Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
D 2001 Nature Publishing Group 0929-1903/01/$17.00/+0 www.nature.com/cgt 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 354 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 355 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. Cancer Gene Therapy, Vol 8, No 5, 2001 356 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 357 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 co ntr ol Ad 5C M V cis pla - -g a tin , 0 l, 0 0, c is p Ad latin 5C M V 0, Ad -p53 5C 5p M fu Ad V,0 5C p5 35 c i s MV p la - p pf 5 u t M in, A 3 5 p CF f d 5C u, c -7 i M ce V- spla lls p5 tin 35 pf u A WEINRIB, LI, DONOVAN, ET AL: AD5CMV-p53 PLUS CISPLATIN IN NPC β p53 p21 C-6661 Western Blots co ntr o Ad l 5C M cis V pla - tin gal Ad 5C M V Ad 5C -p53 M 5p Ad V fu 5C p53 M 10 Ad V 5C -p5 pfu 35 M 46 V 8 c p5 pfu, el l 3 1 c s 0 p ispl a fu , c tin isp lat in B β p53 p21 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. REFERENCES 1. Allday MJ, Sinclair A, Parker G, Crawford DH, Farrell PJ. Epstein - Barr virus efficiently immortalizes human B cells without neutralizing the function of p53. EMBO J. 1995;14:1382 ± 1391. 2. Al - Sarraf M, LeBlanc M, Giri PG, et al. Chemoradiotherapy versus radiotherapy in patients with advanced nasopharyngeal cancer: phase III randomized Intergroup Study 0099. J Clin Oncol. 1998;16:1310 ± 1317. 3. Altun M, Fandi A, Dupuis O, Cvitkovic E, Krajina Z, Eschwege F. Undifferentiated nasopharyngeal cancer ( UCNT ): current diagnostic and therapeutic aspects. Int J Radiat Oncol Biol Phys. 1995;32:859 ± 877. 4. Burger H, Nooter K, Boersma AW, Kortland CJ, Stoter G. Expression of p53, Bcl - 2, and Bax in cisplatin - induced apoptosis in testicular germ cell tumour cell lines. Br J Cancer. 1998;77:1562 ± 1567. 5. Chen X, Ko LJ, Jayaraman L, Prives C. p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev. 1996;10:2438 ± 2451. 6. Cheung ST, Huang DP, Hui AB, et al. Nasopharyngeal carcinoma cell line ( C666 - 1 ) consistently harboring Epstein - Barr virus. Int J Cancer. 1999;83:121 ± 126. 7. Cirielli C, Riccioni T, Yang C, et al. Adenovirus - mediated gene transfer of wild - type p53 results in melanoma cell apoptosis in vitro and in vivo. Int J Cancer. 1995;63:673 ± 679. 8. Clayman GL, El - Naggar AK, Roth JA, et al. In vivo molecular therapy with p53 adenovirus for microscopic residual head and neck squamous carcinoma. Cancer Res. 1995;55:1 ± 6. 9. Codd JD, Salisbury JR, Packham G, Nicholson LJ. A20 RNA expression is associated with undifferentiated nasopharyngeal carcinoma and poorly differentiated head and neck squamous cell carcinoma. J Pathol. 1999;187:549 ± 555. 360 WEINRIB, LI, DONOVAN, ET AL: AD5CMV-p53 PLUS CISPLATIN IN NPC 10. Eastman A. Activation of programmed cell death by anticancer agents: cisplatin as a model system. Cancer Cells. 1990;2:275 ± 280. 11. Effert P, McCoy R, Abdel - Hamid M, et al. Alterations of the p53 gene in nasopharyngeal carcinoma. J Virol. 1992;66: 3768 ± 3775. 12. Fahraeus R, Rymo L, Rhim JS, Klein G. Morphological transformation of human keratinocytes expressing the LMP gene of Epstein - Barr virus. Nature. 1990;345:447 ± 449. 