Purpose: The objectives of this study were to determine the effects of adenovirus-mediated p16 and p53 on growth and apoptosis in ovarian cancer cells and on survival in nude mice implanted with human ovarian cancer cells.

Experimental Design: SKOV-3 ip1 (p53 and p16 null), 2774 (p53 and p16 mutant), and OVCA 420 (p53 and p16 wild-type) cells were used for in vitro studies. SKOV-3 ip1, 2774, and Hey A8 (p53 and p16 wild-type) cells were used in the nude mouse studies. The E1-deleted adenoviruses containing p53, p16, or β-galactosidase cDNA were transfected into the different cell types or inoculated into the nude mice after injection with ovarian cancer cells.

Results: Cell counting, microtetrazolium, and anchorage-independent growth assays on transfected cells demonstrated that p16 and the p16/p53 combination suppressed growth, whereas p53 did not (except in the anchorage-independent growth assay). Although cells infected with the p16/p53 combination had decreased growth compared with cells infected with either tumor suppressor alone, the difference was only statistically significant compared with p53. p16, p53, and the p16/p53 combination all increased apoptosis in the cells. In the nude mice, p16 treatment resulted in the longest survival for all three models, although it only reached statistical significance for the 2774 and SKOV-3 ip1 groups.

Conclusions: Overall, p16 demonstrated greater growth inhibition than p53 both in vivo and in vitro. The p16/p53 combination demonstrated a consistent trend toward increased growth suppression and apoptosis over p16 or p53 alone. Adenovirus-mediated p16 may be a viable future treatment for ovarian cancer.

Ovarian cancer is the second most common gynecological malignancy in the United States and is the fifth leading cause of cancer death among women. The disease is usually diagnosed at an advanced stage, and despite aggressive surgical debulking and chemotherapy regimens, the 5-year survival remains a dismal 11–25% for advanced stages (1). Other forms of treatment are clearly needed, and several recent studies have focused on gene therapy as a viable option. Specifically, the adenovirus-mediated introduction of tumor suppressor genes such as p53 and p16 is effective in slowing ovarian cancer growth in vitro(2, 3).

The tumor suppressor genes p16 and p53 have been shown to regulate the cell cycle through different mechanisms. p53 regulates the cell cycle at the G1 checkpoint and is primarily stimulated by DNA damage. Activation of p53 leads to either G1 arrest or apoptosis; the protein product of p53 binds to damaged DNA and serves as a transcriptional activator. Specifically, p53 causes growth arrest through the induction of p21 and causes apoptosis via activation of Bax. p53 is also regulated by MDM-2; an increase in MDM-2 results in inhibition of p53 activity. In ovarian cancer, p53 mutations have been found in up to 50% of women with late-stage disease (4, 5). In vitro assays have demonstrated that the introduction of the wild-type p53 gene via a recombinant adenovirus can inhibit growth of ovarian cancer cells, regardless of endogenous p53 status (3, 6, 7). Several groups have also demonstrated the efficacy of adenovirus-mediated p53 in slowing ovarian cancer growth in vivo(6, 7, 8, 9). Ongoing trials are evaluating this treatment in women with recurrent ovarian cancer.

p16 encodes a protein that inhibits cyclin-dependent kinase 4 and cyclin-dependent kinase 6 cyclin D kinases. p16 is a member of the INK4 cell cycle proteins, and these kinases are required for phosphorylation of the retinoblastoma gene product (RB). Hypophosphorylated RB functions to inhibit entry of cells into the S-phase. In vitro studies have also demonstrated the efficacy of p16 transfection via an adenovirus vector in the inhibition of growth and induction of apoptosis in ovarian cancer cell lines (2, 10).

The objective of this study was 2-fold. The first objective was to determine whether the transfection of p16 and p53 together resulted in greater growth inhibition and apoptosis in ovarian cancer cell lines than either individually. The second purpose was to evaluate the effect of that transfection on survival in a nude mouse ovarian cancer model.

Cell Lines.

