CISPLATlN CHEMOTHERAPY PLUS ADENOVIRAL pS3

GENE THERAPY IN EBV-POSITIVE AND -NEGATIVE

NASOPHARYNGEAL CARCINOMA

A thesis subrnitted in conformity with the requirements for the degree of Master of Science,

Graduate Department of Medical Biophysics, University of Toronto

O Copyright by Laura M. Weinrib 2000

National Library 1+1 d a n , Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Weilingtm Street 395. rue Wdlingtori OttawaON K I A W ûttawaON K 1 A W Canada Canada

The author has granted a non- exclusive licence ailowing the National Library of Canada to reproduce, loan, distn'bute or sell copies of this thesis in microform, paper or electronic formats.

The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fiom it may be printed or otherwise reproduced without the author's permission.

L'auteur a accordé une licence non exclusive permettant à la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique.

L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.

Cisplatin Chemotheropy plus Adenoviralp53 Gene Therapy in EBV- Positive and -Negative Nasopharyngeal Carcinoma

Laura M. Weinrib Master of Science 2000

Graduate Department of Medical B iop h ysics University of Toronto

ABSTRACT

Nasopharyngeal carcinoma (WC) is a malignant disease of the

headneck region that is endemic to South East Asia, and is associated with the

Epstein-Barr Virus (EBV). Given that current treatments for NPC yield a 5-year

survival rate of only 65%, Our laboratory was interested in explonng the

potential of adenoviral-mediated p53 gene therapy as a novel treatment that

might improve outcorne.

This work examined the effects of exposure to the combination of

cisplatin chemotherapy and p53 gene therapy in human NPC ce11 lines. p53

protein expression was induced only following p53 gene therapy, but both

treatments induced apoptosis. Overall, the combination of the two modalities

appeared to act in an additive manner with respect to decreasing the cells'

clonogenic survival and/or viability. This work also showed that p53 gene

therapy remains a potential therapeutic treatment despite the presence of EBV in

the tumor cells.

Future studies will determine the effect of tumor hypoxia on the efficacy

of p.53 gene therapy.

My sincerest thanks are extended to al1 those who helped me throughout

my graduate work.

1 extend special thanks to my s u p e ~ s o r Dr. Fei-Fei Liu whose support,

encouragement, and infectious enthusiasm made her a pleasure to work with- 1

would also like to thank al1 the members of my lab, both past and present, who

have made my time in the deparunent an enjoyable one. 1 am grateful for

everyone who provided me various combinations of technical assistance,

patience, advice, and good conversation.

Finally, 1 would like to dedicate this thesis to my family. Mom and Dad,

the love and support you have given me over the past two years has never gone

unnoticed. Thank you.

TABLE OF CONTENTS

.. ............................................................................................ Abstract ..II ... ....................................................... Acknowledgements ....................... .. 111 Table of Contents ............................................................................................................. iv .. .............................................. ...................................... List of Figures and Tables .., .. .vu ... . . ......................................................................... List of Abbreviat~ons ............... .- ...... w11

Chapter One: Introduction

............................................................................................... Gene Therapy -2 ......................................................................... 1.1.1 Introduction ....... ... 2

1.1 -2 Recent Gene Therapy Setbacks and Breakthroughs ...................... 3 1.1 -3 Cancer Gene Therap y ............... .... .............................................. -5

Nasopharyngeal Carcinoma ........................................................................ 7 1 . 2. 1 Introduction .......... ,.. .............................................................. -7 1.2.2 Treatment of NPC .......................................................................... 9 1.2.3 EBV and NPC ........................................ -0 1.2.4 p53 and NPC ............................................................................... 11

....................... ............... 1.2.5 NPC Cell Lines ... ..A 2

Cisplatin ....................................................................................................... 13 .............................................................................. 1.3.1 Introduction 13

............................................. 1.3.2 Cisplatin Structure and Mechanism 14

.................................................................................................. Adenovinis 16 1.4.1 Introduction ................................................................................ 16

............................................... 1 .4.2 Adenoviral S tmcture and Genorne 17 ........................................ .................. 1 .4.3 Adenoviral Life Cycle ... 18

................................... 1.4.4 Adenovirus as a Vector for Gene Therapy 19

................................................................................................................ p53 20 ................................................................................. 1 5 1 Introduction 20

1.5.2 p53 Structure ................................................................................. 23 ................................................... 1.5.3 p53 Siabilization and Activation 24

................................ 1.5.4 p53 Downstream Effects - Ce11 Cycle Arrest 27 ........................................... 1.5.5 p53 Downstream Effects - Apoptosis 28

...................................................................... 1 S.6 p53 Gene Therapy -32 ................................................................................................ 1.5.7 p73 34

.................................................................. Rationale and Project Outline -36

.................................................................................................... References 37

Chapter 2: Cisplatin Chemotherapy plus Adenoviral pS3 Gene Therapy in EBV- Positive and -Negative Nasopharyngeal Carcinoma

2.1 Abstract ...................... .. .................................................................................. 47

2.2 Introduction ......... .... ... ,..-. ....................................................................... -48

2.3 Materials and Methods ................................................................................ -49 2.3.1 Cell and Culture Conditions .......................................................... -49

......................... ............................................ 2.3 -2 Virus Propagation ..... -51 2.3.3 Virus Purification ....................... .... ............................................ -51

...*.........*. .................*.................. 2.3 -4 Plaque Assay for V h s Titer .... - 3 2 .......... 2.3.5 Detection of AdSCMV-#%galactosidase Expression .... 3 3

2.3.6 Infection and Cisplatin Treatment of Cells .................................... 54 2.3 -7 Clonogenic Survival Asssay .......................................................... 54 2.3.8 M T ï Assay .................................................................................. 55 2.3.9 Morphological Assessrnent of Apoptosis .......... ..... .................. 3 5

.......................................................................... 2.3.10 Western Blotting 56

2.4 Results ......................................................................................................... 57 2.4.1 Growth Characteristics of Ce11 Lines ............................................ 57 2.4.2 Effect of Cisplatin plus Ad5CMV-p53 on Ce11 Viabiiity ............. 60

.................... 2.4.3 Effect of Cisplatin plus Ad5CMV-p53 on Apoptosis 63 ............................................................................ 2.4.4 Western B loning 67

................................................... .......................*...*....... 2.5 Discussion .... 71

2.6 Conclusion and Future Work ........................................................................ 75

2.7 Re ferences ................................................................................................... 77

Appendix One: Future Directions: The Efkct of Hypoxia on p53 Gene Thempy in Cervical Carcinoma

................................................... A l . 1 Rationale for Proposed Study -82

............................................................... A1.2 Cervical Carcinoma 82

........................................................................... A 1 -3 H ypoxia -84 ............................................................. A1.3.1 Introduction 84

................................................... A 1.3.2 Effects of Hypoxia -85 .................................... A1.3 -3 Hypoxia in Cervical Carcinoma -87

........................................ A1.3.4 Hypoxia, Apoptosis. and p53 88

................................................................... A 1 -4 Proposed Study -90 ............................................. A1 .4.1 Objective and Hypothesis 90

................................................ A1 .4.2 Selection of Ce11 Lines -91 ................................................... A1 .4.3 Infection Eniciency -93

..................... ............... A1 .4.4 Clonogenic Survival Assays .. -93 .................................... A 1.3.5 p53 Gene Therap y Experiments -95

........................................................................ A1 -5 References -98

Chapter 1 Figure 1.1 The interaction of cisplatin with intracellular molecules ............... 14

.. Figure 1.2 DNA adducts produced by cisplatin ................................... .. 15 Figure 1 -3 Stimuli for p53 activation and the resultant consequences ........... -21 Figure 1.4 Structure of pS3 protein ................................................... -24 Figure 1 -5 Effectors of pS3 leading to ceU cycle arrest ............................. -28

................ ...... ........ Figure 1.6 Effectors of p53 leading to apoptosis .. ., -30

Chapter 2 Figure 2.1 CNE-1 cells growth curve ................................................. 58 Figure 2.2 C666-1 cells growth cwve ................................................. 59 Figure 2.3 Clonogenic survival of CNE-1 celis following AdSCMV-p53

plus cisplatin ................................................................. 61 Figure 2.4 Viability of C666- 1 cells following Ad5CMV-p53

................................................................. plus cisplatin 62 Figure 2.5 Morphological analysis of apoptosis .................................... 64 Figure 2.6 CNE-1 celis Western blot analysis ...................................... -68 Figure 2.7 C666-1 cells Western blot analysis ...................................... -69 Figure 2.8 Western blot analysis for p73 ............................................ -70

........................................... Table 2.1 CNE-1 cells assay of apoptosis -65 Table 2.2 C666-1 cells assay of apoptosis ............................................. 66

Appendix 1 Figure A l . 1 Cervical tumor oxygenation influences survival ............. .... .... 88 Figure A1.2 Viability of p53+/+ and p53-/- cells at varying

............................................. oxygenation concentrations -89 .................................... Figure A1.3 HeLa cells infection efficiency assay 94 .................................. Figure A1.4 HeLa cells clonogenic suMval assay 96

vii

LIST OF ABBREVIATIONS

ADA a - m M AO-EB ATM B-gai CAR cisplatin DNA EBER EBNA EBV ECL FBS HZF-1 HF'V HRE L m - 1 mAb m NP40 NPC PBS pfu Rb RNA SCC UNCT VEGF X-gal XRT

5-fluorouracil recombinant adenovbs type 5 carrying a human wild-type p53 cDNA downstream of a cytomegalovirus promoter recombinant adenovinis type 5 camying the bacterial lac Z gene coding for &galactosidase downstream of a cytomegalovinis promoter adenosine deaminase deficienc y a-minimum essential medium acridine orange - ethidium bromide ataxia telangiectasia mutated f3-galactosidase coxsackievirus and adenovhs receptor cis-diamminedichloroplat inum deoxyribonucleic acid EBV encoded RNA Epstein-Barr nucIear antigen Epstein-Barr virus enhanced chemilurninescence fetal bovine serum h ypoxia-inducible transcription factor- 1 human papillomavirus hypoxia responsi~re element latent membrane protein- 1 monoclonal antibody 3-(4,s-dimethylthiazol-2-y1)-2,5-dephenylteom bromide nonidet P40 nasopharynged carcinoma phosphate buffered saline plaque forming unit retinobiastorna ribonucleic acid squarnous ce11 carcinoma undifferentiated carcinoma of the nasopharyngeal type vascular endothelial growth factor 4-bromo-5-chloro-3-indoyl-~-galatopyranoside ionizing radiation

viii

Cha~ter One

Introduction

1.1 GENE THERAPY

1.1.1 Introduction

Gene therapy refers to the treatment of diseases through the alteration of the

genetic information of the cell via foreign coding sequences. The basic method for gene

t herapy involves isolating or manufactunng the desired gene with appropriate regulatory

sequences. This gene is then inserted into a vector that is targeted to specific cells within

the patient where it must produce adequate amounts of its product.

