when injected into the mammary fat pads, generates only one or two pulmonary metastases in 20-30% of mice. SP-1 cells were transfected with a bacterial plasmid containing a dominant selectable genetic marker (pSVzneo) and a retrovirus construct by means of single-step selection procedures, and large numbers of unique genetic markers were introduced to a defined cancer cell population. Southem gel analysis of tumors resulting from the injection of as many as lo4 uniquely marked clones indicated that within the limits of sensitivity of the technique (
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tases, this is in accord with the suggestion (Miller, 1983) that nonmetastatic cells may be “recruited” by metastatic cells to form separate metastases. In essence, the clonal-dominance hypothesis argues that as a primary tumor undergoes progression, “tumors which orginated from multiple independent transformed progenitors can ultimately achieve clonal homogeneity by the time of clinical tumor development” (Kerbel et al., 1988). However, if a tumor cell population is homogeneous or consists predominantly of one type of cancer cell, then on a probabilistic basis it follows that metastases will be generated exclusively or mainly by cells of this type, because they are the “only show in town!” In other words, all of the cancer cells present in tumors of this type have the potential to form metastases, whether or not this is realized, as proposed by Weiss (1979a). Furthermore, if at the clinical stages of disease, clonal homogeneity has been achieved, then the gross disparity between the large numbers of circulating cancers cells and the small numbers of metastases (i.e., metastatic inefficiency) can only be accounted for on a random basis (Weiss, 1983, 1986). V. The Molecular Biology of Metastatic Inefficiency This section is not concerned with the phenomenal advances made in the molecular biology of cancer. No attempt will be made to review exhaustively the molecular biology of metastasis, and my comments are intended only to orient the general reader in this field. The main purpose is to determine, on the basis of present knowledge, the extent to which genetic alterations in cancer cells account for metastatic inefficiency.

A. OVERVIEW A useful overview of the genetic changes inolved in the pathogenesis of bronchial carcinoma is provided by Minna et al. (1988). Carcinogen exposure, probably enhanced by inherited metabolic phenotypes, produces changes in lung neuroendocrine cells resulting in their autocrine growth stimulation, and probably paracrine stimulation of proliferation of other bronchial epithelial cells. Deletions and translocations in the 3p(14-23) chromosomal regions of these stimulated cells generated clonal abnormalities resulting in the exposure of recessive oncogenes. These and other genetic changes, including activation of myc oncogenes and other nuclear protooncogenes (e.g., p53 and c-jun), by mechanisms involving alterations in transcription, can gen-

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erate bronchial CIS. Additional genetic changes including-but not limited to-mutations of the rus family of oncogenes, and further deregulation of the myc family by gene amplification and rearrangements are associated with progression and metastasis. In the present context, at the macroscopic level, progression implies increased “aggressiveness” of a tumor that is manifest over a period of time, as increased rate of growth and local tissue destruction. The question therefore arises whether metastasis is a consequence of increased aggressiveness manifest in growth-associated phenomena and the passage of time, or whether it is essentially a separate, new process. A major breakthrough in understanding the molecular basis for growth control, or lack of it, in normal and cancer cells has resulted from the isolation and characterization of -20 different retroviral oncogenes (v-onc) and -50 of their normal cellular progenitors (c-om), the so-called protooncogenes. Many oncogene products appear to be involved in signal transduction between and within cells, and a number of protooncogenes encode proteins similar or identical to growth factors and their receptors, protein kinases and so on, the expression of which increases in response to extracellular stimuli (Weinberg, 1985; Bishop, 1986; Varmus, 1987; Rauscher et ul., 1988). The products of two protooncogenes, fos and j u n , participate in a nuclear protein complex that appears to regulate gene transcription. This complex responds to external stimuli and modulates expression of genes responsible for phenotypic change. It is the close association between oncogenes and the various identified aspects of transformation, including signal transduction, growth factors, and their receptors (Kinniburgh, 1986),that make it so difficult to discriminate between the role(s) of oncogenes in tumorigenesis and metastasis, and to separate the niolecular basis of each.

B. METASTASIS The term tumor progression implies changes in gene expression (Foulds, 1949, 1969) and the generation of clonal diversity (Nowell, 1976,1982). This engenders heterogeneity among the cancer cell population, with respect to a variety of phenotypes that may include metastatic capability. However, the precise genetic mechanisms involved in progression in general are not well understood, and at present the genetic mechanisms specifically involved in metastasis are even less well understood. Much of the confusion stems from the fact that enhancement of any or all of a very large number of cancer cell interac-

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tions with their host can influence both the extent and rate of metastatic development. The magnitude of this problem is well illustrated by Maslow’s tabulation (1989) of 60 reports of the characteristics of murine B16 melanoma-derived cell lines related to their colonization potential. Maslow has grouped nine metastasis-related traits: enzyme production, synthetic activities, cell surface properties, growth rate, drug resistance, cytoskeletal properties, immunologic properties, invasiveness, and other properties. Only listing once reports in which the same data by the same authors have been published on a number of occasions, but considering contradictory reports on the same cell lines by the same authors, a plethora of characteristics have been correlated with colonization potential. It is difficult at present to account for all of these tabulated differences in terms of a single or very smaIl number of metastasis-specific genetic determinants, even allowing for pleiotropic or cascade effects. Oncogenes and their products influence growth autonomy, which may be associated with the acquisition of metastatic potential (Chadwick and Lagarde, 1988). Although autonomous growth alone is unlikely to be the only requirement for metastasis, it is certainly a major one in that, as a result of metastatic inefficiency, a large input of cancer cells is required to drive the metastatic process. In addition, growth is an obvious requirement for the development of cancer cell emboli into micrometastases and then overt metastases, although this latter step also requires proliferation within the microvasculature, leading to neovascularization. In discussing growth in relation to growth factors it should be borne in mind that their role in regulating the abnormal growth of cancer cells is largely unknown, although the various factors appear to act in a coordinated manner. In the case of transforming growth factor /3 and its receptors, for example, their sequences have not been identified with a viral oncogene. Attempts to clarify the role of oncogenes in metastasis fall into two categories. First, comparisons have been made of cell lines or tumors with differing metastatic potential with respect to expression of oncogene protein (Kerr et al., 1986; Yokota et al., 1986) or gene amplification (Dolnick and Reznikoff, 1984; Pohl et al., 1988a). Second, the metastatic or colonization potentials of fibroblasts, or better, poorly metastatic cancer cells have been determined after transfection with genetic material from metastatic cancers. However, as noted by Nicolson (1987), Pohl et al. (1988b), Ormerod and Hart (1987), and many

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others, the findings are controversial and the mechanisms largely unknown. The existence of a metastatic gene was suggested by the widely guoted observation of Bernstein and Weinberg (1985) that the metastatic phenotype could be conferred on tumorigenic cells via transfection with DNA extracted from a human metastatic cervical carcinoma. These observations of linkage to a specific DNA fragment were not reproducible after many attempts by these workers (Bernstein and Weinberg, 1988). A major interpretive problem in relating oncogene expression to metastasis concerns the aberrant expression of genes and the substitution of one form of growth control by another. For example, Gallick et al. (1985) found that in 9 of 17 primary colonic carcinomas, substantially elevated levels of ~ 2 1 ‘ were ” ~ present compared with adjacent normal tissues. In contrast, in liver and other metastases from these lesions, 9 of 9 had reduced ~ 2 1 ‘ levels. ”~ It was suggested that although elevation of p21ra5may commonly occur in early primary lesions, in metastases there are more autonomous populations of cancer cells in which the role of ~ 2 1 ” ’is supplanted by other oncogenes. Most gene transfer studies to date have been made with oncogenes of the ras family, in which point mutations result in the production of proteins with altered sequences. Such production has been correlated with the acquisition of metastatic or colonization capacity by NIH 3T3 fibroblasts (Bradley et al., 1986; Greig et al., 1985; Thorgeirsson et al., 1985) or into lines of “nonmetastatic” murine mammary carconoma (Vousden et al., 1986;Waghorne et al., 1987).In experiments with 3T3 cells, allowances must be made for the spontaneous development of tumorigenicity and metastatic capacity (Greig et al., 1985), and in all cells for increases in metastasis-related properties produced by the transfection technique itself (Kerbel et al., 1988; Wallace et al., 1988). However, in most reported transfection studies, the key control experiment of insertion of genetic materia1 with “antisense” orientation was not made. Hill et al. (1988) examined individual clones of NIH 3T3 cells, transfected with human bladder cancer (T24) H-ras oncogene, for expression of p21 and “experimental metastatic ability in the immunodeficient chick embryo.” Whatever reservations one may have about the use of the NIH 3T3 lines of fibroblasts in this context, or the interpretation of experimental metastasis in the chick embryo, the results are interesting. They showed that the clones oftransfected cells were heterogeneous with respect to both “metastasis” and p21 expression, and that there was good (r = 0.85) correlation between these two

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parameters. In apparent variance with the high rate of loss of the metastatic phenotype ( 10-’-10-2 per cell per generation) expected from the dynamic-heterogeneity hypothesis (Chambers et al., 1984), highly metastatic variants were selected from metastases in this system, which also had elevated expressions of p21. Although Hill et al. cite a number of clinical reports in which ras gene copy number or p21 levels correlated with degree of malignancy, they also cite other reports that failed to establish these correlations. However, bearing in mind the multiplicity of influences on metastatic capacity in a wide variety of cancers, it would be simplistic to expect a universal, single “metastasis gene.” It is more profitable to ask how cellular p21 influences metastatic behavior in the system in which a positive correlation has been demonstrated; however, the mechanism is obscured at present by too much apparently conflicting information. Liotta (1988) considers that “the ras p21 product alters some general pathway leading to pleiotropic cellular changes,” which include increased degradative (invasion) enzymes, cell motality, and growth. However, enthusiasm for the universality of a metastatic mechanism consequent upon transition from the cellular to the oncogenic form of rus must be tempered by the fact that it is not applicable to all cell types. It is difficult to evaluate the suggestion that degradative enzymes invariably include cancer cell-released type IV collagenase, because although increased secretion was reported in NIH 3T3 fibroblasts transformed with the c-Ha-ras oncogene (Garbisa et al., 1987; Spinucci et al., 1988),Teale et al. (1988)observed no correlation between secretion of this enzyme and metastatic propensity in a well documented fibrosarcoma. In addition, some workers in this field dissociate growth and metastasis (Garbisa et al., 1987; Muschel et al., 1985). In contrast to the ras gene family, gene activation in the myc gene family occurs through gene amplification and rearrangement. Varley et al. (1987)have found independent amplification in c-myc and c-erbB-2 in human breast carcinomas, which correlate with very poor short-term prognosis. As discussed by Nowell and Croce (1988), chromosomal abnormalities, indicating gene amplification units have in a few cases been shown to involve human protooncogenes. This amplification of c-myc and N-myc has been associated with metastasis in certain neuroblastomas (Seeger et al., 1985). Similar amplifications in L-myc and the HER-2/neu oncogenes have been reported in small-cell carcinomas of the lung and adenocarcinomas of the breast, respectively. Verification of the generality of these changes will be of great interest. In more than one-third of 101 cancers examined by Yokota et al.

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(1986),there were alterations of c-m yc, c-Ha-rus, or c-myb oncogenes. The results suggested correlations between amplification of c-myc and “aggressive” primary tumors and metastasis, between allelic alterations of c-myb and tumor progression, and between increasing frequency of deletions of c-ruP’ and metastasis. Multiple oncogene anomalies were found in “particularly aggressive and widley disseminated cancers.” The observations were consistent with the concept of clonal evolution of cancer cell populations that generates variants with selective growth advantages in uivo as advocated by Duesberg (1985) and Klein and Klein (1985). This also serves to indicate the close connection between tumorigenesis and metastasis. While these and other reports undeniably link elevated expression of certain oncogenes with poor prognosis, cancer cell progression per se is not the only event contributing to poor prognosis, and the links between protooncogene activation, prognosis, and metastasis are indeed tenuous on the basis of present evidence. Thus, at present it is quite impossible to generalize on elevated levels of rus gene expression in human malignancy, and such elevations, when they occur, are not limited to cancers (Varley et al., 1987). In addition to rus, a structurally divergent group of serine-threonine or tyrosine kinase oncogenes can also induce the metastatic phenotype in NIH 3T3 cells (Egan et aZ.,1987). Cell lines transformed by the kinase oncogenes m s , r u , src, fes, and fms generally, but not invariably, formed more pulmonary colonies following tail vein injection into nude (BALBlc nuinu) mice than M S transformants. Single-step transformation assays with these kinase oncogenes in general make it impossible to dissociate proliferation from metastastic capacity, particularly in view of the role of these kinases in environmental signal transduction restilting in proliferation. However, Egan et al. suggest that unregulated alterations in the membrane-associated secondmessenger system resulting from the action of the rus or kinase group of oncogenes are important events in inducing metastasis. Regardless of the general validity of the nude mouse model, these experiments are important in showing that genotypic alterations involving a number of structurally diverse oncogenes can affect that postintravasation phase of metastasis. The main focus, to date, in determining the molecular biologic basis of metastasis has been on identifying determiniants associated with the possession and/or acquisition of the multifactorial metastatic phenotype. However, by analogy with loss of alleles in the genesis of retinoblastoma (Cavenee et d.,1983)and Wilms’ tumor (Koufos et uZ., 1984),there is the possibility that rather than being due to genetic gain,

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metastasis could be associated with loss, resulting in the failure of metastasis-inhibiting mechanisms (Schirrmacher, 1985). Metastasis-suppresssors must be viewed against the general background of genes inhibiting the expression of tumorigenic phenotype. In a timely review. Klein (1987)discusses the inhibition of expression evidenced by fusion of normal and cancer cells, in vitro phenotypic reversion of transformants, induction of terminal differentiation, loss or mutational activation of “recessive cancer genes,” regulatory sequences very close to certain oncogenes, and the inhibition of tumor growth products of adjacent noncancer cells. Klein considers that such tumor suppressor genes will probably be recongized as being at least as diversified as oncogenes, with which they may have interrelated functions. Analogous considerations may well apply to metastasis. Fusion of noninvasive, nonmetastatic T-lymphoma cells with normal T cells results in metastatic hybridomas (Roos et al., 1985), in which genes associated with metastasis have been localized to human chromosome 7 (Collard et al., 1987). However, in some systems the nonmetastatic phenotype is dominant in hybrids (Ramshaw et al., 1983).By use of the technique of subtractive hybridization, differences in gene expression were examined between metastatic and very poorly metastatic clones of the rat DMBA-8 mammary adenocarcinoma line, which differ 1000 fold in colonization potential (Dear et al., 1988). A number of homologous mRNAs were expressed at a 20-fold higher level in the “nonmetastatic” than in the metastatic cells. The differential expression was not due to gene amplification; there was no evidence of gene rearrangement, and the differences were not related to artifacts associated with cell culture. Furthermore, since injection of the nonmetastatic and parent lines of DMBA-8 cells into immunosuppressed hosts does not potentiate metastasis, it was considered unlikely that the mRNAs associated with the nonmetastatic phenotype coded for tumor antigen(s). The apparent deletions ofc-Ha-ras or c-rnyb loci observed by Yokota et al. (1986) may be random or related to the malignancy. If related, they are consistent with the view that loss of antioncogenes or suppressor genes (i.e., normal regulatory genes) may be associated with cancer progression (Klein and Klein, 1985).Evidence for a gene associated with low metastatic potential also comes from the observations of Steeg et al. (1988) on murine K-1735 melanoma cell lines and Nnitroso-N-methylurea-inducedrat mammary carcinomas. Thus, RNA levels of the NM23 gene were generally highest in cells and tumors of relatively low colonization potential. However, within five highly “metastatic” cell lines, the low NM23 RNA levels showed “a less

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dramatic correlation with metastatic potential,” which was accounted for by the suggestion that multiple genes or epigenetic factors determined metastatic potential. A notable attempt has been made by Elvin et al. (1988)to isolate and characterize cDNA clones representing mRNAs associated with tumor progression and metastasis in human colorectal cancer. The vast majority of cDNA clones corresponded to RNA sequences shared by normal and carcerous colon and normal liver. The clones identified by Elvin et al. suggest that the development of cancer cell populations in colorectal tumors that metastasize to the liver is d u e to subtle alterations in a number of genetic loci, as distinct from the aberrant expression of one or two genes. The results do not permit distinction between any of the possible macroscopic mechanisms of alterations in genetic expression discussed elsewhere is this review. The fundamental question raised by this discussion is whether metastatic capacity is a separate property over and above tumorigenicity, or whether the two are inseparable. Apart from the progression of in situ lesions to invasive lesions in which, as discussed earlier, the mechanisms underlying the transition are by no means clear, in naturally occurring human cancers tumorigenicity is almost invariably associated with metastatic capacity. The one major exception is this rule is basal cell epithelioma, which is highly invasive but is very rarely metastatic. Thus, although transplantable murine cancer cells have been described that are tumorigenic and reputedly nonmetastatic in mice or rats (Tao and Burger, 1982; Ghosh et al., 1983; Layton and Franks, 1986), their relevance to human disease is uncertain although they may correspond to lesions frozen in the in situ stage. In addition, many reports on the failure of cancers to metastasize or to form colonies after intravascular injections in mice are based on inspection of target organs by the naked e y e or under a low-power dissecting microscope, where tumors <0.1 mm in diameter are missed. Comparatively uncommonly, negatioe reports have been confirmed b y microscopic examination of serial sections, and in the present context, confirmation by bioassay is rare. The necessity for multiple types of assays has been advocated by Tao and Burger (1982). Although emphasis has been on the often apparently contradictory relationship between metastasis and oncogenes, in some reports with sequences specific for metastasis, Mareel and van Roy (1986), among others, have raised the possibility of metastasis-associated genes. The influence of non-cancer- and non-metastasis-specific products on different phases of the metastatic process has been discussed in a monograph (Weiss, 1985a-d).

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C. METASTATIC INEFFICIENCY The evidence briefly reviewed in the previous section indicates that genetic change can result in acquisition or, more usually, enhancement of the metastatic potential of cell populations. Of the many possible mechanisms involved in enhancement, some appear to be initiated by change in individual cancer cells, against the background of paraneoplastic change in the host. The major question to be addressed in this section is the extent to which the degree of nonrandomness introduced by these changes modifies metastatic inefficiency. Particularly relevant to invasive inefficiency are the experiments of Vousden et ul. (1986), in which MTI C1.517 mouse mammary carcinoma cells were transfected with activated C-Ha-rus-l oncogene cloned from EJ/T24 bladder carcinoma, and a &fold increase in metastatic activity compared with controls was observed following their subcutaneous injection into syngeneic mice. This modest increase was associated with an increased level of expression of c-Ha-rus-1-encoded p21 protein in the transfectants and, with one exception, their metastases. However, in contrast to “natural” metastasis, following intravenous injection of pSV2neo and pSV2neo-EJ cells, similar numbers of lung colonies were obtained, implying that the effect of the activated rus gene occurs in events leading to intravasation, although the role of p21 gene products in these events has yet to be defined. However, in the case of MTI C1.37 cells, the effect of the activated gene product is dependent neither on induction of tumorigenicity nor on survival and seeding of intravasated cells, but on the ability of cells to escape from the primary tumor; this property also can be modulated by many factors dependent on the properties of both cancer and noncancer cells (Weiss and Ward, 1983; Weiss, 198%). Vousden et ul. (1986) demonstrated that oncogene products are involved in the “escape” of cancer cells from their parent primary cancer and suggested that similar genetic mechanisms could be involved in the progression of in situ lesions to invasive lesions. Although this mechanism is not expected to be utilized by all cancer cells (Muschel et al., 1985), failure to intravasate has been identified as a cause of metastatic inefficiency (Weiss et ul., 1982). It is not possible to extrapolate these observations to progression from in situ to invasive carcinoma because the time base in CIS for activation of rus or other genes by point mutation, for example, is unknown; as discussed earlier, however, this may take years in humans. It must be borne in mind that the appropriate control cells in fact had metastatic potential and that

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the 3-fold enhancement of metastasis demonstrated by Vousden et al., is very modest in the present context. Observations relevant to the transition from superficial (in situ)carcinomas to invasive lesions come from a study of the relationship of tumorigenic, growth-associated processes to invasion and metastasis. In this, epidermal growth factor (EGF) receptors were compared in invasive (pT3) transitional-cell carcinomas of the bladder and superficial lesions (pT1 and pTa). By means of immunohistochemical techniques based on mAb against the EGF receptor, Neal et al. (1985) observed that significantly more invasive tumors (21/24) stained positively than superficial tumors (7/24). Analogous observations have been made with breast cancers (Sainsbury et uZ., 1985). However, the presence of EGF receptors on cancer cells does not necessarily mean that they are dependent on EGF for growth, and Neal et al. conclude that the presence of receptors represents “one feature of the genetic alteration that orchestrates the behaviour of the malignant cell.” In associating the metastatic phenotype with progression, the fact that large numbers of patients with cancers measuring several centimeters in diameter are cured by local surgical resection indicates that a considerable period of time is required for the development of metastases. On the one hand, this extended time frame could indicate that the acquisition of the metastatic phenotype is a lengthy process, as does transition from in situ to invasive lesions. On the other hand, it could indicate that a very high level of metastatic inefficiency operates on cancer cells with the metastatic phenotype. The observation of Bradley et al. (1986), that the EJ rus gene can confer a lung colonization (“metastatic”) phenotype on 3T3 cells within 3.5 cell generations after transfection, without selection by clonal growth in vitro, is therefore surprising at first sight. Bradley et at. suggest that once a cell acquires enough activated or normal ras gene product to become transfornied, it virtually simultaneously acquires metastatic capacity. “It is as if a pleogenic master switch is thrown that immediately turns on many other genes.” They also emphasize that it is not known whether such rapid switching can occur in vivo or with other systems, and in this ras- mediated, single-step mechanism, it is not possible to discriminate between tumorigenicity and metastatic capacity. One measure of the effect of phenotypic induction on metastatic inefficiency comes from experiments designed to identify sequences associated with a metastatic phenotype. Radler-Pohl et al. (1988)transfected a mouse bladder carcinoma cell line (BL) with DNA fragments isolated from human colonic carcinoma metastases in the liver. It was observed that cell populations carrying these human sequences

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197

caused more metastases in syngeneic mice than controls transfected with calf thymus DNA. It would have been more interesting to have used human liver DNA as a control! Cancer cells isolated from the metastases in mice retained the highly metastatic phenotype on transplantation into new recipients. In male recipients receiving subcutaneous injections of lo6 cancer cells, 30% of animals developed metastases after injections of BL cells, 25% after receiving BL cells transfected with thymus DNA, and 60%and 55.6% after receiving cells transfected with DNA fragments from hepatic metastases. In female mice, the differences were amplified. After intravenous injection (Pohl et al., 1988a), organ colonization was also enhanced. These workers therefore concluded that enhancement of both metastatic and colonization potential rule against the type of change limited to release enhancement as reported by Vousden et al. (1986). The influence of genetic change on the Ievel of metastatic inefficiency can be estimated from studies by Pohl et al. (1988a) in which a murine bladder carcinoma cell line with low colonization potential was transfected with the nuclear oncogene p53. By 24 days after tail vein injection of 5 x lo5 cells, significantly more lung colonies were observed in animals transfected with p53 than in appropriate controls. Analogous results were reported by Egan et al. (1987), in which a number of different transforming genes increased the lung colonization capacity of NIH 3T3 cells following tail vein injections into nude (BALB/c female nu/nu) mice. Both sets of results, which are summarized in Table V, indicate that even among transfectants there is a very high level of inefficiency (>99.9%) in the postintravasation phase of metastasis, in populations in which all injected cancer cells have been transfected and are homogeneous with respect to expression of the transfected genes. The nonrandom increase in lung colony formation is overshadowed by the non-transfection-dependent, presumably random destruction of cancer cells resulting in the high level of inefficiency.

D. PERSPECTIVE In spite of apparently contradictory evidence, there are unmistakable links between genotypic change, dependent and independent alterations in phenotypic expression, and metastatic capacity. Viewed against the magnitude of metastatic inefficiency, in preexisting cancer cells only comparatively modest changes in metastatic capacity can be

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TABLE V EFFECTOF TRANSFECTION ON COLONIZATION INEFFICIENCY

Cell line Bladder carcinoma“ BL gpt (controls) BL gpt CLB (p53 transfectant) BL gpt C L l l (p53 transfectant) NIH 3T3” NIH-3T3 NIH-3T3 clone 7 (MoMuLv) Transforming gene: N(261.8) (c-H-rus) Mos 2 (v-ntos) Mos 3 (v-ntos) Srcl (v-src) Src2 (v-src) Fesl (v-fos) Fms 1 (v-fms)

Numbers of lung colonies per mousec

Lung colonization inefficiencyd

0 (0-14) 3 (0-21) 5.5 (0-115)

>99.9999

0.5 ? 0.3 0.0 I 0.0

99.9998 100

32 I 2 1 62 2 27 1.8 ? 0.8 59 5 25 6.8 I 1.3 178 4 38 110 2 45

99.9893 99.9790 99.9994 99.9803 99.9977 99.9407 99.9633

99.9994 99.9989

Data from Pohl et al. (1988a); 5 x lo5 cells by tail vein injection. Data from Egan et al. (1987); 3 x lo5 cells by tail vein injection. Values for bladder carcinoma cell lines are median (range);valnes for NIH 3T3 cell lines are mean t SE. Percentage of injected cells NOT forming lung colonies. a

accounted for in terms of molecular genetics. Thus, on the basis of present evidence, which is admittedly incomplete, these modest changes do introduce a nonrandom element into the otherwise random process of metastatic inefficiency. The effects of this nonrandom element should not be overstated. Some perspective can be given to the effects of genetic nonrandomness in relation to metastatic inefficiency, by comparison with the effects of nongenetic random changes in cancer cell populations. Thus, a number of observations have shown that clusters of circulating cancer cells have greater metastatic capacity than similar numbers of single cells from the same population. The data of Liotta et al. (1976) on the T241 mouse fibrosarcoma have been analyzed in this respect (Weiss, 1982).Thus, following intravenous injection of lo3single cells, pulmonary colonies developed with a calculated efficiency of 0.01%; following injections of lo3cells in clusters of 5 to 7 cells, the efficiency increased to 1.25%. Changes of this order match those obtained by trans fection.

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VI. Consequences of Metastatic Inefficiency

Although the metastatic process is inefficient on both a temporal and numerical basis, with the exception of basal cell epitheliomas, total inefficiency is seldom (if ever) achieved, and metastasis and its sequelae are the major cause of death in patients with cancer. The long time taken for progression of in situ cancer to invasive lesions provides very wide diagnostic and therapeutic windows, and recognition of this in defined groups of patients has resulted in the recommendation of less frequent diagnostic procedures. Thus, in 1980, the American Cancer Society recommended that “all asymptomatic women age 20 and over, and those under 20 who are sexually active, have a Papanicolaou test annually for two negative examinations and then at least every three years until the age of 65.” This is a change in the previous recommendation of a universal annual test. Occult primary cancers provide an interesting exception to the general rule that metastatic inefficiency leads to the sequential diagnosis of first primary lesions then metastases. In this condition, small primary lesions that are often too small to visualize by standard diagnostic procedures generate diagnosable metastases, which are responsible for the initial signs and symptoms of the disease. These “unknown” primary lesions, which may represent accelerated progression, are not rare and are the eighth and fifteenth most common cancer “site” in two large tumor registries (Douglas, 1982). An important consequence of metastatic inefficiency is seen in the development of organ patterns of involvement. Cancer cell traffic studies in mice indicated that they majority of cancer cells injected into the portal vein were arrested and rapidly killed in the liver, and only very small numbers of viable cells were released from or passed through the liver to seed the lungs via the inferior vena cava (Weiss et al., 198313).It was therefore suggested that in colorectal carcinomas, only subtumorigenic doses of cancer cells made direct transit of the liver to the lungs; moreover, of the few arriving in the lung, most were arrested and killed there, and very very few made direct transit of the lungs to seed others organs via the arterial route. The undeniable fact that in patients with a history of colorectal carcinoma metastases do occur in the liver, lungs, and other organs could be explained by a cascade process of the type formulated by Viadana et al. (1978). In this process, secondary liver metastases seeded from primary colorectal carcinomas act as “generalizing” sites for seeding tertiary lung metastases. In turn, the tertiary lung metastases act as generaliz-

200

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ing sites for seeding (quaternary) arterial metastases in other organs. This sequential pattern of “metastasis of metastases” spares individual cancer cells from running the gauntlet of multiple traumatic encounters with the microvasculature (Weiss et at., 1988a,b) of more than one organ in any one seeding process. The feasibility of this inefficiency-driven metastatic cascade was tested by analysis of 1541 autopsies on cases with a history of colonic carcinoma. If the cascade hypothesis is correct, then in general, lung tnetastases should not be found in the absence of liver metastases, and arterial metastases should not be found in the absence of lung metastases. As shown in Table VI, in 85%of cases (group A) without detectable liver metastases, none were found elsewhere; in 73% of those (group B) with liver metastases and no detectable lung metastases, none were found elsewhere. I n addition, groups were identified with liver and lung metastases, without (group C ) and with (group D) metastases in other organs. In group A, the 15% of cases without detectable liver metastases but with metastases in other sites can be at least partially accounted for by false negative reports of liver involvement (Weiss and Harlos, 1986) and by vascular anatomy (e.g., direct bone marrow seeding via the

TABLE VI

METASTATICPATI-ERNS OBSERVED AT AUTOPSYI N 1541 CASESWITH

A

HISTORY OF COLORECTAL CANCER

Metastases detected in Croup

Liver

Lungs

Other

A (n=869)

-

-

-

-

-

+ +

+ +

+ +

-

-

-

+

c ( n = 112)

+

-

D (n = 140)

+

+ +

-

B ( n = 420)

“ See text discussion.

+

Percentage per group“

85 7 4 4 100 73 27 100

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201

paravertebral venous plexus). In an analogous manner, the 27% of cases in group B, in which metastases in other sites were found in the absence of detectable lung metastases, could be at least partially accounted for in terms of false negative reports of lung involvement. This discrete, sequential pattern of “metastasis of metastases” therefore appears due to metachronous seeding, within the limits of sensitivity of the autopsy procedure, which depends on naked-eye recognition of lesions, supplemented by histologic examination of random samples and clinical data. An alternative explanation for the sequential pattern development is that the liver, lungs, and other sites were seeded at the same time (i.e., synchronous seeding) by progressively diminishing numbers of cancer cells released from the primary lesions. If small enough numbers of cancer cells seeded the lungs and other organs, the resulting lesions might not be detectable by standard diagnostic or autopsy procedures, in the presence of overt hepatic metastases. Human evidence in favor of metachronous seeding is indirect and comes from autopsy data on cases with a history of squamous-cell carcinoma of the esophagus or adenocarcinoma of the rectum or colon. In the groups with a history of colonic (Weiss et al., 1986), upper rectal, or lower esophageal carcinomas, hematogenous dissemination occurs principally via the portal system to the liver, then to the lungs via the systemic circulation, and to “other” sites via the arterial system. In this group, with relatively few exceptions, there was statistically significant correlation between the incidence of metastatic involvement of “other” sites and the blood flow per gram (ml/min/g) in these target organs; this parameter of vascular density could influence both delivery and growth of cancer cell emboli. In contrast, in the groups with primary carcinomas of the lower rectum and upper esophagus, where initial bloodborne dissemination occurs via systemic veins to the lungs, followed by arterial dissemination, no correlation was obtained between the incidence of metastatic involvement of “other” sites and their blood flow per gram. One explanation of the difference in metastatic behavior between the two groups is that growth in the liver, associated with metachronous rather than synchronous seeding, in some way modified the subsequent metastatic behavior of the cancer cells. Such site-induced modifications have been demonstrated with the transplantable colon-26 carcinoma in mice, which was grown in the liver, lungs, or liver-then-lungs, and then injected into the bloodstream of fresh recipients via the left ventricle, portal vein, or tail vein. Different patterns and degrees of colonization of 11 different target organs were observed between cells with histories of prior growth in the different sites (Weiss and Ward, 1988).

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Evidence in favor of synchronous seeding is not generally compelling. Although it has been demonstrated in rodents that tumorigenic cancer cells pass through the pulmonary capillary bed and can be recovered from the arterial circulation (Zeidman and BUSS,1952), the significance of this observation in the present context is not clear. Thus in reviewing the evidence Wallace et al. (1978)conclude that following injection of cancer cells into the systemic circulation, extrapulmonary colonies are rare, and following injection into the portal vein, extrahepatic colonies are also uncommon. The situation is rendered more complex by the observation that some types of cancer cells, when injected systemically, appear to pass through the lungs to seed other organs preferentially (Nicolson, 1988).In our own experience, histologic examination of the lungs in at least some of these cases reveals microscopic colonies not visible upon inspection under the dissecting microscope. However, it seems unlikely that these very small lesions could have acted as generalizing sites for metastasis to other organs. At present, it is not possible to exclude either metachronous or synchronous seeding in the genesis of metastatic patterns; both are driven by metastatic inefficiency. However, there is no reason why the two mechanisms should be exclusive, and their relative importance appears to be determined by the time frame of the observations in relation to other events. Thus, patients may die with a history of primary colorectal carcinoma, with or without detectable hepatic metastasis, and no evidence of involvement of the lungs or other organs. In these cases, micrometastatic disease in the lungs or other organs was of no clinical significance. However, if patients with a history of colorectal cancer receive local therapy for diagnosed or presumptive liver metastases (Weiss and Mayhew, 1985; Aigner et al., (1988), then the presence of metastases in other organs will make the therapy palliative, since with an extended time frame these extrahepatic lesions will become clinically significant. The relative clinical significance of metachronous seeding, which favors curability, and synchronous seeding, which does not, will be determined by the response to effective local therapy of patients in whom overt metastatic disease is limited to first-encountered target organs. VII. Conclusions The vast majority of cancer cells participating in the metastatic process are killed; on the basis of present evidence, killing occurs in a predominantly random manner.