SKOV3ip1 (p16 and p53 null), 2774 (p16 and p53 mutant), and OVCA 420 (p16 and p53 wild-type) cells were grown in MEM supplemented with 10% fetal bovine serum. The OVCA 420 cell line does not grow in vivo, and Hey A8 cells (p53 and p16 wild-type), along with SKOV-3 ip1 and 2774, were used for the nude mouse model studies.

Adenovirus Infection.

Adenoviral vectors contained wild-type p53, p16, or Escherichia coli β-gal3 cDNA and a CMV promoter inserted into the E1-deleted region of modified adenovirus (Ad5CMV). Monolayer cells were grown in MEM with 10% fetal bovine serum and were infected with adenoviral vectors at a multiplicity of infection of 100 for all subsequent experiments. Cells receiving both p16 and p53 were infected with both vectors at an multiplicity of infection of 100.

Growth Suppression Assays.

Cells were initially plated in triplicate in MEM at a density of 2 × 104 cells/well. Cells were either uninfected (mock); infected with Ad5CMVβ-gal, Ad5CMVp53, AD5CMVp16; or both Ad5CMVp53 and Ad5CMVp16. Cells were harvested and counted with a Coulter counter on days 1, 3, 4, 5, 6, and 7 after infection (Coulter Corp., Miami, FL). Each growth assay was completed in triplicate for three different experiments. The average number of cells/ml for each day after infection was calculated by combining data from all nine experiments. Growth was also monitored using dye conversion in a MTT assay as described previously (11).

Anchorage-independent Colony Formation.

Cells from the three cell lines were infected 24 h after plating. Twenty-four h after infection, 1 × 103 cells were plated in triplicate in 0.3% agar in three separate experiments. Plates were incubated at 37°C, and colonies were counted on an inverted microscope at ×4 magnification at 3 weeks after plating. Total numbers of colonies/dish and colony-forming efficiency were calculated.

Protein Expression.

Total cell lysates were prepared by sonicating cells 3 days after infection in RIPA buffer (150 mm NaCl, NP40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mm Tris, pH 8.0). Fifty μg of protein were loaded onto SDS-polyacrylamide gels (gels ranged from 10–13%, depending on protein to be evaluated) and electrophoresed. The protein was then transferred to a Hybond-ECL membrane (Amersham, Arlington Heights, IL). Membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 and probed with antibodies against the proteins of interest. The specific antibodies used were mouse antihuman p53 (Amersham), mouse antihuman p16 (Oncogene Research Production, Cambridge, MA), rabbit anti-Bax (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-Bcl-2 (DAKO, Carpinteria, CA), and mouse anti-RB (Santa Cruz Biotechnology). Appropriate IgG horseradish peroxidase-conjugated goat antimouse or antirabbit secondary antibodies were used, and blots were processed per the manufacturers’ suggestions.

Apoptosis.

Evidence for apoptosis was evaluated via a terminal deoxynucleotidyltransferase-mediated nick end labeling-based assay (Apo-BRDU kit; Phoenix Flow Systems, San Diego, CA). Plates of 1 × 106 cells were either then mock infected or infected with Ad5CMVβ-gal, Ad5CMVp53, AD5CMVp16, or both Ad5CMVp53 and Ad5CMVp16 at the same multiplicity of infection as the growth curves and evaluated 3 days later. For evaluation, cells were initially fixed in 1% formaldehyde and then in 70% ethanol. They were then incubated with terminal deoxynucleotidyltransferase enzyme and Br-dUTP, rinsed, and incubated with fluorescent monoclonal antibody and PI staining buffer (50 mg/ml PI and 15 mg RNase). The percentages of positive cells were determined by flow cytometry (Coulter Epics XL-MCL; Coulter Corp.).

Cell Cycle Analysis.

Cells were incubated with adenoviral vectors for 72 h at a density of 1 × 106 cell/10-ml plate. Cells were then fixed in ethanol and stored at 4°C. At the time of cell cycle analysis, cells were washed and resuspended in PI staining buffer. DNA content was evaluated by flow cytometry (Coulter Epics XL-MCL).

Nude Mouse Model.