Gene therapy was initially conceived as a treatment for monogenic disorders such

as adenosine deaminase deficiency (ADA), where the genetic defect is corrected by

adding the normal gene, thereby 'curing' the patient. In fact, the f ~ s t human gene

therapy trial took place in September 1990, when two children with severe combined

immunodeficiency syndrome caused by ADA were treated using gene therapy

(Anderson, 1992). The human ADA gene was cloned, inserted into a replication-

deficient retroviral vector, and was transferred into lymphocytes isolated from the

children. Although the children were not cured of the^ disease, the re-infusion of the

lymphocytes containing the normal ADA gene did initially improve their immune

function, thereby demonstrating 'proof of principle' for the use of gene therapy to treat

diseases thought to be due to a single gene defect. More recently, the notion of gene

therapy for acquired disorders such as HIV and cancer has been introduced, and in fact

since the mid-1990s the majority of clinical trials for gene therapy have involved cancer

therapy (Tzeng et al., 1996).

The ethics surrounding gene therapy have ken continually debated, but only one

critical aspect will be briefly addressed at this moment: The difference between somatic

ce11 and germ-line modification. Most experiments involve somatic cells, whereby the

genornic alterations are expressed exclusively in the recipients, not their offspring. This

type of transformation has been likened to surgery (Weatherall, 1995). Germ-line

modification, which is prohibited by law with respect to human trials, involves changes

that would affect future generations and alter the gene pool, and is a much more

contentious issue.

1.1.2 Recent Gene Therapy Setbacks and Breakthroughs

During the mid-1990's gene therapy was hailed as a potentially curative

therapeutic method for rnany diseases including inherited monogenic diseases, A D S , and

cancer. Over the past few years, the general attitude towards gene therapy has changed.

It has k e n widely reported that the outcome of clinical trials in terms of gene transfer,

gene expression, and especially clinicai benefit has been disappointing. This partly

reflects the inordinately high profile of the early ciinical studies and the umealistic

expectations of both the public and the scientific cornmunit y. The negative feelings

towards gene therapy peaked in the fa11 of 1999 following the highly publicized death of

a patient who participated in a trial for a rare liver disease and suffered from a massive

immune response following direct intra-hepatic delivery of a high dose of adenoviral-

mediated gene replacement therapy (Hollon, 2000).

While diseases have not yet been cured with gene therapy, significant progress

has been made in a field that is only 10 years old. This progress is highlighted by the

recent publication of two reports containing gene therapy protocols that have met with

great initiai success (Cavazzana-Calvo et al., 2000; Kay et al., 2ûûû). Kay et al. have

published the initial results of a clinical vi-al where three individuals with hemophilia B

received injections of a replication incornpetent adeno-associated viral vector containing

the gene that encodes factor IX, the clotting protein that such patients lack (Kay et al.,

2000). Even though the patients were given a very bw dose of the therapeutic vector, six

mon ths after treatment , two patients continued to experience a signi ficant reduction (50-

80%) in the number of bleeding events, and a corresponding reduction in the amount of

exogenous factor IX required to controi their bleeding episodes. In the two successfully

treated patients, severe hemophiiia B has, in effect, been converted to a milder form of

the disease.

The second major success involves the treatment of three infants with X-linked

severe combined immunodeficiency disease (SCID-XI) (Cavazzana-Calvo er al., 2000).

Affected individuals, who have a yc cytokine receptor deficiency that leads to a block in

T and naturai killer (NK) lymphocyte differentiation, generally die within their first year

of iife unIess they are successfully treated with a bone rnarrow transplant. In an ex vivo

setting, these investigators used a retrovirus to insert a copy of the yc gene into the

patients' CD34+ stem cells, and then re-introduced the stem cells to the patients.

Detectable levels of T and NK cells containing the introduced gene were found in the

blood within 60 days, and their numbers increased progressively until normal levels were

reached. Al1 three children were able to leave the hospital within four months of

treatment, and in the I l months since treatment, the children continue to have functioning

immune systems, exhibit no side effects, and enjoy apparently normal development.

Therefore, these two studies may be the frst evidence of significant clinical benefit for

patients treated with a gene transfer approach.

1.1.3 Cancer Gene Therapy

The genetic changes that lead to the development of cancer have begun to be

elucidated. It is now known that cancers &se as a result of multiple inherited andior

acquired mutations followed by the clonal selection of mutant cells with aggressive

growth characteristics. The two main targets for mutations that result in oncogenesis

appear to be proto-oncogenes and tumor suppressor genes, although mutations in many

other types of genes are also involved in the progression to a malignant phenotype. The

increased understanding of the genetics of cancer have allowed scientists to develop

strategies to treat cancer by correcting the genetic defects and also by manîpulating genes

to carry out tumoncidal activities.

The largest incentive for researching novel cancer treatrnents such as gene therapy

is the fact that current treatrnents, despite their successes, have many limitations. Surgery

is not always feasible given the location of the tumor. When possible, it is invasive and

unable to nd the body of cancerous cells outside the field of surgery (as in the case of

metastasis). Radiation and chemotherapy lack sufficient specificity, and are therefore

toxic to normal tissues. As well, cancer cells kequently develop resistance to

chemotherapeutic agents. Targeting the genetic basis of cancer through gene therapy has

the potential for selective destruction of tumor cells by improving the specificity of the

treatment, while reducing systernic toxicity. It is important to note that if one or more

forms of gene therapy are approved for patient use in the near future, it will almost

certainly be employed in tandem with more conventional therapeutics, and not as a

'magic bullet' that will cure the disease on its own.

The selection of the vector is a crucial step in the development of a gene therapy

strategy because the vector dictates both the eficiency of gene transfer and the duration

of gene expression. There are two major classes of vectors, viral and non-viral, both of

which were recently reviewed by Jain (Jain, 1998). To date, viral vectors have been the

most frequently studied, with an emphasis on retrovinises, and adenovinises, each of

which possess its own advantages and disadvantages. Retroviral vectors integrate their

genomes into host chromosomes, thereby offenng the chance for continued expression of

the transgene. Unfortunately, the integration is random, so host genes rnay be disrupted.

Additionally, retrovüuses infect only dividing celis (Miller et al., 1990). Adenovirai

vectors offer transient gene expression since the transgene is not integrated into the hast

genes (Zhang et al., 1995). Adenoviruses infect both dividing and non-dividing ceils,

and the level of transgene expression can be very high, especially in epithelial cells

(Zhang et al., 1995). Other viral vectors that have been studied include herpes virus,

adeno-associated virus, and vaccinia virus vectors. Due to the concern over inserting

potentially pathogenic viral vectors into an already diseased patient, the prospect of a

non-viral vector is appealing. The most highly researched prospect is the use of cationic

liposomes to package the foreign gene and prornoter (Huang, 1999). The liposomes are

then incorporated into the target cells through endocytosis. Until recently, gene

expression using cationic liposomes was very low, but new advances in targeting the

liposome to cancer cells demonstrate the potential of this technique for the hture (Xu et

al., 1999).

There are a number of different methods that have been explored in terms of gene

therapy for cancer. The major methods include the expression of tumor suppressor

genes, the in hibition of oncogenes, the induction of dmg sensitivit y, chernoprotection of

normal cells, and immunotherapy. These potential strategies are by no rneans a11 that is

king investigated, but represent a sarnple of some of the more established methods. It is

also important to reaiize that each therapeutic method is not necessarily appropriate for

every variety of cancer. Due to their unique genetic makeup and physical manifestations,

different cancers often require distinct types of therapy. Experiments in this field are

ongoing, and new methods and targets are being evaluated ahost daily.

The remainder of Chapter 1 will introduce the topic of nasopharyngeal carcinoma

and its current treatrnents. This will be followed by a discussion of the adenovirus and its

application as a viral vector for delivering target genes, and the p53 tumor suppressor

gene and its use in cancer gene therapy.

1.2 NASOPHARYNGEAL CARCINOMA

1.2-1 Introduction

Nasopharyngeal carcinoma (NPC) is a malignant disease of the headlneck region

that is rare in most areas of the world including Western Europe and North America, but

is endemic to southem China, and common to a number of other geographical regions

(Fandi & Cvitkovic, 1995). NPC is most frequently classified into two histological types:

squamous ce11 carcinoma (SCC), and undifferentiated carcinoma of nasopharyngeal type

(UCNT) (Fandi et al ., 1994).