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The short-term effect of metastatic inefficiency is to delay the onset of metastasis and thus increase the chances of diagnosis of primary tumors in the premetastatic stage, thereby improving the results of treatment by stage migration. The intermediate effects of metastatic inefficiency are to impose a sequential pattern on metastasis, due either to metachronous and/or synchronous seeding of target organs. The former may permit the cure of limited metastatic disease by local therapy; the latter would permit its palliation by such therapeutic modalities. The long-term consequence of metastatic inefficiency is the retardation rather than the prevention of metastasis. ACKNOWLEDGMENTS My thanks are due to Dr. Alan Kinniburgh (Department of Molecular Gentics, RPMI) and Dr. Dorothy Glaves-Rapp (Department of Experimental Pathology, RPMI) for helpful, critical discussion. Some of my own work was supported by grant PDT-273 from the American Cancer Society.

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GROWTH-REGULATORY FACTORS FOR NORMAL, PREMALIGNANT, AND MALIGNANT HUMAN CELLS IN VITRO Meenhard Herlyn, Roland Kath, Noel Williams, lstvan Valyi-Nagy, and Ulrich Rodeck The Wistar Institute of Anatomy and Biology, Philadelphia, Pennslyvania 19104

I. Introduction 11. Growth of Normal Human Cells I n Vitro 111. Human Melanocytic Cells as a Model for Studies on Tumor Progression A. Clinical and Pathohistological Observations B. Phenotypic Characteristics of Melanocytic Cells Isolated from Different Stages of Tumor Progression IV. Growth Factor Independence of Human Tumor Cells from Metastatic Lesions V. Autocrine Growth Stimulation of Human Tumor Cells and Strategies for Growth Inhibition A. Secretion of Growth Factors and Expression of Growth Factor Receptors by Melanoma and Colorectal Carcinoma Cells B. Approaches for Inhibiting Autocrine Growth Stimulation VI. Summary References

I. Introduction Recent advances in tissue culture techniques have allowed the delineation of factors that control the growth of normal human cells isolated from a variety of tissues. The comparison of growth requirements for normal and malignant cells within the same cell lineage has shown that the two cell types differ in their need for exogenous growth and differentiation factors. This review will focus on normal skin cells (i.e., melanocytes, keratinocytes, and fibroblasts), precursor nevus cells, and primary and metastatic tumor cells. The main tumor systems to be discussed are melanoma and colorectal carcinoma. We will summarize the biologic aspects of tumor progression, with emphasis on factors that control growth of cells isolated from lesions at different stages of tumor progression. Whether the same factors that are active in cultured cells also regulate cell growth in vivo is not known. Little is also known about the molecular mechanisms underlying the complete growth autonomy of metastatic cells. The chromosomal abnormalities associated with tumor progression in the melanocytic system (re213 ADVANCES IN CANCER RESEARCH, VOL. 54

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viewed in Balaban et al., 1986) have not yet resulted in the identification of corresponding gene amplifications, rearrangements, or deletions (Linnenbach et al., 1988). Antigenic changes associated with different stages of melanoma tumor progression have been reviewed (M. Herlyn et u l . , 1987a; Herlyn and Koprowski, 1988). II. Growth of Normal Human Cells in Vitro

Normal human cells derived from a variety of tissues have been cultured in recent years (Weinstein, 1983, for review). Originally, skin fibroblasts were studied because of their successful growth in minimal essential medium (MEM) supplemented with high serum concentrations. Standardized procedures were then developed for the culture of epithelial cells from skin (i.e., keratinocytes: Tsao et al., 1982; Shipley and Pittelkow, 1987), lung (i.e., bronchial epithelial cells: Lechner et al., 1982), mucous membranes of the oral cavity (Arenholt-Bindslev et al., 1987; Southgate et al., 1987), kidney (Chang et al., 1986), bladder urothelium (Reznikoff et al., 1983, 1987; Dubeau and Jones, 1987), breast (McGrath and Soule, 1984; Soule and McGrath, 1986; Yang et al., 1987; Hammond et al., 1984), trophoblasts (Truman and Ford, 1986), and epidermal melanocytes (Eisinger and Marko, 1982; Cilchrest et al., 1984; Wilkins et al., 1985; Halaban et al., 1987; M. Herlyn et al., 1987b, 1988). Endothelial cells of capillaries have also been successfully cultured (Rupnick et al., 1988; Stein and St. Clair, 1988; Hoshi et al., 1988). Table I gives examples of components critical for successful cell culture in each of three categories: base medium, growth factors and other supplements, and substrates. Base media consisting of amino acids, vitamins, fatty acids, salts, sugars, and buffer systems have been most successfully developed by Richard Ham and his associates. Ham’s F10 and F12 base media are widely used for a variety of epithelial and nonepithelial cells. Modified versions of Ham’s FlO medium are the MCDB media (MCDB 104, MCDB 153, MCDB 202, MCDB 170, and MCDB 131), which are used for the culture of fibroblasts, keratinocytes, melanocytes, endothelial cells, breast epithelial cells, and bronchial epithelial cells. To support growth at densities suitable for obtaining large cell numbers, other laboratories have mixed these media with “richer” media or increased individual components. Growth factors and supplements most widely used in serum-free media are insulin, epidermal growth factor (EGF), transferrin, and hydrocortisone. Because fibroblast growth factor (FGF) and related

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TABLE I GROWTHSUPPLEMENTS FOR NORMAL HUMAN CELLS Base medium (predominantly used for cells) Minimal essential medium (MEM) and modifications (fibroblasts) RPMI 1640 (peripheral blood cells) Ham's F10 or F12 and mixtures with other media (epithelial cells and others) MCDB 153 with modifications (keratinocytes, melanocytes) MCDB 104 (fibroblasts) M199 (endothelial cells) MCDB 131 (endothelial cells) MCDB 170 (breast epithelial cells)

Growth factors and hormones Insulin and related growth factors Epidermal growth factor Transferrin Fibroblast growth factor and related growth factors Lymphokines (IL-2, GM-CSF, etc.) Hydrocortisone and related hormones Triiodothyronine 0'31, a-melanocytestimulating hormone, estradiol

Other additions CAMPenhancer (cholera toxin, forskolin) Protein kinase C activator (phorbol ester TPA) Ethanolamine Trace elements (selenium, iron, copper) Serum and serum albumin Pituitary and brain extracts

Substrates Gelatin (fibronectin) Collagen Laminin Extracellular matrix of cells and other equivalents

growth factors are major components in bovine pituitary and brain extracts, media for keratinocytes, melanocytes, and endothelial cells often contain crude extracts of either pituitary or brain. However, since many growth factor genes have been cloned, highly purified growth factor preparations have now become available. Substrates are often required for optimal attachment of normal human cells grown in serum-free medium. Epithelial cells including keratinocytes, urothelial cells, and epithelial cells of mucous membranes adhere best to collagen type I preparations, whereas melanocytes and endothelial cells adhere better to fibronectin. Complex extracellular matrix preparations are well suited for attachment of normal cells of different origin but do not necessarily stimulate cell growth.

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Ill. Human Melanocytic Cells as a Model for Studies on Tumor Progression A. CLINICAL AND PATHOHISTOLOGIC OBSERVATIONS

Benign and malignant lesions of human melanocytes present one of the best-characterized model systems for the study of tumor progression in sitzl. Five major histopathologically and clinically identifiable steps of tumor progression have been described, from an initiated and promoted melanocyte to a primary malignant melanoma that has competence for metastatic spread (Clark et al., 1984,1986).Common melariocytic nevi are postulated to represent the first step in this nonobligatory progression pathway (Table 11).Most of these lesions are stable, but a few may develop histologically and cytologically atypical changes characterized as “melanocytic dysplasia” (step 2). Such “dysplastic nevi” are also mostly stable, but a few may progress to form an inexorably growing superficial plaque that is termed a malignant melanoma (step 3 ) . In this stage, the melanoma may be either confined to the epidermis or invasive but does not show signs of rapid focal growth. This stage has clinically been termed radial growth phase (RGP) because the plaque tends to expand outward along the radii of an imperfect circle. In the next stage of a primary melanoma, a nodule appears within the antecedent plaque of poorly proliferating melanoma cells. This nodule is characterized by expansive and massive growth, often elevating the epidermis and/or invading the dermis, a

TABLE I1

TUMOR PROCRESSION I N THE HUMAN h i E L A x w ’ I T I C SYSTEM Step

Melanocytic lesion

I

Couimon acquired and congenital nevus (no cytologic atypia)

2

Dysplastic nevus with persistent architectural and cytologic atypia Radial growth phase (RGP) of primary nielanonia (no competence for metastasis) Vertical growth phase (VGP) of primary melanoma (competence for metastasis) Metastatic melanoma

3

4

5

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process that clinically has been termed the vertical growth phase (VGP)(step 4).Metastasis is the final step oftumor progression (step 5). B. PHENOTYPIC CHARACTERISTICS OF MELANOCYTIC CELLSISOLATED FROM DIFFERENT STAGES OF TUMOR PROGRESSION

For the characterization of cultured melanocytic cells derived from normal, premalignant, and malignant tissues we have chosen the following parameters: (1) morphology; (2) life span in culture; (3) chromosomal abnormalities; (4) saturation density; (5) anchorage-independent growth of cells in soft agar; (6) tumorigenicity in athymic nude mice; (7) expression of melanocyte- and melanomaassociated antigens; (8) requirements for exogenous growth factors, and (9) effect of the phorbol ester tetradecanoylphorbol-1Sacetate (TPA) or its analogs on cell growth. Normal melanocytes have a bipolar to tripolar morphology (Fig. 1A). Cells do not reach densities of >60-70% confluency and appear to be growth-inhibited when contacts with juxtaposed cells increase (M. Herlyn et al., 1985a, 1987a). The addition to the medium of dibutyryl cyclic AMP (dB-CAMP)leads to formation of dendrites (Fig. 1B). Dendritic cells seek initial contact with other melanocytes but avoid numerous contacts. However, dB-CAMP-treated cells are not terminally differentiated and continue to proliferate (M. Herlyn et al., 1988a).The polar morphology of melanocytes may still be seen in nevus cells (Mancianti et al., 1988; Mancianti and Herlyn, 1988 but not in melanoma cells. Approximately 40-60% of primary melanoma cultures have a spindle morphology; others are polygonal (Table 111).Cultures of spindle morphology, however, are not as bipolarly oriented as nonmalignant cells (as illustrated in Fig. 2A on one primary, and in Fig. 2B and C on two metastatic cell lines, established from lesions ofthe same patients). Advanced VGP primary and metastatic melanoma cells grow to higher densities per square centimeter than melanocytes and nevus cells (M. Herlyn et al., 1985b). Pigmentation, in general, decreases with tumor progression (Table 111). The life span of normal melanocytes and nonmalignant nevus cells is limited. Melanocytes and nevus cells from children under the age of 15 years grow for 50-60 doublings. Epidermal melanocytes from adults, on the other hand, grow for only 10 doublings, and nevus cells from adult patients have a variable life span in culture depending on the histology of the lesion. Chromosomal abnormalities are found in melanoma cells but not in normal melanocytes and nevus cells from common acquired and con-

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FIG.1. Growth of'human meIanocytes from newborn foreskin. Cells were maintained in growth medium (A) without or (B) with dB-CAMP at 1 mM for 5 days.

genital nevi. Nonrandom abnormalities have been identified in primary and metastatic melanoma cells on chromosomes 1 , 6 , and 7, but metastatic cells may have additional abnormalities (Balaban et al., 1984, 1986; M. Herlyn et al., 1985b). Normal melanocytes do not proliferate anchorage-independently in soft agar, whereas nevus cells do, with an average colony-forming efficiency of 0.9% (M. Herlyn et al., 1983, 1985a; Mancianti et al., 1988). Melanoma cells, on the other hand, readily form colonies in soft agar with an average of 8% colony-forming efficiency for primary cells compared to 25% for metastatic cells. Anchorage-independent growth

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FIG.1. (continued)

in soft agar correlates well with tumorigenicity in nude mice, as all of VGP primary and metastatic melanoma cells lines tested formed tumors after subcutaneous injection, Little information is available on the properties of dysplastic nevus and RCP primary melanoma cells in uitro, because these lesions are dimcult to culture in quantities necessary for experimental studies (M. Herlyn et al., 1987b). Figure 3 illustrates the growth of normal human melanocytes in a chemically defined medium consisting ofW489 (four parts MCDB 153 medium and one part L15 medium with the addition of 2 mM Ca2'; M.

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MEENHARD HERLYN ET AL.

FIG.2. Growth ofpriman. (A) and metastatic ( B and C) melanoma cells from the same patient (WM 983). All lesions had been removed simultaneously. Cells were seeded at 1 x 104/cmZin protein-free medium and cultured for 7 days.

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TABLE I11 MORPHOLOGIC, BIOLOGIC, AND GENETIC CHARACTERISTICS OF HUMAN MELANOCYTICCELLSAT DIFFERENT STAGES OF TUMOR PROGRESSION’’

Parameter

Nevus (congenital)

Melanocytes

Morphology (%) Bipolar and tripolar, pigmented (100) Life span <60b (doublings) Chromosomal None abnormalities Colony-forming efficiency in soft agar (%) Tumorigenicity in nude mice (% cell lines positive)

Primary melanoma (VGP)

Metastatic melanoma

Bipolar pigmented (100)

Spindle (60), polygonal (40), pigmented (30)

Spindle (40), polygonal (60), pigmented (20)

<5OC

> 100

>100

None

Nonrandom, chromosomes 1,6, and 7 5-20 (average 8)

Nonrandom, chromosomes 1,6, 7 and others 5-70 (average 25)

<0.001

0.001-3 (average 0.9)

0

0

100

100

~~

At least 10 different cell lines from each cell type were tested. Melanocytes from newborn foreskin underwent 50-60 doublings. Melanocytes from adult skin grew to -10 doublings. Most vigorous growth was achieved with congenital nevi from children <15 years old.

Herlyn et al., 1987a) and supplemented with insulin (5pg/ml), basic fibroblast growth factor (bFGF) at 40 ng/ml, TPA for protein kinase C activation at 10 ng/ml, and a-melanocyte-stimulating hormone (a-MSH) for enhancement of intracellular levels of CAMP at 10 ng/ml (M. Herlyn et al., 1988). Gelatin was used as substrate. Depletion of individual components reduced cell growth by 40-50%. This medium allowed the continuous growth of cells for >4 months. The doubling times of melanocytes in serum-free, chemically defined media, however, is only 30-50% of that of cells cultured in optimal medium containing pituitary extract and fetal calf serum (FCS).Therefore, the use of chemically defined medium for normal human melanocytes is generally limited to experiments requiring such conditions. Melanocytes and keratinocytes, which are juxtaposed in uivo, have strikingly divergent requirements for growth. In contrast to melanocytes, the growth of keratinocytes is inhibited by high calcium con-

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n l t

FIG.3. Growth of normal human melanocytes in a chemically defined medium. Medium W48Y consists of MCDB 153 (four parts) and L15 (one part) and 2 mM calcium. Complete W48Y medium is supplemented with insulin (5 pg/ml) or IGF-I (100 ng/ml), bFGF from bovine pituitary (40ng/ml), a-MSH (10 ng/ml), and TPA (10 ng/ml). Bars 1-4 show growth of cells after deleting individual snpplements from media: bar 1, -insulin; bar 2, -bFGF; bar 3, -TPA; bar 4, -a-MSH.

centrations (Boyce and Ham, 1985; Pittelkow and Scott, 1986) and phorbol ester (Wilkie et al., 1985) in medium. Prolonged presence of either component in growth medium of keratinocytes will induce terminal differentiation. Since normal melanocytes in situ are nonproliferative, it is conceivable that keratinocytes produce inhibitory factors for melanocytes. Whether such factors are antagonists for protein kinase C activation or for intracellular CAMPaccumulation remains to be explored. On the other hand, proliferating keratinocytes in vitro produce FGF, which may stimulate melanocytes (Halaban et al., 1988). Nevus cells from common acquired and congenital nevi have growth requirements similar to normal melanocytes with the exception that they require less FGF. The changes in the nevus phenotype brought about by depletion of TPA from the medium are not as dramatic as in normal melanocytes deprived of TPA; that is, nevus cells continue to proliferate slowly and to express nevus-associated antigens (M. Herlyn et ul., 1983, 1985a; Mancianti et al., 1988).

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When primary and metastatic melanoma cell lines are cultured in W489 medium in the presence of FCS, they grow at similar doubling times. However, primary and metastatic melanoma cells show striking differences when FCS or other growth factors are omitted from the medium (Rodeck et al., 1987a).As shown in Fig. 4, none of five primary melanoma cell lines grew in protein-free W489 medium with gelatin as substrate. Primary melanoma cells require at least insulin or insulinlike growth factor I (IGF-I) for continuous growth. Metastatic melanoma cell lines, on the other hand, quickly adapted to protein-free growth conditions and continued to proliferate for >6 months under the same conditions. The response of melanocytic cells to the tumor-promoting phorbol ester TPA reflects the stage of tumor progression (Fig. 5).Depletion of TPA from the medium for normal melanocytes not only leads to decreased cell growth but also to differentiation toward a cell type with flattened morphology, decreased tyrosinase activity, loss of pigmentation, and loss of expression of melanocyte-associated antigens (M. Herlyn et al., 1987a,b). In contrast, all of eight primary and metastatic melanoma cultures tested were growth-inhibited by TPA. Examples of one primary and one metastatic melanoma cell line are given in Fig. 5. In conclusion, the human melanocytic cell system has been exten-

400

T

300

--

% Increase in Cell

Number

*O0

100

.-

--

0 1

WM WM 9028 793 Primary melanoma

WM WM75WM39 115

WWM WM 239A 266-4 373

WM 184

WMWWM9 852

Metastatic melanoma

FIG.4. Growth of primary and metastatic melanoma cell lines in protein-free W489 medium. Cells were seeded on gelatin-coated plastic and cell numbers were determined on days 1 and 8.

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MEENHARD HERLYN ET AL.

% Increase

60

T

--

Melanocytes

News Cells

Melanoma Cells Primary

40

20 Cell Growth

Decrease

0

Metastatic

--

---

-20

--

-40

--

-60

--

-80 J.

FIG.5 . Effect of tumor-promoting phorbol ester TPA on the growth of melanocytic cells isolated froin different stages of tumor progression after 10 ng/ml of TPA were added t o growth medium. Results are expressed as percentage ofcells after 7-day culture in the presence of TPA compared to number of cells in media without TPA.

sively studied clinically anti histopathologically and is ideally suited for experimental investigations on tumor progression. Besides “classical” parameters for characterization of cells from each stage (i.e., life span, anchorage-independent growth, tumorigenicity in nude mice), we have delineated the requirements for exogenous growth factors in chemically defined medium for cells from each stage. This parameter clearly reflects the stage of tumor progression. The following section delineates growth autonomy in culture as a common phenomenon in human metastatic tumor cells of different origin. IV. Growth Factor Independence of Human Tumor Cells from Metastatic Lesions When human metastatic carcinoma cells from colon, rectum, bladder, ovary, and cervix, and melanoma cells were gradually adapted to protein-free medium over a period of 2-10 weeks, 30 of 35 cell lines (85.7%)continued to proliferate and cell lines could be maintained in protein-free medium for >2 years (Table IV). As illustrated in Fig. 6,

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TABLE IV CONTINUOUS GROWTHOF HUMAN TUMOR CELLS FROM ADVANCED STAGES O F TUMOR PROGRESSION IN PROTEIN-FREE MEDIUM” Cell lines

Growthkested

Colorectal carcinoma Bladder carcinoma Ovarian carcinoma Cervix carcinoma Melanoma

4/6b 516 1/2 2/2 18/19 30/35 (85.7%)

a Melanoma cells were cultured in W489 medium with gelatin as substrate; carcinoma cells were cultured in W489 medium (four parts MCDB 202 medium and one part of L15 medium). Cell lines not growing in protein-free medium required insulin as supplement only.

Proteinfree medium

KS

Insulin

IGF-I

EGFnGF-u Rx+

TGF-8

w;F

FIG.6. Doubling times of colorectal carcinoma cells lines in protein-free W489 medium and in the same medium supplemented with either 2% FCS; 5 pg/ml insulin; 100 ng/ml IGF-I; 5 ng/ml EGF or synthetic TGF-a; 10 nglml PDGF; 10 ng/ml TGF-p; or 100 ng/ml bFGF.

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the doubling time of a colorectal carcinoma cell line was 72 h in protein-free medium compared to 48 h in medium supplemented with 2% FCS, indicating that the cells remain responsive to exogenous mitogens. Growth stimulation similar to FCS was achieved with insulin and with IGF-I. All other growth factors tested, including EGF, transforming growth factor a (TGF-a), platelet-derived growth factor (PDGF), basic and acidic FGF, and TGF-P did not significantly stimulate or inhibit cell growth of this colorectal carcinoma cell line. The growth-stimulating effect of insulin and IGF-I was also seen in melanoma cells (Rodeck et al., 1987a). Both growth factors can stimulate cell growth at low cell densities and increase the colony-forming efficiency of a colorectal carcinoma cell line from 20% to 60% after seeding at 60 cells/cm2. A monoclonal antibody (mAb) to the IGF-I receptor can block the mitogenic activity of insulin, indicating that insulin at high concentrations acts predominantly via the IGF-I receptor (Rodeck et al., 1987a). Although EGF and TGF-a are essential for initial tumor cell growth (Singletary et al., 1987),they show little effect on established cell lines under our experimental conditions chosen, and bFGF, PDGF, TGF-P, and lymphokines such as granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukins 1 and 6 (IL-1, IL-6) also show no effect on the growth of melanoma cells in culture. In conclusion, growth autonomy has been observed in human tumor cells of different origin. The adaptation of cells to protein-free medium conditions leads to doubling times that are similar to those in FCScontaining medium. Insulinlike growth factor I and insulin are most frequently mitogenic for malignant cells in protein-free medium, followed by EGF/TGF-a. Least effect was seen with exogenous PDGF and FGF. Growth factors such as TGF-P are inhibitory for normal cells but show no effect on malignant cells. Increased growth factor autonomy of metastatic cells when compared to nonmetastatic cells has also been demonstrated in several murine tumor systems (Ethier and Cundiff, 1987; Chadwick and Lagarde, 1988; Waghorne et al., 1988). Growth autonomy, when defined as reduced response to exogenous growth factors, points to the involvement of endogenous factors for autocrine growth stimulation. V. Autocrine Growth Stimulation of Human Tumor Cells and Strategies for Growth Inhibition

It has been hypothesized that an autocrine mechanism of growth stimulation, involving secretion of endogenous peptide growth factors that stimulate growth of the producer cells via functional surface re-

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ceptors, underlies the relative growth autonomy of tumor cells from advanced stages of tumor progression (Todaro and DeLarco, 1978). Several growth factors have been suggested to function through an autocrine mechanism in cancer cells. Cells of small-cell lung carcinoma, for example, produce and respond to the tetradecapeptide bombesin (Carney et al., 1987; Moody et al., 1981). Antibodies specific for the C-terminal region of bombesin not only block the binding of bombesin to its receptor, but also markedly inhibit the growth of small-cell lung cancer cell lines in uitro and in uiuo (Cuttitta et al., 1985). Platelet-derived growth factor has also been implicated in autocrine growth stimulation, since it is produced by various human tumor cells, including osteosarcomas (Heldin et al., 1980), sarcomas (Betsholtz et al., 1983), gliomas (Nister et al., 1984), melanomas (Westermark et al., 1986), and carcinomas of bladder, breast, and lung (Bowen-Pope et al., 1984; Rozengurt et al., 1985; Betsholtz et al., 1987). Antibodies to PDGF have been used to inhibit the growth of simian sarcoma virustransformed murine tumor cells (Garret et al., 1984; Huang et al., 1984),but these antibodies have no effect on the growth rate of PDGFproducing spontaneous human tumors such as sarcomas and gliomas (Betsholtz et al., 1984; Nister et al., 1986).Despite the ability of human colorectal carcinoma cells to produce TGF-a (Coffey et al., 1986; Hanauske et al., 1987), evidence for an autocrine function of this growth factor has not been found (Coffey et al., 1987). Insulinlike growth factor I has also been suggested as an autocrine growth factor for breast and lung carcinoma cells, since cells in culture produce and respond to it (Huff et al., 1986; Perroteau et al., 1986). A. SECRETION OF GROWTH FACTORS AND EXPRESSION OF GROWTH FACTOR RECEPTORSBY MELANOMA AND COLORECTAL CARCINOMA CELLS

Table V summarizes the production of peptide growth factors by melanoma and colorectal carcinoma cells. Most melanoma cell lines produce PDGF (Westermark et al., 1986).The secretion of TGF-a and TGF-P is variable (-50% positivity of cell lines tested). Basic FGF has been detected in cell extracts of 9 out of 10 melanoma cell lines. Melanoma cells do not secrete IGF-I, IGF-11, or EGF, nor could transcripts be found in mRNA preparations. Normal melanocytes do not secrete PDGF or contain bFGF. Nevus cells extracts, on the other hand, contain bFGF. Colorectal carcinoma cells, similarly to melanoma cells, secrete PDGF, TGF-a and TGF-P. Secretion of IGF-I and IGF-I1 was at the lower detection limit, and production was not found for EGF and bFGF.

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TABLE V GROWTH FACTORS PRODUCED HY hf ELANOhlA AND COLOHECTAL CARCINOMA CELLS MAINTAINED IN PROTEIN-FREE CULTURE hIEDIUh1" ~

~

~~

Growth factor

Melanomab

PDGF

PDGF A + + + + PDGF B +

TGF-cz bFGFf

++Variable

IGF-I IGF-I1

-

++++

TGF-P

+ ++Variable

EGF

-

~

~~

Colorectal carcinomacd

+++@

+++ -

t t

++Variable -

" The production of growth factors was confirmed by Northern blot hybridization analyses using cDNA probes encoding for PDGF A, PDGF B, bFGF, IGF-I, IGF-11, TGF-p, TGF-a, and EGF. "Six cell lines were tested. ' One cell line was tested. * Values are approximations: + + + +, high secretion (>1ng/ml) into culture supernatant; +, positive but lowest limit of detection; -, undetected. Not determined whether PDGF A or PDGF B were secreted. f Isolation from cell extracts by heparin-Sepharose column chromatography.

Growth factor receptors for EGF/TGF-a, IGF-I, NGF (nerve growth factor), and b F G F have been detected on melanoma cells (Rodeck et al., 1987b) (Table VI). The number of EGF/TGF-a receptor sites per cell varies and in melanoma is highest in those cell lines that have an extra copy of chromosome 7 (Koprowski et al., 1985). Whereas detectable levels of the receptors for IGF-I, NGF, and b F G F were found on melanoma cells, there is no evidence for PDGF receptor expression (Westermark et al., 1986). On colorectal carcinoma cells, the E G F receptor is more prominently expressed than on melanoma cells, whereas we found no detectable levels of b F G F receptor and low levels of NGF receptor (Table VI). Melanoma cells and colorectal carcinoma cells either lack P D G F receptors, have levels that are below detection limits using radiolabeled compounds, or have receptors that cannot be detected with currently available cDNA probes. On the other hand, it was found that melanoma cells incorporate P D G F and

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TABLE VI GROWTH FACTOR RECEPTORSON MELANOMAAND COLORECTAL CARCINOMA CELLS Number of sites per cell" Receptor for EGF/TG F-CI ICF-I NGF bFGF PDGF

Melanoma

1000-50,000 <5000 10,000-2,000,000 Low-affinity receptors: 100,000 High-affinity receptors: <5,000 Below detection level

Colorectal carcinoma

1000-200,000 <5000

1000-10,000 Below detection level Below detection level

a Values are approximations and were obtained with mAbs defining EGF and NGF receptor or with radiolabeled ligand (IGF-I, bFGF, and PDGF) using at least four cell lines for each tumor system.

transport it to the nucleus (Rakowicz-Szulczynskaet al., 1986; van den Eijnden-van Raaij et al., 1988). The existence of at least two different PDGF receptor classes has been demonstrated on human fibroblasts (Heldin et al., 1988). The type A PDGF receptors bind all three dimeric forms of PDGF (PDGF-AA, PDGF-BB, and PDGF-AB), whereas the type B PDGF receptors bind PDGF-BB with high affinity and PDGF-AB with lower affinity but do not bind PDGF-AA. It may be speculated that the PDGF receptor on human tumor cells is even more d'iverse. B. APPROACHES FOR INHIBITING AUTOCRINEGROWTH STIMULATION 1. Anti-Growth Factor Receptor Antibodies

Several laboratories have produced mAbs to the EGF receptor. mAb 425 blocks binding of EGF and TGF-(r to the receptor and can inhibit stimulation of fibroblasts by exogenous EGF (Murthy et al., 1987). Growth of a cell line that overexpresses EGF receptor is inhibited by mAb 425, whereas a colorectal carcinoma cell line, which has only 3000 receptor molecules per cell, was not growth-inhibited (Rodeck et al., 1987~).Other colorectal and mammary carcinoma cell lines expressing 50,000 to 100,000 EGF receptor binding sites per cell are inhibited by mAb 425 in the presence of exogenous EGF or TGF-a. Anti-receptor antibodies may be suitable for growth inhibition in subsets of carcinoma cells by inhibiting access of endogenously produced TGF-a to surface receptors.

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2. Growth Factor-Antagonistic Peptides Several laboratories have attempted to use peptides with partial sequence homology to growth factors as antagonists, and it was postulated that peptides may bind to the receptor without eliciting a mitogenic response. However, to date, with the exception of one preliminary report (Nestor et al., 1985), attempts to inhibit growth of tumor cells with peptides of growth factors have failed.

3. Znhibition of Growth of Tumor Cells by Anti-Growth Factor Antibodies Inhibition of growth of human tumor cells by anti-growth factor antibodies has been achieved in several tumor systems as discussed earlier. Our own studies with antisera to PDGF and TGF-a in colorectal carcinoma and melanoma cells cultured in protein-free medium gave conflicting results. Difficulties in inhibiting growth of tumor cells with anti-growth factor antibodies are apparently due to either: batchto-batch differences in the neutralizing activity of polyclonal antibodies, the ability of tumor cells to utilize more than one growth factor for autocrine growth stimulation, or the existence of “intracellular loops” for growth stimulation.

4. Soluble Receptors fo r Competition with Cell Surface Receptors Human immunodeficiency virus 1 (HIV) binds to the CD4 receptor of T cells (see Fauci, 1988, for review). Reports by several laboratories indicate that soluble recombinant CD4 receptor can compete with the cell surface receptor for binding of HIV and can prevent infection (Smith et al., 1987; Fisher et ul., 1988; Hussey et al., 1988; Deen et al., 1988;Traunecker et al., 1988). It is conceivable that the same approach can be applied to growth factor-growth factor receptor competition.

5. Antiidiotype Antibodies Mimicking Cell Surface Antigens Polyclonal antibodies that specifically bind to antigen-combining site-related idiotypes of antitumor mAbs functionally mimic cell surface tumor antigens and can induce an antitumor response in experimentai animals (D. Herlyn et al., 1986, 1987a) and in cancer patients (D. Herlyn et al., 1987b). An antiidiotype antibody to a mAb defining the human EGF receptor may therefore mimic the EGF receptor and can potentially be used as an alternative for a soluble EGF receptor.

6. Active Zmmunization against Peptide Growth Factors The feasibility of immunoneutralization of a growth factor in viuo has been demonstrated with NGF. Application of mouse antibodies against mouse NGF to young mice caused the destruction of NGF-

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dependent neurons in sympathetic ganglia (Businaro and Revolta, 1982). Whether this approach is applicable for human tumors needs to be explored. VI. Summary Normal human cells, cells from nonmalignant proliferative lesions, and primary and metastatic tumor cells can be maintained in vitro and analyzed for requirements for growth in chemically defined media. The human melanocytic cell system with normal melanocytes, precursor nevus cells, and primary and metastatic melanoma cells has been extensively studied for the phenotypic properties of the cells, including their requirements for exogenous growth factors and other mitogens. In high calcium-containing W489 medium, normal melanocytes require four supplements: IGF-I (or insulin); bFGF, TPA, and a-MSH. Nevus cells are largely independent of bFGF. Depletion of TPA from medium is not as detrimental to nevus cells as it is to melanocytes, but the phorbol ester is still essential for maintenance of the typical nevic phenotype. Primary melanoma cells require at least one growth factor, IGF-I (or insulin), for continuous proliferation. On the other hand, metastatic cells of melanoma as well as of carcinomas of colon and rectum, bladder, ovary, and cervix are able to proliferate after a short adaptation period in medium depleted of any growth factors and other proteins. Doubling times of metastatic tumor cells in protein-free medium are only 30-60% longer than in FCS-containing medium. The growth autonomy of human tumor cells is apparently due to the endogenous production of growth factors. Likely candidates for autocrine growth stimulation of human tumor cells are TGF-a, TGF-p, and PDGF. Melanoma and colorectal carcinoma cells express functional EGF/TGF-a receptors, and produce TGF-a, indicating that this growth factor is produced for autocrine stimulation. In addition to the use of anti-growth factor antibodies, other strategies for the inhibition of autocrine growth stimulation include mAbs to growth factor receptors, soluble receptors, receptor-mimicking antiidiotype antibodies, and active immunization against growth factors. Whether any of these therapeutic approaches is clinically feasible will need to be determined in extensive preclinical investigations.