This protocol was approved by the Animal Care and Utilization Committee at The University of Texas M. D. Anderson Cancer Center. Seventy-five female, athymic nude mice (Harlan Sprague Dawley Inc., Indianapolis, IN) were obtained at 6–8 weeks of age and kept in a contained biohazard area. The mice were divided into three groups of 25 mice. Each group was injected i.p. with either Hey A8, SKOV-3 ip1, or 2774 cell lines (5 × 106cells in 200 μl of sterile saline). Within each group, there were five different treatment groups of five mice each. Each group of five mice received i.p. injections of either saline, β-gal virus, p16 virus, p53 virus, or both p16 and p53 adenoviruses. Viral doses (2 × 109 plaque-forming units/200 μl of saline per injection) were given on days 4, 6, and 8 after inoculation with the ovarian cancer cells. Mice were then evaluated daily for morbidity and mortality. Mice were euthanized once the ascites was large enough to preclude movement or if cachexia produced severe morbidity. Ascitic fluid, tumor samples, and liver samples were collected at the time of necropsy. All surviving mice were sacrificed 150 days after initial ovarian cancer cell inoculation. Multiple biopsies of tissue from mice lacking gross evidence of disease at necropsy were evaluated for evidence of cancer by a veterinary histopathologist.

Statistical Analysis.

SPSS 10.0 software (SPSS, Inc., Chicago, IL) was used for statistical analysis. One-way ANOVA was used to compare means between treatment groups and Tukey’s HSD was used to evaluate the statistically significant differences between groups. The Kaplan-Meier method and the log-rank statistic were used to evaluate survival in the nude mouse model. P < 0.05 was deemed significant.

Adenovirus Infection.

Western blot analysis detected p16 and p53 protein products in all cell lines after infections with adenoviral constructs; this was done to confirm expression of infected proteins as well as to establish baseline protein expression patterns. p53 was not expressed in the SKOV-3 ip1 line (p53 null) and expressed at low levels in the 2774 and 420 (p53 mutant and wild-type, respectively) cell lines. p53 was always expressed strongly when it was transfected; there was not a difference in levels between the p53- and the p16/p53-transfected groups. p16 was only expressed in cell lines that were infected with the p16-containing adenovirus; it was expressed less in the p16 group than in the p16/p53 group (Fig. 1).

Growth Suppression Assays.

All data were pooled and analyzed to determine the overall effect of the five treatment types (mock, β-gal, p16, p53, and p16/p53) on growth, as assessed by one-way ANOVA comparing the average cell counts on designated days after infection. There was no significant difference on day 1 (F = 0.297; P = 0.879), however, on days 3, 4, 5, 6, and 7, there was a statistically significant difference between the groups (F = 3.5–26; P = 0.000–0.011). Tukey’s HSD test showed that by day 3, only the p16/p53 group showed decreased growth as compared with mock and β-gal (P = 0.030–0.034). By day 7, both the p16 and p16/p53 had decreased growth compared with the mock, β-gal, and p53 groups (P = 0.000–0.047). The p53 did not demonstrate a statistically significant decrease in cell growth as compared with controls. Although the p16/p53 group did have a lower mean number of cells/ml than the p16 group, it was not statistically significant (P = 0.117–0.995; Fig. 2).

The data were also analyzed separately for each cell type, and only minimal exceptions to the results of the pooled data were found. In SKOV-3 ip1 cells; the only difference was that growth was more suppressed in the p53 and β-gal groups than the mock group by day 7 (P = 0.004 and 0.008, respectively). In OVCA 420 cells, both p16 and p16/p53 groups showed decreased growth compared with the mock and β-gal groups on days 4 and 6 (P = 0.000–0.016), but by day 7, the differences had lost statistical significance (F = 1.709; P = 0.167). In 2774 cells, p16 and p16/p53 groups showed significantly less growth than mock and β-gal groups at days 3 and 6 (P = 0.003–0.039); however, that difference also disappeared by day 7.

