ADENOVIRUSlp53 GENE THERAPY IN NASOPHARYNGEAL CARCINOMA XENOGRAFTS

Stuart Allen Lax

A thesis submitted in conlomity with the requirements for the degree of Master of Seience,

Graduate Department of Medicai Biophysics

University OC Toronto

@ Copyright by Saurt A. Lax (2000)

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C a n a !

ADENOVIRUS-p53 GENE THERAPY IN

NASOPHARYNGEAL CARCINOMA XENOGRAITS

M.Sc. Degree, 2000

Stuart Ailen Lax

Department of Medical Biophysics

University of Toronto

A bstract

Overexpression of the tumor suppressor protein p53 with an adenoviral vector

carrying the p53 gene (Ad-p53) has been used successfully in other reports as an anti-

cancer therapeutic in vitro and following intratumoral injection in vivo. Further, this

thenpeutic strategy has been combined with radiation in animal tumor modets, with

resultant enhanced radiosensitivity observed. We were interested in exiimining the

impact of Ad-p53 on the radiocurability of nasopharyngeal carcinoma (NPC). Severn1

techniques were used in this thesis to examine the in vivo transduction efficiency

achieved following intratumoral injection of an adenoviral vector canying the lac2 gene

(Ad-P-gal). NPC tumor cross-sections and tumor whole mounts were stained with the B-

gal substrate X-Gal. and some tumors were stained with the fluorescent p-gal subsmte

FDG. The results and limitations determined for each technique will be discussed. As

well, data from therapeutic experiments combining Ad-p53 and ionizing radiation in NPC

xenografts will be presented.

. . ........................................................................................................ Abstract .il ... ............................................................................................. Table of Contents 111 ................................................................................................. List of Figures vi ...

List of Tables ................................................................................................ v111 " . ..................*.............................. ...................................... List of Abbrevrations .. ix

Chapter One: Introduction

3 .................................................................................... 1 . 1 Adenovirus ....- 7 1.1.1 Gene Therapy ........................................................................ .-

................................... 1.1.2 The Adenovirus Pathogen ................... .. 3 1.1.3 Adenovirus Structure ........................ ... ...................................... 4

......................................... ................. 1.1.4 Adenovirus Genome ... ... .. 6 ....................................................... 1.1.5 Life Cycle of the Adenovirus 7

............................................. 1.1.6 Use of Adenovirus for Gene Therapy 8

................................................................................................ 1.2 p53 9 1.2.1 Introduction ............................................................................ 9 1.2.2 p53 Rotein Smcture ........................................................... 12

.................................................. 1.2.3 p53-Mediated Ce11 Cycle Arrest 12 ........................................................... 1 . 2.4 p53-Mediated Apoptosis 13

.............................................................. 1.2.5 Use in Gene Therapy -15

........................................................................... 1.3 Radiotherapy 19 1.3.1 Inmduction .......................................................................... 19

........................................... 1.3.2 Biologic Effects of Radiation Therapy 20

33 .................................................................... 1.4 Nasopharyngeal Carcinoma 1.4.1 In~oduction ......................................................................... 22

.................................. ................. 1 .4.2 Epstein-Barr Virus and NPC .. 23 ......................................................................... 1.4.3 pS3 and NPC 24

................................................................. 1.4.4 Treatment of NPC 2 6

.............................................................. 1.5 NPC Ce11 Lines and Xenografts 26 ............................................................... L.6 Rationale and Project Outline 3 8

....................................................................................... 1.7 References 30

Chapter Two: in vivo A d e w v i w Distribution in Nasopharygeai Carcinom Xenografts

2.1 Abstract .............................................. 45 ...................................................................................... 2.2 Introduction 47

........................................................................ 2.3 Materials and Methods 49 2.3.L Cells andCultureConditions ...................................................... 49 2.3.2 Tumor Mode1 ........................................................................ 50

................................................................... 2.3.3 Virus Propagation 51

.................................................................. 2.3.4 Virus Purification -52 2.3.5 Plaque Assay for Virus Titer ..................................................... -53

......................................... 2.3.6 Ad-p-gd and X-Gd: Histology Sections 54 ............................................. 2.3.7 Ad-p-gd and FDG: Flow C ytometry 56

..................................... 2.3.8 Ad-B-gd and &Gd: Tumor Whole Mount 56

2.4 Results ........................................................................................... 58 ............................. 2.4.1 Ad-p-gd transduction in vivo: Histology Sections 58

.............. 2.4.2 Ad-B-gal transduction in vivo: Whole Tumor Flow Cytometry 62 .......................... 2.4.3 Ad-fbgd transduction in vivo: Tumor Whole Mount 66

2.4.3.1 Ad-p-gal transduction in vivo: Tumor Whole Mount - ....................................................... X-Gal Tirnecourse 69

2.4.4 India Ink: Tumor Whole Mount ................................................... 73

....................................................................................... 1.5 Discussion 74

2.6 References ....................................................................................... 82

Chapicr Thiw: Efferts of Ionizing Radiation and AdenovirusgS3 Gene Therapy on Nasopharyngeai Carcinoma Xenogralts

....................................................................... 3.3 Materials and Methods -88 3.3.1 Cells andCulnireConditions ...................................................... 88 3.3.2 TumorModel ........................................................................ 88

............................................... 3.3.3 Vinis Ropagation and Purification 89 3.3 -4 CNE-1 in scid Mice: Therapeutic Experiment ................................. -89 3.3.5 CNE-1 in RAG 1-1- Mice: Higher D o s Therapeutic Experiments .......... 91 3.3.6 Tirnecourse of p53 Expression, Apoptosis, and Necrosis .................... -91

............................................................................................ 3.4 Results 93 3.4.1 CNE-I Xenograft Therapeutic Experiment in scid Mice ..................... 93 3 .4.2 Higher Dose Therap y on CNE-1 Xenografts in RAG 14- Mice ............. 97

........................ 3.4.3 p53 Tirnecourse Following Ad-p53 Injection in vivo 103

.................................................................................... 3.5 Discussion -108

Chapter Four: General Conclusions and Future Directions

.......................................................... 4.1 Conclusions ........................ .. 117 4.1.1 Adenovinis Distribution Following Intratumoral Injection ................. -117 4.1.2 Ad-p53 and Ionizing Radiation in NPC Xenografts .......................... 119

.............................................................................. 4.2 Future Directions 121 ....................................................... The C 15 Xenograft Mode1 122

4.2.1.1 Preliminary Thernpeutic S tudies with the C 15 ...................................................... Xenograft Mode1 -123

......................................................... Adenoviral Distribution -127 ......................................... Tumor Mode1 and Effects of Ad.p53 -127

Improving the Effects of Ad-p53 on NPC in vivo .............................. 128

List of Figures

Chapter One

. . ................................................................ Figure 1.1 Adenovirus infection 5 ............................................... Figure 1.2 Response of p53 to DNA damage 11

79 ............................ Figure 1.3 Clonogenic swiva l following ionizing radiation .,

Chapter Two

Figure 2.1

Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7

Figure 2.8

Figure 2.9

Figure 2.10

Figure 2.1 1

25X magnification of a histology section from the most ................................................... transduced tumor obtained 3 9

............... lOOX magnification of the s m e section shown in Figure 2.1 59 .................. Percentage of positive blue X-Gd-staining tumor sections 61

