Genetic Recombination in Cancer

Genetic Recombination in Cancer

To Shri Sai Baba

Genetic Recombination in Cancer Gajanan V. Sherbet Communications and Signal Processing Research Group, School of Electrical, Electronic and Computer Engineering, University of Newcastle upon Tyne, UK Institute for Molecular Medicine, Huntington Beach, California, USA

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This book is printed on acid-free paper Copyright 2003, Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press An Imprint of Elsevier 84 Theobald’s Road, London WC1X 8RR, UK http://www.academicpress.com Academic Press An Imprint of Elsevier 525 B Street, Suite 1900 San Diego, California 92101-4495, USA http://www.academicpress.com ISBN 0–12–639881-X Library of Congress Catalog Number: 2003102994 A catalogue record for this book is available from the British Library Composition by Genesis Typesetting Limited, Rochester, Kent Printed and bound in Great Britain by Biddles Ltd, Guildford and Kings Lynn 03 04 05 06 07 9 8 7 6 5 4 3 2 1

Contents

Preface

ix

Abbreviations

xi

1 Introduction

1

2 Genetic integrity, DNA repair and recombination Base excision and nucleotide excision repair system RAD genes and their operation in DNA replication checkpoint DNA recombination repair pathway VDJ recombination, the immune system and haematological malignancy SET domain proteins in genetic transcription, growth regulation and myeloma Control of cell proliferation by SET domain proteins The generation of chimeric transcripts of NSD family genes in multiple myeloma and Wolf–Hirschhorn syndrome Ku protein in cell function Regulation of transcription by Ku protein Ku protein and DNA repair Ku protein in the maintenance of chromosomal integrity Ku protein in cell cycle progression and differentiation Ku protein in cancer

3 5 5 8

3 Replication error (RER) and genetic instability Microsatellite instability and cancer progression Effects of microsatellite instability on p53 and growth factor gene expression

9 12 12 14 15 17 18 19 19 21 24 25 28

vi

Contents

Influence of microsatellite instability on invasive behaviour of tumours Microsatellite instability and metastasis suppressor nm23 gene abnormalities Chromosomal fragile sites and the RER phenotype Sister chromatid recombination and fragile sites Microsatellite instability and sister chromatid recombination Genomic stability and chromosome structural dynamics 4 DNA repeats, genetic recombination and the pathogenesis of genetic disorders CAG repeat expansion and genetic instability Microsatellite dinucleotide repeats in insulin-dependent diabetes mellitus and autoimmune thyroid disease Alu repeats in genetic recombination Intrachromosomal recombination in muscular dystrophy and Alzheimer’s disease mediated by Alu repeats Intrachromosomal recombination in tumour suppressor genes in cancer Alu-mediated alterations of cell cycle regulator genes Alu-mediated interchromosomal recombination in leukaemias Alu-mediated interchromosomal recombination in Ewing sarcoma Alu repeats, DNA mismatch repair gene mutation and genomic instability Alu elements in genetic transcription Mode of Alu function in recombination Functional involvement of Alu with signal recognition particles LINE repeats in genetic rearrangement Cognate elements associated with recombination Translin-binding elements Chi sequences in DNA repair and recombination

31 33 34 35 39 39

41 42 49 50 51 52 55 55 56 57 57 58 61 62 64 64 65

5 Chromosomal recombination in cancer Karyotypic abnormalities in cancer DNA aneuploidy and cancer progression DNA ploidy, cell proliferation and growth factor expression Association of DNA ploidy with p53 abnormalities

67 67 68 70 75

6 Chromosomal translocation and its phenotypic effects Chromosomal translocation and signal transduction Deregulation of notch signalling by chromosomal translocation Loss of integrity of notch signalling in leukaemia and lymphoma cells

76 76 77 83

Contents

Genetic abnormalities downstream of notch signal activation Fibulins, notch signalling and cancer invasion Chromosomal translocation and the dynamics of cell population expansion in cancer The modulation of cell proliferation-related gene expression by chromosomal rearrangement Genetic rearrangement and the regulation of apoptosis by bcl2 gene family Bcl2 in p53-induced apoptosis T-cell leukaemia/lymphoma (TCL1) gene in apoptosis The PI-3 kinase/Akt pathway of signal transduction in cell proliferation, apoptosis and differentiation Akt in PTEN tumour suppressor function Akt signalling in cancer growth and invasion Akt in TCL1 function Fusion oncoprotein signalling by PI-3 kinase/Akt pathway Mechanism of transduction of signals by Akt Chromosomal translocation and genetic transcription Chromosomal translocation in synovial sarcomas AML1 fusion proteins in haematopoietic neoplasms Modulation of retinoic acid receptor function by chromosomal translocation TEL transcription-related gene rearrangement in leukaemia and lymphoma Modulation of developmental and transcription factor PAX genes by translocation The role of HOX genes in development, differentiation and neoplasia Chromosomal translocation, fusion proteins and tumour vascularisation Chromosomal translocations, fusion proteins and immunity The association of chromosomal translocation with fragile sites Chromosomal fragility, genetic loss and tumour suppressor genes Fragile histidine triad (FHIT) gene abnormalities in cancer Tumour suppressor function of von Hippel–Lindau gene The ras association domain family RASSF1 gene in tumour suppression Gene amplification and its relationship to genetic instability Chromosomal fragility and micronuclei DNA damage and the generation of micronuclei The involvement of p53 in the formation of micronuclei Centromeric instability, DNA methylation and micronuclei in ICF syndrome The nature and constitution of micronuclei

vii

85 87 89 90 91 92 95 96 97 97 100 100 103 104 105 106 108 109 111 113 117 119 120 121 122 124 126 126 128 128 129 130 131

viii

Contents

7 DNA methylation and genetic instability DNA methylation in normal and neoplastic development Abnormal DNA methylation in ICF and Rett syndromes DNA methylation and genetic instability The CpG Island Methylator Phenotype (CIMP) Influence of DNA methylation on recombination Methylation-associated chromosomal recombination and genetic transcription in childhood leukaemias The dynamics of histone acetylation in cell differentiation, cell proliferation and neoplasia Histone acetylation dynamics in development and differentiation Cell proliferation, apoptosis and histone acetylation dynamics Histone acetylation dynamics and the neoplastic process Synergistic effects of DNA methylation and histone acetylation dynamics on epigenetic gene silencing HDAC inhibitors in the treatment of cancer The effects of p53 methylation on chromosome stability DNA methylation in fragile X syndrome FMR1 gene function in the pathogenesis of fragile X syndrome Epigenetic silencing of FMR1 in fragile X syndrome

133 133 134 135 136 139

8 Telomeric DNA and genetic instability Telomeres and cell proliferation, apoptosis and cell senescence Telomeres in DNA repair Telomere function and ATM kinase Telomeric association The incidence of telomeric association in cancer Telomeric association and gene expression Telomere position effect

154 154 158 158 159 159 161 162

Epilogue

164

References

170

Index

237

140 140 141 143 144 147 148 149 151 151 152

Preface

The generation of genetic mosaicism has mystified, moved and exercised the minds of many. In recent decades advances have been made that have significantly contributed to our understanding of how genetic alterations might generate cellular diversity together with the diversity of cellular behaviour so characteristically displayed by neoplastic cells. Not only has an array of alterations been identified but also a larger picture has emerged of genetic recombination as an important means of producing the panoply of phenotypic properties. This is the general compass of coverage in this book and therefore it is in the nature of things that the discussion should cover embryological phenomena of cell differentiation and morphogenesis, neoplastic disease and its progression as well as a number of genetic disorders, through which runs the common strand of genetic instability and variation. I have described, at various times and in various publications, the evolution of thought relating to tumorigenesis, the appearance of genetic mosaicism within tumours, and the evolution of the various components of the tumour into cell types with diverse biological properties of enhanced cell proliferation, invasion and metastatic deposition at sites distant from the focus of tumour initiation, and the role played by aberrant signalling in these processes (Sherbet, 1978, 1982, 1987, 2001; Sherbet and Lakshmi, 1997). But in none of these works have I afforded myself an opportunity to discuss in detail the role of genetic recombination in the generation of intratumoral diversity in the form of cell variants that impinge greatly upon the growth and dissemination of the tumour. Here I have attempted to do this, but have been, not inordinately, mindful of the enormity of the task that I have ventured upon. So this book does not deal with the mechanics and mechanisms of genetic recombination but focuses da capo el fine on the biological manifestation and phenotypic consequences of recombination.

x

Preface

I am grateful to Dr MS Lakshmi for a critical reading of the manuscript and making helpful suggestions and for providing the Tamil quotation and translation. I would like to record my gratitude to Professor Oliver Hinton for facilitating my research and providing much help and encouragement. Ms Judy Preece of the Audio Visual Centre prepared the figures. I thank her sincerely for her help. Academic Press have always been very receptive to my ideas. They received this book with great enthusiasm. I greatly appreciate their help, cooperation and support. It has given me much pleasure working with them on this project. G.V. Sherbet October 2002

Sherbet GV. (1978). The biophysical characterisation of the cell surface. Academic Press, London. Sherbet GV. (1982). The biology of tumour malignancy. Academic Press, London. Sherbet GV. (1987). The metastatic spread of cancer. Macmillan, Basingstoke. Sherbet GV. (2001). Calcium signalling in cancer. CRC Press, Boca Raton, FL, USA. Sherbet GV and Lakshmi MS. (1997). The genetics of cancer. Academic Press, London.

Abbreviations

3-AB ABL ALL AML ANN APC APL AR AT ATM Bcl2 BH BER bHLH bp BS cAMP CBF CCNU Cdk CFTR CHD CIMP CML CREB CTL DCC

3-aminobenzamide Acute biphenotypic leukaemia Acute lymphoblastic leukaemia Acute myeloid leukaemia Artificial neural network Adenomatous polyposis coli [gene] Acute promyelocytic leukaemia Androgen receptor Ataxia telangiectasia Ataxia telangiectasia mutated [protein] B-cell leukaemia/lymphoma-2 [genes and proteins] Bcl2 homology [domain] Base excision repair Basic helix-loop-helix base pair Bloom’s syndrome Cyclic adenosine monophosphate Core-binding factor 1-(2-chloroethyl)-3-cyclohexyl-nitrosourea Cyclin-dependent kinase Cystic fibrosis transmembrane conductance regulator [gene] Chrome domain CpG island methylator phenotype Chronic myelogenous leukaemia Cyclic AMP response element binding protein Cytotoxic T-lymphocyte Deleted in colon carcinoma [gene]

xii

DHFR DM DMSO DNA-PK DNA-PKc DNMT DRPLA DSB DSBR EBNA EBV ECM EGFr ENU ER ERK FA FANCA FAP FGF FGFr FHIT FMR FNA HAT HBV HCC HDAC HIF HIV HJR HMBA HNPCC HPV HSP HSR HTLV ICF ICM IDDM IFN IGF IL

Abbreviations

Dihydrofolate reductase Double minute [chromosome] Dimethylsulfoxide DNA-dependent protein kinase DNA-PK catalytic subunit DNA methyltransferase Dentatorubral-palliodoluysian atrophy [syndrome] Double strand break [in DNA] Double strand break repair EBV nuclear antigen Epstein–Barr virus Extracellular matrix Epidermal growth factor receptor Ethylnitrosourea Oestrogen receptor Extracellular signal-regulated kinase Fanconi anaemia FA complementation group A [gene] Familial adenomatous polyposis Fibroblast growth factor Fibroblast growth factor receptor Fragile histidine triad [gene] Fragile X mental retardation [gene] Fine needle aspirates Histone acetyl transferase Hepatitis B virus Hepatocellular carcinoma Histone deacetylase Hypoxia inducible factor Human immunodeficiency virus Holliday junction resolvase Hexamethylene bisacetamide Hereditary non-polyposis colon cancer Human papilloma virus Heat shock protein Homogeneously staining region [of chromosomes] Human leukaemia/lymphoma virus Immunodeficiency-centromere instability-facial anomalies [syndrome] Image cytometry Insulin-dependent diabetes mellitus Interferon Insulin-like growth factor Interleukin

Abbreviations

IUdR JAK kb LINE LOH LTR MAPK MECP MJD MLL MMP MMS MN MNU NBS NDP NE NER NGF NOS NSCLC OPMD PARP PBL PCR PDGFr PgR PHA PHD PI PLZF PML PSA RA RAG RAR RCC RER RSV RTK RTS SBMA SCA

Iododeoxyuridine Janus tyrosine kinase kilobase Long interspersed nuclear elements Loss of heterozygosity Long terminal repeat Mitogen-activated protein kinase Methyl-CpG-binding protein [2] Machado–Joseph disease (SCA3) Mixed lineage leukaemia [gene] Matrix metalloproteinase Methyl methane sulphonate micronucleus Methylnitrosourea Nijmegen breakage syndrome Nucleoside diphosphate Neuroendocrine Nucleotide excision repair Nerve growth factor Nitric oxide synthase Non-small cell lung carcinoma Oculopharyngeal muscular dystrophy Poly (ADP-ribose) polymerase Peripheral blood lymphocytes Polymerase chain reaction Platelet derived growth factor receptor Progesterone receptor Phytohaemagglutinin Plant homeodomain [motif] Phosphatidyl inositol Promyelocytic leukaemia zinc finger [protein] Promyelocytic leukaemia Prostate specific antigen Retinoic acid Recombination activating genes [1 and 2] Retinoic acid receptor Renal cell carcinoma Replication error [phenotype] Rous sarcoma virus Receptor tyrosine kinase Rett syndrome [X-linked disorder] Spinobulbar muscular atrophy [X-linked recessive] Kennedy’s disease Spinocerebellar ataxia [syndrome]

xiii

xiv

SCE SCID SCR SH SINE SMC SPF SRP STAT TAS TBP TCL TCR TGF TLC TNF TPE UC VD3 VDJ VDRE VEGF VHL WHS WS XP

Abbreviations

Sister chromatid exchange [SCR] Severe combined immunodeficiency [syndrome] Sister chromatid recombination [SCE] Src-homology [domain] Short interspersed nuclear elements Structural maintenance of chromosomes [proteins] S-phase fraction of the cell cycle Signal recognition particle Signal transducer and activator of transcription Telomeric association TATA-binding protein T-cell leukaemia/lymphoma [gene] T-cell receptor Transforming growth factor Telomerase RNA component [gene] Tumour necrosis factor Telomere position effect Ulcerative colitis Vitamin D3 V (variable), D (diversity) and J (joining) recombination Vitamin D3 response element Vascular endothelial growth factor von Hippel–Lindau [gene] Wolf–Hirschhorn [syndrome] Werner’s syndrome Xeroderma pigmentosum

1 Introduction

Vigorous action is expedient when good can flow from it, If not, open another path. Thiru Valluvar (Tamil Poet, second century, India) Thirukkural, chapter 68, verse 673)

Genetic recombination, as the term implies, is a process of generation of combinations of genes from parental cells that leads to the generation of cell variants possessing characteristic phenotypically distinct cell types. Having generated genetic diversity, it is essential that its integrity is maintained. Both these processes are therefore of fundamental importance in the evolution of species as well as in embryonic development and differentiation. Their importance extends also to pathogenesis of disease. The generation of cell variants possessing different genetic traits and varying, often abnormal, cell phenotypes is an important element in the initiation and development of cancer and its progression to the metastatic state. The loss of genetic integrity also seems to be closely associated with aberrant cell differentiation and the pathogenesis of neoplastic disease. Genetic Recombination in Cancer ISBN 0-12-639881-X

Copyright © 2003 Elsevier Science Ltd All rights of reproduction in any form reserved

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Genetic Recombination in Cancer

One can identify several pathways of evolution of cellular phenotypes and these often correspond with specific events in the biology of the cell. Genetic recombination occurs during mitotic and meiotic cell division. Often gross events such as chromosomal recombination and karyotypic change can be seen to occur in the process of carcinogenesis. Abnormal genetic recombination and inability of the cellular genome to repair genetic damage not only lead to the generation of abnormal phenotypes, but also, as a part of an insidious circle of events, deregulate the progression of the cell cycle and lead to the perpetuation of the formation of abnormal cells. Such deregulation also affects apoptotic destruction of cells and this in turn would lead to an abnormal expansion of a cell population. Finally, the appearance of drug-resistant variant clones of cancer cells and metastatic variants seems to be linked sufficiently closely to suggest that investigation of genetic instability has deep implications for disease progression and therefore for designing appropriate strategies of treatment. I hope that the discussion in the following pages will demonstrate how genetic alterations have been or could be employed to elicit clinically useful information. For, as the above-quoted Tamil Thirukkural would imply, this mode of approach to the seemingly intractable problem should be tested vigorously and aggressively for the potential benefit of patients. It is needless to say that new avenues would need to be opened, however profound the scientific advances might be, if tangible benefits do not flow from the inquiries.

2 Genetic integrity, DNA repair and recombination

The ability to maintain the integrity of the genetic material is a vital and fundamental requirement of cell survival. In general evolutionary terms, a total maintenance of integrity and genetic stability would run counter to the evolutionary advantage that can be derived only by a degree of genetic instability. Genetic instability could be viewed as a mechanism that provides a means of achieving genetic diversity and confers evolutionary advantage. The cell and the organism as a whole have therefore to balance these opposing requirements. The cellular DNA is constantly subjected to damage and the cell has therefore evolved a number of DNA repair mechanisms. On the other hand, the cell has evolved several ways by which potential evolutionary advantage accrues from an inherent instability of the genetic material, which purely on thermodynamic properties can undergo replication and recombination. It is this provision that leads to the generation of variation, regulation of the cell cycle and the associated process of apoptosis. Genetic recombination can occur in both mitotic and meiotic cycles of cell division. The recombination of homologous DNA sequences is a ubiquitous process, which was once believed to be a random and non-specific event. However, there is a large body of evidence that suggests that homologous recombination is indeed a highly regulated genetic feature. The genome contains recombination hotspots, and certain DNA repeat elements have been Genetic Recombination in Cancer ISBN 0-12-639881-X

Copyright © 2003 Elsevier Science Ltd All rights of reproduction in any form reserved

4

Genetic Recombination in Cancer

identified as hotspots of recombination. However, not all of them show comparable levels of recombination. Furthermore, recombination frequency shows a marked enhancement during meiosis. Recombination involves the crossover or a reciprocal exchange caused by the breakage and subsequent rejoining of parental DNA strands. A second type of recombination leads to gene conversion. Both these are explained by the Holliday crossover concept (see below). The major area of focus here is the mode of implication of genetic instability in the pathogenesis of cancer. The initiation, development and growth of neoplasms, and their progression as evidenced by the transition of the localised lesions into the invasive phase, and the further step in transition to the metastatic state, all show phenotypic changes that can be linked with genetic instability, alterations in or deregulation of cell cycle control and apoptotic control of population expansion. Genetic instability can be seen in the form of karyotypic changes, which are often a prelude to cell immortalisation. The DNA repair process itself can be a source of chromosomal rearrangement. The incision of DNA and the presence of bulky lesions can themselves be conducive to and promote chromosomal reorganisation. The efficiency of DNA repair is another factor that enters the picture; less efficient repair could make cells more prone to genetic recombination. This could lead to an increase in the rate of generation of cell variants with altered phenotypic properties. Genetic instability is closely associated with predisposition to cancer, the loss of tumour suppressor genes, or inappropriate expression of genes in a spatial and temporal fashion. Such genetic changes inevitably lead to cell transformation and development of neoplasms, together with abnormalities of cell cycle progression and control of apoptosis, which both determine expansive growth of the primary neoplasm and conceivably lead also into the invasive phase of cancer progression. When cells are proliferating and replicating DNA, errors might be introduced in the replicated DNA. If the damage were to persevere, cellular physiology could go awry, and mutations might accumulate that could lead to cellular abnormalities. Several modes of DNA repair have been recognised. The cell possesses systems that continually scan for damage and repair it. Prominent among these are mismatch repair, base excision repair and nucleotide excision repair. The mismatch repair system is involved in the scanning of newly synthesised DNA for errors made in copying. Incorrectly paired bases are excised, and correct bases are inserted to fill the gap. The investigations of mismatch repair in lower organisms have inspired active efforts to understand the importance of mismatch repair in humans and its involvement in the pathogenesis of human disease. Mismatch repair genes have come into sharp focus, with the identification of some of them with the development of hereditary non-polyposis colon cancer (HNPCC). Overall, human mismatch repair has turned out to be far more complex than in bacteria.

Genetic integrity, DNA repair and recombination

5

Base excision and nucleotide excision repair system The nature of DNA damage and its extent determines which repair pathway is activated. The damage to DNA, sustained by oxidation and other chemical insults, is repaired by the excision repair pathway. This mode of repair removes the damaged base or nucleotide and replaces it by using the complementary DNA strand as a template. In base excision repair (BER), the damaged base is released by a DNA glycosylase and then the AP site is excised by an AP endonuclease. The nucleotide excision repair (NER) pathway recognises bulky lesions in the DNA that are induced by UV irradiation, free oxygen radicals, and a variety of chemical carcinogens and chemotherapeutic agents. An enzyme system hydrolyses the phosphodiesterase bonds on either end of the lesion and the oligonucleotide is excised from the DNA. The excised oligonucleotides are released and the gap is filled in and appropriately ligated. The nucleases taking part here are called excision nucleases or excinucleases. The genetic condition called xeroderma pigmentosum is characterised by a lack of NER and enhanced sensitivity to UV exposure together with a propensity to develop skin cancer. The excinucleases are also capable of repairing O6-methyl guanine and other methylated bases. Three DNA repair proteins, UvrA, UvrB and UvrC, have been identified in Escherichia coli. Their participation in NER is defined in a series of interactions involving, in the first place, the recognition of DNA damage by UvrA. It forms a complex with UvrB. The UvrA then binds to the DNA lesion, unwinds the DNA and changes the conformation of UvrB to enable it to bind to the lesion. UvrA now dissociates from the DNA–UvrB complex. The binding of UvrC to the DNA–UvrB complex is the next event. This leads UvrB to make a 3’ incision, which alters the conformation state of the complex and in turn leads UvrC to make the 5’ incision. A helicase releases the excised oligonucleotide and the gap generated by the excision is filled and ligated. Several human homologous proteins that serve similar functions have been identified. Of the human homologues, XPA is damage recognition protein, which functions in consort with XPF-ERCC1 and RPA. XPB and XPD are helicases. These proteins are subunits of the transcription factor TFIIH. Besides these, TFIIH also contains other DNA repair proteins. Indeed TFIIH is a transcription factor complex and is also involved in the regulation of the cell cycle.

RAD genes and their operation in DNA replication checkpoint The cell must repair the damaged DNA before it is replicated. The maintenance of genomic integrity requires that cells are prevented from entering into mitotic or meiotic division when DNA replication is incomplete. This control of cell cycle progression operates by the institution of DNA replication checkpoints.

6

Genetic Recombination in Cancer

Three DNA damage surveillance checkpoints have been identified and defined. These are the G1 –S transition checkpoint, which restrains cells with damaged DNA from entering the S-phase. The second checkpoint, namely the S-phase checkpoint, monitors the progress of cells through the S-phase and regulates the rate of DNA synthesis. The third checkpoint monitors the G2M boundary (Paulovich and Hartwell, 1995; Paulovich, Toczyski et al., 1997; Weinert, 1998). A family of genes called the RAD genes of Saccharomyces cerevisiae are closely associated with DNA replication checkpoints and with the control of rate of DNA replication. They are required for the repair of DNA double strand breaks (DSB) by homologous recombination. Their inactivation leads hypersensitivity of cells to agents that induce DSBs and to defective recombination. Some RAD genes could be involved also in base excision repair. The RAD proteins are homologues of human excision repair proteins, and have been shown also to interact with cell cycle control p53 protein, as well as with other tumour suppressor proteins. The p53 suppressor gene is mutated in a great majority of human neoplasms. When DNA sustains damage, p53 is induced to express and this results in the restraint of cells from entering into the S-phase. Mekeel et al. (1997) reported that mutations of p53 or p53 (-/-) state resulted in a greatly increased frequency of homologous recombination. Inactivation of p53 function by SV40 large T-antigen or by HPV E6 protein had similar effects on recombination. Obviously, p53 seems to be able to suppress recombination. Possibly, this is related to the interaction of p53 with RAD proteins, themselves capable of regulating the entry of cells into the S-phase and of monitoring the rate of replication. Whereas wild-type RAD cells show tardy S-phase progression in response to DNA damage, RAD mutants show enhanced replication (Paulovich, Margulies et al., 1997). Much evidence for RAD involvement in the regulation of cell cycle progression has come from another line of study. Cell proliferation is associated with a continual erosion of telomeric DNA repeats and this leads to replicative senescence and to apoptosis. The enzyme called telomerase, a ribonucleoprotein composed of telomerase RNA and a reverse transcriptase, restores and maintains telomere length of chromosomes and reinstates the capacity of cells to proliferate. Several reports have now appeared that RAD proteins can substitute for telomerase in its function of telomere maintenance. A progressive erosion of telomeres occurs in RAD mutant Schizosaccharomyces pombe and Arabidopsis thaliana (Dahlen et al., 1998; Gallego and White, 2001). RAD1 over-expression in S. pombe leads to an increase of telomere length, and its disruption decreases at the rate of around 1 nucleotide/generation. But telomere length is restored upon reintroduction of RAD1 (Dahlen et al., 1998). The rate of telomere length restoration is nowhere comparable with that achieved by telomerase, and it appears likely that other proteins besides RAD might be involved in the general phenomenon of telomere elongation occurring in the absence of telomerase. Indeed there might be differences in

Genetic integrity, DNA repair and recombination

7

the pathways of function of different RAD proteins themselves in respect of this function (Le et al., 1999). Overall, there is little doubt that recombination mediated by RAD genes is involved in maintaining telomere integrity and in this way also in cell proliferation and apoptosis. This cell cycle checkpoint pathway, a pathway that has been highly conserved in evolution, involving the operation of RAD proteins, has assumed considerable significance in relation to the development of cancer in populations that carry genetic susceptibility to cancer. Some RAD genes might be residing in the same loci or close to the locus occupied by tumour suppressor genes. Dean et al. (1998) cloned the human homologues of RAD1 and RAD17 genes of Schizosaccharomyces pombe and mapped them to loci that are known to harbour tumour suppressor genes. The BRCA1 and BRCA2 are breast cancer susceptibility genes. BRCA1 seems to make a significant contribution to cell differentiation and embryonic development. Most interesting aspect of the potential role of BRCA1 gene is that its expression might be cell cycle-related. Quite conceivably, the expression of BRCA1 might be linked to the switching of cells from proliferation to the pathway of differentiation and it might indeed regulate this switch (Sherbet and Lakshmi, 1997). Thompson et al. (1995) found that the transition of in situ breast cancer to the invasive phase was associated with a decrease in BRAC1 expression. A loss of heterozygosity of both BRCA1 and BRCA2 is said to be more frequent in metastatic tumour than in primary breast carcinoma (Hampl et al., 1996). The protein products of both BRCA1 and BRCA2 genes appear to function in conjunction with RAD51. A single nucleotide polymorphism in the 5’ untranslated region of RAD51 has been found to affect the development of breast or ovarian cancer in subjects carrying the susceptibility genes. Recently it has been recorded that RAD51- [135C] polymorphism affects BRCA2 carriers to a far greater extent than the carriers of BRCA1; the former show a greatly enhanced risk of developing breast and/or ovarian carcinoma (Levy-Lahad et al., 2001). Although it is conceivable that BRCA proteins might function in conjunction with RAD, one should be mindful of the possibility of other modes of function, as Yarden et al. (2002) showed, by activating kinases that regulate DNA damage-induced G2M checkpoint control of cell cycle progression. Bell, Wahrer et al. (1999) investigated the presence of germ-line mutations of RAD51, 52 and 54 in 100 cases with early onset of breast cancer and 15 human breast cancer cell lines. However, they found no relationship between earlyonset breast cancer and the presence of RAD mutations. Tseng et al. (2001) have reported to the contrary. They found RAD expression in normal breast tissue, but none in invasive carcinoma. When RAD was transfected into MDAMB345 cells that were RAD-negative, the transfectant acquired enhanced growth rate and colony formation in soft agar, clearly demonstrating an association between tumour cell growth and RAD expression. Another interesting finding by these authors is that the small proportion of breast

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Genetic Recombination in Cancer

cancers that continued to express RAD had disseminated to axillary lymph nodes. This suggestion of a relationship between RAD expression and metastatic spread is strengthened by the inhibition of RAD-mediated effects when there is concomitant expression of the putative metastasis suppressor nm23 gene. The provisional conclusion of this study is that the RAD protein is tumorigenic, but nm23 protein might interact with it and negate its perceived effects on supporting metastatic spread into the regional lymph nodes. The conclusion may be regarded as provisional simply because there are numerous studies that nm23 does not influence to any significant degree the metastatic spread of breast cancer; indeed only a few forms of human cancer show such an association. However, as noted earlier, this could be an effect on cell cycle progression, for the nm23/NDP kinase does negatively influence cell proliferation (Sherbet, 2001).

DNA recombination repair pathway Recombination involves the crossover or a reciprocal exchange caused by the breakage and subsequent rejoining of parental DNA strands. A second type of recombination leads to gene conversion. Both these are explained by the Holliday crossover concept. This postulates the formation of the Holliday intermediate, a heteroduplex formed by complementary strands of a pair of parental DNA molecules. These are joined at the so-called Holliday junction. The resolution of Holliday intermediate by endonucleases results in the crossing over of DNA regions flanking the junction, and further the postulate suggests gene conversion to be a result of mismatch of unpaired or mis-paired bases of the heteroduplex. The association between recombination and the formation of the Holliday intermediate and the joining of the heteroduplex at the Holliday junction has received general acceptance over the years. The crossover in recombination is a highly controlled process. A resolution of the Holliday junction can lead to crossover when double strand break repair (DSBR) is completed, but single strand gap repair does not. This is because the Holliday junction is recognised by a number of enzymes, namely the Holliday junction resolvases (HJR) and their complexes (Aravind et al., 2000; Cromie and Leach, 2000). The specificity of these enzyme complexes in controlling the resolution of the 4-way junctions appears to be an attribute of the positioning of the complex with Holliday junctions (Cromie and Leach, 2000). Among other agents associated with the process is the human papilloma virus (HPV) oncogenic protein E6. E6 binds DNA via its C-terminal zinc-binding domain. This domain is mainly responsible for the ability of E6 to recognise Holliday junctions (Ristriani et al., 2001). The mismatch repair protein MSH2 also can recognise and interact with Holliday junctions (Alani et al., 1997). An unstable genome is a characteristic feature of certain syndromes such as the Bloom’s syndrome (BS) and the Werner syndrome (WS). In both BS and WS,

Genetic integrity, DNA repair and recombination

9

mutations occur in BLM and WRN genes. The proteins encoded by these genes are human homologues of recQ helicases. RecQ is a component of the recF pathway of recombination and re-initiation of DNA replication after repair of any lesions. It follows that defects in the processes leading to recombination will entail severe genetic instability (Enomoto, 2001). Karow et al. (2000) have shown that BLM selectively binds Holliday junctions in vitro and might in this way inhibit hyper-recombination. The abnormalities associated with BLM and WRN are thus the primary source of genetic instability encountered in BS and WS.

VDJ recombination, the immune system and haematological malignancy Two recombination pathways, which are developmentally regulated, have been identified in the development of the immune system. These are the switch recombination and the VDJ recombination pathways. The precursor B-cells develop independently of antigen into mature resting cells that express membrane-bound IgM and IgD. Mature B-cells with membrane-bound IgM interact via CD40 receptor with helper T-cells carrying CD40 ligand. This activates B-cells to proliferate. Upon antigenic stimulation, a process of switching of immunoglobulin heavy chain class occurs by means of region-specific rearrangement of the IgH locus, leading to the generation of antibodies with ␥-, ␣- and ␧-heavy chains. Cytokines play an important part in isotype switching (Figure 1). Their participation is by way of regulating the transcription of specific switch sites and by promoting recombination. The switching process involves the rearrangement of the constant region of the heavy chain, and possibly also alternative splicing of mRNA in the germ line. Isotype switching occurs together with the germinal centre reaction and onset of somatic hyper-mutation. The processes of switch recombination and VDJ recombination are molecularly linked although they do appear as distinct processes. Both involve the generation of double strand breaks and their subsequent repair. DNA-PKc and Ku protein (discussed elsewhere in this book) are possibly also involved in both switch recombination and somatic hyper-mutation. There are also indications that DNA mismatch repair genes might be involved in both switch recombination and hyper-mutation (Ehrenstein and Neuberger, 1999). Another shared feature is that the hotspots of breakpoints seem to correspond with specific sequence motifs, namely a pyrimidine-rich motif either RCTYT or CCYC, and RGYW associated with hotspots of hyper-mutation (Kong and Maizels, 2001). The generation of antigen receptor diversity in developing immune systems involves genetic rearrangement of immunoglobulin and T-cell receptor (TCR) loci. Germ-line genes do not code for antigen receptors, but genes that are produced by recombination of gene segments do. TCR␣/␦ locus contains a large number of V, D, J and C gene segments, which by recombination generate

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Genetic Recombination in Cancer

Figure 1 Isotype switching in the developing immune system. The Figure summarises the generation of antibody diversity by isotype switching in the developing immune system. Cytokines participate by regulating the transcription of specific switch locations and by promoting recombination. Interleukins (IL-2, 4, 5, 6 and 13), TGF-␤ and IFN-␥ promote certain class switches. But TGF-␤ and IFN-␥, and IL-12 can inhibit the expression of some isotypes.

antigen receptor diversity in developing immune systems. This recombination is generally referred to as V (variable), D (diversity) and J (joining) or VDJ recombination. VDJ recombination is developmentally regulated during lymphocyte differentiation in the thymus. VDJ recombination is not a random process and not related to the proximity of gene segments involved, but it is a site-specific process and is influenced by recombination signal sequences, which are involved in targeting the VDJ recombinase activity (Livak et al., 2000). The first step in VDJ recombination involves the generation of site-specific double strand breaks in the DNA. VDJ recombination involves a number of genes, e.g. recombination activating genes RAG1 and RAG2, IL-7-␣ receptor gene, CD45, gamma-c, and Janus kinase-3, among others. RAGs 1 and 2 are

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lymphoid-specific components of the recombination system. They are capable of site-specific recognition and generation of site-specific double strand breaks in the DNA between the recombination signal sequence and adjacent coding DNA. It is the RAG heterodimers that cleave the DNA. Not only this, high RAG expression is consistent with marked recombinase activity. The RAG proteins are essential for the production of immuno-competent T- and B-cells and in the absence of their proper functions the maturation process is inhibited. The cytokine IL-7 controls the recombination process by regulating the access of VDJ recombinase. In the recombination at TCR-␥ locus, IL-7 is suggested to increase the acetylation status of histones and produce an open configuration of the chromatin, which provides access to the transcription machinery and RAG proteins (Huang and Muegge, 2001). The second step is DSB repair and recent studies have shown that DNA-PK is required for the operation of the VDJ recombination machinery. The adenovirus oncoprotein E4 can inhibit VDJ recombination. Furthermore, E4 immuno-precipitates with DNA-PK suggesting a direct role for DNA-PK in the recombination process (Boyer et al., 1999). The region between the VDJ and c-mu of the Ig heavy chain locus is believed to display a high frequency of mutation (Middleton et al., 1991). Gy et al. (1992) reported some time ago that certain chromosomal translocations encountered in ALL and AML might involve VDJ recombinase. But other translocations such as the t(12; 21) (p13; q22) involving the TEL gene do not seem to involve the VDJ recombinase (Romana et al., 1999). The deregulation of the switch-mediated recombination and VDJ recombination pathways might occur differentially in myeloma. Isotype switch-mutated plasma cells progress into multiple myeloma, and progression is associated with increased IgH translocations with chromosomal partners involving 11q13 (cyclin D1), 6p21 (cyclin D3), 4p16 (FGFr3 and WHSC1/MMSET) and 16q23 (c-maf) undergo error-prone recombination (Bergsagel and Kuehl, 2001; Shaughnessy et al., 2001) leading to abnormal expression of these growth and cell cycle regulatory factors. Ho et al. (2001) appear to be suggesting that illegitimate recombination might reflect differences in the pathways of pathogenesis of the disease. This might indeed be the case. Chesi et al. (1996) found abnormal switch recombination in 2/3 myeloma cell lines that might have been brought about by a (11;14) chromosomal translocation, whilst in mantle-cell lymphoma errors could have occurred in VDJ recombination. The isotype switch recombination might be an important feature of diffuse large B-cell lymphoma. Although typically these tumours are likely to express a single isotype, some cells might express alternative isotypes representing intraclonal evolution of cellular phenotypes (Ottensmeier and Stevenson, 2000). These authors have in fact suggested that secondary mutations might be occurring after neoplastic transformation. The t(11;14) translocation, which occurs in childhood T-ALL leukaemia, involves the LMO2 gene located on chromosome 11p13. This gene encodes a haemopoiesis-related transcription factor. The translocation leads to aberrant LMO2 expression and potentially this could result in secondary mutations and

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culminate in pathogenesis. Dynan et al. (2001) found T-cell tumorigenesis in transgenic LMO2 mice to be independent of RAG recombinase expression. However, fragile sites of LMO2 and other genes implicated in lymphoid malignancies could serve as VDJ recombination breakpoints, albeit functioning with different degrees of efficiency that might determine the frequency with which the translocation occurs (Raghavan et al., 2001). The syndrome called severe combined immunodeficiency (SCID) syndrome is associated with defective VDJ rearrangement. The equine as well as human SCID syndrome has been studied in much detail in the past few years. Lymphocytes of SCID mice express a defective VDJ recombinase. The lack of VDJ rearrangement in SCID mice causes a failure of humoral and cellular immune systems to mature. One can cite much indirect evidence to implicate VDJ recombinase-mediated translocation in the pathogenesis of haematopoietic malignancies. In SCID mice there is a high incidence of B-cell lymphomas. Nonetheless, there is ample recognition that SCID is associated with mutations in other genes such as RAG 1 and 2, IL-7 receptor-␣, Janus kinase 3, CD45 among others. Inevitably of course, DNA-PK deficiency contributes significantly to the SCID phenotype. There is little doubt that illegitimate VDJ recombination might be involved in the pathogenesis of haematological malignancies, but one should add a rider that there is no exclusivity in the association between recombination and the pathogenesis of neoplastic disease. This clearly emerges from the study of certain autosomal recessive conditions such as the Bloom’s syndrome (BS), the Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT). All these conditions are hypersensitive to radiation-induced damage, accompanied by marked immunodeficiency and a proneness to develop cancer. In spite of the similarities of phenotype, they do differ significantly in respect of VDJ recombination. Kaneko et al. (1999) found no aberrant VDJ recombination in haemopoietic cells derived from BS patients, but abnormal recombination is detectable in AT cells. Cells from NBS cells are prone to develop lymphoid tumours involving translocations of Ig and TCR loci, but they carry out errorfree VDJ recombination efficiently (Yeo et al., 2000).

SET domain proteins in genetic transcription, growth regulation and myeloma Control of cell proliferation by SET domain proteins SET domain-containing proteins form a class of chromatin-associated proteins that are frequent targets of growth regulatory signals and signals of oncogenic transformation and appear to be involved in gene silencing. The protein products of several genes such as the TRX (trithorax) and Polycomb families of genes regulate the transcription of developmental genes by resorting to modification of chromatin structure. There is general recognition that gene loci

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that can be transcribed might be shielded by inactive heterochromatin. Therefore, chromatin is organised into a higher order structure that is capable of influencing the transcription of genes by epigenetic mechanisms and of chromosome function. This involves a covalent modification of histone tails and the formation of sub-domains that are modified by non-histone factors. Deregulation of SET domain-containing proteins interferes with this higher order chromatin organisation (Melcher et al., 2000). The SET domain is a 130-aminoacid motif that appears to have been highly conserved in evolution. The SET motif might occur together with chrome and zinc finger motifs. Both SET and chrome motifs seem to function by modulation of chromatin structure. The SET domain protein of Drosophila called TRX binds to core histones and nucleosomes, and mutations in the SET domain affect histone binding (Katsani et al., 2001). The human SET protein SUV39H1, the murine Suv39h1 homologue of the Drosophila protein Suv(var)3–9 and cir4 of Schizosaccharomyces pombe are histone H3-specific methyltransferases and produce site-specific modification of H3 tails (Rea et al., 2000). The mammalian protein SUV39H1 contains both SET and chrome domains and it is capable of repressing genetic transcription. Furthermore, this SET protein appears to undergo cell cycle related phosphorylation at the G1 –S transition of cells, and when experimentally induced to express it leads to growth suppression (Firestein et al., 2000). SUV39H1 shows centromere-specific distribution in mitotic cells (Aagaard et al., 1999). Naturally its aberrant expression would be expected and indeed does lead to defective mitosis and segregation of chromosomes (Melcher et al., 2000). SET proteins are involved in centromeric and telomeric gene silencing. The Set1p of yeast is a SET domain protein whose inactivation inhibits telomere position effect (TPE) of gene silencing. Corda et al. (1999) have shown that the SET domain interacts with Mec3p and that deletion of Mec3p enhances TPE and enhances telomere length. The telomere is eroded at cell division and is believed to lead to chromosomal instability and to illegitimate chromosomal recombination. Telomeric integrity is essential for cells to continue to replicate, for in its absence cells enter a phase of senescence. The chrome domain (CHD) proteins are involved in the regulation of DNA transcription, its degradation and in the remodelling of chromatin. The centromere plays an essential role in chromosome segregation. Chrome proteins show a close association with the centromere and have been implicated in centromere function (Doe et al., 1998). In fission yeast, genetic transcription is repressed in the vicinity of the centromeres and telomeres via the agency of chrome domain proteins (Thon and Verhein-Hansen, 2000). They form a family that can be subdivided into the CHD1 and CHD3/4 sub-groups. CHD1 might be involved in the remodelling of nucleosomes, whilst CHD3/4 regulates in vitro transcription by modulating chromatin structure (Tran et al., 2000). A great deal remains to be elucidated vis a ` vis the mode of function of chrome domain proteins, but the discovery of a number of chrome interacting

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proteins and their structural considerations suggests that they might act as adaptor molecules (Ball, Murzina et al., 1997). Aside from their involvement in genetic transactions, chrome proteins might be involved in the pathogenesis of autoimmune diseases such as rheumatic conditions and systemic lupus erythematosus; pathogenesis seems to result from an autoimmune response to the chrome motif (Iwai et al., 1996; Muro et al., 1996). Some TRX and Polycomb group proteins contain the PHD (plant homeodomain) motif in addition to the SET domain. The PHD motif occurs in a large number of eukaryotic proteins, many of which are regulators of transcription. Abnormalities in the PHD domain, e.g. mutation in or deletion of the domain, are believed to lead to autoimmune disorders and myeloid leukaemias. The proteins containing zinc-binding domains RING, LIM (Mec3p) together with proteins containing the PHD domain form a structurally related family with a consensus C6HC pattern (Van der Reijden et al., 1999). These authors believe that these proteins are probably functionally related to one another and possibly also with SET/chrome proteins involved in chromatin remodelling, as evidenced by the interaction of Set1p in TPE and maintenance of telomere integrity. Indeed there is wide-ranging interaction of DNA checkpoint proteins among themselves as well as with SET and chrome domain proteins. The RAD17, RAD24, DDC, Mec and RAD9 are Saccharomyces cerevisiae checkpoint genes that are closely involved in arresting the cell cycle in response to DNA damage. Interactions between and a co-ordinated functioning of the products of these genes seem to be essential in DNA damage recognition and cell cycle arrest. Another possible link with control of cell proliferation is indicated by the possibility that PR domain of certain proteins might be a derivative of the SET domain. It is known that PR domain-containing protein might interact with Rb cell cycle regulatory protein. Besides, the PR domain may be disrupted as a result of chromosomal translocation in myeloid leukaemia.

The generation of chimeric transcripts of NSD family genes in multiple myeloma and Wolf–Hirschhorn syndrome A family of genes referred to here as the NSD family contains a number of genes that are characterised by presence of the SET domain and PHD fingers in a shared and conserved region (Angrand et al., 2001) and are called NSD1, NSD2 and NSD3. NSD2 is also known as the WHS (Wolf–Hirschhorn syndrome) C1/MMSET. The t(4;14)(p16.3;q32) translocation is frequently involved in multiple myeloma leading to the deregulation of FGFr3 and WHSC1/MMSET genes. Approximately a quarter of multiple myeloma patients carry the translocation. The 4p16.3 locus is critical for WHS syndrome and in the pathogenesis of multiple myeloma. The translocation breakpoint occurs at 4p16 within the

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5’-introns of MMSET. This results in the formation of hybrid transcripts of IgH/ MMSET (Chesi et al., 1998). Chimeric transcripts have been encountered in around 20% (11/53) of multiple myeloma cases as compared with only 6% (1/16) of monoclonal gammopathy (Malgeri et al., 2000). Furthermore, FGFr3 occurs 50–100-kb telomeric to the breakpoint and seems an obvious cause of FGFr3 deregulation. FGFr3 has been recognised with the potential to inflict genetic skeletal abnormalities. Thus both WHSC1/MMSET and FGFr3 genes might be contributing significantly in multiple myeloma. The contribution by FGFr3 to growth deregulation might be only one-half of the equation. According to Otsuki et al. (1999), antibodies against FGF-4 also were able to inhibit growth. FGF-1 and FGF-4 both function as ligands for FGFr3, which suggests that an autocrine mechanism of growth regulation might be operating in the myeloma cell lines that they were investigating. Angrand et al. (2001) have reported that the related NSD3 is amplified in breast cancer tissue and cell lines. Whether the IgH-WHSC1/ MMSET hybrid transcript is exclusively the causal event is unclear. For instance, it is not known whether the translocation affects the gene silencer function of the SET domain. It would be worthwhile mentioning here that t(4;14) is also encountered in primary amyloidosis, albeit the pattern of generation of hybrid transcripts could be different (Perfetti et al., 2001). In cyclin D3-over-expressing cells, the translocation brings ␥-4 switch sequences into close proximity of cyclin D3 gene. However, Ho et al. (2001) encountered switch translocation and upregulation of cyclin D and FGFr in only 57% of patients. Besides, they found no differences in survival or differences in the expression of prognostic markers such as ␤2-microglobulin and serum thymidine kinase between patients with progressive disease and with or without switch translocation. It would not be appropriate to comment further on any potential significance of switch recombination in relation to malignancy based on these isolated findings.

Ku protein in cell function The DNA-dependent protein kinase (DNA-PK) is a multi-function enzyme. It participates in a number of cellular processes by virtue of its ability to interact with DNA. DNA-PK consists of a catalytic component (DNA-PKc) and a regulatory component. DNA-PKc is a serine/threonine kinase of 460 kDa molecular size. The so-called Ku protein forms the regulatory component. The Ku protein targets DNA-PK to the DNA. Ku protein is highly conserved in evolution. But there are probably small differences between Ku from different species as suggested by differences in their electrophoretic mobility (Koike, Kuroiwa et al., 2001). Ku takes part in cell differentiation, maintenance of chromosome integrity as well as in DNA transcription. Ku also contributes to genomic integrity by supporting homologous recombination as well as to the non-homologous end-rejoining pathway of

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double strand break (DSB) repair (Figure 2). Hence, it is constitutively expressed in quantities adequate for proper functioning in DSBR. Ku is a heterodimer of two subunits, Ku70 and Ku80. Its participation in cellular function is attributed to its ability to bind to DNA ends with high specificity. Ku is now known to bind to nicks in the DNA, as well as gaps and hairpins. It binds to double strand breaks and promotes repair by non-homologous end rejoining. Its mode of function is not by directly interacting with the DNA bases or with the sugar-phosphate backbone. The two subunits form a ring around the DNA duplex. The binding covers two full turns of the duplex but encircles only 3–4 base pairs. The Ku ring determines the through path of the duplex by virtue of being able to fit sterically into the major and minor grooves thus supporting the broken strands for end processing and ligation (Walker et al., 2001). It seems possible that cellular proteins might interact with dimerised Ku and inhibit its binding to DNA (Muller et al., 2001). Mutation analyses show that changes in the N-terminal two-thirds of the protein result in loss of Ku70/80 interaction and also loss of DNA end binding. Mutations in C-terminal region do not affect DNA binding but they do affect DNA-PKc activation. The C-terminal domain of DNA-PKc is said to be essential for DNA-PK activity (Jeggo et al., 1999). The formation of heterodimeric Ku is essential for its function. Jin and Weaver (1997) have identified molecular areas of Ku70 required in the dimerisation of Ku. Osipovich et al. (1997) identified a 28-aminoacid sequence (449–477), which they claim is important for Ku80 interaction with Ku70. Mutations in this sequence abrogate the ability of Ku80 to interact with Ku70. These authors also categorically rule out that Ku subunits form homodimers at all. But more recently it has been suggested that homodimers might have restricted ability to cross the nuclear membrane. Although how heterodimerisation is involved in its function is not precisely known, it appears possible that the protein might be stabilised in dimeric form. But more importantly, heterodimerisation seems to be essential for the protein to enter the nucleus. Koike, Shiomi et al. (2001) transfected Ku into Ku-deficient cells and demonstrated that the exogenous Ku70/80 accumulated in the nucleus. They also generated a mutant not carrying the signal sequence for nuclear localisation, which failed to show nuclear localisation. However, Koike, Shiomi et al. (2001) found nuclear location of the mutant with signal dysfunction, if the wild-type Ku was present simultaneously. This suggests that homodimerisation of these subunits with dysfunctional subunits might promote nuclear localisation. DNA-PK appears to be activated by structured DNA such as hairpin ends, and possibly also by damage sustained by DNA. Soubeyrand et al. (2001) believe that hairpin ends are powerful activators of DNA-PK. The kinase might be inactivated by autophosphorylation, thus blocking the phosphorylation of heterologous substrates. Ku binds also to RNA, albeit with less affinity than to DNA. Yoo and Dynan (1998) have explored the RNA-binding properties of Ku.

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Figure 2 DNA-PK/Ku proteins in non-homologous end-joining DNA repair. This Figure is a diagrammatic representation of the involvement of DNA-dependent protein kinase (DNA-PK) in non-homologous end joining DNA repair. Double strand breaks (DSB) activate DNA-PK. Ku70/80 heterodimer is the DNA targeting regulatory subunit of DNA-PK, and by virtue of its function as a helicase participates in the resolution of aberrant DNA structures. Among cellular proteins that interact with the Ku heterodimer is the Werner’s syndrome protein WRN. This interaction with Ku enhances the exonuclease activity of WRN, which is crucial in the repair pathway. The Ku proteins are involved in cell differentiation, maintenance of telomeres and chromosomal integrity and in the regulation of gene transcription. The Figure shows the participation of Ku in cell proliferation and the apoptotic process. The expression of DNA-PK is modulated in a number of human cancers. Based on references cited in the text and Orren et al. (2001) and Shen and Loeb (2001).

Regulation of transcription by Ku protein DNA-PK is known to phosphorylate several factors that regulate DNA replication and transcription. Prominent among them are SV-40 large T-antigen, p53, heat shock protein (HSP) 90, topoisomerases and RNA-polymerase II as well as transcription factors such as c-jun, c-fos, oct-1, c-myc, Sp-1 and the heat shock transcription factor HSF1. Chibazakura et al. (1997) showed that DNA-PK phosphorylates the general transcription factors TATA-binding protein (TBP) and TFIIB and possibly stimulates basal RNA-polymerase II transcription by phosphorylating these transcription factors.

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HSF1 can bind to both Ku70 and Ku80 and more weakly to DNA-PKc (JR Huang et al., 1997). DNA-PK-deficient cells show a marked reduction of transcription in vitro due either to the lack of Ku80 or DNA-PKc. Such decrease occurs with several promoters. Addition of extracts from cells that contain DNA-PK restores transcription in cells deficient in Ku80 or DNA-PKc (Nueda et al., 1999). However, Ku represses glucocorticoid-induced DNA transcription in mouse mammary tumour virus (MMTV) by sequence-specific binding to the NRE1 element in the viral long terminal repeats (LTR). Alu core sequence elements of RNA-polymerase III modulate both DNA replication and transcription. Ku appears to be able to bind to Alu core element of Raji cells (Tsuchiya et al., 1998), but the functional significance of this binding is yet to be determined. The EBV-mediated immortalisation of B-cell occurs via transcription of CD23. Shieh et al. (1997) have identified an EBV-responsive enhancer element in intron 1 of CD23. They have shown also that Ku binds with high specificity to this EBV-responsive element. Ku binding is seen only in EBV+ but not in EBV– nuclear extracts. Furthermore, the binding is enhanced by protein phosphorylation, thus implicating a functional aspect to the binding between Ku and the EBV-responsive element.

Ku protein and DNA repair There are two pathways by which double strand break (DSB) repair of DNA proceeds. In mammals and higher eukaryotes, DSB repair (DSBR) can take place by non-homologous end rejoining as a primary mechanism of DSBR. In the event that a cell is unable to do this, strand break repair takes to the homologous recombination pathway. The latter will not normally be evident unless the non-homologous end-rejoining pathway is not functional (Pluth et al., 2001). The non-homologous end-rejoining pathway of DSBR requires several factors such as Ku protein, DNAP-PKc catalytic subunit of DNA-PK, the DSB repair protein XRCC4 and DNA ligase IV. DNA-PK deficiency correlates with reduced repair and increased radiation sensitivity. Radiosensitive BALB/c mice are less efficient in the repair of DSBs induced by ␥-radiation than other strains of BALB/c or C57BL mice. Besides, radiation sensitivity and reduced repair appear to relate to reduced DNA-PKc expression and activity (Okayasu et al., 2000). However, the relative importance of DNA-PKc and the Ku cannot yet be ascertained with certainty. Kienker et al. (2000) seem to suggest that DNA-PKc alone might be sufficient to activate DSBR and render the cells radiationresistant. One ought to make a note of the lack of correlation between DSBR and the expression of Ku or DNA-PK activity, whilst cell survival was related per se to DSBR capacity (Kasten et al., 1999). These authors have stated that Ku70 and Ku80 mRNA signal densities did not vary between the cell lines that they tested. A significant element here is the stability of the transcribed messages. It

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is an open question whether this has any bearing on the activation of DNA-PKcs in the experimental system employed by Kasten et al. (1999). Therefore it might be premature to evoke some other pathway as a plausible explanation for their findings. Nonetheless, there are indications that heat-induced radiosensitivity might not be related to the DNA-PK pathway of DSBR (Woudstra et al., 1999). DNA-PK activity is essential for DSBR and this seems to be regulated by phosphorylation of the component units of DNA-PK. DNA-PKc, Ku70 as well as Ku80 can be phosphorylated, which leads to inactivation of the enzyme. Phosphorylation of the enzyme is a reversible process and is in fact regulated by protein phosphatase-1 or PP2A both in vitro and in vivo (Douglas et al., 2001).