The results from the MTT assay were remarkably similar to those in the cell counting assays. Using the pooled data, for all 4 days (days 1, 3, 6, and 7) significant differences were found among treatment groups (F = 5.3–39.8; P = 0.000–0.001). The p16 and the p16/p53 groups showed significantly decreased cell growth than mock and β-gal on all days tested (P = 0.000–0.031). The p53 group did not differ from controls, although it was always significantly different from both the p16 and p16/p53 groups. Although the p16/p53 group always had a lower mean uptake than the p16 group, it was only statistically significant on day 3 (P = 0.011; Fig. 3).

The MTT data for the individual cell lines were almost identical to the pooled data, except that the data on day 1 were not significantly different among the groups. In the SKOV-3 ip1 cell line, the p16 and p16/p53 groups differed by day 7 (P = 0.001). In the 420 cell line, growth in the p53 group was decreased compared with that of controls by day 7 (P = 0.005–0.020).

Anchorage-independent Colony Formation.

One-way ANOVA revealed a significant difference between the mean colony-forming efficiencies of the various treatment groups (F = 100; P = 0.000). All three intervention groups resulted in decreased colony-forming efficiency compared with both the mock-infected and the β-gal-infected groups (P = 0.000 for p16, p53, and p16/p53). Cells containing the β-gal construct did have a lower mean colony-forming efficiency than the mock control, but it was not statistically significant (P = 0.206). The p53 group had more growth inhibition than the controls; however, the p16 and the p16/p53 groups had even greater growth inhibition than the p53 group (P = 0.000 for both). Although the p16/p53 group had a fewer colonies than the p16 group, the difference was not statistically significant (Table 1).

Protein Expression.

The retinoblastoma gene product was expressed in all three cell lines and did not appear affected by the transfection with p16 or p53. Bax expression was found in both controls and infected cells; however, it was enhanced after transfection with p53 and suppressed after transfection with p16. Bcl-2 expression was demonstrated only in the 2774 cell line and was unaffected by transfection with the various adenoviral constructs. p19, another member of the INK4 protein family, expression was not detected in any of the cell lines tested. p21 expression was induced in the cell lines after infection with p53 (Fig. 1).

Apoptosis.

When data were pooled, one-way ANOVA showed that the p53, p16, and p16/p53 groups had a significantly increased percentage of cells in apoptosis than mock and β-gal controls (F = 31.1; P = 0.000). The p16/p53 group had significantly more apoptosis than the p16 group (P = 0.000) but not more than the p53 group, although it showed a trend toward significance (P = 0.107). These trends were consistent with those when each cell line was analyzed individually; the only exception was in the SKOV-3 ip1 line in which only the p53 group demonstrated a significant increase in apoptosis (P = 0.020; Table 2).

Cell Cycle Analysis.

The pooled data from all three cell lines revealed a significant difference in the percentage of cells in G1 (F = 4.32; P = 0.002). Further analysis revealed that the major difference was a lower percentage of cells in G1 in the β-gal group (44%) than the mock (56%), p53 (54%) or p16 (57%) groups (P = 0.005–0.038). The percentage of cells in G1 also differed between the p16/p53 and the β-gal groups but was not significant (53% versus 44%; P = 0.067). When the three cell lines were analyzed individually, the β-gal group consistently had a lower percentage of cells in G1 than did the other groups; otherwise, the individual data were inconsistent with the pooled data. For example, in the SKOV-3 ip1 cells, the p16 and the p53 groups had a higher G1 percentage than mock (P = 0.038 and 0.001, respectively; the p16/p53 did also but it did not reach statistical significance (P = 0.066). In 2774 cells, the p53 and p16/p53 instead had lower G1 percentages than mock (28% and 41% versus 53%; P = 0.000 and 0.029, respectively). Finally, in the 420 cells, only the β-gal significantly decreased the percentage of G1 cells in comparison with the other groups (Table 3).

Nude Mouse Model.

For all three cell lines, the p16 group had the longest survival and the most mice that showed NED at the completion of the experiment. (Table 4; Fig. 4). No control mice in any of the cell lines were found to be histologically NED at the completion of the experiment. No statistically significant differences were found between treatment groups with regard to weight or amount of ascites at the time of necropsy.