........... Average percent blue tumor in sections with some blue stoining 62 Ruorescence of cells denved from three plates ............................... 64

.......................... Ad-@-gal-infected CNE- 1 cells stained with %Ga1 65 Ruorescence following digestion into a single cell suspension and

........................ exposure to 7AAD . with or without addition of FDG 66 Tumor pieces from Day 1 and Day 2 CNE-1 tumor whole mounts stained with X.Gal ................................. ......... 68 Dûy 1 CNE-22 tumor whole mounts stained with X-Ga1

........................................................ and analyzed immediately 69 Pictures of an Ad-p-gal-injected tumor and Tris-injected tumor

..................................... iifter 6, 10 and 18 hours of X-Gai staining 72 Whole mount of a CNE-1 tumor injected with India Ink ..................... 73

Chapter Three

Figure 3.1 Figure 3.2 Figure 3.3 Figure 3 -4

Figure 3.5

Figure 3.6

Figure 3.7

Wgure 3.8

Figure! 3.9 Figure 3 -10 figure 3-11

Tumor weight versus tumor + leg diameter ................................... ..89 Effects of radiation +I- Ad-p53 on CM-1 in scid mice ...................... 95 Estimated mean tumor doubling time and time to 1.6 g ...................... 97 Effects of a higher dose of radiation and Ad-p53 on CNE-I

.................................................................... in EtAG 1-1- mice 98 Repeat experiment to detennine the effects of a higher dose of radiation +/- Ad-p53 on CNE-1 in RAG 14- mice ........................ 99 Third experiment to determine the effects of a higher dose of radiation +!- Ad-p53 on CNE1 in RAGIJ- mice ........................... 100 Tumor doubüng time and time to 1.6 g for al1 three CNE-1

.............................................. RAG 14- therapeutic experiments 102 p53 MC on sections h m a Tris-injected tumor and an . . .......................................................... Ad-p53-injected tumor 103

................ Percentage of p53-positive cells using IHC .. .. ... .... 104 ........ H & E section displaying apoptosis in an Ad-p534njected turnor 105 .......... Percentage of apoptotic iumor cells b m H & E tumor sections 106

Figure 3.12 H & E section displaying necrosis in an Ad-p53-injected tumor ........ -107 Figure 3.13 Amount of necrotic tumor area h m H & E tumor sections ............... 108

Chapter Four

Figure 4.1 C 15 tumor growth in scid rnice versus RAG 14- mice ................... .i 23 Figure 4.2 Cl5 tumor growth following radiation and Ad-pS3 ........................ 124 Figure 4.3 C 15 tumor growth following a lower dose of radiation ..................... 135

Chapter One

......................... Table 1.1 Pubüs hed Ad-pS3 + radiation animal tumor models 17

Chapter Two

................... Table 2.1 Adenovirus distribution following intratumoral injection 48 ................................ Table 2.2 ~d-p-gd distribution using histology sections 60

.................................... Table 2.3 Ad-b-gal distri bution using w hole mounts 68 ............................................. Table 2.4 X-Gd staining duntion expriment 70

Chapter Three

.............................. Table 3.1 Protocol for CNE- 1 scid thenpeutic experiment 90 ........................ Table 3.2 Protocol for CNE-1 RAG 14- therapeutic experiment 91

...................................... Table 3.3 Ad-pY + radiation published protocols 112

P-go1 cDNA DNA EBV ms FDG

Pm mL mM NP40 LMP1 WC PBS -1- efu RAG 14- S.C.

SCC scid UCNT X-Gd

7-amino-actinomycin-D Adenovirus Recombinant adenovirus type 5 canying the bacterial lac2 gene coding for fbgalactosidase downstream of a cytomegaiovirus promoter Recombinant adenovirus type 5 carrying a human wild- type p53 cDNA downstream of a cytomegdovinis promo ter &galactosidase Complimentary DNA Deoxyribonucleic acid Epstein-Barr virus Fetal bovine senun Ruorescei n di-p-D-galactop yrmoside Needle gauge Grms Unit of radiation dose, 1 Gy = absorbed dose of 1 Joule 1 kg of irradiated materid Hematoxylin and eosin Intramuscular Immunohistochernistry Kilogram Kilovolts Molar Microli ter Micrometer MilIiliter MilIimolar Nonidet P40 EBV latent membrane protein 1 Nasopharyngeal carcinoma Phosphate Buffered Saline -~a '+ - M ~ ~ + Plaque fotming units Recombinase Activating Gene 1 knockout mouse Subcutaneous Squamous ce11 carcinoma Severe combined immune deficient mouse Undifferentiated carcinoma of nasopharyngeal type 4bromo-5-chloro-3- indoly l -~-D-galûcto~

Cha~ter One

Introduction

1.1 Adenovirus

1.1.1 Gene Thera~y

Research in gene therapy has developed rapidly in the last 15 years. It includes gene-

replacement therapy, immunothenpy and insertion of a gene whose protein can convert a

pro-dnig into a cytotoxic rnolecule. Mile a focus of gene thenpy research in the 1980's

was the correction of inheritable diseases (Anderson, 1984). the field has gradually

shifted towards examination of its usefulness as an anticancer therapeutic (Rosenberg et

al., 1990), Even before this had been attempted, the ethical implications of human

somûtic gene therapy had been extensively debated, with an eventual consensus of

acceptance (Richter and Bacchetta, 1998; LilX and Liu, 1999). The result of this

acceptance has ken a npid expansion of a wide variety of anti-cancer somatic gene

therapy clinical trials worldwide, especially with the increased support from large

pharmaceutical and biotechnology f imis (Martin and Thomas, 1998).

There are severd basic compnents one should consider when pursuing gene therapy

research. It is important to focus on (a) a discase site where there is a potential for

benefit, (b) an appropriate gene or genes to deliver to the target population. and (c) an

appropriate delivery vehicle and method. There are several gene therapy vectors

currentiy in use, including plasmid DNA. liposome-DNA complexes, and retroviral

vectors. Each vector has its own advantages and disadvantages. Plasmid DNA generally

has low transduction eniciency as compared to virai vectots. but this has ken enhanced

sornewhat by incorporation into various types of liposomes (Felgner et al.. 1987; Lax and

Liu, 1999). Liposomes have the advantage of king able to carry large amounts of DNA

(Strauss et al., 1993) as well as the potential for repeated use without the immune

rejection that cm occur with viral vectors (Liu et al., 1995b). However, the transduction

efficiency of liposomes is still lower than viral vectors (Hsiao et al., 1997). Most

retrovirai vectors, while capable of delivenng high transduction efficiencies as compared

to naked DNA and liposomes, do not infect non-dividing cells efficiently (Miller et al.,

1990; Benchimol and Minden, 1998; Lax and Liu, 1999). This may be a disadvantage in

anti-cancer therapeutics, where tumors an comprised of cells at various stages of the ce11

cycle (Mendelsohn, 1960). Cunently, one of the most popular gene delivery vehicles in

clinical trials is the adenovirus (Roth and Cristiano, 1997; Dube, 1998; Lax and Liu,

1999). The following section will discuss the characteristics of adenoviral vectors and

some of the reasons for their popularity as gene therapy vectors, as well as present the

rationale for their use in the experiments described in this thesis.