In areas of the world where NPC is rare, most populations exhibit an incidence of

0.5 to 2 per 100,000 persons per year (Altun et al., 1995), although the incidence is

higher in citjes with large Asian populations, such as Toronto. In North America and

Western Europe, NPC occurs sporadically and is primarily related to exposure to the

classic head and neck cancer risk factors, such as aicohol and tobacco (Vokes et al.,

1993). SCC accounts for 35%-50% of NPC in low risk populations, and this type of NPC

is sirnilar to carcinomas that arise from other sites of the head and neck. (Fandi et al.,

1994).

NPC is endemic to Southern China where the incidence ranges fiom 30 to 80 per

100,000 persons per year. (Fandi & Cvitkovic, 1995). Other populations or regions with

high incidence include Eskimos from Alaska and Greenland, and the general populations

in South East Asia, North Africa and regions surrounding the Mediterranean basin, and

the Caribbean nations. In these populations, SCC accounts for less than 5% of NPC that

is diagnosed, while an overwhelming majority of patients (over 95%) have UCNT (Fandi

& Cvitkovic, 1995). Unlike SCC type NPC, UCNT bears no apparent relationship to

either alcohol or tobacco consumption. The age distribution for UCNT is much younger

than for cancer of other head and neck sites, including SCC type WC. The rnean age at

which W C occurs in the high risk populations is 40 years, which is 20 years younger

than the rest of the world, and males are three times as likely to develop NPC as females

(Altun et al., 1995).

The etiology of endemic NPC is not well understood, but a nurnber of factors

have been implicated, with Epstein-Barr virus (EBV) king the most widely studied (Hsu

& Glaser, 2 0 ) . Other etiological factors likely include genetic susceptibility, early

exposure to wood fres, herbal medicines and the consumption of preserved foods,

especially salted fish (Zheng et al., 1994).

1.2.2 Treatment of NPC

The current treatment regimen for NPC (both SCC and UNCT) yields a 5-year

survival rate of approximately 65% (Al-Sarraf et al., 1998; Altun et al., 1995; Payne et

al., 1996). Treatment for NPC is administered with the intent to cure for patients with

locally and regionally confined tumors, with radiotherapy being the primary treatment

since surgery is not an option given the anatomical location of the disease.

Although NPC is highly curable in its early stages, the clinical warning signs such

as nose bleeds, a stuffed nose, and enlarged glands in the neck are easily missed and

frequently misdiagnosed (Liu, 1999; Vokes et al., 1997). In fact, only 25% of patients

are diagnosed in stages 1 or II of the disease, where the 5-year survivai is between 75%

and 95% (Liu, 1999).

In recent years, the combination of chemtherapy plus radiotherapy has been the

subject of intensive research in an effort to improve survival, especially in the more

advanced cases in which radiotherapy alone has a lower control rate (Al-Sarraf et al.,

1998; Chan et al., 1998). Cisplatin is by far the most fiequently used chemotherapeutic

agent in the treatment of WC. Kt has been utilized either done or in combination with

another agent, such as 5-fluorouraçil (S-FU).

A recent randornized study demonstrated that concomitant chemo-radiotherapy

demonstrated an improved disease-free and overail 2-year survival rate for patients with

stage Iiï or IV NPC (Al-Sarraf et al., 1998). Ba& on these survival data, the

administration of concomitant cisplatin and radiotherapy has becorne the standard of care

for NPC in the United States (Vokes et al., 1997). A number of small studies examining

chemotherapeutic regimens (ali of which include cisplatin) in the treatment of metatsatic

disease indicate that chemotherapy can prolong survival, and that a subset of the patients

may achieve long term survival (Chan et al., 1998).

1.2.3 EBV and NPC

EBV is a double-stranded DNA virus of 172 kb belonging to the herpesvirus

family. Oropharyngeal epithelial cells were once thought to be the site of primary

infection, but it is now believed that EBV targets B-cells located in pharyngeal tissues

(Hsu & Glaser, 2000). EBV is transmitted through saliva, and seroepidemioiogic studies

indicate that over 90% of adults worldwide are infected with EBV (Hsu & Glaser, 2000).

Pnmary infection with EBV usuaily occurs in childhood, where a mild self-limiting

illness may or may not be detected. If infection is delayed until adolescence or

adulthood, infectious mononucleosis results in approximately 50% of cases (Vokes et al.,

1997). Following the initial infection, where the virus is in its lytic phase, the virus

continues to exist as an episome in a small number of latently infected B-cells.

Therefore, most individuals are asymptornatically infected for life (Hsu & Glaser, 2000).

Aside fkom NPC, a growing number of cancers appear to be associated with EBV

infection, including Burkitt's lymphoma, Hodgkin' s disease, and gastric carcinoma.

Although individual studies Vary in the exact percentage of NPC patients expressing EBV

in their NPC cells, reports suggest that between 80% to over 95% of NPC patients are

positive for EBV (Hsu & Glaser, 2000; Liebowitz, 1994; Raab-Traub, 1992).

The method by which EBV enters NPC cells remains unclear, but it appears that

EBV might play a causal role in the development of rnalignant WC. The malignant

epithelial cells of NPC are infected with EBV, but the surrounding normal epithelial cells

have not k e n s h o w to harbor the virus (Young, 1996). As well, in a longitudinal study

of archiva1 patient samples with pre-invasive or dysplastic legions, in which some

patients subsequently deveIoped NPC, latent gene products of EBV such as EBV

encoded RNAs (EBERs), and Latent Membrane Protein 1 (LMP-1) were identified in

these precursor lesions. The EBV DNA within individual patients was also observed to

be clonal, in contrast to the variability of the EBV genome within infected B-cells of the

same patient (Pathrnanathan et al., 1995). This finding suggests that the viral genomes

are derived from a single virus present pnor to rnalignant transformation and expansion

(Hsu & Glaser, 2000). In terms of tumor progression, LMP-1 has been consistently

shown to be transfonning in vitro (Fahraeus et al., 1990), and also to be oncogenic

(Moorthy & Thorley-Lawson, 1993).

The EBV that is expressed in NPC cells is in the latent forrn, where only a small

number of its more than 100 genes are expressed. There is some evidence that two latent

EBV protein products, BZLF-L and EBNA-5, may alter p53 function by binding to p53,

possibly making the NPC cells more aggressive, andfor refiactive to therapy (Szekely et

al., 1993; Zhang et al., 1994).

1.2.4 p53 and NPC

There is currently some controversy with regard to p53 status in human NPC.

The majont y of reports suggest that the p53 gene is not mutated in the pnmary tumor, but

is more likely to be mutated in metastases and derived cell lines (Effert et al., 1992; Lo et

al., 1992; Spruck et al., 1992; Sun et al., 1992). However, two reports that examined p53

expression using imrnunohistochemical techniques indicate that the majonty of human

WC's overexpress the p53 protein (Chen & Cooper, 1996; Porter et al., 1994)-

suggesting a b n o d function. As was mentioned in the previous section, latent protein

products of EBV have been shown to bind to p53 (Szekely et al., 1993; Zhang et al,,

1994). The lünctional significance of this protein binding is controversiai, but it might in

part account for the conflicting observations with regard to pS3 in human NPC, given that

NPC is genotypically wild type for p53, but functionally appears to be mutant for p53.

1.2.5 NPC Ce11 Lines

The establishment of NPC ceil lines has proven to be very difficult, for reasons

which are unclear (Chang et al., 1989)- Our Iab has been unable to generate any ce11 lines

from biopsy sarnples. Over the past twenty years a handful of groups have successfuily

established a few NPC lines as well as a number of rnouse xenograft rnodels (Busson e t

al., 1988; Chang et al., 1989; Huang et al., 1980; Hui et al., 1998; Sizhong et al., 1983;

Yao et al., 1990; Zeng, 1978).

Our lab O btained the CNE- 1 W C ce11 line (along with the CNE-22 NPC ce11 line)

from the Chinese Academy of Medical Sciences in Beijing, China (Sizhong et al., 1983;

Zeng, 1978). The CNE-1 ce11 line has a heterozygous mutation in the p53 gene within

exon 8, resulting in a change in the nucleotide sequence €rom AGA to ACA at codon 280

(Effert et al., 1992; Lo et al., 1992). The mutation in p53 has been found to produce a

dominant negative mutant p53 protein, which can block the transcriptional activities of

wild-type pS3 (Sun et al., 1992).

As was stated in a previous section, there is close association of EBV and WC.

But most of the NPC ce11 iines established over the past two decades had shed their EBV

during in v i t ro propagation. Recently, our lab acquired an EBV positive NPC ce11 line,

C666-1, from the Chinese University of Hong Kong (Cheung et al., 1999; Hui et al.,

1998). The C666-1 cell Iine was derived from the NPC xenograft xeno-666 that was

established from an undifferentiated NPC biopsy (Hui et al., 1998). This line has k e n

shown to consistently express the EBV-encoded RNAs LMP-1 and LMP-2, as well as the

EBNA-1 protein (Cheung et al., 1999). Our lab has demonstrated that there is a mutation

in the p53 gene of C666-1, a deletion in Exon 7 of p53 resulting in a premature stop

codon. The mutation is Iikely heterozygous in nature (Li et al., 2000).

1.3 CISPLATIN

1.3.1 Introduction

Cisplatin (cisaiarnrninedichloroplatinum) was first discovered as a biological

agent in 1965 foIIowing the observation that an electric current delivered to a bacterial

culture via platinum electrodes led to the inhibition of bacterial growth (Rosenberg et al.,

1965). The active compound was subsequently found to be cisplatin. Since the initial

discovery, cisplatin has becorne a widely used anticancer dmg. Cisplatin based

chemotherapy is curative for rnany patients with testicular carcinoma, and is also used in

the treatment of a variety of solid tumors, including ovarian, bladder, cervical, lung, and

head and neck cancers (Perez, 1998) (see Chapter 1.2.2).