ACKNOWLEDGMENTS We thank Andrea Barol for secretarial assistance. These studies were supported, in part, by grants from the National Institutes of Health CA-25874, CA-44877, and CA10815.

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THE LYMPHOPOIETIC MICROENVIRONMENT IN BONE MARROW Paul W. Kincade lmmunobiology and Cancer Program, Oklahoma Medical Research Foundation,Oklahoma City, Oklahoma 73104

I. Introduction Evolution of Experimental Approaches Long-Term Bone Marrow Cultures Differentiation Steps and Lineages Lymphocytes in Long-Term Cultures initiation of Long-Term Cultures Essential Cells of the Microenvironment Recognition and Adhesion between Cells IX. Some Cytokines That Affect Lymphohemopoiesis X. Interleukin 7 XI. Localization of Cytokines in the Microenvironment XII. Transforming Growth Factors p XIII. Cytokine Responses Involving Stromal Cells XIV. Adipogenesis XV. Additional Perspective References 11. 111. IV. V. VI. VII. VIII.

I. Introduction Lymphocyte precursors and other cells in bone marrow are frequent targets of malignant transformation and can be altered in many other disorders. Also, there are many challenging basic questions about how multipotential stem cells normally give rise to cells of eight different lineages in a coordinated manner. The composition and function of bone marrow has thus been an object of study for some time. Information has steadily accumulated about the differentiation events that occur as functional lymphocytes emerge from early precursors, and progress has been particularly rapid in learning about cellular interactions and molecules that regulate that process. Although it is premature to draw conclusions about many of the complex aspects of bone marrow function, some general themes will form the basis of this review. The concept of a “hemopoietic inductive microenvironment” was proposed by Trentin and colleagues to account for localized areas of hematopoiesis that could be observed under some experimental circumstances (Trentin, 1971). It now seems unlikely that a specialized 235 ADVANCES IN CANCER RESEARCH, VOL. 54

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organ comparable in all respects to the avian bursa of Fabricius exists in mammals. That is, B lymphocytes are formed in close proximity to maturing cells of several other lineages, rather than in discrete “follicles.” However, very close cellular interactions, together with limited availability and/or accessibility of regulatory molecules, produce a microenvironment for lymphopoiesis that has now been partially defined. “Stromal” cells are being studied and characterized as cloned lines, and it is already clear that they are a pivotal and multifunctional component of the microenvironment. They elaborate myeloid colonystimulating factors (CSF) under steady-state circumstances, and also in response to exogenous inflammatory stimuli. A recently discovered growth factor for B-lymphocyte lineage precursors (interleukin 7 or 1L-7),a factor affecting stem cells (interleukin 6 or IL-6), and a potent inhibitor of lymphohematopoiesis (transforming growth factor @ or TGF-P) are among the other regulatory substances made by stromal cells. Complex networks of interacting cytokines can now be envisioned, and it is interesting that stromal cells seem to respond to several substances that they themselves can make. Stromal cells can undergo dramatic morphological and functional changes as they become adipocytes, and it is possible to influence that process in culture. The precise localization of stem cells and maturing lymphocyte precursors in bone marrow has not yet been achieved. However, a framework concept is provided by electron-microscopic and other studies, and a few generalizations are now possible. Despite their high mitotic activity, large accumulations of lymphocytes have not been found in marrow. Stem cells and their immediate progeny are thought to be preferentially located in the subendosteal area of the bone, whereas mature blood cells of all lineages egress via more centrally located venous sinues. This suggests a continual movement of dividing hematopoietic precursors, and definition of the chemotactic factors and/or adhesion molecules that mediate this process should be given a high priority. The rapid pace of work in this field can be appreciated b y a brief review of experimental approaches that have been and are being brought to bear on these questions. A synopsis of B-cell differentiation steps will be followed by a consideration of events that can be duplicated in long-term culture systems, cellular components of the microenvironment, and cytokines that they make. It is interesting to compare and contrast one growth fiactor (IL-7) that is made in limiting quantities and an antagonist of its action (TGF-P) that is made in

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abundance in an inactive precursor form. The multifunctional nature of stromal cells is indicated by their ability to generate fat cells. Finally, studies of genetically determined abnormalities and polymorphisms of inbred mice reveal an additional degree of complexity to the lymphopoietic microenvironment. II. Evolution of Experimental Approaches The extraordinary progress being made in understanding lymphopoiesis is directly attributable to the number and quality of experimental models that are now available. It is therefore interesting to reflect on how and when these approaches were developed. Fundamental concepts about central lymphoid organs and the separation of B and T lymphocytes derived from studies of chickens. An appreciation of developmental and functional differences in these cells followed Glick’s discovery of the importance of the bursa of Fabricius to humoral immunity (Glick et al., 1956; Cooper et al., 1966). Later studies revealed a programmed switching of immunoglobulin heavy (IgH) chains in emerging B lymphocytes, and the model continues to be important for investigating the origin and migration of stem cells as well as mechanisms for generating antibody diversity (Kincade et al., 1970; Kincade, 1981; Dieterlen-Lievre, 1975; LeDouarin, 1986; Weill and Reynaud, 1987). However, fundamental differences in the timing and means of utilization of Ig genes have been found in birds and mammals. Unlike the situation in mammals, heavy and light chains are simultaneously expressed during development in the bursa of Fabricius. Also, unique events occur to allow exploitation of limited numbers of variable-region genes. This review will emphasize studies performed with experimental mice. Radioautographic techniques have been extremely important for investigating events that occur within the bone marrow of mammals (Rosse, 1976; Osmond, 1986).This approach was first used to establish that it is a major site for small-lymphocyte production, that those lymphocytes were destined to produce Ig, and that a size progression occurs from large to small precursors (Osmond and Nossal, 1974; Landreth et d., 1981).For example, large proliferating lymphocytes in marrow incorporate label after a single-pulse injection of [3H]thymidine and these subsequently give rise to small pre-B cells and then to small B cells. Variations on this approach, including double labeling with fluorescent lectins and monoclonal antibodies (mAb) as well as metaphase arrest techniques, have provided essentially all that

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is known about the kinetics of lymphocyte formation in bone marrow (Osmond et al., 1984; Park and Osmond, 1987; Opstelten and Osmond, 1983, 1985). Another important development was recognition that the immediate precursors of B cells contain p heavy chains, but not light chains, of Ig (Raff et al., 1976). This made it possible to enumerate these “pre-B” cells and follow their fate in fetal and adult hematopoietic tissues. It then became possible to identify still earlier cells in the lineage with mAb and to enrich them from human and murine hematopoietic tissues (Kincade et al., 1981b; Coffman and Weissman, 1981; Coffman, 1982; Landreth et al., 1982,1983).Depletion of cells with these reagents was then used together with transplantation of immunodeficient and irradiated recipient mice to investigate relationships between lymphoid and myeloid progenitors (reviewed in Kincade, 1981).Application of multiparameter cell sorting and new combinations of mAb has now made it feasible to track cells from the very earliest steps in the lineage (Spangrude et al., 1988). A new approach to dissecting lymphocyte differentiation events has been provided by recombinant-DNA technology. Several laboratories have isolated probes to genes that are preferentially, or exclusively, expressed in cells of the B lineage without reliance on the existence of a mAb to the gene product (Kudo et al., 1987; Kudo and Melchers, 1987; Bauer et al., 1988; Sakaguchi et al., 1988; Hermanson et al., 1988a,b). These should provide powerful tools for “phenotyping cells from the inside,” and information about structure may be immediately informative as to their possible function (Letarte et al., 1988). Oncogenes can be expressed in a stage-restricted as well as a lineagerestricted manner (Zimmerman et al., 1986; Bender and Kuehl, 1987; Graninger et al., 1988). It is also possible to isolate murine B-lineage genes that were originally discovered in studies of human lymphocytes (Tedder et al., 1988a,b). Information about the basic organization of bones suggests that the lymphopoietic microenvironment is only microns in size and must be viewed in dynamic terms. Multipotential stem cells are preferentially localized in the endosteal area just beneath the bone cortex, whereas all of their progeny must leave the marrow via more centrally located venous sinuses (Shackney et al., 1975; Lord et al., 1975; Tavassoli and Shaklai, 1979). Developing erythroid cells are arranged around a central macrophage forming erythroblastic islands (Bessis and BretonGorius 1962; Bessis et al., 1978; Crocker and Gordon, 1985). Similarly, immature myeloid cells are closely associated with nonphagocytic, alkaline phosphatase-positive, adventitial reticular cells that radiate

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away from venous sinuses (Westen and Bainton, 1979; Lichtman, 1981, 1984; Weiss and Sakai, 1984; Weiss, 1976). B-Cell precursors in fetal liver are scattered (Kamps and Cooper, 1982), and their proximity to other components of that organ has not yet been established. As hematopoiesis shifts from liver to bone marrow during the neonatal period, foci of B-lineage lymphocytes are found in the liver and the cells are clonally related (Rossant et al., 1986).Large accumulations of lymphocytes are not found in mammalian bone marrow. However, early lymphoid progenitors and pre-B cells tend to be more concentrated in the subendosteal region than in the center of the marrow shaft (Osmond and Batten, 1984; Batten and Osmond, 1984; Hermans et al., 1988). Recent immunoelectron-microscopic studies reveal cells bearing Blineage markers associated with thin membranous processes of other, as yet unidentified cells (D. Osmond, personal communication). It seems likely that they are intimately related to “stromal” cells, which are now being characterized with long-term culture techniques (see later). Cloned transformed lines in erythroid, myeloid, and lymphoid lineages have been found that are responsive to inductive stimuli (Koeffler, 1983; Lebien et al., 1982; Paige et ul., 1978). These provide homogeneous models in which to explore cytokines that might normally regulate proliferation and differentiation, as well as molecular events associated with those processes. For example, ion flux associated with transmembrane signaling, changes in chromatin state, activation of DNA-binding proteins in the nucleus, transcription, and translation have all been studied with an inducible pre-B lymphoma cell line, 70213 (Rosoff and Cantley, 1985; Stanton et al., 1986; Parslow and Granner, 1982; Perry and Kelly, 1979; Wall et al., 1986; Sen and Baltimore, 1986).It is likely that established tumor cell lines are intrinsically different in some respects from their normal counterparts. However, our experience with the 70Z/3 cell line suggests that it is usually a more valid model than the “normal” untransformed lymphocytes that can be propagated for extended periods in long-term cultures. Cell lines established with the aid of transforming viruses have been extremely useful because they spontaneously rearrange and sequentially express Ig genes (Alt et al., 1986).They have also helped in establishing patterns of surface marker expression (Davidson et al., 1984; Holmes et al., 1986, reviewed in Kincade, 1987; Kincade et ul., 1988). B cells are spontaneously produced within 1day in liquid cultures of muk-ine bone marrow that have been depleted of mature cells. This process is diminished when the cells are dispersed in semisolid agar or

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when adherent cells are removed by passage of the suspensions over G-10 Sephadex columns. Emergence of B cells is increased when appropriate soluble factors are added to either semisolid or liquid cultures. Such simple experimental designs provided the first indications that close interactions between cells enhanced B-cell formation and made it possible to identify some candidate regulatory molecules (reviewed in Kincade, 1987). However, it has only recently become possible to explore which cells elaborate such factors and how the requisite communication between cells is mediated (see later). Initial studies of stimuli that influence lymphopoiesis involved crude sources of activity in conditioned medium, serum, and urine, as well as nonphysiological stimuli such as lipopolysaccharide (LPS) and phorbol esters. Recombinant-DNA technology has provided a means for amplification of extremely rare cytokines and obtaining them in purified form (Metcalf, 1986). Considerable information is also being obtained about the effects of these molecules in uiwo. This is made possible b y retroviral-mediated gene transfer, overexpression of cytokine genes in transgenic mice, and infusion of recombinant proteins to experimental animals and patients (Metcalf et al., 1987; Lang et al., 1987; Clark and Kamen, 1987). Ill. Long-Term Bone Marrow Cultures

At least some components of the lymphopoietic microenvironment can be grown in culture, where they can be dissected, characterized, and independently manipulated. The experimental approaches that made this possible were first developed by Dexter and colleagues (Dexter and Lajtha, 1974; Dexter and Testa, 1976; Dexter et al., 1977, 1984) and modified in important ways by Whitlock and Witte 1982; Whitlock et al., 1984, 1985). Dexter cultures favor survival of multipotential stem cells and active production of granulocytes and macrophages. Significant numbers of lymphocytes are not made under these conditions, which involve low temperature, high concentrations of supporting serum, and use of either horse serum or steroids. Whitlock and Witte devised conditions that were permissive for only lymphoid growth [i.e., low concentrations of selected fetal calf serum (FCS), inclusion of 2-mercaptoethanol (2-ME), incubation at 3TC, and absence of steroids]. A number of other technical innovations permit “switching” between these conditions, establishment of adherent layers devoid of hematopoietic precursors, selective depletion of cells from marrow suspensions that can form adherent layers, cloning of stromal cell-dependent lymphocytes, and cloning of stromal cells from

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the adherent layer (Whitlock et al., 1983, 1984; Witte et al., 1986; Denis and Witte, 1986; Dorshkind, 1986; Dorshkind et al., 1986b; also see later). The current rapid progress in understanding the bone marrow environment is attributable to advances in these experimental approaches. A substantial literature is accumulating on long-term culture of murine lymphocytes (Kincade et al., 1988).Comparable success has been elusive in studies of human marrow, and even myelopoiesis is not as efficient or extensive in human cultures (Eaves et al., 1987). IV. Differentiation Steps and Lineages Formation of B cells can be viewed as a continuum of differentiation steps that progressively limit the options of multipotential hematopoietic stem cells to become first lymphocytes, then B lymphocytes, and finally individual B-cell clones. It is convenient to draw these events diagrammatically, but this implies a rigidity and level of understanding that may not exist. In the murine system, for example, we know that from 16 days of gestation and throughout adult life, all B-lineage precursors express a particular epitope of the leukocyte common antigen (Ly-5/CD45)family, and it is clear that this marker is acquired before IgH chains are synthesized. However, pre-B cells found early in embryonic life, some tumors and transformed cell lines, and some of the pre-B and B cells grown in long-term cultures lack this marker (Kincade et al., 1988). Furthermore, it is not certain that an absolute order is followed for expression of all of the B-cell genes and there is reason to believe that some branching in the pathway occurs. Some of these complications have been considered in previous reviews (Kincade, 1987; Kincade et al., 1988), and only a simplified overview will be given here. A chromatin change in, and transcription from, germ-line IgH chain genes may be the earliest recognizable event in cells destined to become B cells (Alt et al., 1986; Yancopoulos and Alt, 1985). It has been argued that this renders the DNA accessible to the rearrangement events that follow. Still unclear is whether this occurs before or after lineage commitment (Ford et al., 1988).Synthesis of the nuclear enzyme terminal deoxynucleotidyltransferase is another early event that may occur in “lymphoid” stem cells or in uncommitted precursors, which have the option of becoming either B or T lymphocytes (Park and Osmond, 1987; Opstelten et al., 1986; Deenen et al., 1987). The enzyme is thought to contribute to diversification of antibody-combining sites by mediating insertion of non-germ-line nucleotides (Baltimore, 1974; Desiderio et al., 1984). A later differentiation

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event leads to display of an isoform of the common leukocyte antigen family referred to earlier, and this marker is continuously expressed during all subsequent stages until the B cells differentiate to antibodysecreting plasma cells (Kincade, 1987). Sequential rearrangement events bring D and J segments, as well as V and D segments into a transcriptionally useful unit, and this is followed by rearrangement of genes for Ig light chains ( C o f h a n , 1982). A series of studies involving transplantation and cell separation experiments suggested that precursors of myeloid cells were quite distinct from pre-B cells (reviewed in Kincade, 1981, 1987; Kincade and Phillips, 1985). However, more recent analyses of retrovirally transformed and spontaneously arising tumor lines have been interpreted to mean that macrophages and pre-B cells are closely related (Bauer et al., 1986; Holmes et al., 1986). A particularly impressive study was made of multipoteritial line derived with v-Ha-ras from fetal liver (Davidson et al., 1988). Lipopolysaccharide stimulation resulted in subclones that had many characteristics of either pre-B cells or macrophages. Examples of “lineage promiscuity” or “lineage infidelity” have also been reported in human tumors, and an early study showed that murine pre-B cells could be turned into macrophages with a demethylating agent (Greaves et al., 1986; Boyd and Schrader, 1982). Studies involving introduction of v-ruf into myc-transgenic mice showed that acquisition of macrophage characteristics can occur from several stages in the B lineage (Klinken et al., 1988).This may reflect a default characteristic that occurs in “reprogrammed” cells. While these findings are indeed interesting, it is not clear how informative they are about normal differentiation pathways. Branched and/or parallel lineages of B-cell differentiation have often been postulated to result in functionally restricted progeny, and the most convincing evidence for this possibility relates to cells bearing the Ly-1 (CD5) marker. Ly-l-bearing B cells may derive from self-renewing stem cells at a stage prior to full hematopoiesis in bone marrow. They have a distinctive tissue distribution and have been the subject of some excellent reviews (Hardy and Hayakawa, 1986; Herzenberg et al., 1986). V.. Lymphocytes in Long-Term Cultures Lymphocytes growing in Whitlock-Witte cultures are morphologically quite different from transformed B-lineage cell lines and in fact look rather normal (Whitlock and Witte, 1982; Whitlock et al., 1984). They are small to medium in size and have nearly clear cytoplasms.

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Cell lines such as 70Z/3 have a large “lymphoblast” appearance with basophilic-staining cytoplasms. In spite of this difference, the mitotic index of long-term-cultured lymphocytes is reasonably high. Approximately one-fourth of the cells are in S, Gz, or M stage at any one time (Witte et al., 1986). Furthermore, they usually die within 3 days of removal from supporting stromal cells. In these respects, cultured lymphocytes are relatively normal. They do not typically form tumors when placed in athymic nude mice, and, in the rare cases when serially propagated cells do transform, cell size and morphology are not notably changed (Whitlock et al., 1984; P. W. Kincade, unpublished observations; C. Whitlock, personal communication). Difficult questions thus arise as to the stage of maturation they represent and the degree to which they can respond to normal differentiation stimuli. The cultured cells appear to be exceptional in these respects. All normal B-lineage precursors in late fetal liver and adult bone marrow express an epitope of the leukocyte common antigen family (Ly-5,220) detected by our mAb 14.8. Moreover, all mature B cells express this marker. However, it is not unusual for long-termcultured lymphocytes to be negative, even when they are classified as pre-B or B cells on the basis of other criteria, such as synthesis of cytoplasmic or surface Ig chains (Witte et al., 1986, 1987b). The BP-1 marker is usually present in high density, but can be lost with extended culture (Witte et al., 1987a). In our experience, cultured lymphocytes are unresponsive to many stimuli that affect normal pre-B cells and an inducible pre-B cell line. An apparent isotype switch was achieved in one study with phorbol ester treatment, but many other agents were ineffective (Dasch and Jones, 1986). Early-passage lymphocytes often give a weak proliferative response to IL-7 (see later), whereas cloned, stromal celldependent lymphocytes give a marked, but usually limited response to this cytokine. However, this differs from the behavior of normal Blineage cells in one important respect. Large pre-B cells from normal bone marrow respond to IL-7, but small pre-B cells and surface Igbearing B cells die when exposed to it (Lee et al., 1989). Our longterm-propagated lymphocyte clones, which respond to IL-7, are surface Ig-positive. Thus, the cells are phenotypically B cells, but function like pre-B cells in responding to this factor. Lymphocytes with aberrantly rearranged Ig genes grow well in long-term bone marrow cultures (LTBMC), and there would seem to be no selective pressure against such defective cells (Witte et al., 1987a). Several findings suggest that long-term cultures are relatively pauciclonal. That is, only a small number of the millions of lymphocyte

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precursors placed in culture seem to give rise to all of the cells that expand and can be maintained for an extended period oftime. Comparison of replicate cultures made from a single pooled cell suspension revealed remarkable dish-to-dish variability and the results suggested that this was acquired during the first few weeks (Witte et aZ., 1986). In another study, equal-part mixtures of marked bone marrow suspensions were used to establish long-term cultures. Although surviving cells were initially derived from both donor cell suspensions, this dramatically changed with time (Hayashi et al., 1988). Apparent clonal dominance occurred in many cultures regardless of whether lymphoid (Whitlock-Witte) or myeloid (Dexter)conditions were employed. Relatively simple patterns of Ig gene rearrangements in Whitlock-Witte cultures would also be consistent with this interpretation (Denis et al., 1984; Witte et al., 1987a; Hirayoshi et al., 1987; Yoshida et al., 1988), and several laboratories have been able to isolate cloned lymphocyte lines that remain factor and/or stronial cell-dependent (Whitlock et aZ., 1983, 1987; Hunt et al., 1987; Kennick et al., 1987; Pietrangeli et ul., 1988; Lemoine et al., 1988). The degree to which these clones represent normal differentiation steps has not been established. They may resemble “premalignant” cells that have been selected for efficient growth under the suboptimal conditions of culture. Some, but not all, normal regulatory responses and differentiation potential could be lost. At least some cells pooled from long-term cultures retain differentiation options that can be demonstrated by transfer to immunodeficient or irradiated recipients. Variable degrees of B-cell reconstitution, immune responsiveness, and even some penetration of the T-cell lineage have been reported (Nagasawa et al., 1985; Phillips et al., 1984; Dorshkind et al., 1986a; Denis et ut., 1987). In one early study, previously cultured lymphocytes with Ig genes in germ-line configuration were recovered from the bone marrow of mice transplanted with LTBMC and successfully established again in culture (Kurland et al., 1984). Such results indicate that uncommitted, or at least normal, lymphocyte progenitors persist in long-term cultures for extended periods of time. It remains unclear what fraction of the total cultured lymphocytes these cells represent. Successful long-temi maintenance of early lymphoid precursors in culture with IL-3 has been reported by several investigators (Palacios and Steinmetz, 1985; Sideras and Palacios, 1987; Kinashi et at., 1988; Palacios et al., 1987; McKearn et al., 1985; Spalding and Griffin, 1986; Griffin and Spalding, 1988). Cells with some features of B-lineage

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progenitors, but with Ig genes in germ-line configuration could in some circumstances differentiate further in vivo or in uitro. Details of the requisite stimuli for this transition are not known. Goldschneider and colleagues also developed long-term culture conditions that appear to favor survival and growth of early TdT' lymphocytes (Hayashi et al., 1984; Medlock and Goldschneider, 1987). VI. Initiation of Long-Term Cultures

It is important to know what lymphohematopoietic cells and microenvironmental elements from fresh tissues are required to initiate long-term cultures. While early studies demonstrated the feasibility of relatively short-term cultures of embryonic tissues (Owen et al., 1975; Teale and Mandel, 1980), typical Whitiock-Witte cultures were not easily established with cells from this source. This required transfer of fetal cells onto established adherent layers prepared from adult bone marrow (Denis et al., 1984, 1987; Kincade et al., 1987). This suggests that cells capable of making the appropriate in vitro microenvironment were limiting in embryonic tissues, and the same conclusion has been reached in cultures of human tissues (Riley and Gordon, 1987). However, Hardy et al. (1987) established a preadipocyte stromal cell clone from fetal liver that supported growth of lymphocytes derived from newborn mice. The populations of cells that grew out were unusual in that they expressed Ly-1 (CD5) and a high percentage of them were sIgM+ B cells. Multipotential stem cells develop in extraembryonic as well as intraembryonic sites (Metcalf and Moore, 1971). However, elegant transplantation studies with avian embryos revealed that only those within the embryo normally give rise to the B and T lymphocytes that subsequently develop (Dieterlen-Lievre, 1975). Similarly, while cells isolated from murine yolk sac could reconstitute B cells in immunodeficient recipient mice, the process was much slower and less efficient than when grafts were made with fetal liver cells (Paige et al., 1979). Those findings were extended with a cloned bone marrow-derived stromal cell line (Ogawa et al., 1988).At a critical stage approximated at 9.6 days of gestation, embryonal body, but not yolk sac-derived cell suspensions efficiently generated B-lineage lymphocyte cultures. It was suggested that the precursors later found in yolk sac could have derived from within the embryo. Paige and colleagues have used a multilayer culture system to clone and extensively characterize Blineage precursors from fetal liver (Paige, 1983; Paige et al., 1984,

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1985; Sauter and Paige, 1987; Wu and Paige, 1986). Their approach also yielded information about cytokines that might directly or indirectly participate (Paige, 1985). The general characteristics of lymphocytes grown in our LTBMC have been similar regardless of which cell suspensions were used to initiate the cultures (Kincade et al., 1987).However, it is not yet clear if multipotential stem cells can give rise to B cells in culture and what the requirements for that transition might be. Innovative culture manipulations developed independently by Dorshkind (1986) and Denis (Denis and Witte, 1986) permit cells maintained under Dexter culture conditions to grow out as lymphocytes when the conditions are made permissive (Whitlock-Witte). Self-renewal of stem cells is known to occur in Dexter cultures, and one interpretation is that they differentiate along the B lineage when the cultures are shifted. However, it is equally possible that rare committed progenitors of B cells present in the Dexter cultures simply expand when satisfactory conditions are achieved. Appropriate cells for establishing lymphocyte-producing cultures can be harvested from the marrow of corticosteroid-treated mice (Ku and Witte, 1986). Early hematopoietic progenitor cells can be considerably enriched by electronic sorting for cells that express low amounts of the Thy-1 antigen, but that lack markers found on mature blood cells (MullerSieburg et aZ., 1986,1988). A transient wave of myeloid cells, followed b y B-lineage lymphocytes, emerged when such suspensions were transferred to establish adherent layers. Stem cells of even higher purity were obtained when another cell surface marker was added to the selection protocol, and these gave rise to B cells when transferred to irradiated recipient mice (Sprangrude et al., 1988). It would be interesting to know the potential of purified stem cells when placed on cloned stromal cells. VII. Essential Cells of the Microenvironment Close interactions between lymphoid progenitor cells and other components of bone marrow favor the emergence of B cells in shortterm cultures (Kincade et aZ.,1981a). However, detailed analysis of those relationships only became possible with development of the Whitlock-Witte culture system (Whitlock and Witte, 1982). At least two prominent cell types are present in the adherent layer, which is critical for long-term lymphocyte growth (Witte et al., 1987b). Macrophages are easily identified on the basis of phagocytosis, pinocytosis, expression of nonspecific esterase, absence of alkaline phosphatase,

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uptake of acetylated low-density lipoprotein (aLDL), or staining with several mAb (F4/80, Mac-1, Mac-2, Mac-3). There is reason to believe that macrophages and their products contribute to the final stages of B-lymphocyte maturation (Kincade et al., 1981a; Gisler et al., 1984). However, they are not essential to survival and proliferation of lymphocytes in long-term bone marrow cultures. This was first demonstrated by using aLDL staining and electronic cell sorting to deplete macrophages from established cultures (Witte et al., 1987b). Lymphocyte growth continued in the macrophage-depleted cultures, but it is noteworthy that foci were associated with only a subpopulation of the large adherent cells (see Fig. 3G, in Witte et al., 198713). Many cloned cell lines have been prepared from the adherent layers of LTBMC (reviewed in Kincade et al., 1988). These may represent homogeneous components of the microenvironment, which can be subjected to molecular analysis and provide a powerful means of standardizing experimental conditions in culture. Perhaps the most widely used of many names for these is “stromal cells.” This relatively neutral term is appropriate in light of uncertainties that remain about their lineage derivation, life span, and differentiation potential. Developmental, functional, or phenotypic similarities have been noted between stromal cells and epithelial cells, endothelial cells, adipocytes, and smooth muscle cells. Examples from personal experience with these interesting cells will be emphasized in this review, but rapid progress is now being made in many laboratories in characterizing stromal cells. Pietrangeli and colleagues (1988)isolated >40 stromal cell lines and clones from murine bone marrow and spleen by selective use of the drug 5-fluorouacil. A methylcellulose cloning assay was used to assess quantitatively lymphocyte support capability. A major objective was to determine common features between lines that sustained growth of adherent cell-depleted bone marrow lymphocytes and stromal celldependent lymphocyte clones, which were themselves derived from long-term cultures. This function, and the tissue of origin, were used to name the clones. For example, BMSB is a bone marrow-derived clone that supports lymphocyte growth, whereas the same lymphocytes simply die when placed on the spleen-derived clone SNS1. Subsequent studies suggest that the support-nonsupport designation may not reflect all of the functional heterogeneity that exists between clones. BMSB has provided vigorous lymphocyte support over an extended period of time whereas no lymphocytes have ever grown on SNSl. However, certain of our other stromal cell clones will support particular lymphocyte clones, but not others (P. W. Kincade, unpub-

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lished observations). This important finding suggests that multiple factors probably contribute to this function. Subtle differences were noted when immunoperoxidase staining was used to compare stromal cell clones, but there was no obvious pattern that corresponded to the support function (Pietrangeli et al., 1988).A variety of cDNA probes were subsequently used to compare clones at the level of niRNA transcripts (Gimble et d.,1989). The clones were virtually identical in most respects to each other, and to NIH 3T3 cells. The latter “fibroblast” cell line had previously been shown capable o f temporarily supporting multipotential myeloid cell progenitors (Roberts et aZ., 1987), but lymphocytes do not grow on them (P. W. Kincade, unpublished observations). A noteworthy exception was in the message for IL-7, which was detectable on Northern blots of RNA prepared from our best lymphocyte support clones, but not from 3T3 or our nonsupport clones. Heterogeneity between stromal cell clones that support lymphocyte growth makes it difficult even to know how many kinds of cells are represented. It is clear that serially propagated lines differ from their nornial counterparts in some respects. For example, while Thy-1 was not detectable in primary bone marrow cultures, it is strongly expressed on most of our cloned stronial cell lines (Pietrangeli et al., 1988).Thus, some of the clone-to-clone variability may be more apparent than real. We have therefore focused attention on differences between subclones of an individual cell line in an attempt to determine which features correspond to the lymphocyte support function. Of 10 subclones prepared from BMS2, only one, BMS2.4, completely lacked lymphocyte support capability (P. W. Kincade, unpublished observations). None of our lymphocyte clones survived when placed in liquid or methylcellulose-containing medium over the BMS2.4 subclone. Another laboratory reported an apparent correlation between lymphocyte support capability of a series of stromal cell clones and expression of an antigen normally expressed on pre-B cells (Whitlock et al., 1987).The most efficient of the clones displayed the most staining with the mAb 6C3. Identical, or closely related epitopes are detected by the mAb 6C3 and BP-1 (M. D. Cooper, personal communication), and most of our experience has been with the latter reagent. Nearly all of the cytoplasmic p+, sIg- pre-B cells and many of the newly formed B cells in normal bone marrow express the 6C3/BP-1 antigen (Cooper et al., 1986). The average level of expression initially increases in long-term bone marrow cultures (Witte et al., 1987a), or when IL-7 is used to expand pre-B cells in short-term cultures (Lee et aZ., 1989). Few, if any,

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of the adherent cells in primary Whitlock-Witte cultures stained with BP-1 in a sensitive immunoperoxidase assay, and lymphocytes in some individual cultures totally lost this marker (Witte et al., 1987a,b).Thus, BP-1 was not readily detectable in either the adherent or nonadherent components of some long-term cultures. Weak expression of BP-1 was found on two of our cloned stromal cell clones, but not on another (Pietrangeli et al., 1988). It will be very interesting to learn if expression of 6C3/BP-1 corresponds to function in our BMS2 subclones. All of these observations suggest that “stromal” cells constitute a critical component of the lymphopoietic microenvironment. However, we still do not know how specialized they are to conduct this particular function and how restricted they might be with respect to localization. In adults, typical pre-B cells are only found within bone marrow, but there is reason to believe that appropriate microenvironmental elements may be present in a quiescent state in other tissues. Functional stromal cell clones have been isolated from adult spleen (Pietrangeli et al., 1988),fetal liver (Hardy et al., 1987),and other tissues (D. Rennick, personal communication). The abundance of IL-7 mRNA is actually less in bone marrow than it is in spleen (Namen et al., 198813). Pre-B cells may not be conspicuous in the spleen because the IL-7responding precursors are absent from that tissue, or because their expansion is prevented by antagonistic cytokines (see later). Extramedullary stromal cells might become functionally active during conditions of marrow failure, such as in myelofibrosis (Gordon and Barrett, 1985), or during regeneration following myeloablative drug therapy. The expansion of lymphoid tumors could also be influenced by IL-7-producing stromal cells, and it is possible that this interaction is not limited to the bone marrow. We recently isolated functional stromal cells that had apparently infiltrated a subcutaneous lymphoid tumor growing in athymic nude mice (K. Medina et al., unpublished observations). If further studies document that these were indeed of host origin, it will be interesting to learn whether they migrated from the bone marrow, or were present in the peripheral tissues. There may be a slow progression of cells in typical Whitlock-Witte cultures, but it has been repeatedly stressed by many investigators that production of B lymphocytes is limited (Whitlock et al., 1985; Kincade, 1987). Furthermore, normal pre-B cells maintained in IL-7 show little tendency to acquire mature B-cell characteristics (Lee et al., 1989). This suggests that we have more to learn about microenvironmental elements that control the final events in this lineage.