1.1.2 The Adenovirus Pathogen -

There are over 100 serotypes of adenovirus (Ad), with over 40 of these tropic for

humans (Graham and Revec, 1991). The most ftequently used human serotype in gene

therapy is type 5. Adenoviruses have not ken linked to human tumorigenesis (Horwitz,

1996), and the safety of Ad type 5 as is gene therapy vector is demonstrated by its use as a

human vaccine for decades without detectable side-effects (Siegfried, 1993).

Approximately 3% of al1 infections in Noah American civilian populations are caused

by adenovinises. It accounts for 7% of febrile ilhesses (Fox et al., 1969), and antibodies

to Ad type 5 are identified in 40% - 60% of children studied (Brandt et al., 1969). The

prevalence of Ad and its immmunogenicity are potential obstacfes to adenovirus gene

therapy, since the immune system mounts a relatively rapid response against so-cailed

"first generation" adenovirus vectors, which have ntained most of the adenovinis

genome. Most of such viruses are typically cleared boom the body within several weeks

following administration (Morsy et al., 1998).

1-1.3 Adenovinrs S truc tu^

The adenovirus is an icosohedrai DNA virus (Figure 1.1). approximately 70 - LOO nm

in diameter when fully assernbled (Home et al.. 1959). The assembled viral particle is

mostly protein and includes a protein capsid surrounding a linear double-stranded DNA

genome of approximately 36 kb (Chroboczek et al.. 1992). The penton base, made up of

five copies of polypeptide m. is located at the vertices of the capsid. From it extends a

polypeptide IV fiber protein that is responsible for binding to the Ad receptor (Figure

1.1). The capsid is primarily made up of hexons (Figure 1.1), which are trimers of

polypeptide O[; five hexons surround the penton base. The nmaining capsid

polypeptides, ma, VI, Mn, and [X serve to connect and stabilize the hexon protein with

con protein and DNA (Shenk, 1996).

The viral core inciudes the genome dong with four known associated proteins, V, W.

the terminal protein, and mu. Polypeptide V appears to link the viral genome with the

capsid through interaction with the penton base. Rotein W is the most abundant core

protein and appears to act as a histone-like protein, mpped b y DNA (Lischwe and Sung,

1977). The terminal protein is associated with the 5' end of each strand of viral DNA. It

acts as an initiation site for DNA replication and facilitates attachent of wal DNA to

the nucleus of an infected ce11 (Shenk, 1996). The hction of the mu protein is

iinknown*

integrins on the ce11 d a c e , specifically 0 4 3 3 and %Pr, promote Ad intemalization

into the ce11 (Wiclrham et al., 1993) (Figure 1.1). AdewWus penton base contaias five

kg-Gly-Asp (RGD) motifs, typically recognized by integrins and containeci in ce11

adhesion molecules such as fibronectin (Wickham et al., 1993). It was found that Ad

attachent and intemalization could be separated, with attacbment occmhg in the

absence of inteprin-promoteâ intemalization, suggesting that an additional Ad receptor

might be involved. Indeed a receptor for Ad types 2 and 5 was discovered and termeci the

coxsackievim and a d e n o h receptor (CAR). It was found to bind to the Ad fiber

protein (Bergelson et al., 1997).

1.1.4 Adenovirus Genome

Genes in the Ad genome have been categorized into groups based on their time of

transcription following Ad infection. There are five early mRNA groups (designated

ELA, ElB, E2, E3, E4). two delayed early uni& (IX and Iva2) and five late mRNA

groups (LI, L2, L3, LA, L5) derived from the Ad genome's major late unit. Roteins

encoded by regions deleted in the Ad vector used in this thesis will be discussed.

The ElA protein is capable of immortalizing rodent cells, but only inefficiently

without concomitant expression of EIB (White, 1995). This is thought to occur because

even though ELA has been found to bind pRB and stimulate cellular proliferation (Whyte

et al.. 1989). it is also capable of stimulating p53-dependent apoptosis (Debbas and

White, L993). E1B encodes a 55 kD and a 19 kD protein. The former inhibits p53-

activated transcription (Martin and Berk, 1999). The latter protein appears to behave

similarly to Bcl-2 by binding and inhibiting the pro-apoptotic Bax protein and

suppressing p53-medinted apoptosis (Han et al., L996).

This concept has been exploited recently by the development of a tumor-targeted Ad,

the ONYX-015 virus, which is missing only the gene encoding the EIB 55 kD protein

(Bischoff et al., 1996). The rationale behind the use of this virus was that in cells with

wild-type p53, replication of ONYX45 would be inhibited bccause p53 would remain

active. However, in cells lacking functional p53, such as tumor cells, the virus would be

able to replicate, lyse its host cell, and infect and replicate in adjacent cells also lacking

wild-type p53. This concept has encouraged a number of other groups to develop other

so-called bboncolytic Wuses" (Coffey et al., 1998; Pennisi, 1998). Even though the

ONYX415 virus has met with some success in clinicd triais (Pennisi, 1998). the

mechanism of its action and selectivity has ncently k e n disputed (Hall et al., 1998).

These new findings suggest that even though the virus may kill cancer cells, its tumor

targeting ability may have little to do with hep53 status of a tumor.

The E3 region encodes several proteins that are not ~quired for virai replication

(Graham and Prevec, 199 1; Zhang et al., 1995). They appear to be partial1 y responsible

for an Ad anti-immune system nsponse. The 19 kD msmembrme glycoprotein, W-

gp NkD, is retained in the endoplasmic reticulum (Paabo et al., 1987) and is capable of

binding major histocompatibility clnss 1 antigens (Burgert et al., 1987) that are required

for presenting foreign antigens to cytotoxic T lymphocytes for cell lysis. It is thought

that binding in this way reduces plasma membrane expression of MHC class 1 antigens

and thus cytotoxic T lymphocyte response (Burgert et al., 1987).

1.1.5 Life Cvcle of the Adenovirus

Adenovirus internalization occurs via receptor-mediated endocytosis (Varga et al.,

1991) following binding to the CAR receptor and a& or &Br integins (Figure 1.1).

Once bound, the virus is rapidy intemalized, reaching the endosornes within minutes

(Leopold et al., 1998). It is thought that the virus then ruptures the early endosome

before it becomes a lysosome (Figure 1.1). The mechanism for this is not clear, but this

process requires endosomal acidification (Seth et al., 1984; Leopold et al.. 1998), and the

penton base of the virus also appears to be involved (Seth, 1994). Approximatel y 90% of

the virus is able to rapidly escape into the cytoplasm, probably before endosome-

endosome fusion occurs (Leopold et al., 1998).

The virus then proceeds to gradually dismanile itself as it travels towards the nucleus

(Gretter et al., 1993). More and more of the capsid proteins are degraded and lost to

prepare the virus for insertion of its DNA through nuclear pore complexes @ales and

Chardonnet. 1973) (Figure 1.1). The virus moves through the cytosol to the nucleus in a

pattern and speed suggestive of rnicrotubular assistance (Leopold et al., 1998). In about

I hour, 80% of the virus has reached the nucleus (Ltopold et al., L998), whcre a

constitutively active viral ElA promoter transcribes the ElA proteins responsible for

activating transcription of the rest of the viral genome (Osborne and Berk, 1983).

