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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 !
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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.
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. .
........................................................................................................
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
........................................................................
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................................... 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
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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
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............................................................................................
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
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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
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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
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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
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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~
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Cha~ter One
Introduction
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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
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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).
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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.
-
Abraham, J., Spaner, D. and Benchimol, S., 1999, Phosphorylation
of p53 protein in response to ionizing radiation occurs at multiple
sites in both normal and DNA- PK deficient cells. Oncogene, 1%
1521-7.
Ai-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. and Ensley, I. F., 1998, Chemoradiotherapy venus radiotherapy in
patients with advanced nûsopharynged cancer: phase III randomized
Intergroup study 0099. J Clin Oncol, 16, 1310-7.
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,
Benchimol, S. and Minden, M. D. (1998) Viruses, oncogenes, and
tumor suppressor genes. In The basic science of oncology, (eds. 1.
F. Tannock and R. P. Hill), pp. 79-105, McGraw-Hill, New York.
Bennett, M., Macdonaid, K., Chan, S. W., Luzio, J. P., Simari,
R. and Weissberg, P., 1998, Cell surface trafficking of Fas: a
rapid mechanism of p53-mediated apoptosis. Science, 282,290-3.
Bergelson. J. M., Cunningham, I. A., Droguett, G., Kurt-Jones,
E. A., kithivas, A., Hong, 1. S., Horwitz, M. S., Crowell, R. L.
and Finberg, R. W., 1997, Isolation of a cornmon receptor for
Coxsackie B viruses and adenoviruses 2 and 5. Science. 275,
1320-3.
Bemheim, A., Rousselci, G., Massaad, L., Busson, P. and Tursz,
T., 1993, Cytogenetic studies in t h e xenografted nasopharyngeal
carcinomas. Cancer Genet Cytogener, 66,114.
Bischoff, I. R., Kim, D. H., Williams, A., Heise, C., Hom, S.,
Muna, M., Ng, L., Nye, J. A., Sampson-Johannes, A., Fattaey, A. and
McCormick, F., 1996, An adenovinis mutant that repücates
selectively in p53kficient human tumor cells. Science.
274,373-6.
Bouvet, M., Ellis, L. M., Nishizaki, M., Fujiwara, T., Liu, W.,
Bucana, C. D., Fang, B., Lee. J. J. and Roth, J. A., 1998,
AdenoWus-mediated wild-type p53 gene transfer down-regulates
vascular endotheliai growth factor expression and inhibits
angiogenesis in human colon cancer. Cmcer Res, 58,2288-92.
Brandt, C. D., Kim, H. W. and Vargosdo, A. 1.. 1969. Infections
in 18,000 infants and children in a contmlled study of respiratory
tract disease. 1. AdenoWus
-
pathogenicity in relation to serologic type and illness
syndrome. Am J Epidemiol, 90,484-500.
Bristow, R. G. and Hili, R. P. (1998) Molecuiar and ceilular
basis of radiotherapy. In The Basic Scimce of Oncology, (eds. 1. F.
Tannock and R. P. Hill), pp. 295-321, McGnw-HiU, New York.
Brooks, L., Yao, Q. Y., Rickinson, A. B. and Young, L. S., 1992,
Epstein-Barr virus latent gene transcription in nasopharynged
carcinoma cells: coexpression of EBNAI, LMPl, and LMP2 transcripts.
J Virol. 66,2689-97.
Burgert, H. G., Maryanski, l. L. and Kvist, S ., 1987, "E3119K"
protein of adenovinis type 2 inhibits lysis of cytolytic T
lymphocytes by blocking cell-surface expression of
histocornpatibility class I antigens. froc Nat! Acad Sci U S A, 84,
1356-60.
Busson, P., Ganem, G., Flores, P., Mugneret, F., Clausse, B.,
Caillou, B., Braham, K., Wakasugi, H., Lipinski, M. and Tursz. T.,
1988, Establishment and characterization of three transplantable
EBV- containing nasopharyngeal carcinomas. Int J Cancer,
42,599-606.
