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Cisplatin chemotherapy plus adenoviral p53 gene
therapy in EBV-positive and -negative
nasopharyngeal carcinoma
Laura Weinrib,1,2 Jian-Hua Li,1,2 Jeffrey Donovan,1,2 Dolly Huang,3 and Fei-Fei Liu1,2,4,5
1
Department of Experimental Therapeutics, Princess Margaret Hospital/Ontario Cancer Institute, University
Health Network, Toronto, Ontario, Canada; 2Department of Medical Biophysics, University of Toronto, Toronto,
Ontario, Canada; 3Chinese University of Hong Kong, Shatin, Hong Kong, China; 4Department of Radiation
Oncology, Princess Margaret Hospital/Ontario Cancer Institute, University Health Network, Toronto, Ontario,
Canada; and 5Department of Radiation Oncology, University of Toronto, Toronto, Ontario, Canada.
We have previously shown that the introduction of human recombinant wild - type p53 mediated by an adenoviral vector ( Ad5CMV p53 ), either alone or delivered in combination with ionizing radiation, was cytotoxic to two nasopharyngeal carcinoma ( NPC ) cell
lines. To further explore the potential therapeutic role for gene therapy, the combination of Ad5CMV - p53 and cisplatin was
examined in two NPC cell lines, CNE - 1 and C666 - 1. The C666 - 1 cells are particularly relevant because they express Epstein - Barr
virus latent gene products analogous to human NPC in situ. Cells were infected with 5 pfu / cell of Ad5CMV - p53 or Ad5CMV - - gal,
followed by exposure to increasing doses of cisplatin. Clonogenic and MTT assays were used to assess the sensitivity of cells to these
treatments, and apoptosis was also quantified. The combination of Ad5CMV - p53 and cisplatin resulted in approximately 25%
greater cytotoxicity compared to that observed with cisplatin alone in either cell line. Apoptosis was induced in approximately 50%
of cells following administration of both Ad5CMV - p53 and cisplatin, but was induced in considerably fewer cells following either
treatment alone. The two modalities appeared to interact in an additive manner. Ad5CMV - p53 gene therapy resulted in the
expression of biologically active p53 protein, shown by induction of p21WAF1 / CIP1. Cisplatin treatment showed little effect on either
p53 or p21WAF1 / CIP1 expression. Therefore, both p53 gene therapy and cisplatin chemotherapy demonstrated cytotoxicity mediated
by apoptosis despite the presence of EBV gene products in the C666 - 1 cells, but it appears that the two modalities induce cytotoxicity
by independent pathways. Cancer Gene Therapy ( 2001 ) 8, 352 ± 360
Key words: p53; nasopharyngeal carcinoma; Epstein - Barr virus; cisplatin chemotherapy; apoptosis; adenovirus vector.
N
asopharyngeal carcinoma (NPC ) is a malignant disease
of the head/neck region that is endemic to Southeast
Asia, where it affects a predominantly young population.3
The current treatment regimen of radiation therapy (XRT ),
even combined with cisplatin ( cis -diamminedichloroplatinum( II )) chemotherapy, yields a 5 - year survival rate of
approximately 65%.2,3 Therefore, the study and the development of novel therapeutics are warranted.
The introduction of wild - type p53 (wtp53) -expressing
plasmids into tumor cells can induce overexpression of
recombinant wtp53 protein and directly drive cells into either
growth arrest or apoptosis.35,38 Recently, replication -deficient adenoviruses, which express human recombinant
wtp53 ( Ad5CMV- p53), have demonstrated cytotoxic
effects on human cancer cells bearing different p53
genotypes, both in vitro and in vivo.7,8,18,28,45 A strategy to
Received March 22, 2001.