Ku protein in the maintenance of chromosomal integrity Ku participates in the repair of telomeric DNA. Ku70, Ku80 and DNA-PKc are associated with telomeric DNA. The inactivation of Ku70/80 leads to shortening of the telomeres in mouse cells, whereas in contrast, deficiency of proteins such as XRCC5 or FDNA ligase IV does not produce diminution of telomere length (Di Fagagna et al., 2001). Ku does not bind telomeric DNA directly, but interacts with great specificity with the telomere binding protein TRF (Hsu et al., 1999, 2000). It has been reported to activate telomerase (Lansdorp, 2000; Peterson et al., 2001). Ku appears to play a key role also in telomere position effect (TPE) or in the silencing of genes proximal to the telomere. This probably involves an interaction of Ku with the protein called HP1-␣. HP1 is a telomere-associated protein that is capable of suppressing genetic transcription in mammalian cells (Song et al., 2001). RAD50 seems to be involved in Ku-mediated double strand break repair and TPE, although RAD50 itself does not influence TPE (Boulton and Jackson, 1998). These observations are compatible with reduction of Ku expression in cells undergoing replicative senescence, and enhanced expression in immortalised cells. It is of interest to note in this context that BLM and WRN proteins have also been implicated in the repair of telomeric DNA.

Ku protein in cell cycle progression and differentiation DNA-PK, being a kinase, would be quite obviously capable of regulating cellular processes by phosphorylation of substrate proteins. DNA-PK can efficiently phosphorylate from structured DNA proteins such as the p53, the cell cycle control protein, which bind to single stranded DNA. It follows therefore that Ku protein will be involved in cell cycle progression and the allied processes of cell differentiation and in the pathogenesis of cancer, which has often rightly been described as a disease of differentiation.

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The expression and activity of Ku protein have been studied in relation to cell cycle progression. Nilsson et al. (1999) found that DNA-PK activity was low in G1-phase of the cell cycle but rose sharply in the S-phase and remained high through the G2-phase. The levels of the expression of the enzyme were unchanged, but the enzyme was relocated from the cytoplasm to the nucleus at the transition of cells from G1 to S-phase. This is roughly compatible with the report by SE Lee et al. (1997), who found peak DNA-PK activity at both G1/Sand G2-phases. Furthermore, lack of DNA-PK at G1S correlated with increased sensitivity to radiation. Although these findings indicate an association of DNAPK activity with cell cycle progression, no functional correlation is apparent at present. However, a distinction has to be made between the DNA-PKc and the Ku protein while assessing the significance of DNA-PK to cell cycle progression. Koike et al. (1999) state that Ku is diffuse in its distribution in the cytoplasm and occurs at the periphery of condensed chromosomes. DNA-PKc in contrast is nuclear in distribution but not associated with condensed chromosomes. Koike et al. (1999) appear to suggest that Ku associated with metaphase chromosomes might be involved in G2M transition of cells. These findings are based on immunohistochemistry and merely indicate the presence of the proteins and do not necessarily relate to their function. This is an important proviso for drawing any conclusion about their involvement in the cells cycle traverse. However, since DNA-PK can efficiently phosphorylate the cell cycle control protein p53, the differential activity of the enzyme does assume some significance. Expression of p53 is induced in response to DNA damage and there is indirect evidence that this process might involve DNA-PK (Boyer et al., 1999). Especially interesting is the possibility that DNA-PK might influence p53 function of checkpoint control at the G1-S transition. Some years previously, Sullivan et al. (1997) studied the progression of the cell cycle in cells derived from patients with the Nijmegen breakage syndrome. This syndrome is characterised by immunodeficiency, growth inhibition and susceptibility to cancer. Sullivan et al. (1997) found that cells exposed to DNA damage by ionising radiation failed to arrest progression at the G1S checkpoint, which is controlled by p53. It would be interesting to see if there is any parallelism between the phosphorylation status of p53 and DNA-PK activity levels, indeed at both G1-S and G2-M transition checkpoints both under p53 control. Admittedly, other proteins such as S100A4 have been implicated at both these checkpoints and stathmin at G2-M transition (Cajone and Sherbet, 1999; Sherbet, 2001). The participation of Ku protein in the early stages of embryonic development emerges with clarity from some experiments carried out by Kanungo et al. (1999). Ku antibodies injected into 2-cell stage embryos of the sea urchin Lytechinus pictus lead to developmental block but antibodies against Ku alone produced no developmental arrest. Kanungo et al. (1999) have also reported differences in the location of DNA-PK. The enzyme occurs in the cytoplasm of early developmental stages but in the nucleus of late stages of development.

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They conclude that the antibodies might be interfering with the translocation of the kinase to the nucleus. These findings are somewhat surprising since one would have expected that the early stage of embryonic development is mainly a division of the fertilised egg to form the cell mass with the blastocoel subsequently leading to morphogenetic movements and to the differentiation of the germ layers. We know that DNA-PK activity shows a marked relationship with the cell cycle, and hence it is rather odd that Kanungo et al. (1999) found no nuclear localisation of Ku proteins. The association of Ku with cell proliferation is further supported by the finding that cells that are undergoing replicative senescence show a reduced expression of both Ku subunits with a parallel reduction in PARP function. In contrast, immortalisation of cells with SV-40 leads to an increased expression of DNA-PK (Salminen et al., 1997). Another line of evidence for the involvement of Ku in cell immortalisation is the EBV-mediated activation of CD23 leading to B-cell immortalisation (Shieh et al., 1997). Here, Ku binds specifically to the EBV-responsive enhancer element of CD23. The participation of Ku in cell differentiation as well as in the apoptotic pathway is an area of considerable interest. Some years ago, Ajmani et al. (1995) reported that Ku was not detectable in neutrophils, but it was present in G0-lymphocytes. However, HL60 promyelocytic leukaemia cells that were induced to differentiate by dimethylsulfoxide (DMSO) into neutrophils were Ku+. Furthermore, cells in G0/G1-, S- and G2M-phase of the cell cycle all stained for Ku. More recent studies using DMSO or all-trans-retinoic acid (RA) on HL60 as well as NB4 cell lines have indicated that Ku levels remain unchanged during exposure to the differentiating agents, but there is reduced binding to double stranded DNA ends (Muller et al., 2001), probably attributable to other factors. Ajmani et al. (1995) also postulated that cells on the apoptotic pathway might not be expressing Ku, for they noticed that cells with hypodiploid DNA did not contain Ku. This may be so, also in view of the association of the stringent requirement of Ku for DSB repair (DSBR). The presence of DSB and the activation of repair processes, e.g. as exemplified by the presence of PARP, is an indication that cells are undergoing apoptosis (Sherbet, 2001). They have indeed suggested that Ku might be actively degraded in cells undergoing apoptosis. It is not surprising therefore that DNA-PK is inactivated with the onset of apoptosis. The loss of kinase activity appears to be a result of cleavage of DNA-PKc, but the Ku protein remains stable. The introduction of bcl2 anti-apoptosis protein prevents the cleavage of the catalytic subunit. Similarly, inhibition of the ced3-like protease, which cleaves PARP, also prevents DNA-PKc cleavage (Le Romancer et al., 1996).

Ku protein in cancer The implication of Ku in cell proliferation and developmental processes has inevitably suggested the possibility that it might also play a part in the

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pathogenesis of neoplastic lesions and their progression. There are many indications that the expression of Ku proteins might be related to the aggressiveness of cancer. A reduction in Ku70/Ku80 binding to DNA has been encountered in advanced stages of breast and bladder carcinoma (Pucci et al., 2001), and invasive carcinomas of the breast expressed Ku at lower levels as compared with normal breast epithelium (Moll et al., 1999). A more telling correlation is the reported reduction of Ku70/Ku80 in oral melanoma with metastatic spread (Korabiowska et al., 2002). Evidence towards the possible involvement of Ku protein in tumorigenesis has come from some early work on BRCA1 tumour suppressor protein and its mediation of DSBR via DNA-PK function. Critchlow et al. (1997) found that the XRCC4 protein, a substrate for DNA-PK in vitro interacted with ligase IV via its C-terminal extension. This contained two tandem motifs that bear homology to the C-terminal domain of BRCA1 protein. This suggests the tumour suppressor function of BRCA1 might be related to its ability to participate in DNA-PK-mediated DSBR. However, the role of BRCA proteins needs to be investigated further, especially in the light of contradictory findings of Wang et al. (2001). They found no differences in DSBR of radiation-induced strand breaks in pancreatic carcinoma cell lines with wildtype or mutated BRCA2. These cell lines showed similar DSBR capacity whether by DNA-PK-dependent or -independent means. Wang et al. (2001) also examined cell lines derived from human breast cancer with mutated BRCA1 that showed normal end rejoining of breaks induced by radiation. These findings do not necessarily detract from the proven potential of BRCA proteins as tumour suppressors, but nonetheless might be significant since breast cancer management relies quite heavily on radiotherapy. Tumours that contain only a small number of cells expressing Ku might be more radiosensitive, as Wilson et al. (2000) have noticed in a study of cervical carcinomas, and hence such low expression is likely to correlate positively with improved survival. Increased Ku activity does indeed render cells resistant to ionising radiation (Frit et al., 1999). This could be due to the induction of the protein called clustrin, which is implicated in the induction of apoptosis by radiation. In fact, clustrin might bind Ku70/Ku80 as indicated by its co-immunoprecipitation with Ku (CR Yang et al., 2000). These observations are compatible with the finding that Ku deficiency increases the susceptibility of cells to apoptosis induced by chemotherapeutic agents (SH Kim et al., 1999). A marked reduction in DNA-PK activity has been found in peripheral mononuclear cells from lung cancer patients as compared with cells from cancer-free subjects. Corresponding differences also occur in bronchial epithelial cells of cancer patients (Auckley et al., 2001). It appears that there might be differences in the pattern of expression of Ku protein in early stage disease and cancers that have progressed to the metastatic stage. Reduced DNA-binding or expression of Ku70/80 has been associated with tumour progression to the metastatic state (Pucci et al., 2001; Korabiowska et al., 2002). This is consistent with the earlier observations that

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invasive breast cancers contained less Ku80 and DNA-PKc than did normal breast epithelia (Moll et al., 1999). How this relates to kinase activity is uncertain, especially since others have found no differences in Ku70, Ku80 or DNA-PKc in tumour and normal tissues, nor was their expression related to radiosensitivity (Sakata et al., 2001). The use of immunohistochemistry in the latter work has provided information of the presence of these proteins in the nucleus, whilst Western blotting that Pucci et al. (2001) carried out did not possess the benefit of demonstrating any cell compartment-related expression. Bjork-Eriksson et al. (1999) investigated the expression of DNA-PKc as well as Ku70/Ku80 in a series of head and neck cancers and found no relationship between the levels of expression of DNA-PKc or Ku 70/80 and tumour histology and stage, nor was there any correlation with location of the tumour. Neither did levels of p53 show any correlation with DNA-PKc or Ku. Bjork-Eriksson et al. (1999) have also pointed out that they examined the tumour fraction surviving exposure to 2 Gy-radiation. There was no relationship between the surviving fraction and the DNA-PK subunits, although one would have expected radiation-resistance to be associated with high levels of DNA-PKc or the regulatory Ku. One has to attach a caveat to these findings. They are mainly based on immunohistochemical assessment of expression, and one should be mindful of the somewhat subjective nature of determination of levels of expression by this method. Furthermore, by these means one is looking at protein levels and not the activity of the kinase. Furthermore, there is clear in vitro evidence that differential sensitivity to irradiation is associated with DNAPK activity. Polischouk et al. (1999) studied two cell lines, UMSCC-1 and UMSCC-14A, of human squamous carcinoma that displayed marked differences in radiosensitivity. The resistant line UMSCC-1 repaired X-ray-induced DNA breaks more efficiently than did UMSCC-14A. Furthermore, UMSCC-1 showed 1.6-fold higher constitutive levels of DNA-PK than UMSCC-14A. We know very little at present about possible mechanisms that could lead to reduced expression of DNA-PK in cancer cells. Galloway et al. (1999), while investigating the expression of the kinase in the human malignant glioma cell line M059J, found low levels of DNA-PKc transcripts in the cells. They found no genetic alterations in the DNA-PKc gene, but the stability of DNA transcripts had substantially reduced. This certainly could account for the differential expression observed in certain neoplasms, but in mane alia this does not lead very far where a link-up between aberrant DNA-PK function and neoplastic alternation is concerned. This is quite surprising in view of the discovery of the kinase as far back as the mid-1980s.

3 Replication error (RER) and genetic instability

The genome has accumulated a vast amount of repetitious DNA sequences over the eons of evolution. DNA repeats are found all along phylogenetic evolution. Highly repetitive DNA occurs in many forms and locations in the genome, and varies enormously in respect of the lengths of repeat tracts. The repeat sequences can vary from single nucleotide repeats to complex ones with varying polymorphism of length of the repeat tracts. ZL Gu et al. (2000) estimate that approximately 40% of autosomes and approximately 51% of sex chromosome constitute repetitious DNA elements and that there are about 4.0×106 repeat elements in the genome. The generation and incorporation of repeat elements is a continuing process. There is general recognition now that genetic instability is brought about by either or both chromosomal instability in the form of allelic loss and microsatellite instability. Microsatellite repeat sequences of varying lengths have been found within or between genes. They tend to be inherently unstable and this instability manifests as variation in lengths of the repeats. Two modes of instability can be distinguished and identified, namely instability attributable to mismatch repair deficiency generally described as the RER phenotype, and instability due to the presence of di-, tri- and tetranucleotide repeats in other regions of the genome. The repeat or microsatellite sequences are prone to replication error which results from inactivating mutations of mismatch repair genes. Mismatch repair gene mutation has been postulated to occur in two steps. Primary mutations Genetic Recombination in Cancer ISBN 0-12-639881-X

Copyright © 2003 Elsevier Science Ltd All rights of reproduction in any form reserved

Replication error (RER) and genetic instability

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inactivate certain genes. This leads to the mutation and to a second stage of mismatch repair gene inactivation, which compounds the mismatch repair deficiency sustained as a result of the primary mutation (Perucho, 1996). Mismatch repair deficient cells tend to accumulate somatic mutations in genes that are involved in important pathways of DNA damage response, cell cycle regulation and tumour suppressor function. Both microsatellite instability and chromosomal instability manifested as karyotypic abnormalities are regarded as phenotypic manifestations of genetic instability and have provided a focus for extensive studies on their association with the evolution of variant cell types leading to cancer progression (Sherbet and Lakshmi, 1997). Genetic instability engendered by nucleotide repeats is associated with a number of neurological disorders and inherited genetic syndromes, and it is increasingly being associated also with cancer progression. They are discussed here in separate chapters in order to give due weight to the two modes of expression and operation of genetic instability. The RER phenotype represents the microsatellite instability attributed to mutations of the so-called mismatch repair genes. Mismatch repair genes are highly conserved in evolution from bacteria to man. The MutL and MutS represent two families of genes encoding mismatch repair proteins. These mismatch repair genes were cloned some years ago (W Kramer et al., 1989; Prolla et al., 1994). The proteins encoded by these genes subserve different functions. The MutS protein recognises mismatch repair, whilst another protein called the MutH introduces a nick in the target strand. MutL seems to be the mediator of interaction between MutS and MutH. Several human homologues, hMLH1, hMSH2, hMSH4, hMSH6, PMS1 and PMS2, have been cloned (Bronner et al., 1994; Nicolaides et al., 1994). Genes containing microsatellite sequences become susceptible to replications error that could lead to allelic alterations, loss of heterozygosity (LOH), loss or gain of function mutations, and abnormal gene expression. Mismatch repair proteins have been attributed with another property, which might also lead to the stabilisation of the genetic material. There is an increasing awareness that they might be capable of inducing apoptosis of cells that have damaged DNA. The net result of such a function would be the maintenance of a cell population with intrinsically stable DNA. GM Li et al. (1999) have suggested that mismatch repair proteins participate in the recognition of DNA damage, and initiate the apoptotic pathway by activating kinases that phosphorylate and activate p53.

Microsatellite instability and cancer progression Microsatellite instability has been encountered in many forms of human cancer and its possible involvement in their growth, invasive and metastatic behaviour has been recognised (Sherbet and Lakshmi, 1997). Much evidence is now available that suggests a close relationship between the RER phenotype and

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progression of cancers to invasive and metastatic phases. De Marchis et al. (1997) had shown that microsatellite instability is not encountered in benign fibroadenomas of the breast. A third of breast carcinomas showed abnormal microsatellite alleles at one or more foci. The RER+ tumours tended to be larger in size than RER– ones. Furthermore, the RER+ phenotype correlated significantly with the presence of metastatic tumour in regional lymph nodes. Thus 14 of 19 RER+ tumours were node positive, but only 15 of 59 RER– tumours showed nodal spread. It is also notable that invasive behaviour might be associated with RER phenotype. Aldaz et al. (1995) had earlier found that 9 of 23 invasive breast carcinomas were RER+, whereas the RER+ phenotype was found in only 7 of 52 breast cancers with ductal differentiation. In contrast, poorly differentiated tumours tend to be RER+ (Kobayashi et al. 1995; Seruca et al., 1995). The delineation of the path traversed by tumours from initiation to tumour development, clonal expansion, invasion and metastatic dissemination is a major objective in scientific research. The biology of tumour development and progression has often indicated that tumours can take different routes towards dissemination. One of the reasons for this is that metastasis is a very inefficient process, and this is in turn attributable to the incompatibility of genes becoming functional at the various stages of progression. Furthermore, whereas some gene function has a cumulative effect on tumour progression, the expression of others has to be switched off in order that the tumour cell can transit from one stage of progression to the next. The genetic changes associated with the progression of normal colonic epithelium from the stage of neoplastic transformation to the development of overt carcinomas have been elucidated in the past few years and this is the only model of carcinogenesis that has been characterised with regard to the activation of specific genes accompanying appropriately defined phenotypic changes. The early phase of hyper-proliferation is accompanied by mutations in suppressor gene such as the APC (adenomatous polyposis coli) gene and results in the development of adenomas. In the late adenomatous period, mutations seem to occur in the ras gene and the adenomas tend to develop carcinomatous foci. Further genetic changes occur during this period and these involve the DCC and p53, and these appear to result in the development of overt carcinomas. This pathway has provided a suitable model for investigating whether the genetic changes are produced by or associated with microsatellite instability. Microsatellite instability does not appear consistently to accompany the genetic changes all along the defined pathway of progression. According to D Shibata et al. (1994), microsatellite instability is encountered at the stage of neoplastic transformation and persists after transformation. But, APC protein, which is apparently functionally important at the early stages of tumour development following neoplastic transformation, is reported to be unrelated to microsatellite instability (Heinen et al., 1995). Also, there is no discernible association

Replication error (RER) and genetic instability

27

of the RER phenotype with progression towards the end stages of the path of progression. Ishimaru et al. (1995) showed that the RER+ phenotype occurs in the same proportion of primary carcinomas and their metastatic deposits. Of the two suppressor genes implicated to contribute to the development of overt carcinomas, p53 has shown no positive correlation, but, indeed showed a negative correlation with RER status. Cottu et al. (1996) found no p53 mutations in 4/4 RER+ colonic cancer cell lines, but 15 of 17 RER– cell lines contained mutations of p53. In a similar vein are the findings of Yamamoto et al. (1998) relating to DCC. The loss of DCC or a reduced expression of the gene was less frequent in RER+ carcinomas. Furthermore, reduced expression of DCC occurred in all RER– colorectal carcinomas with liver metastasis (Yamamoto et al., 1998). Allen (1995) had suggested that colorectal carcinogenesis might take either of two pathways, namely loss of heterozygosity of the genes involved or the RER pathway. Yamamoto et al. (1998) seem to subscribe to this view. However, the association of RER+ phenotype is not unambiguous and, furthermore, none of the genes demonstrated to date to be able to drive colonic tumours along the path of progression seem to be affected by RER status. Besides, clinical aggressiveness of the disease and overall survival of patients seem to be independent of RER (Ishimaru et al., 1995). Indeed, RER+ colonic carcinomas have been claimed to be less aggressive and to have better prognosis (Ko et al., 1999). In endometrial cancers too, there is no apparent association between RER+ status and muscular invasion by the tumour and lymph node metastasis (Kihana et al., 1998). Therefore the nature of RER involvement with tumour progression is yet to be clarified. This statement should be read with the caveat that the progression of other tumours might be RER linked. To give an example, both hyperplastic and adenomatous polyps of the stomach are said to be RER+. But the highest frequency of RER+ phenotype occurs in adenomatous polyps that contain carcinomatous foci (Nogueira et al., 1999). In oral cancer, RER+ status shows a marked correlation with lymph node metastasis (Ogawara et al., 1997). It could be argued that the development of metastasis is a result of a selective dissemination of a clone of cells, which have acquired special biological properties required for metastatic spread. Ishimaru et al. (1995) found that primary colorectal cancer and its metastatic deposits in the liver showed microsatellite instability at the same locus. This is an isolated instance, although found in three cases. The putative metastasis suppressor gene called nm23 that encodes nucleoside diphosphate (NDP) kinase occurs on chromosome 17q21.3. Many studies have suggested that a loss of expression of nm23 or the NDP kinase is consistent with high metastatic potential. The metastasis suppressor function of nm23 has not yet been proved beyond reasonable doubt (Parker et al., 1991; Albertazzi et al., 1998; also reviewed by Sherbet and Lakshmi, 1997 and Sherbet, 2001), but abnormalities of the gene are associated with several human neoplasms. In this context, it is worth recording here that the nm23 locus is a focus of genetic instability. Patel et al. (1994a) showed not only that alterations in microsatellite repeats occurred in

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colonic tumours but also that the locus often showed LOH. However, the germ of the possibility that genetic instability could lead to metastatic dissemination of the primary tumour cannot be disregarded. At the other end of the spectrum, Kang et al. (1999) found no relationship between early and advanced stages of gastric carcinoma, nor did they find any relationship between RER status and tumour differentiation or host cell infiltration in the tumours. Nevertheless, the findings of Nogueira et al. (1999) are highly significant in relation to the actual pathway of progression and they do suggest strongly that progression of the adenomatous polyps to frank carcinomas might be associated with changes to the RER+ phenotype. In fact, the clonal evolution of RER+ phenotype with attendant progression of the neoplastic process is patently indicated in a study of prostate carcinomas (Miet et al., 1999). These authors investigated the RER status of 172 carcinomatous foci using ten microsatellite markers. The RER+ phenotype occurred in 42% of carcinomas. In pre-cancerous intra-epithelial neoplasia and in carcinomatous foci the RER+ phenotype occurred with roughly comparable frequency. In comparison, the RER+ phenotype was encountered in only one of 26 nondysplastic glandular hyperplastic prostate tissues. Here microsatellite instability seems to occur early in the neoplastic process and apparently related to the progression of neoplastic foci to carcinomatous state. It might be recalled here that less than a sixth of sporadic colorectal cancer without any familial history of the disease might show microsatellite instability (Bubb et al., 1996). The RER-mediated instability of the genome may be a pathway of tumour progression occurring quite independently of chromosomal instability and recombination-mediated genetic alterations. Possibly, either pathway is not adopted to the exclusion of others, and indeed, as suggested by some investigators, it is fairly reasonable to assume that pathways of progression might be switched dependent upon the nature of genetic instability subsisting at a given point in progression.

Effects of microsatellite instability on p53 and growth factor receptor gene expression Microsatellite instability can influence the expression of genes essential for cell proliferation and this can potentially impinge upon tumour progression. Sherbet and Lakshmi (1997) reviewed and assessed the evidence and have concluded that the evidence can at best be described as ambivalent. This view has lately been strengthened by several studies relating to the association of the expression of and abnormalities in the cell cycle control gene p53 with the RER phenotype. There is now a general consensus not only that p53 abnormalities are not associated with the RER phenotype but also that microsatellite instability and mismatch repair has no influence on p53. Distinctive chromosomal anomalies might occur with RER+ and RER– phenotypes, but frequency of

Replication error (RER) and genetic instability

29

incidence of chromosomal defects was low in RER+ phenotype. Neither did the p53 status correlate with chromosomal anomalies (Curtis et al., 2000). Yoshida et al. (2000) found an inverse correlation between microsatellite instability and LOH of a number of genes including p53, in tumours of the gall bladder. Shen et al. (2000) found that although in situ and invasive carcinomas of the breast differed significantly in respect of microsatellite instability, there was no correlation with p53 expression, nor with the expression of other markers of tumour growth and aggressiveness, such as oestrogen and progesterone receptor (ER and PgR) proteins. RER is not related to p53 mutation in thyroid lymphomas (Takakuwa et al., 2000), non-small cell lung cancer (NSCLC) (Caligo et al., 1998), and colorectal cancers or cancer derived cell lines (Cottu et al., 1996; Olschwang et al., 1997; Eshleman et al., 1998), among others. Mancuso et al. (1997) looked for, but were unable to find RER+ related mutations in codon 248 of p53, which is frequently mutated in colon carcinomas. In sharp contrast are a few studies that appear to support a correlation between genetic instability and p53 abnormalities. Peng et al. (1996) looked for mutations in exons 5–8 of the p53 gene in mucosa-associated lymphoid tissue lymphomas. They found mutations in 11/40 cases. Giarnieri et al. (2000) have compared the expression of the mismatch repair genes MSH2 and MLH1 in noninvasive and invasive squamous cell carcinomas of the uterine cervix. They have reported a marked downregulation of both genes in invasive tumours as compared with the non-invasive carcinomas. The loss of expression of the mismatch repair genes correlated significantly with an over-expression of p53. There is much support for this view. For example, Forster et al. (1998) found loss of p53 correlated with microsatellite instability. Valentini et al. (2002) found that colorectal cancers that over-expressed p53 tended to be microsatellite-stable. Thus although a substantial body of evidence leads one to conclude that p53 mutations are not associated with replication error deficiency, the dissenting data are of sufficient weight and hence deserve due recognition. Arguably, there is a reasonably large body of evidence supporting a lack of relationship between p53 and the RER phenotype, but it is far from allowing a justifiable conclusion that microsatellite instability and loss of heterozygosity or mutations resulting from genetic instability reflect separate and independent pathways of tumorigenesis, as some authors have tended to suggest. Albertoni et al. (1998) have found that in a human glioblastoma and a cell line derived from it, both show a reciprocal translocation t(17;20) and deletion and of both p53 alleles as a consequence. It may be that other means than RER+ might be involved in the emergence of p53 abnormalities. For instance, epigenetic changes such as methylation, which has a marked effect on DNA stability, might be an alternative mode that can be implicated. With the linking of p53 mutations in CpG islands within the gene, it seems possible that abnormalities in p53 might indeed lead to genetic instability. A variety of other target genes have been tested for mutations. The RER phenotype does not seem to affect other genes that affect tumour growth and

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apoptosis, e.g. transforming growth factor-␤ receptor, c-erbB2, insulin-like growth factor receptor, or bax (Caligo et al., 1998; Nogueira et al., 1999; Schmitt et al., 1999). Nor does the RER phenotype influence ER and PgR expression, as mentioned above. It is worth noting here, however, that the reduced expression of TGF␤RII gene noticed by Jiang et al. (1998) in RER+ colon carcinoma cell lines is a consequence of decreased stability of the mutant mRNA. They have concluded that a frame shift mutation occurring in the receptor gene results in a premature stop codon in the 5’-half of the mRNA in the RER+ cells. Mutational inactivation caused by microsatellite instability is a recognised pathway of inactivation of this receptor gene, but its transcriptional inactivation has also been advocated (SJ Kim et al., 2000). Other growth factor receptors such as the epidermal growth factor receptor (EGFr) are yet to be examined from the standpoint of RER status. This is presumably partly due to the inverse relationship that generally exists between EGFr and ER/PgR and lack of influence of the RER phenotype on ER/PgR. Koul et al. (1999) have reported that somatic mutations of the tumour suppressor genes BRCA1 and also BRCA2 occur in association with the RER+ phenotype in endometrial cancers. The deletion mutation results in the loss of the N-terminal transactivation domain of BRCA2, and further this confers growth advantage on RER+ cancers. BRCA2 might also be functioning as a regulator of genetic transcription. Siddique et al. (1998) found that exon 3 of BRCA2 can activate transcription and this is linked with the ability of BRCA2 protein to alter the acetylation status of histones H3 and H4. Furthermore, the carboxyl terminal domain of BRCA1 interacts and associates with HDAC1 and HDAC2 (Yarden and Brody, 1999). The loss of heterozygosity at loci of both BRCA1 and BRCA2 genes occurs frequently in breast cancer, and the transition of breast cancer from in situ to the invasive stage accompanies a reduced expression of BRCA1 (Thompson et al., 1995). Hampl et al. (1996) found that metastatic breast cancer shows more genetic alterations in BRCA1 than the corresponding primary tumour. The inactivation of both these suppressor genes enhances cell proliferation and tumour growth. Other suppressor genes such as the PTEN may also show abnormalities in association with microsatellite instability. There is a report concerning PTEN abnormalities in primary cutaneous T-cell lymphoma (Scarisbrick et al., 2000). PTEN is located on chromosome 10q23. It codes for a phosphatase that causes cell cycle arrest at G1 with the mediation of p27kip1 cyclin-dependent kinase (cdk) inhibitor, and reduces growth and saturation densities of cells in culture. PTEN is regarded as a potential tumour suppressor (see Sherbet, 2001). The expression of p27kip1 itself seems to be amenable to alteration by RER at chromosome 12p. Another inhibitor of cdk, namely p16ink4, which is located on chromosome 9p, is also a suggested target for abnormalities attributable to microsatellitemediated instability (Nishimura et al., 1999). It would be highly rewarding to investigate whether genetic abnormalities that appear to be associated with

Replication error (RER) and genetic instability

31

PTEN relate to genetic instability of cancers instigated by microsatellite sequences. Whether mutation of the K-ras gene, which is an important component of G-protein mediated signal transduction cascade, occurs in the context of microsatellite instability is another question that has been asked frequently. The findings seem to be equally divided into groups that claim to have noticed ras mutations and those who found none. K-ras mutations have not been found in endometrial carcinomas (Sakamoto et al., 1998) and colorectal tumours (Olschwang et al., 1997; Eshleman et al. 1998). The incidence of ras mutations and LOH of the putative metastasis suppressor nm23-H1 in pancreatic and colorectal cancers roughly paralleled the levels of microsatellite instability encountered in them (Caligo et al., 2000). Takakuwa et al. (2000) reported that 4 out of 5 diffuse large B-cell lymphomas, which were RER+, also contained mutations of ras. Furthermore, 56% of RER+ and only 14% of RER– endometrial carcinomas revealed the presence of K-ras mutations (Duggan et al., 1994). Although the later study of Sakamoto et al. (1998) did not confirm this finding, there is one import element in the investigations of Duggan et al. (1994) that needs to be emphasised, and that is that, albeit in a solitary case, the ras mutation was detected after cells acquired the RER+ phenotype as a result of clonal expansion. This provides a more persuasive argument in favour of the involvement of the RER phenotype in being responsible for mutations of specific target genes than the experimental manipulation described by Anthoney et al. (1996). Anthoney et al. (1994) transfected mutant p53 but detected no changes in the RER phenotype. The premise of these experiments is that mutated genes would induce RER+ phenotype, whereas the correct direction of phenotypic change would be from changes in the RER phenotype producing instability in the genome, leading to mutations of target genes that in turn would alter the behavioural phenotype of cells. Although the discussion so far provides a very negative picture of RER influence on cell proliferation-related genes in relation to cancer progression, it should be borne in mind that a negative conclusion may be unwarranted, especially in view of the variety of physiological features of the cell that actively take part in cancer invasion and metastatic spread. One might legitimately inquire whether other molecular features of the cell that are important in cell adhesion and invasive behaviour are influenced or controlled by the RER phenotype.

Influence of microsatellite instability on invasive behaviour of tumours Tumour dissemination is mediated by subtle alterations in intracellular adhesion and the release of tumour cells from the primary tumour, their invasion of surrounding normal tissue components, adhesion to and intravasation into and

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exit from the vascular system at distant metastatic sites. Several membraneassociated glycoproteins mediate this variety of biological processes. The role played by many of these glycoproteins has been elucidated and the degree and nature of their participation in tumour cell adhesion and invasion has been defined. Of the several glycoproteins on which much attention has been focused is a family of proteins called cadherins. Cadherins are transmembrane glycoproteins that mediate Ca2+-dependent adhesion of cells. The extracellular domains of cadherin self-associate via Ca2+ binding and bring about intercellular adhesion. The cytoplasmic domain of the glycoprotein binds several cellular proteins, e.g. ␣- and ␤-catenin and plakoglobin, which link it to the cytoskeletal system (see Sherbet and Lakshmi, 1997; Sherbet, 2001). In this way cadherin forms an important part of a cell adhesion and signal transduction mechanism. Much has been understood about its participation in intercellular interactions and the transduction of the extracellular signals into the cell, and its loss in neoplastic transformation leading to the conferment of invasive behaviour upon transformed cells. E-cadherin, for instance, is not expressed in cancer cells. There is currently a large body of evidence upon which it has been suggested that E-cadherin subserves the function of an invasion suppressor. But very little is known about the possible mechanisms by which such a loss of expression of cadherins occurs in cancers. Microsatellite instability could be a pathway for actuating the loss of cadherins and this possibility has been studied recently. It has been reported that microsatellite instability produces certain changes in the gene coding for E-cadherin. Efstathiou et al. (1999) found a low level of E-cadherin mutation in colon carcinoma cells. In three RER+ cell lines frame-shift single base deletions occurred in exon 3 in the repeat regions of the gene that are similar to microsatellite repeats. This seems to have led to the truncation of the protein at codon 216 in the three RER+ cell lines. At the phenotypic level, this mutation was accompanied by loss of expression as well as function of E-cadherin. When these cells were transfected with full-length E-cadherin cDNA, there was full restoration of E-cadherin function in the form of increased intercellular adhesion and induction of differentiation, together with inhibition of cell proliferation and tumorigenicity. Furthermore, Efstathiou et al. (1999) noticed that E-cadherin mutation occurred more frequently in RER+ (70%) than in RER– (7%) cells. Ilyas et al. (1997) found alterations in E-cadherin expression in 38% of colorectal cancers, but these occurred at similar levels in RER+, RER– and ulcerative colitis-associated cancers. Also, allelic loss occurred at the E-cadherin gene locus but there were no differences in the levels of allelic loss between the three groups. New alleles at exon 16 were detected in 14% (9 of 22) RER+ tumours, whilst none was found in RER– or ulcerative colitis-associated tumours. Overall therefore, Ilyas et al. (1997) have concluded that there is no apparent relationship between replication error phenotype and the loss of cadherin function. Recent work by Shinmura et al. (1999) seems to support this conclusion, for they detected no correlation between RER+ phenotype and

Replication error (RER) and genetic instability

33

E-cadherin expression in gastric cancers. Downstream in the cadherin-APC-␤ catenin pathway of signal transduction, colorectal tumours might show APC and ␤-catenin mutations, but only infrequently (O Muller et al., 1998). Muller et al. (1998) did find one RER+ tumour with ␤-catenin mutation, but no germ-line mutations in patients with familial adenomatous polyposis (FAP). Most of the studies discussed above focus on abnormalities of E-cadherin gene in association with the RER phenotype. Another possible means to establish a relationship between the E-cadherin expression and genetic instability is to look at possible epigenetic changes of the gene. An aberrant methylation of E-cadherin gene is said to occur in AML, ALL and in breast cancer (Corn et al., 2000; Nass et al., 2000). The methylation of CpG islands in ER and E-cadherin genes was far more frequent in metastatic tumour than in ductal carcinomas in situ of the breast (Nass et al., 2000). It should be borne in mind that epigenetic changes of DNA methylation can significantly alter genetic stability. But neither study has had a look at the methylation status of mismatch repair genes.

Microsatellite instability and metastasis suppressor nm23 gene abnormalities It ought to be recognised as quite paradoxical that abnormalities associated with a gene locus may not always correspond with the intrinsic instability indicated by microsatellite markers. The nm23 is a putative metastatic suppressor gene occurring on chromosome 17q22 and its expression is reported, albeit not consistently, to be inversely related to metastatic potential of murine tumours as well as certain human cancers (see Sherbet and Lakshmi, 1997; Sherbet, 2001). Although transfection of nm23-H1 and H2 into cells has resulted in suppression of metastatic ability (Miyazaki et al., 1999), this putative function of the gene or its relevance of 5-year survival of patients (Nesi et al., 2001) is still a matter of debate. Loss of heterozygosity at the nm23 locus and/ or mutations of nm23 have been correlated with cancer progression to the metastatic stage, and inevitably a correlation has been sought between these genetic alterations of nm23 and genomic instability. Caligo et al. (2000) found no LOH of nm23-H1 in pancreatic tumours even though 41% (of 34) of the tumours showed microsatellite instability, and this is in spite of instability associated with 17q22 at which nm23 is located. Gomez et al. (1996) have expressed the view that allelic loss on chromosome 17, on which nm23, p53, BRCA1, and erb-B2 are located, may not be relevant in melanomas. These reports support the earlier findings of Patel et al. (1994a, 1994b) that there is no relationship between LOH of nm23 and microsatellite instability in colorectal or breast cancers, and the findings of Hayden et al. (1997), who had reported no microsatellite instability in association with allelic loss of nm23 in gastric adenocarcinomas. However, according to Indinnimeo et al. (1998), nm23 mutations in rectal carcinomas correspond with microsatellite instability.

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Table 1 Mutation status of mismatch repair genes, proliferation-related and growth factor receptor genes Mutation status Mismatch repair genes High Low

Apoptosis genes

Cell cycle regulatory genes

Growth factor receptor genes

DNA damage response genes

++ +

++ +

+++/++ +

+ ±

This table summarises the data discussed in the text and those collated by Duval and Hamelin (2002) and shows the apparent correlation between mutation status of mismatch repair genes and that of groups of genes associated with tumour growth. High mutation of mismatch repair genes correlates with these genes in some forms of human cancer. Mutation frequency + <21%; ++ 21–50%; +++ >51%; ± ambiguous.

Bosnar et al. (1997) had noticed that LOH of nm23 in renal carcinomas was closely related to microsatellite instability. Such discordant findings are often recorded in respect of nm23 and are in general compatible with the views often expressed that nm23 abnormalities may not be related to metastatic spread. This does not suggest that nature has undertaken a futile exercise of genetic alteration but simply that nm23 may not exercise the metastasis suppressor function that has often been attributed to it. These changes might be related to some other feature of the cancer cell such as, for instance, the control of telomere dynamics. One cannot gainsay the fact that microsatellite instability is associated with genetic alterations in a number of genes that affect certain biological processes characterising tumour development and expansion. It is also quite obvious from the preceding discussion that mutation of mismatch repair genes corresponds closely with mutation of some of these genes, especially those closely implicated in tumour growth, e.g. genes controlling cell proliferation and apoptosis and those encoding growth factors (Table 1). It should be recognised though that there is little evidence to date that microsatellite instability is associated with the processes of angiogenesis, tumour invasion and distant deposition of cancer cells and their growth into overt metastases. Since dormant metastatic deposits could be triggered by inappropriate expression of growth factors into forming overt metastatic disease, it would be prudent not to exclude that possibility.

Chromosomal fragile sites and the RER phenotype The mammalian chromosome contains sites, which are called fragile sites, that are visualised as gaps or breaks in the chromosome. The fragile sites may be

Replication error (RER) and genetic instability

35

constitutively conserved sites or may be induced. Fragile sites are active loci for genetic recombination, such as chromosomal translocations, inversions, and sister chromatid exchanges (SCE). A variety of chemical carcinogens and mutagens induce a large number of recurrent fragile sites, and a significant proportion of these occur at the location of important genes that have been consistently implicated in carcinogenesis (Yunis et al., 1987).

Sister chromatid recombination and fragile sites Sister chromatid exchanges (SCE) are a form of genetic recombination that involves the exchange of homologous double stranded DNA segments between chromatids. The occurrence of sister chromatid recombination (SCR) is an indication that genetic recombination is activated. SCR is enhanced in cells transformed by chemical carcinogens, which seem to generate SCR-inducing lesions in the DNA. Enhanced levels of recombination are also found in a variety of human malignancies and syndromes resulting from DNA repair abnormalities. The induction of SCE by ethylnitrosourea and methylnitrosourea appears to be closely related to the ability of the cells to repair the DNA damage in the G1-phase of the cell cycle (Ganzalez-Beltran and Morales-Ramirez, 1999). Deficiency of DNA repair occurs in certain autosomal recessive disorders, e.g. xeroderma pigmentosum (XP), ataxia telangiectasia (AT) and Bloom’s syndrome (BS). Both XP and AT syndromes are associated with deficiency of repair of DNA damage induced by ionising radiation. Bloom’s syndrome is characterised by chromosomal fragility, which is reflected in a marked increase in the incidence of SCEs (see Sherbet and Lakshmi, 1997). This seems to be caused by the loss of a nuclear protein encoded by a gene called BLM, which is not expressed in BS. Ellis et al. (1999) have demonstrated this by transfecting normal BLM cDNA into BS cells. The transfected cells expressed BML protein at high levels and this was accompanied by a marked reduction in SCE frequency as compared with BS cells transfected with only empty vector. The BLM gene codes for a DNA helicase, and this gene is mutated in BS, which results in increased abnormalities of homologous recombination (GB Luo et al., 2000; Van Brabant et al., 2000). The chromosomal fragility seems to derive from deficiency of a repair enzyme called DNA ligase 1 (Willis and Lindahl, 1987). The repair of DNA damage also involves the synthesis of poly (ADP-ribose) polymerase (PARP). PARP is a nuclear enzyme that is synthesised in response to DNA damage. It is activated upon binding to DNA strand breaks. PARP binds to DNA via its zinc finger domains, links poly (ADP-ribose) to the nicks in the DNA and maintains its structural integrity until excision repair of DNA is carried out (see Sherbet, 2001, for references). SCR occurs under conditions where DNA repair is inhibited. It is to be expected therefore that PARP would be involved in SCR.

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Indeed, over-expression of PARP brought about by using transfecting hamster cells with PARP-1 leads to the inhibition of SCR induced by alkylating agents (Meyer et al., 2000). Compatible with this negative regulation of recombination, the PARP inhibitor 3-aminobenzamide (3-AB) greatly enhances SCR incidence (Morgan and Cleaver, 1982). Although the effects of 3-AB have always been attributed to an inhibition of PARP, there might be other mechanisms involved, as in the case of its effects on SCE enhancement in Down’s syndrome (Caria et al., 1997). Nonetheless, in investigations using a variety of strategies such as PARP inhibitors (Morgan and Cleaver, 1982; Burkle et al., 1990), dominant negative mutants (Schreiber et al., 1995), and employing antisense PARP RNA expression (Ding et al., 1992; Ding and Smulson, 1994), PARP inhibition is demonstrably associated with enhanced SCE, as well as other nuclear abnormalities such as the formation of micronuclei, enhanced DNA strand breakage and recombination, and gene amplification, all features generally regarded as indicators of genetic instability. Historically, a high level of incidence of sister chromatid recombination has been described in a large number of human cancers and cancer derived cell lines (see Sherbet, 1987). In metastatic variants of the B16 murine melanoma, SCR frequency seems to reflect metastatic potential (Sherbet, 1987; Lakshmi et al., 1988), with B16–ML8 line derived from a pulmonary metastasis of the B16–BL6 showing the highest incidence of SCR (Table 2). Furthermore, SCR appears to be closely related to and, in fact, occurs in, aneuploid cell populations (Lakshmi et al., 1988). In the context of human neoplasia, equally important is that the level of SCE occurrence could provide valuable information about the progression of the disease. There is evidence that SCE incidence is far greater (9.24 per metaphase) in peripheral blood lymphocytes (PBL) of patients with prostate carcinoma than in control subjects (5.94 per metaphase) (Dhillon and Dhillon, 1998). High Table 2

Sister chromatid recombination frequency in relation to metastasis

Tumour cell line

Metastatic potential

SCR frequency % of cells scanned

SCR/chromosome

B16–F1 melanoma B16–F10 B16–BL6 B16–ML8 IJKt human astrocytoma G-UVW astrocytoma

Low Moderate High High N.D. N.D.

3.0 12.5 28.9 40.1 2.0 4.0

0.07 0.09 0.09 0.07 0.1 0.04

Source: Based on Lakshmi et al. (1988); Sherbet (1987) N.D.: Not determined

Replication error (RER) and genetic instability

37

frequency of SCE incidence in Hodgkin’s disease appears to relate to the development of a second tumour (Strom et al., 1998). This has potential significance in patient management. Furthermore, PBL from patients with ulcerative colitis (UC) show a far greater incidence of SCE than do lymphocytes from control subjects. Generally telomeric associations (TAS) and chromosomal aberrations were far more frequent in UC patients than in controls. A few of the breakpoints involved in chromosomal aberrations corresponded with breakpoints found in colorectal carcinomas (Cottliar et al., 2000a). There is a thinly veiled suggestion here that the SCE status of UC patients could be reflecting an intrinsic chromosomal instability that could lead to development of colonic cancer. In other words, the high SCE in UC patients might indicate a predisposition to carcinogenesis. Roy et al. (2000) have amply demonstrated such a predisposition to breast cancer pathogenesis. They examined PBL from hereditary breast cancer (HBC) patients, their healthy relatives (HBR) and a group of unrelated control subjects, for chromosomal aberrations as well as for SCE incidence. Their results make interesting reading. The incidence of both chromosomal aberrations was far greater in HBC and HBR than in the control group. Chromosomal aberrations were 2.5-fold greater in HBC patients than in the HBR group. But the two groups were much closer to each other in respect of SCE incidence. This work suggests that susceptibility to develop breast cancer in subjects with familial history of the disease may be reflected in the level of SCE. Furthermore, these findings suggest that enhanced chromosomal instability might be a predisposing factor in hereditary breast cancer. SCRs may involve exchanges of equal chromatid segments; exchange can infrequently be unequal or inverted. In these cases, SCR can lead to altered patterns of gene expression and amplification of genes occurring at the sites of exchange. The incidence of double minute chromosomes (DM), which are

Table 3 Relationship between sister chromatid recombination frequency and incidence of double minute chromosomes Cell type RPMI melanoma MEL57 melanoma G-UVW astrocytoma B16–BL6 melanoma B16–F1 B16–ML8 B16–F10 IJKt astrocytoma

SCR/chromosome

DM/chromosome

0.01 0.0355 0.045 0.07 0.07 0.07 0.0914 0.098

0.014 0.022 0.016 0.023 0.038 0.043 0.059 0.031

Source: Data from Lakshmi and Sherbet (1989)

38

Genetic Recombination in Cancer

thought to represent gene amplification, show a very close correlation with that of SCR (Table 3). Sherbet and Lakshmi (1997) have given a schematic presentation as to how DMs can occur as a consequence of unequal SCR. However, an occurrence of an unequal recombination has not yet been demonstrated. Nonetheless, these findings are compatible with the view that gene amplification is an event often found to be associated with metastatic ability. SCR are non-random events. Albeit using only a few tissue culture cell lines of human astrocytomas and melanomas, Lakshmi and Sherbet (1990a) have recorded that some chromosomes appear to possess a greater propensity to undergo SCR than others. Furthermore, a vast majority of SCR breakpoints were concentrated at chromosomal fragile sites (Tables 4 and 5). The recombination event also involves a number of important oncogenes and growth factor genes (Lakshmi and Sherbet, 1990a). At the time this was described as nothing more than could be expected. However, there is now adequate evidence that this

Table 4

Sister chromatid recombination at chromosomal fragile sites

Cell line

RPMI 5966 MEL 57 IJKt GUVW

SCRs detected

SCRs associated with fragile sites (%)

Fragile sites with no SCRs

108 41 70 11

76 47 73 72

33 17 33 14

Source: Data from Lakshmi and Sherbet (1990)

Table 5 lines

The non-random occurrence of SCRS in melanoma and glioma cell

Chromosome 2 4 5 13 14 15

RPMI 5966

MEL 57

IJKt

GUVW

+ + +

+ + + + + +

+ +

+ +

+

+

+ +

+ = chromosome showed more SCRs than expected in relation to chromosome length. Source: Data from Lakshmi and Sherbet (1990)

Replication error (RER) and genetic instability

39

correlation, identified a decade ago, indeed supports the currently held view that not only SCR but also other forms of chromosomal recombination are initiated by chromosomal fragility.

Microsatellite instability and sister chromatid recombination It is therefore of considerable interest to examine whether microsatellite instability is involved in the recombination events. In early experiments, Foucault et al. (1996) were unable to detect any relationship between the mutation frequency of two hyper-mutable microsatellites and SCE incidence in BS cells. Limoli et al. (1997) generated genetically stable and unstable clones from a human–hamster hybrid cell line exposed to X-ray damage. They found no differences between these clones in the incidence of SCE, delayed mutation or in mismatch repair. Durant et al. (1999) have approached this question by using the relationship between loss of mismatch repair and the development of resistance to DNA damaging agents. They found that in human ovarian tumour cells, the loss of hMLH1 correlates not only with cisplatin resistance but also with increased cisplatin-induced SCE. However, the induction of SCE by methylnitrosourea (MNU) appears to be no different in mouse bone marrow cells, which are MSH2 (+/+), than in cells that are MSH2 (+/–) or MSH2 (–/–) (Bouffler et al., 2000). Similarly, neither of the human mismatch repair genes hMSH2 and hMSH1 is involved in conferring chromosomal instability in lymphoblastoid cells derived from hereditary non-polyposis colon cancer (Lindor et al., 1998). Thus overall there is very little support for the thesis that this form of recombination is related to abnormalities of mismatch repair. Nonetheless, a strain of evidence, which does support a link of chromosomal instability with SCR is that based on the involvement of chromosomal fragile sites in other chromosomal recombination events such as translocations.

Genomic stability and chromosome structural dynamics The structural dynamics of chromosomes is a facet of genomic stability that is closely associated with the life of the cell in health and disease. The roles that chromosome structure plays are largely a reflection of the functions subserved by chromosome-associated proteins. A major family of chromosome-associated proteins is composed of proteins known as the ‘structural maintenance of chromosome’ (SMC) proteins. These proteins have been highly conserved in evolution and occur in prokaryotic as well as eukaryotic chromosomes. SMC proteins have been implicated in the performance of a variety of functions related to the maintenance of the structural integrity of the chromosome and in

40

Genetic Recombination in Cancer

genomic stability. They have been associated with the cohesive bonding between sister chromatids, chromosomal condensation, gene dosage compensation as well as in DNA recombination and repair and cell cycle progression. SMC proteins may be sub-grouped into what are descriptively called ‘condensin’ and ‘cohesin’. They represent heterodimers of four SMC proteins, namely SMC1–4. The condensin sub-group, composed of complexes of SMC2 and SMC4, takes part in chromosome condensation and sex chromosome dosage compensation, whilst cohesin, which is made up of SMC1 and SMC3, participates in the cohesion of sister chromatids and in genetic repair and recombination (see Strunnikov, 1998; Strunnikov and Jessberger, 1999; Ball and Yokomori, 2001). Both condensin and cohesin complexes show cell cyclerelated distribution in the cell. In the interphase, the cohesin complex is associated with chromatin and condensin is found mainly in the cytoplasm. In mitosis, cohesin dissociates from chromosomes but condensin associates with them. SMC1 and SMC3 may show specific association with meiotic chromosomes (Eijpe et al., 2000; Revenkova et al., 2001). DNA repair involving SMC1 seems to be mediated by ATM kinase, which itself functions as a tumour suppressor protein being intimately involved in DNA damage response and initiation of apoptosis. ATM phosphorylates specific serine residues of SMC1. This phosphorylation may also be carried out by other kinases. Also required are the ATM substrates BRCA1 and NBS1 (ST Kim et al., 2002). BRCA1 has already been associated with DSBR mediated by DNA-PK. Both BRCA1 and BRCA2 are tumour suppressor proteins. They appear to function in conjunction with RAD51, a DNA repair and recombination protein, a paralogue of which has been described as influencing sister chromatid cohesion (Godthelp et al., 2002). Similarly RAD50 function is akin to SMC (Hartsuiker et al., 2001). From this point it is not a far cry to cell transformation. From some recent work by Ghiselli and Iozzo (2000), a concept is emerging of the participation of SMC proteins in cellular transformation. These authors transfected full-length cDNA of SMC3 into Balbc/3T3 and NIH 3T3 cells and demonstrated that the transfectants displayed anchorage independent growth in vitro together with enhanced expression of SMC3. Interestingly, they also report enhanced expression of SMC3 in murine and human colon carcinoma cells and human colonic tumour specimens. Although these findings are interesting, further work is required for the elucidation and link-up of SMC participation and the nature of its perceived participation in the neoplastic process.