Busson, P., McCoy, R.. Sadler, R., Gilligan, K., Tursz, T. and
Raab-Traub, N., 1992, Consistent transcription of the Epstein-Barr
virus LMP2 gene in nasophqgeal carcinoma. J Virol, 66,3257-62.
Chan, A. T.. Teo, P. M., Leung, T. W. and Johnson, P. J., 1998,
The role of chemotherapy in the management of nasopharyngeal
carcinoma. Cancer, 82, 1003-12.
Chang, Y. S., Lin, S. Y., Lee, P. F., Durff, T., Chung, H. C.
and Tsai. M. S.. 1989, Establishment and characterization of a
tumor ce11 line from human nasopharyngeal carcinoma tissue. Cancer
Res, 49,6752-7.
Chellappan, S. P., Hieben, S., Mudryj, M., Horowitz, J. M. and
Nevins, J. R., 1991, The E2F transcription factor is a cellular
target for the RB protein. Cell, 65,10534 1.
Chen. P. HeT Omelles, D. A. and Shenk, T., 1993, The adenovirus
U 23-kiloâalton proteinose cleaves the amino-terminal head domain
from cytokeratin 18 and disrupts the cytokeratin network of HeLa
cells. J Virol, 67,3507-14.
Chen, W . and Cooper, N. R., 1996, Epstein-Barr virus nuclear
antigen 2 and latent membrane pmtein independently transactivate
p53 through induction of NF- kappa activity. J Virol.
70,4849-53.
Chen, W., Huang, S. and Cooper, N. R., 1998, Levels of p53 in
Epstein-Barr virus- infected cells determine ce11 fate: apoptosis,
ceIl cycle arrest at the GUS boundary without apoptosis, ce11 cycle
m s t at the G2/M boundary without apoptosis, or unrestricted
proliferation. Virology, 251,217-26.
Chen, X., Ko, L. J., Jayaraman, L. and Prives, C.. 1996, p53
levels, functional domains, and DNA darnage determine the extent of
the apoptotic response of tumorcells. Genes Dev, 10,2438-51.
Chow, W. HaT McLaughlin, J. K., Hnibec. Z., Nam, J. M. and Blot,
W. J., 1993, Tobacco use and nasopharyngeal carcinoma in a cohort
of US veterans. Int J Cancer, 55, 5384.
Chroboczek, J., Bieber, F. and Jacrot, B., 1992, The sequence of
the genome of adenovirus type 5 and its cornparison with the genome
of adenovinis type 2. Virology, 186,280-5.
-
Cirielü, C., Riccioni, T., Yang, C., Pili, R., Gloe, TeT Chang,
J., Inyaku, K., Passaniti, A. and Capogrossi, M. C., 1995,
Adenovim-mediated gene tmnsfer of wild-type p53 results in wlanoma
ce11 apoptosis in vitro and in vivo. Int J Cancer, 63,673- 9.
Clayman, G. L., el-Naggar, A. K., Lippman, S. M., Henderson, Y.
C., Fredenck, M., Memtt, J. A., Zumstein, L. A., Timons, T. M.,
Liu, T. J., Ginsberg, L., Roth, I. A., Hong, W. K., Bniso, P. and
Goepfert. H., 1998, Adenovims-mediated p53 gene transfer in
patients with advanced recurrent head and neck squamous ce11
carcinoma. J Clin Oncol, 16,2221-32.
Clnyman, G. L., el-Naggar, A. K., Roth, J. A., Pimg, W. W.,
Goepfert, H., Taylor, D. L. and Liu, T. 1.. 1995, In vivo molecular
therapy with p53 adenovirus for microscopic residud head and neck
squamous carcinoma. Cancer Res, 55, 1-6.
Codd, J. D., Salisbury, I. R., Packham, G. and Nicholson, L. J.,
1999, A20 RNA expression is associated with undifferentiated
nasopharyngeal carcinoma and poorly differentiated head and neck
squarnous ce11 carcinoma. J Pathol, 187,549- 555.
Coffey, M. C., Strong, I. E., Forsyth, P. A. and Lee, P. W.,
1998, Reovirus therapy of tumon with activated Ras pathway.
Science, 282, 1332-4.