Address correspondence and reprint requests to Fei - Fei Liu, Department of Radiation Oncology, Princess Margaret Hospital / Ontario
Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada
M5G 2M9. E-mail address: fei - [email protected]
352
combine adenovirus -mediated p53 gene transfer with DNA damaging agents, such as ionizing radiation or chemotherapy, has been studied in lung, colorectal, ovarian,
esophageal, and head /neck cancers, with additional cytotoxicity or tumoricidal effects being consistently
observed.14,17,26,30,33
A complicating factor in studying NPC in the laboratory is
the challenge of obtaining NPC cell lines that harbor the
Epstein -Barr virus ( EBV ). In greater than 80% of patients
with NPC, the latent form of EBV is present in the NPC
cells.24,34 The previous cell lines that we have studied, CNE 1 46 and CNE - 2Z,39 were NPC cell lines that have shed their
EBV. To determine whether Ad5CMV- p53 gene therapy has
a potential application in human patients with NPC, it was
necessary to evaluate this experimental therapy in an EBVpositive model. This model was particularly important
because there is some evidence that EBV protein products
may alter p53 function.42,47 The NPC cell line C666- 1,
which has consistently been demonstrated to harbor the
latent form of EBV-1 and to express a number of EBV latent
mRNAs including EBNA -1 and LMP - 1, is one of the few
such lines available in the world.6,16
Cancer Gene Therapy, Vol 8, No 5, 2001: pp 352 ± 360
WEINRIB, LI, DONOVAN, ET AL: AD5CMV-p53 PLUS CISPLATIN IN NPC
Our laboratory has previously demonstrated that the
introduction of wtp53 mediated by the adenoviral vector
into the NPC cell lines, CNE -1 and CNE -2Z, resulted
in significant cytotoxicity when administered either alone
or in combination with ionizing radiation.21,22 In the
current study, the effects of combining cisplatin chemotherapy with Ad5CMV- p53 gene therapy in both EBVpositive and EBV-negative NPC cell lines were
evaluated.
MATERIALS AND METHODS
Cells and culture conditions
The NPC cell line CNE -1 was obtained from the Cancer
Institute / Chinese Academy of Medical Science in Beijing,
China.46 It has a heterozygous mutation of p53 at codon 280
with a transversion from G to C.11,25,46 CNE -1 cells had a
doubling time of around 20± 24 hours, and were maintained
in - MEM supplemented with 10% fetal bovine serum
( FBS; CanSera, Canada).
The NPC cell line C666- 1 was obtained from the Chinese
University of Hong Kong.6,16 The p53 gene in C666- 1 cells
has been determined in our laboratory to harbor a deletion of
codon 249 ( exon 7).23 Their doubling time was around 3.5
days, and appeared to require a minimum density before
subpassaging to maintain monolayer growth. C666 - 1 cells
were propagated in RPMI 1640 supplemented with 10%
FBS. All experiments were conducted when the cells were in
an exponential growth phase.
The human embryonic kidney 293 and cervix HeLa cell
lines ( American Type Culture Collection, Rockville, Maryland, USA ) used for the adenoviral studies were both
propagated in -MEM with 10% FBS.
Recombinant adenovirus vectors
The adenovirus vectors, which have been previously
described,21,22 were obtained from Dr. Frank Graham at
McMaster University ( Hamilton, Canada ). The Ad5CMVp53 is a replication- deficient adenovirus vector containing a
cytomegalovirus ( CMV ) promoter upstream from a human
recombinant wtp53 minigene. The control adenovirus vector
used in this study was Ad5CMV- - gal, an adenovirus
vector containing the -galactosidase gene. The vectors
were propagated in human embryonic kidney 293 cells,
purified by two cesium chloride (CsCl) density gradients,
titered by plaque -forming assay, and determined to be free of
replication- competent viruses using HeLa cells as previously described.48
Detection of Ad5CMV - - gal expression
To evaluate the infection efficiency and transgene
expression after exposure to the adenovirus vector,
Ad5CMV- -gal ±mediated expression of - galactosidase
activity was determined using X -gal staining, as previously described.21,22 Briefly, cells were seeded onto 24 well culture plates; after incubating ( 1 day for CNE - 1
cells, 3 days for C666- 1 cells), the cultures were exposed
to various concentrations of Ad5CMV- -gal (1 ±50 pfu /
Cancer Gene Therapy, Vol 8, No 5, 2001
353
cell ) in culture medium containing 2% FBS. One hour
later, the serum concentration was increased to 10% FBS
and the cells were incubated for 48 hours. Cells were
subsequently washed with PBS ± Ca2 + ± Mg2 + , fixed with
2% formaldehyde /0.2% glutaraldehyde for 5 minutes at
48C, and then stained with X - gal solution following
washing with PBS /0.02% NP40 buffer. A blue - green stain
indicated the cells in which - galactosidase was
expressed. The number of positively and negatively stained
cells was counted, and infection efficiency was scored as
the percent of positively staining cells.