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VIII. Recognition and Adhesion between Cells

The distribution of lymphocytes in long-term cultures is distinctly nonrandom (Kincade, 1987).Although some of them are free-floating or easily detached into the medium, most are found in foci, associated with cells of the adherent layer. Some specificity to the interaction is suggested by the fact that lymphocytes never bind directly to macrophages, and it is reestablished within 1 hr when it is deliberately disrupted (Witte et al., 1987b). The strength of the adhesion between lymphocytes and stromal cells seems to vary with the age of culture and depends on which stromal cells are used. However, it is sufficient to withstand the pull of gravity in inverted cultures for at least 1month (P. W. Kincade, unpublished observations). Identification of adhesion molecules responsible for this interaction could be informative with respect to how recognition and organization is achieved in the bone marrow environment. Many lymphocytes can be detached from stromal cells by treatment with EDTA, or with an enzyme specific for the glycosyl-phosphatidylinositol (G-PI) linkage (Witte et al., 1987b). A requirement for divalent cations has been shown for several cell adhesion systems. One ligand for LFA-1, which is found on lymphocytes, is thought to be ICAM-1 (Rothlein et al., 1986; Dustin et al., 1986; Marlin and Springer, 1987). Addition of mAb to LFA-1 had no consistent effect on lymphocyte binding in LTBMC, and it has not yet been possible to demonstrate mRNA transcripts for ICAM in stromal cells with a human cDNA probe (P. W. Kincade, unpublished observations). Cadherins represent another well-Characterized family of adhesion molecules (Takeichi, 1988), and stromal cells seem to express at least one of these (unpublished collaborative studies with Dr. M. Takeichi). However, proof that this is functionally significant will await development of appropriate reagents for study of murine cells. Lymphocytes can also bind to cryptic sites in fibronectin (Fn) fragments (Bernardi et d., 1987), but we did not observe inhibition of binding in cultures with synthetic Fn peptides (Witte et al., 1987b). Some isoforms of the N-CAM family of adhesion molecules were known to be anchored to the membrane via G-PI, and we found that N-CAM was readily demonstrable on our cloned stromal cells (He et al., 1986; Hemperly et al., 1986; Thomas et al., 1988). At least seven N-CAM transcription species result from alternative-exon utilization, and this leads to tissue-specific synthesis of protein isoforms (Owens et al., 1987; Cunningham, et aZ., 1987; Dickson et al., 1987).For example, embryonic or regenerating muscle cells express a unique form of

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N-CAM, which has a domain not found in other tissues, and two of the isoforms are G-PI-linked. Only one isoform of N-CAM is G-PI-linked on neural cells, and a unique domain can also be made in that tissue. Similarities and possible differences from those patterns have been revealed by our analyses of stromal cells. The mRNA transcripts are of similar size to those made by the G7 myoblast line and were estimated at 2.9,5.2, and 6.7 kh (Gimble et al., 1989).This pattern is interesting in light of similarities noted between stromal cells and smooth muscle cells in human bone marrow cultures (Charbord et al., 1985).Immunoblots revealed one major (155 kDa) and two minor (79 and 40 kDA) protein species, and only the smallest appeared to be G-PI-linked to the membrane (Thomas et al., 1988). Lymphocytes from long-term cultures lacked N-CAM, so it is unlikely that these molecules mediate their adhesion to stromal cells in a homotypic fashion. However, N-CAM has a binding site for heparan sulfate (Cole et al., 1986a,b),and this could be important for immobilizing hematopoietic growth factors that stromal cells make (Roberts et al., 1988; Gordon et al., 1987). The interaction of recirculating lymphocytes with specialized cells lining high endothelial venules (HEV) has been extensively studied (Woodruff et al., 1977; Gallatin et al., 1986). Methods have been reported for establishing HEV-like cells from rat lymph nodes in monolayer cultures (Ager and Mistry, 1988; Ise et al., 1988).Mature lymphocytes bind to and migrate beneath these large adherent cells in a fashion that is morphologically similar to the interaction previously described for thymus- and bone marrow-derived stromal cells and lymphocytes from those tissues (Nishi et al., 1982; Hiai et al., 1985; Kincade, 1987; Witte et al., 1987b). Other similarities include the absence of common leukocyte antigens, presence of Thy-1 antigen, synthesis of laminin and collagen type IV, and requirements for divalent cations for lymphocyte binding. It will be interesting to learn if stromal cells and HEV are developmentally or functionally related. The basis for the curious phenomenon termed “pseudoemperipolesis” is not known. Lymphocytes might recognize some polarity to the highly spread stromal cells in culture and/or migrate beneath them because of chemotaxic factors they release. The extent to which it occurs in our hands depends on the stromal cell and lymphocyte clones that are used and does not obviously relate to lymphocyte growth. Recognition and adhesion between cells frequently involves molecules from distinctly different families as well as alternative forms of those proteins (Edelman, 1986; Takeichi, 1988; Springer et al., 1982). In the case of bone marrow, progress is also being made in understanding how maturing erythroblasts interact with macrophagelike nurse

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cells (Crocker and Gordon, 1986; Pate1 and Lodish, 1986) and how immature granulocytes bind to matrix-associated proteins (Campbell et al., 1987; Campbell and Wicha, 1988).This is critical to learning how hematopoietic cells are retained in appropriate niches within the microenvironment, how their timely release is linked to maturation, and how dysfunction of these processes might contribute to marrow dysfunction and malignancy (Gordon, 1988; Gordon and Barrett, 1985). IX. Some Cytokines That Affect Lymphohematopoiesis

Erythropoietin and macrophage colony-stimulating factor (M-CSF) are cytokines that selectively and efficiently stimulate production of red blood cells and macrophages, respectively (Metcalf, 1984). Some years ago, it seemed likely that specific poietins would exist for each of the other blood cells, including lymphocytes, but a much more complex picture is emerging. To date, at least seven well-characterized cytokines have been shown to influence stem cells and precursors of B lymphocytes in culture. None of them are totally lineage-restricted in action, and it is difficult to imagine how normal steady-state lymphopoiesis is the net result of so many molecules. Two recently described ones of these, IL-7 and TGF-P, merit particular attention because of their potency in ijitro. They also serve to indicate quite different mechanisms of synthesis, availability, and action. Interleukin 7 is probably only made in trace quantities, but in immediately active form. In contrast, large amounts of TGF-P molecules are present in bone marrow and closely associated with cells that are potentially responsive to them. They are expressed in inactive precursor forms that can be activated by limited proteolysis or acidification. There is reason to believe that still other cytokines influence hematopoiesis only under circumstances of unusual demand or systemic response to inflammation. Techniques for enrichment of multipotential stem cells and assaying them in culture have made it possible to determine which cytokines affect their proliferation and differentiation (Metcalf, 1987; Bertoncello et al., 1989; Bartelmez et al., 1989; Ikebuchi et al., 1987, 1988).So far, it appears that several may act on cells at that early stage. Granulocyte-macrophage CSF (GM-CSF) and IL-3 can both act as multipoietins, whereas IL-1 and IL-6 are effective cofactors. Interleukin 3 has been used in a number of studies to derive multipotential progenitor cell lines, including cells that appear destined to become lymphocytes (Greenberger et al., 1983; Dexter et al., 1980; Palacios and Steinmetz, 1985; Kinashi et al., 1988). However, it is not yet clear

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that this cytokine participates in normal bone marrow function. Repeated attempts to demonstrtate IL-3 in long-term bone marrow cultures have been negative (e.g., Zipori and Lee, 1988),and some strains of apparently healthy mice are virtually unresponsive to it (Lee, 1988). Studies with a cloning assay for early precursors also indicates that IL-3 may have an indirect effect on lymphopoiesis (Paige, 1985). The helper T lymphocytes that make IL-3 may be infrequent or quiescent in normal bone marrow. However, they could participate in inflammatory responses by release of this multifunctional cytokine. Short-term cultures and an inducible pre-B cell line have been useful for identifying factors that affect the final stages in B-lymphocyte formation. Interleukin 1 (IL-1) and tumor necrocis factor (TNF) are constituents of tumor necrosis serum, which was first found to be active in culture (Hoffman et al., 1977; Kincade, 1987). Purified IL-1 was subsequently shown to induce d i g h t chain synthesis in a pre-B cell line and to accelerate maturation of normal precursors in cultures of fresh bone marrow suspensions (Giri et al., 1984; Stanton et al., 1986). The possibility that IL-1 may also work via an indirect mechanism is suggested by findings that it caused one cloned stromal line to make IL-4, which in turn stimulated pre-B cell maturation (Kinget al., 1988). Interleukin 4 does not enhance expression of light chains on pre-B cells, but it does induce class I1 antigens on certain pre-B cell lines (Polla et al., 1986a,b; 1988; Lee et aZ., 1987).A pH-sensitive cytokine that also stimulated pre-B cells was subsequently identified as immune interferon (IFN-y) (Giri et al., 1984; Paige et al., 1982; Sidman et al., 1984; Weeks and Sibley, 1987). Two potentially novel cytokines have been purified from the serum of young NZB strain mice that also influence events at this stage of the lineage (Jyonouchi et al., 1985, 1988). The first substances found to influence the production of pre-B cells in culture from earlier precursors were isolated from the urine of cyclic neutropenic patients (Landreth et aZ., 1985). In a standardized assay, this material stimulated pre-B cell formation from marrow cell suspensions that had been extensively depleted of recognizable B-lineage cells. Using the same system, an active substance was identified in the supernates of a cloned stromal cell line and subsequently obtained in recombinant form (Landreth and Dorshkind, 1988; K. Landreth, personal communication). While little is known about effects of IL-6 on committed precursors of B cells, it plays a critical role in preparing multipotential stem cells for stimulation by other cytokines (Ikebuchi et aZ., 1987). At the other end of the lineage, it is an essential replication factor for plasma cells

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(Muraguchi et aE., 1988). Synthesis of IL-6 by stromal cell clones appears to be under positive autocrine regulation (Gimble et al., 1989). Interleukin 5 is another cytokine that merits further study. It is the only factor known that can selectively stimulate eosinophil formation, and it can influence pre-B cells in long-term cultures (Yamaguchi et al., 1988; K. Takatsu, personal communication). X. interleukin 7

Stromal cell clones produce extremely small quantities of one or more cytokines that affect pre-B cells (Hunt et al., 1987; Whitlock 1987; Landreth and Dorshkind, 1988; Lemoine et al., 1988).Although these were demonstrable in conditioned medium, purification and molecular cloning have thus far been achieved for only one replication-inducing factor, which has come to be known as IL-7 (Namen et al., 1988a,b). The standardized bioassay for IL-7 utilizes a unique IL-7-dependent lymphocyte clone that was developed from long-term culture. It, and a similar clone we have prepared, are unusual in that they will grow continuously with this lymphokine as the only stimulus. Our other lymphocyte clones, like lymphocytes freshly isolated from primary Whitlock-Witte cultures, give a prompt proliferative response and then die unless stromal cells are present (K. Medina, unpublished observations). The effect of IL-7 has now been evaluated with cells from normal hematopoietic tissues (Lee, 1988; Lee et al., 1989). This factor caused marked proliferation when added to liquid cultures of whole bone marrow, and cell separation experiments revealed that the immediate response was attributable to large pre-B cells. Small pre-B cells, newly formed bone marrow B cells, and B cells in peripheral tissues simply died when placed in IL-7. In contrast, large pre-B cells, which were probably already in cycle, usually continued to divide for 2-3 weeks before exhaustion in IL-7-containing cultures. The selectivity of this response was then exploited to devise a cloning procedure for pre-B cells. Whole bone marrow, or enriched fractions, gave rise to compact colonies of 52000 cells when plated in semisolid agar with IL-7. A linear dose response was achieved, suggesting that the factor acts directly on responsive cells of the B lineage. There was little tendency for pre-B cells replicating in IL-7stimulated semisolid or liquid cultures to mature (Lee, 1988; Lee et al., 1989). Few, if any cells in pre-B colonies expressed K-light chains. Enriched B-lineage cells were held as long as possible in liquid culture so that their fate could be closely monitored. Display of Thy-l and

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class I1 antigens were significant, but otherwise the phenotypes were consistent with long-term-cultured lymphocytes. Virtually all of them were positive for the BP-1 antigen, and variable numbers displayed the common leukocyte family epitope detected by our mAb 14.8. Although no pre-B colonies were generated with marrow from immunodeficient SCID (severe combined immunodeficiency) mice, they did give rise to BP-1+, Thy-l+ lymphocytes after an interval in liquid culture with IL-7. Some early lymphocyte progenitors may be responsive to this cytokine (Lee, 1988; Lee et al., 1989). Marrow suspensions were extensively depleted with mAb 14.8 and cultured with IL-7. After a significant delay, typical B-lineage lymphocytes emerged. It is possible that time was required to appreciate the replication of a few large pre-B cells that were not eliminated from the preparations. The cytokine may also have facilitated the survival of early precursors, which then spontaneously achieved a degree of maturation that would permit them to replicate in IL-7. Ongoing studies with enriched hematopoietic cells and embryonic tissues may be informative in this regard. Effective lymphocyte growth support by stromal cell clones and subclones appears to correlate with IL-7 gene expression. This suggests that it may be a critical replicative stimulus for pre-B cells. However, if it were the only limiting factor, one should be able to classify all of our stromal cell clones on the basis of support or nonsupport of pre-B cells. This has proved not to be the case thus far (P. W. Kincade, unpublished observations). It is also easy to invoke the existence of another factor to explain why normal pre-B cells do not divide indefinitely with this stimulus alone. At least some memory B-cell clones have extensive replicative potential (Williamson and Askonas, 1972), and this should be the case with a subpopulation of normal pre-B cells. Loss of the requirement for a putative second factor would explain the appearance of very rare lymphocyte clones that can grow in IL-7 alone. One of these now has no apparent dependence on either stromal cells or IL-7, and it readily formed tumors in nude mice (K. Medina, unpublished observations). XI. Localization of Cytokines in the Microenvironment

Few of the cytokines that influence lymphohematopoiesis are lineage-specific, and this complicates an understanding of how production of each of the eight types of blood cells can be coordinated and independently controlled. However, several observations bear on this question. A conserved AU-rich sequence in the 3’-untranslated region

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of certain messages is thought to be recognized by specialized RNases (Shaw and Kamen, 1986; Beutler et al., 1988). This results in a very short half-life of the mRNA, which could not only limit the abundance of cytokines, but also provide a tight coupling of stimulus to response. That is, elicited factor production would last only so long as an exogenous stimulus such as endotoxin or systemically released cytokine were present. Many factors may be accessible only to cells within the immediate vicinity of factor-producing cells. Not all cytokines are released in soluble form. For example, there may be membrane-associated forms of M-CSF, GM-CSF, TNF, and IL-1 (Rettenmier et al., 1987; Gough et al., 1985; March et al., 1985; Bakouche et al., 1988; Kurt-Jones et al., 1985). In the case of M-CSF, the factor can be made as a transmembrane molecule that is subject to proteolytic cleavage and release (Rettenmier et al., 1987). Other cytokines are probably secreted and immediately bound to components of the ECM (Gordon et al., 1987; Roberts et al., 1988). This may be similar to the situation with other types of growth factors, which are known to be immobilized and stabilized by heparan sulfate (Lobb et al., 1986; Saksela et al., 1988).It is possible that bone marrow stromal cells elaborate not only regulatory cytokines, but also matrix elements to bind and present them to hematopoietic progenitors. Complex components of the matrix have been studied in myeloid long-term bone marrow cultures, where they undoubtedly contribute to the microenvironment (Wight et al., 1986; Campbell et al., 1985; Campbell and Wicha, 1988). Experiments employing permeable membrane separations in longterm cultures demonstrated the importance of close interactions between stromal cells and lymphocyte precursors (Kierney and Dorshkind, 1987). Negative regulation, which can be appreciated with adherent layer cells from mutant moth-eaten mice, also seems to require cell contact because conditioned medium consistently lacked inhibitory activity (Hayashi et al., 1988). As mentioned earlier, only lymphocytes that remained bound to stromal cells in an inverted microculture system remained viable (P.W. Kincade, unpublished observations). All of these observations indicate that spatial relationships are important for positive and negative control of lymphopoiesis in longterm cultures, and the same is presumably important in situ. Xll. Transforming Growth Factors p

Progress has been rapid in identifying cytokines that stimulate proliferation and maturation of B-lymphocyte lineage precursors, but much less is known about substances that act as antagonists of these

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responses. Transforming growth factors /3 (TGF-P)in mice and humans are virtually identical and can be neutralized by a mAb that recognizes both (Dasch et al., 1989). This extraordinary conservation suggests these factors mediate vital functions, and many have been described (Spom et al., 1987; Cheifetz et al., 1987; Heine et al., 1987).Effects on lymphohematopoietic cells are particularly relevant to this review, and TGF-/3 are known to be localized in bone marrow as well as in hematopoietic foci of developing embryos (Ellingsworth et al., 1986; Wilcox and Derynck, 1988). As little as M concentrations of active TGF-Pl or TGF-P2 blocked the LPS-induced K-light chain expression in the 70Z/3 pre-B cell line (Lee et al., 1987). The selective nature of this inhibition also revealed complexity in the transmembrane signaling pathways that influence this critical differentiation event. Very little inhibition of the response to IFN-y was noted, although this stimulus induced K-light chain transcription as effectively as LPS (Briskin et al., 1988). Furthermore, a K enhancer-binding protein, NFK-b, was activated by LPS but not by IFN-y. These findings are consistent with previous studies by Sibley and colleagues, who showed that variants of the 70Z/3 line can selectively respond to one or the other of these agents (Weeks and Sibley, 1987; Mains and Sibley, 1983).It appears that two independent signaling pathways lead to the same response: transcription of the K-light chain gene. Induction by LPS is preferentially sensitive to TGF-P. The highly selective nature of TGF-P inhibition was also obvious in the response of pre-B cells to IL-4. This cytokine induces expression of class I1 antigens, but not surface Ig, and this is unaffected by TGF-P (Polla et al., 1986a,b, 1988; Lee et al., 1987). Similarly, ongoing Ig synthesis in mature resting B cells was insensitive to TGF-P. That these cells had active receptors was demonstrated by ablation of the mitogen-stimulated proliferative response by this factor (Lee et al., 1987). Proliferation of pre-B cells driven by IL-7 was also sensitive to TGF-P, whereas a number of other cytokines had no effect (Lee et aE., 1989). These findings are consistent with reports of discrete responses of a wide variety of cell types to this interesting family of mediators (Spom et al., 1987; Massague, 1987). In contrast to other regulatory molecules, TGF-P are relatively abundant and can even be demonstrated on the surface of pre-B cells by immunoperoxidase staining (P. W. Kincade, unpublished observations). Similarly, synthesis of this factor is known to occur in resting and stimulated human B cells (Kehrl et al., 1986). We used the same approach, as well as Northern blot analysis, to determine that these molecules were associated with both the adherent and nonadherent

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phases of long-term bone marrow cultures (Hayashi et al., 1989).This inspired experiments to determine the effects of TGF-P addition to long-term bone marrow cultures. If the cytokine was present only during the first week of establishment, neither myeloid (Dexter) nor lymphoid (Whitlock-Witte) cultures were subsequently productive. In the case of Dexter cultures, the adherent layer was markedly deficient in adipocytes. Various treatment protocols were then used to determine that microenvironmental elements as well as hematopoietic progenitors were affected. M yelopoiesis was ablated in Dexter cultures with one treatment regimen, but the cultures could still give rise to pre-B cells when conditions were made lymphocyte-permissive. Lymphocyte precursors, as well as the microenvironmental elements necessary for their expansion, survived the period of TGF-@exposure, even though neutrophil production was arrested. Multipotential stem cells, are known to be sensitive to this cytokine (Ottmann and Pelus, 1988; Sing et al., 1988; Keller et al., 1988). This raises the possibility that lymphocytes that emerged from the switch cultures after treatment derived from rare cells that were precommitted to this lineage. Transforming growth factors P are known to be synthesized as high molecular weight precursors, and most of the material stored in platelets and other tissues is biologically latent (Sporn et al., 1987; Massague, 1987). They are experimentally activated by acidification, but it is not known when and how this step is achieved in uiuo. There would be a number of consequences for lymphohematopoiesis should this occur spontaneously, following trauma, in disease, or associated with an inflammatory response. Myelopoiesis would be preferentially aborted, but higher concentrations and durations would also result in cessation of pre-B cell replication and differentiation. Stromal cells would be prevented from giving rise to adipocytes, and synthesis of ECM components would also be affected (Gimble et al., 1989; Ignotz and Massague, 1985; Seyedin et al., 1987; Bassols and Massague, 1988). Activation of TGF-P might also occur as bone marrow cell suspensions are prepared or as platelets disintegrate in culture. This might have a significant effect on the types of events that can be reproduced in uitro. Studies involving infusion of active TGF-P and a neutralizing mAb may reveal the contribution of this cytokine to normal hematopoiesis and its influence on experimental systems (L. Ellingsworth, personal communication). Other factors may act in competing fashion to regulate events in the B lineage. Prostaglandins inhibit induction of class I1 antigens on pre-B cells by IL-4 (Polla et al., 1986a,b). Interleukin 4 can stimulate pre-B cell maturation while inhibiting their replication on stromal

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cells (King et al., 1988; Rennick et al., 1987).The 70Z/3 line is responsive in culture to a number of cytokines, and it preferentially homes to the bone marrow when injected intravenously (Pietrangeli and Kincade, 1987). It might be expected that the cells growing in uiuo would be substantially different with respect to surface markers from the cells that were transplanted. However, this was not the case and the fact that normal mouse serum blocks many responses suggests that endogenous inhibitors could be involved. It will be interesting to learn if these represent novel cytokines and, if so, to investigate their specificity and origin. XIII. Cytokine Responses Involving Stromal Cells

Studies of cloned stromal cell lines demonstrate that they can make and respond to multiple cytokines in culture. The biological implications of these findings will be clear only when we know which ones are produced under normal steady-state conditions in bone marrow and which ones can be elicited under unusual circumstances. However, it is already clear that some factors are constitutively made in culture, and details are being revealed as to how production of others can be manipulated experimentally. In all cases where biological or mRNA assays were performed, production of macrophage colony-stimulating factor (M-CSF/CSF-1) has been found in uninduced stromal cells (see review of clones in Kincade et al., 1988).Attempts by many investigators to demonstrate production of the multipoietin IL-3 have, in contrast, been negative (see, for example Eliason et al., 1988; Zipori and Lee, 1988).Two other myeloid colony-stimulating factors GM-CSF and G-CSF, can be made by stromal cells, but generally have to be elicited by some stimulus (Beutler et al., 1985; Yang et al., 1988; Quesenberry et al., 1984; Broudy et al., 1986a; Kohama et al., 1988; Gimble et al., 1989). Thus, all stromal cells, regardless of whether they support lymphopoiesis, can provide a microenvironment for myeloid cell growth (Collins and Dorshkind, 1987; Hunt et al., 1987; whitlock et al., 1987; Rennick et al., 1987; Pietrangeli et al., 1988). The question then arises as to whether functionally bipotential stromal cells simultaneously support cells of both lineages. Culture conditions that support myelopoiesis reportedly favor release of myeloid CSF (Johnson and Dorshkind, 1986), and the difference in Dexter versus Whitlock-Witte culture systems might be explained in terms of direct effects on stromal cells. However, in our experience, production of mRNA for CSF in a cloned stromal cell line has not been obviously influenced by hydrocortisone,

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or any other constituent of the two types of culture medium (P. W. Kincade, unpublished observations). Furthermore, release of biologically active CSF from the same cells in Whitlock-Witte medium was readily detected with a coculture system (Pietrangeli, 1989). The presence of steroids and absence of 2-ME from Dexter culture medium may simply preclude lymphocyte survival. Receptors for IL-4 have been demonstrated on stromal cell clones, and at least one stromal cell line can be induced to make this cytokine (Lowenthal et al., 1988; King et al., 1988). In addition, addition of exogenous 11-4 affects production of other factors by that particular clone (K. Landreth, personal communication). We have thus far not been able to demonstrate IL-4 mRNA in resting or induced BMS2 cells (P. W. Kincade, unpublished observations). Interleukin 4 has been shown to have differential effects on lymphohematopoietic cells (Rennick et al., 1987; Peschel et al., 1987). It partially inhibited responses stimulated by IL-3, facilitated responses of erythroid and myeloid cells to other factors, and blocked the functional interaction of pre-B cells with a stromal cell clone. Several cytokines influence the growth of stromal cells in culture and their ability to support lymphopoiesis tended to be inversely related to replication (Pietrangeli et aE., 1988). Small amounts of IL-6 mRNA were detected in all of our stromal cell clones, and this was dramatically upregulated by several exogenous stimuli (Gimble et al., 1989). Effective agents included IL-6 itself, as well as LPS, IL-1, IL-7, TNF, and epidermal growth factor (EGF). These responses were abolished by actinomycin, consistent with a requirement for new mRNA synthesis and enhanced by treatment with cycloheximide. The latter effect has been attributed in part to interference with degradation of the message (Shaw and Kamen, 1986). As mentioned before, the presence of conserved UA-rich sequences in the 3’-untranslated region of some cytokine mRNAs corresponds to very short half-lives (Shaw and Kamen, 1986; Beutler et al., 1988).Many similarities can be noted between responses elicited in stromal cells and those independently studied with fibroblasts, endothelial cells and macrophages (Kohase et al., 1987; Van Obberghen-Schilling et al., 1988; Walther et al., 1988; Broudy et al., 1986b; Segal et al., 1987; Seelentag et al., 1987; Thorens et al., 1987; Vallenga et al., 1988; Koeffler et al., 1988). In bone marrow, all of these cell types, in addition to activated T lymphocytes from other tissues, could contribute to systematic inflammatory responses. Pre-B cell numbers were observed to reach abnormally high values during periods of myeloid progenitor cell deficiency in a cyclic neu-

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tropenic patient (Engelhard et al., 1983).This seemed to correlate with the appearance in urine of factors that could stimulate pre-B-cell formation in culture (Landreth et d., 1985). In an animal model, granulocytosis corresponded to pre-B-cell suppression (Fulop et al., 1985). This reciprocal relationship suggests that a common feature, such as competition for an essential stimulus, might be involved. Stromal cells are probably pivotal to production of cells in both lineages, and a better understanding of which cytokines are produced under different circumstances may be informative as to how an appropriate balance is achieved in situ.

XIV. Adipogenesis Bone marrow is known to include nonproductive areas that are laden with fat, and this can be reversed under conditions of anemia (Tavassoli, 1984). It is interesting that most stromal cell clones have been described as being preadipocytes (Kincade et al., 1988). For example, our BMS2 clone spontaneously accumulates large fat-containing vacuoles, and this is accelerated by exposure to hydrocortisone and certain other agents (Gimble et al., 1989).On the other hand, TGF-P and TNF are inhibitors of adipogenesis. The precise role of fat within bone marrow merits further study, as does the function of stromal cells in preadipocyte and adipocyte forms. This dramatic change in morphology must involve a series of coordinated differentiation events, and it highlights the multifunctional and multipotential nature of stromal cells. XV. Additional Perspective

The validity of any model or experimental approach requires direct testing, and application of long-term culture techniques to studies of genetic defects has revealed some limitations of these methods. When and if it becomes possible to duplicate the lymphopoietic microenvironment in vitro, dysregulated conditions observed in vivo will be faithfully represented. This is presently not the case, but the patterns that have been observed are informative about the complexity of intact bone marrow. There is substantial evidence for hyperactivity associated with preB-cell formation in embryonic and young neonatal NZB strain mice (Kincade et al., 1982). Precursors appear earlier, and in larger numbers, than in other strains of mice, and in young mice there are factors in serum that promote pre-B-cell maturation. No evidence for this

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activity was found in serum of adult NZB mice, and at that stage there was a deficiency of precursors detectable in functional assays or by immunostaining. Thus, a good correlation was found between dysregulation in uiuo and observations made with short-term culture procedures. Unexpectedly, these markedly unusual features were not reflected in the behavior of NZB marrow cells in long-term cultures (Kincade et al., 1987; Yoshida et al., 1987). Other than a tendency to make somewhat higher numbers of B cells, the results were similar to those obtained with BALB/c mice. Moth-eaten mice have a severe autoimmune disease that is attributable to one of two recessive mutations (Schultz and Sidman, 1987). However, B-lineage lymphocytes are made in these mice and their marrow contains a nearly normal incidence of myeloid progenitor cells. It was therefore surprising to find that their marrow cells were nonproductive in three types of long-term cultures (Greiner et al., 1986; Medlock et aE., 1987; Hayashi et al., 1988).Regardless of culture conditions, a population of macrophagelike adherent cells dominated the cultures and prevented outgrowth of lymphohematopoietic cells. This also occurred when wild-type cells were mixed with moth-eaten cells and placed in culture, However, many attempts to demonstrate production of a soluble inhibitor by the moth-eaten adherent layers were negative. This suggests that direct contact with cells from the mutant mice negated responses that normally lead to growth in longterm cultures. The reason for the growth of suppressive cells in moth-eaten cultures is unclear. Macrophage growth factor (M-CSF) is produced, but not in exceptional quantities, and addition of neutralizing antibodies to this factor has no effect on adherent-layer composition. While macrophage markers are expressed on the predominant cells in culture, there is not an increased frequency of macrophage precursors in moth-eaten bone marrow, and it is not possible to grow macrophage colonies from adherent-layer cells subcultured with CSF-1. It seems possible that a negative regulatory network is being revealed by this mutation, and that it normally operates in concert with other mechanisms in uiuo. The relative simplicity of long-term cultures apparently permit this to be appreciated. Other subtle effects of mutated or polymorphic genes in intact animals become exaggerated in long-term cultures. The Xid mutation of CBAlN mice preferentially affects cells of the 3 lineage (Scher, 1982)and makes them developmentally dependent on the presence of T cells (Wortis et al., 1982; Mond et al., 1982; Sprent and Bruce, 1984). It was therefore surprising to find that Xid actually causes accelerated

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establishment of growth in long-term bone marrow cultures (Hayashi et al., 1988). The effect was clearly caused by this X chromosomelinked mutation and seemed to be expressed via an influence on cells in the adherent layer. There have been other indications that the product of this gene affects nonlymphoid cells (Scher et al., 1979; Clayberger et al., 1985; Jyonouchi et al., 1983; Teale, 1983), but normal numbers of lymphoid and myeloid cells are produced within the mutant mice (Scher, 1982; Kincade et al., 1982; Reid and Osmond,

1985). Genetic polymorphisms can profoundly affect the behavior of cells in culture. We found that proliferation of lymphohematopoietic cells from BALB/c mice is much better than those from CBA/H strain animals (Hayashi et al., 1988). This was first indicated by comparison of the kinetics and efficiency of cell growth in long-term cultures prepared from marrow of the two strains. When equal-part mixtures were made and placed in culture, BALB/c origin cells dominated within a short period of time. There is no evidence that CBA/H mice are any less normal than BALB/c mice, and other investigators have appreciated an influence of the pedigree of mice on the in uitro performance of their cells (Sakakeeny and Greenberger, 1982). These influences are presumably neither detrimental or significant in uiuo. Interleukin 3 is a potent multipoietin in uitro and may influence early lymphoid progenitor cells (Ihle and Weinstein, 1986). However, hematopoietic progenitors from some strains of mice are virtually unresponsive to this cytokine (Lee, 1988). There must be redundancy with respect to the normal function of IL-3, because the nonresponder mice are not obviously compromised. Mice with heritable SCID make very few cells in either B or Tlymphocyte lineages, and this has been attributed to problems with rearrangement of T-cell receptor and Ig genes (Bosma et aZ., 1983; Schuler et al., 1986, 1988; Okazaki et al., 1988; Kim et al., 1988). The fate of the intrinsically defective lymphocytes in SCID mice is not known, and it seems possible that their failure to expand in significant numbers is due to some “quality ~ o n t r ~ lsystem. ’’ In contrast to their behavior in uiuo, one can easily grow SCID lymphocytes in long-term bone marrow cultures (Witte et al., 1987a; Hirayoshi et at., 1987). A11 markers are normally expressed in that circumstance, and presumably abortive rearrangements affect the Ig genes. In addition, IL-7responsive lymphocytes can be expanded in culture from SCID marrow suspensions (Lee et al., 1989). All of these findings suggest that the normal bone marrow microenvironment is more complex, and subject to more interacting regulatory

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networks, than are long-term bone marrow cultures. This is an advantage for dissecting individual elements, and it may someday be possible to construct more realistic models with appropriate combinations of recombinant molecules and cloned cell lines. In the meantime, information is being obtained about how the growth of lymphoid precursors is normally regulated, and this should be relevant to cancer research in general.

ACKNOWLEDGMENTS The published and unpublished observations from our laboratory were supported b y grants A1-19884 and A1-20069 from the National Institutes of Health, as well as by a fellowship from the Leukemia Society of America.