Cusack, I. C., Spitz, F. R., Nguyen, De, Zhang, W. W., Cnstiano,
R. 1. and Roth, J. A., 1996, High levels of gene transduction in
human lung tumon following intralesional injection of recombinant
adenovirus. Cancer Gene nter, 3,245-9.
Dales, S . and Chardonnet, Y., 1973, Early events in the
interaction of adenoviruses with HeLa cells. IV. Association with
microtubules and the nuclear pore complex during vectorial movement
of the inocdum. Virology, 56,465-83.
Dalla-Favera, R.. Bregni. M., Erikson, J., Patterson, D.. Gallo,
R. C. and Croce, C. M., 1982, Human c-rnyc onc gene is located on
the ngion of chromosome 8 that is translocated in Burkitt lymphoma
cells. Proc Nail Acad Sci U S A, 79,7824-7.
Dawson, C. W., lückinson, A. B. and Young, L. S., 1990,
Epstein-Barr virus latent membrane pmtein inhibits human epithelial
ceIl differentiation. Nature, 344,777- 80.
Debbas. M. and White, E., 1993, Wild-type p53 mediates apoptosis
by ELA. which is inhibited by ElB. Genes Dev, 7,546-54.
Deng, C.. Zhang, P., Harper, S. W., Elledge. S. I. and Leder,
P., 1995, Mice lacking p21wAF11C*1 undergo normal development, but
are defective in G1 checkpoint control. Cell. 82,675-684.
Dittmer. D., Pati, S., Zambetti, G., Chu, S., Teresky, A. K.,
Moore, M., Finlay, C. and Levine, A. J., 1993, Gain of function
mutations in p53. Nat Genet. 4,42-6.
Donehower, L. A., Harvey, M., Slagle. B. L*, McArthur, M. J.,
Montgomery, C. A., Ir., Butel, I. S. and Bradley, A., 1992, Mice
deficient for p53 are developrnentally normal but susceptible to
spontaneous tumours. Nature. 356,215-21.
Dube, 1. (1998) Opening address: The past and the future of gene
therapy in Canada. In 3rd Canadian Gene Xherupy Symposium, Monmai,
Quebec.
Duiic, V., Kaufmann, W. K., Wilson, S. J., Tlsty, T. D., Lees,
E., Harper, J. W., Elledge, S. J. and Reed S. I., 1994,
p53-dependent inhibition of cyclindependent kinase activi ties in
human fibroblasts during radiation-induced G1 amst. Cell. 76, 10
13- 23.
-
Edwards, R. H. and Raab-Traub, N., 1994, Alterations of the p53
gene in Epstein-Barr virus-associated immunodeficienc y-related 1
ymphomas. J V h l . 68, 1 309- 1 5.
Effert, P., McCoy, R., Abdel-Hamid, M.. Flynn, K., Zhang, Q.,
Busson. P., Tursz, T., Liu, E. and 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 downstnmgenes. Sentin
Cancer Biol, 8,345- 57.
el-Deiry, W. S., Tokino, T., Velculescu, V. E., k v y , D. B.,
Parsons, R., Trent. J. M., Lin, D., Mercer, W. E., Kinzler, K. W.
and Vogelstein, B., 1993, WAFl, a potential mediator of p53 tumor
suppression. CeIl, 75,8 17-25.
Elkind, M. M. and Sutton, H., 196û, Radiation response of
mammalian cells grown in culture: 1. Repair of x-ray damage in
surviving Chinese hamster cells. Rodiat Res, 13,556493.
Elkind, M. M. and Sutton, H. G., 1959, X-ray darnage in recovery
in mamalian cells in culture. Nature, 184, 1293-1295.
Epstein, M. A., Achong, B. G. and Barr, Y. M., 1964, Virus
particles in cultured 1 ymphoblasts from Burkitt's 1 ymphoma.
Lcrncet, 1,702.
Fahraeus. R., Fu, H. L., Emberg, L, Finke, I., Rowe, M., Klein,
G., Falk, K., Nilsson, E., Yadav, M., Busson, P. and et ai., 1988,
Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal
carcinoma. Int J Cancer, 42,329-38.