4 DNA repeats, genetic recombination and the pathogenesis of genetic disorders A characteristic feature of DNA repeats that is of major consequence to the disease process is their fixed and irreversible nature and transmission to descendants. DNA repeats are associated with a variety of human genetic disorders and this association can be attributed to their involvement in genetic reorganisation, mutations and deletion or duplication of DNA segments. The formation of extra-chromosomal DNA bodies called double minute chromosomes or their integration into the genome resulting in the cytologically demonstrable homogeneously staining regions of the chromosome can occur from homologous or non-homologous recombination involving repeat elements. From such genetic modifications flow a variety of consequences to developmental mechanics, cellular physiology, cell proliferation and apoptosis, which are at the basis of the pathogenesis of an array of human genetic disorders. DNA repeats may be found in a tandem arrangement, such as the trinucleotide repeats occurring in a number of genetic disorders. Repeat elements that are interspersed in the genome are known as short interspersed nuclear elements (SINE) or long interspersed nuclear elements (LINE). Other forms of DNA repeat of consequence to human health are transposable elements, such as those integrated into human genome in the form of retroviruses and often associated with neoplastic transformation of cells. The association between DNA repeats and human genetic disease is shown in Genetic Recombination in Cancer ISBN 0-12-639881-X

Copyright © 2003 Elsevier Science Ltd All rights of reproduction in any form reserved

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Genetic Recombination in Cancer

Table 6

Association of DNA repeat elements in human genetic disorders

DNA repeat-associated genetic recombination Intrachromosomal

Interchromosomal

Deletions Muscular dystrophies Neoplasms Cell cycle regulatory genes Mismatch repair genes Tumour suppressor genes Duplication Neoplasms

Translocation Gene fusion

DNA repeat-associated genetic disorder

SCA Huntington’s disease DRPLA SBMA Fragile X syndrome HNPCC OPMD Myotonic dystrophy Friedrich’s ataxia

DRPLA, Dentatorubral-palliodoluysian atrophy; HNPCC, hereditary non-polyposis colon cancer; OPMD, oculopharyngeal muscular dystrophy; SBMA, spinobulbar muscular atrophy (Kennedy’s disease); SCA, spinocerebellar ataxia.

Table 6, and here the discussion is focused on the nature of genetic changes engendered by the repeat tracts and possible mechanisms and pathways of pathogenesis of human disease with which they are implicated.

CAG repeat expansion and genetic instability The multitude of DNA repeats occurring in the genome is an important focus of genetic instability and mutation. Some repeats, such as CAG, appear to be unstable in DNA replication, others constitute chromosomal breakpoints and become a source of chromosomal translocations and genetic recombination, and yet others such the Alu sequences, may cause mismatch defects during the replication of DNA. These nucleotide repeats possess the potential to function as transposable elements and produce insertion mutations. It is little wonder, therefore, that in many genetic diseases an expansion of triplet nucleotide repeats within the disease locus engenders genetic instability. As alluded to earlier, microsatellite sequences tend to be focal points of genetic instability. We have noted that microsatellites are short nucleotide sequences of 1 to 6 bases, which are repeated at tandem. The triplet nucleotide repeats may be regarded as a particular class of microsatellites. An expansion of the CTG triplet occurs in myotonic dystrophy, an autosomal dominant disorder that affects distal limb, and facial and neck muscles. However, it is uncertain whether the length of repeats is related to the morphological abnormalities associated with the dystrophy; but severe dystrophy seems often to be

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 43

associated with high numbers of repeat elements. Indeed there is a view that high repeat frequency might correlate with predisposition to the disease (Gennarelli et al., 1999). CAG repeat tracts are characteristic of spinocerebellar ataxia (SCA). Several variants (SCA1–7, SCA12) of SCA are known. SCA are an autosomal dominantly inherited neurodegenerative disorder. Associated with the disorder are a family of proteins called ataxins, voltage-gated sub-type P/Q calcium channel protein, and the regulatory subunit of the holoenzyme protein phosphatase 2A, PP2R2B (Table 7). Quite obviously the clinical manifestations of CAG repeat disease syndrome are attributable to a number of factors. The ataxin-1 gene SCA1 and ataxin-3 encoded by the gene that causes SCA3, also known as Machado–Joseph disease (MJD), contain expanded polyglutamine (CAG repeat) tracts within the genes. A similar expanded polyglutamine tract of Huntingtin protein characterises Huntington’s disease. This expanded polyglutamine constitutes a gain of function mutation of the encoded protein and the mutant protein is believed to cause neuronal degeneration. The polyglutamine-containing mutant proteins are found in nuclear inclusions with other proteins that might be functioning as transcription factors. A sequestration of these will have obvious implications for gene transcription. Polyglutamine repeat tracts are also found in the transmembrane ␣1 subunit, which is the pore-forming protein of P/Q calcium channels. This abnormality might be expected to alter calcium channel activity of nerve terminals and also affect neurotransmitter release. The protein phosphatase called PP2A is intricately involved in the regulation of cell proliferation and motility and calcium signalling as well as in the regulation of gene transcription (see Sherbet, 2001). PP2A is a holoenzyme and is composed of three subunits, namely the catalytic C subunit, structural A subunit and the regulatory B subunit. The core complex consists of subunits A and C. The B subunit binds the core complex and regulates the activity of the enzyme. The occurrence of CAG repeats in the 5’ region of the gene encoding isoform of PP2R2B of the regulatory subunit is associated with SCA (Holmes et al., 1999). With a wide-ranging function, it is conceivable that abnormalities associated with PP2A could result in the pathogenesis of SCA12, but a direct causal relationship is yet to be demonstrated. Possibly, PP2A might affect the phosphorylation status and thereby influence the function of the transcription factor, the cAMP response element binding protein (CREB). On the other hand, McCampbell et al. (2000) showed that in spinobulbar muscular atrophy (SBMA) and in SCA3, CREB is sequestered in neuronal intranuclear inclusions, and in this way influences CREB function. They demonstrated a reduction in CREB levels in cells expressing CAG repeat expansion. Furthermore, enhancing the expression of CREB negates polyglutamine-induced cytotoxicity. As stated in a later section, CGG repeat motifs occur in the FMR1 gene of fragile X syndrome. The expression of FMR1 is lost in fragile X and this is by all accounts related to epigenetic alterations in the repeat elements.

44 Table 7

Genetic Recombination in Cancer DNA nucleotide repeats and their association with genetic diseases

Nucleotide repeat motif

Associated disease syndrome

(CAG) (n)

Huntington’s: Normal Disease state carcinoma of the prostate, breast and endometrium

Mutation range Repeats (n)

6–35 38–180

Pathogenesis and related mechanism

Polyglutamine containing mutant toxic proteins, e.g. huntingtin, and ataxins. Wildtype huntingtin reduces toxicity of mutant protein; involvement in RNA metabolism; interaction with Ca2+-binding proteins; found in intranuclear inclusions, sequestration of CREB transcription factor and affects gene transcription, CAG repeat instability in gametes; in yeast instability associated with PMS1 and MSH2 mutations Ataxin-1

SCA1: Normal Disease state

6–39 40–88

SCA2: Normal Disease state

14–32 33–77

SCA3 (MJD): Normal Disease state

12–40 55–86

SCA6: Normal Disease state

4–18 21–31

SCA7: Normal Disease state

7–17 34–306

SCA12: Normal Disease state

<29 66–93

DRPLA: Normal Disease state

3–36 49–88

Ataxin-2

Ataxin-3 Stability of repeat tract related to configuration of (CAG) (n); length correlates with age at onset and clinical phenotype Mutated P/Q calcium channel ␣1 transmembrane poreforming protein subunit, altered calcium channel activity Ataxin-7

Protein phosphatase PP2A regulatory subunit PP2R2B Atrophin-1

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 45 Table 7

Continued

Nucleotide repeat motif

(CTC) (n) (CGG) (n)

Associated disease syndrome

Mutation range Repeats (n)

Pathogenesis and related mechanism

Repeats in androgen receptor gene, abnormal activation of T-type Ca2+ channel, induction of apoptosis

SBMA: Normal Disease state

9–36 38–65

SCA8: Disease state

107–127

Fragile-X: Premutation allele Full mutation Normal subjects

55–<200 >200 6–54

Loss of FMR1 expression Length of repeat tract in cell lines related to hMLH1 and hMSH2 mutation

Premutation range

Repeat lengths related to hMLH1 abnormality

Non-coding expansion

(CGG) (n)

HNPCC and normal subjects (peripheral blood leucocytes)

(CGG) (n)

Early embryogenesis

Stability coincides with methylation

(CTG) (n)

Myotonic dystrophy: Severe dystrophy Intermediate–mild Normal

80–4000 35–80 5–35

High frequency of repeats could be indicative of predisposition to disease; in cell lines related to hMLH1 and hMSH2 mutation

OPMD Recessive/ homozygous Dominant Normal

7 8–13 6

Friedrich’s ataxia: Full mutation Normal

200–900 7–22

(GCG) (n)

(GAA) (n)

Loss of mitochondrial protein (frataxin); overload of mitochondrial iron and oxidative stress

The nucleotide sequences CCG, CGG and GCC are identical, so also are AGC and CAG. Although only CAG are indicated in the Table, SCAs are caused by expansion of CAG or CTG motifs. DRPLA, Dentatorubral-palliodoluysian atrophy; MJD (SCA3), Machado–Joseph disease; HNPCC, hereditary non-polyposis colon cancer; OPMD, oculopharyngeal muscular dystrophy; SBMA, spinobulbar muscular atrophy (Kennedy’s disease); SCA, spinocerebellar ataxia. Sources: Data collated from references cited in text: Benton et al. (1998); Burman et al. (1999); Ellerby et al. (1999); Evert et al. (2000); Fusco et al. (2000); Gennarelli et al. (1999); Hilditch-Maguire et al. (2000); Holmes et al. (1999); Klockgether and Evert (1998); Koeppen (1998); Koob et al. (1999); Leavitt et al. (2001); McCampbell et al. (2000); Riess et al. (1997); Sculptoreanu et al. (2000); Vig et al. (2000); Wheeler et al. (1999); Yamada et al. (2000); Yue et al. (2001); and DNA Repeat Sequences and Disease at http://www.neuro.wustl.edu/neuromuscular/mother/dnarep.htm

46

Genetic Recombination in Cancer

Caspases are cysteine-proteases that are involved in neuronal apoptosis. Caspase inhibitors inhibit neuronal apoptosis induced by ischaemic injury. The over-expression of certain caspases induces apoptosis of prostate cancer cells (see Sherbet, 2001). A super family of nuclear receptors composed of androgen receptors (AR), oestrogen receptors (ER), progesterone receptors (PgR), retinoic acid receptors and vitamin-D3 receptors, among others, participate in transducing extracellular ligand signals to the nucleus and function as transcription factors. These receptors also function in cell differentiation, cell proliferation and apoptosis, and it is needless to say that they are involved in the development of cancers in their target organs. It is of much interest to note, in this context, that exon 1 of the AR gene contains tracts with CAG tracts. The lengths of the CAG repeats show some relationship to cancer development, although the nature of correlation seems to depend upon tumour type. Short CAG repeats have been reported to carry with them a higher risk of developing prostate cancer (Giovannucci et al., 1997; Ingles et al., 1997). Besides, Cude et al. (2002) found that the presence of shorter CAG repeat lengths correlated with tumour grade but not with disease progression or patient survival. But Shibata et al. (2002) have failed to find any correlation between CAG polymorphism and histological grade, nor with other established markers such as PSA. The APS gene that encodes PSA is regulated by androgens and therefore one would expect a modulation of PSA expression if there were influences of CAG polymorphism in AR function. Attenuation of CAG repeats appears to be associated also with colonic cancer. Normal colonic mucosa expresses two isoforms of AR, namely 110 and 87 kDa. However, in colonic cancer only the shorter isoform is expressed. This 87 kDa AR is truncated by the deletion of an N-terminal region, which also contains the CAG repeats (Ferro et al., 2002). In contrast, shorter CAG repeat tracts might confer protection from breast cancer (Rebbeck et al., 1999; Elhaji et al., 2001; Giguere et al., 2001). The presence of longer CAG repeat tracts seems to be associated not only with breast cancer but also with endometrial carcinoma (Sasaki et al., 1999). An intensive investigation by Haiman et al. (2002) has indicated that women with one or more long CAG repeats (≥22) do not necessarily carry a higher risk of breast cancer. Also they found no relationship between breast cancer risk and average repeat length. However, longer repeats may be associated with higher risk in patients who have a familial history of breast cancer. This in some way goes towards reconciling the apparent dichotomy of function of CAG repeats in AR in the pathogenesis of breast cancer. Quite obviously other factors, possibly genes such as BRCA1, might be a complicating familial factor together with the CAG repeat polymorphism. This is patently obvious from the finding that women who have longer CAG repeats carry a higher risk of developing ovarian cancer (Santarosa et al., 2002), again indicating potential BRCA1 involvement in the disease process. The possibility that a similar situation might subsist in the pathogenesis of prostate cancer has not been addressed with equal vigour.

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 47

There might be more to the question of CAG repeats and functioning of AR than meets the eye. Shorter CAG repeat lengths also seem to be associated with the pathogenesis of hepatocellular carcinoma (HCC). There is a suggestion that CAG polymorphism might function by entirely different mechanisms in the pathogenesis of HCC in men and women (MW Yu et al., 2002). Also, the presence of hepatitis B virus infection appears to be an important element that has to be taken into the reckoning (Yeh et al., 2002; MW Yu et al., 2002). One has to take account of the fact that in the normal population CAG repeat length ranges from 6 to 40 repeats and <40 occur in a variety of genetic conditions (Table 7). It seems imperative that whenever repeat lengths are defined the corresponding values for a comparable normal population should also be made available. One cannot over-emphasise the importance of appropriate design and definition of study together with stringent sample comparability. CAG expansion occurs with SBMA syndrome. Ellerby et al. (2000) have articulated these arguments with the finding that expression of AR with increased CAG tracts increases apoptosis, and furthermore caspase-3 mediated cleavage of AR is associated with apoptosis. The association of CAG repeat attenuation in AR with pathogenesis might be a reflection of the modulation of the mitogenic function of androgen by CAG polymorphism of AR in the target tissues. Thus several pathways can be identified leading to neuronal cell loss associated with CAG repeat syndromes. The activation of the caspase cascade might be the crucial factor. Brooks et al. (1997) had earlier transfected a neuronal cell line with AR cDNA containing 24 or 65 CAG repeats. In both transfectants, the AR functioned normally, notwithstanding the differences in the numbers of repeats carried. However, the level of expression of AR with 65 repeats was distinctly lower than that of AR containing 24 repeats. This suggests that levels of AR per se are important, and it is here that activation of the caspase cascade can bear significantly on neuronal apoptosis. Basically, the presence of CAG repeats seems to affect the ability and competence of AR as a transcription factor (Mhatre et al., 1993; Beilin et al., 2000). Finally, the CGG repeats in fragile X and microsatellite repeats are further examples of this phenomenon of association of triplet microsatellites with disease conditions. The trinucleotide-repeat tracts are often unstable in transmission, and the degree of instability seems to be related to the lengths of the repeat tracts. The reasons for the instability are not well understood. One possible factor could be the influence of the mismatch repair genes. As noted earlier, abnormalities in these genes affect the stability of microsatellite repeats in several biological systems. If this were the source of instability then one can view increased frequency of repeat tracts as evidence of destabilisation. On the other hand, stability might be conferred on them by mechanisms that involve the formation of loops and folds, possibly also aided by methylation. Indeed, methylation per se appears to be conducive to the stability of these repeats. Not infrequently,

48

Genetic Recombination in Cancer

the acquisition of stability by the repeat tracts is coincidental with methylation, as demonstrated for early embryonic development. Contrary to expectation, FMR1 alleles in the full mutation range appear to be intrinsically stable. This could be a result of the repetitive DNA sequences participating in loop-folding patterns. Nadel et al. (1995) noticed that (CGG) (n) tracts of FMR1 show intramolecular folding forming hairpin structures. The (CCG) (n) tract of fragile X and the (GAA) (n)/(TTC) (n) tract of Friedreich’s ataxia show loop folding. The formation of these seems to confer stability on the DNA molecule. Possibly, the loop structures might be stabilised by Hoogsteen hydrogen bonding or Hoogsteen and Watson–Crick bonding. The longer the (CCG) (n) tract the greater is the probability that they will form a hairpin loop. Furthermore, the hairpin upstream of the FMR1 is more prone to methylation than its duplex, leading to intramolecular stability and epigenetic suppression of its transcription (Catasti et al., 1999). In MJD, a polymorphism CGG→GGG downstream of (CAG) (n) stretch affects inter-generation stability of the repeat tract; the fidelity of replication of the latter is affected. This effect is not only cis to the disease chromosome but also on the normal chromosome, which suggests that the normal and the expanded alleles interact (Maciel et al., 1999). Other factors such as the configuration of (CAG) (n) repeat tract could be operating in generating instability and determining the length of the repeat tract, as Matsumura et al. (1996) showed some time ago. They reported that the (CAG) (n) tracts occur in two configurations, either followed by C or G. In normal alleles, tract length is between 13 and 17 repeats. In MJD, with one expanded repeat allele, the tract length had increased to 64–84 repeats. In these patients, expanded alleles of only (CAG) (n)C configuration were found, whilst normal subjects and MJD with normal alleles had both (CAG) (n)C and (CAG) (n)G configurations. Matsumura et al. (1996) have suggested on the basis of these findings that the configuration of the repeats tracts also determines the stability of the repeats. The influence of mismatch gene expression has been investigated in relation to repeat tract stability or otherwise in many systems. Kramer et al. (1996) reported that mutations of mismatch repair gene hMLH1 and hMSH2, that contribute so much to genetic instability, have no influence on the instability of CGG or CTG repeat tracts of cell lines derived from fragile X or myotonic dystrophy patients. However, in hereditary non-polyposis cancers, the CGG tract length appeared to relate to the occurrence of mutated hMLH1 (Fulchignoni-Lataud et al., 1997). Schweitzer and Livingston (1997) examined the stability of CAG repeats in yeast strains that carried PMS1 and MSH2 mutations. In the wild-type cells the repeats were stable when CAG served as the lagging strand template but comparatively unstable when CTG served as the lagging strand template. However, when accompanied by PMS1 and MSH2 mutations, the repeats gained increases in tract length. It would appear therefore that the mismatch repair system does contribute to the stability of the repeat tracts.

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 49

Microsatellite dinucleotide repeats in insulin-dependent diabetes mellitus and autoimmune thyroid disease Microsatellite markers are also valuable tools in mapping genetic linkage and recombination. Allelic diversity and heterozygosity are important features for the establishment of microsatellite markers for linkage studies. The notch signalling pathway plays a major role in the differentiation of many cell types. The interactions of cell types leading to the determination of cell fate during embryonic development involve notch proteins and their ligands. The deregulation of notch signalling is a key factor in the pathogenesis of haematological malignancies. Several factors, e.g. interacting pathways and regulators of the pathway, influence the flow of information along the notch signalling pathway. The human SEL1L gene, which has been postulated and not infrequently and not indisputably assumed to be a negative regulator of the notch signalling pathway, occurs on chromosome 14q24.3-q31 (Field et al., 1996) proximal to the locus that is believed to confer susceptibility to insulin-dependent diabetes mellitus (IDDM)-11. SEL1L expression seems to be confined to adult pancreatic cells, both acinar and ␤-cells (Biunno, Appierto et al., 1997; Donoviel et al., 1998; Harada et al., 1999). Furthermore, sel-1 in C. elegans (Grant and Greenwald, 1997) and Hdr3 in S. cerevisiae (Hampton et al., 1996) have putatively been assigned a role in protein processing and degradation. Defects in SEL1L in IDDM patients could lead to inappropriate processing of or failure to degrade an islet antigen. SEL1L has been implicated also in Grave’s disease, which is an autoimmune thyroid disease. Tomer et al. (1999) have reported evidence that the susceptibility gene for Grave’s disease is located in proximity of IDDM11 locus. This leads to the hypothesis that this region could represent a clustering of different genes related to different autoimmune phenomena, albeit without being responsible for associated pathogenesis. Chiaramonte, Sabbadini et al. (2002) recently determined the allele set frequency and average heterozygosity of two intragenic cytosine-adenosine microsatellite repeats, CAR/CAL and RepIN20, which have been identified in SEL1L gene. These are located in intron 2 and 20 respectively (Biunno et al., 2000). Dinucleotide microsatellite repeats tend to be more polymorphic than tri- or tetranucleotide repeats. Both CAR/CAL and RepIN20 microsatellite repeats are highly polymorphic in nature (Table 8) and in the light of their intragenic location Chiaramonte, Sabbadini et al. (2002) have suggested that they could provide an effective means for linkage analysis to clarify the potential role of SEL1L in conferring susceptibility to IDDM or Grave’s disease. However, there is a report that SEL1L might not be related to IDDM (Pociot et al., 2001). Furthermore, Ban et al. (2001) have excluded the involvement of SEL1L in autoimmune thyroid diseases as well. Nonetheless, these findings emphasise the importance of investigating the integrity of the notch signalling pathways in these disease

50

Genetic Recombination in Cancer

Table 8 Allele frequency of dinucleotide CA repeats CAR/CAL and RepIN20 polymorphisms in normal individuals from Northern Italy CAR/CAL (bp) 207 209 211 213 215 217 219 221 223 225

Allele frequency

RepIN20 (bp)

Allele frequency

40.43 1.08 0 29.79 2.13 1.06 2.13 22.34 0 1.06

237 239 241 243 245 247 249 251 253 255

3.19 2.13 26.6 1.06 1.06 8.51 30.85 18.09 5.32 3.19

Source: Chiaramonte, Sabbadini et al. (2002). (Reproduced with kind permission of Kluwer Academic Publishers.)

conditions. The possibility that the presence of these highly polymorphic repeats might mutate genes in the particular locus resulting in the deregulation of notch signalling as well as other phenomena related to the function of the locus deserves to be fully investigated.

Alu repeats in genetic recombination Alu repeats constitute a family of the most abundant of SINE elements occurring in the genome of primates. There are around 1.0–1.4 ×106 Alu repeats interspersed in the haploid genome. The density of Alu repeats is 2- to 3-fold greater in GC-rich than in AT-rich DNA (ZL Gu et al., 2000). Structural considerations suggest that they may be derived from the 7SL RNA component of signal recognition particles (SRP), reverse transcribed and integrated into the genome. Alu repeats are amplified by a mechanism called retroposition. They are transcribed by pol III, replicate within the genome via the agency of an RNA intermediate. The retroposition activity of Alu repeats is determined by the flanking as well as internal regulatory elements and by genes in the neighbourhood that regulate genetic transcription and transcription stability. Alu are GC-rich repeats of 300 base pairs (bp). Several subfamilies of Alu have been identified. Alu have been implicated in a wide range of functions, including DNA replication, regulation of genetic transcription, and signalling. Their mode of function is not fully understood, but what has been fully appreciated to date is that Alu repeats influence the genomic structure and

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 51

reorganisation, and are associated closely with intra- as well as interchromosomal recombination leading to gene fusion, duplication, deletion and mutations (see Deininger and Batzer, 1999; Rowold and Herrera, 2000). DNA sequences occurring in the neighbourhood of Alu repeats seem to be prone to duplication (Jurka and Pethiyagoda, 1995). Such duplication might be arranged as tandem repeats or as inverted repeat insertions produced by homologous recombination of Alu elements. The consequences of these genetic modifications have been investigated on several physiological phenomena and in a variety of genetic diseases and in neoplasia.

Intrachromosomal recombination in muscular dystrophy and Alzheimer’s disease mediated by Alu repeats The intrachromosomal recombination events mediated by Alu can lead to deletions or duplication of genetic material. In several human diseases, intragenic deletion is associated with Alu or LINE repeats. For example, Duchenne and Becker muscular dystrophy are characterised by large deletions in the dystrophin gene. Either or both Alu and L1 elements occur flanking the dystrophin gene. The deletion within the gene extends from exon 2 to 7. Hu et al. (1991a) reported some years ago that a duplication of exon 3 occurs by an Alu-mediated homologous recombination. Unequal sister chromatid exchange has also been implicated in other duplications detectable in the dystrophin gene (Hu et al., 1991b). However, there has been no subsequent evidence that can be adduced in support of this contention. Intron 7 is especially prone to deletions. It is a large intron of 112 kb size and 40% of its sequences are recognisable as inserts of LINE-1 and THE-1/LTR transposon-like elements. Alu sequences also occur in this intron. Intron 8 is shorter with 1.1 kb and contains no detectable insertions (McNaughton et al., 1997). The insertion of these repeats could be the source of instability of intron 7. Suminaga et al. (2000) have identified a breakpoint in Alu repeat of intron 1, which ligates with an L1 repeat in intron 7, with the deletion of the intervening 430 kb stretch encompassing exons 2 to 7. Intron 11 also could be a focus of Alu-mediated rearrangement. Ferlini and Muntoni (1998) have described a cluster of Alu, LINE1 and THE repeats in a 5’ position of intron 11 of dystrophin gene. Ferlini et al. (1998) have reported dystrophin gene abnormalities in a family with X-linked dilated cardiomyopathy. Here an Alu sequence occurring 2.4 kb downstream of intron 11 is involved in the rearrangement of the gene. This leads to the generation of several transcripts with intron 1a spliced between exons 11 and 12. These transcripts are truncated by the numerous stop codons in the Alu sequence. Ferlini et al. (1998) also report the interesting finding of what appears to be a tissue-specific expression and translation of these abnormal transcripts.

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A non-homologous intrachromosomal recombination has also been reported in the presenilin gene in Alzheimer’s disease. Two genes called PS1 (presenilin)-1 and PS2 are associated, in a mutated form, with the onset of Alzheimer’s disease. The wild-type PS protein is found in the endoplasmic reticulum and may be involved in calcium homeostasis. Mutation of these proteins could conceivably be interfering with calcium homeostasis and the calcium-mediated process of neuronal apoptosis (see Sherbet, 2001). Hiltunen et al. (2000) have found a 4.6 kb deletion in PS-1 corresponding to exon 9. The deletion is mediated by an Alu sequence in intron 8 and probably nonAlu repeats in intron 9. The autosomal recessive disorder Fanconi anaemia (FA) is characterised by severe alterations in the FA complementation group A gene (FANCA). Levran et al. (1998) identified a number of mutations in FA patients, and they described two deletions of 1.2 and 1.9 kb deletions in FANCA gene; both deletions included exons 16 and 17. Introns 15 and 17 were rich in Alu repeats. Furthermore, the same Alu repeat of intron 15 harboured the breakpoint, but breaks occurred in different repeats of intron 17. There is a general confirmation of the mediation by Alu in FA recombination. Morgan et al. (1999) investigated 26 cell lines, all of which carried mutations of different types. Intragenic deletions constituted 40% of mutant alleles, and the massive deletions excluded up to 31 exons. They found several breakpoints involved in the deletions. Interestingly, the number of breakpoints that occurred within an intron correlated highly significantly with the numbers of Alu repeats it contained. Aside from the genetic changes described above, one should take into account the implications for differential gene expression engendered, however indirectly, by LINE elements. The presence of L1 elements in fibroblasts derived from rheumatoid arthritis synovial fluid seems to render genomic DNA susceptible to hypomethylation with serious implications for genetic expression (Neidhart et al., 2000). Modification of genetic expression in this way could conceivably bring about changes in the behaviour of the cellular elements leading to pathogenesis. Neidhart et al. (2000) reported alterations in c-met expression. The c-met gene encodes the tyrosine kinase receptor for hepatocyte growth factor. Abnormalities of c-met expression are associated with invasive behaviour of tumour cells.

Intrachromosomal recombination in tumour suppressor genes in cancer Genomic deletions occur in tumour suppressor genes and genes regulating cell proliferation and such mutations are encountered frequently in the pathogenesis and progression of cancer. As discussed elsewhere in this book (see Chapter 3), homologous and non-homologous intrachromosomal recombination or

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 53

unequal exchanges and recombination between chromatids can bring about the duplication of genetic material, which is often visualised as HSRs or DMs. Drug resistant cell lines contain DMs and HSRs, which represent amplification of the multi-drug resistance gene. These DMs not only contain amplified DNA but they also harbour Alu repeats (Sognier et al., 1994), and one could tacitly assume that Alu might have played some part in the amplification process. The BRCA1 gene is associated with susceptibility to develop breast and ovarian cancer. It may be described as a classical suppressor gene, shows LOH in cancer families and somatic mutations may also occur in breast and ovarian cancers. The wild-type gene appears to act as a negative regulator of tumour cell proliferation and growth, and its expression might be altered in tumour progression. The predisposition to develop cancer is related to mutations and the abrogation of suppressor function of the gene. The BRCA1 protein is a zinc finger protein and therefore might participate in the regulation of gene transcription. Germ-line mutations of BRCA1 seem to result in total loss of transcription or lead to the production of a truncated product, which might be functionally impaired. BRCA1 mutations and deletion mediated by Alu repeats occur in a significant proportion in families susceptible to breast and ovarian cancer. A deletion of 14 kb fragment encompassing exons 1a and 1b involving transcription start sites, and exon 2 was reported in a family carrying mutation of BRCA1. The deletion seemed to result from an unequal crossover of Alu repeats (Swensen et al., 1997). Rohlfs et al. (2000) have found a 7.1 kb germline deletion in two families with breast and ovarian cancer. The deletion of exons 8 and 9 occurs as a result of homologous recombination between an Alu repeat in intron 7 with one in intron 9, leading to a frame-shift mutation. Another frame-shift mutation in BRCA1 occurs as the result of a 3-kb exon 17 deletion, again with the apparent involvement of two closely related Alu repeats (Montagna et al., 1999). However, Payne et al. (2000) reported that a complex mutation consisting of an inverted duplication and deletion in the gene was not identified with any breakpoints and homologous recombination at Alu repeats. Germ-line mutations of the APC gene are a significant feature of hereditary colon carcinoma. APC is a tumour suppressor gene and truncating mutations of this gene are associated with the early stages of colon cancer progression. The APC protein might interact with the transmembrane cell adhesion protein cadherin and, therefore, APC protein might also be involved together with cadherin in intercellular adhesion as well as in the signal transduction pathway (Sherbet and Lakshmi, 1997; Sherbet, 2001). Alu repeats seem to be involved in many APC mutations. Su et al. (2000) have found a large number of APC mutations in FAP families, rearrangements and deletions that appear to result from homologous or non-homologous recombination involving Alu repeats. A truncating mutation of APC is caused by the insertion of a 337-bp Alu into codon 1526 and the insertion has a poly (A) tail at the 3’ end, which suggests that the insertion occurs by retroposition (Halling et al., 1999).

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The nm23-H1 and H2 genes, which code for nucleoside diphosphate (NDP) kinases, have been regarded as putative metastasis suppressor genes. These genes are human homologues of nm23 first identified as a suppressor gene in murine tumours. The levels of expression of nm23 or the NDP kinase show an inverse correlation with metastatic behaviour in many experimental tumour models as well as in human cancers. Equally there is much disagreement about the metastasis suppressor function of nm23 (see Sherbet, 2001). Lau et al. (1997) propagated NSCLC tissue by serial subcutaneous transplantation of primary tumours as well as their metastatic deposits. They state that nm23-H1 and H2 were detected in early passages of the tumours, but the genes seemed to be deleted in later passages. The genes were deleted in all metastatic propagation. The authors also noticed that human Alu sequences had also been deleted in the tumours that had lost nm23. This implicates Alu repeats in the deletion of nm23. However, it might be well to remember that the loss of nm23 could be due to other recombination events. For Lau et al. (1997) found that mouse DNA had been incorporated into the NSCLC grafts. The 13q14 locus harbours a number of tumour suppressor genes that have been implicated in the pathogenesis of leukaemias. LOH or homozygous loss of 13q14 occurs in a majority of CLL and also in mantle-cell lymphomas and multiple myeloma. Bullrich et al. (2001) have sequenced a 790 kb segment corresponding to the region harbouring putative suppressor genes related to CLL pathogenesis. They found that the centromeric section of this region contained twice as many Alu sequences as LINE1 elements, whilst in contrast LINE1 elements were preponderant in the telomeric section. The congregation of most known suppressors at the centromeric section of the locus suggest the possibility that the preponderance of Alu elements in this region might have had a bearing on the genetic stability of the region leading to genetic alterations or loss. The loss of chromosome 3p is a frequent occurrence in cancer of the lung, breast, kidney and ovaries, and in cervical and head and neck cancers. Chromosome 3p contains many suppressor genes such as the von Hippel– Lindau (VHL) gene, FHIT, TGF␤-RII and the RASSF1A gene of the ras domain family. The FHIT gene has been putatively attributed with tumour suppressor function, or at any rate, its loss appears to confer a proliferative advantage on cells that have forfeited FHIT expression. The FHIT locus, 3p14.2, encompasses the fragile site FRA3B. The reason for mentioning this gene is simply to point out the operation of the LINE elements rather than the Alu in a large deletion in this gene. Nine out of 10 allelic deletions resulted in the loss of exon 7, and in 7 deletions the breakpoints occurred close to LINE elements, but no Alu sequences were involved (Mimori et al., 1999). Nevertheless, it seems possible that genetic instability might result from contribution of both Alu and LINE elements. This is exemplified by the 13q14 locus where Alu and LINE1 repeats are only modest, around 20% as compared with other genomic sites, with Alu composing as high as 56% (International Human Genome Consortium, 2001),

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 55

suggesting a potential loss of equilibrium between them in the generation of instability (Bullrich et al., 2001).

Alu-mediated alterations of cell cycle regulator genes Mutations of the tumour suppressor and cell cycle regulator gene p53 are a most common event in cancer. Cells that have sustained DNA damage are restrained by wild-type p53 from entering the S-phase, until the DNA is repaired or the cells are induced to adopt the apoptotic pathway. Mutations of p53 seem to abrogate this G1-S checkpoint control function of the suppressor protein. Several hotspots of single base mutations have been identified, but the inactivation of p53 also seems to result from a rearrangement of the gene. Magewu and Jones (1994) had noticed that codon 248 in exon 7 of the gene was prone to extensive methylation and this applied also to the Alu sequences occurring in the neighbourhood. Slebos et al. (1998) have recorded a complex mutation involving deletion and inversion, which implicates Alu elements in the deletion of exons 2 to 4. A 175-bp Alu element is inserted between the breakpoints that culminated in the genomic deletion of exons 2 to 4. The gene coding for the retinoblastoma susceptibility protein Rb is another regulator of the cell cycle, whose inactivation allows the progression of the cells into the S-phase of the cycle. Rb is another example where the gene appears to undergo a reorganisation by the deletion of a 799-bp sequence in intron 2, which might be due to a homologous recombination of Alu repeats (Rothberg et al., 1997). The significance of this genomic alteration in terms of the cell cycle regulating function of Rb and the possible significance of the alteration to tumorigenesis are still unclear. This deletion polymorphism was encountered at an allele frequency of 2.2% in normal subjects and at 5.2% in brain cancer patients. Rothberg et al. (1997) seemed to be mindful of the possibility that this might be a benign polymorphism. Furthermore, on the basis of the available data it would be premature to attach a possible cancer predisposing function to this mutation. It would have been more informative if the analyses had been between glioma patients and those with non-malignant conditions such as meningiomas or with gliosis. Nevertheless, with the strong involvement of a number of growth factor in the pathogenesis of brain tumours, one cannot rule out Rb mutation of this kind as a contributing factor in the pathogenesis of these tumours.

Alu-mediated interchromosomal recombination in leukaemias The pathogenesis of haematological malignancies is replete with instances of the involvement of interchromosomal translocations and gene fusion resulting in chimeric protein products. Notably, t(9;22) leading to Bcr-abl (Morris et al.,

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1996; Jeffs et al., 1998; Martinelli et al., 2000), t(3;11) leading to BOB1/OBF1-LAZ3/Bcl6 (Galiegue-Zoultina et al., 1996), and the frequently encountered MLL gene rearrangements (Super et al., 1997; Wiedemann et al., 1999; Akao and Isobe, 2000), are all examples of Alu-mediated translocations. We discuss subsequently (see page 109) the abnormalities in TEL gene that accompany haematopoietic malignancies. The translocation t(12;21)(p13;q22) involves TEL (on chromosome 12) and the breakpoint is found in its intron 5. Romana et al. (1999) detected no involvement of Alu repeats in this translocation. Hirai et al. (1999) studied the t(3;21)(q26;q22) in CML. They found 3 CT-rich sequences and 4 Chi-like sequences but no Alu around the breakpoint on chromosome 21 and none of any type on chromosome 3. However, the fragile sites FRA3D and FRA3C occur at 3q25 and q27, in the region of the 3p breakpoint and possibly these fragile sites might have some involvement in this translocation. Although interchromosomal recombination is a highly characteristic feature of leukaemias, there is evidence that tandem duplication occurs in the ALL1 gene of AML. The duplication results from the fusion of intron 6 or 8 with intron 1. The breakpoints of this intrachromosomal recombination have been characterised in 9 AML patients with duplication of exons 2 to 6. They occur in introns 1 and 6 in Alu elements and lead to homologous recombination that results in tandem duplication of the exons (Strout et al., 1998). A tandem duplication of exons 2 to 6 of MLL can occur with break and fusion taking place within Alu repeats (So et al., 1997).

Alu-mediated interchromosomal recombination in Ewing sarcoma Gene fusion is a phenomenon arising from interchromosomal reorganisation. The fusion of RET and ELE1 genes occurs in radiation-induced thyroid carcinomas. We know from the investigation by Nikiforov et al. (1999) that the breakpoints culminating in the formation of the fusion gene seem to be distributed in the introns of the genes in a rather random manner. However, they do point out that there is a cluster of breakpoints in the Alu repeats of ELE1 gene. A characteristic reciprocal translocation t(11;22) has been described in Ewing’s sarcoma and in the Ewing’s-related neuroepitheliomas and in Askin’s tumour. This is also a most frequent constitutional translocation in man. Two t(11;22) translocations occur, of which one (q24;q12) results in the fusion of EWS occurring on chromosome 22 with FL11 gene on chromosome 11. Obata et al. (1999) have described five types of chimeric EWS-FL11 protein resulting from fusion between exons 7 or 10 of EWS with exons 5, 6, or 8 of FL11. They also describe the occurrence of a consensus sequence, which flanks all these breakpoints, but in addition, Alu repeats and topo II cleavage sites are found in close vicinity of or even encompass the breakpoints. A second

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 57

t(11;22)(q12;q12) brings about fusion of EWS with an ETS family gene. The breakpoint occurs in an Alu-like repeat in the ETS gene (Ishida et al., 1998). The incidence of t(11;12) breakpoints on both chromosomes in Alu repeats tracts has been again confirmed recently, and therefore this translocation appears to be Alu-mediated (Hill et al., 2000). Hill et al. (2000) have also made a rather striking observation that in five samples in which they mapped the breakpoints, the latter showed very little variation as to their location in the Alu tract, and notably the break occurred within 32 bp and 22 bp respectively in derivative chromosomes 11 and 22. This would suggest that Alu elements are far from being non-specific foci of breakpoints, but breaks occur with a hitherto unknown degree of selection. This is in sharp contrast with other findings, especially that of Nikiforov et al. (2000), which suggest no specificity as regards the location of breakpoints within the Alu tract, and indeed, gives no indication that the recombination was induced by Alu, as some authors tended to describe these interchromosomal transfers. At any rate, how this specificity can be achieved can, at present, be only a matter of speculation. On the other hand, there are also clear indications that the formation of these chimeric genes could be mediated by other consensus oligomeric sequences, as indeed it seems possible from the reports mentioned above (Ishida et al., 1998; Obata et al., 1999).

Alu repeats, DNA mismatch repair gene mutation and genomic instability We noted earlier that genetic instability could be attributed to epigenetic silencing of human DNA mismatch repair genes. A clear correlation has emerged between mismatch repair gene hypermethylation and genetic instability. Conceivably, another dimension might be added to the effect of DNA mismatch repair gene expression on genetic stability of Alu repeats. The hMLH1 gene of certain HNPCC families shows mutations with a 3.5 kb deletion in exon 16, and another larger 22 kb deletion corresponding to exons 12 to 16. These deletions appear to be mediated by homologous recombination of Alu sequences (Nystrom-Lahti et al., 1995; Mauillon et al., 1996). Whilst mutations of mismatch repair genes per se may not be the causal factors of HNPCC, they might offer another mechanism that produces DNA mismatch repair deficiency leading to genetic instability and its multifarious consequences.

Alu elements in genetic transcription Alu elements were once regarded as functionally inert repeats. It was recognised some time ago that they might be functionally active. The presence of Alu sequences in the promoter regions of many genes has suggested a role in

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genetic transcription. For example, transcription factor binding elements have been identified within Alu sequences. The sequences have been seen as a means of regulating the expression of RNA polymerase II. The frequency and the numbers of such sites encountered have led to the postulation of a role for Alu elements in RNA polymerase-dependent gene transcription (Kazakov and Tomilin, 1996). On the other hand, other Alu-binding proteins might suppress RNA polymerase-mediated transcription (Kropotov et al., 1999). The promoters of certain genes that contain binding sites for thyroid hormone receptor, ER and RA receptors also contain Alu repeats. Norris et al. (1995) reported that a class of Alu elements seemed to function as enhancers of oestrogen-dependent transcription, and further that they might have diverged into a special class of repeats. Norris et al. (1995) found that anti-oestrogens abolished this enhancer activity. Li, Yeh et al. (2000) have characterised the ER␤ promoter and have identified an Alu sequence containing an enhancer of ERdependent transcription. However, there is a possibility that Alu repeats might be involved in other pathways of oestrogen function, namely via inactivation of progesterone receptors. Alu insertions may be found in PgR (Rowe et al., 1995). The thyroid hormone receptors, ER and RA bind to the nucleotide sequence AGGTCA. This motif may be duplicated and distributed with defined spacing and orientation, and they occur in Alu repeats (Babich et al., 1999). The identification of Alu repeats in transcription silencer elements of the promoter regions of genes, as in BRAC2 for example (Sharan et al., 1999), does provide some footing for suggesting a direct role for Alu elements in gene transcription. A negative regulation of BRCA1 and BRCA2 is characteristic of breast cancer derived cell lines and Sharan et al. (1999) note that this negative regulation of BRCA2 by the silencer element is not seen in non-breast cancer cell lines. Furthermore, breast cancer cell lines contain specific proteins that bind these silencer elements. The information that would be decisive would be whether these proteins are Alu binding or Alu RNA binding proteins.

Mode of Alu function in recombination The close association between the occurrence of Alu elements and genetic recombination, on the other hand, may be deemed to be well established. The phenotypic effects of recombination in the onset as well as degree of clinical manifestation of genetic disorders are beyond dispute. However, we do not yet fully understand the mechanisms that bring about the replication and insertion of DNA repeat elements in the genome. Nor do we know the modus operandi of Alu and other DNA repeats in bringing about recombination. The readiness with which this retro-positioning of Alu repeats occurs is not evidence that the insertion is a functional manoeuvre. Alu and other elements are very frequently detected in regions flanking the breakpoints or breakpoints occur within the repeat tracts. Some authors have suggested a selectivity of recombination,

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 59

indeed a preferential recombination, mediated by Alu elements. An essential justification would be to demonstrate that break and recombination occurs in Alu elements in stochastic preference to other repeats that are prone to breaks. This does not appear to be the case. On the other hand, can it be that retroposition of Alu is a selective process? If it is not, it should occur as a random process. This is not patently the true situation either. Alu elements containing ER binding motifs are found in ER-positive but not in ER-negative cells. No doubt this can be due to the presence of other trans-acting factors, but nonetheless it would be legitimate to enquire how such tissue specificity can flow from a random event. There are tissue-specific differences in Alu methylation, which could impinge upon chromatin condensation, organisation, genetic stability and gene expression. The methylation of CpG islands within Alu can alter recognition by Alu binding proteins and in this way alter gene expression (Cox et al., 1998). Furthermore, Alu RNA levels increase in response to cellular insults together with increase in protein synthesis, which provides another link in the potential role of Alu in physiological function (see Schmid, 1998). Alu RNA levels also appear to be influenced by mutations and the 3’ sequence variations. Obviously, this would allow some elements a selective advantage over others (Aleman et al., 2000), which could lead to a definition of their link with specific cellular events. Alu repeats are derived from the 7S RNA component of the signal recognition particle (SRP). Both nascent SRP and Alu are processed before their transport from the nucleus (YH Chen et al., 1998). There is now an increasing awareness of the presence of translin binding sequences within or in the neighbourhood of Alu repeats. It has been suggested that translin binding activates recombination. But, far from providing a viable explanation of recombination, this simply inserts another potential causal element in the stream of recombination events. It ought to be recognised not only that translin does not occur with Alu elements as an invariable feature but also that translin might be a lymphoid-specific protein. Therefore, translin cannot be invoked as participant in genetic recombination in general, for recombination occurs across the phylogeny. We know of other repeat tracts such as certain triplet repeat elements that might be rendered unstable because of mutation of DNA mismatch repair genes. The status of mismatch repair genes has not been investigated in relation to Alu-mediated recombination, but this would certainly be worthwhile. Some circumstantial evidence may be cited in this context. The promoter of the gene encoding the human corticotrophin releasing hormone contains repeat elements bearing much homology to Alu repeats, and coincidentally, this gene occurs at the same locus as the chromosomal fragile site FRA8F on chromosome 8 (Vamvakopoulos and Chrousos, 1993), which is liable to the interpretation that the Alu elements could be conferring fragility on the chromosome. Keaveney et al. (1993) also opined that the presence of a large number of repeat elements, including Alu, in the 3’ region of the ER gene, could lead to inherent instability.

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Figure 3 A schematic representation of p53 abnormalities and Alu repeats in genetic recombination. The tumour suppressor p53 mutations are a most common event in cancer. Wild-type p53 restrains cells that have sustained DNA damage from entering the S-phase until that damage is repaired or the cells are induced to adopt the apoptotic pathway. Mutated p53 does not exert the G1 –S checkpoint control function. This Figure represents schematically the possibility that might be involved in Alu transcription and insertion into the genome thus providing genetic instability and foci for recombination. Inactivation of p53 can also result from a rearrangement of the gene and from extensive methylation, also on account of Alu sequences occurring in the neighbourhood. This Figure postulates a mechanism in which p53 abnormalities and the continued repair of DNA damage, replication and insertion of Alu and other DNA oligomeric repeats might generate genetic disorders and cancer pathogenesis.

Abnormalities of p53 are a major feature of human cancers. These include mutations and aberrant patterns of localised methylation, and it has been recognised that mutations of p53 and of other genes occur mainly in methylated CpG islands within the gene. The methylation of CpG islands of p53 could be cumulative, and in turn generate cumulative effects on genetic stability. DNA damage is not only the activator of p53 expression but it can also activate transcription and insertion (Rudin and Thompson, 2001). Furthermore, we have noted that in some cases p53 might itself become inactivated by Alumediated rearrangement. Thus one can envisage a mechanism in which p53 abnormalities and the continued replication of DNA damage, replication and

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 61

insertion of Alu and other DNA oligomeric repeats might perpetuate the expansion of aberrant clone of cells with enhanced proneness to genetic recombination that can have powerful phenotypic consequences (Figure 3).

Functional involvement of Alu with signal recognition particles We noted earlier that structural considerations have suggested that Alu repeats are a derivative of the 7SL component of signal recognition particles (SRP). The SRP are ribonucleoproteins with two component domains called the S-domain and an Alu domain. The S-domain contains unique SRP RNA sequence and four SRP proteins. SRP are involved in the targeting of membrane proteins and nascent proteins to the endoplasmic reticulum for appropriate processing. The 7SL RNA of SRP contains a motif that can activate RNA polymerase III (pol III). Like 7SL RNA, Alu repeats contain promoters for RNA polymerase III (pol III) and are transcribed by the polymerase. Like the 7SL RNA component, Alu RNA is processed and adenylated in the nucleus. The Alu elements participate in the SRP functions of controlling the nascent protein elongation and translocation of proteins to and across the endoplasmic reticulum. The region of SRP containing Alu together with two 9 and 14 kDa polypeptides (SRP9 and SRP14) appears to regulate the elongation of nascent protein. These polypeptides bind as a heterodimeric complex to Alu of 7SL RNA (Bovia et al., 1995; K Hsu et al., 1995). The interaction of the SRP9/SRP14 heterodimer with 7SL RNA involves the C-terminal region of SRP14. The ability of SRP to arrest protein elongation is lost if the C-terminus of SRP14 is mutated. Mason et al. (2000) demonstrated this by using a C-terminus truncated mutant SRP14 derived from Saccharomyces cerevisiae. The mutation also affected the chaperoning and protein targeting abilities of SRP. Mutations in the 7SL RNA region with which SRP14 interacts also reduce the binding of the polypeptide heterodimer (Chang et al., 1997). The SRP constituent protein subunit (the bacterial equivalent called Ffh) as well as the subunit SR-␣ (FtsY) of the receptor of the recognition particle is a GTPase. The interaction of the SRP signal peptide binding subunit (the mammalian SRP54 or bacterial Ffh) with the SRP receptor leads to stimulation of GTPase activity. The signal peptide binding subunit recognises the N-terminal signal sequence of nascent proteins and targets them to the cell membrane of prokaryotes or to the endoplasmic reticulum of eukaryotes. The targeting of the nascent proteins for translocation is regulated by GTPases. The SRP subunit and its bacterial homologues show a conserved general organisation into three domains, namely a hydrophilic N-terminal domain, a methionine-rich M-domain at the C-terminus and the G-domain, which is the GTPase domain in the middle. The M-domain binds to both SRP RNA and signal peptides.

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Although the discussions above relating to SRP seem like a digression, it is not so. The SRP proteins are also known to bind to Alu-containing RNAs, which suggests a potential role for them in the control of genetic expression at the translational level (Bovia et al., 1995). The Alu domain of SRP is believed to retard the elongation of the SRP subunit before they come to be associated with the protein translocation machinery of the cell. Albeit currently in the realm of speculation, this ability of SRP proteins might be relevant in controlling the expression of messages that have been modified by Alu-mediated recombination, implicitly with considerable implications in neoplastic development. This potential dual role for Alu elements is certainly worth focused attention and research, especially in leukaemias and lymphomas.

LINE repeats in genetic rearrangement LINE elements occur at high density but with uniform distribution in AT-rich regions of the genome, in contrast with Alu, which occur at a 2- to 3-fold higher density in GC-rich than in AT-rich regions (ZL Gu et al., 2000). Like Alu, LINE elements might be capable of retro-positioning and therefore potentially capable of mediating genetic rearrangement. Several years ago, Filippov et al. (1992) concluded from sequence studies of the so-called Tu80 and NOV1 clones derived from mouse chromosome 17 that their sequence might have been derived from an ancestral clone through the mediation of LINE1 elements. LINE can occur together with other repeat elements, as for instance in intron 11 of the dystrophin gene where LINE1 occurs with Alu and THE elements (Ferlini and Muntoni, 1998), although there is no indication from either Ferlini et al. (1998) or Ferlini and Muntoni (1998) that they participate in any way with the rearrangement of dystrophin gene. Biunno, Rogozin et al. (1997) mapped the Xp27.1 locus and have reported the occurrence of 5 LINE1 repeats (4.8%), 8 Alu elements (6.2%) and, together with others, the repeat elements constituted one-fifth of a 35 kb human DNA sequence. Whether this high density of repeat elements has any influence on the organisation of genes residing in that locus is unclear. A potential benefit that could flow from this is repair of genetic damage. LINE elements seem to integrate into DNA lesions and participate in DNA repair by virtue of their retroposition function (Morrish et al., 2002). LINE1 repeats are reportedly hypomethylated in murine liver tumours and also in human hepatocellular carcinomas (HCC). The hypomethylation of HCC is restricted to tumour tissue alone and none is detected in the normal tissue surrounding the tumour foci (Takai et al., 2000), although Lin et al. (2001) found comparable levels of LINE1 transcripts in HCC, non-HCC and in normal liver. Nonetheless, an oblique suggestion is that diminished methylation of LINE repeats might render them unstable and prone to transposition in the genome, which could potentially bring about reorganisation of genes with consequences to their expression. Possibly, such a state of increased instability might be the

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 63

reason for the detection by Lin et al. (2001) of truncated LINE1 transcripts. Such a situation could be subsisting in rheumatoid arthritis. Retroviruses often have LINE and THE-like sequences and related murine repeats inserted into the viral genes (Smit, 1993; Kulski et al., 1999). For instance, synovial fibroblasts derived from synovial fluid of patients with rheumatoid arthritis contain viral open reading frame (ORF-2) with L1 elements. Neidhart et al. (2000) found that transcripts of ORF-2/L1 occur in cartilage and bone at the sites of destruction caused by the synovial fibroblasts. Comparable tissues derived from patients with osteoarthritis do not show their presence. Neidhart et al. (2000) transfected synovial fibroblasts that did not contain L1 with functional L1 constructs and noticed that the transfectant cells now differentially expressed a number of genes, including human stress-activated protein kinase-2, and c-met gene, among others. In this case also, synovial fibroblasts with L1 seem more susceptible to demethylation, which could account for the enhanced and differential gene expression. This is a most interesting outcome. The c-met gene codes for a tyrosine kinase receptor protein to which the hepatocyte growth factor binds and this might be relevant in the context of the invasive behaviour of cancers. The c-met gene and its protein product show enhanced expression in a number of carcinomas and could be associated with tumour invasion and metastatic progression (see Sherbet and Lakshmi, 1997). It is conceivable that LINE elements might similarly be associated with the invasive behaviour of cancers and this is worthy of further inquiry. The amplification and reintegration of exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene seems to occur with the mediation of LINE1 elements. Rozenmahel et al. (1997) isolated cDNA clones of CFTR gene that hybridised with genomic DNA and analysed the sequence of these clones. This study has revealed that exon 9 and the flanking introns have LINE1 elements in juxtaposition. These gene sequences seem to be reintegrated into the genome at different sites together with the LINE repeats. Rozenmahel et al. (1997) also suggest on the basis of the similarities of the sequences that the multiplicity of reintegration might have occurred from a single event of retro-transposition and amplified subsequently. Herein contained is a powerful argument that LINE elements participate in genetic reorganisation. Admittedly, however, there is little functional attribution to the multiple repeats of exon 9 of CFTR gene. On the other hand, does the total depletion of LINE elements have any particular connotations? Apparently it does not. The runt domain proteins are a family of transcription regulating factors that are involved in a variety of developmental processes and in the formation of blood and bone in mammals. The RUNX1 gene, resident on chromosome 21q22.12, codes for a transcription factor RUNX1 (also called AML1 and CBFA). This gene belongs to the runt domain protein family, of which runt gene of Drosophila is the patriarchal gene. To this family belongs the core-binding factor (CBF), a heterodimeric transcription factor composed of RUNX1 and CBFB. RUNX1 is the DNA-binding subunit. CBF induces transcription or repression of genes if

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their promoters or enhancer elements contain the consensus sequence TGT/ CGGY. CBFB, the second subunit of the transcription complex, is not involved in DNA binding. CBF regulates genetic transcription in a tissue-specific manner, as for instance in the expression of osteocalcin gene in osteoblasts (Javed et al., 2000), or in the mediation of proliferative effects of oestrogen in osteoblasts (Sasaki-Iwaoka et al., 1999), and this is regarded as a reflection of the differential response of the gene promoters. Several alternatively spliced cDNAs of RUNX1 gene have been identified; besides a large number of alternatively spliced isoforms of the RUNX1 protein with a range of molecular sizes are expressed in cells. RUNX1 regulates haemopoiesis and is the focus of chromosomal translocations. But interestingly, the introns of the genes are notable for the lack of repeat elements especially of the LINE type. Furthermore, certain high frequency translocations involve the transfer of a 555-bp segment FLI1 gene from chromosome 11 into intron 3.1 of RUNX1 and the same introns contain the breakpoint for the t(8;21) and t(3;21) that are encountered in AML (Levanon et al., 2001).