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STRUCTURE AND FUNCTION OF THE B-LYMPHOCYTE EPSTEIN-BARR VIRUS/C3d RECEPTOR Glen R. Nemerow, Margaret D.Moore, and Neil R. Cooper Research Institute of Scripps Clinic, Department of Immunology. La Jolla, California 92037

I. Introduction 11. Identification and Characterization of CR2 A. Functional Relationship of the EBV Receptor and the C3d Receptor B. Immunochemical Analysis of the EBV/C3d Receptor (CR2) 111. Structure of CR2 A. Isolation and Characterization of cDNA Clones Encoding CR2 B. CR2-A Member of a Multigene Family C. Expression and Analysis of Recombinant CR2 IV. Structure and Function of the CR2 Ligands A. C3d and C3dg B. The gp350/220 Envelope Proteins of EBV C. Sequence Similarity of C3dg and gp 350/220 and Its Role in CR2 Binding V. Cellular and Tissue Distribution of CR2 A. Lymphoid Cells B. Thymocytes C. Follicular Dendritic Cells D. Epithelial Cells E. CR2 Expression during B-Cell Development, Activation, and Differentiation VI. Functional Properties of CR2 A. Role of CR2 in Ligand Internalization B. Role of CR2 in B-Cell Activation C. Role of CR2 in Complement Activation and Regulation VII. Summary and Future Prospects References

1. Introduction

The cell surface molecule(s) used by viruses to initiate infection of human cells has been identified for only a few viral pathogens. The best-known example of this situation is the human immunodeficiency virus (HIV-1),which uses the CD4 cell surface glycoprotein as a receptor to infect T lymphocytes. The putative natural ligands of CD4 are the class I1 antigens of the major histocompatibility complex (MHC). Epstein-Barr virus (EBV), another example of a virus that uses a normal host cell glycoprotein as a receptor during infection, is a human oncogenic herpesvirus that is the etiological agent of infectious mononucleosis and is implicated in the pathogenesis of Burkitt’s lymphoma 273 ADVANCES IN CANCER RESEARCH, VOL. 54

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and nasopharyngeal carcinoma. The receptor for EBV is complement receptor type 2 (CR2), a molecule that is primarily expressed on the surface of B lymphocytes but is also the receptor for the complement C3d and C3dg fragments (thus its designation as complement receptor type 2). Thus EBV and HIV have established a biological niche by binding to host cell molecules that are primarily restricted to cells of the immune system and that play a role in immune recognition or immunomodulatory reactions. Studies of CR2 structure and function, as with CD4, have been greatly stimulated by the prospect of elucidating the molecular basis of virus-host cell interactions as well as by the opportunity of determining how these receptors function in cellular immunity. This review integrates current knowledge of the structure of the EBV/C3d receptor (CR2)with the functions ofthe molecule not only in terms of its ability to initiate EBV infection but also in its involvement in B-lymphocyte activation and other immunomodulatory reactions.

Ii. Identification and Characterization of CR2 A. FUNCTIONAL RELATIONSHIP OF THE EBV RECEPTOR AND THE C3d RECEPTOR The identification of a lymphocyte receptor specific for the complement C3d fragment was first reported independently by three laboratories (Eden et al., 1973; Ross et al., 1973; Okada and Nishioka, 1973). The receptor was defined by its functional ability to form rosettes with erythrocytes bearing C3d molecules. Subsequent studies by Jondal and Klein (1973),Jondal et al. (1976), Yefenof et al. (1976), Yefenof and Klein (1977), and Klein et al. (1978) showed aclose functional relationship between the EBV receptor and the C3d receptor on B cells. Epstein-Barr virus as well as C3d-bearing particles bound to the same cell lines, and both receptors cocapped independently of other cell surface markers. In competition studies, C3d binding was blocked by EBV and anti-EBV complexes. Later studies by Hutt-Fletcher et al. (1983)and Magrath et al. (1981)showed that EBV absorption to B cells could be blocked by C3d-dependent rosetting. Magrath et al. (1981) also demonstrated that C3d receptors and EBV receptors could be coinduced with theophylline in undifferentiated lymphoma cells. Rosenthal et al. (1978) used fluorescence polarization analysis to show that C3d blocked EBV fusion with B-cell membranes. Taken together, these earlier studies provided compelling evidence that the EBV and C3d receptors were closely related if not identical.

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B. IMMUNOCHEMICAL ANALYSIS OF THE EBV/C3d RECEPTOR(CR2) The development of monoclonal antibodies (mAb) to cell surface molecules played a decisive role in the identification of the EBV/C3d receptor (CR2). Iida et al. (1983), using the B cell-specific mAb, antiB2, developed by Nadler et al. (1981), demonstrated that the C3d receptor isolated from detergent extracts of Raji B lymphoblastoid cells or tonsil B cells was a 140-kDa protein having an isoelectric point of 8.2. These properties were identical to those of molecules isolated by affinity chromatograpy on C3d-Sepharose columns. The anti-B2 mAb blocks C3d rosetting in the presence of a secondary anti-Ig antibody, indicating that this mAb binds to an epitope of CR2 that is distinct from the precise ligand-binding epitope. Subsequently, Weis et al. (1984) used the HB5 mAb developed by Tedder et al. (1984) to demonstrate similar functional and biochemical properties for CR2. Fingeroth et at. (1984) carried out important experiments using the HB5 antibody to reveal the identity of the EBV and C3d receptors. In these studies CR2 was immobilized on staphylococcal protein Abearing particles containing HB5. These complexes were then shown to bind to radiolabeled EBV, thus indicating that CR2 is both a C3d receptor and an EBV receptor. Subsequently, Nemerow et al. (1985b) and Rao et al. (1985) showed that the OKB7 mAb produced by Mittler et al. (1983) directly blocked both EBV- and C3d-dependent binding to B cells. Infection by EBV was also blocked by the OKB7 mAb (Nemerow et al., 1985b), as well as by polyclonal anti-CR2 antibodies (Frade et al., 1985a), thus further indicating the CR2 plays a primary role in virus infection. A number of other studies have used C3d affinity chromatography to characterize CR2. Micklem et al. (1984) isolated a 145-kDa molecule from human tonsillar B cells using this technique. In contrast, Lambris et al. (1981) had earlier identified a 72-kDa C3d-binding protein from spent culture medium of Raji cells. Subsequent studies by Micklem et al. (1985), Myones and Ross (1987),and Petzer et al. (1988)showed that this smaller molecule is likely a proteolytically derived fragment of the 145-kDa molecule. Complement receptor type 2 is modified following translation in vivo and contains -20% carbohydrate by weight. Endoglycosidase studies by Siaw et al. (1986) and biosynthetic studies by Weis and Fearon (1985) indicated that CR2 is derived from a 111-120 kDa nonglycosylated precursor. The study by Weis and Fearon indicated that CR2 contains a number of N-linked oligosaccharides that do not contribute to the C3d-binding activity ofthe molecule but do extend its half-life on the plasma membrane.

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The immunochemical and biosynthetic studies cited earlier have enabled further analysis of the dual ligand-binding activity of the receptor. Complement receptor type 2 isolated by immunoaffinity chromatography (Nemerow et al., 1986) was shown to bind directly to immobilized EBV and C3dg, to the EBV gp350/220 envelope protein (Nemerow et al., 1987),or to C3d-Sepharose (Weis et al., 1986a).Mold et al. (1986) have also incorporated purified CR2 into liposomes by detergent dialysis and demonstrated that these artificial lipid vesicles bound to EBV and to C3d-bearing particles 111. Structure of CR2

A. ISOLATION AND CHARACTERIZATION OF cDNA CLONES ENCODING CR2 Weis et n2. (1986b) first demonstrated that CR2 has a similar amino acid composition to the receptor for C3b/C4b (CRl), a finding that suggested that these two receptors are highly related. Using this information, these authors probed a tonsil B-cell library in hgtll with a CR1-specific cDNA probe at low stringency in order to isolate CR2specific cDNA clones (Weis et al., 1986a). Several partial clones for CR2 were identified, enabling further verification of the relatedness of CR1 and CR2. Subsequently Moore et al. (1987) isolated a full-length CR2 cDNA clone from a Raji B-cell library. This clone contained the entire coding region of CR2, which allowed the determination of the nucleotide sequence of the receptor. The deduced amino acid sequence of CR2 (Moore et al., 1987) indicates that the entire extracellular domain of the receptor is made up of 16 repeating subunitsreferred to by Weis et al. (1986a) as SCR (small consensus repeats)each of which is composed of 60-75 amino acids. The subunits have -25-35% identity with each other and are delineated by four highly conserved cysteine residues as well as a number of other residues. These subunit repeats are also present in a large number of C3- and C4-binding proteins (Fig. 1). Diagon computer analysis also shows a higher order of repeating structure for CR2 in that the repeat subunits of the receptor occur in clusters of four. Approximately 10% of the sequence of CR2 has been verified by sequencing of tryptic peptides derived from purified CR2. This has allowed identification of the amino terminus (isoleucine) and carboxyl terminus (serine) of the molecule. Complement receptor type 2 is anchored in the membrane of B cells hy a 28-amino acid hydrophobic transmembrane domain that is linked

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FIG.1. Alignment of the consensus sequences representing the repeat elements composing the 60mer family of proteins. See text for explanation of abbreviations. Single letters encode individual amino acid residues. Boxed residues indicate the position of highly conserved amino acids; the shaded boxes indicate the location of the four cysteine residues.

to a short, 34-amino acid cytoplasmic tail, which has several potential sites for serine or threonine phosphoryIation. Weis et al. (1988) have also isolated several full-length CR2 cDNA clones from a tonsil B-cell library and determined their sequences. One of five clones contains a 16-repeat form of the receptor while four of the clones encode a 15-repeat structure. This finding suggests that CR2 may be encoded by different allelic genes or that its mRNA is alternatively spliced. Recent work on analysis of the CR2 gene (Fujisaku et al., 1989) indicates that multiple allelic forms of CR2 corresponding to the 15- and 16-repeat forms are present in the human genome.

B. CR2-A

MEMBEROF A MULTIGENEFAMILY

A comparison of the nucleotide and amino acid sequences of CR2 (Moore et al., 1987; Weis et al., 1988) with other C3- and C4-binding proteins including CR1 (Wong et al., 1985; Klickstein et al., 1987),

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decay-accelerating factor (DAF)(Caras et d., 1987; Medof et al., 1987), C4-binding protein (C4bp) Chung et al., 1985; Kristensen et al., 1986), factor H (Kristensen et al., 1986), C2 (Bentley and Porter, 1984), factor B (Morley and Campbell, 1984; Mole et al., 1984), and membrane cofactor protein (MCP) (Lublin et al., 1988), as well as other complement proteins such as C7 (DiScipio et al., 1988; Stanley and Luzio, 1988), C l r and C l s (Leytus et al., 1986),indicate that this receptor is a member of a family of proteins that contain one or more of the repeating subunits found in CR2 (Fig. 2). A number of these proteins appear to be mosaic in nature in that they also contain other unrelated structural elements. The largest member of this 60mer repeat family, CR1,

Number of 6Omer repeats CR2, Complement Receptor 2 CRI, Complement Receptor 1 Factor H C4-Binding Protein Factor Xlllb MCP Decay-Accelerating Factor p,- Gtycoproleln I Interleukin 2 Receptor (p55) c2 Factor B Clf ClS c7 Cartilage Proteoglycan Core Protein

15 or 16

30 20 8 10 4

I 5 2

3 3 2 2 2 1

FIG.2. Schematic representation of the 60mer family of repeat proteins. The insert includes a key for symbols representing the various structural domains of the proteins. EGF, Epidermal growth factor; LDL, low-density lipoprotein.

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has 30 repeat elements, while other molecules such as the terminal complement proteins C6 and C7 have as few as 2 repeats. Although the majority of the 60mer proteins are C3- and CCbinding proteins, dearly others have different ligand-binding properties such as the 55-kDa chain of the interleukin 2 (IL-2) receptor, or IL2R (Leonard et al., 1985), the &- glycoprotein I or P2gpI (Lozier et al., 1984), and bloodclotting factor XIIIb (Ichinose et al., 1986). Kotwal and Moss (1988) have shown that vaccinia virus encodes a secretory protein that is highly related to the 60mer family. They suggest that this molecule may play a role in regulating complement activation by vaccinia and thus facilitate infection by the virus. Given the heterogeneity of the different proteins that contain the 60mer repeats, it is likely that the repeat elements serve primarily as a framework for the overall protein structure and that the ligand-binding domains are probably contained within only a few of these repeat elements. Klickstein et al. (1988)have analyzed truncation mutants of recombinant CR1 and have identified ligand-binding regions of the molecule. The C4b-binding epitope maps to the first two repeat elements of the amino terminus, while C3b-binding epitopes are present on two other separate elements within the molecule. Klickstein et al. (1987)had earIier postulated that CR1 exists in an extended conformation that serves to position the ligand-binding domain of the receptor away from the cell surface. Physical evidence for this kind of structural motif has been revealed for C4-binding protein or C4bp (Dahlbach et al., 1983), another member of the 60mer family. Preliminary studies with recombinant forms of the extracellular domain of CR2 also indicate that this receptor is an extended molecule. As might be expected from the high degree of sequence homology among some members of the 60mer family, several of the genes encoding these proteins are linked together in the human genome. Studies by Rodriquez et al. (1985), Rey-Campos et al. (1987), ( J. H. Weis et al. (1987), and Carrol et al. (1987), have shown that this linkage group, referred to as RCA (regulator of complement activation) and including CR1, CR2, C4bp, DAF, and factor H (Vik et al., 1989) are on chromosome 1.Complement receptor type 2 has been further mapped to band 32 of the short arm of chromosome 1 (lq32) J. H. Weis et al., 1987). Studies of the CR2 gene by Weis et al. (1988)have indicated that the exons encoding the receptor are contained within 25 kb of genomic DNA. More extensive studies by Fujisaku et al. (1989) have revealed several features of the genomic organization of CR2. Three types of exons are present in the CR2 gene. These include exons that encode

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single repeat elements, those that encode two elements, and those that are split to encode parts of two separate repeats. The order of exons in the CR2 gene is in a repeated array of four, indicating that the contemporary CR2 gene likely evolved from an ancestral gene that contained four repeats. Restriction-fragnient-length polymorphism (RFLP) studies by Fujisaku et al. (1989) also identified allelic variants of the CR2 gene as well as potential sites for alternative mRNA splicing. Thus far no disease association has been linked to CR2 RFLP.

C. EXPRESSION AND ANALYSIS OF RECOMBINANT CR2 1. Expression of Membrune-Associated CR2 in Rodent Cells

Although immunochemical and functional analyses of purified Bcell CR2 have provided strong evidence that CR2 is the major EBV receptor, more definitive proof of this conclusion has recently been obtained by expression of CR2 in cells that lack endogenous receptors. M. D. Moore, N. R. Cooper, and G. R. Nemerow (unpublished) have constructed a eukaryotic-expression vector containing the CR2 cDNA downstream of the immediate early gene promoter of cytomegalovirus (CMV). Murine L-cell fibroblasts were transfected with this construct and a number of cell lines expressing CR2 were identified using fluorescence-activated cell sorting (FACS) analysis. As shown in Fig. 3, one of these CR2-expressing cell lines was capable of binding C3dg, recombinant gp350/220 (Whang et al., 1987), the EBV ligand for CR2 (Nemerow et al., 1987; Tanner et al., 1987), as well as the anti-CR2 mAb HB5. These results verify the dual ligand-binding activity of CR2 and should permit further analysis of CR2 function. Ahearn et al. (1988) have also transfected mouse L cells with the CR2 gene. Highlevel CR2 expression was selected by FACS. It was shown that CR2producing cells bind to both EBV and C3d. In addition, a small proportion of the cells (- 1/200)were infectible by the virus as ascertained by fluorescence staining of the EBV nuclear antigen (EBNA). However, the small proportion of EBV-expressing cells did so transiently. This is likely due to the inability of the ori-P (origin of EBV plasmid replication) to function in rodent cells (Yates et al., 1985). These studies nevertheless indicate that CR2 expression in cells that normally lack this receptor renders them susceptible to virus infection. Undoubtedly other cellular and viral factors play an important role in controlling the outcome of EBV infection. Expression of CR2 in receptor-deficient primate cells may also facilitate further analysis of CR2’s role i n uiuo.

B-LYMPHOCYTE EPSTEIN-BARR

Control

V I R U S / CRECEPTOR ~~

Transfected

28 1

RAJl

Log Relative Fluorescence FIG.3. Stabile expression of CR2 in mouse L cells. Under control of the CMV IE promoter, CR2 cDNA was transfected into L-cell fibroblasts using calcium phosphate. After selection oftransfected cells with G418, the cells were analyzed for CSdg, gp350, and monoclonal antibody HB5 binding by FACS. Control cells were transfected with a plasmid containing pSVneo alone.

2. Expression of Soluble Forms of CR2 in the Baculovirus

Vector System An inherent problem in studying the structural features of CR2 is its relative low abundance on B cells. We have begun to explore the utilization of an efficient eukaryotic expression system for producing relatively large amounts of soluble CR2. Baculovirus, an insect virus, has been adapted for the high-level expression of mammalian proteins (Miyamoto et al., 1985; Smith et al., 1985). Using this system three different forms of the extracellular region of CR2 cDNA (lacking the transmembrane and cytoplasmic domains) were placed downstream of the strong polyhedrin promoter in the baculovirus transfer vector, pAc373 (Fig. 4).The first form of recombinant CR2, containing the 4 N-terminal repeats, was derived by introduction, by site-directed mutagenesis, of an internal BamHI site and translation termination codon.

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282

Eem

Erm

EcoRV

FIG.4. CR2/baculovirus expression vectors. Complement receptor type 2 cDNA containing the first 4 repeats (left diagram) or the entire 16 repeats (middle), or 10 repeats were placed downstream of the polyhedrin gene promoter in the baculovirus transfer vector pAc373. The full-length CR2 lacks the natural internal BamHI site as a result of deletion by site-directed mutagenesis.

The second form of CR2, containing the entire 16 repeats of CR2 followed by a stop codon and BarnHI site, was also generated by site-directed mutagenesis. The third form of the receptor contained 10 repeats derived by using the natural BamHI site. Since this latter construct places CR2 in the correct reading frame with the polyhedrin gene and does not contain a stop codon, the expressed molecule would be expected to be a fusion protein. Each of these cDNA constructs has been used to cotransfect SF9 insect cells in the presence of wild-type baculovirus DNA. Recombinant viruses have been isolated and the expressed secreted proteins analyzed by immunoprecipitation. The analysis with the full-length extracellular form of CR2 is shown in Fig. 5. A 125-kDa molecule, which is close to the predicted size of this form

FIG.5. lmmunoprecipitation analysis of CR2 (1-16) expressed in baculovirus. Infected cells were pulse-labeled with [%]methionine and the spent medium was analyzed for secreted CR2 by immunoprecipitation with monoclonal antibody HB5 (lane l), ROS-1 (lane 2), or OKB7 (lane 3). A control nonrecombinant virus (lanes 4-6) was also analyzed with the same antibodies.

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FIG.6. Dot-blot immunoassay for recombinant CR2 binding to EBV and C3dg. Various ligands in duplicate including gp350/220, EBV, R-MuLV, OVA, and C3dg were blotted onto nitroceIlulose. The blots were then incubated with SF9 culture supernate containing secreted forms of CR2 or an irrelevant molecule (CD4).Receptor binding was detected by sequential incubation with HB5 monoclonal antibody, biotinylated antimouse Ig, streptavidin-horseradish peroxidase, and nondiffusible substrate.

of the receptor, was detected with two anti-CR2 mAb, whereas a control mAb did not react with this molecule. A nonrecombinant virus also did not react with the CR2 antibodies. The full-length CR2 recombinant protein is functionally active, since it binds to C3dg and gp350/ 220 in an enzyme-linked immunosorbent assay (ELISA, not shown). In similar studies, we have examined the function of the other truncated forms of recombinant CR2. As shown in Fig. 6, both the 1-4 and 1-10 repeat forms of CR2 react with EBV gp350/220 and C3dg. These results indicate that the ligand-binding domains of CR2 can be tentatively assigned to the first 4 repeats of the molecule.

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IV. Structure and Function of the CR2 Ligands

A. C3d AND C3dg

Human complement component C3 plays a central role in humoral immunity to infectious microorganisms by virtue of its ability to be cleaved into a number of biologically active fragments by the classical (antibody-mediated) and alternative (properdin) complement pathways (reviewed by Cooper, 1988).Cleavage of C3 by either of these two pathways generates two major activated fragments, the anaphylatoxin C3a (Hugli, 1986),and C3b, an opsonin that is the ligand for the C3b/C4b receptor, CR1 (reviewed in Fearon and Wong, 1983). C3b itself is susceptible to cleavage by factors H and I to yield iC3b (Ross et al., 1982). Further cleavage of this component by limited digestion with trypsin or elastase yields a 30-kDa fragment known as C3d (Davis et al., 1984),a ligand for CR2. Although multimeric display of C3d on erythrocytes exhibits a high affinity for CR2, it is probably not the physiological ligand for CR2. Several investigators have shown that a somewhat larger fragment of C3, designated C3dg ( M , 43,000), which is generated in plasma by cleavage with factor I and CR1 (Ross et al., 1982; Lachmann et al., 1982; Medicus et al., 1983; Medof et al., 1982), is likely to be the natural ligand for CR2. This fragment has specificity for CR2 (Ross et al., 1982; Vik and Fearon, 1985) and requires a multimeric display for efficient binding to B cells. The cDNA encoding human C3 has been sequenced, allowing determination of its amino acid sequence (DeBruijn and Fey, 1985) and localization of the different functionally active cleavage fragments.

B. THEgp350/220 ENVELOPE PROTEINS OF EBV As a member of the herpesvirus family, EBV contains a lipid envelope in which are located four proteins (Edson and Thorley-Lawson, 1983). Two of these highly glycosylated proteins, gp350 and gp220, identified by their apparent sizes on sodium dodecyl sulfate (SDS)polyacrylamide gels, are encoded by a single EBV gene (Beisel et al., 1985). The difference in the molecular weights of the two proteins appears to be due to differences in mRNA splicing in productively infected cells. Several lines of investigation have indicated that these molecules play an important role in the earliest phases of EBV infection. Monoclonal (Thorley-Lawson and Geilinger, 1980) or polyclonal human antibodies (Thorley-Lawson and Poodry, 1982) reactive with gp35OI22O were shown to neutralize the virus both in citro and in vivo.

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Incorporation of the purified gp350/220 protein into liposomes was also shown to induce neutralizing antibody in mice (North et al., 1982) or in cottontop tamarins (Epstein et al., 1985).Wells et al. (1982),using partially purified envelope proteins derived from infected cells, showed that both gp350 and gp220 bound to B cells, suggesting that these molecules mediated receptor binding. The development of mAb directed against gp350/220 has led to the purification and further analysis of these proteins in CR2 binding. Nemerow et al. (1987) demonstrated that purified gp350/220 derived from B95-8 cells bound to CR2. Tanner et al. (1987) also showed CR2 binding to recombinant gp350/ 220 (Whang et al., 1987).Tanner et al. (1988)have reported that recombinant gp350/220 competitively blocks EBV binding and infection of B cells. These authors also reported that gp350/220 has a relatively high affinity as a monomer for CR2 ( K d = 1.2 x M). C. SEQUENCE SIMILARITY OF C3dg AND gp350/220 AND ITS ROLEIN CR2 BINDING The recognition that C3dg and gp350/220 both bind to CR2 raised the question of whether these proteins contained similar amino acid sequences. A comparision of the two sequences (Nemerow et al., 1987; Tanner et al., 1987)was made possible by the complete sequencing of the entire EBV genome by Baer et al. (1984). The protein comparison revealed two small regions of similar amino acid sequences, one of which is near the N terminus of gp3501220 (EDPGFFNVE) and is similar to a region in C3dg (EDPGKQLYNVE). This region in C3dg was previously demonstrated by Lambris et al. (1985)to mediate C3dg binding to CR2. In order to examine the potential role of the C3d-like region in gp350 in binding to CR2, Nemerow et al. (1989) have used synthetic peptides. As shown in Fig. 7, one of these peptides corresponding to the region near the N-terminus of gp350/220 bound to purified CR2 in an ELISA, whereas other related peptides lacked this activity. Specificity of the ligand binding was demonstrated by examining the binding of fluorescent microspheres bearing the peptide to CR2-positive but not CR2-negative B or T cells (Nemerow et al., 1989). The EDPGFFNVE peptide bound to CR2-positive cells, and this activity could be blocked by monoclonal or polyclonal antibodies to CR2. EDPGFFNVE peptides coupled to albumin to yield a multivalent complex also blocked gp350/220 and C3dg binding to B cells (Fig. 8). These conjugates also abrogated EBV infection of B cells as measured by inhibition of B-cell transformation (Fig. 9). Taken together these studies indicate that the N-terminal region of gp350/220 plays an im-

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gp350 T I O S L I H L T G E D PG--F F N V E I P E C3dg L T T A K D K N R W E O P G K O L Y N V E A T S

.7

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.6

.5 YI

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2

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0

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pg PeptidelWell FIG.7. Binding of purified CR2 to gp350/220 peptides. Synthetic peptides having varying degrees of sequence similarity (underlined residues) to C3dg were coated onto 96-well plates. Binding of CR2 to peptide-coated wells was determined by an ELISA using the HB5 monoclonal antibody as described in Fig. 6.

portant role in receptor interaction. They also imply that a common region in CR2 is responsible for interaction with both ligands. In order to define further the CR2-binding domain in gp350, Tanner et al. (1988)have examined the functional activity of several truncation and deletion mutants of gp350/220. A deletion mutant protein lacking the VE residues of EDPGFFNVE did not bind to CR2 even though it retained the ability to bind to several anti-gp350/220 mAb. However, since the rate of release of this protein from cells transfected with the mutant gp350/220 gene was significantly lower than the rate of the wild-type recombinant protein, it may have other structural alterations that make conclusions from these data about the role of the N terminus of gp350 in CR2 binding uncertain. Work by Bare1 et al. (1988) using anti-CR2 mAb suggests the existence of separate domains for gp350 and C3d binding to CR2. These findings do not rule out the possibility that common regions in gp350/ 220 and C3d could react with different epitopes in CR2 because of the presence of repeat elements in the receptor. The precise identification of ligand-receptor domains most likely awaits x-ray crystallographic structural analysis.

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FIG.8. The N-terminal gp3501220synthetic peptide-albumin complex inhibits binding of fluorescent microspheres bearing gp350/220 or C3dg to Raji B cells. Raji B cells were preincubated with varying amounts of HTLGEDPGFFNVEC-BSA (w) or KCKWTLTSGTPSGCE-BSA (&--A)complexes prior to addition of gp350I220-coated fluorescent microspheres (A) or C3dg-bearing microspheres (B). Cell binding was quantitated by flow cytometry.

V. Cellular and Tissue Distribution of CR2

A. LYMPHOID CELLS

1. B Lymphocytes Henle et at. (1967)had first reported that EBV selectively immortalized human leukocytes. The presence of C3d receptors on B cells that coexpressed with EBV receptors was later recognized by Jondal and Klein (1973). As the development of more specific reagents for detection of EBV receptor expression were developed, accurate detection of CR2 on different cell types became possible. Tedder et al. (1984), using the HB5 anti-CR2 mAb, showed that most mature B cells express CR2. Aman et al. (1984) subsequently reported that EBV primarily infects mature, high-density resting B cells, although not all the cells become immortalized. Inghirami et al. (1988) used biotinylated EBV to study receptor expression on various subpopulations of B cells.

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t’ t T

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0

10

5

w

c

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0.1

1.0

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pg BSA-Peptide

pg BSA-Peptide

KCKWTLTSGTPSGCE

HLTGEDPGFFNVEC

FIG.9. The N-terniinal gp350/220 peptide-albumin complex inhibits transformation of peripheral-blood B cells (PBL). Human PBL were preincubated with 0.1, 1.0, or 10.0 pg of the peptide-BSA conjugate before addition of EBV. The cells were then washed and cultured in the presence ofcyclosporin A for 14 days before enumerating the transfomied R cells.

These workers showed that EBV binds to all classes of immunoglobulin (Ig)-bearing B cells including y , a, p, and 6. Although many Burkitt’s 3 lymphoblastoid cells and other transformed B cells also express CR2, (Nadler et al., 1981), many EBV genome-negative B-lymphoma cells do not (Nilsson and Klein, 1982; 1987; Calender et al., 1987). Nonetheless, these cells can Cohen et d., be induced to express high levels of CR2 following EBV infection (Calender et nl., 1987). Whether these cells originally had very low levels of CR2 prior to infection or whether virus infection occurred via a different receptor has not been determined.

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Various immunoenzymatic assays have been used to quantitate the number of CR2 molecules on normal or transformed B cells. Rasmussen et al. (1988) found normal peripheral-blood B cells (PBL) expressed 12,000 molecules per cell while tonsil B cells have 34,000 per cell; Raji B cells were found to express 20,000-35,000 receptors per cell. Weis and Fearon (1985) have previously shown that CR2 is rapidly transported to the cell surface of B cells following synthesis, indicating that there is not a large intracellular pool of receptor in these cells. The estimated half-life of CR2 on immortalized B cells is -14 h (Weis and Fearon, 1985). 2. T Lymphocytes The majority of normal peripheral-blood T lymphocytes as well as monocytes, granulocytes, and natural killer (NK) cells lack antigenically detectable CR2 (Tedder et al., 1984; Nadler et al., 1981) and fail to react with biotinylated EBV (Inghirami et al., 1988).A C3d-binding structure unrelated to CR2, however, is present on phagocytic cells (Frade et al., 198513;Vik and Fearon, 1985). A number of other studies have provided evidence for expression of CR2 on some T cells. Molt-4 T-lymphoblastoid cells bind C3d (Ross and Polley, 1975) and EBV (Menenzes et al., 1977),and react with HB5 mAb (Tedder et al., 1984). These cells, though expressing detectable CR2, are nonetheless not susceptible to EBV infection because of the failure of the virus to fuse after binding (Menenzes et al., 1977). Fingeroth et al. (1988) later showed that several human T-lymphoblastoid cell lines react with HB5 and anti-B2 mAb as well as with EBV. The molecule responsible for these activities was isolated from the HPB-ALL T-cell line and found to have the same molecular weight and N-terminal amino acid sequence as B-cell CR2 (Fingeroth et al., 1988).

-

B. THYMOCYTES Tsoukas and Lambris (1988) have also identified CR2 on human thymocytes, primarily at the immature stage (CD1-positive) of differentiation. Thymocyte CR2 also has physiochemical properties similar to B-cell CR2. Evidence that CR2 may be present on some T cells was indicated from the discovery of EBNA-positive/EBV genome-positive T-lymphoma cells in a patient with Kasaki disease who had chronic active EBV infection (Kikuta et al., 1988).These T cells, however, did not react with anti-B2 antibody, although it is possible that they did so prior to EBV infection. Taken together, these studies raise the possibility that CR2 on T cells could also provide a target for infection by EBV.

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C. FOLLICULAR DENDRITECELLS Studies by Campana et al. (1985) and Reynes et al. (1985) demonstrated that follicular dendritic cells in the germinal centers of lymphoid tissues react strongly with anti-CR2 mAb. These cells are nonphagocytic accessory cells that reside in areas of B cell-dependent lymphoid development. Their ability to retain antigen on their surfaces for prolonged periods of time suggests that one of their primary functions is the generation of memory B cells. Whether CR2, which would be expected to bind to immune complexes bearing C3dg, plays a role in this process remains to be determined.

D. EPITHELIAL CELLS The presence of the EBV genome in malignant, undifferentiated epithelial cells in patients with nasopharyngeal carcinoma (review in Tosato, 1987) suggests that these cells must also possess EBV receptors. Initial attempts to demonstrate EBV receptors on nasoepithelial cells or to infect these cells in uitro with laboratory strains of EBV were unsuccessful (Shapiro and Volsky, 1983). However, these cells could be infected by direct transfection of viral DNA thus bypassing receptor requirements. Subsequently, Sixbey et al. (1983) demonstrated that freshly isolated epithelial cells were infectible by EBV obtained from patients. Young et al. (1986)and Sixbey et al. (1987) have used the HB5 mAb to demonstrate the presence of CR2 on the less-differentiated cells of epithelial tissues. Whether the molecule reactive with HB5 is immunochemically identical to B-cell CR2 has not yet been reported. In addition, the role of this molecule in EBV infection of epithelial cells also remains to be established. E. CR2 EXPRESSION DURING B-CELLDEVELOPMENT, ACTIVATION, AND DIFFERENTIATION Lymphoid tissue stained with different mAb has revealed that CR2 is differentially expressed on these tissues. Anti-B2 reacts weakly with B cells of the peripheral area of primary lymphoid follicles and strongly with B cells in the germinal centers (Bhan et al., 1981).These results have also been confirmed with other mAb (Cohen et al., 1987; Campana et al., 1985). The HB5 mAb, however, reacts strongly with both peripheral and germinal B cells. The failure of anti-B2 and other mAb to react with all B cells may be due to differential epitope exposure rather than absolute levels of CR2 expression, as indicated by immunoprecipitation analyses (Siaw et al., 1986).