Fandi, A., Altun, M., Azli, N., Armand, J. P. and Cvitkovic, E.,
1994, Nasopharyngeal cancer: epidemiology, staging, and matment.
Semin Oncul, 21,382-97.
Fandi, A. and Cvitkovic, E., 1995, Biology and treatment of
nasopharyngeal cancer. Curr Opin Oncol, 7,255-63.
Farrell, P. I., Man, O. J., Shanahan, F., Vousden, K. H. and
Crook, T., 199 1, p53 is frequently mutated in Burkitt's lymphoma
ce11 lines. EMBO J. 10,2879-87.
Fearon, E. R. and Vogelstein, B., L990, A genetic model for
colorectal tumorigenesis. Cell, 61,759-67.
Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W.,
Wenz, M., Northrop, I. P., Ringold, O. M. and Danielsen, M., 1987,
Lipofection: a highly efficient, lipid- mediated DNA-transfection
procedure. Proc Nat1 Acad Sci U S A, û4,7413-7.
Fields, S. and Jang, S. K., 1990, W n c e of a potent
transcription activating sequence in the p53 protein. Science, 249,
1046-9.
Fol kman. J., 1992, The role of angiogenesis in tumor growth.
Seniin Cancer Biol, 3,65- 71.
Fox, J. P., Brandt, C. D., Wassermann. F. E. and al, e., 1969,
The Virus Watch Rograrn: a continuing surveillance of viral
infections in metroplitan New York fami lies. VI. Observations of
adenovirus infections: Wus excretion pattems, antibody response,
efficiency of surveillance, patterns of infection and relation to
illness. Am J Epidemiol, 89,2540.
Frank, D. K., Frederick, M. J., Liu, T. J. and Clayman, O. L.,
1998, Bystander effect in the adenovinis-mecbated wild-type p53
gene therapy model of human squamous cell carcinoma of the head and
neck. Clin Cmcer Res. 4,2521-8.
Freedman, D. A. and Levine, A. J., 1998, Nuclear export is
required for degradation of endogenous p53 by MDM2 and human
papillomavinis E6. Mol Cell Biol. 1% 7288-93.
-
Fries, K. L., Miller, W. E. and Raab-Traub, N., 1996,
Epstein-Barr vinis latent membrane protein 1 blocks p53-mediated
apoptosis through the induction of the A20 gene. J Virol,
70,8653-9.
Fu, L. and Benchimol, S., 1997, Participation of the human p53
3'UTR in translationai repression and activation following
gamma-irradiation. EMBO J, 16,4117-25.
Fuks, Z., Haimovitz-Friedman, A. and Kolesnick, R. N., 1995, The
role of the sphingomyelin pathway and protein kinase C in
radiation- induced ce11 kill. Imporrant Adv Oncol,, 19-3 1.
Gallardo, D., Drazan, K. E. and McBride, W. H., 1996,
Adenovirus-based transfer of wild-type p53 gene increases ovarian
tumor radiosensitivity. Cancer Res, 56, 489 1-3.
Graham, F. L. and Revec, L. (1991) Manipulation of adenovirus
vectors. In Methods in Molecular Biology, Vol. 7, (ed. E. J.
Murray), pp. 109-128, The Huma Press Inc., Clifton, New Jersey.
Gratama, J. W., Zutter, M. M., Minarovits, J., Oosterveer, M.
A., Thomas, E. D., Klein, G. and Ernberg, I., 1991, Expression of
Epstein-Barr virusencoded growth- tnuisformation- associated
proteins in lymphoproliferations of bone-marrow transplant
recipients. Int J Cancer, 47, 188-92.
Greber. U. F.. Willetts, M., Webster, P. and Helenius, A., 1993,
Stepwise dismantling of adenovirus 2 during entry into cells. Cell,
75,477-86.
Gu, Y., Turck, C. W. and Morgan, D. O., 1993, Inhibition of CDKZ
activity in vivo by an associated 20K regulatory subunit. Nature,
366,707-10.
Hail, A. R., Dix, B. R., SI, O. C. and Braithwaite, A. W., 1998,
p53-dependent ce11 deatMapoptosis is qui red for a productive
adenovinis infection. Nat Med, 4, 1068-72.