Infection and cisplatin treatment of cells
CNE - 1 cells were maintained in either culture flasks or
culture plates (Nunc, Roskilde, Denmark ) at a density of
1.5105 cells per T-25 flask in the appropriate medium.
After 24 hours, the cultures were infected with 5 pfu /cell of
either Ad5CMV- p53 or Ad5CMV- -gal in fresh medium
containing 2% FBS at 378C for 1 hour. Subsequently,
medium with 10% FBS was added, and the infected cultures
were incubated for 24 hours. Cells were then exposed to
cisplatin chemotherapy for 24 hours, with cumulative doses
ranging from 0 to 8 g /mL (Faulding, Quebec, Canada ).
Finally, the cells were harvested at different time points for
the clonogenic survival assay, MTT assay, morphological
analysis of apoptosis, or protein extraction.
C666 -1 cells were treated in a manner similar to the
CNE - 1 cells, but due to their requirement of a minimum
plating density, plus their relatively slow doubling time, they
were plated at a density of 1106 cells for each T-25 flask,
or 2104 cells for each well in 96- well plates, and were
incubated for 3 days before the initial infections.
Clonogenic survival assay
After treatment, CNE -1 cells were detached using trypsin
and plated onto 100- mm2 dishes ( Nunc ) in -MEM plus
10% FBS culture medium at a density of 102 ± 104 cells/dish.
The dishes were incubated in a 378C /5% CO2 incubator for
10 days. The plates were then fixed and stained with
methylene blue in 50% ethanol, and the number of visualized
colonies ( >50 cells) was counted. The plating efficiency of
the CNE -1 cells was approximately 80%. Triplicate dishes
were set up for each condition, and each experiment was
conducted on three separate occasions.
MTT assay
The nature of C666 -1 cells precluded the use of the
clonogenic survival assay; therefore, the MTT (3 -( 4,5dimethylthiazol -2 - yl -2,5 -diphenyltetrazolium bromide )
assay was used to assess the effect of viral infection and /
or cisplatin on cell viability, as previously described.22
Briefly, the cells (in 96- well plates ) were incubated for 6
days following the completion of the prescribed treatments.
Subsequently, MTT (Sigma, St. Louis, Missouri, USA ) was
dissolved in PBS at 5 mg /mL and filtered using sterile
techniques. Ten microliters of the MTT stock solution plus
90 L of PBS was added to each well, and the plates were
incubated at 378C for 3 hours. Acid ±isopropanol with 0.01
N HCl was added to all wells and mixed thoroughly to
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WEINRIB, LI, DONOVAN, ET AL: AD5CMV-p53 PLUS CISPLATIN IN NPC
dissolve the blue MTT formazam crystals. The plates were
then read on a BioRad 3350 microplate reader (BioRad
Laboratories, Hercules, CA, USA ) at a wavelength of 570
nm. Triplicate wells were set up for each condition, and each
experiment was repeated at least three times.
Morphological assessment of apoptosis
Apoptosis was evaluated morphologically using acridine
orange -ethidium bromide (AO -EB ) (Sigma ) fluorescence
staining. CNE - 1 cells were assayed 24 hours after the
completion of their treatments, and C666 -1 cells were
assayed 3 days after the completion of their treatments. Cells
were washed with PBS, pelleted gently, re - suspended in 1
mL PBS, and then mixed with 20 L of AO -EB stock for a
final concentration of 2.5 M. The stained cells were
centrifuged to remove the supernatant and re -suspended in
30 L of 10% glycerol in PBS. The cells were then placed
onto glass slides and immediately visualized using fluorescent microscopy ( Leica, Heerbrugg, Switzerland ). Cells
were scored as apoptotic if morphological features such as
chromatin condensation, loss of nuclear envelope, membrane blebbing, and /or apoptotic were observed. For each
slide, 10 fields containing approximately 50 cells each were
counted. The experiments were performed three times.