S imi lar to other chemotherapeutic dmgs, patients ueated with cisplatin may

experience a number of side effects including nausea and vorniting, hearing loss, and

neurotoxicity. Unlike many chemotherapeutic agents, cisplatin causes little toxicity to

the bone marrow on its own, but it can add to the toxic effects of other drugs. The dose-

limiting toxicity is that of kidney damage, although the effects on the kidneys may be

minirnized through saline infusion in order to maintain rapid urine output during

treatment (O'Dwyer P., 1997).

1.3.2 Cisplatin Stmcture and Mechanism

Cisplatin is a neutral square-planar compound. The chloride ligands are stable at

extracellular chloride concentrations, but after diffusion into a cell, the lower chloride

concentration facilitates the exchange of the chloride ligands for water or hydroxyl

groups. This exchange produces a bifunctional charged electrophile that can react with

nucleophilic sites in the ce11 (Roberts & Thomson, 1979) (See Figure 1.1).

Figure 1.1 The interaction of cisplatin with intracellular moiecules- (From Eastman, 1990)

Although most of the cisplatin interacts with protein, RNA, and small thiol compounds in

the cell, approximately 1% reacts with genomic DNA, and it is DNA that has been

implicated as the critical target for cisplatin rnediated toxicity (Eastman, 1990). The

potential contribution of cisplatin induced RNA or protein damage to cyîotoxicity is less

clearly defined (Perez et al., 1997).

Cisplatin's bifunctional binding to DNA (one attachrnent for each chloride ligand

that is displaced) can potentially produce many different adduct structures (see Figure

1 3 , but evidence indicates that majority of lesions are DNA-intrastrand cross-links

(Eastman, 1987). The major lesion is a cross-link between adjacent guanines on the same

strand; these represent approximately 65% of the lesions formed between DNA and

cisplatin. A further 25% of the lesions are cross-links between a neighboring guanine

and adenine, and the remaining intrastrand cross-links are between two guanines

separated by one base. Cisplatin induced DNA-interstrand cross-links and DNA-

intermolecular cross-links (such as DNA-protein cross-links) represent approximately 1 %

of the platination of DNA, and cisplatin induces even fewer monofùnctional Iesions.

Figure 1.2 Structures of the various adducts produced in DNA by cispIatin. (From Eastman, 1990)

Although it is known that the formation of cisplatin induced DNA-crosslinks that

remain unrepaired lead to ce11 death (Roberts & Thomson, 1979), the speciflc signaling

pathway by which the cells die remains unknown. It has been proposed that cells anest

in the G2 phase of the ce11 cycle foilowing cisplatin exposure, and those ceils that are not

repaired go on to die (Sorenson & Eastman, 1988). Considerable evidence indicates that

cisplatin kills cells by apoptosis (Eastman, 1990; Sorenson et al., 1990), but the specific

mechanism(s) that trigger apoptosis in response to cisplatin have not yet been defined.

The topic of cisplatin, apoptosis, and the involvement of p53 are further discussed in

Section 2.5.

1.4 ADENOVIRUS

1.4.1 Introduction

Adenovimses are DNA viruses that predominantly infect epithelial cells. Lytic

infection results in ce11 death and the production of 10,000-1,000,000 progeny vinises per

cell. Human adenovirus infections are ubiquitous, although there are slight variations in

the more than 40 human serotypes causing various syndromes in different parts of the

world. Most of the population has experienced infection with an adenovirus by the t h e

they are 10 years old (Baum, 1990).

Adenovirus infection most ffequently causes rnild upper respiratory tract

infections in children (Le. a cornmon cold), but it has also been implicated in more

serious respiratory diseases, infantile diarrhea, conjunctivitis, and central nervous system

infect ion (Baum, 1990). There are no specific therapeutic measures available for the

treatrnent of adenovirus infectious, but most of the syndromes are rnild and self-limiting.

Some adenoviruses are dso capable of transforming prirnary rat and mouse ceils, but the

virus has not been implicated in the development of human cancers (Baurn, 1990).

1.4.2 Adenoviml Stnicture and Genome

Adenoviral DNA is double stranded and linea., and the intact virion has a

diameter of about 70 nm (Home er al., 1959). The vud genomic DNA is 36 kb in length

(Chroboczek et al., 1992), and is contained in a protein coat, known as a capsid. The

capsid is arranged in an icosahedral structure that has 20 sides and 12 vertices. The

majority of the capsid proteins are hexons, which have six nearest neighbors, while the

twelve vertices are occupied with pentons, which have five nearest neighbors. Finally, a

rodlike structure with a knob at the end, hown as a fiber, projects from each penton.

The hexons, pentons, and fibers differ from one another immunologically as well as

morp hologicall y (S henk, 1996).

The viral core is composed of the adenoviral genome plus a number of associated

proteins. Polypeptide V links the viral genome with the capsid, polypeptide VI1 acts as a

histone-like protein that is wrapped in the viral DNA, and the terminal protein acts as an

initiation site for DNA replication and facilitates the attachrnent of viral DNA to the

nucleus of infected cells (Shenk, 1996). The genes that rnake up the adenoviral genorne

have been divided into two groups based on their tuw of transciption. There are five

early gene groups (E 1 A, ElB, E2, E3, and E4) and five late gene groups (LI-L5) (Shenk,

1996).

1.4.3 Adenoviral Life Cycle

The life cycIe of the adenovhs is divided into eady and late phases, the latter

beginning at the onset of DNA replication. The v w s enters the cell by means of two

receptors. The viral cycle begins with the attachment of the fiber to a cellular receptor,

the coxsackievirus and adenovirus receptor (CAR) (Bergelson et al., 1997). The

attachment is followed by the interaction of the penton base with the surface integrins

a& and a& that serve as receptors that mediate the internalization of the virus

(Wickham et al., 1993). Adenovirus pentons contain five Arg-Gly-Asp (RGD) motifs

which are recognized by a, integrins, and are often contained in ce11 adhesion molecules

suc h as fibronectin (Wickham et al., 1993). Following receptor-mediated endoc ytosis,

penton mediated activity ruptures the endosornai cornpartment, and the virus is released

into the cytoplasm. The viral core migrates to the nucleus where the viral DNA enters

through nuclear pores. The majonty of viruses reach the nucleus in approximately 1 hour

(Leopold et al., 1998).

The first rnRNA, and protein, to be made after infection is the EarlylA (HA)

protein. E1A is a transcnptional regulatory factor that is necessary for the transcnptional

activation of the rernaining early genes (Osborne & Berk, 1983). The second protein

made is EIB. ElA and E1B together are capable of transforming primary cells in vitro

(Whyte el al., 1989). ELA binds to pRb and can stimulate cellular proliferation (Whyte et

al., 1989), while ElB binds to p53 and can inhibit p53-activated transcription and p53-

mediated apoptosis (Han et al., 1996; Martin & Berk, 1999). Both Rb and p53 are tumor

suppressor proteins involved in the regulation and control of the ce11 cycle, and the

binding of EIA and El% to these proteins leads to an increase in cellular proliferation

and a decrease in ce11 death. As a result, adenoviruses are able to enhance their own

survival,

At the onset of adenoviral DNA replication, the pattern of transcription changes

from the early to the Iate genes, which are transcribed fiom the major late promoter. The

late proteins are structural proteins involved in virus assembly (Shenk, 1996).

1.4.4 Adenovinis as a Vector for Gene Therapy

Adenoviral vectors for gene therap y are adenoviruses that have been geneticall y

modified by introducing deletions in the viral genome to create space for the foreign gene

to be inserted, and to create a replication defective virus to achieve greater safety (Zhang

et al., 1995). The most frequently used adenovirus serotype in gene therapy is type 5,

which has k e n used for many years as a human vaccine without detectable side-effects

(Siegfried, 1993). For the construction of the fist generation of adenovims vectors, the

adenovirus is rendered repiication incompetent by deletion of the ELA and E1B genes.

These genes are replaced by the therapeutic gene of interest and any appropriate

regdatory sequences, such as promoter and enhancer elements. Rernoval of the non-

essential E3 gene creates more space, thereby allowing the insertion of up to 7.5 kb of

exogenous DNA (Graham & Prevec, 199 1).

There are a number of advantages associated with the use of an adenovirus vector

system for cancer gene therapy: 1. Adenovirus vectors can be prepared at much higher

titres compared to many other vector systems. 2. The vectors can infect both àividing

and non-dividing cells. 3. The adenovirus exhibits a high infection efflciency in many

target cells. 4. Finally, adenovirus genomes do not integrate into the host ce11

chromosomes, and are suited for directing transient gene expression, which is al1 that

shouid be necessary to ablate turnor ceUs (if that is the goal).

As with any system, there are also a few disadvantages to the use of the

adenovirus vector: 1. Adenovïrus can induce an inflammatory response, and repeated

administration can elicit an anti-viral response that may limit the therapeutic effect. 2.

The adenovirus can infect a wide range of different ceils, making it difficult to deliver

genes to specific target cells by intravenous administration (Channon & George, 1997).

A new generation of adenoviral vectors have k e n designed, and are in the

process of king tested. The newer generation of vectors have further deletions in

various regions of the E2 or E4 genes (Channon & George, 1997). These deletions allow

for insertion of more exogenous DNA, and should also reduce the host immune response,

compared with the first generation vectors.

1.5 p53

1.5.1 Introduction

The p53 tumor suppressor gene and its corresponding protein have becorne one of

the most intensely studied molecules in cancer research since their discovery over two

decades ago (Lane & Crawford, 1979). p53 has since k e n denoted as 'the guardian of

the genome', and 'gatekeeper of the cell', for its d e in preventing the accumulation of

genetic alterations through the regulation of criticai checkpoints in response to distinct

stresses (Lane, 1992). The p53 protein, a transcription factor, is stabilized and activated

in response to a number of stressfbl stimuli including exposure of cells to DNA damaging

agents, h ypoxia, growt h factors, or activated oncogenes (el-De-, 1998) (see Figure 1.3).