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Binding to CR2 by EBV, C3d, and anti-CR2 antibody is primarily restricted to mature B cells (Tedder et al., 1984; Bofill et al., 1985; Hansson et al., 1983). B cells do not bind these ligands, although rare cells from such tissues that do so have been reported. Terminally differentiated plasma B cells are also CR2-negative (Bhan et al., 1981; Tedder et al., 1984). Expression of CR2 on B cells is susceptible to modulation by mitogens. B cells activated with pokeweed mitogen exhibit transiently increased CR2 expression by day 3 and this deceases to undetectable levels by day 5 as assayed with the anti-B2 mAb (Stashenko et al., 1981). A more comprehensive study by Inghirami et al. (1988) also showed that CR2 expression as well as EBV binding simultaneously decreased at 72 h following activation of PBL with anti-Ig. Whether the loss of receptor expression is due to internalization of receptor has not been determined. Triggering of B cells to enter the cell cycle has previously been shown to reduce the susceptibility of these cells to transformation by EBV (Aman et al., 1984; Roome and Reading, 1987). Taken together these studies indicate that CR2 is lost upon activation of B cells and entry into the cell cycle. Loss of receptor expression is associated with decreased EBV binding and transformation. VI. Functional Properties of CR2 A. ROLEOF CR2 IN LIGAND INTERNALIZATION

As noted before, CR2 on B cells serves as the primary receptor for EBV and the gp350/220 envelope protein. A number of studies by several groups have also indicated that receptor binding by CR2 ligands induces receptor internalization. Nemerow and Cooper (1984a), used immunoelectron microscopy (IEM) to show that EBV is endocytosed into large uncoated vacuoles of PBL. In contrast, the virus fuses directly with the plasma membrane of Raji B lymphoblastoid cells. Thus EBV binding to CR2 can lead to two separate pathways of internalization. The underlying biochemical basis for the two different modes of virus entry has yet to be identified. Tedder et al. (1986) have confirmed the IEM studies using a separate approach. A CR2-specific immunotoxin was shown to cointernalize with EBV during virus infection of PBL whereas in contrast it remained on the cell surface of Raji cells following EBV binding and penetration. Ingrihami et al. (1988) have also shown that nonspecific B-cell activation probably leads to CR2 internalization, thus abrogating EBV binding and infection. In later studies, Tanner et al. (1987) showed the recombinant gp350/220

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coated onto small polyacrolein beads induced capping and patching of CR2 followed by endocytosis. The cointernalization of EBV and CR2 appears to be regulated by calcium-dependent cellular processes. Nemerow and Cooper (1984b) showed that a number of calniodulin antagonists block EBV internalization into PBL. Since CR2 triggering by various ligands including EBV leads to calcium mobilization and protein kinase C (PKC) translocation (discussed later), these biochemical processes may play an important role in the earliest stage of EBV infection. A close relationship between CR2 and membrane-associated IgM on I3 cells has been reported by Tanner et nl. (1987).These authors found the CR2 ligands and IgM cocap. Tsokas et al. (1988) showed that association of CR2 and surface IgM requires the occupancy of both molecules b y the appropriate ligand. These in vitro studies suggest that immune complexes bearing both C3dg and IgM antibody in vivo could potentially provide the stimulus for B cell-triggering reactions. B. ROLEOF CR2 IN B-CELLACTIVATION In addition to its role in legand binding and internalization, CR2 transduces signals to the interior of B cells. Evidence accumulated from a number of different approaches using CR2 ligands to stimulate cellular responses have indicated that receptor triggering leads to B-cell activation. Melchers et al. (1985) showed that prestimulated murine B cells proliferate after exposure to insoluble niultimeric forms of C3d. In contrast, soluble monomeric C3d was found to block B-cell proliferation. The human equivalent of CR2 has yet to be identified and characterized on murine B cells, but these cells clearly possess C3d receptors. Studies by Hatzfeld et al. (1988)and Schulz et al. (1987) have indicated that soluble monomeric C3dg induces Raji cells, initially cultured at very low density, to proliferate in serum-free media. Epstein-Barr virus has long been recognized as a T-independent activator of B cells (reviewed b y Tosato, 1987). The nontransforming strain of EBV, P3HR1, or UV-irradiated transforming B95-8 EBV bind to CR2 and induce B-cell activation in the absence of cell transformation as indicated by thymidine incorporation (Hu et al., 1986; Hutt-Fletcher, 1987; Tosato and Blaese, 1983). This activity appears to require participation by T cell-derived factors. Following EBV binding and infection, B-cell proliferation is accompanied by polyclonal Ig secretion (Bird and Britton, 1979; Kirchner et al., 1979). Triggering of CR2 via certain mAb also induces B-cell activation. Mittler et al. (1983) first described the ability of the OKB7 antibody to

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enhance Ig secretion of mitogen-activated B cells. The specificity of the OKB7 antibody with regard to CR2 binding had not yet been established at that time, however. Subsequently, several other groups showed that CR2 triggering by certain but not all mAb stimulates B-cell activation (Petzer et al., 1988; Wilson et al., 1985; Nemerow et al., 1985a; Bohnsack and Cooper, 1988). The underlying biochemical mechanisms leading to CR2-induced B-cell activation are still in the early stages of investigation by many laboratories. Several studies have indicated that calcium mobilization may play a central role in these reactions as mentioned before. Increased calcium modulates transmembrane signaling in B cells and other cells (reviewed by Cambier and Ransom, 1987). Carter et al. (1988) showed that crosslinking of CR2 together with IgM induces a synergistic increase in free cytoplasmic calcium and a low but significant increase in thymidine incorporation in B cells. Dugas et al. (1988) have carried out more extensive studies in this area and shown that EBV binding also induces a cellular rise in free calcium, an effect that can be abrogated by the calcium channel blocker, verapamil. Calcium mobilization induced by EBV binding was also accompanied by PKC translocation from the cytosol to a membrane-bound compartment as well as by an increase in production of phosphatidylinositol metabolites. This latter finding may be even more significant in light of the ability of CR2 to be phosphorylated by activators of PKC (Changelian and Fearon, 1986). The deduced amino acid sequence of CR2 indicates that the cytoplasmic tail of the receptor contains several potential sites for serine and threonine phosphorylation by PKC (Moore et al., 1987; Weis et al., 1988). Whether PKC, which mediates anti-IgMinduced B-cell activation, also regulates CR2-induced B-cell activation, however, remains to be established. The participation of calmodulin-regulated enzymes such as PKC in EBV internalization is consistent with this pathway of B-cell activation. ACTIVATIONAND REGULATION C. ROLEOF CR2 IN COMPLEMENT

As reviewed earlier, CR2 is a member of a family of molecules that regulate complement activation. However, unlike some of these proteins, CR2 lacks cofactor activity for factor I-mediated cleavage of soluble C3b as well as the ability to accelerate the decay of alternative and classical C3 convertases (Weis et al., 1986b). One report, however, has indicated that purified CR2 serves as a cofactor for factor I-mediated cleavage of particle-bound iC3b (Mitomo et al., 1987). Thus, CR2 has at least one regulatory function present in the other CR2-like proteins.

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A number of earlier studies had indirectly indicated that a complement receptor present on Raji cells played a role in activation of the alternative complement pathway (Budzko et al., 1976; Theofilopoulos and Perrin, 1977). Good correlation between CR2 expression on B cells and complement-activating ability was found by Ramos et al. (1985)and Kai et al. (1988).The presence of CR2 on Molt-4 cells is also consistent with receptor-mediated alternative-pathway complement activation (Praz and LeSavre, 1983). Subsequently, Mold et al. (1988) have shown that CR2 or Raji cells serves as the primary site of covalent and noncovalent binding of C3 during alternative-pathway activation. Since these properties are not blocked by the OKB7 mAb, the ligandbinding epitope of CR2 does not appear to be involved in this activity. Purified CR2 isolated from Raji cells was also shown to activate the alternative pathway. Thus CR2 not only is a regulator of complement activation but also is itself an activator. The physiological relevance of these reactions remains to be determined, however. Complement receptor type 2-mediated deposition of C3 on EBV-transformed Blymphoma cells might serve to mark these cells for destruction by cells of the reticuloendothelial system. VII.. Summary and Future Prospects

Complement receptor type 2, a cell surface receptor of B cells, serves to focus infection of EBV onto B cells and probably epithelial cells. Since this receptor is primarily expressed on B cells, it is also in a position to trigger immunoregulatory reactions following EBV or C3dg binding. At the present time, many of the precise molecular details involving receptor structure, ligand binding, and cell activation remain to be elucidated. However, a significant amount of current research in this area has begun to bring some of these details into focus. The molecular cloning of CR2 cDNA has revealed some of the basic features of its structure, including the 60mer repeating elements that constitute its extracellular domain. The identification of the important ligand-binding epitopes within the repeat structure remains to be accomplished, although these regions are likely to be positioned in the first four repeats. Large-scale production of soluble forms of recombinant CR2 in various eukaryotic expression systems such as baculovirus should also provide an opportunity to analyze further the overall structure of CR2 using such techniques as x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Since a region in the major envelope protein of EBV, gp350/220, involved in CR2 binding has been identified, this information may also lead to identification of the localization of ligand-binding epitopes in CR2 by crystallizing the

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receptor in the presence of its ligands. Identification of an EBVbinding site in CR2 as well as the receptor-binding epitope of gp350/ 220 could provide rationales for the production of an EBV vaccine in which blockade of endogenous receptors would be the major target. Such approaches have been demonstrated in vitro with soluble forms of CD4 in blocking HIV binding and infection of T cells. The sequence of the short cytoplasmic region of CR2 has revealed several potential sites for phosphorylation, a process that may be an integral part of receptor-induced B-cell activation. How this reaction leads to B-cell stimulation awaits further study. Of particular interest is the identification of the biochemical mediators involved in CR2induced B-cell activation. The ability to incorporate recombinant receptors into various cell lines and to identify molecules that interact with the cytoplasmic domain of the receptor may provide new information in this area. The recent expression of recombinant CR2 in rodent cells that lack endogenous receptors has allowed initial studies of the role of CR2 in EBV infection. Future studies including introduction of the CR2 gene into primate cells will undoubtedly lead to a greater understanding of the biology of the receptor. The identification of host cell factors that influence EBV infection and cell immortalization may also be forthcoming from such transfection studies. In addition to analysis of CR2-mediated B-cell activation, transfection of CR2 in different cell types that may have special biological properties is of additional potential value if they lead to the establishment of EBVinduced immortalized cells lines. Although technical difficulties may hinder the exploration of some of the questions described here, preliminary studies have already yielded promising results in some of these areas. Future investigation of this receptor should begin to uncover further molecular details of how a human herpesvirus interacts with a normal host cell receptor in the earliest stages prior to infection and how this process leads to B-cell activation and cell immortalization.

ACKNOWLEDGMENTS This research (Publication Number 5766-IMM) was supported by NIH grants CA36204, CA14692, AI17354, and the PEW Scholars Award for Biomedical Science.

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Baer, R., Bankier, A. T., Biggin, M. D., Deininger, P. C., Farrell, P. J., Gibson, T. J., Hatfield, G., Hudson, G. S., Satchwell, S.C., Sequin, C., Tuffnell, P. S.,and Barrel], B. C. (1984). Nature (London)310,207-211. Barel, M., Fiandino, A., Delcayre, A. X., Lyamani, F., and Frade, R. (1988).J. Immunol. 141,15W-1595. Beisel, C., Tanner, J., Matsuo, T., Thorley-Lawson, D., Kezdy, F., and Kieff, E. (1985). J. Virol. 54,665-674. Bentley, D. R., and Porter, R. R. (1984). Proc. Natl. Acad. Sci. U.S.A.81,1212-1215. Bhan, A. K., Nadler, L. M., Stashenko, P., McClusky, R. T., and Schlossman, S. (1981). J. E x p . Med. 154,737-749. Bird, A. C., and Britton, S. (1979).Imniunol. Reo. 45,41-67. Bofill, M., Janossy, G.. Janossa, M.,Burford, G. Seymour, G., Wernet, P., and Kelemer, E. (1985).J. Imtnunol. 134, 1531-1538. Bohnsack, J. F., and Cooper, N. R. (1988).J. Immunol. 141,2569-2576. Budzko, D. B., Lachmann, P. J., and McConnell, I. (1976).Cell. Irnmunol. 22,98-116. Calender, A., Billand, M., Aubry, J. P., Banchereau, J., Vuillaume, hl., and Lenoir, G. M. (1987). Proc. A’atl. Acad. Sci. U.S.A. 84,8060-8064. Cambier, J. C., and Ransom, J. T. (1987).Annu. Rei;. Immunol. 5, 179-199. Campana, D., Janossy, G., Bofill, M., Trejdosiewicz, L. K., Ma, D., Hoffbrand, A. V., Mason, D. Y., Lebaco, A. hl., and Forster, H. K. (19%).J. Immunol. 134, 1524-1530. Caras, I. W., Davitz, M. A., Rhee, L., Weddell, G., Martin, D. W., and Nussenzweig, V. (1987). Nature (London),325,545-549. Carroll, M. C., Alicot, E. A,, Katzman, P., Klickstein, L. B., and Fearon, D. T. (1987). Complement 4,141. Carter, R. H., Spycher, M. O., Ng, Y. C., Hoffman, R., and Fearon, D. T. (1988). J. Immunol. 141,457-463. Changelian, P. S., and Fearon, D. T. (1986).J. E x p . Med. 161, 101. Chung, L. P., Bentley, D. R., and Reid, K. B. (1985). Biochemistry 23, 133-141. Cohen, J. H., Fischer, E., Kazatchkine, M. D., Lenoir, G. M., Lefevre-Delvin Court C., and Revillard, J. P. (1987).Scand. J. Immunol. 25,587-598. Cooper, N. R. (1988). I n “Balliere’s Clinical Immunology and Allergy: International Practice and Research. Complement and Immunological Disease” (M. Kazatchkine, ed.), Vol. 2, pp. 263-293. Harconrt Brace Jovanovich, London. Dahlbach, B., Smith, C. A., and Muller-Eberhard, H. J. (1983). Proc. Natl. Acad. Sci. U.S.A.80,3461-3465. Davis, A. E., Harrison, R. A., and Lachmann, P. J. (1984).J. Immunol. 132, 1960-1966. DeBruijn, M. H. L., and Fey, G. H. (1985).Proc. Natl. Acad. Sci. U.S.A.82,708-712. DiScipio, R. G., Chakravarti, D. V., Miiller-Eberhard, H. J., and Fey, G. H. (1988).J.Biol. Chem. 263,549460. Dugas, B., Delfraissy, J. F., Calenda, A., Peuchmaur, M., Wallon, C., Rannou, M. T., and Galanand, P. (1988).J. Immrrnol. 141,4344-4351. Eden, A., Miller, C. W., and Nussenzweig, V. (1973).J. C h i . Irloest. 52,3239-3242. Edson, C. hi., and Thorley-Lawson, D. A. (1983).J. Virol. 46,547-556. Epstein, M. A., Morgan, A. J., Finerty, S.,Handle, B. J., and Kirkwood, J. K. (1985). Nature (London) 318,287-289. Fearon, D. T., and Wong, W. W. (1983).Aritirt. Rev. [nrrnutiol. 1,243-271. Fingeroth, J. D., Weis, J. J., Tedder, T. F., Stroniinger, J. L., Biro, P. A., and Fearon, D. T. (1984).Proc. Natl. Acud. Sci. U.S.A.81,4510-4514. Fingeroth, J. D., Clabby, M. L., and Strominger, J. D. (1988).J. Virol. 62, 14421447.

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THE OPPORTUNISTIC TUMORS OF I MMUNE DEFlClENCY Harry L. loachim Department of Pathology, Lenox Hill Hospital and College of Physicians and Surgeons of Columbia University, New York. New York 10027

I. Introduction 11. States of Immune Deficiency 111. Opportunistic Infections IV. Opportunistic Tumors A. Tumors Associated with Primary Immune Deficiencies B. Tumors Associated with Acquired Immune Deficiencies V. Distinctive Features of Opportunistic Tumors VI. Spontaneous Regression of Opportunistic Tumors VII. Stromal Reaction of Tumors VIII. Immune Deficiency and Immune Surveillance of Tumors References

I. Introduction

The environment of living organisms contains an immense number and variety of aggressive agents. Viruses, bacteria, fungi, and protozoans may invade, colonize, multiply, and kill other organisms. As a result of protracted evolutionary processes under the pressures of environmental agents, organisms have developed their systems of defense. Lower animals are protected against infectious organisms by soluble molecules and by phagocytic cells. Vertebrates carry out the defense against pathogenic agents through a complex immune system made up of a multitude of interdependent cellular and humoral components. Because of the extreme efficiency of this system, most infections are of limited duration and leave little permanent damage (Roitt et al., 1985). Deficiencies in any part of the system expose the individual to a greater risk of infection, although some mechanisms for compensation are built in. A host of pathogenic or potentially pathogenic agents can take advantage of temporary or permanent lowering of defenses. These are the opportunistic microorganisms, which either produce de novo infections or are reactivated from a state of saprophytic latency. The opportunistic infections differ substantially from the infections common to the general population in their high incidence, uncommon 301 ADVANCES IN CANCER RESEARCH, VOL 54

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severity, and unusual features. As various states of immune deficiency were identified, it became apparent that those suffering from their effects are prone to the development of not only opportunistic infections but also neoplasia. Like the infections, the tumors developing in immunodeficient individuals are characterized by unusually high incidence, severity, and specificity. Accordingly, the designation of opportunistic tumors for the group of neoplasms associated with immune deficiency is perfectly appropriate. The objectives of the present paper are to review the types of opportunistic tumors, to identify their particular features, and to examine their etiology and pathogenesis.

II. States of Immune Deficiency The immune system includes (1)a first line of defense, the innate immunity manifested by nonspecific, general reactions, and (2) a secondary line of defense, and adaptive immunity characterized by specific reactions and by immunological memory. There are multiple types of immune deficiency, the classification of which is difficult because of the great variability of immunological findings. These findings, in turn, are the expression of abnormal function of the various components of the immune system, and their study has contributed significantly to a better understanding of its normal structure and function. There are two major categories of immune deficiencies: primary and secondary. The primary group is defined by developmental deficits of the various elements of the immune system. Therefore, almost all primary immune deficiencies are congenital and the majority of patients are children. In secondary immune deficiencies the immune system is intact; however, as a result of disease processes or medical intervention, the host’s immune defenses are temporarily or permanently impaired. The primary immune deficiencies are a large diverse group of syndromes that were initially named and classified by a special committee of the World Health Organization (Fudenberg et d , 1971). They were assigned, according to which major system was mainly affected, into cellular (T-cell) and humoral (B-cell) types. Subsequently, as new syndromes were described, subtypes were included in the classification, which became increasingly complex (Stiehm, 1980). A congenital thymic deficiency results in a T-cell immune deficiency in which antibody synthesis is not affected, such as in congenital thymic hypoplasia. A peripheral lymphoid tissue impairment may pro-

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duce a B-cell defect with normal cellular immunity such as the Xlinked agammaglobulinemia. An abnormality affecting the stem cells results in deficiencies of both T and B cells leading to concomitant failures of cellular and humoral immunity, as in severe combined immune deficiency or SCID (Steihm, 1980).Immune deficiencies involving the B-cell system, expressed as antibody immunodeficiencies, are the most common, representing -50% of all primary immune deficiencies. The next largest group, -40%, are immune deficiencies of the T-cell system; three-fourths of these have associated B-cell (i.e., antibody) deficiencies and are therefore far more severe, for example the combined immune deficiencies. Phagocytic immune deficiencies represent 6%of the total and include impairments of polymorphonuclear leukocytes or of mononuclear phagocytes. Disorders of the complement system make up the remaining 4% of the total (Medical Research Council Working Party, 1969). In a British study of primary immune deficiency, 17%of cases were diagnosed in infants <1 year old and 41% in children from 1 to 15years of age. Immune deficiencies are more common in males, and in this study they constituted 62%of all cases (Medical Research Council Working Party, 1969). Acquired immune deficiencies comprise a broad spectrum of disorders, some occurring spontaneously and others as the result of medical intervention. Similarly to the congenital immune deficiencies, those acquired are also heterogeneous and consist of diverse dysfunctions that involve various components of natural immunity, eventually leading to the deregulation of the entire immune system. They include autoimmune diseases such as systemic lupus erythematosus (SLE)and Sjogren’s syndrome, malignant neoplasms such as Hodgkin’s disease and Kaposi’s sarcoma, the aftereffects of radiotherapy, chemotherapy, or organ transplants, and finally, the acquired immune-deficiency syndrome (AIDS).Whether congenital or acquired, the immune-deficient state-once established-determines the conditions that are favorable to the development of opportunistic infections and tumors.

Ill. Opportunistic Infections Individuals with immune deficiencies both cellular and humoral are prone to infections. The infections are frequent and recurrent, occurring singly or in combination. Most patients, particularly those with primary immune deficiencies, have long medical histories usually consisting of repeated respiratory infections that began in childhood. The infectious agents consist of a broad spectrum of pathogens including a variety of bacteria, viruses, fungi, and protozoans. However, not all

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microorganisms are equally represented. The types of agents involved are related to the type of immune deficiency present and are generally different from those encountered in immunologically normal persons. The microorganisms controlled by cellular or humoral immunity will be those involved in the opportunistic infections occurring in individuals with cellular or humoral deficiency. In AIDS, for example, a state of immune deficiency caused by the insufficiency of the helper T-cell population, a characteristic constellation of opportunistic infections is noted. Thus in AIDS patients severe, generalized infections with Mycobacteriuin auium-intracellulare (MAI) are frequent and primary infections with Mycobacterium tuberculosis (MT) are rare, in contrast to the normal population in which infections with MA1 are almost unknown while infections with MT are prevalent. Pneumocystis carinii, a protozoan parasite with worldwide distribution, causes severe pneumonia in 65% of AIDS patients; indeed it is responsible for most of the fatalities while hardly ever causing disease in the normal population. A large part of the opportunistic infections associated with AIDS are in fact reactivations of latent infections acquired in childhood. Thus histoplasmosis and coccidioidomycosis are often seen in AIDS patients who have temporarily lived in areas where these infections are endemic (Ioachim, 1988). In almost all cases, infections in AIDS patients are considerably more aggressive than their counterparts in inimunocompetent individuals. The clinical manifestations are more severe, organs not commonly affected are involved, and early dissemination is common. Resistance to treatment, frequent recurrences, and short survival are characteristically associated with the AIDS infections. IV. Opportunistic Tumors

Like infections, tumors are more common in persons with immune deficiencies than in the normally immunocompetent individuals of the general population. Case reports in the medical literature have occasionally documented a relationship between immune deficiency and neoplasia. Strong evidence supporting this relationship became available after cancer registries for neoplasms occurring in individuals with congenital immune deficiencies and in recipients of organ transplants were initiated in the early 1970s in Minneapolis and in Denver, respectively (Kersey et al., 1973; Schneck and Penn, 1971). It soon became apparent that the incidence of tumors in immunodeficient patients was far above that recorded in the age-matched general population. The risk of developing a malignant tumor is 4% in persons with

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genetic immune deficiencies; that is 10,000 times higher than the risk in their normal counterparts. The occurrence of neoplasia in immunosuppressed renal-transplant recipients is even higher, reaching 6% in a review of 1767 tumors recorded in the transplantation register (Penn, 1977). The present AIDS epidemic has revealed a new aspect of the relationship between immune deficiency and neoplasia, with specific tumor types occurring in ever-increasing incidences. Kaposi’s sarcoma, a neoplasm with an incidence of only 0.02% in the general population of the United States, may develop in as many as 36% of homosexual patients with AIDS (Selwyn, 1986). Not only are these tumors more common in immunodeficient individuals, but their histological type, location, clinical course, and response to treatment also tend to be different from those seen in the population at large. The aim of the present study is to review the spectrum of tumors that accompany the various states of immune deficiency. Egamination of the characteristics of these tumors will reveal that thef form a fairly uniform group with numerous features in common regardless of the type of immune deficiency with which they are associated. It is also noteworthy that in their behavior, the tumors associated with immune deficiencies display strong similarities to the opportunistic infections affecting the same populations of individuals.

A. TUMORS ASSOCIATEDWITH PRIMARY IMMUNE DEFICIENCIES Combined immunodeficiency disease or Swiss-type agammaglobulinemia is the most severe form of congenital immunological failure because it includes deficiencies of both the T- and B-cell systems. The thymus is depleted of lymphocytes and lacks Hassal’s bodies. In the absence of a functional thymus the T-cell system remains undeveloped, while the B cells fail to mature, resulting in agammaglobulinemia. The disease becomes apparent soon after birth and consists of recurrent septic episodes including bacterial pneumonitis due to the B-cell defect and P . carinii pneumonia due to the T-cell defect. Overwhelmed by repeated infections caused by a variety of agents to which no immune response is mounted, infants with SCID usually do not survive beyond 2 years of age (Ammann and Hong, 1980). Notwithstanding the short life span of these children, neoplasms may develop in association with SCID; by 1987 there were 42 such cases recorded by the immunodeficiency cancer registry (Filipovich et al., 1987). Of these, 31 were non-Hodgkin’s lymphomas and only 2 were tumors of nonhematological tissues. All these tumors occurred in infants or very young children, which is in sharp contrast with the virtual absence of

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lymphomas in immunocompetent children of this age (Waldmann et al., 1972). Di George’s syndrome, a deficiency of T cells or cellular immunity, is the result of a defect in the development of the third and fourth pharyngeal pouches during embryogenesis. This maldevelopment produces thymic hypoplasia along with hypoparathyroidism and various cardiovascular abnormalities. The disease is very rare and life expectancy very short; however, a glioma is reported to have occurred in one case (Ammann and Hong, 1980). In patients with infantile X-linked agammaglobulinemia, also known as Bruton’s type agaminaglobulinemia, the defect involves the B-cell system. A failure in the differentiation of B-cell precursors, probably in the gut-associated lymphoid cells, results in lack of plasma cells and failure of humoral immunity with frequent recurrent pyogenic infections. Malignancy, mostly leukemia and lymphoma, may develop in as many as 6% of boys with this sex-linked recessive abnormality (Kersey et al., 1973). More common than agammaglobulinemias are dysgammaglobulinemias, in which defects in the B-cell system result in marked decrease or total absence of only one or two classes of immunoglobulins. In addition to infections and allergic diseases, neoplasias have also been associated with these states. In IgA deficiency, adenocarcinomas of the stomach and colon, carcinomas of the lung and esophagus, leukemias, and lymphomas have been recorded (Waldmann et al., 1972; Ammann and Hong, 1980.) A remarkable case report concerns a 10-year-old girl who had serum IgA levels <20% of normal and who over a 9-year period developed a variety of malignant tumors including thymoma, carcinomas of various organs, and eventually a fatal cerebral astrocytoma. Her brother had total absence of IgA and died of lymphoma of the central nervous system at 16 years of age (Hammondi et al., 1974). The Wiskott-Aldrich syndrome, which is genetically transmitted and X-linked, consists of thrombocytopenia, eczema, and severe recurrent infections that kill these children in their first years of life (Hitzig, 1976).The female carriers appear healthy and cannot be detected. The deficiency involves primarily the T cells, with thymic hypoplastia and depleted T-cell zones in the lymph nodes (Snover et al., 1981). Infant boys with the Wiskott-Aldrich syndrome had a median survival of 8 months before the introduction of antibiotics, so that by 1980 median survival had increased to 10 years (Perry et al., 1980). As patients lived longer, their risk of developing malignant tumors increased propor-

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tionally. In fact, the Wiskott-Aldrich syndrome is associated with an estimated >10% incidence of malignancies-the highest of all immune deficiencies (Waldmann et al., 1972).The majority of tumors are lymphomas, which as in other immune deficiencies are more often extranodal, with a predilection for the gastrointestinal tract and the brain (Ammann and Hong, 1980). The histological types are of highgrade malignancy, and as in other immune deficiencies including AIDS, the lymphomas are preceded by persistent, indolent, unexplainable lymphadenopathies (Gatti and Good, 1971). Ataxia telangiectasia, an inherited multisystem syndrome, is characterized by the triad of progressive cerebellar ataxia, mucocutaneous telangiectasia, and frequent sinopulmonary infections. Mental retardation, growth failure, and atrophy of the ovaries and testes are often associated (Peterson et al., 1964; Hitzig, 1976). The immunological deficiency involves both cellular and humor systems, including depletion of T-cell areas and Ig abnormalities (Waldmann et al., 1972). It is believed that the cause may be an early failure in tissue differentiation, impairing interactions between endoderm and mesoderm and thus preventing the development of the thymus (Peterson et al., 1964; Waldmann et al., 1972). The occurrence of malignancy is frequent and includes a variety of tumors. About half of the 145 cases recorded by the registry are lymphomas, but the others comprise cerebral glioma, cerebellar medulloblastoma, gastric carcinoma, hepatoma, ovarian dysgerminoma, and breast cancer (Filipovich et al., 1987).Acute leukemias and lymphomas eventually develop in -10% of all individuals with ataxia telangiectasia (Kersey et al., 1973; Toledano and Lange, 1980). They are invariably of high-grade histological type and show unfavorable clinical features (Frizzera et al., 1980). The X-linked lymphoproliferative (XLP) syndrome is characterized by severe, usually fatal lymphoproliferative diseases. In the original report, six boys in the Duncan family died within a 13-year period of infectious mononucleosis, agammaglobulinemia or malignant lymphoma (Purtilo et al., 1975).All were infected by Epstein-Barr virus (EBV),as demonstrated by positive staining of EBV nuclear antigen (EBNA) in involved organs, and by cRNA-DNA and vDNA-DNA hybridization studies (Purtilo et al., 1981). The immune deficiency, transmitted by females, affects males exclusively and is characterized by T-cell depletion and inability to form antibodies to EBV. As a result, the proliferation of EBV-infected B cells progresses uncontrolled, advancing from a polyclonal benign to a monclonal malignant cellular growth.

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€3. TUMORS ASSOCIATEDWITH ACQUIREDIMMUNE DEFICIENCIES

The medical literature of the past 20 years contains numerous case reports relating to autoimmune diseases and neoplasia. In a series of 70 consecutive cases of SLE followed for a median interval of 6.8 years, eight patients developed malignant tumors including lymphoma, multiple myeloma, and carcinoma of the uterine cervix (Canoso and Cohen, 1974). In nine patients with Sjogren’s syndrome, nonHodgkin’s lymphoma developed after an average of 10 years (Zulman et al., 1978). Most published reports refer to individual cases, however, in that large, controlled series are difficult to assemble because of the low incidence and protracted course of most autoimmune diseases. The relationship between organ transplantation and development of neoplasia is clearly established based on the large number of cases recorded in the Transplant Tumor Registries (Penn, 1988). The incidence of cte novo tumors in series of patients with renal transplants, each ,500 cases, was between 2 and 7%, 100 times greater than in the age-matched general population (Penn, 1979). In the Cincinnati Transplant Tumor Registry, 3251 neoplasms occurred in 3040 organ transplant recipients (Penn, 1988).The average age ofthese patients was 40 years-relatively young-and the average interval from organ transplantation to tumor diagnosis was 60 months. It is interesting to note that the time intervals were different for various tumors, at an average of 23 months for Kaposi’s sarcoma, 37 months for lymphoma, and 100 months for carcinomas of the vulva and perineum (Penn, 1988). The most common tumors of the general population (i.e., carcinomas of the lung, breast, colon, and rectum) have not shown an increase in incidence in the recipients of organ transplants. As in other types of imniune deficiencies, carcinomas of the skin and lips, carcinomas of the vulva and perineum, Kaposi’s sarcoma, and non-Hodgkin’s lymphomas are the tumors most commonly observed (Penn, 1988). The risk of developing a lymphoma in patients with renal transplants is 35 times higher than in the general population (Hoover and Fraumeni, 1973). Lymphomas represent 26%of all tumors occurring in this population, while in the general population lymphomas occur only rarely, representing only 3-4% of all neoplasms (Penn, 1979). Hodgkin’s disease, which constitutes 18-34% of all lymphomas in the population at large, accounted for only 2-3% in transplant recipients (Penn, 1983). Similarly, the ratio between nodal and extranodal lymphomas was reversed in favor of the latter, again showing that the location of tumors is also affected by the background of immune deficiency (Ioachini, 1987). A favored organ among the extranodal locations is the

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brain, which is the major or unique site of non-Hodgkin’s lymphoma in 46-73% of renal-transplant recipients as compared with only 0.041.5%in the general population (Schneck and Penn, 1971). Neoplasms in recipients of organ transplants generaIIy display considerable aggressiveness and a tendency for early dissemination. Kaposi’s sarcoma in recipients of renal transplants involved internal organs, causing gastrointestinal bleeding and death in a third of the patients in one series of cases (Stribling et al., 1978). In another series the mortality rate from Kaposi’s sarcoma following renal transplants was 10 times higher than in the spontaneous form (Harwood, 1984). Skin cancers in transplant recipients affect younger persons, are multiple in most cases, and comprise predominantly squamous-cell carcinomas-a more malignant tumor than the basal-cell carcinomas that are more common in the population at large (Penn, 1979). Melanomas are more common, more malignant, and more often located on unexposed skin-in contrast to their appearance in the general population (Greene. et al., 1981). Some tumors, particularly in heavily immunosuppressed patients, appeared after unusually short intervals. A 14year-old boy developed primary lymphoma of the brain only 4 months after cardiac transplantation (Krikorian et al., 1978). Cyclosporin, the most powerful immunosuppressant thus far used in organ transplantation-with a selective inhibitory activity on T-cell differentiationhas been most frequently associated with tumor development. Lymphomas are the most common, often accompanied by an increase in anti-EBV, particularly anti-viral capsid antigen (VCA) (Nagington and Gray, 1980). The acquired immune-deficiency syndrome now commonly occurring after infection with HIV offers a new illustration of the relationship between immune deficiency and neoplasia. As in other states of immune deficiency, patients with highly depressed amounts of Thelper lymphocytes are at great risk of opportunistic infections and tumors (Ioachim, 1984). Kaposi’s sarcoma and non-Hodgkin’s lymphoma are most commonly seen, occurring in as many as 40% of all AIDS patients (Friedman-Kien et al., 1982). Kaposi’s sarcoma shows still-unexplained differences of incidence between various types of immune deficiency. Thus in congenitally immunodeficient persons Kaposi’s sarcoma is rare, while it occurs in 5.5%of transplant recipients and in 35% of AIDS patients (Longo et al., 1984). Within the population of AIDS patients, Kaposi’s sarcoma is present in 45%of homosexual males but in only 443% of nonhomosexual individuals (Longo et al., 1984). A substantial decrease in the incidence of Kaposi’s sarcoma in AIDS homosexuals has been noted recently, possibly related to a

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parallel decrease in the rate of some opportunistic infections (Selwyn, 1986). Lymphomas are also more common in persons with AIDS and show a reversed Hodgkin’s/non-Hodgkin’s ratio with the latter far exceeding the former, in contrast to the general population in which Hodgkin’s disease is more common-particularly in young adults. In our updated series of 72 lymphomas in patients with HIV infection, 66 had nonHodgkin’s and only 6 had Hodgkin’s lymphoma (Ioachim et al., 1985). There is also a reversal of the nodal/extranodal location ratio, with more tumors appearing in viscera than in lymph nodes; this is in diametric opposition to the distribution in the general population. Of the extranodal locations, the most commonly involved are the gastrointestinal tract and the brain. Although in the general population lymphomas involve the central nervous system in no more than 2% of cases, in AIDS patients the incidence of brain lymphoma ranged between l l and 42%of cases (Ziegler et d.,1984; Ioachim et al., 1985). Unusual tumor locations have been frequently noted, such as the primary lymphomas of the anus and rectum, a newly recognized category of tumors in the homosexual patients with AIDS (Ioachim et al., 1987). Lymphomas occurring in AIDS patients have been highly aggressive and have uniformly involved multiple organs. Upon autopsy extensive tumor involvement was evident, including organs not commonly affected such as the heart, kidneys, testes, and skin (Ioachim, 1988). Upon histological examination, the non-Hodgkin’s lymphomas almost exclusively belong to high-grade categories. The most common type is an undifferentiated lymphoma of Burkitt’s or BL-like type (Ioachim, 1988). Chromosomal studies performed in some cases showed a predominance of t(8 : 14) translocation, which is characteristic for BL (Kalter et al., 1985).The phenotype was monoclonal B cell or non-B, non-T cell in virtually all cases (Ioachim, 1988).Nonneoplastic lymphadenopathies showing typical AIDS-related morphological changes precede the lymphomas in a large number of cases (Ioachim et aE., 1983). Reflecting the immature tumor cell type and the defective immune response, the course of lymphomas associated with AIDS is highly malignant. Despite a positive initial response to chemotherapy, almost all patients relapse and the survival is generally very short (Ioachim and Cooper, 1986). A third group of tumors frequently seen in individuals with AIDS include condyloma, carcinoma in situ or Bowen’s disease, and invasive squamous-cell carcinoma of the anus (Ioachim, 1988). These tumors, uncommon in the general population, are seen with increased frequency in homosexuals. Koilocytotic atypia, a cellular alteration asso-

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ciated with papilloma virus infection, was seen in 71% of cases of carcinoma in situ in our series (Lauwers et al., 1989). V. Distinctive Features of Opportunistic Tumors

The preceding review of opportunistic tumors reveals a number of distinctive features that they share in common. Although the states of immune deficiencies with which they are associated represent a broad and heterogeneous spectrum, the tumors are fairly homogeneous, resembling each other in histological types and clinical behavior (Ioachim, 1987). Regardless of the type and cause of immune deficiency, non-Hodgkin’s lymphomas, Kaposi’s sarcomas, and carcinomas of the skin and the cloacogenic area are the prevailing tumors (Ioachim, 1988). These tumors of very low frequency in the general population reach incredibly high incidences in individuals who are immunodeficient. The selectivity and rarity of tumor types occurring in immunodeficient persons are reminiscent of the infections seen in the same group of patients. Moreover, like the infections associated with immune deficiencies, the tumors are unusually aggressive, are often in unusual locations, and have a strong tendency for generalization, resistance to treatment, and recurrence. Like the opportunistic infections, they appear in the context of immune deficiency and therefore deserve the designation of opportunistic tumors.