Cognate elements associated with recombination A number of genetic elements besides Alu may be found in recombination events, either functioning as cognate elements or subserving a subsidiary role in the process. Thus, Hirai et al. (1999) have reported the presence of CT-rich sequences and topo II binding and cleavage consensus sequences near the breakpoints, but not Alu repeats, in t(3;21)(q26;q22) in CML. The t(11;14)(p13;q11) in T-cell ALL cell line is reportedly mediated by Alu repeats found at the chromosome 11 breakpoint, and what is of interest here is the identification of tetranucleotide repeats encompassing 70 bp (WF Dong et al., 1995). What role, if any, these elements play in events leading up to recombination is uncertain at present, for some occur together with Alu repeats and therefore one cannot make functional differentiation and attributions to them. On the other hand, there should be a clear cognisance of the importance of those elements that occur independently of Alu.

Translin-binding elements Two important elements, the translin-binding elements and Chi (Crossover hotspot instigator)-like elements might both be functioning as activators of recombination. Translin gene was identified in connection with translocations encountered in lymphoid malignancies (Aoki et al., 1995, 1997). Its protein product associates with the translin-like protein known as TRAX, which contains a sequence that targets it to the nucleus. TRAX bears a marked sequence homology to translin and the TRAX and translin genes show a

DNA repeats, genetic recombination and the pathogenesis of genetic disorders 65

similarity in organisation (Meng et al., 2000). Translin in native form has a ring structure and binds to sequences found only at the single stranded ends of DNA. DNA damaging agents reportedly stimulate nuclear translocation of translin (Kasai et al., 1997). This conceivably provides a physical mode of initiation of recombination by translin, that is, of recognition of single stranded DNA ends generated by DNA damage. The integrity of the highly conserved leucine-zipper motif of the protein is important in its interaction with DNA; for point mutations in this motif produce a loss of DNA binding together with loss of its multimeric structure. Mutations in the ring structure of the molecule totally inhibited DNA binding without affecting the multimeric structure, thus closely identifying the ring structure with DNA binding (Aoki et al., 1999). Translin also binds RNA in vitro and in the latter context might inhibit the translation of mRNA that possesses the appropriate binding motifs. Translin might have other functions, such as in the process of mitosis. Translin associates with centrosomes and is redistributed during the cell cycle (Castro et al., 2000). It can also bind to microtubules (Wu and Hecht, 2000). Translin binds consensus sequences in the neighbourhood of breakpoints of chromosomal translocations in haematological malignancies (Aoki et al., 1995). The presence of translin-binding sequence, together with or within Alu repeats, has been detected in the formation of the bcr-abl fusion gene in CML (Jeffs et al., 1998; Martinelli et al., 2000). Translin-binding motifs occur in liposarcoma showing t(12;16) (q13;p11) in the close vicinity of the translocation breakpoint (Hosaka et al., 2000). TRAX binding sequences have been found within about 150–250 kb of the translocation breakpoint at 1q42.1, which dislocates and segregates the DISC1 and DISC2 candidate genes in patients with schizophrenia (Millar et al., 2000). The alveolar rhabdomyosarcoma is characterised by t(2;13)(q35;q14) and a fusion gene PAX3-FKHR results out of this translocation. The breakpoints in the derivative chromosome 13 contain sequences that resemble those of translin (Chalk et al., 1997). We have noted earlier the presence of translin-binding sequences within or in the neighbourhood of Alu repeats. These findings have led to the suggestion that translin binding might activate recombination. However, as stated earlier, not only translin does not occur with Alu elements as an invariable feature but also it is believed to be a lymphoid-specific protein. Therefore, it cannot be invoked as a participant in genetic recombination across the phylogenetic cascade.

Chi sequences in DNA repair and recombination Chi (Crossover hotspot instigator) elements are oligomeric sequences in DNA, which play an important role in DNA repair by homologous recombination. Chi elements are specific for individual organisms. Their genomic distribution might not be random. The Chi sequence of Escherichia coli is 5’-GCTGGTGG-3’ and occurs on the leading strand within the so-called recombination islands. Chi is orientation-specific; its orientation corresponds with DNA replication

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and transcription. The major pathway of recombination-mediated DNA repair in E. coli involves the heterotrimeric RecBCD protein. The latter possesses the ability of fast DNA unwinding and cleavage. RecBCD recognises the recombination hotspot Chi of double-stranded DNA, switches the polarity of DNA cleavage, produces single stranded DNA and stimulates recombination of sequences in the vicinity. Chi-like recombination-activating signal sequences occur near the breakpoint of translocation involving chromosome 21 in CML (Hirai et al., 1999). A heptameric sequence CCCACCC or its abbreviated version CCACC derived from mammalian cells seem to be able to activate recombination. Upon insertion into retroviral vectors, these sequences seemed to initiate repair and recombination of the vector. However, mutant versions of the sequences did not possess this ability (Olson and Dornburg, 1999). The presence and the putative role of Chi-like elements in the mammalian genome are matters still subject to debate. It is possible that they might serve as adjuncts in recombination, viral integration and in the function of viral genomes as transposable elements. Taking an overall view, one cannot unduly emphasise the importance of Alu repeats and categorically exclude the participation of other consensus sequences and signalling factors in these genetic rearrangements.

5 Chromosomal recombination in cancer

A marked heterogeneity of the chromosome complement of the component cells of a tumour is a striking feature of heterogeneity of cancers in respect of several cellular, biological and biochemical features. The heterogeneity that accompanies tumour development and progression has been studied extensively in the past few decades. In many ways, karyotypic heterogeneity reflects the evolution of diversity of cell type in the tumour and very often the heterogeneity of the karyotype can be related to the acquisition of certain phenotypic behavioural features. Furthermore, the generation of this heterogeneity is attributable to abnormal genetic recombination as well as to deficiencies in the maintenance of the integrity of the genome by efficient and accurate repair of DNA damage. In hindsight, therefore, the extensive study of chromosomal abnormalities over the past years has been invaluable for the interpretation of their mode of involvement in the pathogenesis of tumours with regard to the abnormal and inappropriate activation and expression of genes, which have a direct bearing on the initiation, development, and invasion and metastasis of tumours.

Karyotypic abnormalities in cancer Karyotypic abnormalities of cancer can be structural or numerical or both. The heterogeneity of the complement of chromosomes can be in the modal number Genetic Recombination in Cancer ISBN 0-12-639881-X

Copyright © 2003 Elsevier Science Ltd All rights of reproduction in any form reserved

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of chromosomes in the complement, i.e. chromosomal ploidy and DNA ploidy, in the presence of specific marker chromosomes, and in chromosomal damage and rearrangement. Rearrangement can be interchromosomal, such as reciprocal translocations and insertions, or intrachromosomal, as in inversions and insertions. In general, karyotypic variability arises from genetic instability. The effects of this are discernible as loss of heterozygosity, changes in chromosomal numbers, chromosomal non-disjunction, chromosome breakage and abnormal recombination, reciprocal translocations between chromosomes, equal and unequal sister chromatid exchange, and double minute chromosomes representing gene amplification. Double minute chromosomes might be specifically targeted and reintegrated in the chromosome and visualised as homogeneously staining regions. Homogeneously staining regions may also be the result of intrachromosomal amplification of genetic material. Chromosomal fragile sites are another feature of genetic instability and can be a source of the genesis of some forms of chromosomal aberrations. The formation of micronuclei is also believed to result from genetic instability.

DNA aneuploidy and cancer progression DNA aneuploidy is often regarded as an indicator of tumour malignancy. Many early reports have indicated a high frequency of DNA aneuploidy in several human neoplasms. This has inevitably led to detailed investigations of the influence of DNA aneuploidy on cancer invasion and metastatic progression and its potential for predicting prognosis. DNA ploidy reportedly correlates with the myometrial invasion by primary endometrial carcinomas. Aneuploid primary tumours also recur more frequently as compared with diploid tumours (Giovagnoli et al., 1995). Sun et al. (1993a) found DNA ploidy to be a very important and independent prognostic indicator in patients with stage A–C colonic adenocarcinomas. The survival of patients with prostatic adenocarcinoma has also been correlated with DNA ploidy (Azua et al., 1996). Yuan et al. (1992) had reported some years ago that DNA ploidy was significantly predictive of relapse of disease as well as overall survival in a series of patients with node-negative breast cancer. Azua et al. (1997) measured the DNA ploidy in fine-needle aspirates (FNAs) of breast cancer patients. They found DNA quantification to be significantly related to survival times. In another study involving a large series of breast cancer, Gilchrist et al. (1993) found that DNA ploidy strongly correlated with lymph node metastasis and early death. DNA ploidy correlated with the presence of tumour in the axillary lymph nodes, sometimes with distal metastases in a small series of breast cancer (Sherbet and Lakshmi, 1997). In a study of gastric cancers, Baba et al. (2002) noticed that DNA aneuploidy correlated with high invasive and metastatic potential and with poor prognosis. Ioakim-Liossi et al. (2000) investigated a series of superficial transitional cell bladder carcinomas. It may be recalled that a

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proportion of superficial tumours recur as invasive tumours. In this context it is of much interest to note that Ioakim-Liossi et al. (2000) found a highly significant correlation between DNA aneuploidy and tumour recurrence. Contrary to this, pancreatic carcinomas with diploid DNA content are said to be less aggressive than corresponding aneuploid ones. Furthermore, patients with diploid tumours have shown decreased median survival (Linder et al., 1995; Southern et al., 1996). Similar findings have also been reported in respect of colorectal cancer (Sampedro et al., 1996) and hepatocellular carcinoma (Bottger et al., 1996). Esteva et al. (2001) found no relationship between DNA ploidy and prognosis in lung cancer, although others, e.g. Cibas et al. (1989), had previously reported such a relationship. Similarly, neither overall survival nor disease-free survival of patients with head and neck cancers correlated with DNA ploidy (Tamas et al., 1999). Igarashi et al. (1999) noticed no changes in the state of DNA ploidy in the progression of early gastric cancer to advanced stages of the disease. Thus, overall, there is little consensus concerning the value of DNA ploidy as a prognostic marker (Camplejohn et al., 1993; Hedley et al., 1993; Wenger et al., 1993). It should also be noted here that hyperdiploidy has been associated with good prognosis and haploid state with poor outlook in childhood leukaemia (Salford et al., 1996; Harrison, 2000; also see IHN Wong et al., 2000). Presumably, it is not merely the relationship between the general state of chromosome number or DNA ploidy to disease progression that one needs to take into account, but more importantly whether individual genes or cohorts of genes are amplified into multiple copies. It is not reasonable to assume that the chromosomal complement is amplified as a whole. But it is possible that specific chromosomes might be increased in number with each abnormal cell proliferation cycle. Abnormal cell cycle progression leads to hyperdiploidy. Thus we know that over-expression of both cyclin D3 and p21waf1/cip1, itself subject to regulation by cyclin D3 and p53, is associated with enhanced ploidy (Kikuchi et al., 1997; Zimmet and Ravid, 2000). Non-random alterations of chromosomes occur at a high level in hypodiploid AML and indicate poor survival. This is in sharp contrast with hyperdiploid AML, which has low nonrandom chromosomal changes and good prognosis (Ramos et al., 2000). ALL patients with trisomy of chromosomes 10, 17 and 18 have better prognosis than those without trisomy. Trisomy of chromosome 5 is associated with worse prognosis (Heerema et al., 2000). Aneuploidy has been reported to correlate with the expression of aberrant markers in both AML and ALL. EksiogluDemiralp et al. (1999) described a series of 61 AML and ALL patients of whom 18 were aneuploid and aberrant markers were expressed in 20 patients. Seven (of 18) patients who expressed abnormal markers had aneuploid DNA content, and had short survival of three months. It is inconceivable that aneuploidy should be studied on its own merit for its relevance to prognosis of leukaemia, especially without reference to the S-phase fraction (SPF). The latter could be an important parameter to include in these studies and indeed might be an

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important prognostic factor (Kute et al., 1995). Hyperdiploid ALL is more sensitive to antimetabolite therapy (Kaspers et al., 1995), and this could be due to high SPF attendant upon the hyperdiploid state. Presumably for the same reason hypodiploid multiple myeloma is refractory to chemotherapy.

DNA ploidy, cell proliferation and growth factor expression There are fundamental arguments for suggesting that DNA ploidy is a reflection of some basic processes gone astray during cell proliferation. It has been suggested that DNA aneuploidy might be a consequence of cells entering the S-phase prematurely. The close association between aneuploidy and the size of the S-phase fraction (SPF) seems to suggest this. According to Wenger et al. (1993), aneuploid tumours show a virtual doubling of the S-phase fraction. Esteva et al. (2001) have claimed that diploid lung cancers might have a lower mean SPF value of 5.01% than aneuploid cancers, in which SPF is 2-fold higher, at 10.84%. Stal et al. (1994) reported some years ago that the growth factor encoding c-erbB2 gene was grossly over-expressed in breast cancer cells. The gene was expressed also at a far greater frequency (63–81%) in aneuploid colorectal adenocarcinomas as compared with only 53% of diploid tumours (Sun et al., 1995). Ottesen et al. (2000) have confirmed recently that DNA aneuploid breast carcinoma in situ show a strong association between DNA ploidy, cell proliferation and over-expression of c-erbB2. Furthermore, enhanced association of p53 suppressor protein (see below) with aneuploid tumours also correlated with enhanced cell proliferation (Midulla et al., 1999; Raybaud et al., 2000). Early investigations using image cytometry (ICM)-based measurements of metastatic variants of the B16 melanoma, and cell lines derived from non-malignant hamster lymphoma, a malignant primary lymphoma and its metastatic tumour, have shown no relationship between DNA ploidy and size of the S-phase fraction (Lakshmi and Sherbet, 1990b; Hallouche et al., 1992). The high metastasis variant B16-BL6 was more aneuploid than the low metastasis variant B16-F1 but they were identical as regards the size of SPF. Sherbet and Lakshmi (1997) have stated that DNA ploidy of breast cancer FNA cells was totally unrelated to SPF. Similarly, DNA ploidy of squamous cell carcinomas of the oral cavity bears no relationship to SPF (Oya and Ikemura, 2002). There is little new evidence to support either point of view with the exception of two recent studies on breast cancer. Bakhtawar et al. (2001) found that size of SPF correlated with the presence of tumour in the regional lymph nodes but not to the numbers of nodes so involved; whilst Chassevent et al. (2001) found medium to high SPF correlated with shorter disease-free survival. Furthermore, in node-negative patients, SPF seemed to be the only predictor of disease-free survival and in node-positive patients high SPF indicated a high risk of recurrence of the disease. Overall, it

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would be reasonable to suggest that the size of the S-phase fraction, together with DNA ploidy and other prognostic markers generally appears to serve as powerful predictors of early relapse, albeit with reservations as to its significance in certain tumour types (Berek et al., 1993). Oestrogens exert physiological effects on many tissues; the major targets are the reproductive organs and female secondary sexual characteristics. Oestrogens stimulate their growth and maintenance. They bind specific intracellular receptors, the oestrogen receptors (ER). There are two ER subtypes called ER␣ and ER␤, which are expressed in a wide variety of tissues; in some tissues either ER␣ or ER␤ might be expressed while in others both subtypes might be expressed. Upon ligand binding the receptors are translocated into the nucleus where they initiate the transcription of responsive genes resulting in appropriate physiological function. ER mediates transcription via oestrogen responsive elements of the target genes or by the agency of AP-1 composed of c-fos and c-jun proteins. The presence of ER indicates the state of tumour differentiation. Loss of ER (ER␣) has been encountered in a number of human cancers. The absence of ER in breast and endometrial cancers is generally regarded as an indicator of poor prognosis, since ER-tumours are resistant to anti-oestrogen therapy, continue rapid growth and result in poor outcome for patients. It was demonstrated sometime ago that the loss of expression of ER in breast cancer was due to hypermethylation of the ER gene (Falette et al., 1990; Piva et al., 1990; Lapidus et al., 1996; Ottaviano et al., 1996). In breast carcinoma not only ER but also PgR (progesterone receptor) gene is downregulated by hypermethylation. Hypermethylation of ER gene and a downregulation of its expression also occur in tumours of the prostate and in colorectal, lung, endometrial and haematopoietic neoplasms (Piva et al., 1989; Issa et al., 1993, 1994, 1996, 1998; Li, Chiu et al., 2000). In some cases a relationship between the silencing of the ER gene by hypermethylation and progression of cancer is obvious. The poor prognosis of ER-negative breast cancers is compounded, not infrequently, by the presence of epidermal growth factor receptors (EGFr). It has been shown that a subset of ER-negative breast cancers tends to be EGFr positive. Therefore, Andronas et al. (2003) recently measured DNA ploidy and SPF in another group of breast cancer aspirate cells by image cytometry and examined whether these relate to oestrogen receptor status of the primary tumours. In this study a series of 46 breast cancer fine-needle aspirates were investigated, divisible into three groups, namely ER+/PgR+, ER+/PgR– and ER– /PgR–. Inter-group comparisons were made of DNA ploidy, SPF and the pattern of cell cycle distribution defined by the ratio G0G1/G2M, and these have enabled the investigators to determine the influence of ER and PgR on the respective cell features. PgR seemed to influence both DNA ploidy and SPF in a bimodal manner. ER had no effect on either feature. Spiethoff et al. (2000) reported that ER/PgR-positive tumours were peri-diploid and therefore the effects of the receptors seem to be minimal. Leers et al. (2000) have also

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reported the association of higher DNA ploidy in ER-positive breast carcinomas and also that aneuploid ER-negative tumours contained >10% of cells in the SPF. Boman et al. (1995) had reported some years ago that ER-negative endometrial cancers had higher SPF than ER-positive carcinoma and also that PgR-positive tumours tended to be aneuploid. This latter finding is consistent with the finding of Andronas et al. (2003). Nonetheless, it would be worthwhile noting that Andronas et al. (2003) have been able to separate the potential effects of ER from those exerted by PgR. There have been suggestions in the past that the expression of aneuploidy in cancers is a result of abnormal cell cycle progression. One would expect therefore that the expression of these two features might be inter-related. Baba et al. (2002) recently reported that aneuploid gastric tumours show high proliferation. Such a relationship has not been encountered in squamous cell carcinomas of the oral cavity (Oya and Ikemura, 2002). Although these reports are few and far between, the work of Andronas et al. (2003) has attempted to throw some light on this aspect. They have looked at how ER and/or PgR might affect the expression of DNA ploidy and SPF relative to each other. They have demonstrated that ER does affect the inter-relationship between DNA and SPF. In the absence of ER, a decrease of SPF is seen together with increase in DNA ploidy. Andronas et al. (2003) also state that ER also influences the relationship between SPF and G0G1/G2M ratio (Table 9). Of note from this study of Andronas et al. (2003) are the analyses of prediction of nodal involvement and 5-year disease-free survival, which involved combining ER/PgR status with DNA ploidy, SPF and G0G1/G2M ratios. The analysis predicted correct nodal status for 13/16 for Group ER+/PgR+ and 11/16 for the ER–/PgR– group. The accuracy that they have achieved for Table 9 The influence of oestrogen and progesterone receptor expression on DNA ploidy, cell cycle distribution and proliferation of breast cancer aspirate cells Parameter

ER

PgR

DNA ploidy SPF DNA ploidy and SPF DNA ploidy and G0G1/G2M G0G1/G2M and SPF

– – + + +

+ + – – +/–

DNA ploidy, SPF and cell cycle distribution were determined by image cytometry. G0G1/G2M represents the ratio of cells occurring in the two cell cycle peaks. Normally only around 10% of cells are found in the G2M peak and >10% in this peak can be regarded as indicating hyperdiploidy. Source: Andronas et al. (2003)

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predicting 5-year disease-free survival is most impressive. Andronas et al. (2003) have correctly predicted the survival of group ER+/PgR+ patients in 15/16 cases, 12/14 for the ER+/PgR– group and 14/16 for the ER–/PgR–. From these investigations, these authors conclude that ER and PgR exert differential effects on the cell features examined, and therefore suggest that a combination ER/PgR status with DNA ploidy, SPF and cell cycle distribution might provide a powerful marker for disease prognosis (Andronas et al., 2003). In recent years many factors that reflect the biological features of neoplastic disease related to the state of its progression have been identified. The massive body of information available and its interpretation has become a testing task and the various statistical techniques available to date, such as univariate, multivariate and proportional hazards paradigms, have not contributed significantly to the application of data on the numerous cellular markers for assessing cancer prognosis (Naguib and Sherbet, 2001b). The complexity that accompanies statistical analyses of the significance and weight of individual markers from the range of factors that impinge upon prognosis has necessitated the employment of other methods, such as artificial neural networks, for the analysis of cell measurements in relation to their relevance for cancer progression. Indeed, cellular features such as DNA ploidy, size of SPF, cell cycle distribution, and nuclear pleomorphism of breast cancer cells from fine-needle aspirates (FNA) have been analysed using artificial neural networks (ANN). ANN is an artificial information processing system whose organisation is akin to information processing and performance characteristics of biological neurones (Naguib and Sherbet, 2001a, b). This was with a view to determining whether they can be successfully used to predict sub-clinical metastatic disease (Naguib et al., 1999). In that study, DNA ploidy ranged from 2n to 12.5n, the median being 4n and 82% of the tumours were hyperdiploid. The relative distribution of cells between the G0G1 and G2M phases of the cell cycle, namely the G0G1/ G2M ratio, is regarded as an aspect of DNA aneuploidy; but only 25% of samples were aneuploid by this criterion. Nonetheless, by neither criterion did DNA ploidy show any relationship to nodal status. In ANN-mediated prediction, DNA ploidy measured by nuclear DNA content did not appear to be a significant marker in the analysis, since its omission had no bearing whatsoever on the different statistics. However, when the G0G1/ G2M ratio is omitted, the results are worsened, indicating the positive effect that the ratio has on the neural outcome prediction and the importance of its inclusion in the analysis. A statistical analysis of DNA ploidy distribution by either criterion did not relate to nodal spread. This contrasts with the ANN analysis, which indicated a positive effect on neural-based prediction. The ratio G0G1/G2M indeed reflects a facet of DNA aneuploidy. The presence of >10% of cells in the G2M peak indicates a hyperdiploid state of a cell population (Tribukait et al., 1982; Scott et al., 1989). This possibly provides a more reliable and stringent technique for the determination of aneuploidy than by using the criterion of DNA index (Naguib and Sherbet, 2001c).

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Neural analysis demonstrates that a high degree of accuracy can be attained for the prediction of nodal involvement, based on DNA ploidy together with additional cellular parameters such as SPF and nuclear pleomorphism, measured by ICM techniques. Prediction of lymph node involvement reaches an overall prediction accuracy of 87%, with equally high sensitivity and specificity values (70% and 95%, respectively). We have also analysed the effect of individual parameters on the neural analysis. The omission of SPF results in an increased sensitivity, but all other parameters are of lower values when compared with those of the combined markers. Therefore, it could be concluded that the inclusion of SPF in the analysis results in a negative effect on sensitivity but appears to be a highly significant factor for predicting nodenegative status. In a separate study, Mat-Sakim et al. (1998) have compared the results derived from the neural approach with those obtained using logistic regression and demonstrated that SPF was an independent prediction marker to identify nodenegative patients. This is in concordance with the findings of Muss et al. (1989) that SPF or DNA index is an independent prognostic factor for survival analysis. Based on the ␹2 value, the ANN and logistic regression analyses resulted in identical results (ANN ␹2 test = 10.7989, p = 0.9992). Determinations of DNA ploidy using ICM and conventional flow cytometry have shown significant concordance in several studies (Baretton et al., 1994; Gandour-Edwards et al., 1994; Papadopoulos et al., 1995; Epp et al., 1996). In a further investigation, the Fuzzy K-Nearest Neighbour algorithm (FKNN), regarded as a powerful classifier, has been employed to analyse histological type, grade, and ICM-based measurements of DNA ploidy, SPF, G0G1/G2M ratio and nuclear pleomorphism, in order to determine whether they can provide a basis for making prognostic predictions of survival of breast cancer patients. The subset containing tumour grade, histological type, DNA ploidy, and SPF emerged as the dominant subset predictive of survival (Seker et al., 2000). Odusanya et al. (2002) used a genetic algorithm to evolve prognostic rules for breast cancer. The task for the genetic algorithm system was to establish one or more rules that would perform breast cancer prognosis. In a series of 91 breast cancer patients, the classifiers evolved in their study classified all 81 surviving patients correctly and a varying number of non-survivors correctly. The best classifier that was evolved had an accuracy of 93.41%. Seker et al. (2002a, b) have recently carried out a further evaluation of breast cancer cellular features measured by ICM for their relevance to patient survival. As in their previous studies the cellular features analysed were DNA ploidy, SPF and nuclear pleomorphism. Seker et al. (2002b) analysed the ICM data by ANN-based multilayer feed-forward back-propagation method, FKNN classifier algorithm and conventional logistic regression-based technique. All three methods identified SPF as most significantly related to 5-year survival of patients. These studies show beyond reasonable doubt that artificial neural network approach to analysing ICM data can allow one to identify cellular factors that

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could provide a sensitive and accurate way of predicting tumour progression. Although much progress is conceivably achievable in the coming years, the value of machine learning methods and image cytometric measurement of features of cancer cells is amply exemplified and emphasised by these studies and these should provide a fillip to further investigations along these lines.

Association of DNA ploidy with p53 abnormalities One of the important factors that support the view of a basic relationship between DNA ploidy and premature entry of cells into the S-phase of the cell cycle is the general observation that DNA ploidy is associated with the expression of genes encoding growth factor and hormone receptors. Also the expression of cell proliferation-related genes such as p53 and metastasis-related genes such as S100A4 has been found to correlate with DNA ploidy. Therefore it may be argued that some credence should be given to the apparent association of DNA ploidy with SPF and with possible abnormalities associated with the G0G1 cell cycle checkpoint (Sherbet and Lakshmi, 1997). The suppressor gene p53 has been widely investigated for possible association with aberrant DNA ploidy, on account of the suggested relationship of abnormal expression of p53 with genetic instability. Some early findings, e.g. Sun et al. (1993b) and Yahanda et al. (1995), suggested an association between p53 expression and DNA ploidy. These findings are amply supported by much recent work relating to genomic abnormalities as well as aberrant expression of the gene. In colorectal carcinomas, allelic loss of 17p, which includes the p53 locus, is said to occur predominantly in aneuploid rather than in diploid cancers (Cianchi et al., 1999; Sugai et al., 2000). The levels of p53 protein detected by immunohistochemistry correlates with aneuploidy in several forms of cancer, as for instance, in oral and pharyngeal carcinomas (Raybaud et al., 2000), breast cancers (Midulla et al., 1999; Ottesen et al., 1999) and colorectal carcinomas (Sugai et al.,1999). Equally, there are reports that p53 expression bears no relationship to DNA ploidy (Garcia et al., 1999; Rihet et al., 2000). It ought to be recognised, however, that there are certain problems intrinsically associated with immunohistochemical detection of p53 protein expression. It is essential that stringent criteria are defined and established for p53 positivity, besides ensuring that more than one observer assesses the degree of staining. It should also be pointed out here that the enhanced staining of p53 can be attributed to either stabilisation of p53 by other cellular proteins or to an increase in the half-life of the protein by mutation. The genetic imbalance introduced by allelic loss may encompass more than one gene, and the effects of LOH of p53 ought to be interpreted in the light of the possibility of allelic loss of other genes in the proximity. But then, there are other lines of evidence that do suggest that genetic instability arises in the wake of aberrations in and allelic loss of p53.

6 Chromosomal translocation and its phenotypic effects

Chromosomal translocation and signal transduction Chromosomal translocations have been seen consistently in a number of human neoplasms, especially those of haematopoietic origin. It was recognised many years ago that translocation of genetic material within a chromosome or a reciprocal exchange between different chromosomes can result in the inappropriate activation of genes leading to the initiation of processes, many of which might be associated with tumour development and progression. Thus the expression of several genes involved in the cell proliferation cycle, cell differentiation and in the regulation of genetic transcription, is altered by genetic rearrangement brought by chromosomal translocation. Growth factors and inducers or suppressors of differentiation and cell motility impart specific signals to the cell by binding to appropriate receptors present on the cell surface. These signals are then transduced to specific intracellular compartments by a complex machinery of molecules that form links in the chain of a cascade of signalling events. Chromosomal translocation can affect genes encoding elements of the signal transduction machinery and also genes coding for transcription factors, which are essential for the transcription of genes associated with cell proliferation, differentiation, and apoptotic regulation of growth. A deregulation of their expression will inevitably lead to deregulation of signal transduction. An abnormality even in a single link in this signalling Genetic Recombination in Cancer ISBN 0-12-639881-X

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chain could deregulate the flow of information along the cascade, and will eventually affect genetic transcription that will have serious consequences for the life of the cell.

Deregulation of notch signalling by chromosomal translocation The notch transmembrane protein plays an important part in the survival of precursor cell as well as in determining the cell differentiation pathway during haemopoiesis. The deregulation of notch signalling leads to inhibition of differentiation, maintenance of the undifferentiated or precursor state and enhancement of cell proliferation. Although one can envisage how such abnormal states achieved through deregulated signalling can lead to the development of neoplasia, the functioning of the intracellular pathway and the factors that influence its normal functioning are still only poorly understood. Notch signalling has been an area of intense interest in the wake of this gene being involved in consistent chromosomal translocations involving its locus. Four notch genes, namely notch1/TAN-1, notch2, notch3 and notch4/int-3, have been identified and these are expressed in unique developmental patterns (Williams and Lardelli, 1995; Lindsell et al., 1996). The notch proteins bind ligand proteins in a non-preferential fashion. These are encoded by the DSL (Delta/Serrate/Lag2) family of genes, which include the vertebrate homologues of Jagged1 and Jagged2 (Lindsell et al., 1995; Valsecchi et al., 1997) and Delta1, Delta2, Delta3 and Dll4 (Joutel and Tournier-Vasserve, 1998; Gray et al., 1999; Shutter et al., 2000). The notch pathway of signalling plays a major role in the differentiation of many cell types as well as in the pathogenesis of human diseases such as leukaemias and certain hereditary conditions. Notch ligands are upregulated in cervical neoplasia. The notch signal transduction pathway is deregulated in the congenital autosomal dominant condition known as the Alagille syndrome and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, abbreviated as CADASIL (Viitanen and Kalimo, 2000). Notch expression is upregulated in many human neoplasms. T-cell lymphoblastic leukaemia/lymphoma and the Alagille disorder are known to accompany mutations in notch1, notch3 and Jagged1 genes (Joutel and Tournier-Vasserve, 1998). In CADASIL patients these mutations have been suggested to be a reason for the frequently encountered accumulation of the extracellular domain of notch 3. The mutations could be affecting proteolytic cleavage of the extracellular domain (Joutel et al., 2000). Notch is able to inhibit the differentiation of haemopoietic cells into myeloid lineage (Milner et al., 1996). Intercellular signals often converge to generate the requisite biological responses. Notch signalling is no exception to this and it has other ramifications

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Figure 4 A scheme of notch protein and the translocation breakpoint affecting its function. The organisation of the transmembrane notch protein is shown. The chromosomal translocation t(7;9)(q34;q34.3), discussed above, is often encountered in T-cell lymphoblastic leukaemias. The notch gene occurs at this chromosomal translocation breakpoint and the translocation leads to the generation of a truncated notch transcript (Ellisen et al., 1991). The truncated notch protein appears to be constitutively activated and is capable of transforming cells in vitro (Capobianco et al., 1998). The RAM domain, which occurs close to the transmembrane domain and ankyrin-repeats domains of the intracellular segment of notch protein bind the transcription factor RBP-J to activate transcription of target genes involved in the signalling cascade. The EGF repeats of the extracellular notch domain seem to be required for Delta and Serrate ligand-mediated signalling in wing development of Drosophila (Lawrence et al., 2000).

by being able to interact with other signal transduction pathways. The notch pathway interacts with p53 pathway of cell cycle arrest and apoptosis, and the wnt/␤-catenin and ras-mediated pathway of signal transduction in regulating the processes of cell differentiation and morphogenesis. In certain T-cell lymphoblastic leukaemias the t(7;9) (q34; q34.3) translocation is encountered. The notch gene occurs at this chromosomal translocation breakpoint (Figure 4). Indeed, the breakpoint occurs within 100 bp of an intron in notch, and the translocation seems to lead to the generation of a truncated notch transcript (Ellisen et al., 1991). The truncated notch protein appears to be constitutively activated and is capable of transforming cells in vitro (Capobianco et al., 1997). The notch family genes encode highly conserved transmembrane proteins, composed of an extracellular ligand binding domain (EC) and a transmembrane signalling domain (IC). Notch signalling involves the proteolytic cleavage and translocation of the intracellular domain into the nucleus. The interaction of the two domains of the molecule is essential in signal transduction. When the interaction between the two domains is broken, the extracellular domain is shed. This results in notch activation (Rand et al., 2000). The IC domain seems to be constitutively active but the EC domain inhibits it. The release of the EC relieves this inhibition and activates notch signalling. Mutation of the notch protein leads to the formation of structurally distinct notch extracellular and intracellular domain proteins, and in the shedding of the extracellular domain (Hoemann et al., 2000).

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Figure 5 The notch signal transduction pathway. Represented here is the pathway of notch signal transduction, initiated by the binding of notch ligands to the transmembrane protein leading to the release and translocation of its IC into the nucleus, activation of CBF1 transcription factor and expression of the effector HES proteins. The transduction of the signal along this pathway leads to an inhibition of differentiation and the maintenance of undifferentiated state together with an increase in cell proliferation. SEL-1 has been postulated to possess a negative regulatory effect on notch signalling. EBNA2 and adenovirus E1A protein can bypass this regulatory control and activate HES by activating the CBF1/RBP-J␬ transcription factor. This transcription factor interacts with the RAM and ankyrin repeats domain (see Figure 4) to activate genetic transcription. The notch pathway of signal transduction seems to interact with other pathways such as the p53-mediated pathway and may result in cell cycle arrest. Overall the flow of information along the notch pathway seems to be related to histogenetic origin, as occurring between cells of haemopoietic origin and neuroendocrine descent. Based on references cited in the text and Ansieau et al. (2001); Hsieh et al. (1996, 1999); Kageyama and Ohtsuka (1999); Kurooka and Honjo (2000); Sakai et al. (1998); Sherbet (2001); Strobl et al. (1997); Tani et al. (2001).

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There is now a considerable body of evidence that activation of notch pathway is indicated by activation of HES proteins, which are the effector proteins. The IC is translocated to the nucleus where it interacts with the CBF1/ RBP-J transcription factor and activates it. This transcription factor is in the form of a complex with HDAC co-repressor complex. This interaction displaces HDAC and activates with CBF1/RBP-J transcription factor. The latter activates HES1, because the HES1 promoter is responsive to CBF1/RBP-J (Figure 5). Notch signalling is probably regulated by a number of factors, such as regulatory genes or by other signalling systems that interact with the notch pathway. It is needless to say that the factors that regulate these interacting pathways would also influence notch signalling. Equally, notch signalling might antagonise or block information flow in the interacting pathways. The human SEL1L gene is a homologue of C. elegans sel-1, which has been regarded on empirical grounds as a negative regulator of notch signalling pathway in C. elegans (Sundaram and Greenwald, 1993). SEL1L is located on human chromosome 14q24.3-q31 (Field et al., 1996) in the vicinity of the insulindependent diabetes mellitus (IDDM11) locus. SEL1L shows highly specific expression only in adult pancreatic cells and it is not detected in other tissues. SEL1L has also been implicated in Grave’s disease. The human SEL-1 has also empirically been suggested as a negative regulator of notch signalling. For, inactivating mutations of SEL-1 have been reported to increase notch activity. SEL-1 interacts with the notch receptor and downregulates it (Grant and Greenwald, 1996, 1997). However, such a role has been repudiated in recent studies on notch signalling and the expression of HES-1 in leukaemia and lymphoma cells (Chiaramonte, Calzavara et al., 2002a, 2003). Nonetheless, a differential expression of these genes does occur in tumour cells (Cattaneo et al., 2000), which suggests that notch regulation might be an important factor in neoplastic development. The EBV nuclear antigen (EBNA) can circumvent this cascade of signalling. EBNA2 is itself capable of trans-activating the CBF1/RBP-J transcription factor and activating HES. Similarly adenovirus E1A antigen is also capable of activating this transcription factor and HES1. The integrity of the notch signalling can be established by examining the expression of notch, SEL1L, HES1 as well as the EBNA status (Figure 5). It ought to be recognised, however, that notch signalling could generate opposite effects on cell proliferation and growth. Whilst notch signalling inhibits the differentiation of myeloid precursors in response to different cytokines (Milner et al., 1996; Bigas et al., 1998; Li et al., 1998), it can produce cell cycle arrest and apoptosis in B-cells under certain conditions (Morimura et al., 2000). When infected with adenovirus carrying activated notch, SCLC cells seem to manifest cell cycle arrest accompanied by enhanced expression of the cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1. Furthermore, the signalling downstream seems to follow the ras/raf/MAPK pathway (Sriuranpong et al., 2001). Ronchini and Capobianco (2001) used an oestrogen inducible

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notch (IC)/ER chimeric construct and demonstrated that hormone-induced activation of notch intracellular domain led to constitutive expression of cyclin D1 and in this way influenced cell proliferation and possibly cell transformation. There is apparent here an interaction of notch signalling with the p53-dependent pathway of cell cycle regulation. The p53 family proteins, namely p63 and p73, both required for the cell cycle regulatory function of p53, have been found to be able to upregulate Jagged1 and Jagged2, which encode notch ligands. Jurkat cells expressing notch1 at high levels show a marked upregulation of the downstream effector HES1 of notch signalling, when these cells are co-cultured with cells transfected with p63 (Sasaki et al., 2002). Besides, the induction of p53 in response to DNA damage leads to the activation of the MAPK cascade via ras/raf mediation, thus bringing into a single fold the p53, ras and notch pathways. More indirect evidence for this comes from the relationship between Siah-1, the human homologue of the Drosophila gene Sina (Seven in Absentia) and cell proliferation and apoptosis pathways. According to Susini et al. (2001), Siah-1 can bring about the redistribution of notch from the cell membrane into the cytoplasm and then into the nucleus and activate notch signalling. There are indications from the work of Tewari et al. (2000) of interaction of notch signalling with the p53- and Rb-mediated cell cycle regulation in cervical epithelia. They have described an increase of cell proliferation in dysplastic tissues with parallel increases in p53 and notch1 expression. The expression of HES1, which is the effector of notch signalling, could have a clear role to play in conferring neuroendocrine (NE) phenotype on SCLC. The human counterpart of the achaete-scute (hASH1) is associated with the normal development of pulmonary NE cells and with establishment of NE phenotype of SCLC and other tumours with NE phenotype (H Chen, Udelsman, 1997). H Chen, Thiagalingam et al. (1997) found that induction of HES1 expression in SCLC cells leads to the downregulation of hASH1. This inverse relationship between HES1 and murine hASH1 may be related to the appearance of NE differentiation, as shown in the case of murine ASH1 and HES1. Mice deficient in HES1 display an enhanced murine ASH1 expression together with an increased number of NE cells (Ito et al., 2000). It is obvious that marked differences exist between the haemopoietic cells and cells of neuroendocrine descent with regard to the flow of notch signalling in determining the differentiation, phenotype and in the control of proliferation. Notch interaction with ras signalling is another powerful indicator of cross talk between signalling pathways in determining the degree of cellular responses. In the differentiation of Drosophila mesoderm EGF and FGF receptors activate the ras/MAPK pathway. It would not be out of place to mention here that the EGFr-related transmembrane tyrosine receptor kinase erbB2, which is amplified and over-expressed in many human cancers, might be regulated by notch signalling. Notch activation in turn activates the transcription factor RBP-J␬ initiating further events downstream. Apparently, RBP-J␬ is

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able to initiate erb-B2 transcription (YY Chen et al., 1997). Furthermore, the erb family growth factor receptor signalling is channelled via PI-3 kinase and Akt. This is another point of intersection between notch and other signalling pathways (see Figure 5). Interestingly, ras also induces notch, the notch ligand Delta and the EGFr antagonist Argos (Carmena et al., 2002). Not only this, but the notch mediated neoplastic transformation process seems to require ERK/ MAPK, which are themselves downstream effectors of ras signalling (Fitzgerald et al., 2000). Notch pathway activation plays a significant role in the pathogenesis of T-ALL leukaemia by interacting with other pathways such as ras signalling and apoptosis regulating proteins. Notch pathway could be involved in the enhanced resistance of T-ALL to apoptosis. Hexamethylene bisacetamide (HMBA), a differentiation-inducing agent, appears not only to downregulate notch but also to increase apoptosis (Chiaramonte, Calzavara et al., 2002b). HMBA also inhibits cell growth, which might be related to increased apoptosis. On the other hand, HMBA is known to upregulate the expression of the Cdk inhibitor, p27kip1, and suppress the kinase activity of Cdk (Baldassarre et al., 1999) and HMBA mediated growth inhibition runs parallel to downregulation of Cdk expression (Shirsat et al., 2001). This could be happening via notch activation, since notch can influence cell cycle progression by interfering with the function of cell cycle regulatory proteins. Notch activation itself has been found to downregulate p27kip1 and upregulate Cdk, and this occurs in parallel with continuous induction of cell cycling (Cereseto and Tsai, 2000; Classon et al., 2001). Whilst the correlation between notch downregulation and enhanced apoptosis is significant, it is possible that HMBA might function by suppressing the expression of the anti-apoptotic Bcl2 gene (Siegel et al., 1998). Besides, HMBA is believed to inactivate the JAK/STAT transduction pathway (Arcangeli et al., 2000), which is closely involved in cell proliferation and apoptosis and the pathogenesis of haematological malignancies (see Figures 9 and 10). A co-operation between notch4 and ras pathways has been suggested for breast cancer cell lines. Moreover ras protein is frequently activated in T-ALL patients as compared with B-ALL patients, leading to the hypothesis that ras activation might contribute to the more aggressive nature of the former. An active role has been proposed for ras pathway in the growth of T-ALL cell lines. Consistent with this, Chiaramonte, Calzavara et al. (2002b) have noted a marked and widespread activation of Akt. MEK activation seems to be more limited in nature. This accords with the ability of Akt to inhibit MAPK. Furthermore, inhibitors of the ras signalling affect tumour cell proliferation rate. Finally, rasmediated transformation was earlier shown to be accompanied by changes in notch expression levels (Ruiz-Hidalgo et al., 1999). This lends further support to the view that deregulation of notch and ras signalling might generate convergent effects of cell transformation occurring in the pathogenesis of leukaemia. Thus several factors appear to be involved in the autoregulatory mechanism of different interacting pathways. Indeed, it may be that both activated ras and

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notch might function by downstream signalling mediated by Akt and in consort with HPV oncogenes, which participate in the pathogenesis of cervical neoplasms (Rangarajan et al., 2001) (see also Figure 10). The interaction of notch signalling or its interconnection with other pathways of signal transduction is not an unusual phenomenon. This could be a mechanism of mutual regulation of information flow with resultant opposing function of notch. The wnt pathway is a good example of this. The wnt family proteins are morphogenetic proteins. They are associated with segmentation in Drosophila, cell proliferation and migration as well as in carcinogenesis. Notch and wnt proteins seem to generate opposite effects in the morphogenesis of epithelial cells. In this system wnt seems to be able to counteract the inhibition of morphogenesis by notch (Uyttendaele et al., 1998). On the other hand, notch and wnt co-operate in neuronal development in embryogenesis (Cotter et al., 1999). A proper functioning of notch seems to be essential for wingless (wnt) signalling of morphogenesis and development of the wing and other tissues of Drosophila (Hing et al., 1994). The wnt signalling system has serious repercussions on the adhesive faculty of cells via the modulation of expression of ␤-catenin linked in turn to the transmembrane cadherins that are closely involved with intercellular adhesion (Sherbet, 2001). One should anticipate therefore that all these interacting pathways of signalling would inevitably modulate cell motility or the invasive behaviour of cancer cells. Furthermore, with the participation of notch in regulating cell proliferation it is not inconceivable that the p53-mediated pathway of cell cycle arrest and apoptosis is most likely another pathway that interacts with notch in determining phenotypic outcome.

Loss of integrity of notch signalling in leukaemia and lymphoma cells Chiaramonte, Calzavara et al. (2003) examined the integrity of notch signalling cascade in a number of leukaemia and lymphoma cells in culture (Table 10). They defined three groups of leukaemia and lymphoma cell lines in respect of operation of notch signalling, namely group 1 in which the pathway appears to be subject to regulation by SEL1L. This group included control cell lines and a majority of lymphoma cell lines. Group 2 included T-ALL established cell lines, in which notch signalling is constitutively active and this is characterised by HES1 expression in the absence of notch and in the presence of SEL1L expression. Group 3 contains the cell lines, in which notch seems to be constitutively functional due to the intervention of EBNA, which circumvents the notch signalling cascade and leads to HES1 activation. Farrel et al. (1997) showed that EBNA mimics the notch pathway. Furthermore, certain proteins encoded by EBV genes have recently been shown to interact with and influence notch IC domain-mediated signalling (Zhang et al., 2001; Kusano and

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Raab-Traub, 2001). The findings of Chiaramonte, Calzavara et al. (2003) support the view that EBNA can and does abnegate notch signalling. However, it is apparent that EBNA seems unable to bypass the negative regulation of HES1 expression in BL135 and AS283, which might suggest that EBNA might function also by means other than interaction with the IC domain of notch protein. This is compatible with the finding that EBNA2 can function as a homologue of notch IC and the notch IC can transactivate EBNA2-regulated viral promoters that contain CFB1/RBP-J␬ recognition elements (Hoefelmayr et al., 1999; Cotter et al., 2000; Kusano and Raab-Traub, 2001).

Table 10 Expression of component genes of the notch signalling pathway in leukaemia and lymphoma cell in culture Cell line

Group 1a PMBC Daudi*/EW36

Tumour EBNA2 EBNA SEL1L TAN-1 TAN-1 HES1 Notch type gene protein (wt) (truncated) pathway

Control

ND

–

+

+

–

–

Intact

Lymphoma

+

–

–

–

–

–

Intact

Lymphoma

+

++

+/–

–

–

+

CA (EBNA)

Group 3a SupT1; FRO

T-ALL

–

–

+

+/–

+

+

CA

Group 3b MOLT4, CEM; KE37

T-ALL

–

–

+

+

–

+

CA

Group 1b BL135; AS283; VAL; LY8; BL41 Group 2 Raji; BC2

The table summarises the expression of genes involved in the notch pathway in some leukaemia and lymphoma cell lines. Column 3: – and + signs indicate absence or presence of EBNA2 in cell genome. In the other columns: – sign indicates no detectable expression, +/– low level of expression, and ++ high expression of the genes. EBNA: Epstein–Barr virus nuclear antigen-2; CA: constitutively activated; CA (EBNA) EBNA-mediated constitutive activation; ND: not determined. *Daudi cell line is reported to be EBV infected (ATCC catalogue). Truncated TAN-1 was expressed in SupT1 as a result of a translocation found in T-ALLs and widely studied by Ellisen et al. (1991), causing the loss of most of the extracellular domain of the protein with a consequent constitutive activation. Source: Based on Chiaramonte, Balordi et al., 2001; Chiaramonte, Calzavara et al. (2003)

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The data published by Chiaramonte, Calzavara et al. (2003) are also amenable to the interpretation that notch signalling might differ between T-ALL and B-lymphoma, as in the former it is due to an alteration in the notch pathway itself, whereas, in some B-lymphomas, notch activation seems to be due to the intervention by EBNA2. In others, EBNA2 does not result in notch activation. In other words, signalling in B-lymphomas is not invariably associated with EBNA2 expression. Therefore the presence of EBV in the genome of a B-cell is not sufficient to activate the proliferation by genes such as HES1. These results seem to suggest that tumorigenesis is not invariably correlated or associated with EBV infection. Whilst the present findings support the view that EBNA can abnegate notch signalling, the constitutive activation of the pathway in the absence of any interference from EBNA, as well as the inability of EBNA to be an invariable activator of the notch pathway, raises the highly pertinent question of whether there are other factors that can abrogate the regulation of notch signalling. As stated already, EBNA2 activates the CBF/RBP-J␬ transcription factor and activate HES1, since the HES1 promoter is responsive to CBF1/RBP-J transcription factor (Hsieh et al., 1996; Strobl et al., 1997). Interestingly, B- and T-lymphocytes differ markedly with regard to interaction between notch IC and CBF1/RBP-J␬. B-cells show no notch IC interaction with the transcription factor, but such association does occur in T-lymphocytes (Callahan et al., 2000). This further adds weight to our finding of constitutive notch activation in T-ALL, but differential effect of EBNA2 in B-lymphomas. Perhaps one should also consider the possibility that this might occur by interaction with other developmentally regulated signal transduction pathways such as the wnt- or ras-mediated pathway of signal transduction.

Genetic abnormalities downstream of notch signal activation It would be needless reiteration to say that a signalling process can be deregulated by abnormalities of any one of the components of the machinery. The human SEL1L gene shows sequence homology to sel-1 gene of Caenorhabditis elegans. Sel-1 has been reported to negatively regulate the activity of lin-12 gene, which is implicated in the processes of cell differentiation. It has been suggested on an empirical basis that notch signalling is also negatively regulated by SEL1L gene, which is the homologue of the Caenorhabditis elegans sel-1. It has been postulated that SEL1L interacts with notch receptor and downregulates it (Grant and Greenwald, 1996, 1997). Mutations of sel-9 enhance expression of lin-12 and glp-1, which code for members of the notch family receptors (Wen and Greenwald, 1999). Sel-1 mutations similarly increase lin-12 activity (Grant and Greenwald, 1997). Indeed, it has been suggested that Sel-1 might interact with lin-12 and glp-1 receptors or their ligands to downregulate signalling. These findings have

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provided grounds for formulating the concept that SEL1L negatively regulates notch signalling. Equally, however, it may be argued that this concept is still unproven. Notch signalling culminating in the activation of HES1 would be expected to inhibit differentiation and promote cell proliferation. It follows therefore SEL1L expression should correlate with differentiated state of tissues and should relate inversely to cell proliferation. Unexpectedly, however, SEL1L expression is high in pancreatic and gastric carcinomas, and even more incongruous with its postulated role, it shows similar expression between normal adult and neoplastic tissues (Biunno et al., 1997; Cattaneo et al., 2000). SEL1L expression is said to be downregulated in breast carcinomas and indeed related to clinical aggressiveness of the disease (Orlandi et al., 2002). Orlandi et al. (2002) found high expression correlated with good prognosis, albeit only marginally significant statistically. Implicit in these studies is the assumption of a regulatory role for SEL1L in notch signalling. However, Chiaramonte, Calzavara et al. (2002a) noted that leukaemia and lymphoma cells expressed HES1 irrespective of SEL1L expression and found no inverse relationship between SEL1L expression and the status of notch signalling. As we have discussed elsewhere in this book, highly polymorphic CA dinucleotide repeats occur in this gene (Chiaramonte, Sabbadini et al., 2002). These occur in introns 2 and 20. Although their presence in the introns will have no bearing on the expression of the transcript, it does raise the possibility that SEL1L might be liable to undergo abnormal genetic alternations that could contribute to the neoplastic process. It ought to be pointed out, however, Chiaramonte, Sabbadini et al. (2002) found no structural abnormalities in SEL1L in the lymphoma and leukaemia cell lines they had investigated. Taken together, the findings of Chiaramonte, Calzavara et al. (2002a) do not support the postulated negative regulatory role for SEL1L in notch signalling. The SEL1L gene is located at 14q31 and this locus harbours a number of genes, notably the thyroid stimulating hormone (TSH) receptor gene. Significantly, a gene residing at this locus encodes a 230 kDa protein, which seems to be capable of binding the cell cycle regulatory Rb protein as well as the TSH receptor. The Rb protein could be regulating cellular response to TSH via the Rb-interacting protein (KH Chang et al., 1997). It is not known at present whether Rb affects the transcription of other genes in the locus including SEL1L. A gene whose expression is related to the myelination of the CNS (Ono et al., 1999) and genes belonging to the fibulin family are also present at the locus. The protease inhibitor kallistatin gene has been assigned to chromosome 14q31–32.1 as also genes coding for ␣-1-antichymotrypsin, ␣-1-antitrypsin, and protein C inhibitor (Chai et al., 1994). These proteins are associated with cancer in some way or other. For example, protein C is an important component of the anticoagulation pathway and could be at the root of coagulation disorders encountered in cancer patients. Deficiency of protein C and resistance to activated protein C are major risk factors of coagulation

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disorders in the general population as well as in cancer patients (Nijziel et al., 1999; Paspatis et al., 2000). Other factors such as the endothelial cell protein C receptor and thrombomodulin have been implicated in the functioning of protein C. Both the protein C receptor and thrombomodulin are expressed frequently in cancer cell lines and in breast cancer (Tsuneyoshi et al., 2001). ␣-1-antitrypsin was shown many years ago to possess immunosuppressive properties, besides being associated with certain human neoplasms as a paraneoplastic marker. It was even suggested to relate to the state of progression of carcinoma of the cervix (Sherbet, 1982). Recent work indicates a link-up between ␣-1-antitrypsin deficiency with genetic instability. For instance, a fifth of colonic carcinomas with high microsatellite instability were deficient in ␣-1 antitrypsin. In contrast, colonic tumours with low microsatellite instability were comparable with a control group in respect of antitrypsin deficiency (P Yang et al., 2000). The obvious conclusion is that there is a link between expression of antitrypsin alleles and genetic instability. It is also worthwhile noting that smokers carrying antitrypsin deficiency alleles carried a 20-fold greater risk of developing colorectal cancer with genetic instability (Yang, Cunningham et al., 1999). The antitrypsin gene Pi locus is highly polymorphic, and heterozygous individuals carrying a defective Pi allele are prone to develop various neoplasms, e.g. lung carcinoma (Yang, Wentzlaff et al., 1999). We also know that PiZ type of antitrypsin deficiency strongly correlates with the incidence of hepatic tumours. Patients who were heterozygous for PiZ carried a higher risk of developing hepatic neoplasms (Zhou et al., 2000). Higher levels of antitrypsin also occur in patients with biliary cancer as compared with benign disease (Hedstrom et al., 1999). Antitrypsin might be involved in a paracrine mechanism of growth regulation in cancers. Albeit risking a minor digression, these findings emphasise the importance of the potential of genetic changes to the neoplastic process.