Statistical analysis was performed using an unpaired
SURVIVING FRACTION
1.
cisplatin
cisplatin + p53 (5pfu/cell)
p53 (5pfu/cell) + cisplatin
0.1
0
2
4
6
8
10
CISPLATIN DOSE (ug/ml/24 h)
Figure 1. The effect of cisplatin with or without Ad5CMV - p53 on the clonogenic survival of CNE - 1 cells. CNE - 1 cells were treated with cisplatin
doses of 0, 2, 4, 8 g / mL for 24 hours, either before ( cisplatin + p53 ) or after ( p53 + cisplatin ) infection with 5 pfu / cell of Ad5CMV - p53 for 24
hours. A clonogenic cell survival assay was performed. Each data point represents the mean and standard error from three separate experiments.
Cancer Gene Therapy, Vol 8, No 5, 2001
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WEINRIB, LI, DONOVAN, ET AL: AD5CMV-p53 PLUS CISPLATIN IN NPC
Student's t test to compare untreated cells to cells subjected
to the combination treatments.
Western blotting
Similar to the apoptosis assay, CNE -1 and C666 -1 cells
were harvested 24 hours and 3 days, respectively, following
completion of their treatments. The cultured cells were
washed three times with cold PBS, and lysed in cold lysis
buffer [0.1 M Tris ± HCl ( pH 8.0 ), 1% sodium dodecyl
sulfate (SDS ), 10 mM EDTA, and 2 mM DTT ]. The cell
lysate was centrifuged at 14,000 rpm for 10 minutes.
Samples containing 10 g of protein were denatured by
boiling for 3 minutes in loading buffer [20% glycerol, 5% -
mercaptoethanol, 4% SDS, 125 mM Tris ( pH 6.8 ), 0.1 mg
bromophenol blue ] and then electrophoresed on 10% SDS
polyacrylamide gel electrophoresis ( PAGE) for 120 minutes
at 100 V. The protein was transferred onto a nitrocellulose
membrane and probed with the primary antibodies as
follows: 0.01 g /mL of p53 monoclonal antibody (mAb )
( DO - 1; Santa Cruz Biotech, Santa Cruz, CA, USA ) and 1
g /mL of p21WAF1 / CIP1 mAb (Oncogene Science, Cambridge, Massachusetts, USA ). After washing, the blots were
incubated with horseradish peroxidase conjugated to a
secondary antibody. Specific complexes were visualized
using chemiluminescence reagents ( DuPont, Boston, MA,
USA ). MCF - 7 cells were used as a positive control for p53
and p21 protein expression in the CNE -1 experiments; 468
VIABLE FRACTION
1.
cisplatin
p53 (5pfu/cell) + cisplatin
0.1
0
2
4
6
8
10
CISPLATIN DOSE (ug/ml/24 h)
Figure 2. The effect of cisplatin with or without Ad5CMV - p53 on the viability of C666 - 1 cells. C666 - 1 cells were infected with 5 pfu / cell of
Ad5CMV - p53 for 24 hours, followed by treatment with cisplatin doses of 0, 2, 4, 8 g / mL for 24 hours. Cell viability was analyzed using the MTT
assay. Each data point represents the mean and standard error from three separate experiments.
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WEINRIB, LI, DONOVAN, ET AL: AD5CMV-p53 PLUS CISPLATIN IN NPC
cells served as positive control for p53 protein expression in
the C666 -1 experiments.
RESULTS
Effect of cisplatin combined with Ad5CMV - p53 on cell
viability
Adenoviral infection efficiency for both the CNE - 1 and
C666- 1 cell lines was high, with almost 95% and 100% of
cells, respectively, transduced with 2 pfu /cell of Ad5CMV - gal ( data not shown ).