VI. Spontaneous Regression of Opportunistic Tumors

It is of great interest that partial or total regression of malignant tumors, an occurrence almost never seen in the general population, has been reported in immunosuppressed persons following the correction of their immune deficiency. Thus Kaposi’s sarcoma completely regressed in three patients with cutaneous tumors and one patient with oral tumors after the cessation of chemotherapy. In two of these patients who later died of unrelated causes, no residual tumor was found at autopsy (Stribling et al., 1978). Seven recipients of kidney, three of liver, one of heart, and one of heart-lung homografts developed malignant lymphomas that regressed after reduction or discontinuance of immunosuppressive therapy (Starzl et al., 1984). In some of these patients in whom immunosuppression was stopped or reduced, no remaining tumor could be found upon repeat surgery 2.5-5.5 weeks later. In other patients who had no irradiation and who had extensive tumors that required surgery to relieve bowel obstruction or perforation, the simple measure of stopping or reducing cyclosporin and

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prednisone treatment led to prompt and seemingly permanent resolution of tumors (Starzl et al., 1984). Repeated reports in the medical literature of spontaneous regression of tumors led to an international symposium devoted to the examination and discussion of this problem (Lewison, 1974). The tumors most commonly observed to regress spontaneously are renal carcinomas, neuroblastomas, malignant melanomas, choriocarcinomas, and BL, although almost all types of neoplasms have been occasionally reported to regress. Although the causes for spontaneous regression of tumors could not be determined in most cases, involvement of host immune response was generally suggested (Cole, 1976; Strauchen et aZ., 1987). VII. Stromal Reaction of Tumors

Malignant tumors are commonly associated with inflammatory cells, mostly lymphocytes, that infiltrate the tumor tissues and appear to be involved in the disintegration of tumor cells (Ioachim, 1976). This phenomenon, referred to as the stromal reaction of tumors, has long been observed; however, its systematic study has only been sporadically pursued. The reason lies in the inherent difficulty of assessing both the identity and the function of the infiltrating cells within the tumor tissues. Two major approaches to this problem are available. The study of cells in situ on histological sections provides valuable information on the cell types involved and on their relationship with the tumor cells but cannot estimate their functions, whereas the study of cells in vitro, extricated from the tumor tissue, may allow evaluation of their function but fail to determine their topography. Despite the difficulties, such studies have revealed new aspects of the local reactions to tumor growth (Ioachim, 1976; Ioachim et al., 1976; Vose et al., 1977; Vose and Moore, 1979).Thus, patterns of cell reaction that are characteristic for different histological types of tumors were recognized, and the various cell types taking part in the antitumor reactions were identified and quantified (Ioachim et al., 1976; Rabinovich et al., 1987; Nakamura et aZ., 1988).,4 significant observation is that considerable lymphocytic infiltrates regularly accompany the changes of severe dysplasia and carcinoma in situ. In such diverse neoplastic processes as carcinoma in situ of the bronchial epithelium, larynx, and uterine cervix, intraductal carcinoma of the breast, Bowen’s disease of the skin, and Paget’s disease of the nipple-although the tumor cells have not yet crossed the basement membranes and are still strictly intraepithelial-substantial collections of lymphocytes usually border the lesions and separate them from the underlying stroma (Ioachim, 1976,

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1980). Malignant melanoma, a tumor commonly associated with a strong lymphocytic reaction, is also known to undergo occasionally partial or total spontaneous regression. It is in the regressing tumors that lymphocytic infiltration is particularly abundant, resulting in tumor cell disintegration, phagocytosis, and fibrosis (Clark et al., 1969). It is reasonable to assume, with regard to their characteristic features, that the accumulation of lymphocytes and other inflammatory cells at the tumor site represents an immune reaction to the tumor growth. Strengthening this belief is the observation that melanomas developing in immunodeficient renal-transplant patients are devoid of the usual infiltrates of lymphocytes and macrophages (Greene et al.,

1981). VIII. Immune Deficiency and Immune Surveillance of Tumors

The concept that host immune mechanisms are in place and are able to recognize and restrain the development of tumors has been the subject of active controversy (Moller and Moller, 1975; Ioachim, 1976). From the wide popularity in the 1960s of the hypothesis of ThomasBurnett postulating the strict surveillance of antigenically modified tumor cells, to the total rejection of the concept in the 1970s by workers with serially transplanted, usually nonantigenic tumors, a variety of interpretations have been put forward to explain the pathogenesis of tumors (Burnett, 1970; Hewitt et al., 1976; Woodruff, 1982). As redefined by Klein, immune surveillance is indeed a powerful protective mechanism against neoplasms but with a limited range, being effective only against virus-induced neoplasms (Klein, 1976). According to this concept, immune mechanisms fixed by evolution and transmitted genetically are effective in the prompt recognition of oncogenic viruses and of virus-determined tumor-associated antigens. Infection with EBV provides the best illustration for the theory of immune surveillance against viruses and virus-induced tumors. Epstein-Barr virus is adapted to B-cell membrane receptors, thus infecting a considerable population of B lymphocytes (Jondal and Klein, 1973). The immune response, both humoral and cellular, includes a succession of antibodies against various components of the virus as well as activated suppressor T cells against the EBV-carrying B cells (Purtilo, 1984). This effective immune survellance ensures a lifelong latency for the virus in spite of an 85-90% rate of infection in the general population (Henle et al., 1974). The existence of an immune-surveillance system is further documented by its failure under conditions of immune deficiency. Boys

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with the XLP syndrome are highly vulnerable to the EBV because of an inherited defective lymphoproliferative control locus on the X chromosome (Purtilo, 1984). Following infection with EBV, chronic and frequently fatal infectious mononucleosis or lymphoproliferative disorders, usually BL, develop in such individuals as the result of their failing immune surveillance. The high incidence of EBV-associated BL in other congenital immune deficiencies, in organ transplant recipients, and recently in individuals with AIDS is explainable by the inability of the deficient T-cell system to control the proliferation of EBV-transformed B cells. The hypothesis of immune surveillance of virus-induced tumors thus provides a satisfactory explanation for the frequent generation of EBV-associated lymphomas in immunodeficient individuals. There are other neoplasms, however, that like lymphomas show uncommonly high incidences in immunodeficient persons, yet for which the same explanation is not readily applicable. Kaposi’s sarcoma, the neoplasm most commonly associated with immune deficiency-particularly AIDS-still has an obscure etiology. Epidemiological evidence based OR serological studies indicated an association between cytomegalovirus (CMV) and Kaposi’s sarcoma; however, the very high rates ( > W o ) of seropositivity for CMV in healthy homosexual men make this association less significant (Giraldo et al., 1972; Drew et al., 1981).In a subsequent study, CMV sequences were identified by in situ hybridization in various organs of AIDS patients with Kaposi’s sarcoma, but in very few cases was CMV present in the Kaposi’s sarcoma cells themselves-and then in only a few isolated cells (Grody et al., 1988). Moreover, in cases of classic, non-AIDSrelated Kaposi’s sarcoma, the CMV in situ hybridization was entirely negative. Bowen’s disease and squamous-cell carcinoma of the anus are far more common in people with immune deficiency, especially AIDS (Lauwers et al., 1989).The presence of human papilloma virus (HPV) in these lesions has been demonstrated by in situ hybridization, though far more frequently in the nonmalignant and the in situ lesions than in the clearly malignant, invasive carcinomas (Ioachim et al., 1989).These findings may indicate that some oncogenic viruses are present in such tumors in a state not detectable by the in situhybridization technique or by other methods currently used. Tumor cells may contain CMV, HPV, and perhaps other oncogenic viruses not yet identified in a latent state, with only few copies of their transforming sequences integrated in the host cellular genome (Grody et al., 1988).

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The concept that tumorigenesis is regulated by immune mechanisms has evolved with the progress of cancer research. The allencompassing theory of immune surveillance of tumors is presently conceived as applying mainly to the highly antigenic tumors induced by oncogenic viruses and by ultraviolet radiation (Kripke, 1988).The dramatic increase in incidence and capacity for dissemination of such tumors in association, in AIDS and other immune deficiencies, with the collapse of the T-cell system that normally controls their proliferation, clearly confirms the theory of immune surveillance of tumors at least in relation to the virus-related neoplasms. No similar association has as yet become apparent between immune deficiency and some other tumors common in the general population, such as cancers of the lung, colon, breast, and prostate. Reports in the literature, however, record cases of tumors in persons with AIDS that are unusual by their occurrence in young individuals, their peculiar location, or their uncommon aggressiveness. Documentation on the course of tumors other than those known to be regulated by the T-cell system in individuals with immune deficiencies, though of great interest, is not yet available. The apparent lack of relationship between immune deficiency and the inception and progression of tumors not considered to be virus-related does not necessarily imply the absence of a tumor surveillance system. It may only indicate a present failure to understand the mechanisms, which are probably different from the better-known systems of viral oncogenesis. In fact, the absence of defense mechanisms against such common tumors would be unlikely in view of the high efficiency of immune surveillance against the virus-induced tumors. Far from denying the concept that the development of neoplasms is controlled by effective defense mechanisms, the knowledge acquired from the study of neoplasia in immune deficiency, particularly AIDS, strongly suggests the existence of sophisticated surveillance systems able to restrain, regulate, or totally inhibit the growth of malignank tumors. REFERENCES

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f’ilipovich, A. H., Heinitz, K. J., Robison, L. L. and Frizzera, G. (1987).Am./. Pediatr. Heniatol. Oncol. 9, 183-184. Friedman-Kien, A. E., Laubenstein, L. J., and Rubinstein, P. (1982). Ann. Intern. Med. 96,693-700. Frizzera, G., Roasi, J., and Dehner, L. P. (1980).Cancer (Philadelphia)46,692. Fudenberg, H., Good, R. A., and Goodman, H. C. (1971). Pediatrics 47,927. Gatti, R. A,, and Good, R. A. (1971). Cancer (Philadelphia)28,89. Giraldo, G., Bethe, E., Coeur, P., Vogel, C. L., and Dhru, D. S. (1972).J.Natl. Cancer Inst. (U.S.) 49, 1495. Greene, M. H., Young, T. I., and Clark, W. H., Jr. (1981).Lancet 1,1196. Grody, W. W., Lewin, K. J., and Naeim, F. (1988).Hum. Pathol. 19,524-528. Ilammondi, A. B., Ertel, I., and Newton, W. A., Jr. (1974). Cancer (Philadelphia) 33, 1134. Harwood, A. R. (1984).I n “AIDS: The Epidemic of Kaposi’s Sarcoma and Opportunistic Infections” (A. E. Friedman-Kien and L. J. Laubenstein, eds.), pp. 41-44. Masson, New York. Henle, W., Henle, G. E., and Honvitz, C. A. (1974).Hum. Pathol. 5,551. Ilewitt, H. B., Blake, E. R., and Walder, A. S. (1976).Br. I. Cancer 33,241-259. Ilitzig, W. H. (1976).Pathobiol. Annu. 5,163. Hoover, R., and Fraumeni, J. F., Jr. (1973).Lancet 2,55. Ioachim, H. L. (1976).J.Natl. Cancer Inst. (US’.)57,465-475. Ioachim, H. L. (1980). Contemp. Top. Zmmunobiol. 10,213-238. Ioachim, H. L. (1984).Lab. Inoesf.51, 1-6. loachim, H. L. (1987).I n “Pathology Annual” (P. P. Rosen and R. E. Fechner, eds.), pp. 177-222. Appleton & Lange, New York. Ioachim, H. L. (1988).“Pathology of AIDS.” Lippincott/Gower, Philadelphia, Pennsylvania. loachim, H. L., and Cooper, M. C. (1986). Lancet 1,96. Ioachim, H. L., Dorsett, B., and Paluch, E. (1976). Cancer (Philadelphia)38,2296-2308. loachim, H. L., Lerner, C. W., and Tapper, M . L. (1983).JAMA,J.Am. Med. Assoc. 250, 1306. loachim, H. L., Cooper, M. C., and Hellman, G. C. (1985). Cancer (Philadelphia) 56, 2831. loachim, €I. L., Weinstein, M. A,, Robbins, R. D., Sohn, N., and Lugo, P. N . (1987). Cancer (Philadelphia)60,1449-1453. Ioachim, H. L., Lauwers, G., Cronin, W., and Weinstein, M. A,, and Sohn, N. (1989). Cancer Detect. Preu. (in press). Jondal, M., and Klein, G. (1973).J.E x p . Med. 138, 1365. Kalter, S. P., Riggs, S. A., and Cabanillas, F. (1985). Btood 66,655-659. Kersey, J. H., Spector, B. D., and Good, R. A. (1973).Znt.1. Cancer 12,333. Klein, G. (1976).Natl. Cancer Inst. bfonogr. 44, 109. Krikorian, J. G., Anderson, J. L., and Bieber, C. P. (1978)./AMA,J. Am. Med. Assoc. 240, 639. Kripke, A l . L. (1988)./. Natl. Cancer Inst. 80,722-727. Lauwers, G., Weinstein, M.,Sohn, N., and Ioachim, H. L. (1989). Lab. Inuest. Lewison, E. F., ed. (1974).Natl. Cancer Inst. Monogr. 44. Longo, D. L., Steis, R. G., and Lane, H. C. (1984). Ann. N . Y. Acad. Sci. 437,421. Medical Research Council Working Party (1969). Lancet 1, 163. Moller, G . , and Moller, E. (1975)./. Natl. Cancer Inst. (U.S.)55,755-759. Nagington, J . . and Gray, J . (1980). Lancet 1,536.

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Nakamura, H., Ishiguro, K., and Mori, T. (1988). Cancer (Philadelphia)62,2489. Penn, I. (1977). Transplant. Proc. 9,1121. Penn, I. (1979). Transplant. Proc. 9,1047. Penn, I. (1983). Transplant. Proc. 15,2790. Penn, I. (1988).Annu. Rev. Med. 39,63-73. Perry, G. S., Spector, B. D., and Schuman, L. M. (1980).J.Pediatr. 97,72. Peterson, R. D. A., Kelly, W. D., and Good, R. A. (1964).Lancet 1,1189. Purtilo, D. T. (1984). Lab Invest. 51,372-385. Purtilo, D. T., Cassel, C., and Yang, J. P. S. (1975). Lancet I, 935. Purtilo, D. T., Sakamoto, L., and Saemundsen, A. (1981). Cancer Res. 41,4226. Rabinovich, H., Cohen, R., Bruderman, I., Steiner, Z., and Klajman, A. (1987). Cancer Res. 47,173-177. Roitt, I. M., Brostoff, J., and Male, D. K. (1985). “Immunology,” MosbylCower Med. Publ., St. Louis, Missouri. Schneck, S. A., and Penn, I. (1971). Lancet 1,983. Selwyn, P. A. (1986). Hosp. Pract. 21,119-153. Snover, D. C., Frizzera, G., and Spector, B. D. (1981). Hum. Pathol. 12,821. Starzl, T. E., Porter, K. A., and Iwatsuki, S. (1984).Lancet 1,583. Stiehm, R. E. (1980).In Immunologic Disorders in Infants and Children” (R. E. Stiehm and V. A. Fulginiti, eds.), pp. 183-218. S a n d e r s , Philadelphia, Pennsylvania. Strauchen, J. A., Moran, C., Goldsmith, M., and Greenberg, M. (1987). Cancer (Philadelphia) 61,1872-1875. Stribling, J., Weitzner, S., and Smith, G. V. (1978). Cancer (Philadelphia)42,442. Toledano, S. R., and Lange, B. J. (1980). Cancer (Philadelphia)45,1675. Vose, B. M., and Moore, M. (1979). Znt. J . Cancer 24,579-585. Vose, B. M., Vanky, F., and Klein, E. (1977). Znt.]. Cancer 20,895-902. Waldmann, T. A., Strober, W., and Blaese, R. M. (1972).Ann. Intern. Med. 77,605. WoodruK M. (1982). Br. J . Cancer 46,313-322. Ziegler, J. L., and others (1984).N. Engl. J . Med. 311,565. Zulman, J., Jaffe, R., and Talal, N. (1978).N. Engl.J.Med. 299,1215.

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A NOTE ON CONCOMITANT IMMUNITY IN HOSTPARASITE RELATIONSHIPS: A SUCCESSFULLY TRANSPLANTED CONCEPT FROM TUMOR IMMUNOLOGY Graham F. Mitchell The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050,Australia

I. Introduction 11. Some Examples of Concomitant Immunity in Parasitized Mice A. Taenia taeniaeformis Infection B. Leishmania major Infection C. Schistosoma mansoni and Schistosoma japonicum Infections 111. Concluding Comments References

I. Introduction

The term concomitant immunity, taken from tumor immunology, has been very usefully applied by investigators interested in the immunology of host-parasite relationships. The term was introduced into the parasitology literature by S. R. Smithers and colleagues in 1969 in the context of the helminth (worm) disease, schistosomiasis (Smithers and Terry, 1969; Smithers et al., 1969).Concomitant immunity refers to a common situation in natural host-parasite relationships, namely that hosts already infected with a parasite are partially or wholly resistant to reinfection with the “homologous” organism. In this short article, in which immunology is emphasized rather than parasitology, I will highlight some of the features of concomitant immunity in several helminth and protozoan parasite infections that we have studied in this laboratory and that may be of interest to tumor biologists. The phenomenon of concomitant immunity, in which high-level resistance can be demonstrated in the face of existing, often light, infection, has several alternate descriptors; for example “premunition” in malaria (Sergent, 1963) and “nonsterilizing immunity” (Cohen, 1974).This particular type of resistance to reinfection is best detected in helminth infections where the established parasite, unlike in most protozoan infections, is not proliferating, has a relatively restricted location, and can be clearly differentiated from the infective form of the parasite. 319 ADVANCES IN CANCER RESEARCH, VOL. 54

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

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In their original papers, Smithers et al. cite Bashford et al. (1908)and Gershon et ul. (1967)as relevant early references on concomitant immunity in tumor systems. I am not familiar with how the concept has advanced in tumor immunology, but papers continue to appear with good descriptions of the phenomenon-that is, failure of a tumor graft to establish in a second (cutaneous) site in a mouse bearing that same tumor (Vaage, 1971; North, 1984; Gorelik, 1983). Many parasitic infections are acquired early in life and are chronic. Clearly, for the parasite population, there is little evolutionary purpose in being particularly accomplished at evading protective immune responses in the host to the extent that the very existence of the host species is threatened. High mortality in prereproductive life of the natural host species is not a feature of a balanced host-parasite relationship. Successful chronic parasitism is a compromise in which partially effective host-protective immune responses are balanced by partially effective immune-evasion mechanisms (Burnet and White, 1972; Capron et al., 1968; Damian, 1964; Dineen, 1963; Mims, 1976; Mitchell, 1979, 1982a; Sprent, 1962). Thus concomitant immunity makes “good biological sense”: in order to prevent superinfections that prejudice the well-being of the already-infected host, the established (resident) parasites should induce and maintain immune responses that are highly effective against establishing (invasive) parasites. The susceptibility of the invasive parasite and the resistance of the resident parasite are ensured if immune-evasion mechanisms are inefficient in the former and highly developed in the latter-that is, they develop during the course of initial infection. II. Some Examples of Concomitant Immunity in Parasitized Mice

A. Taenia taeniaefomis INFECTION An excellent example of concomitant immunity, in which some clues are available on likely mechanisms, is provided by the larval cestode parasite of mice and rats T. tueniaeformis, which causes murine cysticercosis (Mitchell, 1982b; Rickard and Williams, 1982). This is a parasitic helminth that parasitizes two host species: tapeworms are found in the intestines ofcats and liver cysts are found in rodents. Cats are infected by ingestion of these liver cysts, and rodents are infected by ingestion of eggs voided in the feces of cats. Taenia taeniaeformis is a very successful parasite in which immunology has been shown to play a decisive role in the intermediate (rodent) host. Infected mice and rats cannot be reinfected by oral

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administration of eggs harvested from tapeworm segments. They contain serum antibodies that can transfer complete resistance to naive recipients. Small volumes of sera taken from mice infected for some weeks are highly host-protective, even when injected into very susceptible hypothymic nude mice at about the time of egg challenge (Mitchell et al., 1980).Despite some effort, little is yet known about the specificities of these antibodies (Bowtell et al., 1983; Lightowlers et al., 1984). They include T-helper ( T H ) cell-dependent, protein Abinding, complement-fixing IgG antibody isotypes. Decomplementation of recipients with cobra venom factor reduces the efficacy of passively administered antibody in both rat and mouse systems (Musoke and Williams, 1975; Mitchell et al., 1977). The target of hostprotective antibody is the invading larva that hatches from eggs in the intestines, since antibody must be administered at about the time of egg challenge. There is also evidence that intestinal IgA antibodies contribute to resistance in mice (Lloyd and Soulsby, 1978). Anticomplementary activities, not yet fully characterized, have been described in established T. taeniaeformis cysts (Suquet et al., 1984; Hammerberg and Williams, 1978; Letonja and Hammerberg, 1983). From the data obtained in these laboratory model systems, the statement is warranted that the immunology of the rodent-T. taeniaeformis host-parasite relationship may be relatively simple. One hastens to add that no evolved parasite population will have invested in a single mechanism of immune evasion, just as no evolved host population will have invested in a single immune-effector mechanism. Neverthelss, the principal events in this relationship may be antibodydependent, complement-mediated destruction of invading larvae, with established liver cysts protecting themselves against such attack by elaboration of anticomplementary factors. Establishing parasites differ from established parasites in that they do not have an efficient anticomplementary immune-evasion mechanism in place. Mouse strains differ in susceptibility to T. taeniaeformis infection, the resistant genotypes being earlier responders in terms of hostprotective antibody production. Presumably, in a primary infection in genetically resistant mouse strains, titers and specificities of complement-fixing antibodies are appropriate for elimination of establishing parasites prior to the full expression of anticomplementary activities in the invading parasite. This “race against time” in the early stages of infection is lost by the genetically susceptible mouse strains (e.g., C3H/He). One complication in this story is that the most resistant mouse strain is the C57BL/6 that is known to show a delay in switch from IgM to IgG isotypes following immunization with various anti-

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gens (Silver et al., 1972;Winn, 1965;Chapman et al., 1979).That is, the C57BL/6 mouse strain is a high and prolonged producer of IgM antibodies, these being poor fixers of (mouse) complement (Klaus et al., 1979).Another feature of the rodent-T. taeniaeformis systems is that, besides the existence of shared antigens presumed to be involved in the maintenance and expression of concomitant immunity, there are clearly stage-specific antigens in larvae and cysts. Thus immunization with larval extracts of T. taeniaeformis efficiently inhibits initial infection, whereas immunization with cyst extracts results in severe retardation of cyst growth rather than efficient inhibition of parasite establishment (Bogh et al., 1988). It seems that concomitant immunity is only one form of immunologically based resistance in this infection. In regard to concomitant immunity, there is suggestive evidence (but no proof) that the established parasite need only maintain a state of sensitization that leads to accelerated responsiveness upon contact with the challenge larvae. That is, protective antibodies need not be of protective titer at the time of challenge as long as such titers are attained quickly after challenge. Presumably, this is assured by maintenance of memory in both T H - and B-cell populations (Mitchell, 1989). A prediction from this type of immunity is relevant to tumor immunology. A very low-level “trickle” infection will be able to establish itself without eliciting a rapid or anamnestic response that is necessary to eliminate the invading organism. In primary infections with T . taeniaeformis, naive genetically resistant (C57BL/6)mice are more resistant to high-dose egg challenge than they are to low-dose challenge (Mitchell et aZ., 1980).Of course, one good way to accelerate responses is to vaccinate or preimmunize with appropriate antigens. Even the most susceptible mouse strains can be protected completely by injections of crude antigen with no stringent requirement for adjuvants (Rajasekariah et d.,1980). Perhaps appropriately in view of all the evidence that antitaeniid immune responses can be highly protective and that concomitant immunity is well developed, the first molecularly defined vaccine proven to be S O % effective in a natural host species has been developed in cysticercosis (Johnson et aZ., 1989). With the assumption that a tumor may be susceptible to complement-fixing antibodies, it would seem unlikely (though not entirely farfetched) that the environment of an established localized tumor would be anticomplementary, whereas an inoculum of cells of that same tumor, or fresh tumor implant, would be susceptible to complement-mediated, antibody-dependent destruction. The next example, Leishmania infections, may be more relevant to tumor systems.

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B. Leishmania major INFECTION The cause of Old World or zoonotic cutaneous leishmaniasis in humans, L. major, is a protozoan parasite that is transmitted by sand flies as a flagellated promastigote and that resides in macrophages as a nonflagellated, proliferating amastigote. The mouse is a good model for the human disease, and the immunological aspects of the relationship between L. major (plus related species) and mice has been studied in some detail during the 1980s (see Louis and Milon, 1987, for reviews). All indications are that expression of resistance to chronic infection (this infection manifesting as a cutaneous lesion with some dissemination of the parasite) requires an involvement of CD4’ T cells with specificity for antigens that are associated with the infected macrophage and that are probably shared with the infective form of the parasite, the promastigote. Moreover, compelling evidence exists that CD4’ T cells can exacerbate disease in murine cutaneous leishmaniasis. Resistance and susceptibility in this model reflect an interplay or balance between resistance-promoting T cells and disease-promoting T cells, both of the CD4 phenotype. Some contribution of CD8’ T cells to the outcome of infection in mice has been suggested, but the contribution is likely to be minor (Titus et al., 1987). Mice that have a large cutaneous lesion (together with regional lymph node and more systemic involvement) can be difficult to reinfect in another cutaneous location using promastigotes of the same isolate of L. major (Mitchell and Handman, 1983). Good examples of concomitant immunity have been described in guinea pigs infected cutaneously with the parasite Leishmania enriettii. These animals can be relatively resistant to reinfection in that promastigotes deposited in another location may not result in lesion development (Bryceson et al., 1970; Poulter, 1979).Recently, J. Mauel (personal communication) has described a situation in which an L. major lesion in the ear apparently inhibits very effectively the development of a second lesion on the dorsum of the mouse. The failure of second-lesion development after promastigote challenge in infected animals is not absolute, and observations in humans indicate that resistance to homologous challenge in cutaneous leishmaniasis does not precede healing of the primary lesion (Dostrovsky et al., 1952; Adler, 1963). Interestingly, in the more severe leishmaniasis, espundia, resistance to reinfection does precede healing (Bryceson, 1970). Certainly cutaneous “metastases” are not rare in leishmaniasis. Indications of concomitant immunity that is only partially effective can be accommodated readily within what is currently the most attrac-

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tive explanation for T cell-dependent resistance and T cell-dependent disease exacerbation in murine cutaneous leishmaniasis. Following the work of Mossman and Coffman (1987) and others (see Bottomley, 1988) on the two broad categories of CD4' T cells-namely TH1 and TH2-Locksley et al. (1987) have proposed that resistance to L. major results from effector activities (presumably macrophage activation) of TH1 cells that secrete y-interferon (IFN-y). By contrast, disease exacerbation follows from preferential induction of TH2 cells that secrete interleukin 4 (IL-4).This lymphokine seems entirely inappropriate for elimination of intramacrophage infection, although it may be appropriate for expression of resistance to some helminths, for example. Locksley et al. (1987) have demonstrated, through hybridization approaches, a predominance of mRNA for IL-4 in regional lymph nodes of genetically susceptible mice bearing a lesion and increased amounts of IFN-y mRNA in lymph nodes of genetically resistant mice in which the lesion is resolving. In regard to concomitant immunity in this infection, it is possible that disease-promoting T cells are enriched relative to resistancepromoting T cells in locations where parasitized macrophages are plentiful rather than where newly injected promastigotes are located after cutaneous deposition. There is another, less likely possibility (Hoover and Nacy, 1984; Moll and Mitchell, 1988): macrophages newly parasitized by L. major are more vulnerable to IFN-.)Iand any other activating mediators than parasitized macrophages in longestablished lesions. Both resistance-promoting and disease-promoting T cells can certainly be found in infected mice. [From work in this laboratory (Handman and Mitchell, 1985; Mitchell and Handman, 1986), we believe the specificities of the two CD4' T-cell types are different and that carbohydrates are included in the range of target epitopes recognized (Moll et al., 1989).This, of course, is a contentious issue with respect to class I1 major histocompatibility complex (MHC)-restricted recognition by CD4' T cells (or class I MHC-restricted recognition by CD8' T cells), peptide recognition only by T cells being the current dogma. The lack of reactivity of TH cells to bacterial capsular polysaccharides has been extrapolated to carbohydrates in general. Why such an important arm of the adaptive immune response would neglect a universal class of molecules of pathogens is extraordinary. Nevertheless, until a T-cell line is produced that reacts with a synthesized oligosaccharide, perhaps containing a lipid moiety (Robertson et al., 1982), then the issue of T-cell recognition of carbohydrate epitopes in leishmaniasis will remain controversial]. In L. major and related infections in mice, a loss of MHC-encoded

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molecules on macrophages of genetically susceptible mice has been demonstrated (Handman et al., 1979; Reiner et al., 1987). In the case of class I1 MHC this event should be instrumental in reducing the sensitivity of such macrophages to T cell-mediated aggressive immune attack. More speculatively, the loss of MHC may influence the type of ) is recruited, stimulated, or indeed, CD4' T cell (i.e., T H 1 or T H ~that induced by the infected macrophage. It is said that large amounts of MHC-associated antigen, of the type that specific B cells may be able to display, will preferentially promote the T H (inflammatory) ~ type of CD4' T cell rather than the T Htype ~ (Bottomley, 1988). At this stage, the mechanisms underlying reduced expression of MHC molecules on infected macrophages of genetically susceptible mice in leishmaniasis remain unknown as do the consequences of this loss. There are some remarkable similarities between murine cutaneous leishmaniasis and several systems involving immunogenic transplanted tumors (Awwad and North, 1988; Bursuker and North, 1985). This is particularly so in regard to disease characteristics (e.g., lesion development and regression) in reconstituted sublethally irradiated (Howard et al., 1981) or nude mice (Mitchell, 1983). For example, BALB/c mice are very susceptible to L. major, yet BALB/c nude mice given a Eow number of T cells (e.g., lo6 cells) from normal BALB/c donors are highly resistant. Nude mice given a high number of T cells are more susceptible. When minimally reconstituted BALB/c nude mice are given an additional low (or high) number of T cells from chronically infected BALB/c mice, such recipients revert to high susceptibility. This fits with the notion that resistance-promoting TH1 cells (or their precursors if they are induced after stimulation by antigen delivered in a particular way) are at high frequency in normal BALB/c mice relative to disease-promoting T Hcells. ~ In the lymphoid organs of chronically infected mice, this ratio is reversed. In the tumor systems referred to already, the competition appears to be between CD8' T cells ( ? cytotoxic cells) and CD4+ T cells (North and Awwad, 1987). In conclusion, the concomitant immunity that can be demonstrated in cutaneous leishmaniasis and that does not have the hallmarks of a very efficient host-protective immunity, might well parallel some of the phenomena of concomitant immunity in tumor systems. The postulated mechanisms underlying this partial resistance to reinfection involve an interplay between T-cell subpopulations producing different mediators and probably with different specificities. Concomitant immunity must reflect a relatively unimpeded effect of resistancepromoting T cells and their mediators in the second site relative to the firmly established original lesion or tumor mass.