Activation of p53 allows it to function as a tumor suppressor through a number of growdi

controiIing endpoints. The most widely studied downstream effects of p53 activation

include growth arrest and apoptosis, but senescence, differentiation, and anti-

angiogenesis have also been implicated in p53 activation (el-Deiry, 1998). The structure

and activation of p53, as well as its growth arrest and apoptotic downstream responses

will be further discussed in the following sections.

CELLULAR STRESSES Including

Geno toxicity Hypoxia Hea t S hock Hyperoxia Growth Factors Cytokines Metabolic Changes Anchorage Cell-ce11 contact Activated oncogenes Others

CeU and rissue ope specific nzodfiers

ADAPTIVE RESPONSES Including

Apoptosis - Growth Arrest - Other???

Figure 1 3 The many stimuIi that activate pS3 and the potential resultant consequences. (Adapted from Rives & Hall, 1999)

Germ-line mutations in one allele of the p53 gene were fust described in members

of families who were identified with Li-Fraumeni syndrome (Malkin et al., 1990).

Although the syndrome is rare, the risk of developing cancer in these families can reach

90% by the age of 50. Somatic ceii mutations in the p53 gene occur in more than 50% of

human malignancies and in diverse tumor types (Greenblatt et al., 1994). Abnormalities

of p53 appear to be the most prevalent molecular abnormalities in human cancer, with

missense point mutations in the DNA binding dornain being particularly prevalent (Prîves

& Hall, 1999). Loss of p53 function is also observed due to overexpression of the

cel1ula.r oncoprotein Mdm2, which blocks p53 function, or the inactivation of pl4ARF,

which binds to and inhibits Mdm2 function (Pich, 1998; Prives & Hall, 1999). Findly,

p53 is a frequent target for inactivation by virally encoded proteins such as the HPV

oncoprotein E6, which is associated with cervical carcinoma, and the EBV proteins

EBNA-5 and BSLF-1, which rnay be involved in nasopharyngeal carcinoma (Pich,

1998). Therefore, it may be that the p53 pathway is functionally inactivated due to one or

more mechanisms in virtually al1 human cancers.

The clinical significance of p53 mutations remains a controversial topic.

Although exceptions exist, the pattern and fkequency of p53 mutations in various tumor

types roughly correlate to the tumor's responsiveness to therapy (Peller, 1998). For

example, tumor types displaying a high frequency of p53 mutations are generaily not as

responsive to either chemotherapy or radiotherapy as tumors that rarely harbor p53

mutations (ie. breast cancer versus testicular cancer) (Lowe et al., 1994). Within the

same tumor type, such as breast carcinoma, studies have associated p53 mutations with

poor patient prognosis (Lowe et al., 1994). If the cytotoxicity of anticancer agents is

determined by a p53-dependent mechanism, then tumor cells with p53 mutations would

be more resistant to chemotherapy. But, anticancer agents can induce apoptosis in the

absence of p53 function in a number of ce11 lines, so clearly, the p53 protein is not

essential for cancer ce11 killing (Bracey er al., 1995). Nonetheless, the potential clinical

utility of replacing p53 in cancer cells is obvious, and the use of p53 gene therapy will be

discussed in Section 1.5.6.

1.5.2 p53 Structure

The human p53 gene is localized to the short arm of chromosome 17. The

product of the pS3 gene is a 53-kDa nuclear phosphoprotein that is composed of 393

amino acids. The functional molecule is a tetramer that acts as a transcription factor. p53

has been highly conserved during evolution, underscoring its importance to cellular

function. Through cross-species cornparison of the amino acid sequences of p53

proteins, five highly conserved regions (more than 90% homology) within the arnino acid

residues 13-23, 1 17- 142, 17 1- 18 1, 234-250, and 270-286 have been identified (Soussi et

al., 1990).

The human p53 protein has been divided stnicturally and functionally into four

dornains (see Figure 1.4). The first 42 amino acids at the N-terminus constitute the

transcriptionai activation domain. It is this region that interacts with the basal

transcriptional machinery in positively regulating gene expression (Lu & Levine, 1995).

pS3 transcriptionai activation is negatively regulated by the adenovirus ElB-55kDa

protein and the human Mdm2 protein, both of which bind to the N-terminus of p53 (Lin

et al., 1994).

The sequence-specific DNA binding domain of p53 is localized between amino

acids 102 and 292, and can recognize and bind to consensus target sequences. It is this

domain that contains four out of the five highly conserved regions, and it is also within

this domain where 80-901 of p53 mutations have been identified (May & May, 1999).

The native p53 protein is a tetramer in solution, and it is the C-terminus of p53

that is responsible for the formation of this structure. The oligomerization domain is

contained within arnino acids 323-356. Adjacent to the oligomerization domain is a

region (amino acids 363-393) which has k e n referred to as a transcriptional regulatory

domain which regulates sequence specific DNA binding and a DNA damage recognition

domain (Wang & Prives, 1995). Also spread throughout the C-terminal region of p53 are

three nuclear localization signals @ang & Lee, 1989).

Activation domain

Sequence specific DNA Tetramerization Regulatory binding domain domain domaîn

1 II m IV v Evolutionarily highly conserved regions

Figure 1.4 A representation of the structure of p53 protein. The figure highlights five highly conserved regions, and four domains which are involved in different functions.

1.5.3 p53 S tabiiization and Activation

In normal mammalian cells, p53 is present at extremely low levels because the

protein is very rapidly degraded

minutes (Kubbutat et al., 1998).

following synthesis,

p53 is targeted for

exhibiting a half-life of 20-30

degradation by the proteosorne

following ubiquitination. Mdm2 participates in the regulation of the stability of p53 by

24

helping to rnediate this degradation. It has been reported that Md& can interact with

p53 in undamaged cells and target it for ubiquitin mediated degradation (Kubbutat et al.,

1997).

Mdm2 also binds to the p53 protein and inhibits the ability of pS3 to act as a

transcription factor. Mdm2 binds to the N-terminus of p53, within the transactivation

dornain where p53, as a transcription factor, contacts other components of the basai

transcript ional machinery. The binding of Mdm2 inhibits normal function of this region

of p53, reducing the ability of p53 to activate gene expression (Momand et al., 1992).

Interestingly, the promoter of the mdm2 gene contains a p53 binding site and is

transcribed in a p53-dependent manner (Barak et al., 1993). This has led to a mode1 in

which p53 up-regulates the Md& protein, therefore providing a negative feedback

regulatory loop for p53 activity. The control that Mdm2 exerts over p53 is essential for

normal development. This has been demonstrated by embryonic lethality in Mdm2-

deficient rnice which can be rescued by the simultaneous deletion of p53 (Jones et al.,

1995).

As previously mentioned, the levels of p53 rise dramatically in response to many

foms of stress including DNA darnage, oncogene activation, hypoxia, and changes in pH

or temperature. This change in p53 level appears to be achieved for the rnost part

through pst-translational modifications of the p53 polypeptide (Kastan et al., 1991).

Following DNA damage, such as that induced by ionizing irradiation or chemotherapy,

p53 is stabilized and activated. The events that occur upstream of p53 have only recently

begun to be elucidated, and this pathway is still in the process of k i n g mapped. Some of

the advances in the field of p53 activation were reviewed in Lakin & Jackson (Lakin &

Jackson, 1999).

Phosphorylation of p53 in response to DNA damage appears to be one important

mechanism by which its activation is modulated- p53 is phosphorylated at several senne

residues within its amino- and carboxy-terminal domains, and many of these

phosphorylations are inducible upon DNA damage. For example, the phosphorylation of

serines 15, 20,37, and 392, among others, are induced by various forms of DNA damage

including XRT and W (ultraviolet) light (Lakin & Jackson, 1999). Many protein

kinases, sorne of which have been shown to be involved in the detection, signaling,

andior repair of DNA damage, c m phosporylate p53 in vitro and/or in vivo including

ATM kinase, ATM-related kinase (ATR), c heckpoint kinases (Chkl and Chk2), DNA-

dependent protein kinase @NA-PK), and casein kinase II (cm) (Lakin & Jackson,

1999). While the consequences of p53 phosphorylation at all the sites have not yet been

determined, experimental evidence suggests that the phosphorylation of either serine 15

or 20 can reduce the ability of Mdm2 to negatively regulate p53 (Shieh sr al., 1997;

Unger et al., 1999). Therefore, p53 stability in response to certain DNA darnaging agents

is believed to occur, at least in part, by phosphorylation of p53 at serine 15 andior 20,

thereby dismpting the MdmUp53 cornplex, and increasing the half-life and

transcriptional activation properties of p53.

Another mechanism through which p53 activation may be controlled is

acetylation. For example, CBPIp300 has been shown to be able to acetylate p53 at

lysines 373 and 382 in vitro, and the acetylation of these residues has been found to

activate p53 sequence-specific DNA binding (Lakm & Jackson, 1999). It is possible that

the activation of p53 is regulated by a combination of phosphorylation and acetylation

events, and that p53 activation is stimulus specific and ce1 type specific. Additionally,

this area of investigation is relatively new, and more than iikely, not dl, or even most of

the molecuies involved in translating DNA damage into a p53 dependent response have

k e n identified.

Although the effects of p53 stablization and activation manifest themselves in

numerous downstrearn effects, the following two sections will deal only with ceU cycle

arrest and apoptosis, two of the major consequences following DNA damage.