Fibulins, notch signalling and cancer invasion The extracellular matrix (ECM) plays a major part in cellular motility, cell differentiation and morphogenesis and in signal transduction. It has been recognised that remodelling of the ECM is an essential feature of neoplastic transformation and altered configuration and spatial distribution of the components of the ECM confer the capacity for invasion and the formation of metastasis at distant sites. Among ECM components of note are fibronectin, laminin, integrins, tenascin and a variety of intercellular adhesion-mediating components such as cadherins and cell adhesion molecules (Sherbet and Lakshmi, 1997). The fibulin family of proteins are Ca2+-binding ECM glycoproteins. Fibulins are known to associate with another ECM protein, namely fibronectin (YC Gu et al., 2000). By virtue of the RGD motif that they possess, fibulins can interact

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with surface integrins. Yanagisawa et al. (2002) suggest that fibulins might subserve the function of a scaffold protein, a function mediated by their RGD motif. Thus fibulins can influence cell adhesion and cellular motility. They also participate in developmental processes and organogenesis. They might subserve specific functions, as for instance in the differentiation and development of the heart, skeletal and neural structures (Miosge et al., 1996). Certain fibulins interact with integrins but it is uncertain if this has any functional significance (YC Gu et al., 2000), although one should consider their function in terms of integrin-mediated signal transduction. As Yanagisawa et al. (2002) have shown, fibulin-5 interacts with elastin, which can bind EGF receptors. They might indeed function as a link in the EGF signalling machinery of the cell. Fibulins are oestrogen-regulated proteins (Rochefort, 1999). They are believed to be able to influence cell growth. Gallagher et al. (1999) found that the fibulin called MBP1 interacts with mutant p53 and enhances cell transformation and growth rate. Colonic tumours have been shown to express 3- to 7-fold higher levels of fibulin-4 mRNA than control cells. Oestradiol greatly enhances the secretion of fibulin by ovarian cancer cell lines and ovarian cancers (Clinton et al., 1996). The expression of the glycoprotein increases markedly from normal ovarian epithelium to serous carcinomas (Roger et al., 1998). The nature of its participation in the neoplastic process has naturally been the subject of some studies. Fibulins seem to inhibit the invasion of ER+ breast cancer and ovarian cancer cell lines (Hayashido et al., 1998; Rochefort et al., 1998). Of the fibulin family, fibulin-1, 2 and 4 have been assigned to chromosomes 22q.13.2-q13.3, 3p24-p25 and 11q13 respectively (Mattei et al., 1994; Zhang et al., 1994; Gallagher et al., 2001). However, it is of some interest, in the present context of SEL1L function, that fibulin-5 gene occurs at the SEL1L 14q31 locus (Kowal et al., 1999). Unfortunately, fibulin-5 has not been investigated for its potential function of influencing cell adhesion and cancer cell motility. With this background information of the involvement of other members of the fibulin family in inhibition of ER-mediated motility of cancer cells, it might be worthwhile investigating fibulin-5 to provide a possible link with cancer cell motility with notch signalling. The question naturally arises whether any genetic alterations were associated with SEL1L locus in parallel with neoplastic development and progression. A few human tumours have been investigated from this viewpoint. As stated earlier, the relationship between SEL1L and cancer behaviour is not beyond question. SEL1L is expressed abnormally in some human neoplasms, but expression can be at comparable levels between normal adult and neoplastic tissues (Biunno et al., 1997; Cattaneo et al., 2000). On the contrary, SEL1L expression is said to be downregulated in breast carcinomas and indeed related to clinical aggressiveness of the disease (Orlandi et al., 2002). There has been some focus of attention on whether any genomic abnormalities are present at the fibulin/SEL1L locus. From some early work Lee

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et al. (1997) described LOH of 14q31–32.1 in human head and neck squamous cell carcinomas. They found frequent LOH at that locus, and specifically the loss of 4–7 cM regions of 14q31–32.1. But Bockmuhl et al. (2000) encountered over-representation of 14q31 and 14q32, among other loci. In both studies, the genetic alterations were associated with reduced patient survival. Cheng et al. (1997) reported LOH at 14q31 locus in 7/21 nasopharyngeal carcinomas that they had investigated. LOH was frequently encountered in adenocarcinoma of the oesophagus and gastric cardia, and interestingly LOH occurred more frequently in adenocarcinomas of Barrett’s oesophagus than of gastric cardia (Van Dekken et al., 1999). In most of these studies genetic changes occurred also in other chromosomes, and as a consequence it is difficult to evaluate the contribution of changes at the 14q31 locus to the pathogenesis of the disease. Recently Chiaramonte, Calzavara et al. (2002a) failed to notice any genetic abnormalities with SEL1L in leukaemia and lymphoma cells. However, two intragenic cytosine-adenosine microsatellite repeats, CAR/CAL and RepIN20, which have been identified in SEL1L gene in intron 2 and 20 respectively (Biunno et al., 2000; Chiaramonte, Sabbadini et al., 2002). Dinucleotide microsatellite repeats tend to be more polymorphic than others. One should recognise that these highly polymorphic repeats might mutate genes present in that particular locus and deregulate signalling pathways. Finally, O’Connell et al. (1999) made some remarkable observations in their study of breast cancer. They found that LOH at 14q31 was more frequent in breast cancer with no nodal metastasis than carcinomas that had spread to the axillary lymph nodes. This suggests that the locus might be harbouring a gene that promotes metastatic spread. In the light of these various findings, notwithstanding the contradictory nature of some, it seems there is much scope in pursuing the potential role of genes functioning downstream of notch activation in the signalling cascade in relation to neoplastic development and dissemination.

Chromosomal translocation and the dynamics of cell population expansion in cancer Tumour growth and expansion are the net outcome of the forces of cell proliferation and apoptosis. Normal cell proliferation is an exquisitely controlled process and abnormalities in the genetic material induce the expression of genes that control the progression of the cell in the division cycle and direct it into the apoptotic pathway. A number of genes that may be implicated in growth stimulation are induced to express at requisite levels or induced to express at inappropriately high levels as a consequence of chromosomal reorganisation involving them. Also affected in this way are certain genes that regulate the response of cells to apoptosis signals.

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The modulation of cell proliferation-related gene expression by chromosomal rearrangement An early demonstration of the effects of chromosomal translocation on the pattern of genetic expression is the transposition of the cell proliferationrelated c-myc gene, on chromosome 8, into the proximity and control of transcriptional elements of the immunoglobulin gene occurring on chromosome 14, which leads to the activation of the former. This is a major feature of murine plasmacytoma and Burkitt lymphomas. The Philadelphia (Ph) chromosome is formed by a reciprocal translocation between chromosomes 9 and 22. In CML, the result of this translocation is the production of a chimeric protein by the fusion of bcr gene product and the abl proto-oncogene. In ALL, other breakpoints might be involved and this leads to the formation of a different fusion protein with different biological properties. Subsequently, a large number of proto-oncogenes have been found to be so activated, or transcribed in an aberrant protein structure, as a consequence of translocation, deletion or inversion of chromosomal material. In recent years, many chromosomal breakpoints and translocation products have been characterised at the molecular level and this has led to the identification of molecular features of haematopoietic neoplasms that could be used to devise new and improved strategies of treatment. The activation of c-myc in Burkitt lymphomas might seriously affect the regulation of the cell cycle. As an example, the work of Pajic et al. (2000) may be cited here. These authors established an EBV-EBNA1-positive cell line called P493–6, in which the myc gene is placed under the control of a tetracyclineregulated promoter. These cells showed cell cycle arrest G0G1 when myc was switched off. When the gene was reactivated the cell cycle was activated, together with the expression of cyclins D and E and cdk4. The activation of myc also led to the activation of cyclin E kinase and hyper-phosphorylation of the Rb protein. Thus a trail of massive alterations occurs in the expression of several regulatory molecules that control cell cycle progression. The erb genes encode a family of transmembrane receptor tyrosine kinases (RTK), which function as receptors for a number of peptide growth factors, among them are EGF, TGF and neuregulins. Binding by ligands induces the receptors to dimerise, which in turn, leads to auto-phosphorylation. These activated receptors are now able to bind to intermediate adaptor proteins containing what are called src-homology (SH) domains. The SH-containing adaptor protein allows the transduction of the signal downstream eventually leading to genetic transcription. The erb ligand called ␥-heregulin is a member of the neuregulin family of growth factors that bind to erb RTK in the initiation of signal transduction. Wang et al. (1999) have shown that ␥-heregulin is the product of a chimeric gene, resulting from the fusion of DOC4 and HGL genes, which occur on chromosomes 11 and 8 respectively. The fusion seems to occur as a consequence of a translocation. The translocation results in the formation

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of the fusion product, ␥-heregulin, that seems to possess the ability to activate the erb signal transduction machinery leading to inappropriate cell proliferation. Similarly, the bcr-abl fusion protein, which results from t(9;22)(q34;q11), seems to be able to downregulate cell cycle regulators and enable cells to enter into the S-phase (see below).

Genetic rearrangement and the regulation of apoptosis by bcl2 gene family A family of genes called bcl (B-cell leukaemia-lymphoma)-2 are closely involved in the regulation and control of apoptotic cell death. Some members of the bcl2 family, e.g. bax, bak, bok/mtd, bik/nbk, bid, bad, etc., promote the apoptotic process, whilst bcl2, bcl-xL, mcl1 and others protect cells from apoptotic disintegration. The regulation and control of apoptosis seems to be achieved by interactions between members of the two sub-groups. The bcl2 proteins contain four BH (bcl2 homology) domains. The domains BH1, BH2 and BH3 form a hydrophobic pocket in the molecule, which can bind to BH3 of another type of bcl2 protein. The relative importance of these domains to the apoptotic function is still unclear. However, there are indications that BH3 alone might be sufficient to induce apoptosis. A fourth bcl2 homology domain called BH4 might mediate the interaction with other pathways of apoptosis. Proteolytic enzymes of the caspase family and the caspase-binding protein called apaf-1 mediate apoptosis. Apoptotic stimuli activate the dimerisation of caspases, which is also related to the oligomerisation of apaf-1. Cytochrome c located in the mitochondria is required for apaf-1 activation. However, activation is prevented because apaf-1 being located in the cytoplasm is physically sequestered from cytochrome c. The pro-apoptotic bcl2 proteins seem to breach the integrity of the mitochondrial membrane and allow cytochrome c to leak out into the extra-mitochondrial cytoplasm. This sets in motion the interactions that activate apaf-1 and the caspases leading to cellular apoptosis. The anti-apoptosis bcl2 could be interacting with pro-apoptosis bcl2 to inhibit the ability of the latter to release cytochrome c. It is also conceivable that anti-apoptosis bcl2 possess the ability to suppress cytochrome c release and this ability might be affected by their interaction with the pro-apoptosis bcl2 (Gross et al., 1999; Strasser et al., 2000; Sherbet, 2001). There is also some evidence suggesting that caspases activated by apoptotic stimuli might be able to convert anti-apoptotic proteins into proteins with pro-apoptotic properties as demonstrated for the removal of BH4 by TNF family cytokines (MC Chen et al., 2000). This phenomenon can be interpreted in terms of the role of BH4 in mediating the interactions between different pathways leading to apoptosis. In diffuse B-cell lymphoma, t(14;18) (q32;q21) translocation occurs frequently. Associated with this is a rearrangement of the bcl-2 gene. Hence translocation and rearrangement might be responsible for its amplification and

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consequent over-expression (Monni et al., 1999). However, bcl-2 expression is not accompanied by t(14;18) translocation in nasopharyngeal carcinoma (Harn et al., 1996). Possibly, therefore, other mechanisms must be considered. Monni et al. (1999) also speculate that over-expression might be due to point mutations and suggest that these mutations might alter its interactions with other cellular proteins. Point mutations in the gene were indeed reported in non-Hodgkin’s lymphoma and lymphocytic leukaemia some years previously (Tanaka et al., 1992; Reed and Tanaka, 1993). Another putative bcl family gene, i.e. bcl-8, has been identified in B-cell lymphoma in association with t(14;15)(q32;q11–13). Bcl-8 expression is found in patients who carry an abnormality at the 15q11.3 locus involved in the translocation (Dyomin et al., 1997).

Bcl2 in p53-induced apoptosis The bcl2 and related genes are subject to regulation by the cell proliferationrelated p53, which is one of the interacting pathways in the control of apoptosis. The wild-type p53 protein has a wide-ranging function, including regulation of cell cycle arrest, in a transient or sustained way, and regulation of apoptosis and cell senescence. DNA damage induces p53. Two kinases phosphorylate the suppressor protein. One of these is ATM, a 370 kDa protein, which is a member of the PI-3 kinase family. The importance of the ATM pathway is underlined by the fact that ATM is itself a suppressor protein of considerable significance in the pathogenesis of cancer being involved closely in DNA damage response and in the activation of the apoptotic process. ATM is mutated in ataxia–telangiectasia (AT) patients. AT characteristically involves progressive neuronal degeneration, enhanced radiation sensitivity and immunodeficiency. Furthermore, AT patients are predisposed to develop lymphoid tumours. ATM is suggested to function as a suppressor gene in non-AT individuals. P53 is also phosphorylated by checkpoint kinase (Chk)1 and Chk2. Chk2 is the human homologue of the kinase Cds1 of Schizosaccharomyces pombe. Both kinases can function at both G1-S and G2-M checkpoints (Bell, Varsley et al., 1999; Liu et al., 2000). Chk itself is activated by phosphorylation by an ATM-dependent mechanism or independently of ATM (Rhind and Russell, 2000; Zhou and Elledge, 2000). The phosphorylation of p53 by ATM (Banin et al., 1998) or Chk2 has serious consequence for p53 function. The modification blocks the binding of p53 to mdm2 and inhibits degradation of p53 (Hirao et al., 2000). P53 can now induce the transcription of the genes involved in bringing about cell cycle arrest and apoptosis. In the opposite direction of the pathway p53 and mdm2 are involved in an auto-regulatory loop that can result in an over-expression of mdm2 that leads to p53 degradation, and progression of the cell cycle and inhibition of apoptosis. Mdm2-mediated degradation of p53 is regulated by mdmx, which shows a high

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degree of homology to mdm2. Mdmx forms heterodimers with mdm2 and in this way it seems to be able to counteract the function of mdm2 (Figure 6). Caspases may act by influencing mdm2-mdmx mediated regulation of p53 degradation. An auto-regulatory route similar to that involving mdm2 and p53 might also be operating between mdmx and p53, for p53 activation has been shown to decrease mdmx levels, possibly by a feedback regulatory mechanism (Gentiletti et al., 2002). Another pathway of mdm2-mediated regulation of p53 is shown in Figure 7. The CDKN2 gene (9p21) encodes two structurally

Figure 6 The pathway of mediation of cell cycle arrest and apoptosis by p53 and mdm2. The Figure illustrates p53-mediated induction of cell cycle arrest and apoptosis and the involvement of mdm2 in the regulation of this function. DNA damage induces p53 expression, which in turn induces the expression of mdm2. This interaction leads to the degradation of p53 and so the progression of the cell cycle continues unhindered and apoptosis of cells is inhibited. However, phosphorylation of p53 by kinases Chk and ATM inhibits the binding to p53 with mdm2 with resultant inhibition of p53 degradation. Now p53 is able to induce genes, such as the p21waf1/cip1, GADD and the bcl2 family pro-apoptosis gene bax-1, and in this way produce cell cycle arrest and apoptosis. Mdm2-mediated degradation of p53 may be counteracted by heterodimerisation of mdm2 with mdmx, a structural homologue of mdm2. Caspases affect both mdm2–mdmx and Chk–ATM mediated control of p53 function. (Chk, Checkpoint kinase; ATM, kinase encoded by ataxia telangiectasia mutated gene.) Based on references cited in the text and Ding and Fisher (1998); Levine (1997); Polyak et al. (1997); Prives and Hall (1999).

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Figure 7 Pathways of cell cycle arrest by p53 and Rb by the mediation of CDNK2 gene encoded proteins. The Figure shows the pathways of p53 and Rb mediated cell cycle arrest involving CDKN2 gene products p14ARF and p16ink4 respectively. These two CDKN2 products are structurally different and are obviously functionally different too. Whilst p16 functions via effect on cyclin D1, p14ARF inhibits the mdm2-mediated degradation of p53, leading to an accumulation of p53 and consequently to cell cycle arrest. Both p53 and Rb pathways are known to be abnormal in certain gynaecological cancers (see Sherbet and Patil, 2003). Several fusion proteins are involved in the differentiation of haemopoietic stem cells, and cell proliferation, apoptosis and survival. The chimeric AML1-ETO is formed from translocation t(8;21). The AML1-ETO has been shown to suppress p14ARF promoter and reduce p14ARF levels (Linggi et al., 2002) and seems to regulate p53 function in this way.

different proteins, namely p16ink4a and p14ARF. They share exons 1 and 2, but differ with regard to exon 1. Exon 1␤ of p14ARF occurs approximately 13–20 kb upstream of exon 1␣ of p16ink4a (Sherr, 1998). They differ functionally too. P16 functions via the cyclinD1 and Rb pathway to arrest the cell cycle, whilst p14ARF inhibits mdm2-mediated degradation of p53. A consequence of this is an increase in the accumulation of p53 leading to cell cycle arrest. ATM is also a substrate for caspases (Hotti et al., 2000) and it is conceivable that interaction of p53 with mdm2 would be affected by modification of Chk/ ATM pathway of phosphorylation. Inactivation of the Chk/ATM pathway can also occur by Chk mutation in human lung cancer (Matsuoka et al., 2001) and mutation of Chk together with ATM-related ATR in stomach cancers (Menoyo et al., 2001). As stated earlier, AT patients are prone to develop lymphoid malignancies. Sporadic lymphoid malignancies also show inactivation of ATM by mutation. A wide variety of ATM mutations have been encountered in breast cancer patients and some of these might be potential contributors to the initiation and progression of the disease (Dork et al., 2001). ATM expression has been described to be low in invasive carcinoma as compared with benign lesions of the breast (Angele et al., 2001). Angele et al. (2001) add the rider that p53

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might have been altered in these patients (Figure 6, and also see below). Equally, some regard somatic mutations in ATM to be a rare event in the pathogenesis of breast cancer. The tumour suppressor protein p53 seems to be involved in bcl2-mediated apoptosis and it appears possible that BH4 domain mediates the interaction between bcl2- and p53-mediated apoptotic pathways. The anti-apoptotic BFL-1 protein belonging to the bcl2 family very effectively suppresses p53-induced apoptosis (D’Sa-Eipper and Chinnadurai, 1998). However, mutations in its conserved N-terminal BH4 reportedly do not affect p53 function of cell cycle regulation, suggesting the possibility that bcl2-mediated apoptosis and p53-dependent cell cycle regulation are independent of each other (DCS Huang et al., 1997). Nevertheless, this needs to be clarified, with the complexity of interaction between the various regulators of cell cycle. Of special interest would be the phosphoprotein mdm2, which interacts with the tumour suppressor p53 and Rb, which are both closely associated with the regulation of cell cycle progression (Sherbet and Lakshmi, 1997). We also know that mdm2 interacts with a number of transcription factors such as E2F (Martin et al., 1995). These transcription factors form a component part of a self-regulatory mechanism. Thus p53 interacts with mdm2 and induces its transcription. With this enhanced transcription, the balance seems to shift in favour of mdm2, which then binds to and inactivates p53 (Barak et al., 1993; Wu et al., 1993). The overall effect is the degradation of p53 and inhibition of E2F-dependent apoptotic process (Oliner et al., 1993; Wu and Levine, 1994; Haupt et al., 1997; Kowalik et al., 1998). E2F, as a homodimer and also as E2F/DP1 heterodimers, binds to DNA in a sequence-specific manner and induces the transcription of E2F-responsive genes, which are necessary for progression of the cell cycle. The cell cycle regulatory Rb protein in the under-phosphorylated state can bind E2F complex and repress its function. Finally, p53 appears to be able to induce the transcription of Rb. Thus both p53 and Rb not only co-operate in regulating the traverse of the cell cycle but may also be jointly implicated in E2F-mediated apoptosis. It is needless to say, however, that cell cycle arrest and apoptosis have been described, which apparently take place independently of p53.

T-cell leukaemia/lymphoma (TCL1) gene in apoptosis With the perceived implication of the bcl-2 family genes, which are related to apoptosis, it is not surprising that genes with potential anti-apoptotic function might be affected in haematological malignancies. The T-cell leukaemia/ lymphoma (TCL1) gene is located on chromosome 14q32.1 (Virgilio et al., 1994). TCL1 encodes a cytoplasmic 14 kDa protein (Virgilio et al., 1998) normally found in T-cell progenitors, pre-B and immature B-cells. There is a decrease in its expression with the maturation process (Thick et al., 1996; Pekarsky et al., 1999, 2001b; Narducci et al., 2000). Its involvement in T-cell

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leukaemias as well as B-cell lymphomas and AIDS-related lymphoma has been amply demonstrated (Virgilio et al., 1994; Narducci et al., 1995, 1997; Thick et al., 1996; Pekarsky et al., 1999). TCL1 activation and over-expression can be related to chromosomal translocation t(14;14)(q11;q32) or inversion inv(14)(q11;q32.1) resulting from breakage at a site close to the TCL1 locus. The activation has been attributed to TCL1 rearrangement with the promoter of T-cell receptor genes. Yuille et al. (2001) found demethylation of specific sites of the TCL1 promoter might be an alternative mechanism of activation in instances where TCL1 activation is seen in the absence of chromosomal rearrangement. Several TCL related genes, TCL1b, TNG1 and TNG2, are found at the TCL locus (Saitou et al., 2000; Pekarsky et al., 2001a, b). The chromosomal rearrangements mentioned above also result in an over-expression of these genes, and in general this cohort shows similar patterns of expression (Hallas et al., 1999). Transgenic mice carrying human TCL1 gene driven by the lck (lymphocyte-specific kinase) promoter have been found to develop AML (Croce, 1999). Thus there is experimental as well as correlative evidence of TCL1 family gene activation as being responsible for the pathogenesis of T-cell leukaemias and B-cell lymphomas. The expression of TCL1 in B-cell lymphomas seems to relate strongly with the state of differentiation. It is expressed in a majority of low-grade B-cell lymphomas, mantle-cell lymphoma and follicular lymphomas. TCL1 expression is infrequent in high-grade diffuse large B-cell lymphoma (Narducci et al., 2000; Nakayama et al., 2000). Implicit in these findings is a relationship between cell proliferation and TCL1 expression. On the face of it, this is the expected negative correlation with differentiation and positive correlation with proliferative state. But some of the above studies suggest the possibility that TCL1 might influence tumour cell expansion by an anti-apoptosis mode of function. The MTCP1 (mature T-cell proliferation 1), which occurs on chromosome Xp28, is also related to the TCL family and is activated, albeit rarely, in mature T-cell leukaemias and also in benign proliferative foci. The activation of MTCP1 seems to be due to t(X;14)(q28;q11) (TB Fu et al., 1994; Thick et al., 1996). Thus the TCL genes together with MTCP1 may be regarded as constituting a family of oncogenes that are closely associated with the pathogenesis of haemopoietic disorders and neoplasms.

The PI-3 kinase/Akt pathway of signal transduction in cell proliferation, apoptosis and differentiation Many extracellular signals are transduced into the cell via specific growth factor receptors and these signals flow along the phosphoinositide (PI-3) kinase/Akt pathway. The induction by oncogenes of tumour vascularisation by means of VEGF induction also involves the PI-3/Akt pathway, which can also be

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implicated in the function of certain tumour suppressor genes (Figure 8). Akt kinase might directly control cell cycle progression by phosphorylationmediated regulation of activators and inhibitors of cyclins and cyclin-dependent kinases closely implicated in cell cycle phase transitions. Thus the PI-3/Akt pathway has come into sharp focus in relation to the acquisition of proliferative potential and protection of cell from apoptotic stimuli and acquisition of motility or invasive ability by cancer cells.

Akt in PTEN tumour suppressor function PTEN (MMAC1/TEP1) gene codes for a phosphatase that regulates the lipid signalling system and inhibits cell proliferation and motility. Not only this, PTEN is able to inhibit Akt kinase and negate the anti-apoptotic effect of the kinase. Loss of PTEN is associated with constitutively high levels of Akt expression. Exogenous PTEN is able to drive cells into apoptosis with concomitant downregulation of Akt. Aside from apoptosis-mediated control of growth, PTEN seems to be capable of inhibiting cell cycle progression by inhibiting phosphorylation of the cell cycle regulatory Rb protein by inhibiting Akt kinase. PTEN-induced growth arrest can be negated by PI-3 kinase and Akt (Paramio et al., 1999). The degree of PTEN expression might be related to differentiation. Adenocarcinomas of the breast that express PTEN at low levels tend to be also low expressers of ER (Perren et al., 1999), which is compatible with the view that low expression or loss of ER is associated with clinically highly aggressive disease. PTEN is often mutated or suffers LOH in tumours. A tumour suppressor function has been putatively attributed to PTEN, although loss of PTEN is not an invariable feature of neoplasia in general. PTEN is over-expressed in some benign stages of neoplasia. This might be viewed as a safeguard against progression of the neoplasm to overt malignancy (see Sherbet, 2001).

Akt signalling in cancer growth and invasion Cancer cells and transformed cells undergo apoptosis when exposed to tumour necrosis factor (TNF). The TNF family of apoptosis-inducing factors include TRAIL, which is A TNF-related apoptosis inducing ligand. TRAIL is a type II integral membrane protein (Wiley et al., 1995; Pitti et al., 1996) and bears a high degree of homology to the Fas ligand, FasL (Nagata, 1997). TRAIL induces apoptosis of cells by binding to specific receptors. Binding of TRAIL to two receptors called TRAIL-R1 and R2 appears to induce apoptosis, whereas binding of the ligand to TRAIL-R3 and R4 seem to inhibit apoptosis by acting as decoy receptors or by activating NF-␬B. R1 and R2 possess the so-called death domains in the cytoplasmic tail whilst R3 and R4 lack these death domains (Degli-Esposti, Dougallo et al., 1997; Pan, Ni et al., 1997; Pan, O’Rourke et al.,

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Figure 8 Signalling pathway involving phosphoinositide-3 kinase and Akt in the regulation of cell proliferation and apoptosis. The Figure shows the PI-3 kinase/Akt pathway of transduction of signals transduced via erbB family of receptors erbB2/B3/B4 and EGFr, PDGF, TNF, Fas, PTEN, TCL1 and TGF␣ leading to the regulation of cell proliferation and survival as well as to the acquisition of cell motility or invasive properties in the context of cancer cells. PI-3 kinase activates Akt as indicated by the inhibition of Akt activation by PI-3 kinase inhibitors. The downstream event following Akt activation is interaction of Akt with and activation of appropriate transcription factors and the modulation of expression of responsive genes (see Table 11). Akt activation results in the protection of cells from apoptosis, which by all accounts seems to be due to inactivation or reduction in the activity of caspases. Angiogenic signal transduction also follows the PI-3 kinase/Akt pathway resulting the regulation of VEGF expression. VEGF increases the permeability of endothelial cells and their proliferation and migration, and this seems to be due to the synthesis of nitric oxide synthase (NOS) (Yiyu et al., 1999), which is activated by Akt-mediated phosphorylation (Fulton et al., 1999). Inhibitors of NOS, PI-3 and MAPK inhibit VEGF-induced vascular permeability (Lal et al., 2001). Akt-mediated modulation of invasive behaviour of cancer cells can be attributed to its being targeted to the plasma membrane and its apparent involvement in the remodelling of the extracellular matrix. The interaction and mutual regulation of signalling pathways is illustrated in Figures 9 and 10. (MAPK, MAP kinase; NOS, nitric oxide synthase; PI3, phosphoinoside-3; TGF, transforming growth factor; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor.) Based on references cited in the text and Blancher et al. (2001); Chaudhary and Hruska (2001); Choudhury (2001); Hatano and Brenner (2001); Hermanto et al. (2001); Okano et al. (2000); Shin et al. (2001); Wei et al. (2001).

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1997). Osteoprotegerin is a fifth receptor. This competitively inhibits the binding of TRAIL to R1 and R2 receptors and in this way it seems to inhibit TRAIL-induced apoptosis (Emery et al., 1998). The differential expression of the pro- and anti-apoptosis receptors of TRAIL could be the reason why tumour cells are generally prone to induction of apoptosis by TRAIL whilst normal cells are not susceptible. Prostate cancer cells that are resistant to the apoptotic effect of TRAIL express Akt at constitutively high levels. Sensitivity to apoptosis is restored when Akt is downregulated by treatment with inhibitors of Akt (Thakkar et al., 2001). Hatano and Brenner (2001) recently confirmed the involvement of the PI3-kinase/Akt in the transduction of apoptosis signals by TNF-␣ and another member of the TNF family, namely Fas protein. Interestingly, TRAIL-R3 is a glycosyl-PI-3 linked receptor (Degli-Esposti, Smolak et al., 1997). Therefore it seems reasonable to conclude that the TNF family cytokine-induced apoptosis is controlled by the PI3-kinase/Akt. Downstream of the activation by phosphorylation of Akt by PI-3 kinase pathway are the caspases whose activity is reduced or inhibited by activated Akt, which in turn leads to protection of cells from apoptosis. The induction of apoptosis by staurosporine as well as by etoposide, both well-established mitochondrial apoptotic stimuli, is accompanied by marked activation of Akt prior to the onset of apoptosis, and the resultant overexpression of Akt indeed greatly delays apoptosis (Tang et al., 2001). It is to be expected that deregulation of the restraint on apoptosis would lead to tumour expansion and this has turned out to be the case. Akt is over-expressed in cancers of the thyroid (Ringel et al., 2001). Akt is said to be constitutively active in non-small cell lung carcinoma cells lines (Brognard et al., 2001). Its activation occurs upon exposure of breast cancer cell lines to oestrogen, irrespective of their ER status (Tsai et al., 2001). Zinda et al. (2001) found no differences in Akt RNA expression of normal and tumour tissues derived from lung, breast, prostate and colon. However, one ought to take cognisance of the possibility that there might yet be differences in protein expression due to regulation at the translational level. At the experimental level, Mende et al. (2001) were able to induce lymphomas in transgenic mice carrying the Akt gene controlled by the lck promoter. There is general acceptance that oncogenic stimuli frequently change the adhesive and motile properties of cells, often by bringing about the targeting of downstream effector molecules to the plasma membrane and by remodelling the extracellular matrix. It is therefore of much interest to note in the context of the behaviour of cancer the apparent relationship of Akt to cell motility flowing from a modulation of the structure of the extracellular matrix. Matrix metalloproteinases (MMP) and their inhibitors are among ECM components whose modulation is frequently associated with changes in invasive ability of cancer cells. XF Kim et al. (2001) found that Akt was localised at the leading plasma membrane edge of migrating cells. They have attributed the perceived promotion of migratory behaviour to an increased expression of MMP-9. Park et

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al. (2001) have confirmed not only that Akt enhances the invasive ability of cells but it also induces MMP-2. Possibly the targeting of Akt to the plasma correlates with changes in ECM composition together with acquisition of motility. Consistent with this Akt is translocated to the plasma membrane in the transduction of erbB2/erbB3 signal (Hellyer et al., 2001). According to Mende et al. (2001), myristoylation of Akt enhanced both its oncogenic property and kinase activity. In the context of Akt localisation this indicates that myristoylation might play a key role in the targeting of Akt to the cell membrane. Besides MMPs, there are specific key proteins, which are closely involved with intercellular adhesion and invasive properties of the cancer cell. Notable among them are fibronectin, laminin and collagen type IV. Li et al. (2001) found that activated Akt not only was targeted to the plasma membrane but it also induced the synthesis of laminin and collagen IV.

Akt in TCL1 function The anti-apoptosis mode of TCL1 function is founded on the finding that Akt aids TCL1 in its function. The anti-apoptotic properties of Akt kinase, also known as protein kinase B, are well established. TCL1 binds to the pleckstrin homology domain of Akt and activates the kinase. Akt bound to TCL1 is severalfold more active than unbound Akt (Laine et al., 2000; Pekarsky et al., 2000). Pekarsky et al. (2000) also showed that when transfected on its own, Akt remained in the cytoplasm but when transfected with TCL1, it was found in the nucleus, clearly indicating that TCL1 promotes the translocation of Akt to the nucleus. Akt phosphorylates specific transcription factors, which then modulate the expression of responsive genes bringing about phenotypic changes, such as proliferation, apoptosis, angiogenesis, cell motility and invasion.

Fusion oncoprotein signalling by PI-3 kinase/Akt pathway Several chimeric oncoproteins have been identified, especially in relation to the pathogenesis of haematological malignancies, and the mode of their function has been elucidated in recent years. These fusion proteins appear to influence cell proliferation, inhibit apoptosis and promote cell survival and in this way contribute to the development of neoplasia. Notable among them and discussed here with the view to their relevance in the context of clonal expansion of haematological malignancies, are the bcr-abl, AML-1/MDE1/EV11 and several TEL (EVT6) chimeric proteins. Among the latter are TEL-PDGFr, TELAML, TEL-Jak, TEL-abl and TEL-ARG (abl-related) fusion proteins. The Philadelphia chromosome is an abnormal shortened chromosome 22 formed out of a reciprocal translocation involving chromosomes 9 and 22. The Philadelphia chromosome occurs frequently in CML. The translocation break-

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Figure 9 Transduction of oncogenic fusion protein signals by PI-3 kinase/Akt pathway. The Figure summarises the pathways and the interaction between different pathways adopted in the transduction of signalling by oncogenic fusion proteins.

point of chromosome 22 occurs in the region known as the breakpoint cluster region (bcr) and in the vicinity of the abl oncogene occurring on chromosome 9. A consequence of the translocation is the generation of a chimeric protein called bcr-abl. The bcr-abl is a constitutively active tyrosine kinase. The t(9;22)(q34;q11) generates several oncoproteins, namely p190, p210 or p230 bcr-abl proteins. Recent work has indicated that bcr-abl proteins are antiapoptotic and promote cell survival and, furthermore, they appear to activate the PI-3 kinase/Akt-signalling pathway (Griffin, 2001). The translocation t(3;21)(q26;q22) generates another fusion protein known as AML1/MDS1/EVIl. Together with bcr-abl, it is able to block myeloid differentiation leading to rapid development of AML (Cuenco and Ren, 2001). Myeloid leukaemias also involve the fusion proteins, that is, the AML1-ETO and AML1-MTG, which possess the ability to support proliferation of haemopoietic stem cells (Mulloy et al., 2002). These fusion proteins function as transcription factors targeting on AML1 target genes and regulating their expression (see below and Figures 9 and 10). Among other fusion proteins that signal via the PI-3 kinase/Akt pathway are the TEL-JAK, TEL-abl proteins. Voss et al. (2001) have attributed MAPK as well

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as ERK kinase, besides PI-3 kinase/Akt in bcr-abl and TEL-abl signalling. The fusion protein TEL-NTRK between TEL and the neurotropin receptor kinase NTRK also follows both ERK and PI-3/Akt pathways (Tognon et al., 2001). According to Constantino et al. (2001), these fusion proteins constitutively activate STAT family mediators. They found that STAT5 interacted with the regulatory domain of PI-3 kinase, which seems to lead to a constitutive activation of PI-3 kinase/Akt pathway. Skorski et al. (1999) have implicated the SH2 and SH3 domains of bcr-abl in STAT-mediated activation of PI-3 kinase/Akt pathway. Subsequently, Nieborowska-Skorska (2000) seem to be suggesting that the SH2/SH3-mediated activation might be distinguished from the bcr-abl kinase mediated activation of the pathway. Hoover et al. (2001) found that not only STAT but ras signalling also plays a part in bcr-abl mediated transformation of haematopoietic cells. The TEL–Jak fusion protein seems to exert its antiapoptotic effects by the agency of NF-␬B transcription factor (Santos et al., 2001). A direct route to the deregulation of cell cycle control is suggested by the work of Gesbert et al. (2000). These authors found that bcr-abl is able to suppress the cyclin-dependent kinase inhibitor p27kip1 and this results in the deregulation and accelerated entry of cells into the S-phase of the cell cycle.

Figure 10 Interactive signal transduction in cell proliferation, differentiation and apoptosis. The cell proliferation, differentiation and apoptotic signals may be transduced by more than one pathway. The Figure illustrates the interactive and co-operative nature of the pathways of transduction via MAPK, Akt and STAT. JAK can regulate all three and furthermore STAT pathway may involve src kinase as a downstream component. (ERK, Extracellular signal-regulated kinase; JAK, Janus tyrosine kinase; STAT, signal transducer and activation of transcription; MAPK, mitogen activated protein kinase.) Based on Dolci et al. (2001); Matsui et al. (2001); Rane and Reddy (2000); Reddy et al. (2001); Sherbet (2001); Tognon et al. (2001).

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Overall, the intricate interplay of the different pathways that are involved in the transduction of signals that actuate cell proliferation and cell survival in the pathogenesis of haematological malignancies is adequately illustrated by these various studies, as summarised in Figures 8–10.

Mechanism of transduction of signals by Akt Can one formulate a putative mechanism by which Akt might activate specific transcription factors that might be associated with specific cellular properties characterising the development of cancer? At least three isoforms of Akt have been identified. There seems to be no differential expression between the Akt 1, 2 and 3, and it is uncertain if they are differentially expressed between normal and neoplastic tissues. Whilst Ringel et al. (2001) found them overexpressed in thyroid carcinomas as compared with corresponding normal tissue, Zinda et al. (2001) encountered no significant differences in their investigations, which included normal and cancers of the colon, lung, breast and the prostate. It should be noted however that EGF might activate a specific isoform of Akt (Okano et al., 2001). A variety of transcription factors are involved in bringing about the phenotypic or physiological effects generated by Akt-mediation (Table 11). Therefore, aside from differential expression one can envisage Akt mediating regulation of transcription via the regulation of the function of transcription factors themselves. This can take the form of subcellular localisation of transcription factors by regulation of their intracellular translocation. In the breast cancer cell line MDA-MB-231, EGF induces Akt, which is found to phosphorylate FKHR and thus exclude the transcription factor from the nucleus (Jackson et al., 2001). In contrast, the fusion protein PAX7-FKHR resulting from a reciprocal translocation seems to resist exclusion and accumulates in the nucleus leading to aberrant gene expression (Barr, 2001). Another potentially significant aspect is the activation of different transcription factors by a single extracellular signalling ligand. For instance, EGF might function possibly by differential activation of the transcription factors FKHR and NF-␬B. Here one can see several elements that can constitute a putative mechanism for the regulation of genetic transcription by ligands through the PI-3 kinase/Akt signalling pathway. The extracellular signals can be transduced via many pathways. In the present context one should also be mindful of the interactive and regulatory involvement of more than one signalling pathway in the generation of phenotypic response of cell proliferation and apoptosis, and differentiation. With special reference to metastatic spread, one must recognise that signal transduction modulating endothelial permeability, cell motility and invasion also involve multiple pathways that interact and mutually regulate the flow of information. These ideas are summarised in Figures 8–10.

104 Table 11

Genetic Recombination in Cancer Transcription factors in Akt-mediated signal transduction

Transcription factor (TF) Forkhead TF AFX

L1 FOXO1

Physiological/phenotypic effects

Cell cycle arrest at G1; p27kip1 upregulation; AFX phosphorylation by Akt results in nuclear exclusion of the TF Cell survival/proliferation promotion by TGF, IL etc. Glucose metabolism

CREB

PTEN suppressor function Insulin and IGF-1 as survival factor for pancreatic beta cells

STAT

IFN-␥ mediated effects

NF-␬B

EGF, PDGF, TNF-␣, Fas, TGF-␣, ␤ in cell proliferation/apoptosis Akt-mediated induction of MMP-9 and increased cell motility/invasion Akt function in Theileria protozoan transformation of leukocytes

E2F

IGF-1 effects of cell growth and anchorage-independent survival and apoptosis; E2F activation or inactivation is related to the phosphorylation state of Rb

Collated from Barthel et al. (2001); Biswas et al. (2000); Brownawell et al. (2001); De Ruiter et al. (2001); Dijkers et al. (2000); Factor et al. (2001); Hatano and Brenner (2001); Heussler et al. (2001); Hirota et al. (2001); H Huang et al. (2001); Kim et al. (2001); Mehrhof et al. (2001); Nakae et al. (2001); Nguyen et al. (2001); Sherbet and Lakshmi (1997); Shin et al. (2001); Yu et al. (2001). In the breast cancer cell line MDA-MB-231, EGF induces Akt, which is said to phosphorylate FKHR and thus exclude the TF from the nucleus (Jackson et al., 2000). Here is a putative mechanism for the regulation of EGF function possibly by differential activation of the transcription factors FKHR and NF-␬B.

Chromosomal translocation and genetic transcription Another important feature of the influence of chromosomal translocation on biological events is its influence on the mechanics of gene transcription. In this scenario translocation of genetic material and its recombination at the new site appears to affect the expression and function of transcription factors. Notably

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the deregulation of their function appears to affect genes that control haemopoietic differentiation or cell proliferation. Many genes coding for transcription activators or repressors are involved in consistent chromosomal translocation and some of these have been identified as major events associated with the pathogenesis of haematological malignancies.

Chromosomal translocation in synovial sarcomas A translocation t(X;18) (p11.2;q11.2) occurs consistently in synovial sarcomas. Several genes are located at the breakpoints in this translocation. They are SYT occurring on chromosome 18, and members of the testis/cancer antigen SSX gene family, namely SSX1, SSX2 and SSX4 on the X-chromosome. The SSX genes are normally expressed in the testis and the thyroid. Of the SSX homologues, SSX1, SSX2 and SSX4 are frequently expressed in human neoplasms. SSX3 is not usually detected. Furthermore, SSX expression is found in many forms of cancer including breast cancer, colorectal cancer, head and neck tumours, melanoma and in lymphomas. No expression of the SSX genes is detectable in leukaemias, thyroid cancers, and seminomas (Tureci et al., 1998). A result of the translocation that occurs in synovial sarcomas is the generation of fusion transcripts of SYT–SSX1 and SYT–SSX2, which are the most frequently detected transcripts (62% and 31% respectively) (Bijwaard et al., 2002). Formation of SYT–SSX4 is comparatively less frequent. The fusion transcripts can be co-expressed (Yang et al., 2002). The prime point of interest is that both SYT and SSX proteins function as transcription repressors (Dos Santos et al., 2001) and, significantly, it has been reported that the fusion genes might regulate the expression of the normal SYT allele. Brodin et al. (2001) found that the expression of SYT transcripts was downregulated by enforced expression of SYT–SSX4. This occurred also in synovial sarcomas expressing SYT–SSX4. SYT itself does not possess DNA-binding domains but seems to be able to bind to other transcription co-activators such as SNF/SW1 (Brett et al., 1997; Thaete et al., 1999). SYT is able to interact with the histone acetyl transferase (HAT) p300 (Eid et al., 2000). SYT also interact with core histones via a C-terminal aminoacid sequence of SSX (Kato et al., 2002). On the other hand, SSX has limited expression in normal tissues but is expressed significantly in tumour tissues. Now SSX has a repressor domain in the C-terminal region. The chimeric protein therefore contains both transcription activator and repressor domains. Hence, these fusion proteins may be seen as regulators of transcription. The precise nature of the involvement of these fusion proteins in the pathogenesis of synovial sarcoma is not yet understood. Higher proliferative potential and poorer prognosis are associated with sarcomas that express SYT–SSX1 than SYT–SSX2 (Inagaki et al., 2000; Dos Santos et al., 2001).

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According to Inagaki et al. (2000), synovial sarcomas with SYT–SSX1 express the proliferation related Ki-67 at a high levels together with high mitotic rates. As stated earlier, SYT forms complexes with p300 HAT, which is intricately associated with the function of p53 as a transcription factor. Acetylation of p53 leads to the upregulation of cdk inhibitors p21waf1/cip1 and p27kip1 leading to a marked inhibition of cell proliferation. We also know that p300 HAT can directly upregulate these inhibitors. Xie et al. (2002) have demonstrated that antisense oligonucleotides to SYT–SSX mRNA caused a marked reduction in synovial cell proliferation together with an increase in cyclin D1, which they suggest could be due to the stabilisation of the cyclin. By and large, the fusion protein seems to complement the transcription activator and suppressor properties of the fusion partners with the potential net result of deregulation of cell cycle progression. Synovial sarcomas are also characterised by high expression of bcl2 and reduced apoptosis (Mancuso et al., 2000; Danura et al., 2002). It is conceivable that the growth of sarcomas is wholly or partly due to the suppression of apoptotic loss of cells. However, it is uncertain at present if the regulatory properties of SYT–SSX extend to bcl2 protein expression as well. There are suggestions of possible involvement of SYT proteins in the control of cell adhesion (Eid et al., 2000). In the context of the reported interaction of SYT with HAT p300, one should recall that p300 also produces changes in the ECM that can lead to enhanced adhesion to the substratum. Sato et al. (2001) have pointed out that the expression of adhesion-mediating molecules such as cadherin as well as catenins might be deregulated in synovial sarcomas. These findings lead to the further suggestion that a deregulation of the wnt/wingless signalling pathway may become deregulated as a consequence. The wnt genes are developmentally regulated genes and known substantially to affect cell proliferation, cytoskeletal dynamics and cell motility. Therefore, it is easy to envisage how the deregulation of wnt/wingless signalling can lead to developmental malformation as well as to the pathogenesis of cancer. So there might exist alternative routes to ECM and ECM-related adhesion phenomena encountered in synovial sarcomas. Nonetheless, SYT proteins could be making a potentially significant contribution to the development and progression of these sarcomas.

AML1 fusion proteins in haematopoietic neoplasms Several fusion proteins are involved in the differentiation of haemopoietic stem cells, and cell proliferation, apoptosis and survival. Notable among them are the chimeric AML1-ETO and AML1-TEL fusion proteins, which are formed from translocation t(8;21) and t(12;21) respectively. These fusion products are important regulators of haemopoiesis. So the generation of aberrant AML1 fusion transcripts by translocation would be expected to deregulate the

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development of haemopoietic cells and contribute to the disease process (McNeil et al., 2000; Yuan et al., 2001). The AML1 gene encodes the transcription factor AML1B, which is actively involved in haemopoietic gene regulation. AML1 binding to DNA seems to result either in the activation or repression of gene transcription. As a result of translocation, there is a fusion of DNA binding domain of AML1 with ETO. The latter, also known as MTG8, is a co-repressor that bears homology to the Nervy protein of Drosophila melanogaster in four domains called Nervy homology (NH)-1, -2, -3 and -4 (Miyoshi et al., 1993; Feinstein et al., 1995; Melnick, Westendorf et al., 2000a). In the chimeric protein, the co-repressor interacting region of AML1 is deleted and the co-repressor interaction domain of ETO is substituted (Gelmetti et al., 1998; Kitabayashi et al., 1998; Lutterbach et al., 1998; J Wang et al., 1998). The NH domains might be involved in the recruitment of co-repressors by the chimeric AML1-ETO. The fusion protein might potentially deregulate the transcription of AML1 target genes; indeed it represses promoters that possess AML binding sites. It seems to achieve this by recruiting the co-repressors mSin3A, N-CoR and SMRT, and histone deacetylases (HDAC). ETO itself does not possess DNA binding sites but has binding sites for mSin3A and N-CoR, with which it is known to interact, and also possesses separate binding sites for HDAC (Gelmetti et al., 1998; Lutterbach et al., 1998; J Wang et al., 1998; Amann et al., 2001). ETO might itself function as a corepressor upon being recruited by the promyelocytic leukaemia zinc finger (PLZF) protein. ETO contains a zinc finger domain (Davis et al., 1999) and associates with PLZF in vitro and in vivo. ETO is capable of enhancing transcriptional repression by PLZF (Melnick, Westendorf et al., 2000a). Here a linkage is apparent between haematopoietic differentiation and cell proliferation. The AML1 encoded transcription factor AML1B has been found to accelerate by up to 4 hours the entry of cells into the S-phase of the cell cycle, but AML1B expression did not sustain cell cycle progression (Strom et al., 2000). In other words, the G1 –S checkpoint control exerted by p53 might be compromised by AML1B. The finding that AML1B is capable of repressing p21waf1/cip1 promoter (Lutterbach et al., 2000) supports this argument. Again this seems to occur with the intervention of the mSin3A co-repressor. On the other hand, Pabst et al. (2001) have recently demonstrated the ability of AML1ETO to downregulate the transcription factor C/EBP␣, which is required for the differentiation of granulocytes. The p53-mediated pathway of achieving cell cycle arrest involves mdm2. Mdm2 can regulate p53 function by degrading p53 and thus controlling its level. This process itself is subject to regulation by p14ARF, which is one of two proteins encoded by CDKN2. P14ARF is known to inhibit mdm2-mediated degradation of p53 and lead to p53 accumulation and to cell cycle arrest. The fusion protein AML1-ETO seems to be able to bind to and suppress transcription and reduce intracellular levels of p14ARF. But AML1 itself stimulates p14ARF transcription (Linggi et al., 2002) (see Figure 6). This illustrates a clear

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modulation of the regulatory function of AML1 by fusion with ETO and how the checkpoint control function of p53 might be compromised. Although the general focus has been on AML1 target genes, one should heed the findings of Shimada, Ichikawa et al. (2000) that these fusion proteins might regulate the expression of other target genes. Overall, the molecular mechanisms of pathogenesis of haemopoietic disorders are increasingly becoming unravelled.

Modulation of retinoic acid receptor function by chromosomal translocation Retinoic acid receptors (RAR) form a class of transcription factors which are actively involved in the regulation of genetic transcription required in the process of cell differentiation, morphogenesis and neoplasia. RARs bind to consensus sequences in the DNA that constitute specific response elements for the receptors. RARs often form heterodimers with other transcription factors and activate genetic transcription and control biological processes, such as cell proliferation. For instance, a type of RAR called RXR forms a heterodimeric transcription complex with the vitamin D receptor and this transcription factor then binds to the vitamin D response element leading to the regulation of cell proliferation (see Sherbet, 1997, 2001). In acute PML (APL), the retinoic acid receptor RAR␣ forms a chimeric protein with PML protein as a result of t(15;17), and with PLZF gene product, as a result of t(11;17) translocation (Z Chen et al., 1996). The PML gene was identified at the t(15;17) translocation point in APL. However, its expression is not restricted to leukaemia but it is also found in adenocarcinoma and squamous cell carcinoma of the lung. In NSCLC, the PML protein has not been detected but its mRNA has been shown to be present (Zhang et al., 2000). The protein is expressed in HCC, liver cirrhosis and liver abscesses (Chan et al., 1998). The PML protein is a ring-finger protein with growth inhibitory and tumour suppressor properties and it also appears to be able to activate apoptotic pathways. The chimeric protein it forms with RAR, however, seems to disrupt and alter its normal mode of function in cell proliferation and differentiation. Lavau and Dejean (1994) recognised some years ago that the fusion protein retained the functional domains of the constituent proteins, but, nonetheless, it showed several new properties. Chang et al. (1995) showed that PML is expressed in a cell cycle-related fashion. Its expression is low in S-, G2- and M-phase but it is high in the G1-phase, suggesting that it might control the transition of cells from G1- to S-phase. Guo, Salomoni et al. (2000) have shown that PML interacts with and co-localises with p53. The p21waf1/cip1 is a p53-regulated protein and it inhibits the cdk–cyclin complex and thereby

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inhibits the entry of the cell into the S-phase of the cell cycle. PML is required for the activation of the p21 gene (Wang, Delva et al., 1998a). Guo, Salomoni et al. (2000) found that the induction of p21 is impaired in PML –/– cells, which indicates the PML protein functions in collusion with p53 to control cell proliferation. PML also takes part in the p53-mediated apoptotic pathway. There are also other pathways of apoptosis, which implicate PML. PML is essential for Fas and caspase-mediated apoptosis. In contrast, the PML-RAR␣ fusion protein seems to confer resistance to apoptosis (Wang, Ruggero et al., 1998). The PLZF protein belongs to the zinc finger family of transcription factor. PLZF is a phosphoprotein, which appears to be involved in the regulation of the cell cycle and is associated with cdk2 (Ball et al., 1999). Cdk2 could be regulating PLZF function by phosphorylation (Costoya et al., 2000). It is also known to interact with Rb protein (Zhu et al., 1998). PLZF serves important functions in limb and skeletal patterning. It also possesses growth regulatory and apoptosis-inducing effects (Barna et al., 2000). A downregulation of cyclins A and B is associated with cell cycle arrest and inhibition of cyclin A leads to growth inhibition. The expression of PML-RAR␣ or PLZF-RAR␣ fusion proteins is associated with an increased expression of cyclin A1. Activated RAR␣ itself negatively regulates cyclin A1 expression. In reporter constructs, PML-RAR␣ activates the cyclin A1 promoter (Muller et al., 2000). Melnick et al. (Melnick, Carlile et al., 2000; Melnick, Westendorf et al., 2000b) have shown that PLZFRAR fusion protein indeed inhibits RAR␣ target genes. Furthermore, the fusion protein can enlist ETO and HDAC to accentuate the suppression of RAR target genes and inhibit myeloid differentiation. Quite conceivably, the regulation of cyclin-dependent kinases could be by means of regulation of their inhibitors. The chimeric association can conceivably modulate the interaction of RAR with other receptors or its interaction with the appropriate response element in the DNA, and this could affect the pattern of genes being transcribed. As alluded to earlier, RAR species are involved in the regulation of vitamin D3 (VD3) function, wherein VD3 receptor forms a complex with RXR. This complex binds to the VD3 responsive element and regulates cell proliferation via its effect on cdk inhibitors. Puccetti et al. (2000) have reported that the PLZF-RAR␣ protein is involved in VD3 signalling pathway. There is also evidence now that this fusion protein might also regulate G-CSF signalling pathway as well (Girard et al., 2000; Puccetti et al., 2000).