13. Fries KL, Miller WE, Raab - Traub N. Epstein - Barr virus latent membrane protein 1 blocks p53 - mediated apoptosis through the induction of the A20 gene. J Virol. 1996;70: 8653 ± 8659. 14. Fujiwara T, Grimm EA, Mukhopadhyay T, Zhang WW, Owen Schaub LB, Roth JA. Induction of chemosensitivity in human lung cancer cells in vivo by adenovirus - mediated transfer of the wild - type p53 gene. Cancer Res. 1994;54:2287 ± 2291. 15. Hamada K, Alemany R, Zhang WW, et al. Adenovirus mediated transfer of a wild - type p53 gene and induction of apoptosis in cervical cancer. Cancer Res. 1996;56:3047 ± 3054. 16. Hui AB, Cheung ST, Fong Y, Lo KW, Huang DP. Characterization of a new EBV- associated nasopharyngeal carcinoma cell line. Cancer Genet Cytogenet. 1998;101:83 ± 88. 17. Kanamori Y, Kigawa J, Minagawa Y, et al. A newly developed adenovirus - mediated transfer of a wild - type p53 gene increases sensitivity to cis - diamminedichloroplatinum( II ) in p53 - deleted ovarian cancer cells. Eur J Cancer. 1998;34: 1802 ± 1806. 18. Katayose D, Gudas J, Nguyen H, Srivastava S, Cowan KH, Seth P. Cytotoxic effects of adenovirus - mediated wild - type p53 protein expression in normal and tumor mammary epithelial cells. Clin Cancer Res. 1995;1:889 ± 897. 19. Kawanishi M. Expression of Epstein - Barr virus latent membrane protein 1 protects Jurkat T cells from apoptosis induced by serum deprivation. Virology. 1997;228:244 ± 250. 20. Knox PG, Li Q - X, Rickinson AB, Young LS. In vitro production of stable Epstein - Barr virus ± positive epithelial cell clones which resemble the virus: cell interaction observed in nasopharyngeal carcinoma. Virology. 1996;215:40 ± 50. 21. Li JH, Lax SA, Kim J, Klamut H, Liu FF. The effects of combining ionizing radiation and adenoviral p53 therapy in nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys. 1999;43:607 ± 616. 22. Li JH, Li P, Klamut H, Liu FF. Cytotoxic effects of Ad5CMVp53 expression in two human nasopharyngeal carcinoma cell lines. Clin Cancer Res. 1997;3:507 ± 514. 23. Li J - H, Huang D, Sun B - F, et al. The efficacy of ionizing radiation combined with adenoviral p53 therapy in EBVpositive nasopharyngeal carcinoma. Int J Cancer. 2000;7:606 ± 610. 24. Liebowitz D. Nasopharyngeal carcinoma: the Epstein - Barr virus association. Semin Oncol. 1994;21:376 ± 381. 25. Lo K - W, Mok C - H, Huang DP, et al. p53 mutation in human nasopharyngeal carcinomas. Anticancer Res. 1992;12:1957 ± 1964. 26. Matsubara H, Kimura M, Sugaya M, et al. Expression of wild type p53 gene confers increased sensitivity to radiation and chemotherapeutic agents in human esophageal carcinoma cells. Int J Oncol. 1999;14:1081 ± 1085. 27. Moorthy RK, Thorley - Lawson DA. All three domains of the Epstein - Barr virus ± encoded latent membrane protein LMP - 1 are required for transformation of rat - 1 fibroblasts. J Virol. 1993;67:1638 ± 1646. 28. Mujoo K, Maneval DC, Anderson SC, Gutterman JU. Adenoviral - mediated p53 tumor - suppressor gene therapy of human ovarian carcinoma. Oncogene. 1996;12:1617 ± 1623. 29. Niedobitek G, Agathanggelou A, Nicholls J. Epstein - Barr virus infection and the pathogenesis of nasopharyngeal carcinoma: viral gene expression, tumor cell phenotype, and the role of the lymphoid stroma. Semin Cancer Biol. 1996;7:165 ± 174. 