Hamada, K., Alemany, R., Uiang, W. W., Hittelman, W. N., Lotan,
R., Roth, J. A. and Mitchell, M. F., 1996, Adenovims-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. and White,
E., 1996, The ElB 19K protein blocks apoptosis by interacting with
and inhibiting the p53-inducible and death-promoting Bax protein.
Genes Dev, 10,46 1-77.
Harvey, M., Vogel, H., Moms, D., Bradley, A., Bernstein. A. and
Donehower, L. A., 1995. A mutant p53 transgene accelerates tumour
developmen t in heterozygous but not nuilizygous p53-deficient
mice. Nat Genet, 9,305-1 1.
Hearing, P., Samulski, R. J., Wishart, W. L. and Shenk, T.,
1987, Identification of a npeated sequence element required for
efficient encapsidation of the adenovirus type 5 chromosome. J
Virol, 61,2555-8.
Hendnckson, E. A., Qin, X. Q., Bump, E. A., Schatz, D. G.,
Oettinger, M. and Weaver, D. T., 1991, A link between double-strand
breakielated npair and V@)J cecombination: the scid mutation. Proc
Nat1 Acad Sci U S A. 88,4061-5.
Henle, O., Henle, W. and Diehl, V., 1968, Relation of Burkitt's
tumor-associated herpes- type virus to infectious mononucleosis.
Proc N d Acad Sci U S A. 59,94-101.
Hinds, P. W., Mitmacht, S., Dulic, V., Arnold, A., Reed, S. 1.
and Weinberg, R A., 1992, Regulation of retinoblastoma pmtein
functions by ectopic expression of human cyclins. Cell.
70,993-1006.
Hockenbery, D., 1995, Defining apoptosis. Am I P d ,
146,164.
-
Hollstein, M., Sidransky, D., Vogelstein, B. and Harris, C. C.,
1991, p53 mutations in human cancers. Science, 253,4943.
Home, R. W., Bonner, S., Waterson, A. P. and Wildy, P., 1959,
The icosahedral fom of an adenovinis. J Mol Biol, 1,8486.
Horwitz, M. S. (1996) Adenoviruses. In Fields Virology, Vol. 2,
(eds. B. N. Fields, P. M. Knipe, P. M. Howley, R. M. Chanock, J. L
Melnick, T. P. Monath and B. Roizman), pp. 2 149-2 17 1,
Lippincott-Raven, Phüadelp hia.
Hsiao, M., Tse, V., Carmel, J., Tsai, Y., Felgner, P. L., Haas,
M. and Silverberg, G. D., 1997, Intracavi tary liposome-mediated
p53 gene transfer into glioblastoma with endogenous wild-type p53
in vivo results in tumor suppression and long-terni survival.
Biochem Biophys Res Commun, 233,359-64.
Huang, D. P., Ho, I. R, Poon, Y. F., Chew, E. C., Saw, D., Lui,
M.. Li, C. L., Mak, L. S., Lai, S. H. and Lau, W. H., 1980,
Establishment of a ce11 line (NPC/HKl) from a differentiated
squarnous carcinoma of the nasopharynx. Int J Cancer, 26,
L27-32.
Hui, A. B., Cheung, S. T., Fong, Y., Lo, K. W. and Huang, D. P.,
1998, Characterization of a new EBV-associated nasopharyngeai
carcinoma ce11 line. Cancer Genet Cytogenet. 101,83-8.
Imai, S., Koizurni, S., Sugiuni, M., Tokunaga, M., Uemura, Y.,
Yamamoto, N., Tanaka, S., Sato, E. and Osato, T., 1994, Gastric
carcinoma: monoclonal epithelial malignant cells expressing
Epstein-Barr virus latent infection protein. Proc Natl Acad Sci U S
A. 91,9 L3 1-5.
Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S.,
Sameshima, M., Hase, A., Seto, Y. and Nagata, S., 1991, The
polypeptide encoàed by the cDNA for human cell surface antigen Fas
can mediate apoptosis. Cell, 66,233-43.