TEL transcription-related gene rearrangement in leukaemia and lymphoma Abnormalities of the TEL (also known as ETV6) gene are found in many haematological malignancies. The TEL protein is a transcription factor of the ETS family. Rearrangement of TEL occurs in both lymphoid and myeloid malignancies. The TEL protein has three domains: an N-terminal domain with a

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helix-loop-helix (HLH) structure, a C-terminal domain, which is the DNA binding ETS (E-26 transforming specific) domain, and an intermediate domain. The HLH domain and the central domain seem to be involved in transcription function (Chakrabarti and Nucifora, 1999). The TEL gene is involved in many translocations. For instance, chromosome 12, on which this gene is located, is involved in the translocation t(12;21)(p13;q22). The breakpoints of this translocation occur within the TEL gene, with the HLH and central domains being unaffected by the translocation. The TEL gene forms a chimeric fusion with AML1 gene. Wiemels and Greaves (1999) have identified the breakpoints associated with the generation of this chimeric TEL/AML1 gene, with breakpoints in exons 5 and 6 of TEL gene and in intron 1 of AML1 gene. It is of considerable significance to note here that Nishimura et al. (1999b) detected genetic instability in chromosomes 9p and 12p in childhood leukaemia and lymphoma specimens. Although replication error was detected in only 5 out of 65 patients, the RER phenotype was associated mainly with the 9p and 12p regions. Loss of heterozygosity and chimeric TEL/AML1 gene occurred in two patients. Also attendant was a reduction in expression of p27kip1, the cyclin-dependent kinase inhibitor, closely involved in the regulation of the cell cycle. However, the TEL translocation does not seem to involve VDJ recombinase, topoisomerase II, Alu sequences or other sequences associated with recombination (Wiemels and Greaves, 1999; Romana et al., 1999). Although there is much empirical evidence for a role for TEL fusion gene in haematological malignancies, no experimental evidence has been forthcoming until lately. TEL fusion proteins are major participants in signal transduction. An important partner in the formation of TEL fusion proteins related to translocation associated with haematological aberrations of lymphoid or myeloid origin is the Janus tyrosine kinase (JAK). The TEL–JAK2 fusion protein arises from t(9;12)(p24;p13) and is formed by fusion of the oligomerisation domain of TEL with the catalytic domain of JAK2, also of JAK1 and JAK3. Cytokines and growth factors transduce their signals via JAK, which is activated when the ligands bind to the appropriate receptors. This leads to the activation of cytoplasmic transcription factors called STAT (signal transducer and activators of transcription), which are activated by phosphorylation and are translocated to the nucleus. The fusion protein TEL–JAK2 is constitutively active as a tyrosine kinase and phosphorylates STAT family proteins, and in this way it is able to promote cell proliferation independently of cytokine and growth factor signalling. Carron et al. (2000) experimentally demonstrated the significance of the TEL–JAK2 in the pathogenesis of leukaemia. Transgenic mice carrying TEL–JAK2 cDNA and their progeny developed leukaemia at the age of 4–22 weeks, together with a selective amplification of CD8+ T-cells, phosphorylation of the fusion protein, and the induction of the STAT proteins. This is compatible with the ability of STAT3 to regulate G1 –S transition of the cells. The fusion protein seems not only to stimulate cell proliferation but also affects the

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invasive ability of leukaemic cells. Carron et al. (2000) found that TEL–JAK2 transgenic diseased animals as well as nude mice implanted with TEL–JAK2 leukaemia cells also displayed invasion of non-haemopoietic tissues by leukaemic T-cells. This work underlines the significance of the translocation leading to the formation of the fusion protein in the development of leukaemia and points to the JAK and STAT mediated signal transduction mechanism as a potential pathway by which cell cycle control at the G1 –S checkpoint might be abrogated. However, the effect of expression of the fusion protein to induce cellular motility needs a closer scrutiny. Other fusion proteins involving TEL have also been identified and these too possess recognisable oncogenic properties. For instance, CML cells carrying t(5;12) translocation generate TEL-PDGFr-␤ fusion protein, which can transduce the growth factor signal by constitutive activation of the protein kinase (Jousset et al., 1997). TEL-PDGFr-␤ also activates the STAT pathway of signalling. It would not be an over-statement that the JAK/STAT pathway has assumed preeminence in the pathogenesis of haematological disorders and malignancies (see Figures 9 and 10). TEL fusion occurs with several other genes, such as MDS1 (Peeters et al., 1997), BTL (Cools et al., 1999), STL (Sato et al., 1997), ACS2 (Yagasaki et al., 1999) and certain homeobox genes (see below) among others, and the resultant fusion proteins would be expected to impinge upon the functioning of a variety of signalling pathways.

Modulation of developmental and transcription factor PAX genes by translocation Homeobox genes code for transcription factors constituting several families of homeodomain proteins. The homeobox is an approximately 180 bp DNA motif that encodes the so-called homeodomain, which has approximately 60 aminoacids. The homeodomain transcription factors bind to the DNA by means of the homeodomain and regulate the transcription of target genes. Among homeobox families identified to date are PAX, HOX, MSX, EMX, CDX and other cognate but divergent genes. These transcription factors control and coordinate the expression of a number of genes that are involved in embryonic development, differentiation and morphogenesis (McGinnis and Krumlauf, 1992; Cillo et al., 1999). Many homeodomain proteins are abnormally expressed in haematological malignancies and a variety of solid human neoplasms. The PAX genes constitute a family of developmental genes whose products function as transcription factors. Several transcription factors of the PAX family have been characterised. The PAX proteins share a domain called the paired domain, which mediates their binding to the DNA (Mansouri, Goudreau et al., 1999). The PAX genes are normally expressed in developing and differentiating systems. A germ layer-specific pattern of association of PAX genes seems to be

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emerging. Thus PAX4, 6 and 8 seem to mediate endodermal organogenesis. PAX4 and PAX6 are concerned with the differentiation of the endocrine component of the pancreas. PAX8 seems to mediate the differentiation of follicular cells of the thyroid (Mansouri, St-Onge et al., 1999). On the other hand, PAX7 shows muscle cell-specific expression. The involvement of PAX genes in biological processes such as cell proliferation and apoptosis was shown some years ago (Stuart et al., 1995; Bernasconi et al., 1996). Stuart et al. (1995) showed that p53 gene contains a PAX binding site in its untranslated exon1, and PAX2 and PAX5 are both able to inhibit the p53 promoter. In human astrocytomas PAX5 expression shows an inverse correlation with p53 expression. Haematological neoplasia over-express PAX together with chromosomal rearrangements brought about by translocations. But the over-expression of these genes is not confined to haematological malignancies. PAX expression is deregulated in a variety of tumours, e.g. rhabdomyosarcoma, brain tumours and Wilm’s tumours. Furthermore, deregulation might possibly be related to tumour progression. The influence of PAX expression on cell motility is also increasingly being appreciated. It follows therefore that PAX genes would be involved in tumour development and progression. PAX5 over-expression has been found to relate to the degree of tumour de-differentiation in transitional cell carcinoma of the bladder. The expression of PAX5 protein was greater in carcinomas than in normal urothelium (Adshead et al., 1999). A higher proportion of invasive tumours (7 of 8 pT1 and 8 of 9 PT2) showed higher PAX5 expression in the series studied by Adshead et al. (1999), but this difference needs to be confirmed with a larger series. The deregulation of their expression often occurs in the wake of translocations involving the PAX gene locus leading to the formation of aberrant fusion proteins, although epigenetic mechanisms such as methylation cannot be excluded from playing a role in their regulation. PAX5 gene, which codes for the B-cell specific activator protein, located on chromosome 9p13, is involved in the translocation t(9,14)(p13;q32) in B-cell non-Hodgkin’s lymphoma and as a consequence it forms a chimeric gene with IgH locus on chromosome 14q32. The structure of PAX remains unaltered. Nonetheless, its expression is deregulated, as evidenced by its over-expression in a large number of B-cell lymphomas as well as in vitro (Amakawa et al., 1999; Ohno et al., 2000). In spite of these findings, Ohno et al. (2000) do point out that PAX overexpression also occurs in the absence of t(9;14) translocation. Therefore one might regard the significance of translocation as a mechanism that alters PAX expression to the development of the lymphoma, as still undefined. However, the fusion proteins that PAX forms as a result of other translocation does suggest PAX might make functional gains out of the transaction. In paediatric alveolar rhabdomyosarcoma, the translocations t(2;13), t(2;15) and t(1;13) result in the formation of fusion of PAX3 and PAX7 (located on chromosome 1p36) with the FKHR gene. The FKHR gene codes for a

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transcription factor, which belongs to the so-called forkhead family (FKHR). The PAX7–FKHR fusion gene shows amplification in 90% of cases, but the PAX3– FKHR is amplified in less than 5% of the cases (Fitzgerald et al., 2000). It was known for some time that the fusion protein PAX3–FKHR binds to DNA in a sequence-specific manner, and functions efficiently as a transcriptional activator. Fredericks et al. (1995) showed that PAX3–FKHR fusion protein was a more powerful transcriptional activator than PAX3. Indeed, at the functional level, the fusion proteins PAX3–FKHR and PAX7–FKHR have a distinct advantage over PAX3 or PAX7 in that the fusion proteins gain the FKHR transactivation domain, which is resistant to repression by N-terminal cis-acting elements. Thus, whereas PAX is inactive, the PAX7–FKHR fusion protein is highly active and is comparable in activity terms with PAX3–FKHR (Bennicelli et al., 1999). It is obvious from the above discussion that chromosomal translocations have a profound influence on the pattern of genetic expression that regulates normal cellular differentiation and development, cell proliferation and apoptosis, as well as tumour development and possibly the progression of the disease. Therefore, the discussion has inevitably to progress towards the factors that cause translocations of genetic material between chromosomes.

The role of HOX genes in development, differentiation and neoplasia The HOX genes were identified some years ago as being associated with normal embryonic development and differentiation, as well as in neoplastic transformation and progression. The several HOX genes so far identified occur in four gene clusters, namely HOX-A, B, C and D. The HOX genes actively participate in pattern formation, morphogenesis and differentiation. Mutation of HOX genes brings about congenital malformation and developmental disorders (Goodman and Scambler, 2001). These genes are a component of the normal scenario of haemopoietic differentiation, and aberrant expression of these genes occurs in haematological malignancies (Van Oostveen et al., 1999). An enhanced expression of HOX genes has been reported in a number of human solid tumours. HOX-B9 and HOX-A10 seem to be over-expressed in lung cancer cell lines (Calvo et al., 2000); HOX-C6, B3 and B4 in neuroblastomas, lung cancer and mammary epithelial neoplasms (Bodey et al., 2000a, b). Care et al. (1996) reported some time ago that HOX-B7 was constitutively expressed in a number of melanoma cell lines that they had examined. Waltregny et al. (2002) found HOX-C8 over-expressed in prostate cancer, whilst LL Xu et al. (2000) had found earlier a correlation between the incidence over-expression of the protein NKX3.1, another homeodomain protein, and metastatic spread. According to these authors a higher proportion of patients with metastatic disease showed NKX3.1 over-expression as compared with patients with tumours confined to

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the prostate. Whether one should be looking to compare the levels of expression in the two groups as a more relevant statistic rather than incidence of protein over-expression and metastatic potential is a moot point. Nonetheless, LL Xu et al. (2000) found that over-expression of the NKX3.1 protein correlated with tumour volume and the amounts of prostate-specific antigen (PSA) in serum. HOX-D10 expression is deregulated in endometrial carcinomas; the HOX-D10 mRNA levels are reduced in the carcinomas as compared with normal endometrium. Furthermore, MRNA levels showed an inverse correlation with differentiation (Osborne et al., 1998). Whilst the above studies encountered over-expression of a host of HOX proteins, in contrast, the reported relative expression of HOX-B8, B8, C8, C9 and Cdx-1 in colon carcinoma and carcinoma derived cell lines (Vider et al., 2000) might suggest differential regulation and expression of HOX proteins. A differential regulation of a developmentally related family of transcription factors should not come as a surprise. Differential expression of HOX protein has been related to phenotypic properties of cancer cells. Cillo et al. (1996) recorded a differential expression of HOX proteins that correlated with the expression of cell membrane components, such as integrins and ICAMs, which markedly affect intercellular adhesion, adhesion to the matrix and influence cell motility and invasion. We know that steroid hormones can regulate HOX gene expression. HOX-A1 expression in MCF7 breast cancer cells is influenced by progesterone (Chariot and Castronovo, 1996). Phippard et al. (1996) demonstrated that MSX-2 expression, but not that of MSX-1, Bmp-1, Bmp-2 or Bmp-4, is reduced by ovariectomy. Besides, MSX-2 expression is influenced by ␤-oestradiol in MCF7 breast cancer cells. In prostate cancer the levels of expression of NKX3.1 correlate with androgen receptor levels (LL Xu et al., 2000). At this stage one can accept in general terms that a deregulation of HOX expression occurs in cancer, but the relationship of deregulation to the degree of tumour differentiation, growth and dissemination is not fully understood. Nonetheless, looking at the spectrum of HOX target genes, we should have reasonable evidence that HOX expression might indeed be related to the state of progression of cancer. Some HOX proteins appear to be able to transactivate cell proliferation related genes like the p53 and the PCNA gene. Oh et al. (2002) have identified CDX-binding sequences in the 5’-flanking region of PCNA and they have shown that PCNA can be activated by CDX-1 and CDX-2 in vitro with attendant increase in cell proliferation. Raman et al. (2000) have detected HOX binding sites in p53 and HOX is indeed able to activate p53 promoter. The expression of HOX-A5 promoted apoptosis in cells carrying the wild-type p53. Raman et al (2000) also state that there is a clear correlation between loss of p53 and HOX mRNA and HOX protein expression. Although the available evidence is rather meagre, in a further step conducive to the dissemination of cancer deregulation of homeobox gene expression appears to be capable of influencing the process of tumour vascularisation and the invasion of the new blood vessels by tumour cells (see below).

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HOX proteins might recruit or co-operate with other homeobox proteins. Such interactions conceivably enhance the recognition, affinity and specificity of HOX binding to DNA. HOX, PBX and Meis1 homeobox proteins are known to interact with one another in cell transformation. Transfection-mediated simultaneous over-expression of HOX-A9 and Meis1 in haemopoietic cells induces AML in mice, but the transfection of individual genes has no such effect (Kroon et al., 1998). The presence of the homeobox protein PBX seems to greatly enhance cell proliferation and transformation induced by HOX-B3 and B4. That interaction between the HOX and PBX is an essential part of transformation is indicated by the requirement of the PBX domain that mediates its binding to HOX (Green et al., 1998; Krosl et al., 1998). The caudal-related homeobox gene CDX is infrequently involved in a reciprocal translocation t(12;13)(p13;q12) involving TEL in AML (Chase et al., 1999). There is insufficient evidence at present whether the chimeric structure influences the transcription function of either TEL or CDX. Another example of a chimeric protein involving a homeobox protein and a transcription factor, is the fusion of homeobox genes with the E2A gene. E2A encodes two proteins, namely E12 and E47, whose function as transcription factors is essential in normal B-cell development. E2A proteins are bHLH family transcription factors. There are indications that they may possess antiproliferation effects although evidence for growth inhibition is not indisputable. The loss of one E2A allele results in the proliferation of pro-B cells with concomitant downregulation of the cyclin-dependent kinase inhibitor p21waf1/cip1 (Herblot et al., 2002). In certain experimental conditions overexpression of E47 seems to result in growth inhibition, albeit by an indirect route (Wilson et al., 2001). B-cell ALL often show translocation t(1;19) that involve E2A and PBX1. The chimeric E2A-PBX1 protein can successfully transform a variety of cell types. Two motifs have been identified as being involved in co-operative binding of the chimeric protein with DNA. CP Chang et al. (1997) suggest that a motif called the HOX co-operative motif (HCM), which is located at the C-terminal to the PBX homeodomain, is identifiable as a prerequisite for cooperative DNA binding. E2A-PBX proteins carrying mutation in this motif can neither function as transcription factors nor transform cells. Green et al. (1998) believe that the HCM motif does increase DNA binding by PBX, but it is not essential for co-operative interaction. These authors suggest an important role for another region, that is N-terminal to the homeodomain, and find that it is essential for co-operative interaction with DNA. Whatever might be the functional domain required for optimal transcriptional activation, it is clear that translocations involving E2A are liable to deregulate the growth inhibitory function of E2A and in this way lead to the development of leukaemia. The wnt family genes are developmentally regulated genes, affecting cell proliferation, cytoskeletal dynamics and cell motility, and regulate many processes of embryonic development. Deregulation of wnt genes leads not only to developmental malformation but also to pathogenesis of cancer. Wnt signal

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transduction interacts with many signalling pathways. Wnt gene expression seems to be closely related to HOX expression. Calvo et al. (2000) found that wnt-7a is often reduced or undetectable in lung cancers. Indeed, loss of wnt-7a occurs concomitantly with loss of HOX-A1 expression. This link-up strengthens the finding that wnt-16 might be a target of activation by E2A-PBX1. Wnt-16 is said to be transcribed at high levels in the bone marrow and cell lines derived from pre-B-ALL patients who show the t(1;19) translocation. When E2A-PBX1 expression is inhibited wnt-16 mRNA levels decrease, demonstrating that E2APBX1 indeed activates wnt-16 transcription (McWhirter et al., 1999). As discussed earlier, HOX proteins co-operate with other homeodomain proteins in the regulation of expression of their target genes. The fusion of HOX with other proteins might generate chimeric proteins with potential for producing changes in their intracellular distribution and translocation, or directly affecting their interaction with DNA. This could be an aid in enhancing the efficacy of HOX function as a transcription factor. HOX-A9, HOX-D13, HOXD11 and PMX1 genes fuse with the nucleoporin gene. A major chimeric partner of HOX is NUP98. The latter is a component of the nuclear pore complex, which occurs on the inner side of the nuclear membrane. NUP98 is involved in the transport of RNAs and proteins between the nuclear and cytoplasmic compartments. The NUP98 gene is involved in reciprocal translocation between chromosome 11p15, where NUP98 is located, and the chromosomes harbouring the HOX genes. As a consequence, a chimeric protein is generated wherein part of the homeodomain fuses with an N-terminal phenylalanineglycine repeat of NUP98 (Borrow et al., 1996; Nakamura et al., 1996, 1999; Raza-Egilmez et al., 1998). Since NUP proteins have a transport function, it may be that the transport of the HOX transcription factors to the nucleus is affected, and presumably also their binding to the DNA could be affected, since the DNA binding homeodomain is involved in chimeric fusion. Their function might be dependent upon their localisation in the required cellular compartment. HOXcontaining chimeric proteins provide some elucidation of the importance of subcellular localisation and translocation between cellular compartments to their function as transcription activators. Not only do these gene fusions occur in haematological malignancies, but experimental work also indicates that these chimeric proteins can induce leukaemias (Kroon et al., 2001). One should recognise here that HOX chimeric proteins might recruit coactivators of transcription downstream in their function of inducing genetic transcription. HOXA9–NUP98, for instance, interacts with CREB-binding protein by means of FG repeats, and this transactivation by FG-repeat segments of NUP98 correlates with binding to the co-activator CREB-binding protein. Thus NUP98 seems to play an essential role in the regulation of transcription of HOX-responsive genes (Kasper et al., 1999). Translocations involving NUP98 are encountered frequently in leukaemias and myelodysplastic syndrome. Taketani et al. (2002a) have presented evidence that NUP98 forms a fusion protein with HOXC11. Furthermore, these authors found that HOXC11 was

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expressed far more frequently in myeloid leukaemia cell lines than in ALL cell lines. HOXD11 is another fusion partner of NUP98 (Taketani et al., 2002b). Furthermore, NUP98 also forms fusion proteins with HOXA9 in AML and in CML. Taketani et al. (2002c) encountered NUP98-HOXA13 chimeric proteins in association with t(7;11)(p15;p15). Even in the face of this somewhat extensive evidence one should be wary of over-emphasising the importance of NUP98– HOX fusion proteins. It is unclear at present to what extent translocations involving NUP98 are spontaneous events. For, they seem to occur frequently as a result of therapy with agents that cause genetic damage. Although these findings might turn out to be significant if confirmed in further studies, it would constitute a substantial leap if one were to attempt to impute any specificity in or relationship to the genesis of myeloid malignancies.

Chromosomal translocation, fusion proteins and tumour vascularisation An important and crucial element of tumour progression is the development of mechanistic means of spread of the tumour. Tumours can metastasise by the haematogenous route or via the lymphatic system. Intratumoral blood vessels or lymphatic vessels are induced and these can anastomose with vessels in the periphery of the tumour. Tumour tissue appears to be able to synthesise angiogenic factors and the appropriate receptors that can induce neovascularisation in and around the tumour and this event is associated with metastatic spread (Pepper, 2001; Mandriota et al., 2001; Van Trappen et al., 2002). Although the available evidence is not overwhelming, it seems that certain chromosomal translocations and the expression of aberrant fusion proteins as a consequence of gene fusion do influence the process of neovascularisation of tumours and facilitate also the invasion of the vascular compartment by tumour cells. Janowska-Wieczorek et al. (2002) investigated the effects of the bcr-abl translocation in CML on angiogenesis. They made bcr-abl constructs and transfected them into murine cells. The transfectant cells secreted four-fold greater amount of VEGF as well as matrix metalloproteinase (MMP)-9 and -2 than corresponding control implants. When implanted into SCID or Balb/c mice, the transfected cells developed into tumour that also induced new vasculature. Compatible with this is their finding that myoblasts isolated from CML patients carrying the Ph1 chromosome also produced 10-fold more VEGF, FGF2, HGF and IL-8, all potent inducers of angiogenesis. These cells were also capable of secreting MMP-9 and the membrane-type MMP (Janowska-Wieczorek et al., 2002). The association between MMP expression and the invasive and metastatic behaviour is well documented. MMPs are induced by cytokines, growth factors and by some oncoproteins and their expression is counterbalanced by the expression of the so-called tissue inhibitors of metalloproteinases. MMPs participate in

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the remodelling of the ECM and in this way aid in the invasive behaviour of cancer cells (Sherbet and Lakshmi, 1997). As stated earlier, deregulation of homeobox genes affects cell proliferation and apoptosis in tumour cells. A further advance is their influence on neovascularization. Taking the cue from the findings that HOX genes can induce endothelial cells to proliferate and form vascular sprouts (Boudreau et al., 1997; Myers et al., 2000), Care et al. (2001) infected SkBr3 cells with retroviral vectors carrying HOX-B7 and showed that it upregulated the expression of VEGF, a highly potent inducer of neovascularisation, IF-8 and angiopoietin-1. Of further interest in relation to invasion of the vasculature by tumour cells is their finding that HOX-B7 also enhances metalloproteinases that aid invasion. When this is read with the earlier report by Cillo et al. (1996) that HOX genes influence the expression of adhesion-mediating molecules such as the integrins and intracellular adhesion molecules (ICAMs), it is hardly surprising that HOX genes should influence the invasive behaviour of cancer cells and potentially their metastatic spread. Unfortunately, there are no definitive experiments to date that demonstrate any modulation of such behaviour by HOX gene products. Neither is there a clear visualisation as to the possible ways in which deregulation of the function of these transcription factors might be occurring that can bring about knock-on effects on cell motility and invasion. Another family of transcription regulators that participate in haemopoiesis and in haematological malignancies is the so-called LIM-only (LIMO) family of proteins. The LIMO proteins are LIM-domain containing proteins that function as haemopoiesis-related transcription factors. The LIM family has at least four members – LMO1, 2, 3 and 4. Their expression seems to follow definable patterns in adult tissues as well in embryonic development. These proteins participate in haemopoiesis during embryonic development. LMO2 and another haemopoiesis-related transcription regulator GATA occur in embryonic regions that produce haemopoietic precursor cells and their expression coincides with determination of cell fate (Manaia et al., 2000). LMO2-deficient animals die early in embryonic development. But when LMO2 expression is restored haemopoiesis is also restored (Y Yamada et al., 1998). LMO2 deregulation seems to be associated with the induction of vascularisation by tumours. LMO2 expression is upregulated in tumour-associated vascular endothelial cells and indeed required in the formation of capillaries until around 9 days of development (Y Yamada et al., 2000, 2002). LMO2 is expressed in an aberrant fashion where this gene is involved in translocations t(11;14)(p13;q11) or t(7;11)(q35;p13), which are encountered in childhood ALL. Admittedly the deregulation of LMO2 expression does seem to be involved in pathogenesis of the disease. However, it ought to be recognised that recombinations can lead to secondary mutations. On the other hand, the mode of function of LMO proteins as regulators of haemopoiesis is being gradually unravelled. LIM proteins bind to a number of other proteins and generate DNA-binding complexes. LDB1 (NLI), a 46 kDa phosphoprotein, is

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such a LIM-binding protein. LMO1 as well as LMO2 interact and bind to LDB1 (Visvader et al., 1997; Valge-Archer et al., 1998). The LMO2–LDB complex can inhibit the differentiation of erythroid cells and maintain the precursor cells in an immature state (Visvader et al., 1997). This complex also inhibits T-cell differentiation that in turn leads to tumorigenesis (Grutz et al., 1998). So the formation of LMO–LDB heterodimer might be an essential part of tumorigenesis. AF6 is another protein with which LMO2 interacts (Begay-Muller et al., 2002). AF6 is a frequent fusion partner of MLL often implicated in childhood leukaemias. A distinction has to be made between the processes of induction of haemopoiesis and the induction of blood vessels. Y Yamada et al. (2000, 2002) do recognise this and have suggested LMO2 could be mediating transcription of genes relating to specific phases of haemopoiesis and angiogenesis, and that this could involve interaction with different proteins. LMO2, they suggest, could be involved not only in the induction of new capillaries but also in the maturation of the capillaries into mature blood vessels. Clearly much further work needs to be done. Especially, does the function of LMO2-interacting protein complex include the induction of endothelial cell proliferation and the migration of endothelial cells to tumour foci that contain angiogenic factors such as VEGF? How does LMO2 participate in the alignment of endothelial cells into venules? Are VEGF, TGF␤ and hepatocyte growth factor genes targets of the LMO transcription regulatory complex? Even though this information is unavailable at present, there is sufficient evidence to link-up the aberrant functioning of LMO2 resulting from translocation involving its locus with the pathogenesis of T-cell neoplasia.

Chromosomal translocations, fusion proteins and immunity A basic requirement for tumours to invade successfully and metastasise is the ability to evade the host’s immunological surveillance system. Tumours do evoke immune responses from the host organisms. This can be attributed to the formation of novel antigens. Experimentally constructed fusion proteins appear to be able greatly to enhance antigen presentation and elicit improved immune responses, both cellular and humoral (Maranon et al., 2001; McCormick et al., 2001; Sulahian et al., 2001). For instance, Liu et al. (2001) have demonstrated HBV core antigen-specific cytotoxic T-lymphocyte (CTL) activity by immunising mice with HBV antigen-HSP fusion protein. In the same fashion, the fusion protein constructed with KMP-11 protein of Trypanosoma cruzi and HSP70 can generate CTL that can lyse cells that carry the KMP11 antigen (Maranon et al., 2001). Naturally generated fusion proteins that are encoded by chimeric genes resulting from chromosomal translocations also seem to function as novel antigens. The ETV6–AML1 fusion protein is generated in childhood AML by

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t(12;21) translocation. Yotnda, Garcia et al. (1998) identified a peptide at the chimeric junction of the fusion protein that binds to HLA-A2.1 molecules and is capable of inducing specific CTL response in healthy individuals. These cytotoxic cells also destroy tumour cells that express the fusion protein. The bcr-abl fusion protein resulting from t(9;22) in CML similarly functions as a novel antigen (MacIntyre et al., 1996; Yotnda, Firat et al., 1998b). Worley et al. (2001) identified certain consistent chromosomal translocations occurring in certain soft-tissue tumours, constructed peptides that corresponded to the chimeric fusion proteins and tested their ability to bind to class I HLA molecules. Indeed, some peptides encoded by regions of the chimeric genes bound class I HLA with high specificity. Furthermore, they could demonstrate that CTL from normal donors could be activated in vitro by these peptides and upon activation they were able to lyse autologous target tumour cells. The formation of the PAX3-FKHR fusion protein has also raised the possibility that this chimeric protein might function as a novel antigen. CTL sensitised with PAX3–FKHR fusion protein have been found to be able to lyse cells transfected with full-length PAX3–FKHR cDNA (Goletz et al., 1996). The tumour cells expressing these novel antigens will not survive the immunological surveillance of the host and therefore the presence of these new antigens will be counter-productive to the cause of tumour progression. At present these findings are conceptually not reconcilable with promotion of tumour progression, which the majority of findings relating to the generation of the fusion proteins would suggest. Nonetheless, much further supporting experimental work might be called for, especially in the light of the possibility that PAX3–FKHR amplification occurs only in a small number of tumours as compared with the frequency of generation of PAX7–FHKR fusion product. Possibly, acquisition of greater or more effective function as transcription factor by the fusion protein might outweigh the potential autoimmune consequences for the cell. Quite apart from considerations as to how these findings might influence tumour progression, they certainly could provide an experimental basis for the hitherto empirical observation that patients are more often than not sensitised to their tumours. There is little doubt that the work of Worley et al. (2001) has much practical value in terms of potential immunotherapy. However, it cannot be gainsaid that their findings do not contribute significantly to the postulate of a role for chromosomal translocations as an aid in tumour progression, but indeed might explain why metastasis is such an inefficient process.

The association of chromosomal translocation with fragile sites Chromosomal fragile sites are often the focal points of loss of heterozygosity or homozygous allelic loss as well as reciprocal chromosomal translocations.

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Several investigators have characterised selected fragile sites and have accumulated a reasonable body of evidence, which suggests that microsatellite instability might be an important factor in the initiation of recombination events. It is also distinctly possible that repeat elements such as Alu might be present in chromosomal loci encompassing fragile sites and these could confer chromosomal instability in the locus. Other repetitive elements can also conceivably contribute to the genetic instability associated with this fragile site (Wang et al., 1997). Paradee et al. (1996) identified two aphidicolin-inducible breakpoints in the fragile site at chromosome 3p14.2, called the FRA3B. Both breakpoints were adjacent to two polymorphic microsatellite sequences (V Shridhar et al., 1997). Indeed, Shridhar et al. (1997) reported that more than 60% of renal cell carcinomas investigated showed LOH at chromosome 3p14 locus, with breakpoints surrounding the FRA3B fragile site. Loss of heterozygosity of markers of the FRA3B locus also occurs in pancreatic carcinomas. LOH occurred within the 3p14.2 locus in 84% of tumours. In comparison, LOH of 3p markers outside the 3p14.2 locus occurred in only 8% of cases (R Shridhar et al., 1996). FRA3B is also the site of a breakpoint of translocation t(3;8)(p14.2;q24.13) occurring in hereditary renal cell carcinoma. Paradee et al. (1996) found that aphidicolin induced breaks at 3p14.2 and these breakpoints occurred in two clusters on either side of a region within FRA3B that is subject to frequent breaks. Similarly, the FHIT locus (see below) within the FRA3B site seems to be susceptible to alterations in RER+ carcinomas of the pancreas (Hilgers et al., 2000). Thus FRA3b is a source of genetic instability and a focus of chromosomal rearrangement. Another fragile site called FRA16D occurring at chromosome 16p23.2 is a common fragile site that seems to be actively involved in a chromosomal translocation often found in multiple myeloma. The translocation t(14;16) (q32;q23) occurs in roughly a quarter of all multiple myelomas. And this translocation involves the fragile site FRA16D. Of much interest is the finding that microsatellite sequences occurring with this fragile site are frequently lost in many cancers (Krummel et al., 2000). This supports the thesis that presence of the microsatellite sequences in the fragile site might be conducive for recombination to occur and might indeed instigate the events.

Chromosomal fragility, genetic loss and tumour suppressor genes The loss or inactivation of tumour suppressor genes shows a close relationship to chromosomal fragility. A number of tumour suppressor genes occur at genetically unstable chromosomal locations characterised by chromosomal fragile sites, highly polymorphic repeat elements, transposable elements, mismatch repair deficiency and abnormally methylated DNA. Among them, of note are the fragile histidine triad (FHIT), the von Hippel–Lindau (HPL) and the

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RASSF1A genes, all occurring on chromosome 3p, which is a focus of frequent allelic loss in a wide spectrum of human neoplasms.

Fragile histidine triad (FHIT) gene abnormalities in cancer The fragile histidine triad (FHIT) located on chromosome 3p14.2 encompasses the FRA3B fragile site. The FHIT transcript is 1.1 kb although the gene encompasses 1.2 Mb of DNA (Ohta et al., 1996). It codes for a protein belonging to the histidine-triad super family of proteins (Brenner et al., 1999). FHIT is frequently altered or its expression reduced in many forms of human cancer, and the loss of expression appears to correlate with progression of the disease, as for instance in carcinomas of the oesophagus (Menin et al., 2000; Shimada, Sato et al., 2000); endometrium (Ozaki et al., 2000; Segawa et al., 2000), the prostate (ZY Guo et al., 2000), uterine cervix (Yoshino et al., 2000), and also in hepatocellular (Yuan et al., 2000) and colorectal carcinomas (Hao et al., 2000). Gastric cancers also show a loss of FHIT expression (Baffa et al., 1998). A loss of heterozygosity of the FHIT locus has been reported in the normal epithelium of the oesophagus and the stomach (Dolan et al., 2000), possibly indicating that this event might be a prelude to the onset of neoplasia. The expression of FHIT gene has no effect on the progression of the cell cycle. For, alterations in its expression induced by the transfection of sense or antisense gene constructs into human 293T cells seem to have had no influence on proliferation (Guo and Vishwanatha, 2000). Similarly, the restoration of fulllength transcripts of FHIT in cervical carcinoma cells that lack endogenous fulllength transcripts, by transfection seemed to produce no effects on anchorageindependent growth or tumorigenicity (R Wu et al., 2000). Werner et al. (2000) have performed similar experiments using two RCC cell lines that lacked endogenous FHIT. They found no change in proliferation of either cell line that had been restored with FHIT expression. One cell line showed delayed tumorigenesis, not total suppression, upon implantation into nude mice; the tumours that developed showed loss of FHIT in a major proportion of component cells. Obviously, the loss of FHIT does confer some selective proliferative advantage. However, in the experimental set-up there is little unequivocal support for the tumour suppressor function. Although the investigations so far suggest a possible tumour suppressor function for FHIT, this is by no means a fait accompli. The suppressor function of FHIT is not infrequently called into question on account of the rarity of point mutations (Huebner et al., 1998) and upon the finding that abnormal transcripts of the gene are often found also in normal tissues (Panagopoulos et al., 1997). R Wu et al. (2000) stably transfected the gene into cervical and lung carcinoma cell lines, but did not find any alterations in the tumorigenicity of the cells. Nonetheless, the fact that FHIT locus encompasses the fragile site FRA3B

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that has intrinsic genetic instability built into it might be viewed as an important factor in the interpretation of the apparent suppressor function of the gene. Another reason for the abnormalities associated with this gene could simply be the operation of the LINE repeat in a large deletion in this gene. For example, nine out of ten allelic deletions resulted in the loss of exon 7, and in seven deletions the breakpoints occurred close to LINE elements of the FRA3B fragile site (Mimori et al., 1999). FHIT is inactivated by intragenic mutations induced by carcinogens. Furthermore, many tumours are characterised by the presence of aberrant transcripts of the FHIT gene. Therefore there is a reasonably sound basis for postulating a suppressor function to it. Abnormal FHIT transcripts occur in primary hepatocellular (Gramantieri et al., 1999), oral squamous cell (Tanimoto et al., 2000), and endometrial (Ozaki et al., 2000) carcinomas. In cervical cancers, FHIT mutations as well as the presence of aberrant transcripts have been encountered (Yoshino et al., 2000; Herzog et al., 2001). Yuan et al. (2000) found that FHIT was downregulated, but detected no aberrant transcripts or mutations of the gene. Normal transcripts of the gene occur in some forms of renal cell carcinoma (RCC) (Bugert et al., 1997). However, loss of heterozygosity at the FRA3B site is quite frequent in RCC. It may be that the gene is also suppressed by some other mechanism. According to Shimada, Sato et al. (2000), in oesophageal squamous cell carcinoma, the gene is silenced by methylation. Menin et al. (2000) found both LOH and the presence of aberrant FHIT transcripts in oesophageal carcinomas. It may be that in some tumours the breakpoints might be occurring outside FHIT locus (Bugert et al., 1997). There is also a view that the loss of aberrant expression of FHIT might be a result of defective mismatch repair. Mori et al. (2001) investigated the expression of FHIT and MSH2 in colorectal carcinomas. They have found that genetic alterations at the FHIT locus and loss of FHIT correlated with loss of MSH2 and both deficiencies seemed to be related to the progression of the tumours. In spite of the large body of evidence available about FHIT in tumorigenesis we have as yet no firm indications concerning its modus operandi. The work of Chaudhuri et al. (1999) has indicated a mode of function. They have reported that FHIT interacts with tubulin. This could conceivably influence cell division. One might recall that in some experiments the transfection of FHIT has not affected cell proliferation. It would be necessary to study the effects of FHIT expression on cell proliferation in some detail, since this could be a useful avenue of approach to understanding the mode of function of the putative suppressor gene product. It would be of much interest to note here that the metastasis suppressor nm23/NDP kinase might influence cell proliferation by a similar mechanism (see Sherbet, 2001). The metastasis promoting S100A4 also seems to function by interfering with the cytoskeletal dynamics of the cell. Overall, how both tumour/metastasis suppressors as well as promoters might function by employing the same target machinery would be an interesting conundrum that deserves further investigation. At any rate, the possibility that

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there could be common strand linking the functioning of both tumour/ metastasis suppressor and promoter genes simply underlines the conservative nature of regulation of biological processes.

Tumour suppressor function of von Hippel–Lindau gene The von Hippel–Lindau (VHL) gene product subserves diverse functions. It is a component of ubiquitin ligase and it is capable of enhancing ubiquitination of cellular proteins. VHL is involved in the proteolysis of the ␣-subunit of the hypoxia inducible factor-1 (HIF-1) transcription factor complex, which mediates transcription of genes related to adaptive response to hypoxia. VHL degrades HIF-1 in the presence of oxygen, ␣-HIF-1 subunit being regulated by oxygen and angiogenic growth factors (Cockman et al., 2000; Semenza, 2000). HIF-1 is expressed constitutively at high levels in VHL (–/–). HIF-1 activates vascular endothelial growth factor and stimulates angiogenesis. Apart from its relevance to tumour growth in hypoxic conditions, VHL has been found to be able to regulate tumour vascularisation with obvious implications for tumour expansion and dissemination by regulating the expression of cellular receptors for angiogenic factors (Gunningham et al., 2001). VHLmediated degradation therefore produces an inhibition of angiogenesis. Loss of VHL leads to enhanced tumour vascularisation together with overexpression of angiogenic factors such as TGF-␣ and VEGF. Kondo and Kaelin (2001) suggest the mediation of HIF in this. De Paulsen et al. (2001) found that hypoxia seems to induce TGF-␣ in VHL (–/–) renal carcinoma cells (RCC) upon reintroduction of VHL. Together with its ability to inhibit angiogenesis is to be viewed the apparent ability of VHL to remodel the ECM, alter the adhesive faculty of cells, stabilise their cytoskeletal organisation and inhibit cell motility (Hoffman et al., 2001; Kamada et al., 2001). VHL also takes part in the control of tumour growth and has been shown to inhibit growth of RCC in vitro. When introduced into VHL (–/–) RCC, VHL can inhibit cell proliferation in vitro. A peptide sequence in the ␤-domain of VHL seems sufficient to inhibit growth of and invasion by renal carcinoma cells (Datta et al., 2001). The expression of VHL also confers regular growth pattern and epithelial organisation of RCC in culture, whilst the absence of VHL results in the formation of disorganised epithelial sheets (Davidowitz et al., 2001). This suggests a differentiation inducing activity for VHL, which is compatible with its ability to suppress tumour growth. Schoenfeld et al. (2000) investigated the effects of VHL expression of apoptosis and cell cycle regulating genes in renal cell carcinoma. VHL-negative cells underwent apoptosis when exposed to UV irradiation and these cells showed a marked reduction in the cyclindependent kinase inhibitors p21 and p27. In contrast, the levels of these cell cycle regulators were unaffected in VHL-positive cells. Schoenfeld et al.

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(2000) also demonstrated that restoration of VHL expression in the VHLnegative cells protected the cells from apoptosis and this was associated with an increased expression of the apoptosis inhibitory proteins bcl2 and bclx(L). These findings are incompatible with the association of VHL in suppression of cell proliferation. Schoenfeld et al. (2000) made no attempt to look at the expression of apoptosis-promoting members of the bcl-2 gene family. Cell survival or apoptosis seem to be regulated by the dimerisation occurring between different members of the family. The regulation of apoptosis would therefore be a consequence of any differential expression of apoptosis promoter and inhibitor proteins. In other words, the levels of these proteins might set up a hierarchy of bcl-2 protein complexes with different degrees of apoptotic function (Sherbet and Lakshmi, 1997). Quite obviously, much further work needs to be done for the evaluation of the VHL expression on cell proliferation and apoptosis. With such a wide spectrum of biological function it is not surprising that VHL should be associated with the pathogenesis of cancer. VHL germ-line mutations are responsible for association with the VHL syndrome of several forms of human cancer, including clear cell renal carcinoma, phaeochromocytoma, CNS haemangioblastoma and retinal tumours. Allelic loss of VHL region 3p25–26 has been noted in breast cancer (Martinez et al., 2001). However, Sourvinos et al. (2001) do not support a role for this gene in breast cancer pathogenesis. There could be a differential involvement of VHL in sporadic versus VHL-associated phaeochromocytoma (Bender et al., 2000). This could be attributed to region-specific mutations of VHL conferring, in a differential manner, the functions of tumour suppression, HIF-1 degradation or ECM remodelling (see Hoffmann et al., 2001). VHL expression is weak or undetectable in poorly differentiated thyroid cancers but is present in nonneoplastic lesions and in well-differentiated neoplasms of the thyroid (Hinze et al., 2000). There is little doubt that the loss of expression of VHL is consistent with germ-line mutations of VHL or loss of heterozygosity, and in many instances with loss of chromosome 3p. Whether loss of the chromosome region containing FHIT has any bearing on the expression of VHL is unclear, but it is possible that there could occur overlapping deletions affecting both these suppressor genes together with other suppressor genes occurring in the region. Epigenetic inactivation of VHL has not been demonstrated to date. In oligodendrogliomas a large number of genes are hypermethylated, but there is no hypermethylation of the VHL promoter (Dong et al., 2001). Superficial multifocal bladder cancer reportedly shows LOH at FHIT as well as VHL loci (Louhelainen et al., 2001). It would be interesting to compare the frequency of LOH at these gene loci in superficial and invasive carcinomas of the bladder. Further, it would contribute significantly to our understanding the process of progression if one could also investigate the status of superficial tumours that recur as invasive ones.

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The ras association domain family RASSF1 gene in tumour suppression A third suppressor gene that has evoked much interest is the RASSF1 gene residing at 3p21.3. Seven isoforms of this gene have been described. Of these, RASSF1A and B seem to be silenced by methylation of the promoter CpG or undergo LOH in a number of tumours including cancer of the breast, lung, ovary and the kidney, nasopharyngeal carcinomas and gastric adenocarcinomas (Dammann et al., 2000, 2001; Agathanggelou et al., 2001; Burbee et al., 2001; Byun et al., 2001; Lo et al., 2001; Yoon et al., 2001). In gastric adenocarcinoma loss of expression of RASSF1A seems to relate to tumour stage and grade. with greater loss of expression occurring in advanced cancers than in early stages of the disease. With the demonstration in vitro that RASSF1A expression inhibits anchorage-independent cell growth and colony formation, and that it inhibits tumorigenicity in vivo, the view has gained ground that RASSF1 might be linked with ras-mediated signalling involved in cell proliferation and transformation. Ras signalling is also implicated in terminal cell differentiation and senescence, both associated with growth inhibition, cytoskeletal dynamics and apoptosis (Bar-Sagi and Feramisco, 1985; Sistonen et al., 1987; Serrano et al., 1997; Downward, 1998). Ras signalling is activated by GTP binding and inactivated by GTP hydrolysis. Mutated ras possesses reduced GTPase activity and this leads to constitutive binding to GTP. The continually present active form of ras/GTP complex causes loss of growth control and becomes oncogenic. Ras seems to use RASSF1 as a downstream effector in its apoptotic signalling pathway (Vos et al., 2000). It seems possible therefore that the RASSF1 family proteins might subserve a wide spectrum of biological functions that depend upon ras signalling. DNA methylation being itself associated with genetic instability and recombination, it is not surprising that the loci of RASSF1, VHL and FHIT would be implicated in the pathogenesis of cancers.

Gene amplification and its relationship to genetic instability Gene amplification can be intrachromosomal and seen as homogeneously staining regions (HSR) of chromosomes or can be extrachromosomal and occur in the form of double minute chromosomes (DM). Whether these are independent manifestations of gene amplification is much debated. Coquelle et al. (1998) suggested that chromosomal fragile sites are prone to DNA strand breakage and these breaks initiate amplification. They postulate that DMs can fuse and the fused DMs are then specifically targeted to the chromosomes and reintegrated to form HSRs. Coquelle et al. (1997) also state that fragile sites occur at the boundaries of amplicons. Thus there is strong support for the

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involvement DNA flexibility or instability at chromosomal fragile sites in gene amplification encountered in tumour progression and in the generation of drug resistance. DMs occur more frequently in tumours than do HSRs (Benner et al., 1991). The incidence of DMs can be very high, as in NSCLC (Nielsen et al., 1993). The incidence of DMs or HSRs can occasionally show an increase corresponding with tumour progression. The appearance of DMs in prostate carcinoma might be a characteristic of the invasive phase (Milasin and Micic, 1994). Bailly et al. (1993), for instance, found that 1 of 4 poorly metastatic melanoma cell lines were found with DMs as compared with 2 of 3 metastatic cell lines. The incidence of DMs in murine melanomas also correlates with metastatic potential. The high metastasis variants of the B16 melanoma show a far higher incidence of DMs as compared with the low metastasis B16-F1 variant (Table 3). Furthermore, the amplification in DMs of several important genes involved in tumour cell proliferation and growth, e.g. EGFr, FGF genes, myc, and mdm2, c-erbB2, cyclin D, c-met (the gene encoding the tyrosine kinase receptor of hepatocyte growth factor), among others, does provide considerable additional support for the view that the phenomenon of generation of DMs might be related to tumour progression. As stated before, the exchange of unequal chromatid segments has been regarded as one of the mechanisms by which gene amplification might occur. In some tumour models the incidence of DMs correlates significantly with SCR. Sherbet and Lakshmi (1987) have presented a schematic postulate as to how homologous but unequal SCR can generate gene amplification. Homologous but unequal recombination of repetitive elements can cause deletions or duplication of exons, and similar unequal homologous exchanges can be a source of the generation of DMs. Unfortunately, at present we have no information on the occurrence of repetitive DNA elements in these extrachromosomal DNA bodies. The successful employment some years ago of AluPCR to characterise DNA does indeed indicate the presence of these SINE elements in DMs (Sen et al., 1992). But subsequent studies have failed to detect the presence of any repetitive elements in DMs. Van Loon et al. (1994) have taken a totally different view. They sequenced the junctions of recombination in extrachromosomal circular DNA and the chromosomal sequences from which the extrachromosomal DNA bodies were derived and concluded that a nonhomologous rejoining of fragmented DNA resulted in the formation of these bodies. Several genes have been found in an amplified form in DMs. For instance, amplification of EGFr and dihydrofolate reductase (DHFR) genes occurs in DMs (Nevaldine et al., 1999; Rizwana and Hahn, 1999). Treatment of cell lines with EGFr-DMs by hydroxyurea results in the loss of EGFr, together with loss of growth and a decrease in tumorigenicity. Hydroxyurea treatment similarly leads to loss of DHFR-DMs and DHFR amplification. The double minute chromosomes of MHH-MED-2, a human medulloblastoma cell line, contain amplified

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c-myc gene, and the cell line also over-expresses myc mRNA and protein (Pietsch et al., 1994). This gene is amplified on DMs in acute myeloid leukaemia (AML) (Tanaka et al., 1993; Fugazza et al., 1997). Malignant mixed tumours of salivary glands also have shown DMs with amplified myc and mdm2 genes (Rao et al., 1998). In gastric adenocarcinomas, c-myc amplification is found mainly as DMs, and also less frequently in the form of HSRs (Hara et al., 1998). Also amplified in these tumours are the c-met, c-erb-B2 and K-sam genes (Werner et al., 2001). The K-sam gene codes for a truncated FGFR2 form of tyrosine kinase, which is activated by keratinocyte growth factor. The C-terminal region of the kinase seems to be preferentially deleted (Ueda et al., 1999a, b). Overexpression of K-sam is related to high proliferative potential and poor differentiation status of gastric cancers. K-sam amplification occurs as both DMs and HSRs. The MLL gene is amplified in acute leukaemia and takes the form of both DMs and the intrachromosomal form of HSR (Ariyama et al., 1998; Cuthbert et al., 2000). Among other genes amplified are the cyclin D1, RINI (ras interaction/interference protein) and FGF-3 and -4 genes in oral squamous cell carcinomas. These amplifications occur as HSRs (Shuster et al., 2000). The formation of DMs could be an indication of recombination occurring as a consequence of chromosomal instability. Genetic rearrangement also not infrequently accompanies gene amplification. The rearrangement can be a simple inverted duplication (Shuster et al., 2000), or can be far more complex. The rearrangement of MLL occurs frequently in acute leukaemias, as chimeric fusions, or in the form of partial tandem duplication (Ariyama et al., 1998; Cuthbert et al., 2000). Rizwana and Hahn (1999) have studied two murine cell lines, which possess multiple copies of DMs carrying amplification of DHFR gene. In the cell line called Mut-F, the DMs contain two unmethylated CpG islands, and these are cleaved easily by methylation-sensitive restriction endonucleases. In the second cell line, called Mut-C, these CpG islands are only partially methylated and are resistant to cleavage. Rizawa and Hahn (1999) also noticed that the unmethylated DMs undergo deletions and dimerisation more easily than the DMs where the CpG islands are partially methylated. Furthermore, the Mut-C DMs showed deletions and dimerisations upon being demethylated by azacytidine.

Chromosomal fragility and micronuclei DNA damage and the generation of micronuclei Chromosomal and DNA damage is often associated with nuclear structures called micronuclei. The formation of micronuclear bodies (MN) is related to the degree of DNA damage and its fragmentation, and the formation of MN correlates with the frequency of DNA strand breaks induced by exposure to chemical agents (Robbiano et al., 1999). DNA cross-linking compounds such as

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CCNU (1-(2-chloroethyl)-3-cyclohexyl-nitrosourea) (Fiumicino et al., 2000) induce the formation of MN. Radiation-induced DNA damage and radiosensitivity often, but probably not invariably (see Akudugu et al., 2000; Emri et al., 2000, for example), correlates with the presence of MN. Exposure to microwave radiation is also believed to induce MN in human PBL (Zotti-Martelli et al., 2000). This is compatible with the reported induction of single and double strand DNA breaks (Lai and Singh, 1997) and the induction of necrosis or apoptosis (Brehmer, 1997) by microwave exposure. Equally, of course, there are reports that deny any correlation between microwave exposure and DNA damage. However, microwave and hydrogen peroxide exposure seem to have a synergistic effect on the genetic material and viability of microorganisms (Kuchma, 1998). Indeed, oxidative DNA damage by hydrogen peroxide also induces MN formation (Villani et al., 2000). There is a good correlation between DNA breaks and the genesis of MN. In folate deficiency a high number of chromosome breaks and MN formation are induced; both effects are reversed by the administration of folate. The reason for this seems to be that folate causes uracil to be incorporated into the DNA and the DNA is transiently nicked during excision repair, a possible scenario for the formation of MN (Blount et al., 1997). We also know that MN are formed under conditions where DNA repair processes are inhibited. Stopper et al. (1997) had studied MN formation in two mutant cell lines from Chinese hamsters. Of these the V-E5 resembled cells derived from AT patients and the XR-V15B showed a diminished ability to repair double strand breaks. Both cell lines showed an increased incidence of MN and furthermore showed higher susceptibility to induction of MN by exposure to methyl methane sulphonate (MMS). The frequency of formation of MN is enhanced in cells depleted of PARP. Furthermore, mismatch repair deficient cells are hypersensitive to agents such as CCNU, which induce MN formation. Indeed efficient mismatch repair can overcome this CCNU sensitivity. Fiumicino et al. (2000) reported an increased induction of MN by CCNU in two clones of HeLa cells deficient in hPMS2.