The cytotoxic effects of Ad5CMV-p53 on CNE - 1 cells
were assessed using the clonogenic survival assay. As shown
in Figure 1, treatment of CNE -1 cells with cisplatin alone
resulted in a dose -dependent inhibition of cell proliferation,
where a cumulative dose of 8 g /mL cisplatin reduced the
surviving fraction of cells to 35%. Ad5CMV- p53 alone ( 5
pfu /cell ) resulted in 76% cell survival, which decreased
significantly with increasing doses of cisplatin. The
Figure 3. Morphological analysis of apoptosis. CNE - 1 cells were infected with 5 pfu / cell of Ad5CMV - p53 or Ad5CMV - - gal for 24 hours, and / or
exposed to 4 g / mL of cisplatin for 24 hours. The cells were stained by AO - EB and then examined under fluorescent microscopy for
morphological changes indicative of apoptosis. A: CNE - 1 control. B: Ad5CMV - - gal. C: Ad5CMV - p53. D: Cisplatin. E: Ad5CMV - p53 followed
by cisplatin. F: Cisplatin followed by Ad5CMV - p53.
Cancer Gene Therapy, Vol 8, No 5, 2001
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WEINRIB, LI, DONOVAN, ET AL: AD5CMV-p53 PLUS CISPLATIN IN NPC
combination of Ad5CMV-p53 plus 8 g /mL cisplatin
resulted in only 20% cell survival. The sequence of
administration of Ad5CMV-p53 and cisplatin did not appear
to influence the clonogenic survival of the CNE - 1 cells.
The cytotoxic effects of Ad5CMV- p53 on C666- 1 cells
were assessed using the MTT assay, which determines the
biochemical viability of cells. This assay was used because
the C666 -1 cells did not readily form colonies under
similar conditions to the CNE -1 cells. Although the
endpoints of these two assays are different, our laboratory
has shown that their results are consistent in a dose dependent manner in a number of cell lines.22 As shown in
Figure 2, the C666 -1 cells treated with cisplatin alone
resulted in a dose -dependent reduction of viability, where
a cumulative dose of 8 g /mL cisplatin reduced the
surviving fraction of cells to 61%. Ad5CMV- p53 alone ( 5
pfu /cell ) led to 78% cell survival, and the combination of
Ad5CMV- p53 plus 8 g /mL cisplatin resulted in only
34% cell survival. Exposure of either the CNE -1 or C6661 cells to Ad5CMV- -gal had a minimal effect on cell
viability (data not shown ).
Effect of cisplatin combined with Ad5CMV - p53 on
apoptosis
Morphological changes characteristic of apoptosis were
investigated using AO -EB fluorescence staining after
Ad5CMV- p53 and /or cisplatin treatment. The number
of cells manifesting morphological features of apoptosis,
as described in Materials and Methods, was counted as
a function of the total number of cells present in the
field.
Figure 3 shows the CNE -1 cells displaying the
morphological features of apoptosis, after Ad5CMV- -
gal or Ad5CMV-p53, with or without cisplatin ( 4 g /
mL ). It is clear that the combination of Ad5CMV- p53
plus cisplatin demonstrated the greatest propensity toward
apoptosis (Panels E and F ). This datum is also presented
in quantitative form in Table 1a, which shows the
percentage of cells undergoing apoptosis following treatment. Under control conditions, 23% of the CNE -1 cells
displayed apoptosis. Apoptosis was induced in only 40%
of CNE -1 cells following treatment with either
Ad5CMV-p53 or cisplatin alone, but was induced in
approximately 55% of the cells when both treatments
were administered (P < .05). Once again, the sequence of
administration of Ad5CMV- p53 and cisplatin did not
make a significant difference with respect to the induction
of apoptosis.
Table 1b represents the percentage of C666 -1 cells
undergoing apoptosis as quantified using the same experimental procedure as with the CNE -1 cells. Under control
conditions, 25% of the C666 -1 cells displayed apoptosis.