The viral DNA is replicated in the host ce11 nucleus. Empty capsids are formed in

association with virai DNA through interaction with a packaging sequence near one end

of the viral chromosome (Hearing et al.. 1987). In order to release assembled infectious

virus, the infected ce11 is lysed. It is thought that virus-induced intermediate filament

disruption facilitates cellular 1 ysis (White and Cipriani, 1989; Chen et al., 1993).

1.1.6 Use of Adenovirus for Gene Thera~y

Wild-type Ad type 5 normaily infects the epithelium of the respiratory tract, making it

a good candidate for use on epithelial malignancies, such as nasopharynged carcinoma.

As a vector for gene delivery to humans, adenoviruses have several advantages: (1) use

of the adenovirus itself appears relatively sde. (2) the viral genome has been well-

characterized, and deletion of the El region renders the virus infectious but replication-

debctive. (3) huther deietion of the non-essential E3 region allows up to 7.5 kb of

exogenous DNA to be inserted (Graham and Revec, 1991). (4) adenoviruses are easily

manipulated and gemrated to high titre, (5) they exhibit high infectivity in a btood host

ninge, and (6) they infect non-dividing as well as dividing cells (Zhang et al., 1995). The

EL, W-deleted Ad, which is the vector used in this thesis, is a '"first-generation" vector

that was referred to earlier (Section 1.1.2).

Adenoviruses also do not need to be incoiporated i n t ~ the host cell's genome, unlike

retroviruses. Therefore, the virus as well as the inserted foreign gene of interest are

episomally expressed, reduting the risk of insertional mutagenesis (Piang et al., 1995).

Ho wever, this, as well as a targeted immune response agains t adenoviruses (Kass-Eisler

et al., 1994), limit the use of first-generation vectors to transient expression of the

thenpeutic gene. In coninst to correction of genetic disorders such as cystic fibrosis,

however, this may not be an obstacle in tumor suppressor gene therapy, where the desired

ablative effects are immediate.

1 2 . 1 Introduction

Tumorigenesis is cumntly recognized as a multifactorial process of accumuiated

DNA dunaging events (Moolgavkar and Knudson, 1981) that may include the

conversion of proto-oncogenes to oncogenes (Dalla-Favera et al., 1982; Taub et al.,

1982), the inhibition of tumor suppressor genes (Knudson, 1993), or some combination

of these events (Fearon and Vogelstein, 1990). These events cm arise €rom genetic

mutations, rearrangements, or deletions. Tumor suppressors include such genes as RB,

pI6, and p53. While protwncogenes undergo activating mutations or reamngements to

becorne overexpressed or constitutively activated oncogenes, tumor suppressor genes

typically undergo inactivating mutations or deletions during tumorigenesis. Tumor

suppressor gene therapy is designed to restore or enhance tumor cells' inherent tumor

suppressor capabilities, hopefully leading to growth inhibition andlor prograrnmed ce11

deaih, or apoptosis.

Apoptosis is a regulated process characterized morphologically by cellular membrane

blebbing, ce11 siuinkage, and chromlitin condensation. Biochemicaily, ii can be detected

by DNA fragmentation visualized with agarose gel electrophoresis (Hockenbery, 1995).

Apoptosis provides a mechanism whereby cells may self-temiinate upon sensing DNA

damage, thus avoiding the risk of becoming potentidly malignant (Lee and Bernstein,

1995).

p53 has been found to induce ce11 cycle amst (Kuerbitz et al., L992) as well as

apoptosis (Lowe et al., 1993a; Lowe et al.. 1993 b) in response to DNA-damaging agents,

such as ionizing radiation (Figure 1.2). p53 was first isolated as a cellular protein which

bound to the simian virus 40 large T antigen (Lane and Crawford, 1979). Inactivation of

p53 by the Iarge T antigen has been found to increase resistance of cells to radiation-

induced apoptosis (McCûrthy et al., 1994). This inactivation is necessary for partial

transformation and extension of the lifespan of sirnian virus 40-auisfected human cells

(Lin and Simmons, 1991).

When functioning nonndly, p53 can act as r tumor suppressor gene. Not only can

inactivation of p53 lead to partial transformation of simian virus 40-infmed cells, but its

inactivation dso leads to increased susceptibili ty to tumorigenesis. Mice lacking wild-

type p53 have been show to be susceptible to a number of different cancers (Donehower

et al., 1992), and p53 mutations have been found to be among the most cornmon

mutations identified in humm maiignancies (Houstein et al., 1991). p53 is a

transcription factor (el-Deiry et al., 1993). and interaction of the large T antigen

oncoprotein with pS3 results in the inhibition of p53-mediated transcription (Jiang et al.,

1993). It is the role of p53 as a sequence-specific DNA-binding transcription factor that

is criticai for its tumor suppressor capabilities (Pietenpol et al., 1994).

Wild-type p53 has a short hdf-life and is typicdly expressed at low levels in cells,

making it difficult to detect using immunohistochemicd (IHC) techniques (Lane and

Benchimol, 1990; Porter et al., 1994). p53 mutations cm extend this half-life, resulting

in an overexpressed phenotype when using K. While low levels of wild-type p53

protein may actually have o protective effect against ce11 death (Lassus et al.. 1996). this

can change as wild-type p53 accumulates and becomes activated in response to DNA

drunage, such as following ionizing radiation (Kasian et al.. 1991; Fu and Benchimol,

1997; Siliciano et al., 1997; Abraham et al., 1999).

Figure 1.2: Reswnse of ~ 5 3 to DNA Damane

DNA Darnage I

Cyclincdks

1.2.2 1353 Protein Structure

The p53 protein can be divided into several general domains. The hydrophobie region

of 42 amino acids at the N-terminus of p53 comprise a transcriptional activation dornain

as efficient as the powerful herpes virus protein VPL6 activation domain (Fields and

Jang, 1990). This is dso the same region where the p53-inhibitor mdm2 binds (Kussie et

ai., 1996). The second central domain fiom amino acid 102 - 293 (Povletich et al.. 1993)

mediates p53's sequence-specific DNA binding ability (Kern et al., 1991). It is this

domain that contains the majonty of reported p53 mutations in human cancers (Hollstein

et al., 1991). The C-teminal region of p53 contains an oligomerization dornain that is

responsible for bringing wild-type p53 into a dimer of dimers (Lee et al., 1994). The C-

terminal end of p53 contains a single siranded DNA recognition region (Bakalkin et al.,

1994; Bakaikin et al., 1995) that rnay be paaially responsible for recognition of DNA

damage by p53 (L.ee et al., 1995; Reed et al., 1995). It may dso positively ngulate p53

sequence-specific DNA binding activity (Jayaraman and Prives, 1995). Nuclear

localization signais are also present in the C-terminus (Shaulsky et al.. L990; Shaulsky et

al., 1991).

L 2.3 ~53-Mediated Ce11 Cvcle Arrest

As has been mentioned, p53 is an initiator of two major cellular pathways: ce11 cycle

arrest, to allow repair of damaged DNA before DNA replication proceeds, and apoptosis

(Figure L.2). The mechanism of the former will be discussed first. Following radiation-

induced DNA damage and p53 accumulation, wild-type p53 can initiate GL arrest by

transcriptional activation of p21WmK" (Kastan et al., 1991; Kuerbitz et al., 1992; Dulic

et al., 1994) as well as perhaps becoming dinctly involved in DNA npair (Smith et al.,

1995). p2 1 WAF"C" is a wild-type p53-inducible cyclin-dependent kinase inhibitor (el-

Deiry et al., 1993; Gu et al., 1993) (Figure 1.2). It has been found to inhibit the cyclin E-

cdk2 complex during radiation-induced Gl arrest (Dulic et al., 1994), and is apparently

essentid for GI ce11 cycle anrst (Deng et al., 1995).