1.5.4 p53 Downstream Effects - Ce11 Cycle Arrest p53 plays an important role in regulating checkpoints during the G l and G2

phases of the ce11 cycle (see Figure 1.5). These checkpoints presumably prevent cells

with damaged genomes fiom undergoing DNA replication or mitosis. The expression of

p21 is controlled by p53 through p53 DNA-binding response elements Iocated within the

p2I promoter. p2I rnediates p53-dependent G1 arrest by binding to cyclin dependent

kinases (CDKs) and inhibiting their kinase activity so that they cannot phosphorylate the

retinoblastoma (pRb) protein (Harper et al., 1993). In its hypophosphorylated form, pRb

sequesters the E2F transcription factor, thereby preventing the transition from G1 to S

phase. Failure to mest in G1 following DNA damage, which can occur in tumor cells

with a mutant p53 gene, leads to DNA replication using a damaged DNA template. This

scenario will therefore contribute to enhanced genetic alterations.

The role of p53 in G2 arrest is less clear. Although p53 deficient cells can arrest

in G2 following exposure to ionizing radiation, it appears that p53 may contribute to G2

amest when it is present in its wild-type form. In this regard, two p53-target genes have

emerged as potential mediators of p53-dependent G2 arrest. 14-3-30 sequesters cdc25, a

phosphatase of the cycIinB/cdkl compIex that is essential for the G2-M transition (Peng

et al., 1997). A second rnediator appears to be GADD45, which directly disrupts the

cyclinBkdc2 complex (Wang et al., 1999). Both 14-3-3a and GADD4S contain p53

DNA-binding response elements.

DNA Damage + I pz1

1 Cyclidcd k complexes

? GADID45 ? 14-3-3a . I

Figure 1.5 The effectors of p53 chat lead to a ce11 cycle arrest response following DNA damage-

1.5.5 p53 Downstream Effects - Apoptosis The p53 tumor suppressor protein can also respond to cellular stress by signaling

the apoptotic machinery to induce programrned ce11 death. Programmed ceIl death, also

know as apoptosis, is a process of ce11 suicide that occurs through characteristic

morphological changes. The apoptotic changes include ce11 shrinkage, nuclear

condensation, DNA fragmentation, and plasma membrane blebbing (Kerr et al., 1972).

These morphological changes result fiom the activity of intraceilular cysteine proteases

called caspases (Salvesen & Dixit, 1997). Although the mechanism by which p53

initiates apoptosis rernains to be fully elucidated, several transcriptional targets of p53

have k e n identified that mechanistically link p53 to caspase activation and apoptosis

(see Figure 1.6).

The frst p53 target gene identified to encode a candidate effector of p53-mediated

apoptosis was bax, a pro-apoptotic protein that is a member of the Bcl-2 family of

proteins (Miyashita & Reed, 1995). The ratio of Bax:Bcl-2 appears to be important in

determining whether cells live or die. Bax and Bcl-2 control apoptosis at the level of

rnitochondrial cytochrome c release; Bax promotes its reIease whereas Bcl-2 blocks the

release of cytochrome c from the mitochondria (Rosse et al., 1998; Yang et al., 1997).

Once released from the mitchondria, cytosolic cytochrome c (in concert with APAFI)

appears to mediate the activation of initiator caspase 9, which triggers a caspase cascade

leading to apoptosis (Srinivasula et al., 1998). The relative contribution of Bax to p53-

mediated apoptosis appears to be cell type dependent. Thus, Bax is not required for

radiation induced, p53-dependent apoptosis in thyrnocytes (Knudson et al., 1995), but

Bax-knockout fibro blasts appear to be compromised in DNA-damage induced apoptosis

(McCurrach et al., 1997).

Ce11 surface death receptors can also transmit rapid apoptotic signals initiated by

the binding of death ligands. Transcription of the death receptor Fas is induced by p53

through a p53-response element. This induction has been show to contnbute to ce11

death by anti-cancer dmgs (fiesen et al., 1996), but as in the case of Bax, the d e of Fas

transactivation in p53-mediated apoptosis appears to be cell type and signal-dependent.

Following DNA damage, p53 can also stimulate the expression of another death receptor,

KILLEWDRS (Wu et al., 1997). When Fas or KILLER/DR5 binds to the extracellular

signaling molecules Fas ligand or TRAIL respectively, they initiate signaling cascades

that result in the activation of caspases, leading to apoptosis.

There are a number of other genes that are induced in response to p53 and rnay be

involved in apoptosis, aithough their roles are not well understood, Briefly, IGF-BP3

binds insulin-like growth factor4 and prevents it tiom initiating anti-apoptotic signals

(Buckbinder et al., 1995). The overexpression of PAG 608, a protein that localizes to the

nucleolus, leads to morphological changes c haracteristic of apoptosis (Israeli et al.,

1997). Finally, a group of PIGs (p53-induced genes) were recently identified that appear

to increase cellular oxidation. When oxidation was blocked, p53-mediated apoptosis was

inhibited, suggest ing that p53 may activate apoptosis via cellular oxidation (Polyak et al.,

1997).

Bax - cytochrome c release m DNA ? DR5 + TRAL - Caspase

p53 * T Fas and Fas Ligand / Activation /\ 1 Damage

Figure 1.6

*AG 608 ? Apoptosis - ? PIGs ?

The effectors of p53 chat lead to an apoptotic ce11 response following DNA damage. (Adapted ffom Kirsch & Kastan, 1998)

It is important to note that there are stimuli that do not require p53 to initiate

apoptosis. These include tumor necrosis factor and Fas ligand, both of which bind cell-

surface death receptors to activate caspases and cause apoptotic ce11 death in p53-nul1

cells (Fuchs et al., 1997; Yoshida et al., 1996). Moreover, stimuli that require the p53

signal to induce an apoptotic death in one ceU type, may not require p53 to initiate

apoptosis in other ce11 types. Thus, the tnggering of apoptosis is a complex and

multifaceted process.

DNA damage activates p53, and p53 in turn can induce either ce11 cycle arrest or

apoptosis. A number of factors appear to determine whether a ce11 responds to ceilular

stress by undergoing either ce11 cycle arrest, or apoptosis. One important determinant

appears to be cell type. p53 induces apoptosis in some ce11 types, but ce11 cycle arrest in

others, in response to the same stimulus. For example, lymphocytes tend to undergo a

rapid p53-dependent ce11 death following DNA damage, while epithelial celis are more

likely to undergo a ce11 cycle arrest (Kirsch & Kastan, 1998). Even within the sarne ce11

type, the celluIar environment can dictate life or death. For example. DNA damage

causes lymphocytes to undergo ce11 cycle arrest in the presence of interleukin-3, but in its

absence, the same DNA damage causes p53-dependent apoptosis (Canman et al.. 1995).

The deletion of p21 can cause cells that would otherwise undergo pS3-dependent ce11

cycle arrest to undergo apoptosis instead, undersconng the major role of genetic

background in determining these cellular responses (Polyak et al., 1996). Many other

variables, such as the efficiency of DNA repair mechanisrns, the Bstx:Bcl-2 ratio, the

extent of DNA damage, and the levels of p53 expression, among other factors, appear to

influence the decision between ce11 cycle arrest and apoptosis.

1.5.6 p53 Gene Therapy

The introduction of wild-type p53-expressing plasmids into tumors cells can

induce over-expression of recombinant wild-type p53 protein and drive cells into either

growth arrest or apoptosis (Ramqvist et al., 1993; Show et al., 1992). Since these initial

findings there have k e n numerous papers examining the effects of exogenous p53 gene

transfer using an adenovïral vector on a variety of cancer ceil lines, both in vitro and in

vivo. The use of AdSCMV-p53 gene therapy in vitro has resulted in a cytotoxic effect in

ce11 lines of many different cancer types that harbr either mutant or deleted p53

(Hamada et al., 1996; Li et al., 1997; Liu et al., 1995; Pirollo et al., 1997) or wiId-type

p53 (Li et al., 1998; Liu et al., 1995). The effect of p53 gene therapy on 'normal' celIs

has been more controversial. Mi l e a number of groups, including our own, have

reported that both normal human fibroblasts and mammary epithelial cells have been

spared from the cytotoxic effects of this treatrnent (Clayman et al., 1995; Katayose et al.,

1995; Li et al., 1997), our lab also dernonstrated that another fibroblast strain, the KS 1

srrain, exhibited a significant decrease in survival following p53 gene therapy (Li et al.,

1999). This discrepancy might be related, in part, to the infection eficiency of the

specific 'normal' cell line king studied. The adenoviral infection efficiency of the KSI

fibroblasts exceeded that of the cancer ceUs being evaluated, suggesting that if sufficient

p53 is produced, even by the 'normal' cells, cytotoxicity can be induced.

p53 may enhance the cytotoxicity of ionizing radiation and some chemotherapy

drugs (Lee & Bernstein, 1993; Lowe et al., 1993). Therefore, the use of p53 gene therapy

in combination with these therapeutic agents has been studied in head and neck, colon,

and esophageal cancer ceLl lines, among others, with additional cytotoxicity king

consistently observed (Kanamori et al., 1998; Li et al., 1999; Ogawa et al., 1997; Pirollo

er al., 1997). p53 gene therapy has also k e n used in cancer xenograft rnodels in

combination with either radiation or chemotherapy. Most of the reported data

demonstrate significant tumor growth inhibition or regression foiiowing invatumoral

injection of Ad5CMV-p53 combined with either radiation or chemotherapy (Badie et al.,

1998; Pirollo et al., 1997; Roth, 1996). However, Our lab observed no additional growth

inhibition of an NPC turnor xenograft beyond that of radiotherapy alone (Lax, 2000). The

lack of tumor growth inhibition was likely due to a very low tumor transduction

efficiency by the virus, which has been also been reported by other groups (Mujoo et al.,

1996; O'Malley et al., 1995).