Jayanman, J. and Prives, C., 1995, Activation of p53
sequence-specific DNA binding by short single strands of DNA
requins the p53 C-terminus. Cell, 81, 1021-9.
Jiang, D., Srhivasan, A., Lmano, G. and Robbins, P. D., 1993,
SV40 T antigen abrogates p53-mediated transcriptional activity.
Oncogene, 8,2805-12.
Kriss-Eisler, A., Falck-Pedersen, E., Elfenbein, D. H., Alvira,
M., Buttrick, P. M. and Leinwand, L. A., 1994, The impact of
developmental stage, route of administration and the immune system
on adenovirus-mediated gene transfer. Gene Ther, 1,395-402.
Kastan, M . B., Onyekwere, O., Sidronsky, D., Vogelstein, B. and
Craig, R. W., 1991, Participation of p53 protein in the cellular
nsponse to DIVA damage. Cancer Res. 51,6304-1 1.
Katayose, D., Gudas, J., Nguyen, H., Srivastava, S., Cowan, K.
H. and Seth, P., 1995, Cytotoxic effects of adenovirus-mediated
wild-type p53 protein expression in normal and tumor mamrnary
epithelial cells. C h Cmcer Res, 1,889-97.
Kem, S. E., Kinzler, K. W., Bruskin, A., Jarosz, D., Friedman,
P., Prives, C. and Vogelstein, B ., 199 1, Identification of p53 as
a sequence-specific DNA-binding protein. Science, 252, 1708-1
1.
Knudson, A. G., 1993, Antioncogenes and human cancer. Pmc Natl
Acuà Sci U S A, !HD, 10914-21.
Kobayashi, M., Kawashima, A., Mai, U and Ooi, A., 1996, Anaiysis
of chromosome 17p13 (p53 locus) alterations in gastric carcinoma
cells by dual-color fluorescence in situ hybndization. Am J Puthol.
149,1575-84.
-
Koff, A., Giordano, A., Desai, D., Yamashita, K., Harper, J. W.,
Elledge, S., Nishimoto, T., Morgan, D. O., Franza, B. R. and
Roberts, J. M., 1992, Formation and activation of a cyclin E-cdk.2
complex during the G1 phase of the human ceil cycle. Science,
257,1689-94.
Kuerbitz, S. J e T Plunkett, B. S., Walsh, W. V. and Kastan, M.
B., 1992, Wild-type p53 is a ceil cycle checkpoint determinant
following irradiation. Proc Nat1 Acad Sci U S A, 89,7491-5.
Kussie, P. H., Gorina, S., Marechai, V., Elenbaas, B., Moreau,
J., Levine, A. J. and Pavletich, N. P., 1996, Structure of the MDM2
oncoprotein bound to the p53 tumor suppressor transactiviition
domain. Science, 274,948-53.
Lane, D. P., 1992, Cancer. p53, guardian of the genorne. Nature,
358, 15-6. Lane, D. P. and Benchimol, S., 1990, p53: oncogene or
anti-oncogene? Genes Dev, 4, 1-
8. Lane, D. P. and Crawford, L. V., 1979, T antigen is bound to
a host protein in SV40-
uûnsformed cells. Nature, 278,26 1-3. Lassus, P., Ferlin, M.,
Piette, J. and Hibner, U., 1996, Anti-iipoptotic activity of
low
levels of wild-type p53. EMBO J, 15,4566-73. Lax, S. A. and Liu,
F. F., 1999, Tumor suppnssor gene therapy. University of
Toronto
Medical Journal, 76,86-92. Lee, S. M. and Bernstein, A*, 1993,
p53 mutations increase nsistance to ionizing
radiation. Proc Nad Acad Sci U S A, 90,5742-6. Lee, J. M. and
Bernstein, A., 1995, Apoptosis, cancer and the p53 turnour
suppressor
gene. Cancer Metastasis Rev. 14, 149-6 1. Lee, S., Elenbaas, B.,
Levine, A. and Griffith, 1.. 1995, p53 and its 14 kDa
C-terminal
domain recognize pnmary DNA damage in the fonn of
insertioddeletion mismatches. Cell, 81, 10 13-20.