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C. Schistosoma mansoni

AND

Schistosoma japonicum INFECTIONS

As mentioned before, it was through studies in schistosome infections-specifically S. mansoni in rhesus monkeys-that the term concomitant immunity became fixed in the parasitology literature. Mice infected with S. mansoni are also solidly resistant to reinfection when exposed a second time to snail-derived cercariae, the infective form of this trematode parasite (reviewed in Smithers et al., 1987).The observation has been confirmed repeatedly, and high-level resistance to reinfection has also been demonstrated during S. japonicum infection in mice (Maloney et al., 1987; Garcia et al., 1984). However, interpretation of events in mice demonstrating concomitant immunity is far from straightforward. Wilson et al. (1983),Dean et al. (1981),and Harrison et al. (1982)first raised the strong possibility that concomitant immunity in infected small laboratory hosts was not just a matter of expression of host-protective immune responses against the invading parasite. Indeed, most of the concomitant immunity in these mouse models may simply reflect an inability of the second infection to establish in the portal system because of changes in the liver induced by the first infection. Schistosomiasis mansoni and japonica are classical immunopathological diseases (Warren, 1982). CD4’ T cell-dependent immune responses of the delayed-type hypersensitivity (DTH) type, and directed to antigens that are secreted by eggs trapped in the liver (and intestinal wall), result in granuloma formation and fibrosis. These in turn lead to increased portal pressure and establishment of a collateral circulation bypassing the liver. It is this altered environment that second-infection schistosomes apparently experience difficulty in colonizing successfully. These interpretations of concomitant immunity in small-animal models have led to the virtual abandonment of these models for the study of immunological correlates of resistance to reinfection in schistosomiasis. However, it is very likely that a component of this type of resistance in S. mansoni and S. japonicum-infected mice is “immunologically based” rather than only “immunopathologically based,” and even if this component is small, it does not follow that it is equally small in human beings. Analysis of resistance to reinfection in humans, most readily studied after pharmacological elimination of existing infection (Butterworth and Hagan, 1987), may well lead to the identification of relevant immune-effector mechanisms and vaccine candidates. A substantial body of literature exists on effector cells and molecules capable of eliminating or damaging schistosomes from the infective form (schistosomule) through to the adult worm (schisto-

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some) (Capron et al., 1987; Smithers and Doenhoff, 1982; James, 1987; Mitchell et al., 1989). This will not be further discussed here except to say that most involve aggressive immune attack at the parasite surface (e.g., antibody-dependent cellular cytotoxicity, ADCC) involving a variety of cell types including eosinophils, neutrophils, and macrophages (the latter not necessarily requiring antibodies in uitro) plus complement-fixing antibodies of particular isotypes and with various specificities. Moreover, it is in schistosomiasis that the best evidence for blocking antibodies in parasitic infections exists (Butterworth et al., 1987; Dunne et al., 1987; Khaliffe et al., 1986; Yi et al., 1986). It is hoped that blocking antibodies of certain isotypes are a major component of immune evasion utilized by schistosomules and schistosomes, because it is one mechanism that should be amenable to vaccine-induced modification. In other words, as means to induce antibodies of desirable (aggressive) isotypes become apparent through studies on TH cell-dependent regulation of isotypes produced by B cells, appropriate vaccination should militate against induction or persistence of the undesirable (blocking) isotypes. In another trematode infection, fascioliasis caused by Fasciola hepatica, concomitant immunity can be partially effective (in rats) or nonexistent (in mice) (see Chapman and Mitchell, 1982). A contribution of population heterogeneity to inefficient concomitant immunity in this system has been virtually excluded using clonal populations of parasites. That is, concomitant immunity is poor whether or not the same or different parasite clones are used for reinfection (Chapman et al., 1981). 111.. Concluding Comments

Systematic studies on concomitant immunity in host-parasite relationships have to spme extent been put aside, whereas methods to characterize and produce parasite antigens through chemical and biological synthesis have assumed priority in molecular parasitology in the mid- to late 1980s. This is appropriate because without pure antigens and their epitopes, quantitation of immune responses, either antibody or cellular, is impossible. Vaccine development dominates immunoparasitology at the present time, and the major constraint to achieving the vaccine objective in the molecular biology era is a deficiency in knowledge on immuneeffector mechanisms against parasites. Even when these mechanisms are known and the target antigens identified, how should the vaccine be delivered in order to induce the appropriate responses in the vari-

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ous host genotypes? A clear demonstration of concomitant immunity that is uncomplicated (i.e., the established parasite induces or maintains an immune response that is highly efficient at preventing reinfection) is obviously very encouraging for the vaccine developer. First, it demonstrates that induction of a response that prevents infection is possible. Second, established forms of the parasite are usually available in abundance (with some exceptions, such as human filarial worms), whereas infective forms are often far more difficult to obtain in the quantities necessary for antigen or mRNA isolation. Two questions about concomitant immunity are of interest to both tumor immunologists and immunoparasitologists. First, in those situations where concomitant immunity is efficient, what is the nature of resistance to immune attack displayed by the established parasite or tumor? If this is understood, then therapeutic vaccinations may become a reality (Mitchell, 1988).In other words, the feasibility of vaccination to neutralize immune evasion employed by established parasites or tumors will be increased by a detailed understanding of this resistance to immune attack that is not displayed by the invasive parasite or second-tumor inoculum. Second, what aspects of the hosttumor and host-parasite relationship militate against efficient concomitant immunity? At least in helminth and protozoan parasitism, four inechanisms of immune evasion are likely to facilitate establishment of the invasive parasite in parasitized hosts.

1. Antigenic variation or population heterogeneity. Here the challenge (or emergent) organism is not identical to the established population. This is known to be, or is likely to be, a major determinant of inefficient resistance to reinfection or concomitant immunity in African trypanosomiasis and falciparum malaria as it is in influenza, AIDS, gonococcal infections, and so on. 2. Blocking antibodies. This was mentioned before in relation to schistosomiasis but should not be a major stumbling block when methods to induce particular antibody isotypes at will become available. 3. Znduction of “tolerance” to key antigens of the invasive parasite by particular forms of the antigen in established parasites. In this situation, antigens are elaborated by the resident parasite that partially, but specifically, depress immune responses potentially induced by the establishing form of the parasite. Though not proven, it is possible that the circumsporozoite-related antigen (CRA) of blood stage P . fakiparum (Coppel et al., 1985; see also Peterson et al., 1988), which contains epitopes cross-reactive with the circumsporozoite protein (CSP) of infective sporozoites, can alter protective immune

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responses against the sporozoite. These protective antisporozoite responses are now believed to involve both anti-CSP antibodies and importantly, CD8’ (cytotoxic ?) T cells (Schofield et al., 1987; Weiss et al., 1988).Orjih and Nussenzweig (1979) demonstrated some time ago that blood stage infection can reduce protective responses to the sporozoite in a mouse malaria. 4. Generalized immunosuppression. This must be rare, since it does not make “biological sense” for the parasitized host to be vulnerable to any other infection, either heterologous or homologous! However, localized immunosuppression or a local block to expression of resistance may be important, particularly in the gut as indicated later. C3HIHe mice exposed to the intestinal protozoan parasite Giardia muris develop a chronic infection that lasts for many weeks; other mouse strains eliminate the parasite from the gut far more quickly (Roberts-Thomson and Mitchell, 1978). After drug cure of the existing infection in C3H/He mice, a challenge inoculum is eliminated rapidly (Underdown et al., 1981; Erlich et al., 1983).Thus, strong resistance to reinfection can be demonstrated provided the existing parasites are removed by chemotherapy, an event that has also been demonstrated in sheep with certain intestinal nematodes (Wagland and Dineen, 1967). A likely explanation comes from studies by Behnke and colleagues on the intestinal nematode of rodents, Nematospiroides dubius (Heligmosomoides polygyrus) (reviewed in Behnke, 1987). Evidence exists in the N . dubius-mouse system that established adult worms in the intestines elaborate factors that prevent expulsion of these worms plus, to some extent, incoming infection either homologous or heterologous. When the worms are removed, the source of these putative antiinflammatory molecules is removed, and resistance to reinfection can be demonstrated for some time after. To conclude, the fortunate theft by immunoparasitologists of the term and concept of concomitant immunity from tumor immunology and application to the study of the immunology of protozoan and helminth parasitism, has probably uncovered little that is immediately applicable to host-tumor relationships. An obvious difference between the parasite systems discussed briefly here and tumors is that the second inoculum in a demonstration of concomitant immunity involves a different form of the parasite from that already established. This is not obviously the case in tumor systems to my knowledge. It is more likely to be within the multiplicity of immune-evasion strategies utilized by parasites and tumors that one discipline can learn from the other in the quest for new methods of stimulating host resistance.

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ACKNOWLEDGMENTS Work referred to in the text from this laboratory is, or has been, supported by the Australian National Health and Medical Research Council, the Rockefeller Foundation Great Neglected Diseases Network, the UNDPlWorld BanklWHO Special Program for Research and Training in Tropical Diseases, the U.S. National Institutes of Health, and the John D. and Catherine T. MacArthur Foundation Biology of Parasitism program.

REFERENCES Adler, S. (1963).In “Immunity to Protozoa” (P. C. C. Gamham, A. E. Pierce, and I. Roitt, eds.), p. 243. Blackwell, Oxford. Awwad, M., and North, R. J, (1988).Cancer Zmmunol. lmmunother. 26,55-60. Bashford, E. F., Murray, J. A., Haaland, M., and Bowen, W. H. (1908).In “Third Scientific Report on the Investigations of the Imperial Cancer Research F u n d ’ (E. F. Bashford, ed.), pp. 262-283. Taylor & Francis, London. Behnke, J. (1987).Ado. Parasitol. 26, 1-71. Bogh, H. O., Rickard, M. D., and Lightowlers, M. W. (1988). Parasite Immunol. 10, 255-264. Bottomley, K. (1988).Immunol. Today 9,268-274. Bowtell, D. D. L., Mitchell, G. F., Anders, R. F., Lightowlers, M. W., and Rickard, M. D. (1983).E x p . Parasitol. 56,416-427. Bryceson, A. D. M. (1970).Proc. A. SOC. Med. 63,1056-1060. Bryceson, A. D. M., Bray, R. S., Wolstencroft, R. A., and Dumonde, D. C. (1970).Clin. E x p . Zmmunol. 7,301-341. Bumet, F. M., and White, D. 0. (1972). “Natural History of Infectious Diseases.” Cambridge Univ. Press, London and New York. Bursuker, I., and North, R. J. (1985).Cancer Immunol. Zmmunother. 19,215-218. Buttenvorth, A. E., and Hagan, P. (1987).Parasitol. Today 3, 11-16. Buttenvorth, A. E., Bensted-Smith, R., Capron, A., Capron, M., Dalton, P. R., Dunne, D. W., Grzych, J. M., Kariuki, H. C., Khalife, J., Koech, D., Mugarbi, M., Ouma, J. H., Arap-Siongkok, T. K., and Sturrock. R. F. (1987).Parasitology 94,281-300. Capron, A., Biguet, J., Vemes, A., and Afchain, D. (1968).Pathol. Biol. 16,121-138. Capron, A., Dessaint, J. P., Capron, M., Ouma, J. H., and Butterworth, A. E. (1987). Science 238,1065-1072. Chapman, C. B., and Mitchell, G. F. (1982).Znt.]. Parasitol. 12,81-91. Chapman, C. B., Knopf, P. M., Anders, R. F., and Mitchell, G. F. (1979).Aust.J. Exp. Biol. Med. Sci. 57,389-400. Chapman, C. B., Rajasekariah, G . R., and Mitchell, G. F. (1981).Am.J. Trop. Med. H y g . 80,1039-1042. Cohen, S. (1974).In “Parasites in the Immunized Host: Mechanisms of Survival” (R. Porter and J. Knight, eds.), pp. 3-17. Associated Scientific, Amsterdam. Coppel, R. L., Favaloro, J. M., Crewther, P. E., Burkot, T. R., Bianco, A. E., Stahl, H. D., Kemp, D. J., Anders, R. F., and Brown, G. V. (1985).Proc. Natl. Acad. Sci. U.S.A.82, 5121-5125. Damian, R. T. (1964).Am. Nut. 98,129-149. Dean, D. A,, Bukowski, M. A., and Clark, S. S. (1981). Am. J . Trop. Med. Hyg. 30, 113-120. Dineen, J. K. (1963).Nature (London) 197,268-269. Dostrovsky, A., Sagher, F., and Zuckerman, A. (1952).AMA Arch. D e n a t o l . Syphilol. 66,665-675.

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INDEX A Acquired immune-deficiency syndrome (AIDS), tumors associated with, 309-31 1 Active immunization against peptide growth factors in autocrine growth stimulation inhibition, 230-231 Acylation, fatty, in membrane localization of yeast RAS proteins, 97-98 Adenylate cyclase in Ras-CAMP pathway, 101-105 biochemical characterization of, 104 and RAS proteins, direct interaction of, 104-105 Ras-responsive domains of, 102-103 structure of, 101-102 Adipogenesis in bone marrow, 261 ADRl as target of CAMP-dependent protein kinase, 117-118 Agammaglobulinemia Bruton’s type, opportunistic tumors associated with, 306 infantile X-linked, opportunistic tumors associated with, 306 Swiss-type, opportunistic tumors associated with, 305-306 AKXD RI mouse strains, 143-151 lymphomas in susceptibility to, 144 viral integration in, variation in repertoire of cellular protooncogenes activated by, 144, 145 myeloid tumors in, Eui-l identification in, 145-151 Alleles, epigenetic inactivation of, in human cancer, 49-58 predisposition and, 51-56 progression and, 56-58 Anti-growth factor antibodies in autocrine growth stimulation inhibition, 230

Anti-growth factor receptor antibodies in autocrine growth stimulation inhibition, 229 Antibody epitopes, yeast Ras proteins and, 95 Antiidiotype antibodies mimicking cell surface antigens in autocrine growth stimuIation inhibition, 230 Astrocytomas, progression of, loss of heterozygosity and, 38-40 Ataxia telangiectasia, opportunistic tumors associated with, 307 Autocrine growth stimulation of human tumor cells, 226-231 inhibition of, 229-231

B cells; see B lymphocytes B-lymphocyte Epstein-Barr virus/C3d receptor, structure and function of, 273-295 B lymphocytes activation of, CR2 in, 292-293 CR2 in, 287-289 development, activation and differentiation of, CR2 expression during, 290-291 Basement membrane as barrier to invasion by cancer, 162 Beckwith-Wiedemann syndrome (BWS), 42-46 Bisphosphatase as target of CAMP-dependentprotein kinase, 115-116 Bladder, carcinoma of, progression of, from in situ to invasive, 165 Bone marrow adipogenesis in, 261 cultures of, long-term, 240-241 initiation of, 245-246 lymphocytes in, 242-245

333

334

INDEX

lymphopoietic microenvironment in, 235-264 differentiation steps and lineages in, 24 1-242 experimental approaches to, evolution of, 237-40 microenvironment of, essential cells of, 246-250 Breast carcinoma, progression of, from in situ to invasive, 164-165 Bruton’s type agammaglobulinemia, opportunistic tumors associated with, 306

C C-terminal methyl esterification in membrane localization of yeast Ras proteins, 99 C3d, 284 C3d receptor and EBV receptor, functional relationship of, 274 CBdg, 284 and gp3501220, sequence similarity of, role of, in CR2 binding, 285-286, 287,288 CAMP-dependent protein kinase in Ras-CAMP pathway, 105-107 targets of, 111-122 ADRI as, 117-118 bisphosphatase as, 115-116 in feedback regulation of Ras-CAMP pathway, 118-120 glutamate dehydrogenase as, 116-117 glycogen phosphorylase as, 114-115 glycogen synthase as, 113-114,115 inositol phospholipid synthesis as, 121-122 phosphofructokinase-2 as, 115-1 16 proteins in carbon metabolism as, 112-118 proteins in growth control as, 120-122 sugar-transport systems as, 117 transcription factors as, 120-121 trehalase as, 112-113 trehalose synthase as, 112-113 YAKl as, 122

Cancer, mixed genome imprinting and, 55-56 loss of heterozygosity and, 42-49, 55-56 Cancer cells arrest of, in metastatic process, 172- 176 death of, postintravasation rapid phase of, 174-176 slow phase of, 176 loss of, invasive delay and, 166 motility of, invasive delay and, 166 Carbon metabolism, proteins in, as target of CAMP-dependent protein kinase, 112- 118 Carbon source of signals for ras genes, 122-125 Carboxyl-terminal modification of yeast Ras proteins, 97-101 Carcinogens, chemical, effects of, on DNA methylation, 11-13 CASl in Ras-CAMP pathway, 110 CDC25 in Ras-CAMP pathway, 108-110 Cell fusion products, short-term analysis of, 64-67 with heterokaryons, 64-67 with reconstructed cells, 67 Cells, tumor, DNA methylation in, 7-8 Cellular immortalization, genetic and molecular studies of, 63-75 Cervix, carcinoma of, progression of, from in situ to invasive, 164 Chemical carcinogens, effects of, on DNA methylation, 11-13 Chromosome region 13q14, deletions of, in retinoblastoma, 30-31 Clonal dominance, randomness and selection in metastasis and, 183, 186-187 Colorectal carcinoma growth factor secretion and growth factor receptor expression by, 227-229 progression of, from in situ to invasive, 165 Complement, activation and regulation of, CR2 in, 293-294 Complement receptor type 2 (CR2) in B-cell activation, 292-293 binding of, C3dg and gp350/220 in, 285-286,287,288

335

INDEX

cDNA clones encoding, isolation and characterization of, 276-277 cellular and tissue distribution of, 287-291 in complement activation and regulation, 293-294 expression of, during B-cell development, activation and differentiation, 290-29 1 functional properties of, 291-294 identification and characterization of, 274-276 in ligand internalization, 291-292 ligands of, structure and function of, 284-286 membrane-associated, expression of, in rodent cells, 280,281 in multigene family, 277-280 recombinant, expression and analysis Of, 280-283 soIuble forms of, expression of, in Baculovirus vector system, 281-283 structure of, 276-283 Concomitant immunity in host-parasite relationships, 319-329 in parasitized mice, 320-327 with Leishmania major infection, 323-325 with Schistosoma japonicum infection, 325-326 with Schistosoma mansoni infection, 325-326 with Taeniae taeniaefomis infection, 320-322 CpG islands, DNA methylation and, 2-4 Cytogenetics of retinoblastoma, 30-31 Cytokines affecting lymphohematopoiesis, 252-254 localization of, in microenvironment, 256-257 responses to, involving stromal cells, 259-261

D Deletions of 13q14 in retinoblastoma, 30-31

Developmental malformations, tumors and, 42-46 Di George’s syndrome, opportunistic tumors associated with, 306 DNA methylation in cancer, 1-19 effects of chemical carcinogens on, 11-13 genomic imprinting and, 16-19 in oogenesis, 14-15 in spermatogenesis, 14-15 in tumor cells, 7-8 in tumor diversification, 13-14 DNA synthesis, senescence and, studies of with heterokaryons, 64-67 with reconstructed cells, 67 Dynamic heterogeneity, randomness and selection in metastasis and, 183, 184-185 Dysgammaglobulinemias, opportunistic tumors associated with, 306

E EIA adenovirus, effects of, on normal cells in microinjection experiments on cellular immortalization, 75 Ectomesenchymoma, loss of heterozygosity in, 48-49 Ectopic viral integration site 1, 141-155; see also Evi-1 Effector domains of yeast RAS proteins, 95-96 Epigenetic inactivation of alleles in human cancer, 49-58 predisposition and, 51-56 progression and, 56-58 Epithelial cells, CR2 in, 290 Epstein-Barr virus/C3d receptor (CFi2) B-lymphocyte, structure and function of, 273-275 immunochemical analysis of, 275276 Epstein-Barr virus (EBV) gp350/220envelope proteins of, 284-285 receptor for, and C3d receptor, functional relationship of, 274

INDEX

Eui-1 encoding of evolutionarily well-conserved zinc protein by, 149-151 identification of in AKXD myeloid tumors, 145-151 retroviral integration in murine myeloid tumors in, 141-155 locus of, proviruses integrated in, location and orientation of, 149 mapping of, to mouse chromosome 3, 147,148 rearrangements of, detection of, in primary NFSiN myeloid tumors and cell lines, 147 relationship of, to other zinc-finger proteins, 151-153 transcription of, activation of, by viral integration in Fim-3, 154 Extravasation in metastatic process, 176-177

F Fatty acylation in membrane localization of yeast RAS proteins, 97-98 Fim-3, activation of transcription of Eci-1 by viral integration in, 154 Follicular dendritic cells, CFU in, 290

Genes, ras, 79-131; see also ras genes Genetics cancer and, 25-58 cancer predisposition and, 29-36 molecular, of predisposition to retinoblastoma, 31-36 Genome imprinting DNA methylation and, 16-19 in epigenetic allele inactivation, 50 in familial Wilms’ tumor, 52-54 in mixed cancers, 55-56 tumor progression and, 56-58 Glioblastomas, progression of, loss of heterozygosity and, 38-40 Glucose, Ras activity and, 122- 125

Glutamate dehydrogenase as target of CAMP-dependen t protein kinase, 116- 117 Glycogen phosphorylase as target of CAMP-dependent protein kinase, 114-1 15 Glycogen synthase as target of CAMP-dependent protein kinase, 113-114,115 Granulocyte-macrophage CSF (GM-CSF), lymphohematopoiesis and, 253 Growth of human tumor cells, stimulation of, autocrine, 226-231 inhibition of, 229-231 of normal human cells in uitro, 214-215 Growth control proteins in, as target of CAMP-dependent protein kinase, 120-122 Ras-CAMP pathway and, 129-131 Growth factor-antagonistic peptides in autocrine growth stimulation inhibition, 230 Growth factor independence of human tumor cells from metastatic lesions, 224-226 Growth factor receptors, expression of, by melanoma and colorectal carcinoma cells, 227-229 Growth factors, secretion of, by melanoma and colorectal carcinoma cells, 227-229 Growth-regulatory factors, 213-231 GTB-binding proteins reIated to ras genes, 79-81 GTP binding and hydrolysis, yeast Ras proteins in, 91-95

Heterozygosity, loss of in mixed cancer, 42-49,55-56 tumor progression and, 37-41 Host-parasite relationships, concomitant immunity in, 319-329

337

INDEX

Hybrids, proliferation potential of from diploid cell fusions, 68-70 from immortal cell line fusions with each other, 72-73 long-term, 68-73 from normal with immortal cell fusion, 70-71

I Immune deficiency(ies) acquired, tumors associated with, 308-31 1 immune surveillance of tumors and, 313-315 opportunistic tumors of, 301-315 states of, 302-303 Immune surveillance of tumors, immune deficiency and, 313-315 Immunity, concomitant, in host-parasite relationships, 319-329; see also Concomitant immunity Immunodeficiency disease, combined, opportunistic tumors associated with, 305-306 In situ carcinomas, progression of, to invasive carcinoma, inefficiency of, 164 Infantile X-linked agammaglobulinemia, opportunistic tumors associated with, 306 Infections, opportunistic, 303-304 Inositol phospholipid synthesis as target of CAMP-dependentprotein kinase, 121-122 Interleukin-1 (IL-l), lymphohematopoiesis and, 253 Interleukin-3 (IL-3), lymphohematopoiesis and, 253 Interleukin-4 (IL-4), effects of, on lymphohematopoietic cells, 260 Interleukin-6 (IL-6), lymphohematopoiesis and, 254 Interleukin-7 (IL-7),254-256 lymphohematopoiesis and, 252 Intravasation failure of, metastatic inefficiency and, 195 in metastatic process, 169-171

Invasion in metastatic process, 161-169 delay of, mechanisms of, 166-169 inefficiency of, in in situ cancers, 164-166 mechanisms of, 161-164 I R A 1 in Ras-CAMP pathway, 110

K Kaposi’s sarcoma AIDS and, 309 renal transplantation and, 307

L Leishmania major infection, mice with, concomitant immunity in, 323-325 Lymphocytes in bone marrow, 242-245 stromal cells and, recognition and adhesion between, 250-252 Lymphohematopoiesis in bone marrow, cytokines affecting, 252-254 Lymphoid cells, CR2 in, 287-289 Lymphoma(s) AIDS and, 310 renal tansplantation and, 308-309 Lymphopoietic microenvironent in bone marrow, 235-264

Macrophages in microenvironment of bone marrow, 246-247 Malformations, developmental, tumors and, 42-46 Melanocytic cells, human anchorage-independent growth of, in soft agar, 218-219 chromosomal abnormalities of, 217-218 life span of, 217 as model for tumor progression studies, 216-224 morphology of, 217 phenotypic characteristics of, 217-224

338

INDEX

Melanoma, growth factor secretion and growth factor receptor expression by, 227-229 Membrane localization of yeast Ras proteins, 97-101 Metastasis capacity for, tumorigenicity and, 194 inefficiency of, 159-203 consequences of, 199-202 documentation of, 160-161 molecular biology of, 187-198 random and nonrandom events in, 178-187 clonal dominance and, 183, 186-187 dynamic heterogeneity and, 183, 184-185 transient metastatic compartment and, 182-184 metastatic subpopulations in, 178-187 process of, 161-177 arrest of cancer cells in, 172-176 extravasation in, 176-177 intravasation in, 169-171 invasion in, 161-169; see also Invasion in metastatic process neovascularization in, 177 Metastatic phenotype, metastatic inefficiency and, 196 Metastatic subpopulations, 178-187 Methylation DNA, in cancer, 1-19; see also DNA methylation signal for, transduction of, 4-7 Molecular genetics of predisposition to retinoblastoma, 31-36 Mouse strains, RI, as model for identifying novel genes, 143-145; see also RI mouses strains mRNA isolated from normal cells in microinjection experiments on cellular immortalization, 73-74 Murine myeloid tumors, retroviral integration in, 141-155 myc gene family in metasatasis, 191-192

Neoplastic disease, zinc-finger proteins impiicated in, 154

Neovascularization in metastatic process, 177 Neutrophil polymorphs, invasion by cancer and, 163 Nutrients, sufficiency of, ras gene response to, 125-127

Oncogenes H-ras, effects of, on normal cells in microinjection experiments on cellular immortalization, 74-75 Oncogenes in metastasis, 189-192 Oogenesis, DNA methylation during, 14-15 Opportunistic infections, 303-304 Opportunistic tumors, 304-31 1 associated with primary immune deficiencies, 305-307 distinctive features of, 311 of immune deficiency, 301-315 spontaneous regression of, 311-312 Organ transplantation, tumors associated with, 308-309 Osteosarcoma, retinoblastoma and, 35-36

Peptide growth factors, active immunization against, in autocrine growth stimulation inhibition, 230-231 Phosphodiesterases in Ras-CAMP pathway, 107 Phosphofructokinase-2 as target of CAMP-dependent protein kinase, 115-116 Phosphorylation, yeast RAS proteins and, 97 Predisposition cancer epigenetic allele inactivation and, 51-56 genetics and, 29-36 to retinoblastoma, molecular genetics of, 31-36 Proliferation potential of hybrids, long-term, 68-73

INDEX

Protein(s) in carbon metabolism as target of CAMP-dependentprotein kinase, 112-1 18 in growth control as target of CAMP-dependentprotein kinase, 120-122 GTB-binding proteins related to rus genes, 79-81 zinc-finger evolutionarily well-conserved, encoding of, by Eui-l,l49-151 implicated in neoplastic disease, 154 relationship ofEui-1 to, 151-153 Protein kinase, CAMP-dependent, in Ras-CAMP pathway, 105-107 Protein phosphatases in Ras-CAMP pathway, 107-108 Proteolytic cleavage in membrane localization of yeast Ras proteins, 99

Radioautographic techniques for investigating lymphopoiesis in bone marrow, 237-238 Has-CAMP pathway adenylate cyclase in, 101-105 CAMP-dependent protein kinase in, 105-107 CASl in, 110 CDC25 in, 108-110 components of, 101-111 feedback regulation of, 118-120 functions of, 128-131 genes of, 84 growth control and, 129-131 I R A 1 in, 110 phosphodiesterases in, 107 protein phosphatases in, 107-108 SCH9 in, 110 YAK1 in, 110 in yeast, 82-83 ras genes GTB-binding proteins related to, 79-81 in Saccharomyces cerevisiae, 79-131 signals triggering, 122-128 in yeast; see also Yeast Ras proteins

339

ras oncogenes in metastasis, 190-191 RASl expression of, 89 functions of, 87-88 RAs2 expression of, 89 functions of, 87-88 RbZ locus, mutation at, in retinoblastoma and osteosarcoma, 34 Recombinant-DNA technology in investigating lymphopoiesis in bone marrow, 2389 Renal transplantation, tumors associated with, 308-309 Repair processes, invasive delay and, 166-167 Restriction-fragment-length polymorphisms (PFLP) in retinoblastoma, 32-34 Retinoblastoma cytogenetics of, 30-31 epigenetic inactivation of alleles in, 49-51 predisposition to, molecular genetics of, 31-36 Retroviral integration in murine myeloid tumors, 141-155 Rhabdomyomatous variant of Wilms’ tumor, loss of heterozygosity in, 47-48 Rhabdomyosarcoma,loss of heterozygosity and, 45-46 RI mouse strains AKXD strains of, 143-151; see also AKXD RI mouse strains as models for identifying novel genes, 143145 for study of molecular genetic basis of neoplastic disease, 143

Saccharomyces cereuisiae, ras genes in,

79-131 SCH9 in Ras-CAMP pathway, 110 Schistosoma japonicum infection, mice with, concomitant immunity in, 325-326

340

INDEX

Schistosoma mansoni infection, mice with, concomitant immunity in, 325-326 Sjogren's syndrome, tumors associated with, 308 Soluble receptors for competition with cell surface receptors in autocrine growth stimulation inhibition, 230 Spermataogenesis, DNA methylation during, 14-15 Stromal cells in bone marrow, 247-250 cytokine responses involving, 259-261 lymphocytes and, recognition and adhesion between, 250-252 Stromal reaction of tumors, 312-313 Sugar-transport systems as target of CAMP-dependent protein kinase, 117 SV40 T antigen, effects of, on normal cells in microinjection experiments on cellular immortalization, 75 Swiss-type agammaglobulinemia, opportunistic tumors associated with, 305-306 Systemic lupus erythematosus (SLE), tumors associated with, 308

T T lymphocytes, CR2 in, 289 Taeniae taeniaefomis infection, mice with, concomitant immunity in,

320-322 Thymocytes, CR2 in, 289 Transcription factors as target of CAMP-dependent protein kinase,

120- 121

Triton tumor, loss of heterozygosity in,

49 Tumor(s) associated with acquired immunodeficiencies, 308-31 1 developmental malformations and,

42-46 diversification of, DNA methylation in,

13-14 growth excess disorders and, 46 immune deficiency and immune surveillance of, 313-315 murine myeloid, retroviral integration in, 141-155 opportunistic, 304-311 of immune deficiency, 301-315; see also Opportunistic tumors with phenotypically distinct elements,

46-49 progression of, loss of heterozygosity and, 37-41 stromal reaction of, 312-313 Tumor cells autocrine growth stimulation of,

226-231 inhibition of, 229-231 DNA methylation in, 7-8 growth factor independence of, from metastatic lesions, 224-26 Tumor progression, studies on, human melanocytic cells as model for,

2 16-224 Tumor suppressor genes, metastasis and,

193 Tumor tissue, uncultured, methylation in, 9-11 Tumorigenicity, metastatic capacity and,

194

Transforming growth factors-p (TGF-P),

257-259 lymphohematopoiesis and, 252 Transient metastatic compartment, randomness and selection in metastasis and, 182-184 Transplantation, organ, tumors associated with, 308-309 Trehalose as target of CAMP-dependent protein kinase, 112-113 Trehalose synthase as target of CAMP-dependent protein kinase,

112-113

Uterine cervix, carcinoma of, progression of, from in situ to invasion, 164

Variable domains of yeast Ras proteins, 96

34 1

INDEX

Wilms’ tumor familial, genome imprinting in, 52-54 rhabdomyomatous variant of, loss of heterozygosity in, 47-48 Wilms’ tumor cells, chromosomal mechanisms of, 44-45 Wiskott-Aldrich syndrome, opportunistic tumors associated with, 306-307

X X-linked lymphoproliferative (XLP) syndrome, opportunistic tumors associated with, 307

Y YAK1 in Ras-CAMP pathway, 110 as target of CAMP-dependent protein kinase, 122 Yeast, M S genes in, 79-131; see also rus genes Yeast Ras proteins, 86-101 activity of, processing and control of, 101

caroxyl-terminal modification of, 91-101 function of, model for, 82-86 functional domains of, 89-97 antibody epitopes and, 95 effector, 95-96 GTP binding and hydrolysis, 91-95 phosphorylation, 97 variable, 96 membrane localization of, 97-101 C-terminal methyl esterification in, 99 fatty acylation in, 97-98 identification of genes involved in RAS protein maturation in, 99-101 proteolytic cleavage in, 99 Rasl as, 87-89 Rase as, 87-89

Z Zinc-finger proteins evolutionarily well-conserved, encoding of, by Eui-l,149-151 implicated in neoplastic disease, 154 relationship of Eui-f to, 151-153

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