Due to the significant cytotoxic effects of AdSCMV-p53 gene therapy on cancer

cells both in vitro and in vivo, clinical trials have k e n initiated in a number of tumor

types (Clayman et al., 1999; Roth, 1996; Schuler et al., 1998). While the clinicai benefit

to patients has been limited, no major side effects have been detected, and Phase II and

III clinical trials are currently k i n g undertaken.

One of the major obstacles of in vivo gene therapy is the difficulty in specifically

targeting transgene expression to the tumor cells. Recently, a number of targeting

strategies have been reported in the literature, including the use of 'oncolytic vinises'

such as the ONYX-015 virus. This tumor targeting virus is an adenovhs that is missing

only the gene encoding the EIB 55 kDa protein, a protein that binds to and inhibits p53-

activated transcription, and is essential for viral replication (Bischoff et al., 1996). The

rationale behind the use of this virus is that in cells with wild-type p53, replication of

ONYX-015 would be inhibited because p53 would remain active. However, in cells

lacking wild-type p53, such as tumor celis, the virus would be able to replicate, lyse the

host cell, and proceed to infect and replicate in adjacent cells also lacking wild-type pS3.

The virus has had some success in clinical trials, and it may, in fact, be more widely

applicable than was originally thought (Pennisi, 1998). It appears as though the virus is

active not only in tumor cells with mutant p.53, but also in tumor cells with wild-type p53,

which most likely have other defects in the p53 pathway (Heise et al., 1997).

Another rnethod of targeting expression to tumor cells takes advantage of the fact

that the expression of viral genes is a hallmark of certain tumors, such a NPC. To direct

gene expression selectively to NPC cells, our lab has recently developed an adenoviral

vector containing promoter elements that are responsive to the EBV-encoded

transcriptional activator, EBNA-1. Preliminary data has demonstrated that viral

expression is indeed selectively induced in EBV-positive NPC cells (Jian-Hua Li,

personai communcation).

1.5.7 p73

Most genes belong to gene families; until recently, p53 proved an exception to

this rule. However, in 1997 a human homologue of p53, narned p73, was identified

(Kaghad et al., 1997). In short succession, several groups identified a third member of

the family, called p63. (It is also known as Ket, p40, p51, and p73L.) Although it is

longer in length due to an extended C-terminal region, p73 displays significant homology

to p53 especially in the regions corresponding to the p53 N-terminal transactivation, core

DNA binding , and C-terminal oligomerization domains.

The p73 gene maps to chromosome lp36, a region that is frequently deleted in a

variety of human cancers including neuroblastoma, melanoma, and colon cancer (Kaghad

et al., 1997). If p73 were behaving as a classical turnor suppressor gene, then one would

expect that the rernaining p73 allele would be mutated in these cancers. But, so far, p73

mutations have been identified in only 2 of 200 neuroblastomas tested (Kaeiin, 1999),

and no p73 mutations have been identified in other cancers. One potential explanation

for the absence of p73 mutations in tumors with lp36 deletions is that the p73 locus is

monoallelically expressed, so that the loss of only the active copy is needed for the loss

of protein function. Although there is sorne initial evidence suggesting monoailelic

expression of p73 (Kaghad et al., 1997), other p u p s have found evidence of biallelic

p73 expression (Mai et al., 1998). In summary, there is presently no clear genetic

evidence that p73 acts as a tumor suppressor.

Early investigations into the functions of p73 have revealed that it can activate

transcription fiom p53-responsîve promoters, and can induce apoptosis when

overexpressed in cells. However, a number of differences between p53 and p73 have

k e n revealed. p73, unlike p53, is not targeted by Wal oncoproteins such as SV40 T

antigen, adenovims E1B-55 kDa, or HPV E6, despite the similarities in the DNA binding

domain of the two proteins (Marin et al., 1998). As well, while Mdm2 can bind to p73

and inhibit its ability to act as a transcriptional activator, Md& does not target p73 for

degradation (Zeng et al., 1999).

Preliminary data suggested that p73 was not induced by DNA darnage,

specifically actinomycin D and W irradiation (Kaghad et al., 1997), but recent repons

have indicated that p73 is a target of the non-receptor tyrosine kinase c-Ab1 in response to

certain fonns of DNA damage. Foilowing either cisplatin or ionizing radiation treatment,

the activation of c-Abl, an increase in p73 protein, and apoptotic ce11 death have been

demonstrated (Agami et al., 1999; Gong et al., 1999). The significance of p73 activation

following DNA damage remains to be determined.

1.6 RATIONALE AND PROJECT OUTLINE

Given the low overall 5-year survival rate for NPC (approximately 65%), and

considering the relatively young age of most of the patients, NPC provides an attractive

target to try to improve treatment, and thereby survival of the patients. Our laboratory has

previously demonstrated that the introduction of wild-type p53 rnediated by the

adenoviral vector (Ad5CMV-p53) into the NPC ceii lines Cm-1 and CNE-22, resulted

in significant cytotoxicity when administered either aione, o r in combination with

ionizing radiation (Li et al., 1999; Li et al., 1997). The current work (Chapter 2) will

examine the effects of combining cisplatin chemotherapy with Ad5CMV-p53 gene

therapy in both EBV-positive and EBV-negative ceIl lines. Appendix 1 will review the

Iiterature surrounding the effects of hypoxia on turnors, and propose future directions

with respect to the effects of hypoxia on gene therapy, with the presentation of some

prelirninary data.

1.7 REFERENCES

Agarni, R., Blandino, G., Oren, M. & Shaul, Y. (1999). Interaction of c-Ab1 and p73alpha and their collaboration to induce apoptosis. Nature, 399, 809-13.

Al-Sarraf, M., LeBlanc, M., Giri, P.G., Fu, K.K., Cooper, J., Vuong, T., Forastiere, A.A., Adams, G., Sakr, W.A., Schuller, D.E. & Ensley, J.F. (1998). Chemoradiotherapy versus radiotherapy in patients with advanced nasopharyngeal cancer: phase ID randomized Intergroup study 0099. J Clin Oncol, 16, 13 10-7.

Altun, M., Fandi, A., Dupuis, O., Cvitkovic, E., Krajina, Z. & Eschwege, F. (1995). Undifferentiated nasopharyngeal cancer (UCNT): Current diagnostic and therapeutic aspects. Int J Radiat Oncol Bi01 Phys, 32, 859-877.

Anderson, W.F. (1992). Human gene therap y. Science, 256,808- 13. Badie, B., Kramar, M.H., Lau, R., Boothman, D.A., Economou, J.S. & Black, KL.

(1998). Adenovims-mediated p53 gene delivery potentiates the radiation-induced growth inhibition of experimental brain tumors. J Neurooncol, 37,217-22.

Barak, Y., Juven, T., Haffner, R. & Oren, M. (1993). mdm2 expression is induced by wild type p53 activity. Ernbo J , l2,46 1-8.

Baum, S.G. (1990). Adenoviruses. In Principles and Practices of Znfectious Diseuses, Mandel1 G., D.R., Bennett J. (ed) pp. 1185-91. Churchill Livingstone Inc.: New York.

Bergelson, J.M., Cunningham, LA., Droguett, G., Kurt-Jones, E.A., Krithivas, A., Hong, J.S., Horwitz, M.S., Crowell, R.L. & Finberg, R.W. (1997). Isolation of a cornrnon receptor for Coxsackie B viruses and adenovinises 2 and 5. Science, 275,1320-3.

Bischoff, J.R., Kim, D.H., Williams, A., Heise, C., Hom, S., Muna, M., Ng, L., Nye, J.A., Sampson-Johannes, A., Fattaey, A. & McCormick, F. (1996). An adenovinis mutant that replicates selectively in p53-deficient human tumor cells. Science, 274,373-6.

Bracey, T.S ., Miller, J-C., Preece, A. & Paraskeva, C. (1995). Gamma-radiation-induced apoptosis in human colorectal adenorna and carcinoma ce11 lines can occur in the absence of wild type p53. Oncogene, 10,2391-6.

Buckbinder, L., Talbott, R., Velasco-Miguel, S., Takenaka, I., Faha, B., Seizinger, B.R. & Kley, N. (1995). Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature, 377,646-9.

Busson, P., Ganem, G., Flores, P., Mugneret, F., Clausse, B., Caillou, B., Braham, K.. Wakasugi, H., Lipinski, M. & Tursz, T. (1988). Establishment and characterization of three transplantable EBV- containing nasopharyngeal carcinomas. Int J Cancer, 42,599-606.

Canman, C.E., Griber, T., Coutts, S. & Kastan, M.B. (1995). Growth factor modulation of p53-mediated growt h arrest versus apoptosis. Genes Development, 9,600-6 I 1.

Cavazzana-Calvo, M., Hacein-Bey, S., de Saint Basile, G., Gross, F., Yvon, E., Nusbaum, P., Selz, F., Hue, C., Certain, S., Casanova, J.L., Bousso, P., Deist, F.L. & Fischer, A. (2000). Gene therapy of human severe combined irnmunodeficiency (SCID)-X1 disease. Science, 288,669-72.

Chan, A.T., Teo, P.M., Leung, T.W. & Johnson, P.J. (1998). The role of chernotherapy in the management of nasopharyngeal carcinoma. Cancer, 82, lOO3- 1 2.

Chang, Y.S., Lin, S.Y., Lee, P.F., Durff, T., Chung, H.C. & Tsai, M.S. (1989). Establishment and characterization of a tumor cell line fiorn human nasopharyngeal carcinoma tissue. Cancer Res, 49,6752-7.

Channon, K.M. & George, S.E. (1 997). Improved adenoviral vectors: cautious optùnism for gene therapy. Qjm, 90, 105-9.

Chen, W. & Cooper, N.R. (1996). Epstein-Barr virus nuclear antigen 2 and latent membrane protein independently transactivate p53 through induction of NF- kappaB activity. J Virol, 70,4849-53.