Lee, W., Harvey, T. S., Yin, Y., Yau, P., Litchfield, D. and h w
s m i t h , C. HeT 1994, Solution structure of the tetrameric
minimum transfonning domain of p53 [published erratum appears in
Nat Stnict Bi01 1995 Jan;2(1):81]. Nat Strm Biol, 1,877-90.
Leopold, P. L., Ferris, B., Grinberg, I., Worgdl, S., Hackett,
N. R. and Crystal, R. G.. 1998, Fluorescent virions: dynamic
tracking of the pathway of adenoviral gene transfer vectors in
living cells. Hum Gene Ther, 9,367-7 8 .
Laheny, C. D., Hu, H. M., Opipari, A. W., Wang, F. and Dixit, V.
M., 1992, The Epstein- Barr virus LMPL gene product induces A20
zinc finger protein expression by activating nuclear factor kappa
B. J Bi01 C k m , 267,2415760.
Li, l. H., Lax, S. A., Kim, J., Klamut, H. and Liu, F. F e ,
1999, The effects of combining ionizing radiation and adenoviral
p53 therapy in nasopharyngeal carcinoma. Int J Radiat Oncol Bi01
Phys, 43,607-16.
Li, J. H., Li, P., Klamut, H. and Liu, F. F.. 1997, Cytotoxic
effects of AdSCMV-p53 expression in two hurnan nasopharyngeal
carcinoma ce11 lines. Clin Cancer Res, 3,507-14.
Li, P., Bui, T., Gray, D. and Klamut, H- J., 1998, Therapeutic
potential of recombinant p53 overexpression in breast cancer ceiis
expressing endogenous wild-type p53. B~east Cmicer Res Trem,
48,273-86.
-
Liebowitz, D., 1994, Nasopharyngeal carcinoma: the Epstein-Barr
virus association. Semin Oncol, 21,376-81.
Lin, C. T., Dee, A. N., Chen, W. and Chan, W. Y., 1994,
Association of Epstein-Barr virus, human papilloma virus, and
cytomegalovirus with nine nasopharyngeal carcinoma cell tines. Lub
Invest, 71,73 1-6.
Lin, J. Y. and Simmons, D. T., 1991, The ability of large T
antigen to complex with p53 is necessary for the increased life
span and partiai transformation of human cells by simian virus 40.
J Virol, 65,6447-53.
Lischwe, M. A. and Sung, M. T., 1977, A histone-like protein
from adenoWus chromatin. Nature, 267,55 2-4.
Liu, T. I., el-Naggar, A. K., McDonnell, T. I., Steck, K. D.,
Wang, M., Taylor, D. L. and Clayman, G. L., 1995a. Apoptosis
induction mediated by wild-type p53 adenoviral gene transfer in
squamous ceIl carcinoma of the head and neck. Cancer Res, 55,3
117-23.
Liu, Y.. Liggitt, D., Zhong, W., Tu. G., Gaensler, K. and Debs,
R., 1995b. Cationic liposome-rnediated intravenous gene delivery. J
Bi01 Chem 270,24864-70.
Lowe, S. W., Ruley, H. E., Jacks, T. and Housman, D. E., 1993a,
p55dependent apoptosis modulates the cytotoxicity of anticancer
agents. Cell, 74,957-67.
Lowe,S. W., Schmitt, E.M., Smith, S. W.,Osbome, B.A.
andJacks,T., 1993b,p53 is required for radiation-induced apoptosis
in mouse thymocytes. Nature, 362,847- 9.
Lu.S. J.,Day, N.E.,Degos,L., Lepage. V., Wang,P.C.,Chan,S.
H.,Sirnons,M., McKnight, B., Easton, D., Zeng, Y. and et al., 1990,
Linkage of a nasopharyngeal carcinoma susceptibility locus to the
HLA region. Nature, 346,470-1.
Lung, M. L., Hu, Y., Cheng, Y., Li, M. F., Tang, C. M., O, S. K.
and Iggo, R. D., 1998, p53 inactivating mutations in Chinese
nasopharyngeal carcinomas. Cancer Lat, 133,89094.
Martin, M. E. and B