The cyclin E-cdk2 complex has k e n shown to be expressed in the G1 phase (Koff er

al., 1992), where it is capable of phosphorylating the pRb protein (Hinds et al., 1992).

While hypophosphorylated, pRb cm bind and inactivate the E2F transcription factor

(Chellappan et al.. 1991). leading to repression of genes responsible for advancing cells

through Gl to the S phase of the cell cycle. Phosphorylation of pRb by cyclin-cdk

complexes inhibits the ability of pRb to bind E2F (Suzuki-Takahashi et al., 1995),

thereby initiating ce11 cycle progression (Hinds er al., 1992).

L -3.4 ~53-Mediated Apoptosis

p53 is an important determinant for apoptosis as well as proliferation. In nsponse to

DNA strand breaks following ionizing radiation, wild-type p53 protein is activated and

accumulates (Kastan et al., 1991; Nelson and Kastan. 1994), possibly by retention of p53

in the nucleus (Freedman and Levine, 1998; Roth et al., 1998a). p53 has k e n found to

initiate apoptosis following ionizing radiation. thus regulating the toxicity of radiation as

well as other cytotoxic agents (Lowe et al.. 1993a; Lowe et al., 1993b). Mutant p53, on

the other hand, may inactivate wild-type pS3 by binding to it (Harvey et al., 1995)- while

some mutants appear to stimulate tumor growth (Dittmer et al., L993), thus revealing

oncogenic potential for certain pS3 mutations. The %cl-2 family of proteins cm regulate

p53-mediated apoptosis through a variety of mechanism. Bcl-2 has been show to

protect cells from apoptosis initiated by various cellular stresses, including ionizing

radiation (S trasser et al., 1994).

Bax, a Bcl-2 family member (Oltvai et al., 1993) (Figure 1.2). appears to be one of

several pro-apoptotic p53-target genes. It can fom homodimers and ûccelerate apoptosis

induced by cytokine deprivrtion as well as bind Bcl-2 and sounteract Bcl-Zmediated

suppression of ce11 death (Oltvai et al., 1993). p53 may downregulate Bcl-2 gene

expression and upregulate Bax gene expression, suggesting a direct role for Bcl-2 and

Bax in the p53 apoptotic pathway (Miyashita et al., 1994) (Figure 1.2). In an

examination of p53-l- and ban-/- mouse fibroblasts, Bax was shown to be an effector of

p53-mediated apoptosis following exposure to chemotherapy and ionizing radiation

(McCumch et al., 1997). However, this same paper reported that bax deficiency did not

account for the sarne resistance to apoptosis found in p53-deficient fibroblasts following

exposure to chemotherapy.

There are two membrane receptoa that rnay also be mediators of p53-dependent

apoptosis, FadAPO 1 and KIUEWDRS (Figure 1.2). Upon activation, the huma. Fas

receptor can cause apoptosis (Itoh et al., 1991). p53 is capable of upregulating Fas

(Sheard et al., 1997; Bennett et al., 1998; Muller et al., 1998), but it is not clea. how

important a role endogenous levels of Fas play in mediating p53-dependent apoptosis

(Reinke and Lozano, 1997). However, overexpression of Fas using an adenoviral vector

has been found to sensitize cancer cells to gene therapy with an adenovirus encoding a

cDNA for human wild-type p53 (Ad-p.53) (Rakkar et al., 1999), demonstrating a

potentiai for this combined treatment in tumors resistant to Ad-p53.

The KILLEWDRS death receptor is another pro-apoptotic protein induced by DNA

damage that appears to be regulated in some instances by p53 (Wu et al., 1997).

Infection of breast, oviuian, and colon cancer cells with Ad-p53 was found to induce

expression of KIUEWDRS (Wu et al., 1997), and induction of KILLEWDRS foilowing

ionizing radiation appears to be dependent on wild-type p53 (Sheikh et al., 1998). It is

not clear which of the p53-tûrget genes mentioned in this section, if any, play a major mie

in mediating p53-dependent apoptosis. More likely, then is a combination of p53-target

genes thnt play various roles in different physiological conditions (el-Deiry, 1998).

The p53 protein hûs been termed the guardian of the genome. It is thought to be a key

derenninant in sensing DNA darnage and either halting proliferation before DNA

replication until the darnage is repaired, or causing the ce11 to undergo apoptosis if the

damage is too great (Lane, 1992). It is currently not well understood why p53 may

choose GL arrest over apoptosis or vice versa. There has been some suggestion ihat this

decision rnay be related to the level of cellular p53 protein present. Low levels of p53

pmtein appear to favor Gl m s t , while higher levels hvor apoptosis (Chen et al., 1996;

Chen et al., 1998). Certainly, our group and others have shown that large amounts of

exogenous wild-type p53 introduced into a wide variety of cancer cells in a gene therapy

context generally causes induction of apoptosis (Liu et al., L995a; Katayose et al., 1995;

Li et al., 1997; Li et al., 1998; Li et al., 1999).

1 2.5 Use of 053 in Gene Thera~ y

Over the past few yean, there have been numemus papers examining the effects of

exogenous p53 gene nansfer on a variety of cancer ce11 iines and "nomal" cells in vitro

alone (Katayose et al., 1995; Li et al., 1997; Li et al., 1998; Li et al., 1999) and in vitro

and in vivo (Cirielli et al., 1995; Gallardo et al., 1996; Spitz et al., 1996; Pirolio et al.,

1997; Badie et al., 1998). Use of Ad-p53 gene therapy in vitro typically resdted in

apoptosis of human cancer cells harboring either mutant or deleted p53 (Liu et al., 1995a;

Gdlardo et al., 1996; Pirollo et al., 1997) or wild-type p53 (Liu et al., 1995~; Li et al.,

1998), while nomal human fibroblasts (Clayrnan et al., 1995; Li et al., 1997; Li et al.,

1999) and mammary epithelial cells (Katayose et al., 1995) were spared from the

treatment. Use of Ad-p53 in humm cancer xenograft animal models resulted in tumor

growth inhibition (CMelli et al., 1995) andlor pathologically complete regression

(Himada et al., 1996).

Since p53 may enhance the cytoxicity of ionizing radiation and some chemotherapy

dmgs (Lee and Bernstein, 1993; Lowe et al., 1993a), the use of pS3 gene therapy in

combination with these therapeutics has also been examined in various cancers (Gailardo

et al., 1996; Pirollo et al., 1997; Nielsen et al., 1998; Li et al., 1999). We have chosen to

snidy the effects of combining Ad-p53 gene therapy plus or minus ionizing radiation on

NPC in an in vivo setting. To date, there have been only a few groups who have

published on the use of combined ionizing racliation and Ad-p53 in an animal tumor

model (Spitz et al., 1996; Gallardo et al., 1996; Pirollo et al., 1997; Badie et al., 1998).