Cheung. S.T., Huang, D.P., Hui, A.B., Lo, K.W., Ko, C.W., Tsang, Y.S., Wong, N., Whitney, B.M. & Lee, J.C. (1999). Nasopharyngeal carcinoma ce11 line (C666-1) consistently harimuring Epstein-Barr virus. Znt J Cancer, 83, 121-6.

Chroboczek, J., Bieber, F. & Jacrot, B. (1992). The sequence of the genome of adenovims type 5 and its comparison with the genome of adenovirus type 2. Virology, 186,280-5.

Clayman, G.L., EI-Naggar, A.K., Roth, J.A., Zhang, W. W., Goepfert, K, Taylor, D.L. & Liu, T. (1995). Zn vivo molecular therapy with p53 adenovirus for rnicroscopic residual head and neck squamous carcinoma. Cancer Res, 55, 1-6.

Clayman, G.L., Frank, D.K., Bruso, P.A. & Goepfert, H. (1999). Adenovinis-mediated wild-type p53 gene transfer as a surgical adjuvant in advanced head and neck cancers. Clin Cancer Res, 5,1715-22.

Dang, C.V. & Lee, W.M. (1989). Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HS WO, and HIV tat proteins. J Bi01 Chem, 264, 18019-23.

Eastman, A. (1987). The formation, isolation and characterization of DNA adducts produced by anticancer platinum complexes. Phamuicol mer, 34, 155-66.

Eastman, A. (1990). Activation of programmed cell death by anticancer agents: cisplatin as a mode1 system. Cancer Cells, 2,275-80.

Effert, P., McCoy, R., Abdel-Hamid, M., Flynn, K., Zhang, Q., Busson, P., Tursz, T., Liu, E. & Raab-Traub, N. (1992). Alterations of the p53 gene in nasopharyngeal carcinoma. J Virol, 66,3768-75.

el-Deiry, W.S. (1998). Regulation of p53 downstream genes. Semin Cancer Biol, 8,345- 57.

Fahraeus, R., Ryrno, L., R h h , J.S. & Klein, G. (1990). Morphological transfomation of human keratinocytes expressing the LMP gene of Epstein-Barr virus. Nature, 345, 447-9.

Fandi, A., Altun, M., Azli, N., Armand, J.P. & Cvitkovic, E. (1994). Nasopharyngeal cancer: epidemiology, staging, and treatment. Semin Oncol, 21,382-97.

Fandi, A. & Cvitkovic, E. (1995). Biology and treatment of nasopharyngeal cancer. Curr Opin Oncol, 7, 255-63.

Friesen, C., Herr, I., Krammer, P.H. & Debatin, KM. (1996). Involvement of the CD95 (APO- I/FAS) receptodligand system in drug-induced apoptosis in leukernia cells. Nat Med, 2,574-7.

Fuchs, E.J., McKenna, K.A. & Bedi, A. (1997). p53-dependent DNA damage-induced apoptosis requires Fas/APO- 1 -independent activation of CPP32beta. Cancer Res, 57,2550-4.

Gong, J.G., Costanzo, A., Yang, H.Q., Melino, G., Kaelin, W.G., Jr., Levrero, M. & Wang, J.Y. (1999). The tyrosine kinase c-Ab1 regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature, 399, 806-9.

Graham, F.L. & Prevec, L. (1991). Manipulation of adenovinis vectors. In Methodr in Molecular Biology, Murray, E.J. (ed), Vol. 7. pp. 109- 128. The Hurnan Press Inc.: Clifton, New Jersey.

Greenblatt, M.S., Bennett, W.P., Hollstein, M. & Harris, CC. (1994). Mutations in the p53 tumor suppressor gene: clues to cancer etiology and rnolecular pathogenesis. Cancer Res, 54,48578.

Hamada, K., Alemany, R., Zhang, W.W., Hittelman, W.N., Lotan, R., Roth, J.A. & Mitchell, M.F. (1996). Adenovirus-mediated transfer of a wild-type p53 gene and induction of apoptosis in cervical cancer. Cancer Res, 56,3047-54-

Han, J., Sabbatini, P., Perez, D., Rao, L., Modha, D. & White, E. (1996). The ElB 19K protein blocks apoptosis by interacting with and inhibiting the p53-inducible and death-promoting Bax protein. Genes Dev, 10,461-77.

Harper, J.W., Adami, GR., Wei, N., Keyomarsi, K. & Elledge, S.J. (1993). The p21 Cdk- interacting protein Cipl is a potent inhibitor of G1 cyclin-dependent kinases. CeII, 75,805-16.

Heise, C., Sampson-Johannes, A., Williams, A., McCorrnick, F., Von Hoff, D. & Kim, D. (1 997). ONYX-0 15, an E 1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antiturnoral efficacy that can be augrnented by standard chemotherapeutic agents. Nat Med, 3,639-645.

Hollon, T. (2000). Researchers and regulators reflect on fmt gene therapy death [news]. Nat Med, 6,6.

Home, R.W., Bonner, S., Waterson, A.P. & Wildy, P. (1959). The icosahedrai form of an adenovirus. J Mol Biol, 1,8486.

Hsu, J.L. & Glaser, S.L. (2000). Epstein-barr virus-associated malignancies: epidemio logic patterns and et io logic implications. Cr2 Rev Oncol Henzatol, 34, 27-53.

Huang, D.P., Ho, J.H., Poon, Y.F., Chew, E.C., Saw, D., Lui, M., Li, C.L., Mak, L.S., Lai, S.H. &= Lau, W.H. (1980). Establishment of a ce11 Iine (NPUHKl) from a differentiated squamous carcinoma of the nasopharynx. In? J Cancer, 26, 127-32.

Huang, L. (1999). Introduction. In Non-Viral Vectors for Gene Therapy, Huang, L. & Wagner, E. (eds) pp. 3-22. Academic Press: San Diego.

Hui, A.B., Cheung, S.T., Fong, Y., Lo, K.W. & Huang, D.P. (1998). Characterization of a new EBV-associated nasopharyngeal carcinoma ce11 line. Cancer Genet Cytogenet, 101, 83-8.

Israeli, D., Tessler, E*, Haupt, Y., Ekeles, A., Wilder, S., Amson, R., Telerman, A. & Oren, M. (1997). A novel p53-inducible gene, PAG608, encodes a nuclear zinc finger protein whose overexpression prornotes apoptosis. Embo J, 16,4384-92.

Jain, K.K. (1998). Vectors for Gene Therapy. In Te~book of Gene Therapy, Jain, K.K. (ed) pp. 35-55. Hogrefe and Huber Publishers: Seattle.

Jones, S.N., Roe, A.E., Donehower, L.A. & Bradley, A. (1995). Rescue of embryonic lethality in Md&-deficient mice by absence of p53. Nature, 378,206-8.

Kaelin, W.G., Jr. (1999). The p53 gene family. Oncogene, 18,7701-5.

Kaghad, M., Bonnet, H-, Yang, A., Creancier, L., Biscan, J-C., Valent, A., Minty, A., Chalon, P., Lelias, J.M., Dumont, X., Ferrara, P., McKeon, F. & Caput, D. (1997). Monoallelically expressed gene related to p53 at lp36, a region fiequently deleted in neuroblastoma and other hurnan cancers. Cell, 90,80949.

Kanamori, Y., Kigawa, J., Minagawa, Y., hie, T., Oishi, T., Shimada, M., Takahashi, M., Nakamura, T., Sato, K. & Terakawa, N. (1998). A newly developed adenovims- mediated transfer of a wild-type p53 gene increases sensitivity to cis- diamminedichloroplatinum (II) in p53- deleted ovarian cancer cells. Eur J Cancer, 34, 1 802-6.

Kastan, M-B., Onyekwere, O., Sidransky, D., Vogelstein, B. & Craig, R.W. (1991). Participation of p53 protein in the cellular response to DNA darnage. Cancer Res, 51,6304-1 1.

Katayose, D., Gudas, J., Nguyen, H., Srivastava, S., Cowan, K.H. & Seth, P. (1995). Cytotoxic efiects of adenovims-mediated wild-type p53 protein expression in normal and tumor mammary epithelial cells. Clin Cancer Res, 1,889-97.

Kay, M.A., Manno, CS., Ragni, MY., Larson, P.J., Couto, L.B., McClelland, A., Glader, B., Chew, A.J., Tai, S.J., Herzog, R.W., Arruda, V., Johnson, F., Scalian, C., Skarsgard, E., Fiake, A.W. & High, K.A. (2000). Evidence for gene transfer and expression of factor IX in haemophifia B patients treated with an AAV vector. Nat Genet, 24,257-61.

Kerr, J.F.R., Wyllie, A.H. & C h e , A.R. (1972). Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 26, 239-257.

Kirsch, D.G. & Kastan, M.B. (1 998). Turnor-suppressor p53: implications for tumor development and prognosis. J Clin Oncol, 16,3158-68.

Knudson, C.M., Tung, K.S., Tourtellotte, W.G., Brown, G.A. & Korsmeyer, S.J. (1995). Bax-deficient mice with lymphoid hyperplasia and male germ ce11 death. Science, 270996-9.

Kubbutat, M.H., Jones, S.N. & Vousden, K.H. (1997). Regulation of p53 stability by Mdrn2. Nature, 387, 299-303.

Kubbutat, M.H., Ludwig, R.L., Ashcroft, M. & Vousden, K.H. (1998). Regulation of Mdm2-directed degradation by the C terminus of p53. Mol Ce11 Biol, 18,5690-8.

Lakin, ND. & Jackson, S.P. (1999). Regulation of p53 in response to DNA damage. Oncogene, 18,7644-55.

Lane, D.P. (1992). Cancer. p53, guardian of the genorne. Nature, 358, 15-6. Lane, D.P. & Crawford, L.V. (1979). T antigen is bound to a host protein in SV4