After treatment with cisplatin ( 4 g /mL ), 36% of cell was
apoptotic. Ad5CMV- p53 ( 5 pfu / cell ) alone induced
apoptosis in 39% of C666 -1 cells, which increased to 49%
after cisplatin administration (P < .05 ). Ad5CMV- -gal
infection induced only a small increase ( 3± 4% ) in apoptosis
compared to cells under control conditions.
Western blotting for p53 and p21WAF1 / CIP1
Protein was extracted from the CNE -1 and C666 -1 cells
after Ad5CMV- - gal, Ad5CMV-p53, and /or cisplatin ( 4
g /mL) treatments. Western blotting was performed to
determine p53 protein expression. p21WAF1 / CIP1 was also
studied to ensure that the exogenously introduced p53
protein was biologically active. The results from the Western
Table 1. Assay of Apoptosis for CNE - 1 and C666 - 1 Cells
Treatment
( a ) CNE - 1 cells
Control
Ad5CMV - - gal
Cisplatin ( 4 g / ml )
Ad5CMV - p53 ( 5 pfu )
Ad5CMV - p53 ( 5 pfu ) + cisplatin
( b ) C666 - 1 cells
Control
Ad5CMV - - gal
Cisplatin ( 4 g / mL )
Ad5CMV - p53
Ad5CMV - p53 + cisplatin
Day 1
Day 2
Day 3 ( count % apoptosis )
±
X
X
±
X
±
Ad5CMV - p53
Cisplatin
±
±
±
X
±
X
Cisplatin
Ad5CMV - p53
23.3 ‹ 1.9
27.5 ‹ 1.5
37.0 ‹ 5.0
38.7 ‹ 4.4
40.3 ‹ 2.3
41.0 ‹ 2.0
54.7 ‹ 5.5**
50.0 ‹ 6.0**
±
X
X
Ad5CMV - p53
Ad5CMV - p53
Ad5CMV - p53
Ad5CMV - p53
±
±
±
±
±
Cisplatin
Cisplatin
25.0 ‹ 1.1
27.5 ‹ 1.4
35.7 ‹ 2.5
39.1 ‹ 1.5
44.6 ‹ 0.7
48.9 ‹ 3.1**
53.3 ‹ 0.9**
( 5 pfu )
( 10 pfu )
( 5 pfu )
( 10 pfu )
The cells were infected with either Ad5CMV - p53 or Ad5CMV - - gal for 24 hours, and / or exposed to 4 g / mL cisplatin for 24 hours. The
proportion of cells undergoing apoptosis was counted. The experiment was conducted three times for each cell line, and the data represent the
mean and standard deviation from 10 fields. ( a ) CNE - 1 cells. ( b ) C666 - 1 cells.X: exposed to treatment. ± : media alone.**P < .05, Student's t
test.
Cancer Gene Therapy, Vol 8, No 5, 2001
358
CNE-1 Western Blots
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WEINRIB, LI, DONOVAN, ET AL: AD5CMV-p53 PLUS CISPLATIN IN NPC
β
p53
p21
C-6661 Western Blots
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Figure 4. Western blot analysis of Ad5CMV - p53 ± mediated gene
transfer and expression, with or without cisplatin, in CNE - 1 and
C666 - 1 cells. The cells were infected with 5 pfu / cell of Ad5CMV - p53
or Ad5CMV - - gal for 24 hours, and / or exposed to 4 g / mL of
cisplatin for 24 hours. Treatments took place over two consecutive
24 - hour periods. This timing is referred to in the figure by separating
days 1 and 2 treatments by a comma ( , ). For each sample, 20 g of
cell lysate was separated on a 10% SDS - PAGE, electroblotted onto
nitrocellulose membrane, and probed with respective mAbs for p53
and p21WAF1 / CIP1. The signals were detected using the ECL method.