The involvement of p53 in the formation of micronuclei Loss of p53 function induces MN formation (Cistulli et al., 1996; Sablina et al., 1998). P53 controls the G1S checkpoint of the cell cycle and holds back cells that carry damaged DNA from entering the S-phase. Therefore, this might indicate that maintenance of damaged DNA in the absence of p53 function could be leading to the formation of MN. The BRCA2 is associated with susceptibility to breast cancer. Loss of BRCA2 function is believed to result in breast cancer. Tutt et al. (1999) have reported that certain mouse embryo fibroblasts, which carry a homozygous mutation of BRCA2, develop MN frequently. This is possibly related to alterations in DNA repair and transcription that is associated with BRCA2 abnormalities. As noted earlier, abnormal p53 has

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been attributed with the ability to induce genetic instability. Such an effect is also compatible with the appearance of MN in cells that carry p53 mutations. The immunodeficiency virus (HIV) has an accessory gene called Vpr. Vpr can induce cell cycle abnormalities as well as the induction of chromosome breaks, gene amplification and MN (Shimura, Onozuka et al., 1999). Shimura, Tanaka et al. (1999) generated Vpr-containing cell clones, in which Vpr expression was under the control of a tetracycline-responsive promoter. When Vpr was induced to express by exposure of cells to doxycycline, the cells developed giant nucleation and also developed micronuclei. These authors have argued that this could be a result of the genetic instability induced by Vpr. Among other agents that induce MN formation are topoisomerase (topo) inhibitors. DNA topoisomerases are enzymes that participate in chromosome segregation by decatenation of the intertwined DNA molecules after DNA replication, and chromosome condensation, and they alter the super helical organisation of the DNA. Not surprisingly, the expression of these enzymes is linked with that of p53. Thus wild-type p53 negatively regulates topo II␣, but mutated p53 correlates with over-expression of topo II␣ (Rudolph et al., 1999). In breast cancer, for example, high topo II␣ is associated with high p53 expression. Several topoisomerase II inhibitors induce MN formation. The ability of amsacrine and camptothecin to induce MN in bone marrow of mice was described several years ago (Holmstrom and Winters, 1992). Record et al. (1995) showed that MN were induced in murine splenic cells in vitro by treatment with genistein, genistin and etoposide. The cytotoxic effects of etoposide on male rat spermatids included the induction of MN (Lahdetie et al., 1994). In addition to etoposide, mitoxantrone, genistein, daunorubicin and m-amsacrine also induce DNA damage and mutation in L5178Y murine lymphoma cells and in human leukaemia cell lines together with the formation of MN (Stopper et al., 1999; Boos and Stopper, 2000). So abnormal p53 and the topo inhibitors could be influencing different facets of topo II function (see below).

Centromeric instability, DNA methylation and micronuclei in ICF syndrome DNA methylation appears to be involved in MN formation, as indicated by their generation in the recessive autosomal condition known as the ICF (immunodeficiency-centromere instability-facial anomalies). ICF patients show several cytogenetic abnormalities, such as decondensation of para-centromeric heterochromatin, multiradial chromosomes, deletion of chromosome arms and MN formation. The para-centromeric region of chromosomes is elongated and these chromosome regions show self-association in the interphase and form micronuclei. Normal eukaryotic satellite DNA, which is distinguishable by a higher A+T/G+C content than in genomic DNA, shows a high degree of cytosine methylation.

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Sawyer et al. (1995b) identified centromeric abnormalities of chromosomes 1 and 16 in ICF patients. Stacey et al. (1995) investigated phytohaemagglutinin (PHA)-stimulated PBL, Epstein–Barr virus (EBV) transformed B-cells as well as peripheral blood smears for MN. They found chromosomes 1 in a larger proportion of MN in PBL and EBV-transformed cells than were chromosomes 16 and 9. The centromere is believed to be rendered unstable on account of hypomethylation of the classical satellites 2 and 3. Satellite 2 of chromosomes 1 and 16 is hypomethylated in Wilm’s tumour (Qu et al., 1999), and that of chromosome 1 in breast cancer (Narayan et al., 1998). Similar centromeric instability in chromosomes 1 and 16 might occur as a consequence of HIV infection (Sawyer et al., 1995a). Tuck-Muller et al. (2000) found chromosomes 1 and 16 of ICF were often abnormal and the para-centromeric heterochromatin of these chromosomes was abnormally hypomethylated. Indeed satellite 2 of chromosome 1 is usually hypomethylated in ICF (Ji et al., 1997). In fact, Ji et al. (1997) demonstrated that inhibitors of DNA methylation induced rearrangements of the para-centromeric region of chromosome 1. DNA methyltransferases are essential for de novo methylation required in early mammalian development. DNA methyltransferases 3a and 3b methylate centromeric satellite repeat sequences (Okano et al., 1999). The DNA methyltransferase 3B gene is mutated in ICF patients (Xu et al., 1999; Hansen et al., 2000). As recognised some years ago, DNA methylation status, which has serious implications for the conformational state of the DNA may have marked effect on the interaction of topoisomerases with DNA (Kirchner et al., 1995) and the distribution of heterochromatin during mitosis. Therefore it is conceivable that DNA methylation could itself influence MN formation via topoisomerase function.

The nature and constitution of micronuclei Heterochromatin is a highly condensed form of chromatin that is inactive transcriptionally and characteristically replicates late in the S-phase of the cell cycle. The centromeric region of the chromosome contains what is known as constitutive heterochromatin, which is always in the condensed form. Heterochromatin is composed of repetitive DNA sequences and is believed to be the focus of induction of genetic instability and the induction of MN. Focal induction of DNA damage of this heterochromatin leads to the formation of MN (Smith et al., 1998). Micronuclei are made up of whole chromosomes, or chromosome fragments often containing the centromere and para-centromeric heterochromatin, so MN can be centromere-positive with several signals or with a single signal, or centromere-negative. Fragments of most chromosomes can be found in spontaneous MN as well as in those induced by iododeoxyuridine (IUdR), but some chromosome fragments appear to be incorporated into MN more than others. IUdR-induced MN contain chromosomes 1 and 9 more

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frequently than others. Furthermore, decondensation of heterochromatin of these chromosomes correlates with their presence in the MN (Fauth and Zanki, 1999). In general there is a restricted parallelism between the processes of formation of MN and apoptosis. At least in the early stages of both processes, there is DNA fragmentation with fragments being incorporated into MN. Both MN formation and apoptosis are markedly influenced by certain agents, such as endonucleases, topoisomerases and protein kinases (see Isaacs et al., 1998; Abend et al., 1999). As we noted earlier, MN formation is facilitated by inefficient DNA damage repair. Likewise, Chiaramonte, Bartolini et al. (2001) have shown that the degree of apoptosis is also related to the efficiency of repair of strand breaks, and there is an inverse relationship between the two features, with less efficient repair being conducive to apoptosis. Furthermore, both appear to be subject to regulation by p53 via the topoisomerases in MN formation and via the bcl-2 and related genes in the regulation of apoptosis. Finally, it is of much interest to note that the kinetics or dose-rate effects of induction of MN and apoptosis run in parallel in human lymphocytes exposed to gamma-radiation (Boreham et al., 2000).

7 DNA methylation and genetic instability

DNA methylation in normal and neoplastic development Methylation of DNA constitutes an epigenetic mechanism, which regulates genetic expression and gene imprinting. Methylation has also been attributed with the property of altering genetic stability, because it is known to alter the conformational state of DNA. The patterns of methylation might be inherited, although not all subscribe to this view. Nonetheless, the association of methylation with developmental processes as well as with neoplastic progression, has provided a focus for investigation of the possibility that, besides controlling individual gene expression as well as expression of a cohort of genes, the methylation state might influence the state of genetic stability. The genome contains a number of repeat sequences, which occur ubiquitously in several genetic locations and within genes, and these, by the nature of nucleotide in the motifs, can be implicated in gene silencing by means of methylation and chromatin condensation. Methylation can not only affect the normal functioning of the mismatch repair pathway but also influence the conformational state of DNA and by virtue of this might alter the normal pattern of gene expression in disease processes. DNA methyltransferases (DNMT), which catalyse the methylation of CpG islands, may be aberrantly or differentially expressed in cancer cells as Genetic Recombination in Cancer ISBN 0-12-639881-X

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compared with their normal counterparts. Lin et al. (2001) investigated a small series of HCC and noticed a 40–50% increase in DNMT-1, 3a and 3b in the carcinomas as compared with normal liver tissue. Ahluwalia et al. (2001) found a 3-fold higher expression of DNMT-1 gene in ovarian carcinomas than in normal ovarian surface epithelium, but DNMT-3a and 3b expression was comparable in normal and neoplastic tissue. However, according to Mizuno et al. (2001) DNMT-1, 3a and 3b show no differences of expression in the chronic phase of CML, but DNMT-1 does display 3- to 4-fold enhanced expression in the acute phase of the disease. It might be worth making a caveat based on the findings of Trasler et al. (1996) that the absolute values or levels of expression of the methyltransferases are probably not a direct reflection of the methylation pattern. Furthermore, the degree of methylation achieved may be a composite effect of more than one DNMT. For instance, Rhee et al. (2002) have shown a clear co-operative functioning of DNMT-1 and DNMT-3b. The disruption of DNMT-1 alone produced a mere 3% reduction in DNA methylation, whilst disruption of DNMT-3b and DNMT-1 reduced DNA methylation by more than 95%. Therefore a direct correlation between their levels and the disease state ought to be viewed with caution. Nonetheless, it cannot be gainsaid that aberrant DNA methylation may be encountered in immortalised cells in culture, neoplastic transformation of cells, and is indeed widespread among cancers, effecting inappropriate gene expression and inappropriate silencing of tumour and cell cycle regulator genes (Sherbet, 2001). Therefore, one has to seriously entertain the notion that modulation of methylation patterns could be a recurring event, which affects many cellular features that characterise tumour development from initiation through to metastatic progression. Besides, it would be somewhat simplistic to try to relate methylation patterns and DNMT expression to tumour progression to the exclusion of other possibilities. For, DNA methyltransferases might directly influence genetic transcription by forming complexes with transcription repressors such as DMAP1. DMAP1 can interact with and form transcription repressor complex with DNMT-1 as well as HDAC (Rountree et al., 2000), thus participating in transcription silencing by DNA methylation and in the remodelling of chromatin involving histone acetylation dynamics (see below).

Abnormal DNA methylation in ICF and Rett syndromes Abnormal DNA methylation is associated with genetic conditions such as fragile X, ICF and Rett syndromes. As we have seen in an earlier section, the ICF syndrome is characterised by instability of the centromere and para-centromeric heterochromatin, which is occasioned by aberrant methylation. This leads to the formation of multiradial chromosomes and micronuclei. The

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abnormalities of methylation are a consequence of mutations in DNA methyltransferases. Another X-linked dominant disorder known as the Rett syndrome (RTS) also results from abnormalities of DNA methylation. RTS is a neuro-developmental syndrome that leads to mental retardation and autism in young females, and constant movements of the hands accompany this. Hemizygous males develop a severe phenotype of RTS (Schanen, 1999). The abnormalities in DNA methylation in these patients are due to mutations in methyl-CpG-binding protein 2 (MECP2). Mutations can be found not only in the MECP2 coding region but also in a conserved sequence in the 3’-untranslated region of the MECP2 gene. These mutations occur together with marked genetic rearrangements (Bourdon et al., 2001). MECP2 is believed to bind to methylated CpG residues and repress genetic transcription, and to mediate chromatin remodelling. We know that MECP2 interacts with histone deacetylases (HDAC) and with transcription co-repressors such as Sin3A (see below). Many of these mutations have been characterised and assessed for their impact on the function of MECP2 (Ballestar et al., 2000; Yusufzai and Wolffe, 2000); some substantially reduce the binding of MECP2 to methylated DNA, whilst others impair its ability to repress transcription and also affect the in vivo stability of the protein. The extent of genetic silencing that occurs in the genesis of RTS is not yet fully appreciated.

DNA methylation and genetic instability The possibility that genetic instability might be related to DNA methylation was recognised many years ago (Hickey et al., 1988). The epigenetic mode of silencing of human DNA mismatch repair genes has since been well documented in several recent studies and a clear correlation has been demonstrated in many human tumours between mismatch repair gene hypermethylation and genetic instability. Several human tumours have been investigated for possible epigenetic silencing of one or more hMLH genes, hMLH1, 2, 3 and hMLH6, but prominently hMLH1. Some early work on neural tumours revealed hypermethylation of certain loci on chromosome 17p in primary brain tumours (Makos et al., 1993). In a short series of endometrial carcinomas, Esteller et al. (1998, 1999) found a majority of tumours with microsatellite instability to have marked hypermethylation of hMLH1 promoter, whereas none of the tumours that was microsatellite-stable displayed any methylation. In comparison, hypermethylation of the gene occurred only in 8 out of 116 cases of endometrial hyperplasia. Salvesen et al. (2000) found nearly a quarter of 138 endometrial carcinomas with hypermethylation of hMLH1 with attendant loss of expression of nuclear hMLH1 protein and microsatellite instability. All unmethylated tumours showed normal hMLH1 expression. These authors state that the loss of hMLH1

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expression or its hypermethylation did not result in aneuploidy. Although aneuploidy and abnormal chromosomal segregation can and do occur in the wake of genetic instability, it would have been useful to employ other endpoint markers. An analogous situation seems to exist in NSCLC. In a study involving 68 patients, 41% of tumours showed microsatellite instability, and a high proportion of these tumours showed loss of hMLH1 protein expression (JW Chang et al., 2000). Kondo et al. (2000) studied hepatocellular carcinoma specimens and normal liver tissue samples from 40 patients. Both normal liver tissue and hepatocellular carcinoma showed DNA hypermethylation in a high proportion (83% and 100% of 40 in each case) of samples tested. The frequency of incidence of microsatellite instability was also roughly comparable. Gastric cancers also show a high degree of correlation between microsatellite instability and hypermethylation of hMLH1 promoter. According to Fleisher et al. (1999) 26 of 65 gastric carcinomas showed microsatellite instability involving one or two microsatellite loci, and of these three-quarters of the specimens also showed hMLH1 promoter hypermethylation. Of the microsatellite stable tumours, only 2.6% displayed hypermethylation. Fleisher et al. (2000) have described similar findings in respect of colorectal neoplasms occurring in patients with ulcerative colitis or Crohn’s disease. In this later study, the authors further confirm that greatly reduced levels of hMLH1 protein were detectable in tumour cell nuclei as compared with nuclei of normal host tissue. The loss of expression of MLH1, hMSH2, PMS1 and PMS2 has been reported in about a third of human melanomas. The loss of expression was prominent in tumours of Clarke grade III and above. Furthermore, it appeared to correlate with poor prognosis (Korabiowska et al., 2000). It would seem therefore that there is a need to extend the studies on genetic instability and DNA methylation to more human tumour models, so that one can establish whether the association between these two nuclear features is general occurrence in neoplasia.

The CpG Island Methylator Phenotype (CIMP) Hypermethylation and the silencing of other genes besides the mismatch repair genes occur frequently. Ahuja et al. (1997) found microsatellite instability in 32% (9 of 47) colonic tumours, and further that several genes were hypermethylated. The p16ink, thrombospondin gene, IGF2, TIMP (tissue inhibitor of metalloproteinase)-3, and HIC-1 were all hypermethylated predominantly in tumours that also showed microsatellite instability (Herman et al., 1995, 1998; Cunningham et al., 1998; Cameron et al., 1999; Toyota et al., 1999a). The finding that several genetic loci are consistently hypermethylated in colonic carcinoma has prompted the postulate that pathogenesis of tumours, especially with respect to colonic cancers, might follow an epigenetic pathway

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called the methylator phenotype. Liang et al. (1999) have found only p16 methylation correlated with RER status in the colon cancer series that they studied. Among other genes investigated from this point of view and from their relevance to breast and colonic tumour progression are the oestrogen receptor (ER) gene and the putative metastasis suppressor E-cadherin gene. Toyota et al. (1999a) have used the term CpG island methylator phenotype (CIMP) for this epigenetic pathway. Toyota et al. (2000) have investigated mutations in k-ras, p53, DPC4 and TGF␤RII in relation to CIMP type. Mutation of k-ras was found in 68% (of 41) CIMP-positive tumours, as compared with only 30% (of 47) CIMP-negative tumours. By contrast, mutation of p53 was 24% (of 41) CIMP-positive tumours and 60% (of 46) of CIMP-negative carcinomas. Thus within the CIMP groups subdivisions appear that relate to the methylation of certain oncogenes. This, they claim, might generate the diversity that is noticed in tumour development. However, several points need to be considered before these findings can be interpreted as related to progression of colonic cancers. There are several genes involved in the progression of these tumours, which could have been and should have been tested. Among them are APC and DCC genes. These, together with ras are activated at different phases in tumour development. So the pattern of methylation in the temporal dimension is of utmost importance, especially in hereditary forms of colorectal carcinoma. Germ-line mutations of APC gene lead to the development of large number of adenomas that progress to overt carcinomas. In hereditary non-polyposis colorectal cancer, the formation of adenomas is infrequent, but germ-line mutations of mismatch repair gene hMLH1 and hMLH2 are encountered frequently (Liu et al., 1996). It is needless to say that these can lead to mutations of other genes involved in the cascade of progression of the disease. There is also the need to recognise the fact that certain genes, especially p53, come into play at a late stage in overt carcinogenesis. There are reports, however, that RER status is not related to LOH of the DCC gene, nor with p53 over-expression (Liang et al., 1999). The CIMP pathway has also been reported as being operational in the development of other tumours. A subset of pancreatic carcinomas shows the CIMP-positive phenotype, since they show simultaneous methylation of four loci (Ueki et al., 2000). An investigation of gastric tumours has revealed methylation of three or more genetic loci in 41% (of 56) tumours (Toyota et al., 1999b). Microsatellite instability together with hypermethylation of hMLH1 occurs prominently in gastric carcinomas (Endoh et al., 2000). The APC gene is said to be inactivated in gastric cancer cell lines, as well as in carcinomas and non-cancerous gastric mucosa (Tsuchiya et al., 2000). Endoh et al. (2000) also found that hypermethylation of hMLH1 promoter correlated with microsatellite instability of gastric foveolar tumours; besides they detected methylation in 71% non-neoplastic tissue surrounding the carcinomas. This seems to suggest that hypermethylation might be an early event in tumorigenesis.

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Kondo et al. (2000), however, found no differences in the methylation status of p16ink4 between hepatocellular carcinoma and corresponding noncancerous liver tissue. The studies by MM Pao et al. (2000) also do not provide much support for the CIMP concept in the progression of colonic tumours. Quite obviously many of these investigations represent early stages of investigation and it would be some while before the significance of phenotypic effects of silencing of these genes could be properly evaluated. The genes that are investigated to define CIMP appear to be selected arbitrarily. Although, admittedly, p53 function is related to that of p16ink4, it would have been more appropriate to look at Rb, mdm2 and waf1/cip1 methylation in relation to p53 status. Indeed, there is much co-operation between p53 and Rb proteins in the control of cell cycle progression. In certain pituitary tumours, loss of Rb and p16ink4 is encountered that can be related to the loss of chromosomes 13 and 9. In some instances, the loss of p16 expression is said to correlate also with the hypermethylation of CpG islands in the gene. Furthermore, experimentally induced expression by transfection of p16 does lead to G1 arrest of cells. These effects were reversible if the constructs were methylated at the CpG sites (Farrell and Clayton, 1999; Farrell et al., 1999). Possibly, p53 could be functioning in this scenario by suppressing Rb phosphorylation by inducing the expression of CDK inhibitors (see Sherbet and Lakshmi, 1997). Colonic carcinoma undoubtedly has provided an excellent model for the study of CIMP. Other human neoplasms such as leukaemia might also provide an ideal model for study, for childhood and adult forms of leukaemia show marked chromosomal abnormalities, including significant translocations and genetic rearrangement as, for instance in the myc, immunoglobulin and T-cell receptor genes, etc. The INK4 family of cyclin-dependent kinase inhibitors, namely p15, p16, p18 and p19, have been the subject of study in patients with leukaemia. A general finding is that p15, but not p16, is frequently methylated in leukaemia but not in normal subjects (Dodge et al., 1998, Chim et al., 2001). However, recently evidence has been presented that both p15 and p16 are hypermethylated in AML, ALL and acute biphenotypic leukaemia (ABL). IHN Wong et al. (2000) found that the promoter of p15 gene was hypermethylated in 58% (of 79) of AML, ALL and ABL patients. In about a fifth of ALL and AML the p15 and p16 were concomitantly hypermethylated, exclusively in M2, M4 and L2 FAB (French/American/British Study Group) subtypes. Furthermore, whilst patients with unmethylated p15 had normal karyotype or hyperdiploid karyotype and better prognosis, those with p15 methylation carried chromosomal deletion or recombination. It may be noted, however, that Chim et al. (2001) seemed uncertain about the relevance of p15 methylation to disease prognosis. Hypermethylation of these genes has been confirmed in another study, but there were no marked increases in the frequency of mutations or deletions of the genes (SX Guo et al., 2000). Although abnormalities of p15 and p16 are consistently associated with leukaemia, there are other genes,

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especially MLL and myc, which might be profitably investigated from the viewpoint of operation of a methylator phenotype. Whilst it is not claimed that the CIMP concept is universally applicable, it is of considerable potential in the study of tumour development, differentiation and progression. The expression of several genes influences a spectrum of biological features of the cancer cell and dictates their biological behaviour. The relationship between the methylation of these genes in the context of the CIMP seems an exciting avenue of approach. Also called for are experimental studies that can secure a firm foundation for the concept. Especially called for are studies into potential link-up between genes that might be concurrently hypermethylated, and the link between such concurrent methylation patterns and progression of cancer. By all accounts to date, cancers seem to display a general defect of genetic methylation, which would inevitably reflect in the methylation of a variety of genes and therefore such studies are essential to dissect out the methylation patterns of genes relevant to the biological process involved in cancer development, invasion and secondary spread.

Influence of DNA methylation on recombination Demethylating agents do influence recombination events, as evidenced by their ability to increase the SCE frequency in cell lines in vitro. Albanesi et al. (1999) point out that there is an important distinction between the initiation of SCR by DNA damaging agents and those that cause demethylation of DNA. They studied SCE induction by a single pulse or by exposure of cells to two cell cycles. Exposure to mitomycin-C (MMC), UV-irradiation and hydrogen peroxide induced SCE under both treatment protocols, whilst demethylating agents require exposure over two cycles to achieve alteration in SCE frequency. Albanesi et al. (1999) therefore suggest that demethylation of the parental strand at the replication fork causes errors in ligation of repaired lesions and an enhanced recombination seen as SCEs. It should be recalled here, nonetheless, that Noguiez et al. (1993) had found no relationship between the high incidence of SCE in BS cells and the state of DNA methylation. The state of methylation of CpG islands appears to be the source of genetic instability that is associated with DMs found in two methotrexate-resistant cell lines with amplified DHFR gene. Rizwana and Hahn (1999) studied two cell lines, the Mut-F with DMs containing two unmethylated CpG islands that are easily cleaved by methylation-sensitive restriction endonucleases, and the MutC line in which the CpG islands are only partially methylated. They found that the DMs with unmethylated CpG islands underwent rapid dimerisation and deletions, in contrast with the DMs containing partially methylated CpG islands. Rizwana et al. (1999) have therefore suggested that methylation is an important factor in controlling mitotic recombination between large molecular DNA.

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Methylation-associated chromosomal recombination and genetic transcription in childhood leukaemias Several genetic rearrangements are encountered in both myeloid and lymphoid leukaemia. Among the notable ones are the fusion genes ETV6–ML1, bcr-abl, rearrangements of myc, immunoglobulin, and T-cell receptor genes in childhood leukaemia. The chromosome locus 11q23 is a major site of chromosomal translocation in acute leukaemia. A gene called MLL occurring at this locus has been implicated. The breakpoint for translocation occurs within MLL and the truncated gene then forms a fusion gene with a gene in the reciprocal translocation partner. The chimeric MLL can lead to tumour formation in chimeric mice, and indeed the truncated gene product successfully contributes to tumorigenesis, irrespective of the fusion partner (Dobson et al., 2000). Wiedemann et al. (1999) have suggested intragenic duplication as an alternative mechanism of MLL rearrangement involving the 5’ end of the gene. MLL rearrangement occurs in a high proportion of childhood leukaemia, and LOH involving 11q23 occurs in adult leukaemia. This locus appears to be a hot spot of genetic instability (Webb et al., 1999), probably closely linked with the genetic rearrangement consistently seen in leukaemia. Chromosomal deletions, e.g. involving the genes p15 and p16 at 9q21, occur frequently in T-cell ALL. IHN Wong et al. (2000) showed that the promoters of both genes are hypermethylated, and p15 methylation seems especially to correlate with chromosomal translocations, inversions or deletions, and they have therefore implicated p15 abnormalities in this. MLL rearrangement also correlates with homozygous deletions of p15 and p16 genes in ALL and AML (Ohnishi et al., 1997). As we shall see presently, methylation-mediated pathway of regulation of chimeric MLL is closely knitted with regulation of gene expression via the pathway of histone acetylation dynamics (Figure 11).

The dynamics of histone acetylation in cell differentiation, cell proliferation and neoplasia Two distinct modes can be identified by which histone acetylation dynamics control cellular function. One of these is regulation of genetic transcription via chromatin remodelling. A second suggested mode is that the proteins involved in histone acetylation and deacetylation might possess a scaffold function to establish transcription complexes at transcription foci (Chan and La Thangue, 2001). The regulation of genetic transcription by histone acetylation dynamics has been established in the context of development and differentiation and cell proliferation, with obvious implications also for the development and progression of neoplasia.

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Figure 11 Methylation-mediated pathway of genetic regulation involving MLL chimeric protein of leukaemia. This Figure focuses on the regulation of p53-mediated transcription by the agency of HAT and HDAC. HAT has also been implicated in the turnover of p53 itself by interaction with mdm2, thus balancing p53 activation and its degradation by ubiquitination (see also Figures 5 and 6). The oncogenic proteins E1A, HPV-E6 might further participate by promoting p53 degradation. The Figure also brings into the fold the role of DNMT1 in effecting genetic repression directly by DNA methylation or indirectly through the involvement of HDAC. The MLL gene is a focus of instability and it is rearranged in adult leukaemias. DNMT1 has a transcription repression domain, which functions by recruiting histone acetyltransferase. The latter bears homology with the repressor domain of MLL protein. Furthermore, EP300 is a chimeric partner of the truncated MLL protein generated in leukaemia from a reciprocal translocation.

Histone acetylation dynamics in development and differentiation A pathway of regulation of genetic expression by DNA methylation that also implicates histone acetyltransferases (HAT) and histone deacetylases (HDAC) is currently being unravelled. This epigenetic mechanism of gene silencing functions involves a co-ordinated functioning of both DNMT-1 and HDACs. The modification of nucleosomal histone by acetylation or deacetylation has marked effects on cell proliferation, regulation of the cell cycle, embryonic development and differentiation as well as in neoplasia. A group of histone acetyltransferases have been identified, and this includes p300, CBP, Pcaf, GNAT, MYST and Gen5 (of murine origin). These actively participate in

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developmental processes and differentiation (WT Xu et al., 2000). Mutation of HAT genes results in developmental malformation. An insertion mutation in a MYST family HAT has been reported to produce craniofacial abnormalities, possibly related to cortical cell differentiation (Thomas et al., 2000). Both EP300 and PCAF seem to be essential for muscle cell differentiation. Anti-PCAF antibodies inhibit the process. Furthermore, exogenous EP300/PCAF induce p21waf1/cip1 expression (Puri, Avantaggiata et al., 1997). The myogenic protein MyoD is a regulatory protein capable of activating muscle specific gene transcription. It also induces cell cycle arrest by activating Rb, cyclin D3 and p21waf1/cip1 (Puri, Sartorelli et al., 1997; Cenciarelli et al., 1999). MyoD activity is dependent upon its association with CBP, i.e. it clearly requires acetylation by CBP or PCAF (Polesskaya et al., 2000, 2001). Acetylation occurs of conserved lysine residues and seems to result in increased affinity for DNA binding and initiate transcription (Sartorelli et al., 1999). Neuronal differentiation induced by NGF also seems to involve HAT-mediated activation of p21waf1/cip1 promoter (Billon et al., 1999). Similarly RA-induced upregulation of p21waf1/cip1 also takes recourse to HAT (Kawasaki et al., 1998). Finally, the control by p300 of the transactivation of androgen receptors (AR) by dihydrotestosterone could be cited as a further line of evidence of the involvement of acetylation dynamics in the process. Fu et al. (2001) found not only p300-mediated acetylation in dihydrotestosterone dependent transactivation of AR but they also showed that mutation of C-terminal lysine-rich acetylation sites of AR led to the abrogation of activation of AR by the steroid hormone The role played by HAT is further elucidated using VD3 and RA mediated differentiation of leukaemia cells. VD3 is a powerful modulator of cell differentiation, apoptosis, and cell proliferation. VD3 induces its receptor VDR to form a heterodimeric complex with retinoic acid receptor RXR. The formation of the heterodimer appears to enhance the binding affinity as well as specificity of VDR to the VD3 response element. Thus VDR–RXR complex functions as a transcription factor, leading to the transcription of VD3 target genes associated with induction of differentiation and inhibition of cell proliferation. Niitsu et al. (2001) established a cell line, SN-1, from a leukaemia patient carrying the t(11;16)(q23;p13) translocation. This cell line expressed a fusion gene MLL with CBP. SN-1 cells were not amenable to induction of differentiation by VD3 or all-trans RA. But the cells showed differentiation when treated with RXR agonists together with downregulation of expression of the fusion gene. Furthermore, when the cells were treated with MLL-CBP antisense oligonucleotides they responded to VD3 and RA by undergoing differentiation. This clearly shows that MLL-CBP expression inhibited differentiation. Niitsu et al. (2001) also observed that sodium butyrate which inhibits histone deacetylase worked in synergy with RA and VD3 in inducing differentiation. Although the effect of sodium butyrate suggests the involvement of histone acetylation in differentiation, for which there is much documented evidence, this cannot be distinguished from the ability of VDRE to activate and

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upregulate the cdk inhibitors p21waf1/cip1 and p27kip1 and in this way inhibit cell proliferation. It can be argued, however, that induction of differentiation is intricately and closely linked with regulation of cell proliferation, the processes being mutually exclusive. VD3 induces HL-60 cells to differentiate into monocytes. In parallel with this, the expression of stathmin is downregulated (Eustace et al., 1995). We know that stathmin is involved with other cell cycle regulatory factors such as p53 and S100A4 at the G2 –M transition checkpoint of the cell cycle (Sherbet and Lakshmi, 1997; Cajone and Sherbet, 1999).

Cell proliferation, apoptosis and histone acetylation dynamics The participation of histone acetylation dynamics in cell cycle regulation is suggested by several strains of evidence. The progression of the cell cycle is subject to control by p53, which exerts controlling influence at G1 –S as well as G2 –M checkpoints. The activation of p53 and its turnover are therefore important components of the cell cycle regulatory mechanism. Histone acetylation dynamics is involved in both these components. P300 is not only a co-activator of p53 but it is also involved in the ubiquitination-mediated degradation of the suppressor protein. The protein called mdm2 binds to p53 and inhibits its activity as a transcription factor and also promotes its degradation by ubiquitination. P300 seems to binds to mdm2 and inhibit p53 activation as well as it degradation (Zhu et al., 2001). The HPV16-E6 and -E7 genes both cause immortalisation of cells upon transfection. The introduction of a single HPV16-E6 gene not only immortalises cells but also sharply reduces expression of p53 protein. The E7 oncoprotein binds to the retinoblastoma protein Rb and such binding is necessary for E7 to immortalise and transform cells (Dyson et al., 1987; Munger et al., 1989; Gage et al., 1990). The suppression of E6 and E7 expression by means of sodium arsenite has been reported to restore the normal cell cycle regulatory function of p53 (Chou and Huang, 2002). In contrast, the HPV-E7 stabilises p53, but it degrades Rb (Scheffner et al., 1991, 1992), which is also closely involved in the regulation of cell cycle and cell proliferation. The E6 oncoprotein seems to be able to target ubiquitin-protein ligase for p53 degradation. Therefore, the concept has emerged that formation of complexes with oncoproteins E6 and E7 with p53 and Rb suppressor proteins might influence cell proliferation and lead to the development of neoplasia. HPV16-E6 binds to EP300 and CBP (Patel et al., 1999) and facilitates its degradation (Zimmermann et al., 1999; Zhu et al., 2001), the latter affording another mode of controlling cell proliferation. An important finding with respect to the linking up of HAT activity with neoplasia and any other phenotypic properties associated with neoplasia is that E6 derived from the low risk HPV-6 showed no interaction or binding with HAT. The adenoviral E1A is known to be able to inhibit growth and induce apoptosis. E1A also

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suppresses cell transformation by erbB2 and other oncogenes, besides being able to suppress invasive and lung colonisation ability of erbB2 transfectant cells. It now appears that these properties could be mediated by HAT. The N-terminal end of E1A binds to EP300 and PCAF. The modus operandi seems to be the regulation of genetic transcription by means of remodelling chromatin. The HAT activity of EP300 is essential for bringing about the phenotypic effects of altered cell proliferation, apoptosis and differentiation status. For instance, Sang et al. (2001) have demonstrated that RACK1, which inhibits HAT activity can also inhibit E1A-induced apoptosis as well as negate the growth inhibitory effect of E1A. Mutations in EP300 domain required for HAT activity affect the activation of p21waf1/cip1 (Ohshima et al., 2001) cyclin-dependent kinase inhibitor that is regulated by p53. Now, Rb, the second tumour suppressor and cell cycle regulatory protein, is in turn closely related to the tumour suppressor and cell cycle regulatory protein p53. Cyclin D1 controls the phosphorylation of Rb and thereby exerts its effects on cell proliferation and differentiation. Deacetylation, on the other hand, is mediated by a group of enzymes known as histone deacetylases (HDAC). This involves the deacetylation of all four forms of histone. It is well known that p53 produces cell cycle arrest and induces cell apoptosis in response to stress stimuli. The acetylation of p53 by EP300 modulates p53 binding to DNA and stimulates the transcription function of p53. In contrast, deacetylation results in the inhibition of p53 function. Juan et al. (2000) have shown that downregulation of p53 by HDAC1 involves the same region of the molecule that is acetylated by EP300. Furthermore, as we noted already, the HAT activity of EP300 might be required for the activation of p21waf1/cip. Also, inhibition of HDAC1 leads to an increase in p21waf1/cip1 expression together with inhibition of cell proliferation and attendant hyperacetylation of histone H4 (Han et al., 2000). These findings firmly establish that p53 function is regulated by acetylation and deacetylation mechanisms. A further aspect of p300 could be the facilitation of degradation of p53 via the mdm2-mediated ubiquitination pathway (Zhu et al., 2001). HDACs also seem to operate through the cell cycle negative regulator protein Rb (Magnaghi-Jaulin et al., 1998a, b; Stiegler et al., 1998). Among other potential cellular proteins with which HDACs might interact is topoisomerase II, which is known to participate in chromatin reorganisation and regulation of transcription (Tsai et al., 2000) (Figure 12).

Histone acetylation dynamics and the neoplastic process It is increasingly being recognised that histone acetylation and deacetylation are closely associated with the neoplastic process. An essential element of tumour development and probably also secondary spread is tumour growth and expansion consequent upon deregulation of cell cycle progression. As noted above, cell cycle progression and cell proliferation might be subject to

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Figure 12 Histone acetyltransferase in the regulation of cell proliferation and tumour suppression. The Figure illustrates how histone acetyltransferase might be involved in the regulation of cell proliferation via p53 acetylation and activation leading to the activation of the cdk inhibitors p21 and p27 and in this way suppressing tumour growth. On the other hand, the oncogenic proteins HPV-E6 and E1A bind to HAT and can potentially reduce p53 acetylation and activation with corresponding effects on cdk activation and cell cycle progression. Another way in which HAT could conceivably affect tumour suppression is indicated by the finding that BRCA1 tumour suppressor works with p300, possibly also by the cdk route. The figure also shows that HAT could participate in ECM remodelling, thereby enhancing the adhesive properties of cells. Such a remodelling of ECM in cancer cells could result in altered cellular migration and invasion.

regulation by the acetylation status of histones. Indeed, acetyl transferases might be subject to regulation as a prelude to the development of neoplasms to initiate the expression of tumour promoter genes or to inactivation of tumour suppressor genes. Loss of heterozygosity at the p300/CBP locus is associated with some neoplasms, and it has therefore been suggested that CBP might function as a tumour suppressor (Kung et al., 2000). However, the lack of such an association specifically in respect of EP300 might indicate the possibility that the CBP locus might be harbouring genes that do function as tumour suppressors. Nonetheless, one might argue cogently enough that the hitherto empirical association of histone acetyltransferases with haematopoietic malignancies and with developmental abnormalities might now indeed be regarded as a fait accompli. HDAC1 interacts with the metastasis-related proteins MTA1, which is expressed at far higher levels in metastasising tumour than in those with low potential for metastasis (Toh et al., 1994, 1995, 2000). This MTA-HDAC1 complex might also involve p53, as shown in the context of MTA2 (JY Luo et

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al., 2000), and the suppression of transcription of p53-dependent activation of genes concerned with the metastatic cascade of tumour dissemination. Two suppressor genes called BRCA1 and BRCA2 are regarded as tumour suppressors, and abnormalities in these genes are associated with susceptibility to the development of breast and ovarian cancers. The BRCA2 protein shows histone acetyl transferase activity and might therefore function as a transcription regulator (Siddique et al., 1998). The tumour suppressor BRCA1 gene product also interacts via a C-terminal domain with HDAC1 and HDAC2, as well as with Rb and Rb-binding proteins (Yarden and Brody, 1999). GM Pao et al. (2000) reported that BRCA1 transactivates the LTR promoter of RSV and that p300 showed a synergistic enhancement of activation. On the other hand, BRCA1 can also downregulate p300 (Fan et al., 2001). Although these findings appear contradictory, it is possible that BRCA1 interaction with p300 might be functionally self-regulatory. Taken together, this body of evidence is sufficiently persuasive to suggest the involvement of the histone deacetylase complex in the regulation of tumour suppressor function of the BRCA1 protein in breast and ovarian cancer. As noted earlier, HAT proteins control cell differentiation. Therefore deregulation of cell differentiation by regulating the functions of acetyltransferases themselves can itself be viewed as a possible factor in tumorigenesis. MyoD is a protein that is required in muscle cell differentiation. Acetylation of MyoD increases its affinity for DNA and enables it to induce the transcription of genes for muscle cell differentiation. P300 is involved in this acetylation process (Sartorelli et al., 1999). The Tax protein of human T-cell leukaemia/lymphoma virus type 1 (HTLV-1) is known to interact with p300/CBP and repress MyoD function. An N-terminal domain of p300 appears to serve as Tax binding site, and its over-expression by transfection abrogates the inhibition of MyoD function by Tax (Riou et al., 2000). Also relevant in this context is the demonstration that the basic helix-loop-helix protein called Twist binds to two distinct domains of p300 and PCAF and inhibits HAT activity. Furthermore, the adenoviral E1A protein also inhibits HAT activity (Hamamori et al., 1999). The remodelling of the ECM figures prominently in cell motility and in the invasive and metastatic behaviour cancers. Several constituents of ECM determine the character and functioning of the ECM as a modulator of cell behaviour. Among ECM constituents whose expression is linked with HAT activity is laminin-5. This assumes much significance when considered in the light of the link-up between laminin expression and invasive ability of certain tumours. Carcinomas of the prostate, but not breast or the colon, persistently show a loss of laminin-5 as well as its integrin receptor (Davis et al., 2001). The expression of ␥-2 is said to correlate positively with invasion as well as nodal dissemination and metastatic spread of oesophageal carcinoma (Yamamoto et al., 2001). According to Hao et al. (2001), invasive prostate carcinomas show a loss of ␥-2 and ␤-3 expression. Loss of ␥-2 chain seems to correlate with invasive

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ability of cervical neoplasms (Skyldberg et al., 1999). Similarly, a downregulation of ␥-2 together with ␤-3 and ␣-3 subunits has been encountered in breast cancer cells (Martin et al., 1998). In the light of the possible participation of laminin-5 in cell adhesion and the correlation between its loss from the ECM and the gain of invasive ability in many, but not in all, human tumours, it is of considerable interest to note that p300 can influence the constitution of the ECM. Miller et al. (2000) generated MCF-10A derivative cell lines that overexpressed p300. These cells displayed reduced adhesion to substratum and this correlated with reduced levels of laminin-5 in the ECM and reduced expression of two genes that code for the ␣-3 and ␥-2 chains forming the laminin heterodimers. The mode of involvement of HAT in the loss of laminin-5 subunits is not clearly understood, except that laminin-5 might be a substrate for metalloproteinases associated with the cell membrane, and they could degrade ␥-2 and in this way impinge upon the motile behaviour of cells (Gilles et al., 2001). In other words, HAT might be involved indirectly with the expression of laminin by means of activation of other membrane-associated components.

Synergistic effects of DNA methylation and histone acetylation dynamics on epigenetic gene silencing DNA methylation plays a key role in silencing genes. Methylation seems to modulate chromatin structure to make it inaccessible to the transcription machinery. It now appears that histone acetylation dynamics are closely associated with DNA methylation in epigenetic silencing. The DNA methyltransferase DNMT1 methylates cytosine residues and is also capable of repressing genetic transcription. DNA methylation functions through the agency of the methyl-CpG-binding proteins (MECP). Several MECPs are known, e.g. MECP2, MBD2 and MBD3, and all of them interact with histone acetyltransferase. MECP2 interacts with methylated DNA. Mutations of MECP2 greatly reduce this interaction and this impairment of interaction leads to the genesis of the Rett syndrome, thus underscoring the significance of MECP2 in gene silencing. It has been shown, for instance, that DNA methyltransferase (DNMT1) not only methylates CpG islands but can also interact with HDAC (Robertson et al., 2000; Rountree et al., 2000). Furthermore, the ability of DNMT1 to repress genetic transcription is dependent on HDAC activity, and the inhibition of DNMT1 and HDAC functions synergistically in initiating re-expression of a number of genes silenced by methylation (Cameron et al., 1999). The need for HDAC is suggested by the fact that methyl CpG-binding proteins recruit HDACs and the deacetylases remove acetyl groups from lysine residues of the core histones H3 and H4. Thus HDAC promotes enhanced ionic interactions between DNA and the positively charged lysine residues of histones resulting in a compact nucleosomal structure that is not conducive to genetic transcription.

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A link between DNA methylation, histone acetylation and transcription is firmly forged by the demonstration that DNMT1 forms a complex with Rb, HDAC1, and the transcription factors E2F and DMAP1, with inhibition of transcription from gene promoters that are responsive to the transcription factor (Robertson et al., 2000; Rountree et al., 2000). The formation of DNMT1/DMAP1 complex appears to enable targeting of the transcription factor to replicating foci throughout the S-phase. Interestingly, the DMAP1/HDAC complex occurs in the late S-phase (Rountree et al., 2000). The recruitment of HDAC at this stage of the cell cycle clearly relates to chromatin condensation and the repression of genetic transcription. These findings indicate the important role that DMAP1 discharges also in chromatin remodelling. It is interesting to note that DNMT1 has a transcription repression domain, which functions by recruiting HDAC1. This repressor domain bears homology with the repressor domain of MLL protein (Fuks et al., 2000). MLL forms chimeric proteins in which p300/CBP (CREB binding protein), TIF2 and the MOZ histone acetyl transferase are frequently the fusion partners. EP300 is a chimeric partner of the truncated MLL protein generated in leukaemia as a result of reciprocal translocation. The abnormal acetylation function engendered by the formation of fusion protein has been suggested to result in the pathogenesis of leukaemia (Lavau et al., 2000; Champagne et al., 2001). EP300 is altered in colorectal and pancreatic tumours, and also in cell lines derived from these tumours as well as from breast cancer. Some of the mutations generate a truncated protein in a proportion of epithelial cancers (Gayther, 2000). Furthermore, Gayther et al. (2000) note that 5 out of 6 cases with truncating mutations also showed inactivation of the second allele. These findings have prompted the postulate that EP300 might function as a tumour suppressor gene.

HDAC inhibitors in the treatment of cancer The important role that histone acetyltransferase and histone deacetylases play in controlling the expression of cellular regulatory proteins has been heavily underscored by their recognition as potential targets for cancer therapy and by attempts to develop new strategies for cancer treatment based on the employment of HDAC inhibitors. HDAC inhibitors induce transactivation of certain genes, repress the expression of others, and overall suppress and arrest tumour cell proliferation, and induce differentiation and apoptosis. This has made them prime candidates for cancer treatment. The HDAC inhibitor FK228, which is derived from Chromobacteriaum violaceum, has received much attention recently. Murata et al. (2000) showed that FK228 induces apoptosis of human leukaemia and lymphoma cells. FK228 seems to be able to inhibit neovascularisation of the tumour, by inhibiting

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endothelial cell proliferation, invasion and migration. Apparently, it suppresses the expression of angiogenic agents such as VEGF and enhances the expression of anti-angiogenic factors like the von Hippel–Lindau factor and neurofibromin2 (Kwon et al., 2002). These anti-angiogenic effects might be partly mediated by the inhibition of nitric oxide synthase (NOS) by the HDAC inhibitors. For the latter are known to inhibit cytokine-mediated induction of NOS (ZT Yu et al., 2002). FK228 is currently in a phase I clinical trial. A number of other inhibitors obtained from biological sources are also being tested at present. Among synthetic inhibitors that are actively being investigated are sulfonamide anilides. Fournel et al. (2002) have shown that MS-275 is able to selectively inhibit the proliferation of tumour cells and markedly inhibit tumour growth. Thus MS-275 might turn out to be a promising and effective anticancer agent. As noted in an earlier section and summarised in Figure 11, several genes that function as transcription activators or repressors are involved in consistent chromosomal translocations in haematological malignancies. We have also noted the ability of these factors to interact with HAT and HDACs. The translocations often involve the formation of chimeric transcription activators or repressors and thus render themselves as targets for inhibition of HDACs. Thus this opens another avenue of approach for selective and effective use of HDAC inhibitors as anticancer agents.

The effects of p53 methylation on chromosome stability There is sustained argument whether genetic instability is the cause or the result of mutations of a single or a cohort of genes. This has in the main taken the form, first, of whether mutations in the cell cycle control gene p53 influences DNA stability, and secondly how DNA methylation corresponds with stability of DNA. Most vexatious is the question of sequence of some of the events involved. We have noted earlier that genetic instability shows a reasonable degree of correlation with p53 abnormalities. Mutation of p53 occurs in a vast majority of human cancers and therefore alternative explanations may be sought for the preponderant occurrence of abnormalities of this gene locus. An observation that might help here is the apparent correlation between deregulation of p53 and DNA methylation. Many human tumours show p53 mutations and aberrant patterns of localised methylation, and it has been recognised that mutations of p53 and of other genes occur mainly in methylated CpG islands within the gene (Guinn and Mills, 1997; Pfeifer, 2000). The CpG islands of p53 are not only targets of methylation but the cells with aberrant p53 also continue to accumulate further genetic abnormalities and methylation (Guinn and Mills, 1997). Makos et al. (1993) had demon-

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strated a link between the deletion of chromosome 17p, which occurs frequently in primary brain tumours, and hypermethylation at a specific locus on that arm of this chromosome. The presence of intragenic deletions in p53 in NSCLC seems to correlate strongly with microsatellite instability of the tumours (JW Chang et al., 2000). Salvesen et al. (2000) found that hypermethylation of hMLH1 in endometrial tumours not only correlated with microsatellite instability but also with loss of over-expression of p53. Kondo et al. (2000) found DNA hypermethylation in both non-cancerous liver tissue and hepatocellular carcinomatous tissue. Both normal and control tissues showed microsatellite instability in roughly similar proportions. However, there was a marked difference in LOH, with LOH in 38% of 40 normal liver specimens as compared with 100% of 40 carcinoma specimens. Kanai et al. (2000) have shown that hypermethylation of DNA is seen only in normal liver tissue from patients with hepatocellular carcinoma (HCC) also with hypermethylated DNA. This suggests the possibility that hypermethylation might be a cause of pathogenesis of the carcinoma. They then read this with the finding that LOH near the E-cadherin locus occurs with high frequency in noncancerous liver tissue from HCC patients only when LOH also occurred in the carcinomatous tissues. Kanai et al. (2000) therefore go on to suggest that hypermethylation not only precedes but might indeed be the cause of HCC pathogenesis. Finally, there is some direct evidence that methylation could affect DNA stability. Duthie et al. (2000) isolated colonic cells from folate and methyl donor deficient rats. They found marked effects on DNA stability in these cells. Murnane (1996) argued that the prolonged genetic instability occurring as a consequence of exposure to ionising radiation or to alkylating agents is not a result of residual DNA damage or mutations alone, but that the involvement of epigenetic changes is a strong possibility. Indeed, one can safely conclude putatively that methylation status of the gene is responsible for the chromosomal instability and associated abnormalities. Warnecke and Bestor (2000) have turned the question around and ask whether methylation of DNA is a cause or a consequence of transformation and clonal selection. The question hardly seems tenable, for the neoplastic process is driven da capo el fine by abnormal genetic expression in a temporal and spatial manner. It is abnormal genetic expression that drives genetic diversity within the tumour and gives selective clonogenic and growth advantage to the cell types that have evolved within the developing tumour. The weight of evidence seems to suggest that epigenetic changes of DNA and mismatch repair deficiency are the major causes of genetic instability. This then leads to aberrant recombination events and onward to genetic aberrations, such as loss of heterozygosity, point mutations or frame-shift mutations, and abnormal gene expression. These abnormalities and inappropriate genetic expression could result in selective expansion of clones that possess certain phenotypic behavioural properties. The genetic changes are then reflected at the phenotypic level (Figure 13).

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Figure 13 A postulated sequence of events associated genetic instability as the focus in neoplasia and genetic syndromes with associated DNA instability. The Figure depicts pathways by which DNA methylation might instigate genetic instability and recombination resulting in chromosomal aberrations, gene amplification and mutation, which in their turn produce phenotypic alterations in cells, initiate neoplasms and their progression. DNA methylation is also shown as bearing directly on deregulation of cell proliferation and the development and progression of neoplasms by silencing tumour suppressor genes, many of them also intricately involved in the control of cell cycle progression.

DNA methylation in fragile X syndrome FMR1 gene function in the pathogenesis of fragile X syndrome The fragile X syndrome is a disorder resulting from an expansion of 5’-CGG (n) –3’ nucleotide triplet in the promoter region and 5’-untranslated region of the fragile X mental retardation (FMR) gene, FMR1, which occurs on chromosome Xq27.3. The expansion can be n = >200, which is regarded as the full mutation range with clinical manifestation of the syndrome, or can be n = 55–<200 in the premutation allele. In normal subjects CGG repeats may vary between n = 6–54. Mental retardation has been encountered in virtually all males and nearly two-thirds of females carrying the full mutation.

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The pathogenesis of fragile X syndrome might be due to the operation of one or more mechanisms. A feature of nucleotide repeat motifs is that they confer structural instability on chromosomes, and they become prone to breakage in the vicinity of the repeats, with consequent genetic rearrangement and abnormalities in FMR1 gene. The FMR1 protein is known to shuttle between the nucleus and the cytoplasm. Bardoni et al. (1999) have demonstrated that FMR1 interacts with and co-localises with another protein, which is a zinc finger protein and which possesses a nuclear localisation signal. Furthermore, FMR1 is associated with polysomes and the endoplasmic reticulum. It is therefore regarded as taking part in the process of translation of mRNA and synthesis of proteins. Although full mutation and premutation levels are generally shown to be related to the clinical manifestation, often the premutation range is not associated with FMR1 abnormalities, but carriers nevertheless show clinical signs of the syndrome. This seems to be related to translational efficiency of the FMR1 protein (Tassone et al., 2000).

Epigenetic silencing of FMR1 in fragile X syndrome The clinical phenotype is believed to arise from hypermethylation of the repeats and nearby upstream elements and consequent suppression of transcription. There is much variability in the degree of methylation, but FMR1 promoter is sensitive to methylation (Genc et al., 2000). Nevertheless, there is a consensus that hypermethylation of the gene is responsible for the loss of FMR1 protein associated with the syndrome. The gene is reactivated in FMR1 full mutation range by demethylation using 5-azadeoxycytidine (Chiurazzi et al., 1998, 1999). There is an indirect demonstration of the importance of methylation on the expression of FMR1. Interleukin (IL)-1␤ suppresses FMR1 expression via the methylation pathway. IL-1 seems to induce nitric oxide (NO) synthesis. This activates DNMT1 and this leads to CpG methylation, as indicated by the reversal of NO-induced methylation and suppression of FMR1 expression by NOS inhibitors (Hmadcha et al., 1999). As discussed previously, MECP2 binds to HDACs and represses gene transcription. MECP2 in fact recruits HDACs to methylated DNA and produces histone deacetylation, as well as chromatin condensation and genetic repression. Besides methylation, histone acetylation also seems to be involved in the regulation of FMR1 expression. Histone acetyl transferase, in contrast to HDAC, enhances gene transcription. This is indicated by the reactivation of the gene in lymphoblastoid cell lines derived from full CGG mutation patients. Histone acetylation seems to function synergistically with DNA demethylation (Chiurazzi et al., 1999). But, there can be striking discrepancies between degree of demethylation and the perceived FMR1

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reactivation (Pietrobono et al., 2002). This could partly explain why there are reports that methylation is not invariable in its effects on FMR1 expression and for the tissue-specific differences and mosaicism in methylation (Stoger et al., 1997; Tussone et al., 1999). But, Coffee et al. (1999) found that FMR1 is associated with H3 and H4 histones of cells from normal subjects but not from fragile X patients.