Table 1.1 lists some of the details of these papers. Each report described a statistically

significant inmased radiosensitivity, as measured by tumor growth, when radiation was

combined with Ad-p53 in vivo in three human cancer models (Spitz et al., 1996; Gallardo

et al., 19%; Pirollo et al., 1997) and one rat glioma model (Badie et al., 1998). In order

to observe significant tumor regression with ionizing radiation and Ad-pS3, total doses of

5 Gy + 7.5 x 109 pfu of Ad-p53 (Spitz et al., 1996). 8 Gy + 6 x 10' pfu (Gallardo et al.,

1996), 20 Gy + 5 x 108 pfu - 1.5 x 109 pfu (PiroLio et al., 1997), or 10 Gy + 1 x 10' pfu

(Badie et al., 1998) were used.

Table 1.1: Published Ad453 + Radiation AnimPL Tumor Models 1 Authors(Year) 1 Cancer 1 p53 1 Injection ~ e t h o d 1 Mouse ModeüTumor Size Spitz el al ( 1996)

1

Gaiiardo et ai (1996)

Colorectal

Pirollo et al (1991)

Not only is there a wide variation in doses and treatment protocols published for Ad-

p53 gene therap y use in animal tumor models (almost one for every paper published), but

as can be seen to some extent in Table 1.1, various injection techniques and injection

volumes are used as well. Few groups have reported on transduction efficiency obtained

following intratumoral injection of virus, and no injection methods to date have resulted

in 100% Nmor transduction. Only one group has reported on king able to achieve over

80% tumor transduction following intratumoral injection of adenovims (Cusack et al.,

1996). Gallardo et al obtained 50% aimer transduction (Gaiiardo et al., 1996), but

Mujoo et al, using the same ovarian cancer ce11 line, were only able to find 10 - 20% of

Ovarian

Badie et al (1998)

Status Mutant

Head & Nec k

Deleted

Glioma

1

0- . . . Triple Injection i.m. tumors in scid flank; (3 sites) 5 - 6 mm diameter tumors

Mutant

Single Injection 27G needle

Mutant

S.C. tumors in nude rnice; 8 mm diarneter tumors

O. lmL injection Single Injection 0.05rnL injection

treated S.C. tumors in nude mice; unknown tumor size treated

Single injection intracranial tumors in rats; unknown tumor size treated ( 1.5 mm diameter at most)

tested sections staining positive for adenoWus transduction (Mujoo et al., 1996).

O'Malley et al could only obtain 1 - 10% tumor transduction (OMalley BW et al., 1995).

One of the major obstacles of gene therapy is the limited distribution of the gene

therapy vector following intratumoral injection (Sandig et al., 1997). The use of the pS3

gene may be able to take advantage of an in vivo 'bystander effect" to overcome this

difficulty, however. The bystander effect occurs when neighboring non-transduced cells

die dong with those that were successfully transduced, effectively increasing the treated

tumor volume. This phenornenon has been observed typically in gene therapy using the

thymidine kinase gene in combination with ganciclovir (Pope et al., 1997).

Recently, however, a bystander effect was reported with the use of pS3 gene therapy

(Frank et d., 1998). p53 has also been shown to downregulate vascular endotheliai

growth factor (VEGF), a stimulant of angiogenesis (Mukhopadhyay et al., 1995). p53

gene thenpy in colon cancer ceIl lines has confimed a corresponding decrease in VEGF

levels (Bouvet et al., 1998). This finding was suggested as a partial explmation for the

mechanism behind a bystander effect in p53 gene therapy (Bouvet et al., 1998), since

tumors need to recruit new blood vessels in order to grow p u t a certain size (Folkman.

1992).

Despite poor transduction of the vector in vivo, the successful cytotoxic and growth

inhibitory effects observed in cancer cells both in vitro and in vivo in the laboratory with

Ad-p53 gene therapy have encouraged the commencement of clinical trials. The results

of some phase 1 trials have been published (Roth et al., 1996; Clayman et al., 1998; Roth

et al., 1998b; Schuler et al., 1998). In these trials, p53 gene therapy generally was found

to be well tolerated. The earliest study used retroviral-p53 gene therapy in non-mal1 cell

Iung carcinoma (Roth et al., 1996). Three of seven patients demonstrated tumor

regression, with two experiencing a complete response at the treated site 1 - 3 months

post-treatment completion. The patients included in this trial eventually died of causes

unrelated to the treated tumor. In an adenovirus-p53 (Ad-p53) phase 1 trial of

unresectable head and neck squamous ce11 carcinoma, two out of seventeen patients

showed tumor ngression pater than 50% following treatment (Clayrnan et al., 1998).

The authors plan to undertake phase II clinicd trials.

L.3.1 Introduction

C. A. Perez et al in Principles and Practice of Radiation (Perez et al., 1997) define

rdation therapy, or radiotherapy, as follows:

"...a clinical modaiity dealing with the use of ionizing radiations in the matment of

patients with malignant neoplasias (and occasionally benign discascs) ...ln addition to

curative efforts, radiation therapy plays a major role in cancer management in the

effective pdliation or prevention of symptorns of the disease: pain can be alleviated,

Iurninal patcncy restorcd, skeletal integrity prtserved, and organ function reestablished

with minimal morbidity in a vaïety of c l in id circumst;inces."

Ever since a year following theù discovery by W.C. Roentgen in 1895, X-rays have been

used to mat cancer (Bristow and Hill, 1998). The F i t patient c m d by radiation therapy

was reported in 1899 (Perez et al., 1997), and a presentation in 1922 at the International

Congress of Oncology in Paris marked the beginning of radiation therapy as a medicd

discipline (Perez et al., 1997).

X-rays are a form of high energy electromagnetic radiation (EM). EM radiation

includes radio waves with wavelengths of about 10' m to X-rays with wavelengths of less

than 10-~ m. and cm be alternatively considered, due to quantum physics, as consisting of

moving particles of photons (Purdy, 1997). X-rays have a shorter wavelength, and thus

higher energy, than ultraviolet rays (Purdy, 1997; Bristow and Hill, 1998).

Ionizing radiation refers to radiation with energy sufficient to remove an orbital

electron from an atom. This electron loss leaves the atom as an ion with a positive charge

(Bristow and Hill, 1998). The energy required to remove an electron in biologic material

is around 10 eV. X-rays with wavelengths of 1 0 - ~ m have energy of roughly LOO eV. So

X-ray photons, but not ultraviolet radiation, have sufficient energies to be considered

ionizing radiation (Bristow and Hill. 1998).

1.3.2 Biologic - Effects of Radiation Therapy

Radiation dose is rneasured in tenns of absorbed energy. One unit of ionizing energy

is L Gy, which refers to an ûbsorbed dose of 1 Joule I kg of irradiated material.

Approxirnately 105 ionization events are thought to occur on average within a ce11

exposed to a dose of 1 Gy of radiation. This number of ionizations leads to about 200

single-strand and 25 - 50 double-strand DNA breaks (Bristow and Hill, 1998). While

ionizing radiation also causes darnage to the plasma membrane that can lead to apptosis

(Fuks et al., 1995), it is DNA damage that is thought to be the critical deterrninant of

ionizing radiation-induced c ytotoxicity (Nunez et al., 1996; Schwartz et al., 1996;

Bristow and Hill, 1998). Radiosensitivity of certain cells has ken conelated with defects

in DNA repair (Hendrickson et al., 199 1). and the number of DNA double-strand breaks

cornlates with ionizing radiation-induced cytotoxicity in several ce11 types Qristow and

Hill, 1998).