30. Ogawa N, Fujiwara T, Kagawa S, et al. Novel combination therapy for human colon cancer with adenovirus - mediated wild - type p53 gene transfer and DNA - damaging chemotherapeutic agent. Int J Cancer. 1997;73:367 ± 370. 31. Okan I, Wang Y, Chen F, et al. The EBV- encoded LMP1 protein inhibits p53 - triggered apoptosis but not growth arrest. Oncogene. 1995;11:1027 ± 1031. 32. Pathmanathan R, Prasad U, Sadler R, Flynn K, Raab - Traub N. Clonal proliferations of cells infected with Epstein - Barr virus in preinvasive lesions related to nasopharyngeal carcinoma [ see comments ]. N Engl J Med. 1995;333:693 ± 698. 33. Pirollo KF, Hao Z, Rait A, et al. p53 - mediated sensitization of squamous cell carcinoma of the head and neck to radiotherapy. Oncogene. 1997;14:1735 ± 1746. 34. Raab - Traub N. Epstein - Barr virus and nasopharyngeal carcinoma. Semin Cancer Biol. 1992;3:297 ± 307. 35. Ramqvist T, Magnusson K, Wang Y, Szekeley L, Klein G. Wild - type p53 induces apoptosis in a Burkitt lymphoma ( BL ) line that carries mutant p53. Oncogene. 1993;8:1495 ± 1500. 36. Roberts JJ, Thomson AJ. The mechanism of action of antitumor platinum compounds. Prog Nucleic Acid Res Mol Biol. 1979;22:71 ± 133. 37. Sato S, Kigawa J, Minagawa Y, et al. Chemosensitivity and p53 - dependent apoptosis in epithelial ovarian carcinoma. Cancer. 1999;86:1307 ± 1313. 38. Show P, Bovey R, Tardy S, Sahli R, Sordat B, Costa J. Induction of apoptosis by wild - type p53 in a human colon tumor ± derived cell line. Proc Natl Acad Sci USA. 1992;89:4495 ± 4499. 39. Sizhong Z, Xiukung G, Yi Z. Cytogenetic studies on an epithelial cell line derived from poorly differentiated nasopharyngeal carcinoma. Int J Cancer. 1983;31:587 ± 590. 40. Sorenson CM, Barry MA, Eastman A. Analysis of events associated with cell cycle arrest at G2 phase and cell death induced by cisplatin. J Natl Cancer Inst. 1990;82:749 ± 755. 41. Sorenson CM, Eastman A. Mechanism of cis - diamminedichloroplatinum( II ) ± induced cytotoxicity: role of G2 arrest and DNA double - strand breaks. Cancer Res. 1988;48:4484 ± 4488. 42. Szekely L, Selivanova G, Magnusson KP, Klein G, Wiman KG. EBNA - 5, an Epstein - Barr virus ± encoded nuclear antigen, binds to the retinoblastoma and p53 proteins. Proc Natl Acad Sci USA. 1993;90:5455 ± 5459. 43. Wang D, Liebowitz D, Kieff E. An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell. 1985;43:831 ± 840. 44. Wu GS, El - Deiry WS. Apoptotic death of tumor cells correlates with chemosensitivity, independent of p53 or bcl 2. Clin Cancer Res. 1996;2:623 ± 633. 45. Yang C, Cirielli C, Capogrossi MC, Passaniti A. Adenovirus mediated wild - type p53 expression induces apoptosis and suppresses tumorigenesis of prostatic tumor cells. Cancer Res. 1995;55;4210 ± 4213. 46. Zeng Y. Establishment of an epithelioid cell line and a fusiform cell line from a patient with nasopharyngeal carcinoma. Sci Sin. 1978;21:127. 47. Zhang Q, Gutsch D, Kenney S. Functional and physical interaction between p53 and BZLF1: implications for Epstein Barr virus latency. Mol Cell Biol. 1994;14:1929 ± 1938. 48. Zhang WW, Alemany R, Wang J, Koch PE, Ordonez NG, Roth JA. Safety evaluation of Ad5CMV- p53 in vitro and in vivo. Hum Gene Ther. 1995;6:155 ± 164. Cancer Gene Therapy, Vol 8, No 5, 2001