A: CNE - 1 cells. B: C666 - 1 cells.
blot Figure 4 ) demonstrated that under control conditions,
both CNE - 1 and C666- 1 cells had minimal p53 protein
expression. CNE -1 cells showed some endogenous
p21WAF1 / CIP1 expression, whereas p21WAF1 / CIP1 was undetectable in C666 -1 cells. No increase in endogenous p53 or
p21WAF1 / CIP1 protein expression was observed after
treatment with cisplatin. Infection with Ad5CMV- p53,
however, resulted in a significant increase in p53
protein expression, along with higher amounts of
p21WAF1 / CIP1 expression in both cell lines. Cisplatin
combined with Ad5CMV-p53 did not cause an appreciable
increase in either p53 or p21WAF1 / CIP1 protein expression,
beyond what was observed with Ad5CMV- p53 treatment
alone.
DISCUSSION
The primary observations in this work are that Ad5CMVp53 gene therapy can promote cisplatin -induced apoptosis
in NPC cells, and that this therapeutic strategy continues to
be efficacious in an EBV- positive NPC cell line.
Previously, we have reported that Ad5CMV- p53 gene
therapy was cytotoxic to NPC cells lines when administered either alone or in combination with ionizing radiation
( XRT ).21,22 The cytotoxic response of NPC cells to XRT
combined with Ad5CMV- p53 therapy suggested that the
interaction between these two modalities was more than
additive.21 The data from the current study imply that the
combination of cisplatin and Ad5CMV-p53 resulted in an
additive cytotoxic interaction. This suggests that the
apoptotic pathways stimulated by p53 overexpression and
cisplatin chemotherapy likely do not intersect in NPC
cells.
Cisplatin is a chemotherapeutic drug that binds to DNA,
causing predominantly intrastrand crosslinking, which,
when left unrepaired, leads to cell death.36,41 The propensity
of human tumor cells to undergo cisplatin -mediated
apoptosis was initially described in 1990,10,40 and many
different groups have since examined cisplatin -induced
apoptosis in a variety of human cancer cells both in vitro and
in vivo.4,14,37,44 However, the precise signaling pathway
through which cisplatin- induced apoptosis occurs is controversial and has yet to be clearly elucidated. The involvement of p53 in cisplatin - mediated apoptosis remains a point
of contention. Some evidence from the literature suggests
that wtp53 is a key factor in mediating cisplatin -induced
apoptosis.17,30,37 A study of tumor samples obtained from
epithelial ovarian carcinoma patients before and after
cisplatin chemotherapy revealed that tumors with wtp53
demonstrated a greater likelihood of response to cisplatin,
with an increased propensity to undergo apoptosis, compared
to tumors with mutant p53.37 As well, Kanamori et al 17
demonstrated that the combination of p53 gene therapy and
cisplatin has a synergistic effect on cytotoxicity in the human
colon cancer cell line WiDr.
Although wtp53 appears to play a role in the apoptotic
response of some cells to cisplatin, cisplatin- induced
apoptosis has also been shown to occur in testicular germ
cell tumor cell lines in the absence of wtp53, obviously
through a p53 -independent mechanism.4 The testicular
tumor cell line NCCIT, which expressed mutant p53, and
the S2 cell line, which expressed no p53, underwent
apoptosis after cisplatin chemotherapy. Therefore, p53 was
not necessary as a mediator in all cases of cisplatin -induced
apoptosis. The results of our current study are consistent with
this observation, in that both CNE -1 and C666 -1 cells
express a mutant p53,11,23,25 and yet exhibit a significant
increase in apoptosis following treatment with cisplatin
alone. It therefore appears that involvement of p53 in
cisplatin -induced apoptosis may be either a tissue type ± or
cell line ±dependent process.
The observations regarding cisplatin and p53 gene therapy
can only be applied in relation to the relatively low doses of
each treatment that was used. The interaction between the
two modalities may differ using higher doses, but this study
Cancer Gene Therapy, Vol 8, No 5, 2001
WEINRIB, LI, DONOVAN, ET AL: AD5CMV-p53 PLUS CISPLATIN IN NPC
focused on the use of clinically relevant and achievable doses
of cisplatin and Ad5CMV- p53.