The classic radiobiological technique for rneaswing cell death following ionizing

radiation in vitro is the clonogenic assay. This assay directly examines the survival of

colony-forming cells by incubating the cells in a low density following treatrnent and

counting the number of cells able to produce driughter cells (Fuks et al.. 1995).

Generally, clonogenic survival is inversely related to the dose of ionizing radiation. A

plot of clonogenic surviving Fraction of mammalian cells on the y-axis of a

semiloguithmic plot, with increasing dose of radiation on the x-axis, generates an inverse

linesr relationship. This curve tends to have two components, a shoulder region at lower

doses and a linear region at higher doses (Figure 1.3). The dope of the linear portion of

the curve is thought to represent the radiation sensitivity of the cells tested. while the

width of the shoulder portion is believed to reflect the capacity of the tested cells to repair

sublethal radiation damage (Elkind and Sutton, 1959; Bristow and Hill, 1998).

Fnctionation (or splitting a dose over tirne) of radiation results in the ability of cells to

tolerate higher total doses of radiation. This is presurned to be due to cellu1a.r repair

capabilities, and a shoulder is generated with each fraction given. Thus. while a single

dose of 12 Gy might result in 0.1% clonogenic survival, a dose of 4 Gy given on three

consecutive days might result in 6% clonogenic survival (EIkind and Sutton, 1960;

Bristow and Hill, 1998) (Figure 1.3).

Figure 13: Clonmenic Survivd Foilowing Ionizine Radiation

Figure 1.3. Clonogenic survival for ctIIs given a single dose up to 12 Gy (bottom iinc) and cells given 3 Gy singIe doses on threc consecutive days up to û totd dose of 12 Gy (top lines).

1.4 Naso~harvn~eai Carcinoma

1.4.1 Introduction

Nasopharyngeal carcinoma (NPC) is an epithelial tumor of the head / neck ana that

can be classified into two histologie categories: squamous cell carcinoma (SCC) and

undifferentiated carcinoma of nasopharyngeal type (UCNT) (Fandi et al.. 1994). NPC

incidence is about 0.5 - 2 / 100,000 people pet year in the world with a median age of

presenting in patients in their fourth decade (Fandi et al., 1994). However, it is much

more common in certain geographic regions, such as in southem China, where the

incidence reaches 30 - 80 1 100,000 people per year. UCNT is the predominant form

found in endemic regions, with SCC king more commonly found outside these regions

(Fandi et ni., 1994; Fandi and Cvitkovic, 1995). In 1990, the= were an estimated 57,500

new cases of NPC worldwide, with 44% of those cases occming in China and 23% h m

southeast Asia (Parkin, 1998). Canada has a much lower incidence of NPC, but

immigration from southeast Asia has added to this number. Currently, cancer centers in

Toronto treat about 90-100 new cases per year. If Toronto cancer hospitals are

considered to serve a population of 3 million, that puis the incidence of W C in this

population at approximately 3 1 100,000 people per year.

While perhaps diet and exposure to domestic wood fires may be associated with

occurrence of UCNT in China (Zheng et al., 1994), tobacco use may be linked to SCC in

North Amenca (Chow et al., 1993). There has also been some evidence of a genetic risk

factor (Lu et al., 1990), and a role for the Epstein-Barr virus (EBV) in NPC

tumorigenesis has always been suspected, although never proven. EBV is consistent] y

observed in association with UCNT (Liebowitz, 1994).

1.42 E~stein-Barr Virus and NPC

EBV is responsible for infectious mononucleosis (Henle et al., 1968). Aside from

NPC, EBV is dso associated with Burkitt's lymphoma, where the virus was fint

observed (Epstein et al., 1964), pst-transplant 1 ymphoma (Gratama et al., 199 1 ), and

gastnc carcinoma (Imai et al., 1994). EBV is a ubiquitous human herpesvinis that exists

in NPC in a latent form (Rickinson and Kieff, 19%). While there are at lest 10 EBV

gene products, expression of EBV proteins in NPC is typically iimited to EBV nuclear

antigen 1, EBNAI; latent membrane protein 1 and 2, LMPl and LMP2 (Fatuaeus et aï.,

1988; Brooks et al., 1992; Busson et al., 1992); and the small nuclear EBV-encoded

RNAs, EBERs (Niedobitek et al., 1992).

It is currently uncleor whether EBV plays a role in NPC tumorigenesis. EBV

expressirn in NPC tumon has been found to be clonal, and in longitudinal studies, EBV

infection has ken observed in premalignant lesions of patients who have proceeded to

develop NPC (Pathmanathan et al., 1995). EBV latent infection is typically thought to

persist in B-lyrnphoid tissue (Niedobitek and Young, 1994). Even though viral infection

has been associated with epithelid malignancies, nonnd epithelium has not yet ken

found to harbor either latent or lytic EBV (Young, 1996). This, dong with the clonai

nature of the virus in NPC as well as its association with premalignant WC, suggests that

the virus is at least associated with the very early stages of NPC tumorigenesis, perhaps

king involved in tumorigenesis itself. LMPl, one of the latent gene products of EBV,

has k e n found to inhibit differentiation of epithelid cells, suggesting a possible

functional link between EBV and undifferentiated NPC (Dawson et al., 1990), but this is

a point of controveny (Nicholson et al.. 1997). However, as will be described in the

following section, LMPl has been found to interact with p53 activity as well (Fries et al.,

1996).

1.4.3 p5.3 and NPC

The consensus in the literature concerning p53 status in NPC is that pnmary tumors

contain wild type pS3 sequence within exons 4-8 (Effert et al., 1992), although there are a

few reports of p53 mutations in NPC (Lung et al., 1998). There are several groups,

however, that have demonstrated overexpression of pS3 using immunohistochemical

(MC) staining of primary NPC biopsies (Porter et al., 1994). In normal B cells, infection

with EBV has been found to increase basal wild-type p53 levels by about IO-fold, which

is thought to be due to LMPl transactivation (Chen and Cooper, 1996). It is currently

unknown if this accounts for overexpnssion of p53 in NPC, or why overexpressed p53

rnight not inhibit NPC tumor progression. One possibility may be that p53 is mutated

outside exons 4-9 in NPC (Porter et al., 1994), so the overexpressed inactivated mutant

protein would not inhibit NPC tumorigenesis. Altematively, an EBV protein may bind to

wild-type p53, thus stabilizing and inactivating it. An EBV protein, EBNA-5, has been

shown to bind p53 in vitro (Szekely et al., 1993). although Porter et al did not observe a

correlation between IgA titre to EBV viral capsid antigen and level of p53

irnmunostaining (Porter et al., 1994).