8 Telomeric DNA and genetic instability Telomeres and cell proliferation, apoptosis and cell senescence Telomeres are nucleoprotein complexes composed of repeat elements that occur at the termini of eukaryotic chromosomes. They maintain the functional and structural integrity of chromosomes. The telomeres cap the ends of the chromosome, and prevent its fragmentation, damage and rearrangement. Although these terminal repeat elements stabilise the chromosome, in interstitial locations they appear to promote destabilisation and rearrangement of the chromosome (Day et al., 1998). The insertion of telomeric repeats into genes can affect expression of the target gene and also induced dramatic genetic rearrangement. The telomeric complexes comprise tandem hexanucleotide repeats. These are highly conserved repeats of 5’-TTAGGG-3’ nucleotide sequences (Moyzis et al., 1988; Meyne et al., 1989). This sequence has been recorded in many primitive plant organisms from angiosperms, gymnosperms and bryophytes (Fuchs et al., 1995) to the animal kingdom, including vertebrates. The telomere repeats are eroded at the cell division and the average length of repeats diminishes by 50–200 repeats. Such a diminution of telomere length is believed to lead to chromosomal instability and to illegitimate chromosomal recombination and degradation (Counter, Kurte et al., 1994). These abnormalities may be visualised cytogenetically as dicentric, multi-centric or ring Genetic Recombination in Cancer ISBN 0-12-639881-X

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chromosomes. The termini of chromosomes and the integrity of telomeres have actively to be guarded and stringently protected in normal cell physiology so that aberrant outcome is obviated. A number of proteins and complexes of proteins seem to be involved or associated with this process of regulation of telomere length and integrity. Among these are the telomere-associated proteins, which include the so-called telomere repeat binding factors, TRF1 and 2, which have been shown to be essential for the maintenance of chromosomal integrity. TRF1 is a negative regulator of telomere elongation (Bianchi et al., 1997; Smith and de Lange, 1997; Van Steensel and de Lange, 1997); TRF2 is involved with the protection of chromosome ends and maintains telomere integrity by a different mode of function (Bilaud et al., 1997; Broccoli et al., 1997). Another factor called MRE11 of yeast and its human homologue, in the form of a complex with RAD50, and the NBS and Xrs2 proteins also regulate telomere length (Ritchie and Petes, 2000; G Wu et al., 2000). Functionally, their association with telomeric DNA results in the regulation of the cell cycle (Zhu et al., 2000). Telomeric association (TAS) of chromosomes is said to result from the absence of TRF2 (Kierszenbaum, 2000). Cell senescence is dependent upon two pathways, namely the cell cycle regulatory pathway with the mediation of p53 and Rb repressor proteins, and a second pathway related to telomeric integrity. When p53/Rb mechanism is activated cells exit the cycle, but their inactivation leads to immortalisation of cells. In contrast, when telomeric integrity is maintained the cells continue to replicate, but when it is lost the cells enter a phase of senescence. Telomere repeats have in fact been shown to influence genetic stability via the activation of the tumour suppressor p53. AT fibroblasts show enhanced p53 activity in parallel with accelerated telomere loss and premature senescence (Vaziri et al., 1997). The introduction of extraneous telomere repeats is said to lead to stabilisation of p53, an increase in p53 transcription and to growth suppression (Milyavsky et al., 2001). The reduction in the replicative lifespan of cells observed in vitro and in vivo is associated with telomere diminution (Harley et al., 1990; Hastie et al., 1990). The continuous cultivation of cells in culture, interspersed with periods of in vivo growth, leads to the formation of dicentric and multi-centric chromosomes with associated senescence and cell death (Gagos et al., 1996). The cutaneous melanoma of Sinclair swine continuously transplanted in vivo does not show TAS in the early passages, but do develop TAS extensively in late passages (Pathak et al., 2000). Clearly, the formation of TAS seems to be related to senescence of cells. The loss of telomeric DNA has been postulated to be the reason for senescence of cells in respect of replication (Harley et al., 1990; Lansdorp, 2000), although senescence might not be triggered by a minimum repeat length. The transfection of HeLa cells with antisense constructs of human telomerase RNA induces the loss of telomeric DNA and early loss of proliferative capacity (Feng et al., 1995). The restoration of telomere length, on the other hand, seems to renew the lifespan of cells (Wright et al., 1996) (Figure 14).

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Figure 14 Telomere integrity in cell proliferation, immortalisation, senescence and apoptosis. This Figure summarises importance of telomere integrity in cell function and the role telomeric DNA plays in directing the cell into senescence, or into the pathways of apoptosis, proliferation, immortalisation and cancer. Although some regard these pathways to function independently of one another, here some emphasis is placed on the possibility of how these pathways could be interlinked. There are also indications that telomere position effect mediated via histone modifications and alterations of chromatin structure produced by histone deacetylase and histone acetyl transferase, might also be involved in the telomere associated effects of cell proliferation and senescence.

Telomeric diminution results from replication inefficiency, often referred to as the end replication problem. An enzyme called the telomerase, which replaces the lost DNA sequences, reinstates the integrity of the telomere. Telomerase is a ribonucleoprotein with a component telomerase RNA encoded by the so-called TLC1 gene and a reverse transcriptase protein, the latter being the component that regulates telomerase activity. Mutations of TLC1 lead to telomere shortening and loss of cell viability. Experimentally induced expression of the reverse transcriptase component leads to telomere elongation and immortalisation of cells that otherwise would have only a limited lifespan (Cerni et al., 2000). The protein component of telomerase of Euplotes aediculatus and Saccharomyces cerevisiae contains reverse transcriptase motifs. The EST2 protein of yeast loses its ability to catalyse the elongation of the telomere upon mutation of the reverse transcriptase motifs (Lingner et al., 1997). At least four EST proteins have been identified to date, namely EST1, EST2, EST3 and CDC13 (Hughes et al., 1998). Another protein SGS1 has been implicated in telomere elongation. SGS1 is the homologue of

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the human Werner’s syndrome WRN and Bloom’s syndrome BLM proteins belonging to the RecQ DNA helicase family. SGS1 maintains genomic stability in S. cerevisiae (Watt et al., 1996). Werner and Bloom’s syndromes arise from defects in the WRN and BLM proteins. Cell lines derived from WRN and BLM patients show marked chromosomal instability (Watt et al., 1996). In the absence of SGS1, S. cerevisiae survival takes recourse to specific RAD proteins (PH Huang et al., 2001). The EST proteins bind single-stranded telomeric DNA. Chandra et al. (2001) have suggested a dual role for these proteins. CDC13, for instance, recruits telomerase to the replicating site, and secondly, when the C-strand is replicated, suppresses the overhang G-strand synthesis. Telomerase is known to shield cells from undergoing apoptosis and ensure continued proliferation. This is compatible with its identification with the immortalisation of cells (Counter et al., 1992; Counter, Botelho et al., 1994), and its presence in human neoplasms (Counter, Kurte et al., 1994; Nilsson et al., 1994; Hiyama, Hiyama et al., 1995; Hiyama, Yokoyama et al., 1995; Hiyama et al., 1996). The expression of the enzyme is approximately 5-fold greater in Fanconi’s anaemia than in corresponding controls subjects (Leteurtre et al., 1999). Telomerase is expressed in a cell cycle-dependent fashion. Also, its expression inhibited when cells are induced to differentiate (Sharma et al., 1995; Zhang et al., 1996; Xu et al., 1996). Furthermore, cell cycle regulator p53 protein might be involved in telomerase-mediated effects on cell proliferation and apoptosis. On the other hand, Mukhopadhyay et al. (1998) have observed that p53 can induce a diminution of telomeric repeats, which in turn leads to the formation of dicentric and multicentric chromosomes by a process of telomeric association (TAS). Pathak et al. (1994) had predicted some years ago that these chromosomal changes portend the onset of apoptosis. These abnormal chromosomes do indeed undergo DNA fragmentation and apoptosis (Mukhopadhyay et al., 1998; Pathak et al., 2000). The immortalisation concept has been extended to cancer progression with the demonstration of an association of telomerase activity with cancer invasion and metastasis (Sherbet and Lakshmi, 1997). Equally, progression of cancer seems to be accompanied by certain chromosomal abnormalities that arise from telomeric association, apparently instigated by the loss of telomeric repeats. These results seemingly take contradictory positions. If one were to consider the mechanism by which the telomere is replicated, these apparent contradictions can be reconciled. Telomere replication and repeat length extension is initiated by the recognition by telomerase of an overhang of G-rich strand. Telomerase extends this G-rich repeat by a non-template-based synthesis. A complementary C-rich strand is now synthesised by DNA polymerase-primase, which uses the extended G-rich strand as template (Shampay et al., 1984). The DNA polymerase ␣-primase is able to expand telomere repeats in vitro (Nozawa et al., 2000) and possibly other DNA polymerases can subserve the same function.

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Telomeres in DNA repair The importance of telomeres in cellular physiology is further emphasised by the recognition that they take part in the repair of DNA and chromosomes. Telomeric repeats are often inserted during the repair of double strand breaks. Extraneous telomeres appear to be able to provide the full complement of DNA repair machinery. The involvement of telomeres in DNA repair is exemplified also by the enhanced radiosensitivity shown by mice with telomere dysfunction and loss of telomerase. KK Wong et al. (2000) reported that telomerase RNA gene (–/–) mice were radiosensitive in later generations and showed higher mortality. There was also a higher rate of apoptosis of gastrointestinal crypt stem cells and primary thymocytes. Wong et al. (2000) also found a diminished DNA repair capability together with chromosomal malformation and DNA fragmentation, compatible with the onset of apoptotic degradation of cells.

Telomere function and ATM kinase The maintenance of genomic stability has often been shown to involve telomere integrity. As discussed previously, deficiency of telomere repair and integrity can result in accelerated senescence and also lead to genetic instability. The autosomal recessive syndrome ataxia telangiectasia (AT) is a prime example of the significance of telomere integrity and the relationship between telomere function and genetic stability. AT is characterised by genomic instability accompanied by enhanced radiation sensitivity and carries a high cancer risk. The AT syndrome also includes cerebellar degeneration as well as immunological deficiency. The syndrome results from mutation of the ATM gene. Telomere-related abnormalities have been encountered in AT cells (Xia et al., 1996). Also recognised some years ago was the fact that mutation of TEL1, which is homologous to the ATM, produced yeast with shortened telomeres (Greenwell et al., 1995). Experimentally, the involvement of ATM in telomere integrity has been obvious from the transfection of fragments of ATM containing the leucine-zipper motif into colorectal tumour cells. These ATM fragments induced the transfected cells to display the AT phenotype together with telomere erosion and enhanced TAS. The transfected cells showed a greater propensity to generate TAS in response to ␥-radiation (Smilenov et al., 1997). The ATM protein is a kinase belonging to the PI-3 kinase family. Human ATM shows marked homology to MEC1, TEL1 and Rad3 of yeast. TEL1 can compensate defective ATM and suppress hyper-recombination and radiationinduced apoptosis and telomere loss (Fritz et al., 2000). It has therefore been suggested that defective ATM could lead to defects in telomere maintenance (Pandita, 2001). ATM (–/–) mice have markedly shortened telomeres (Hande et

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al., 2001). Defective ATM is associated with accelerated telomere loss and hypersensitivity to radiation, and cells enter mitosis and undergo apoptosis upon sustaining DNA damage. The telomeric protein TRF1 is a substrate for the ATM kinase. Kishi, Zhou et al. (2001) found that exposure to radiation increases phosphorylation of TRF1 in an ATM-dependent manner. An upregulation of TRF1 reduces telomere length and induces apoptosis of cells containing short telomeres, possibly facilitated by caspase-3 (Kishi, Zhou et al., 2001). Kishi, Wulf et al. (2001) also noted that breast cancers express TRF1 at a reduced level. This could have an overall effect of tumour expansion by partly reducing loss of cells by apoptosis. Nonetheless, an examination of the TRF2 would have been worthwhile to complete the story, especially in view of the statement by Kishi, Wulf et al. (2001) that apoptosis is not induced by TRF1 over-expression in cells with long telomeres.

Telomeric association Cellular senescence is characterised by chromosomal changes such as telomeric association (TAS), which is believed to result from the loss of telomeric DNA. Mukhopadhyay et al. (1998) transfected p53 into H1299 cells and demonstrated a diminution of telomeric DNA and the induction of TAS. Sprung et al. (1998) have associated the absence of telomeric repeats at the sites of TAS. Whether TAS is a consequence or the cause of genetic instability is a matter for debate. There are clear indications, nevertheless, that the formation of TAS is closely associated with cancer progression, which is contrary to what one would expect from the direct correlation often reported to exist between telomerase expression and cancer progression. But as Sherbet and Lakshmi (1997) have pointed out, the correlation of telomerase expression with cancer malignancy cannot be regarded as a fait accompli, merely because most investigations of the subject have restricted their attention to telomerase alone without taking into cognisance the part played by DNA polymerases in telomere replication. In fact, there is much more convincing evidence now that TAS formation might be indicative of the state of progression of cancer, which reiterates the view that the restoration of telomere length and its postulated contribution to cell immortalisation is not an invariable event associated with cancer progression.

The incidence of telomeric association in cancer Telomeric association is not exclusively associated with cellular pathology. Pathak et al. (1994) reported the incidence of TAS in normal fibroblasts. TAS is also found in other conditions, such as chronic pancreatitis. In PBL from

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patients with chronic pancreatitis, TAS incidence was 11±2.3 as compared with the control value of 1.09±0.3 (Cottliar et al., 2000b). With the general recognition that genetic instability might be a driving force in cancer progression, it is inevitable that cytogenetic abnormalities of transformed cells and cancers were investigated for possible TAS incidence. Indeed, Pathak et al. (1994) described some of their pioneering work on the incidence of cytogenetic abnormalities of chromosomes in the form of dicentric chromosomes in EBV-transformed cell lines and cell lines derived from human tumours. Although these abnormalities arising from TAS were not confined to neoplastic cells alone, Pathak et al. (1994) emphasised the use of TAS as a cytogenetic marker of cells entering apoptosis. Compatible with their thesis that the formation of TAS is a prelude to apoptosis, Pathak et al. (2000) found that spontaneous regression that occurs in Sinclair swine cutaneous melanoma was closely associated with telomere diminution and a virtual absence of telomerase activity. Pathak et al. (2000) also noticed that, in parallel with these, an increase of TAS also occurred together with the onset of apoptosis. The incidence of TAS has been examined in PBL of women with breast and cervical cancer. Pazy-Mino et al. (1997) found that TAS was 100-fold greater in breast cancer patients than in control subjects, and that in PBL of patients with cervical cancers, approximately 40-fold greater. TAS incidence is high also in PBL of patients with oesophageal squamous cell carcinoma as compared with control PBL (Xiao et al., 1998). Sawyer et al. (1996) have studied TAS incidence in six paediatric tumours and have reported the involvement of several chromosomes in TAS. However, it is difficult to relate the incidence of TAS in tumours from histological divergent pathogenesis to disease progression. There is some indirect evidence that might support the thesis that interference with telomere restoration can influence cancer progression. The nm23 gene, which codes for nucleoside diphosphate (NDP) kinase, is believed to be a metastasis suppressor gene. Two homologues of this gene – nm23-H1 and nm23-H2 – have been cloned (Rosengard et al., 1989; Stahl et al., 1991). The metastasis suppressor property of the nm23 homologues has been demonstrated in a few human tumour types, subject further to many caveats (see Sherbet, 2001). Nonetheless, it is of much interest to note, albeit even in its putative relationship with cancer progression, NDP kinase is apparently a player in the scenario surrounding the maintenance of telomere integrity. Nosaka et al. (1998) found that NDP kinase encoded by nm23-H2 is capable of binding to single stranded TTAGGG repeats, but not to double stranded repeats. This suggests the possibility that it might interfere with, indeed suppress, telomeric replication and effectively inhibit the restoration of telomere erosion. This, as we have noted earlier, would lead to apoptotic death of cells. The NDP kinase seems also to be able to bind to the TRF1 protein, which binds to and inhibits telomerase activity. Thus one can envisage a means by which nm23NDP kinase might conceivably regulate the maintenance of telomere reconstruction, and in this way influence or suppress clonal expansion of cells.

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Overall, the findings described above suggest the possibility that intrinsic instability occasioned by the erosion of telomeric repeats leads to TAS and the formation of abnormal dicentric, multi-centric and ring chromosomes. It has been argued that TAS might be only a secondary phenomenon. This is probably true, but arguably TAS represents dramatic genetic alterations that might be expected to have severe pathogenic consequences to the cell.

Telomeric association and gene expression The phenotypic significance of TAS has naturally to be judged in terms of its influence on gene expression. The erosion of telomeres appears to occur in a random fashion and therefore the incidence of TAS could be a random event too. The work of Sawyer et al. (1996) has, however, suggested it to be stochastically non-random in occurrence. They investigated a number of paediatric tumours and noticed that TAS involving 1p occurred in 3 out of 6 cases. Chromosome 4p was involved in 3 of 6 tumours, of which 2 represented telomeric association of 4p with 22q. Many years ago, Kuwano et al. (1992) induced TAS in PBL by exposing them to aphidicolin. They found that 1q, 2q, 3q, 6p, and 16q were statistically more frequently involved in TAS than other chromosome arms, and also noted non-random association of 1q/2q, 2q/2q, 4q/4q, 6q/6q, and 6p/6p in TAS. According to Cottliar et al. (2000b), chromosomes 9p, 20q, 16p, 21q and 9q were preferentially involved in TAS in chronic pancreatitis. Earlier, Najfeld et al. (1995) described certain unbalanced translocations, called jumping translocations, of 1q and 7q to chromosomes 1, 8, 15, 21 and 22 in leukaemic cells. Najfeld et al. (1995) at the time suggested the involvement of telomeric repeats in these whole-arm translocations. Reddy and Murphy (2000) reported a jumping translocation of 9q with 19p and less frequently with 8 and 9. Furthermore, using telomere-specific probes they demonstrated the presence of interstitial telomeric repeats in derivative chromosomes 19 and 8, thus confirming the role of these repeats in bringing about the translocation. In both these studies there is a clear hint of a nonrandom nature of chromosomal reorganisation. There is little doubt that further studies will take place with a view to confirming the possible specificity and non-random nature of TAS formation. The evidence available at present indicates that genetic reorganisation involved in TAS, and, together with the demonstration that intrachromosomal telomeric repeats are hotspots of genetic recombination (Day et al., 1998), it does lend itself to interpretation that they might impinge significantly on genetic expression. There is now increasing evidence that at the intragenic location, telomeric repeats can and do alter gene expression. Kilburn et al. (2001) inserted an 800-bp TTAGGG repeat into the second intron of adenosine phosphoribosyl transferase gene and detected an enhanced expression that depended on the orientation of the insert.

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Telomere position effect Genetic transcription is greatly influenced by histone modification. Histone deacetylase (HDAC) and histone acetyl transferase (HAT) are enzymes that influence genetic transcription. HDAC deacetylates the ␧-NH2 groups of lysines located at the N-terminus of core histones, whilst HAT acetylates them. Chromatin that is transcriptionally active is known to be associated with hyperacetylated histones, but in transcriptionally silent chromatin histones are hypoacetylated. Telomeres can repress the transcription of neighbouring genes, a phenomenon that has been called the telomere position effect (TPE). This seems to take place by the agency of histone deacetylases (HDAC) called the Sir (silent information regulator) family enzymes, which participate in the maintenance of telomeric heterochromatin. TPE appears to be a result of hypo-acetylation or deacetylation of the sub-telomeric heterochromatin. The Sir proteins are localised at telomeric sites. They appear to be targeted to telomeres by the agency of the Rap1 protein, which interacts with Sir proteins (Moretti et al., 1994; Cockell et al., 1995), and this has been attributed with the initiation of TPE (Lustig et al., 1996). The Sir2p is a deacetylase involved with telomeric heterochromatin. Several Sir proteins have been identified in the yeast; also identified is the human homologue of the Sir2p (Afshar and Murnane, 1999). The Sir2p has been strongly implicated in transcriptional silencing. A deletion of Sir genes seems to eliminate TPE. The telomeric TGrepeat binding protein Rap1 also plays a role in TPE, together with other proteins such as the Ku protein. The Ku is an important agent in the pathway of double strand break repair and maintenance of telomere length, besides participating in TPE. Indeed, Ku requires Sir proteins for its DNA repair as well as transcription silencing functions (Boulton and Jackson, 1998). These proteins might be involved in the recruitment or activation of Sir proteins in TPE of yeast telomeres (Mishra and Shore, 1999). Paradoxically however, Sir proteins do not invariably influence cell growth (Afshar and Murnane, 1999), which one would expect from their close association with Ku-mediated DNA repair and recombination processes. As we have noted earlier, telomeric integrity is closely involved with the regulation of the cell cycle. It is not surprising to note a consistent association of a number of cell cycle regulatory proteins with telomeric sites. The cell cycle regulatory proteins p53 and Rb both need to be linked in some way with telomere integrity and cell proliferation. The HDAC and HAT enzymes could conceivably bring these together. We know for instance that Rb protein is capable of influencing chromatin structure by recruiting HDAC. When Rb is phosphorylated there is a dissociation of Rb from its association with repressor complexes; this leads to activation of S-phase genes. The cyclic AMP-binding protein CBP/p300, in contrast, functions as a histone acetyl transferase. Furthermore, its HAT activity enhances when it is phosphorylated, and this

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therefore leads to G1 –S transition by the activation of appropriate genes. Similarly, cellular senescence attendant upon telomeric diminution could be a consequence of the acetylation status of histones. As discussed elsewhere in this book, DSBR requires several factors. The Ku proteins and DNAP-PKc, the catalytic subunit of DNA-PK, figure prominently in this process. Ku proteins participate in cell differentiation, maintenance of chromosome integrity as well as in DNA transcription. They contribute to the genomic integrity by supporting homologous recombination as well as to the non-homologous end-rejoining pathway of double strand break repair. Ku participates in the repair of telomeric DNA. With their frequently perceived involvement with the progression of the cell cycle, it is not surprising that Ku70, Ku80 and DNA-PKc are associated with telomeric DNA.

Epilogue

The generation of genetic diversity is a basic and inalienable requirement of evolution of the species and for the life of the cell. Genetic recombination is the prime mover for transmission of genetic traits, in embryonic development and organogenesis, and in the development and maturation of the immune system. Genetic diversity is also a basic feature of pathological processes, and, as often emphasised, genetic recombination is responsible for the generation of cellular diversity and further intraclonal variation in the progression of neoplastic disease (Figure 15). Aside from neoplastic disease, several human afflictions can be attributed to inappropriate genetic recombination. Cell proliferation and apoptosis, which are greatly influenced and regulated by recombination events, are both deregulated in cancer development and progression, and often, aberrant recombination lies at the root of deregulation of cell proliferation and apoptosis. Deregulated recombination can bring about abnormal progression of the cell cycle by affecting cell cycle checkpoints, as well as by upregulated expression of other controlling factors such as the cyclins, growth factors and their receptors, and signal transduction mediators. Genetic recombination can constitutively activate signalling pathways. Fusion proteins arising from chromosomal recombination can severely deregulate signal transduction and confer upon cells the propensity to proliferate independently of growth factors and cytokines, inhibit apoptotic control of tumour mass and promote cell survival. Such deregulation also severely impinges upon the initiation and evolution of neoplastic processes. Genetic Recombination in Cancer ISBN 0-12-639881-X

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Figure 15 An overview of genetic recombination in tumour development and progression. The Figure provides a general overview of the many pathways by which genetic instability and recombination function in the progression of tumour from inception and development through to metastatic dissemination. The Figure shows that the major target of influence is the phase of tumour proliferation and expansion. The phase of induction of angiogenesis, acquisition of invasive behaviour and formation of metastatic tumour appears in comparison to be less influenced. However, there are no compelling reasons to believe that genetic recombination is not a driving force in these processes too. Present indications are that genes required to be expressed in this phase are also activated by genetic recombination, but the conundrum is how such gene activation can occur during clonal evolution and yet the phenotypic effects can be suspended until a later stage in progression.

The versatility of genetic recombination as a source of diversity is also amply illustrated and emphasised by the potential of VDJ recombination together with hyper-mutability to generate a vast repertoire of antibodies and TCR with different specificities and precise functions. Naturally therefore aberrant recombination can result in immune deficiency syndromes and onset of allergies with their attendant pathologies. VDJ recombination can drastically affect and

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deregulate the expression of genes, which are brought under the domain of control of immunoglobulin gene enhancers. The expression of many so-called oncogenes is modulated in this way, contributing significantly to tumour development, especially in the pathogenesis of haematological malignancies where one might encounter genetic changes possibly driven by VDJ recombination. Chromosomal instability is frequently encountered under conditions where DNA repair is inhibited and manifests itself in the form of reciprocal translocations and unequal translocations, with severe consequences on account of the involvement of important gene loci in these exchange processes. These can cause over-expression of the genes or lead to their amplification. It is needless to say that in lymphomas these translocations occur in conjunction with VDJ recombination that leads also to the generation of diversity of the Ig gene. At the other end of the spectrum of differentiation, cell proliferation and apoptosis are crucial cellular events in embryonic development and organogenesis and it is not too difficult to visualise developmental and differentiation-related abnormalities should a genetic recombination event go awry. Whilst one cannot predicate complete exclusivity of recombination events in normal differentiation, or its exclusivity in the pathogenesis of human disease, efficient repair of DNA damage is essential for the life of the cell. Several autosomal recessive genetic disorders are characterised by immunodeficiency, enhanced radiation sensitivity and a predisposition to develop cancers. The recessive syndromes such as Bloom’s syndrome and the Nijmegen syndrome both show immunodeficiency but in neither condition is VDJ recombination affected. But in ataxia telangiectasia, another autosomal recessive syndrome manifesting immune deficiency, hypersensitivity to radiation and higher cancer incidence, not only is DNA repair impaired but cells also show higher abnormal recombination of TCR. The life of a cancer cell can be demarcated into three main compartments. The first compartment constitutes the transformation of the cell into a precancerous initiation, progressing into a state with high proliferative potential together with the ability to generate diversity. The evidence is overwhelming that genetic recombination activates and/or deregulates signalling pathways that control cell proliferation. The second compartment is the phase of angiogenesis and invasion of the vascular system. Numerous cell clones with diverse characteristics evolve during the development of neoplasms. One cannot assert with certainty that some of these clones might possess enhanced membrane-associated features that can lead to their release from the primary tumour and enable them to invade the circulatory system and to disseminate to distant sites. The third compartment is the metastatic phase in which tumour cells that have lodged at distant foci proliferate and form secondary tumours. The question arises whether genetic recombination occurring during the initiation of the neoplastic process carries with it a temporal activation mechanism that is required in the expression of genes whose products are essential for the progression of the tumour to the phase of induction of new

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vascular structure and its invasion as a prelude to distant dissemination. Taking a cue from haematological neoplasia, it seems reasonable to suggest that certain genes involved in the induction of new blood vessels and haemopoiesis might be inappropriately activated and expressed following recombination. But we have virtually no information how in the progression of solid tumours these genes, albeit being activated at a far earlier stage, can now begin to exert their effects of angiogenesis. In other words, this scenario requires one to invoke a temporal mechanism in the generation of these specific phenotypic properties. The magnitude of the work being carried out on the genetics of tumour expansion does not mean that efforts at understanding the genetics of invasive and metastatic phases of progression have been feeble. It is the availability of appropriate models for study that seems to hamper progress. The progression of colonic cancer might offer a reasonable approach to elucidating this riddle. It is only when this riddle is resolved that one can with some safety suggest that the events occurring in the first compartment do indeed lead to the generation of clones with heightened invasive and metastatic capacity. The events of note in the metastatic phase are more likely to be related to the extravasation of tumour cells into the parenchyma of target organs, which would be followed by a renewed phase of proliferation and expansion into metastatic tumour. This brings one to pose the inevitable question of how cancer cells evade the immunological surveillance of the host. One can view genetic recombination as a source of novel antigens. Indeed fusion proteins can occasionally be more antigenic than their unaltered components. Whilst these might afford a handle to tackle neoplasms by appropriate immunotherapy, it is difficult to envisage a way by which they might contribute to the process of tumour initiation, development and progression. From the immunological point of view, the generation of novel antigens might lead to the recognition of neoplasms as nonself and theoretically lead to the destruction of the tumour. There is little doubt that tumours are in fact recognised as non-self entities. For instance, intracranial gliomas are potentially highly malignant tumours that can invade extensively in the local environment, but they are not generally known to metastasise to extracranial sites. Nonetheless, the immune system of the host is more often than not sensitised to the presence of the non-self neoplastic tissue. So tumours do generate novel antigens that are recognised by the host. Indeed, vast numbers of tumour cells that are released from the primary tumour into the circulatory system are destroyed. A small number of cells do succeed in evading the immune system and are disseminated to distant sites where they lodge, temporarily remain dormant, or expand to form metastatic deposits upon stimulation to proliferate. It seems therefore a most important element of tumour progression is the appearance of factors at the opportune time point. It is here that one would expect genetic recombination to enter into the picture by activating specific genes that encode growth factors, their specific receptors, or cellular factors that control cell cycle progression. Genetic recombination can also lead to the

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inactivation of tumour suppressor genes and initiate neoplastic transformation of cells and the expansion of transformed clones and emergence of tumours. By all accounts it would seem that the first compartment or the primary phase of tumour development seems to be the all-important phase. For the events occurring here seem to determine the course of tumour progression, whether they grow as localised, encapsulated benign conditions or whether they acquire high invasive ability to disseminate via induction of tumour vasculature and upon lodgement at the metastatic site to grow into secondary deposits. Tumour dormancy, a phenomenon little studied to date, might yet turn out to be an important aspect of tumour biology. Metastatic deposits will remain dormant when a positive balance exists in favour of expansion of tumour cell population with cell proliferation preponderating over cell loss by apoptosis. In the absence of vascularisation therefore the metastatic deposits will not display net growth because this balance is reversed in favour of apoptosis. Consistent with this argument are the several demonstrations of tumour dormancy under conditions where angiogenesis is inhibited. Disseminated individual tumour cells also can remain lodged in a dormant state over prolonged periods. A majority of the tumour cells that enter the vascular and lymphatic systems are destroyed and only a few cells survive to extravasate into the stroma of target metastatic sites or are held in a state of limbo. A breakdown of this immunological surveillance or control will undoubtedly lead to successful dissemination of the tumour. On the other hand, the inefficiency of the metastatic process might equally strongly be attributed to events occurring in the early metastatic deposits. The overwhelming possibility is that this state of individual tumour cell dormancy also results from a balancing of the proliferative ability and apoptosis. But unlike activation of metastatic growth by induced vasculature, an activation of cell proliferation rather than inhibition of apoptosis is the more likely event that contributes to expansion of the cell population. One cannot exclude the possibility that neovascularisation might be required in the transport of growth factors to the micrometastases. Nor can one state categorically that growth factors generated at the metastatic site are not playing a role in reversing dormancy. This is suggested by different patterns of metastasis displayed by different tumours and the target organ specificity they achieve quite independently of the mode of spread. Finally, an activation of autocrine growth control as well as autocrine regulation of induction of neovascularisation could also be factors that need to be taken into account. In other words, single cell dormancy and micrometastasis dormancy could be following different pathways. At the present time no evidence can be presented that suggests any correlation between the appearance of proliferation and apoptosis controlling factors as a consequence of genetic recombination and initiation of de novo genetic transcription. It follows from this that the secondary tumour is not quite the independent entity that one assumes it to be because of its clonal origin from the primary tumour. Factors present in the primary tumour might

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be controlling the switch that diverts micrometastases from the apoptotic pathway to the proliferative pathway. The Folkman group regards angiostatin as a major factor involved in metastatic dormancy. Whatever might be the switch, there is little doubt that extirpation of the primary tumour indeed results in growth imbalance of dormant micrometastases. Nonetheless, one must envisage that the potential for evolving clonal heterogeneity might yet be intrinsic in the secondary tumour mass as it demonstrably is in the primary tumour. Apparently the neoplasm has completed a full cycle of progression and begins again at the beginning.

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Index

Adenomatous polyposis coli (APC) gene, 26 AFX (Forkhead transcription factor), 104 Akt pathway see Phosphoinositide-3 kinase/Akt pathway Alagille syndrome, 77 Alu repeats, 50–62 and BRCA suppressor gene, 53, 58 and cell cycle regulator genes, 55 and DNA mismatch repair gene mutation, 57 and nm23 suppressor gene deletion, 54 in familial adenomatous polyposis, 53 in genetic transcription, 57–8 and genomic instability, 57 interchromosomal recombination in Ewing sarcoma, 56–7 interchromosomal recombination in leukaemias, 55–6 muscular dystrophy and Alzheimer’s disease, 51–2 recombination, 58–9 and signal recognition particles, 59, 61–2 Alzheimer’s disease, 51–2

AML1 fusion proteins, 106–7 AML1 gene, 107 Androgen receptors, 46, 47 CAG repeats in, 46–7 and apoptosis, 47 Aneuploidy, 68–70 and S-phase fraction, 70 in AML, 70 Antitrypsin and genetic instability, 87 APC gene, 53 APC protein, 26–7 Apoptosis, 21 gene mutation status, 34 and histone acetylation dynamics, 143–4 p53-induced, 91–2 PI-3 kinase/Akt pathway, 96–104 regulation by bcl2 gene family, 91–2 role of telomeres in, 154–7 TCL1 gene in, 95–6 TRAIL-induced, 97 Arabidopsis thaliana, 6 Artificial neural networks, 73 and analysis of cell features, 73–5 analysis of prognostic factors, 73–5

238 Askin’s tumour, 56 Ataxia telangiectasia, 12, 35, 92, 158 Ataxins, 44 ATM (protein) kinase, 92 and cell cycle regulation, 92–5 as substrate for caspases, 94 and telomere function, 158–9 homologues, 158 in SMC function, 40 Atrophin, 44 Autoimmune thyroid disease, 49–50

Base excision repair, 5 bax gene, 30 bcl2 gene family, 91–2 in p53-induced apoptosis, 92–5 bcr-abl fusion protein, 91 Becker muscular dystrophy, 51 Bladder cancer, 22, 125 Ku proteins and progression of, 22 loss of heterozygosity of FHIT in, 125 loss of heterozygosity of VHL in, 125 BLM gene, 9, 35 Bloom’s syndrome, 8, 12, 35, 166 sister chromatid recombination in, 35–6 BRCA1 gene, 7, 30, 53, 58, 146 BRCA1 tumour suppressor protein, 22 BRCA2 gene, 7, 30, 58, 129, 146 Breast cancer, 15, 26, 29, 53, 58, 146 CAG repeat tracts in, 46 c-erbB2 gene expression in, 70 DNA ploidy, 68 downregulation of SEL1L in, 86 hereditary, 37 Ku proteins in, 22–3 metastatic, 30 oestrogen receptor expression in, 71, 72 progesterone receptor expression in, 72 prognosis, 74 PTEN gene expression in, 97 RAD genes in, 7–8 see also BRCA1 gene; BRCA2 gene Breast fibroadenoma, 26 Burkitt lymphoma, 90

Index Cadherins, 32, 87 Caenorhabditis elegans, 85 CAG repeat expansion, 42–8 and androgen receptor function, 46–7 in CREB transcription factor, 43 in endometrial cancer, 47 in hepatocellular carcinoma, 47 in protein phosphatases 2A and 2B, 43 in spinobulbar muscular atrophy, 45 in spinocerebellar ataxia, 43–5 CAG repeat lengths, 46 Calcium homeostasis, 52 cAMP response element binding protein see CREB protein Caspases, 46, 91–2, 94 Catenins, 32 CDKN2 gene family, 93–4 Cell adhesion molecules, 87 Cell cycle checkpoint pathway, 7 Cell cycle progression, 19–21 Cell cycle regulator genes Alu-mediated alterations, 55 mutation status, 34 Cell differentiation, 19–21 Cell proliferation, 89–96 and chromosomal rearrangement, 90–1 and DNA ploidy, 70–5 role of telomeres in, 154–7 Cell senescence, 154–7 Cellular adhesion, 32 Centromere, 13 Centromeric instability, 130–1 c-erbB2, 30 in breast cancer, 70 Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), 77 Cervical cancer, 29 CFTR gene, 63 CGG repeats, 40–5 in fragile X syndrome, 45 Chi sequences, 65–6 Chrome domain proteins, 13 Chromosomal fragility see Fragile sites Chromosomal instability see Genetic instability

Index Chromosomal integrity, 19 Chromosomal recombination see Recombination Chromosomal translocation, 76–132 and cell population expansion, 89–96 and fragile sites, 120–1 fusion proteins and immunity, 119–20 and tumour vascularisation, 117–19 and genetic transcription, 104–17 PI-3 kinase/Akt pathway, 96–104 and retinoic acid receptor function, 108–9 and signal transduction, 76–89 in synovial sarcomas, 105–6 Chromosome-associated proteins, 39 CIMP see CpG Island Methylator Phenotype Clustrin, 22 c-met gene, 52, 63 Cohesin, 40 Collagen IV, 100 Colon (colorectal) cancer, 4, 28, 31, 114, 123, 138 CIMP in, 138 hereditary non-polyposis, 4 HOX genes in, 114 RER phenotype and, 26–8 Condensin, 40 CpG Island Methylator Phenotype (CIMP), 136–9 CREB protein, 43, 104 Crossover hotspot instigator elements see Chi sequences Cyclins, 15 Cytokines, 9, 110

Death domains, 97 Dentatorubral palliodoluysian atrophy, 44 and atrophin-1, 44 CAG repeats in, 44 Dihydrofolate reductase (DHRF) genes, 127 DNA damage response genes, mutation status, 34 DNA-dependent protein kinase, 15, 16, 18, 19, 23

239

DNA methylation, 134–53 chromosomal recombination and genetic transcription in childhood leukaemias, 140, 141 CpG Island Methylator Phenotype (CIMP), 136–9 fragile X syndrome, 151–3 and genetic instability, 135–6 histone acetylation dynamics, 140–9 in ICF and Rett syndromes, 134–5 in normal and neoplastic development, 133–4 p53 methylation and chromosome stability, 149–51 and recombination, 139 DNA methyltransferases, 133, 147 DNA nucleotide repeats, 44–5 DNA ploidy, 68–70 artificial neural network based analysis, 73–5 and cell proliferation, 70–5 and growth factor expression, 70–5 and oestrogen/progesterone receptors, 72 and p53 abnormalities, 75 DNA recombination, 3 Alu repeats in, 50–62 Chi sequences in, 65 cognate elements associated with, 64–6 DNA recombination repair, 8–9 DNA repair base excision, 5 BRCA1 and R in, 22 Chi sequences in, 65–6 Ku protein in, 18–19 nucleotide excision, 5 telomeres in, 158 DNA repeats, 41, 42 and genetic recombination, 41–8 in genetic disorders, 41–8 polymorphism of dinucleotide, 49–50 DNA replication checkpoints, 5–8 Double minute chromosomes, 41, 68, 126–8 Alu elements in,53 and sister chromatid recombination, 37

240

Index

Double strand break repair, 16, 18 Down’s syndrome, 36 Drosophila, 13, 63, 83, 107 DRPLA see Dentatorubral palliodolyusian atrophy Duchenne muscular dystrophy, 51 Dystrophin gene, 51, 62

E2A gene, 115 E2F protein, 104 E6 protein, 8 EBNA, 18, 80 in notch signalling 80, 83–5 EBV, Ku binding to, 18 EBV nuclear antigen, see EBNA E-cadherin, 32, 33 Endometrial cancer, 31 CAG repeat tracts in, 46 Epigenetic gene silencing, 147–8, 152–3 Epstein-Barr virus see EBV erb genes, 82, 90 Escherichia coli, 5, 65–6 EST2 protein, 156 Euplotes aediculatus, 156 Ewing’s sarcoma, 56–7 Extracellular matrix, 87

Familial adenomatous polyposis, 26, 33 FANCA gene, 52 Fanconi anaemia, 52 FHIT see Fragile histidine triad Fibronectin, 87, 100 Fibulins, 87–9 FK228, 148 FKHR gene, 112–13 FKHR protein, 104, 113 FL11 gene, 56 FMR1 gene, 43, 151–3 CGG repeats in, 43–5 epigenetic silencing of in fragile X syndrome, 152–3 in pathogenesis of fragile X syndrome, 151–2 Forkhead see FKHR FOXO protein, 104

FRA3B fragile site, 121 Fragile histidine triad, 54, 121 abnormalities in cancer, 122–4 Fragile sites, 121–6 and chromosomal translocations, 56, 120–1 and micronuclei, 128–32 and RER phenotype, 34–5 sister chromatid recombination at, 35–9 Fragile X syndrome, 43, 48 DNA methylation in, 151–3 epigenetic silencing of FMR1 in, 152–3 FMR1 gene function in pathogenesis of, 151–2 Frataxin, 45 Fusion proteins, 100–3 in haematopoietic neoplasms, 106–8 and immunity, 119–20 in synovial sarcomas, 105–6 and tumour vascularisation, 117–19 Fuzzy K-Nearest Neighbour algorithm, 74

Gall bladder tumours, 29 Gastric cancers, 28 DNA ploidy in, 68 Gene amplification, 126–8 Gene expression, and telomeric association, 161 Gene mutation, 24–5 Gene silencing, 12, 13 Genetic abnormalities, 85–7 Genetic instability, 3, 4, 24–40, 25 and Alu repeats, 39 and CAG repeat expansion, 42–8 and DNA methylation, 134–53 and gene amplification, 126–8 and p53 methylation, 149–51 and telomeric DNA, 154–63 Genetic integrity, 3 Genetic loss, 121–6 Genetic recombination see Recombination Genetic transcription see Transcription Genomic stability, 39–40

Index Glioma cells, sister chromatid recombination in, 38 Grave’s disease, 49, 80 Growth factor expression, and DNA ploidy, 70–5 Growth factor receptor genes expression, 28–31 mutation status, 34 Growth regulation, SET domain proteins in, 12–15

Haematological malignancies, 9–12 PAX expression by, 112 see also Leukaemias Haemopoietic neoplasms, 106–8 Hairpin ends, 16, 48 hASH1 protein, 81 Head and neck cancers, 23, 69 Heat shock proteins, 17 Hepatocellular carcinoma, 47, 62, 150 Hereditary non-polyposis colon cancer, 4 ␥-Heregulin, 90 HES proteins, 80, 84, 85 Heterochromatin, 131 Heterozygosity, loss of, 25 Hexamethylene bisacetamide, 82 Histone acetylation, 140–9 and apoptosis, 143–4 and cell proliferation, 143–4 in development and differentiation, 141–3 and neoplastic process, 144–7 synergistic effects on epigenetic gene silencing, 147–8 Histone acetyltransferases, 141, 145, 162 Histone deacetylases, 141, 144, 162 interaction with MTA1 proteins, 145–6 Histone deacetylase inhibitors, 148–9 hMLH genes, 48, 135 hMSH genes, 48 Hodgkin’s disease, 37 Holliday junction crossover concept, 8–9 resolvases, 8 Homeobox genes, 111–17 and cell proliferation related gene expression, 114

241

and chimeric proteins, 115 and tumour vascularisation and invasion, 118 deregulation of, 118 Hoogsteen binding, 48 HOX cooperative motif, 115 HPV16-E6 gene, 143 HPV16-E7 gene, 143 Human immunodeficiency virus, 130 Human papilloma virus, 8 Huntingtin protein, 43 Huntington’s disease, 43, 44

ICAMs, 114 ICF see Immunodeficiency-centromere instability-facial anomalies Immune system, 9–12, 119–20 Immunodeficiency-centromere instability-facial anomalies, 130–1 abnormal DNA methylation in, 134–5 INK4 cyclin-dependent kinase inhibitors, 138 Insulin-dependent diabetes mellitus, 49–50 Insulin-like growth factor receptor, 30 Integrins, 87, 114 Interchromosomal recombination Ewing sarcoma, 56–7 leukaemias, 55–6 Intracellular adhesion molecules see ICAMs Intrachromosomal recombination Alzheimer’s disease, 50–2 muscular dystrophy, 50–2 tumour suppressor genes, 52–5 Isotype switching, 10

Jagged 1 gene, 77 JAK see Janus tyrosine kinase Janus tyrosine kinase (JAK), 10, 12, 82, 110

Karyotypic abnormalities, 67–8 K-ras gene mutation, 31 K-sam gene, 128

242

Index

Ku protein, 15–23 in cancer, 21–3 in cell cycle progression, 19–21 and chromosomal integrity, 19 and DNA repair, 18–19 regulation of transcription, 17–18 structure, 16 L1 protein, 104 Laminin, 87, 100, 146–7 Leukaemias chromosomal recombination and genetic transcription in, 140, 141 genetic transcription in, 140, 141 hyperdiploidy in, 69 interchromosomal recombination in, 55–6 loss of integrity of notch signalling, 83–5 ras proteins in, 82 t(11;14) translocation in, 11 TEL transcription-related gene rearrangement, 109–11 LIM-only proteins, 118 LINE elements, 41, 54, 123 in genetic rearrangement, 62–4 LMO2 protein, 11, 118–19 Long interspersed nuclear elements see LINE elements Lung cancer, 29 and DNA ploidy, 69 Lymphoma loss of integrity of notch signalling, 83–5 TEL transcription-related gene rearrangement, 109–11 Lytechinus pictus, 20 Machado-Joseph disease, 43, 48 Mantle-cell lymphoma, 11 Matrix metalloproteinases, 99, 117–18 mdm2 protein, 93, 107 Melanoma cells, sister chromatid recombination in, 38 Metastasis breast cancer, 30 and sister chromatid recombination, 36

Metastasis-related proteins (MTA1), 145 Metastatic suppressor genes, 33–4 Methyl-CpG-binding proteins, 147 Micronuclear bodies, 128–30 Micronuclei and chromosomal fragility, 128–32 and DNA damage, 128–9 in ICF syndrome, 130–1 nature and constitution of, 131–2 p53 in formation of, 129–30 Microsatellite dinucleotide repeats, 49–50 Microsatellite instability, 24–5 and APC expression, 26 and cancer progression, 25–8 and growth factor receptor gene expression, 28–31 and metastasis suppressor nm23 gene abnormalities, 33–4 and p53 expression, 28–31 and sister chromatid recombination, 39 and tumour invasiveness, 31–3 in colonic cancer, 26–8 Mismatch repair, 4, 24–5, 39 gene mutation status, 34, 57 MLL gene, 140, 141 Mouse mammary tumour virus, 18 MTCP1 gene, 96 Multiple myeloma, 14–15 SET domain proteins in, 12–15 switch and VDJ recombination in, 11 Muscular dystrophy, intrachromosomal recombination, 51–2 MutL mismatch repair gene, 25 MutS mismatch repair gene, 25

Nasopharyngeal carcinoma, 89 Nervy protein, 107 Neuregulins, 90 NF-␬B protein, 104 Nijmegen breakage syndrome, 12, 20, 166 Nitric oxide synthase, 149 nm23 gene, 8, 27, 31, 33–4, 54, 160 Notch genes, 49, 77–8 Notch proteins, 49, 78

Index Notch signalling and EBNA, 83–5 deregulation by chromosomal translocation, 77–83 fibulins in, 87–9 genetic abnormalities, 85–7 loss of integrity in leukaemia and lymphoma cells, 83–5 transduction pathway, 79 NSD gene family, 14–15 Nucleoside diphosphate kinase, see nm23 Nucleotide excision repair, 5 NUP98 gene, 116

Oestrogen receptors, 46, 71 and Alu repeats, 58 and DNA ploidy, 72 Open reading frames (ORFs), 63 Oral cancer, 27 Osteoprotegerin, 99 Ovarian cancer, 39, 46, 53, 146

p53 protein, 6, 17 and Alu repeats, 60 apoptosis, 92–5 and DNA ploidy, 75 in formation of micronuclei, 129–30 methylation and chromosome stability, 149–51 and microsatellite instability, 28–31 Paediatric alveolar rhabdomyosarcoma, 112 Pancreatic carcinoma, 69 PAX3-FKHR fusion protein, 120 PAX genes, 111–13 Peripheral blood lymphocytes hereditary breast cancer, 37 sister chromatid exchange in, 36 Philadelphia chromosome, 90, 100 Phosphoinositide-3 kinase/Akt pathway, 96–104 cancer growth and invasion, 97–100 fusion oncoprotein signalling, 100–3 PTEN tumour suppressor function, 97 signal transduction mechanism, 103–4 TCL1 function, 100

243

Plakoglobin, 32 Plant homeodomain motif, 14 PLZF protein, 109 PML protein, 108–9 Poly (ADP-ribose) polymerase (PARP), 35–6 Polycomb gene family, 12, 14 PP2A protein phosphatase, 43 Presenilin gene, 52 Progesterone receptors, 46 and DNA ploidy, 72 Prostate cancer, 28, 46, 146 HOX genes in, 113 Prostate specific antigen, 46 Protease inhibitor kallistatin gene, 86 Protein C receptor, 87 PSA see Prostate specific antigen PTEN gene, 30, 97

RAD protein, 6–9, 19 and nm23 function in breast cancer, 8 and telomere integrity, 6 RAD genes, 5–8 and BRCA suppressor gene function, 7 RAG genes, 10 RAG proteins, 11 ras gene, 26 ras proteins, 82 RASSF1A genes, 122, 126 ras signalling, 126 Receptor tyrosine kinases, 90 Recombination, 67–75 cell proliferation, 70–5 cognate elements associated with, 64–6 Chi sequences, 65–6 translin-binding elements, 64–5 DNA aneuploidy, 68–70 and DNA methylation, 139 DNA ploidy, 70–5 and p53 abnormalities, 75 and genetic transcription in childhood leukaemias, 140 growth factor expression, 70–5 interchromosomal, 68 Ewing sarcoma, 56–7 leukaemias, 55–6

244

Index

Recombination – continued intrachromosomal, 68 Alzheimer’s disease, 50–2 muscular dystrophy, 50–2 tumour suppressor genes, 52–5 karyotypic abnormalities, 67–8 and tumour development, 165 Recombination activating genes see RAG genes recQ helicases, 9 Rectal carcinoma, 33 Replication error phenotype see RER RER, 24–40 phenotype, and fragile sites, 34–5 Retinoblastoma, 55 Retinoic acid receptors, 46, 108–9 Retroposition, 50 Rett syndrome, 134–5

S100A4 protein, 20 Saccharomyces cerevisiae, 61, 156 RAD genes, 6, 14 SCA see Spinocerebellar ataxia Schizosaccharomyces pombe, 6, 7, 13, 92 SCID see Severe combined immunodeficiency syndrome sel-1 gene, 85 SEL1L gene, 49, 80, 83, 85 SET domain proteins, 12–15 Severe combined immunodeficiency (SCID) syndrome, 12 and VDJ recombination, 12 Short interspersed nuclear elements see SINE elements Signal recognition particles, 50, 59 and Alu repeats, 61–2 Signal transducer and activator of transcription protein see STAT protein Signal transduction, 76–89 phosphoinositide-3 kinase/Akt pathway, 103–4 see also Notch signalling Sina gene, 81 SINE elements, 41 Sir proteins, 162

Sister chromatid exchange, 35–7 Sister chromatid recombination, 35–9 at chromosomal fragile sites, 38 and double minute chromosomes, 37 and metastasis, 36 and microsatellite instability, 39 in ataxia telangiectasia, 35 in Bloom syndrome, 35–6 non-random occurrence in melanoma and glioma cells, 38 SMC proteins see Structural maintenance of chromosomes proteins S-phase fraction, 69, 70 Spinobulbar muscular atrophy (SBMA) syndrome, 47 Spinocerebellar ataxia, 42, 43 ataxins in, 43–4 CAG repeat expansion in, 43–5 src-homology domains, 90 Stathmin protein, 20 STAT protein, 104, 110 Staurosporine, 99 Structural maintenance of chromosome proteins, 39–40 and DNA repair, 40 and genetic stability, 39–40 Synovial sarcomas, 105–6 Systemic lupus erythematosus, 14

t(1;13) translocation, 112 t(1;19) translocation, 115–16 t(2;13)(q35;q14) translocation, 65, 112 t(2;15) translocation, 112 t(3;18)(p14.2;q24.3) translocation, 121 t(3;21)(q26:q22) translocation, 56, 64, 101 t(5;12) translocation, 111 t(7;9)(q34;q34.3) translocation, 78 t(7;11)(p15;15) translocation, 117 t(7;11)(q35;p13) translocation, 118 t(8;21) translocation, 64, 106 t(9;12)(p24;p13) translocation, 110 t(9;14)(p13;q32) translocation,112 t(9;22)(q34;q11) translocation, 90, 91, 101, 120 t(11;14)(p13;q11) translocation, 64, 118 t(11;14)(q24;q12) translocation, 56

Index t(11;14)(q26;q22) translocation, 64 t(11;14) translocation, 11, 118 t(11;16)(q23;p13) translocation, 142 t(11;17) translocation, 108 t(11;22) translocation, 56–7 t(12;13)(p13;q12) translocation, 115 t(12;16)(q13;p11) translocation, 65 t(12;21) translocation, 106, 110, 120 t(14;14)((q11;q32) translocation, 96 t(14;15)q32;q11–13) translocation, 92 t(14;16)(q32;q23) translocation, 121 t(14;18)(q32;q21) translocation, 91 t(15;17) translocation, 108 t(17;20) translocation, 29 t(X;14)(q28;q11) translocation, 96 t(X;18)(p11.2;q11.2) translocation, 105 TAS see telomeric association TATA-binding protein, 17 Tax protein, 146 T-cell leukaemia/lymphoma gene see TCL1 gene T-cell receptor loci, 9 TCL1 gene, in apoptosis, 95–6 TCL1 protein, 100 TEL-abl protein, 101–2 TEL gene, 109–11 TEL-JAK protein, 101–2, 110–11 Telomerase, 6, 157 Telomere binding protein, 19 Telomere position effect, 13, 162–3 and Ku protein, 19 Telomeres, 154–63 and apoptosis, 154–7 ATM kinase, 158–9 and cell proliferation, 154–7 and cell senescence, 154–7 in DNA repair, 158 Ku proteins in integrity of, 19 RAD and integrity of, 6 Telomeric association, 37, 159 and gene expression, 161 incidence in cancer, 159–61 Tenascin, 87 TFIIB, 17 Thrombomodulin, 87 TRAIL-induced apoptosis, 97 Transcription Alu repeats in, 57–8

245

and chromosomal translocation, 104–17 regulation by Ku protein, 17–18 SET domain proteins in, 12–15 Transcription factors, 17, 104 Transforming growth factor-␤ receptor, 30 Translin, 59 Translin-binding elements, 64–5 Trinucleotide repeat tracts, 47 Trithorax gene family, 12, 14 TRX gene family, 12, 13 Tumour dormancy, 168 Tumour invasiveness, 31–3 Tumour metastasis, 117–19 Tumour necrosis factor, 97 Tumour suppression, 97 RASSF1 gene in, 126 Tumour suppressor genes, 121–6 intrachromosomal recombination, 52–5 Tumour vascularisation, 117–19 Von Hippel-Lindau protein and, 124–5 LMO2-mediated transcription and, 119 VEGF and, 98 Ulcerative colitis, 32, 37 Variable diversity joining recombination see VDJ recombination Vascular endothelial growth factor signalling via PI3/Akt pathway, 98 VDJ recombination, 9–12, 166 VEGF see Vascular endothelial growth factor Vitamin D3 receptors, 46, 109 Von Hippel-Lindau (VHL) gene, 54, 121–2 tumour suppressor function of, 124–5 and tumour vascularisation, 124–5 Watson-Crick binding, 48 Werner’s syndrome, 8, 17 Wilm’s tumour, 131 Wnt gene family, 115–16 Wolf-Hirschhorn syndrome, 14–15 WRN gene, 9, 17 Xeroderma pigmentosum, 5, 35

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