In the majority of human patients with NPC, the EBV in
its latent form is exclusively present in the NPC cells.24,29,34
The specific role of EBV in causality and /or progression of
nasopharyngeal cancer remains controversial. EBV DNA
has been observed to be clonal in cells from patients with
NPC.32 As well, LMP -1 has been consistently shown to be
both transforming and oncogenic.12,27,43 These properties
suggest that some of the latent gene products of EBV may
play a role in both the development and progression of NPC.
The concern regarding efficacy of Ad5CMV-p53 gene
transfer into a NPC cell line harboring EBV gene products is
not unwarranted. There is controversial evidence suggesting
that EBNA - 5 and BZLF 1 Ð EBV latent proteins Ð can
physically interact with and perhaps inhibit p53 function,42,47 although others have not corroborated their functional interference with p53.1 There are also several reports
that LMP -1 13,19,31 can inhibit apoptosis. The majority of
these reports concentrated on lymphocyte or lymphoma cell
lines.19,31 The only report which studied an epithelial cell
line demonstrated that a human lung cancer cell line, which
was doubly transfected with a temperature - sensitive p53
plasmid and LMP -1, was less likely to undergo apoptosis in
the presence of LMP - 1.13 Resistance to apoptosis has also
been associated with the expression of A20, a putative
antiapoptotic protein of EBV that can be, but is not only,
induced by LMP -1.9
One approach to address the influence of EBV status on
cisplatin or p53 gene therapy would be to use a pair of
isogenic cell lines with differing EBV status. Only one group
had successfully infected an epithelial cell line with EBV, but
our laboratory was unable to duplicate these results.20
However, the primary goal of our study was to determine
whether p53 gene therapy might be a relevant treatment
modality in EBV-positive NPC. Indeed, our data demonstrated that in the presence of increased exogenous p53
expression, the EBV- positive C666- 1 cells can be induced
to undergo p53- mediated apoptosis, despite the possible
presence of antiapoptotic factors associated with EBV.
The precise extent of EBV latent protein expression in the
C666- 1 cell line is not clear. Cheung et al 6 were able to
detect the expression of EBNA - 1 protein by Western blot
analysis, but cells stained only weakly positive for LMP - 1
using immunohistochemistry, and were negative by Western
blot analysis. Therefore, the expression of LMP -1 in C6661 cells is likely low. This finding is not dissimilar to the
clinical situation where it has been shown that EBNA - 1 is
universally expressed in undifferentiated NPC, whereas
LMP - 1 is expressed in approximately 65% of cases.29
The mechanism by which cells die following Ad5CMVp53 infection, with or without other therapy, is not well
understood, as the exact pathway leading from p53
expression to apoptosis has not been elucidated. However,
it has been demonstrated that high levels of p53 protein will
promote an apoptotic response.5 Although there is debate
about the presence and effect of EBV protein products in the
C666- 1 cell line, it is reasonable to believe that Ad5CMVp53 would work even in the presence of a p53 -inhibiting
agent, such as LMP -1. A gene transfer strategy using p53
Cancer Gene Therapy, Vol 8, No 5, 2001
359
was cytotoxic to cervical carcinoma cell lines that expressed
the human papillomavirus ( HPV ) protein E6, which binds to
p53 and targets it for degradation.15 Although the mechanism of p53 inactivation differs for EBV- and HPV- infected
cell lines, the amount of p53 expression /overexpression due
to p53 gene therapy could potentially overwhelm the
activities of various p53 - inhibitory proteins, thereby leading
to p53 -induced cell death.
CONCLUSION
We demonstrated that Ad5CMV-p53 gene transfer, in
combination with cisplatin chemotherapy, results in an
additive cytotoxic effect that appears to be mediated through
apoptosis in our NPC model. As well, we showed that our
therapeutic strategy continues to be effective despite the
presence of EBV in the C666 -1 cell line. To further increase
the cytotoxic effect of our combination therapy, it would be
necessary to continue to unravel the apoptotic pathway
induced by cisplatin, and to harness this knowledge to
enhance cancer cell killing.
ACKNOWLEDGMENT
This work was supported by funds from the Medical
Research Council of Canada.
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