However, LMPl has k e n also found to upregutate the A20 protein through the NF-

KB transcription factor (Laheny et al., 1992). The A20 protein has been subsequently

shown to inhibit p53-dependent apoptosis following induction by LMPl in epithelial

cells (Fries et al., 1996). Further, A20 mRNA has recently been observed to be

consistently expressed in NPC tissue, whereas A20 mRNA was not detected in normal

squamous epithelial tissue (Codd et al., 1999). It is of note that LMPl is expressed in

NPC and post-transplant lyrnphoma (Gmtama et al., 1991), where p53 is wild-type

(Effen et al., 1992; Edwards and Raab-Traub, 1994), whereas LMP 1 expression is not

obsewed in either Burkitt's lymphoma (Rowe et al., 1987) or gasûic carcinoma (Sugiura

et al., 1996), where p53 is frcquently mutated (Famli et al., 1991; Edwards and Raab-

Traub, 1994; Kobayashi et al., 1996). This may suggest that the= is no need for NPC

tumoa to mutate p53, since the p53 pathway has aiready ken abrogated by EBV (Fries

et al., 1996). This concept is potentially supported by observations in NPC ce11 lines that

tend to be initial1 y EBV positive and wild-type for p53, but then shed EBV and develop

mutations in p53 with subsequent passaging (Lin et al., 1994). Consistent ovenxpression

of wild-type p53 in NPC might not seem counterintuitive, therefore, if the apoptotic

pathway downstream of p53 is also inhibited.

1.4.4 Treatment of WC

The primary modality for treating NPC is cumntly radiotherapy (Fandi et al., 1994;

Mendenhall et al., 1994). Surgery cannot be undertaken due to the anatomic proximity of

the iumor to the skull base, making it technically difficult to operate (Mendenhall et al.,

1994; Vokes et al., 1997). although large nodes in the neck can be resected (Mendenhail

et al., 1994; Vokes et al., 1997). The overall 5-year survival rates in response to

radiotherapy range from 70 - 80% for early stage patients down to 20 - 40% for stage IV patients (Vokes et al.. 1997).

Recent efforts have focussed on combining ionizing radiation and chemotherap y (AI-

Smaf et al., 1998; Chan et al., 1998; Taamma et al., 1999), although on1 y one triai (Al-

Sarraf et al.. 1998) has demonstrated a benefit. Hence, given the modest survival rate in

a relatively young population, there is a need to develop a modality in addition to

radiotherapy that is capable of improving locoregional control.

1.5 NPC Ce11 Lines and Xenomafks

The generation of human NPC ce11 lines has proven very dificuit. One group has

reported obtaining one ce11 line from 117 biopsies (Chang et al.. 1989). The difficulty

appeiirs to be related to overgrowth of fibroblasts and perhaps lymphoid cells h m

biopsy sarnples (Chang et al., 1989). Our [ab has attempted unsuccessfulIy to establish

W C ce11 lines from biopsy materials. However, there are several labs that have ken

successful (Huang et al., 1980; Zhang et al., 1982; Sizhong et al., 1983; Chang et al.,

1989; Yao et al., 1990; Hui et al., 1998). Once an W C ce11 line becomes established and

propagated in vitro, there i s a tendency for the cells to shed the EBV originally associated

with the tumor (Lin et al., 1994). Therefore. the establishment of an NPC ce11 line that

has retained EBV after multiple passages is of significant interest and value. Several

groups have been able to accomplish this (Busson et al.. 1988; Chang et al., 1989; Yao et

al., 1990; Hui et al., 1998).

We originally obtained the NPC ce11 lines CNE-1 and CNE-22 from the Chinese

Academy of Medical Sciences (Zhang et al., 1982; Sizhong et al., 1983). The bulk of the

work presented herein utilizes the CNE-1 ce11 line. CNE-I was established fiom a tumor

biopsy of a well-differentiated squamous NPC from a northern Chinese patient (Zhang et

al., 1982). CNE-22 was established from a 68-year-old Cantonese male with stage III

poorly differentiated NPC (Sizhong et al., 1983). Both harbor the same pS3 point

mutation at codon 280 in exon 8 of AGA to ACA, changing an arginine to a threonine

(Spruck et al., 1992).

Even though mutations in p53 appear to be rare in primary NPC tumors, the sarne

mutation mentioned above was found as a heterozygous mutation in a primary tumor

fiom a patient in Hunan Province (Sun et al., 1992). It is not clear if the p53 mutation in

both CNE-1 and CNE-22 was caused by establishment of the ce11 iines or was already

present in the pnmary tumor (Spruck et al., 1992). This mutation has ken charactenzed,

however, and found to be responsible for producing a dominant negative p53 protein.

The mutant protein was able to inhibit the ability of wild-type p53 to drive transcription

of a p53-responsive reporter gem as well as block wild-type p53-mediated inhibition of

ce11 growth (Sun et al., 1993).

We have mon recently been able to obtain the EBV-positive NPC xenograft Cl5 from

Piem Busson at the Institut Gustave Roussy (Busson et al., L988). I will discuss in this

thesis some preliminary experiments using this xenograft as well. The CL5 xenograft has

been continuously passiiged in nude and scid mice since its establishment (Busson et al.,

1988). It was original1 y obtained from the primary W C tumor of an untreated 13-year-

old girl (Busson et al., 1988). The histology of the xenograft was found to be epithelial.

and the presence of EBV proteins and EBV genome through Southem blot andysis was

confinned as well (Busson et al.. 1988). C 15 was identified dso to have retained wild-

type p53 by sequence andysis (Effett et al., 1992; Bernheim et al., 1993). This xenograft

therefore represents an additional useful tool to determine the efficacy of gene thenpy in

WC. It is especially pertinent since it may be a more accurate mode1 of a primary NPC

tumor in human patients due to the EBV association and wild-type p53 status.

1.6 Rationale and Proiect Outline

Since the overail 5-ycar survival for NPC is currentiy only 6596, the focus of this work

is on the potential use of adenovirus-p.53 gene therapy as an adjunct to radiotherapy in the

matment of this diseme. Towards that end, and encouraged by evidence in our lab that

Ad-p53 plus or minus ionizing radiation produced a more-than-additive effect on NPC

cells in vitro (Li et al., 1999), we have examined the effects of the combination of Ad-

p53 and ionizing radiation using a xenogtafi in vivo model.

The first section of this thesis (Chapter Two) will discuss the detemination of the

infection effciency of the adenovirus vector in intramuscular NPC xenogdt tumors in

mice following intratumord injection. The second section (Chapter Three) will focus on

the results of Ad-p53 4- ionizing radiation therapeutic expenmentç in a CNE-1 xeno@t

model. The concluding chapter will summarize the findings presented in this thesis as

well as pmposed future directions. including the presentiition of some preliminary data

from the C 15 NPC xenograft.

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Anderson, W. F., 1984, Rospects for humm g n e therapy. Science. 226,4û 1-9. Badie, B ., Kramar, M. H., Lau, R., Boothman, D. A,, Econornou, 1. S. and Black, K. L.,

1998, Adenovinis-mediated p53 gene delivery potentiates the radiation-induced growth inhibition of experirnental bnin tumors. J Neurooncol, 37,217-22.

Bdcalkin, G., Selivanova, G., Yakovleva, T., Kiseleva, E., Kashuba, E., Magnusson, K. P., Szekely, L., Klein, G., Terenius, L. and Wiman, K. G., 1995, p53 binds single- stranded DNA ends through the C-terminal domain and intemal DNA segments via the middle domain. Nucleic Acidr Res, 23,362-9.

Bakalkin, G., Yakovleva, T., Selivanova, G., Magnusson, K. P., Szekely, L., Kiseleva, E., Klein, G., Terenius, L. and Wiman, K. G., 1994, p53 binds single-s~mded DNA ends and cataiyzes DNA renaturation and strand transfer. froc Natl Acad Sci US A, 91,413-7,

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