In the mice injected with the Hey A8 cell line, all tumor suppressor treatment groups had longer survival than either β-gal or mock controls. The differences, however, did not attain statistical significance using the Kaplan-Meier log-rank statistic (2.09; P = 0.791) or using ANOVA for length of survival (F = 0.555; P = 0.698). One p16-treated mouse survived 150 days and at the time of necropsy did not have any gross evidence of tumor, and histopathology of numerous biopsies confirmed the absence of microscopic disease.

In the SKOV-3 ip1 cell line, the p16 group again demonstrated the longest survival; however, there was not a statistically significant difference among the groups using the Kaplan-Meier method and the log-rank statistic (4.40; P = 0.3544). However, ANOVA analysis found a significant difference in average survival length between the treatment groups (F = 2.989; P = 0.044). Further analysis with Tukey’s HSD revealed that the difference stemmed from the longer survival of the p16 group compared with the p53 group (143 and 85 days, respectively; P = 0.033). At the completion of the experiment, 7 mice (1 control mouse, 2 β-gal mice, 1 p53 mouse, 2 p16 mice, and 2 p16/p53 mice) were grossly free of tumor at necropsy. On final histopathological review of multiple biopsies, the surviving control mouse was found to have adenocarcinoma, whereas all of the surviving treatment mice were still disease free.

In the 2774 cell line, the Kaplan-Meier survival curves were different between the groups (log-rank statistic, 9.56; P = 0.0485). Specifically, the β-gal group and p16 groups had a higher survival than mock controls (P = 0.0018 and 0.0277, respectively). ANOVA did not show a significant difference in survival length between groups, although it demonstrated a trend (F = 2.318, P = 0.092). An independent samples t test comparing average survival length between mock and p16 revealed a significant difference (43 and 127 days; P = 0.007). A total of 8 mice survived until the completion of the experiment (1 β-gal mouse, 4 p16 mice, 1 p53 mouse, and 2 p16/p53 mice), and all were free of disease both at necropsy and on histopathology.

In light of the poor prognosis for ovarian cancer, the search continues for innovative and efficacious treatment modalities. Along with surgical and chemotherapeutic regimens, gene therapy has emerged as one of the leading contenders for a place in the treatment armamentarium. Several groups have reported both in vitro and in vivo success in introducing tumor suppressor genes to slow the growth of ovarian cancer and induce apoptosis (2, 3, 6, 7, 8, 9, 10).

The majority of gene therapy research in ovarian cancer has focused on p53. p53 is abnormally expressed in approximately 50–67% of ovarian cancer patients and also appears to be the most frequent genetic alteration in the disease (4, 5). Earlier work by our laboratory and others has demonstrated that p53 introduced via an adenovirus vector can suppress growth and induce apoptosis in ovarian cancer cell lines (3, 7). The data in this study showed that adenovirus-mediated p53 increased apoptosis and decreased anchorage-independent growth in the three ovarian cancer cell lines tested. In contrast to some of the previous data in our lab, p53 was not effective in slowing ovarian cancer growth in the cell counting or MTT assays. This seeming contradiction may be partially explained by the use of a lower multiplicity of infection in the SKOV-3 ip1 line compared with previous work (100 versus 250), and the other possibility may stem from potential inconsistencies in viral titers from different viral batches. Unfortunately, p53 did not increase survival in the nude mouse model, either alone or in combination with p16; this may be because of the decreased magnitude of growth inhibition by p53 when compared with p16 and the p16/p53 combination. Several studies have shown that both p53 and β-gal introduced via an adenovirus vector result in increased survival over controls in mouse models (6, 9); one study has shown that p53 increased survival over both β-gal and controls (8).

In ovarian cancer, mutations of the p16 gene are relatively rare, although p16 mutations may approach 50% in ovarian cancer cell lines (12, 13, 14, 15, 16). Recently, alterations in p16 protein expression have also been demonstrated (17, 18). One group found no p16 protein expression in 26% of ovarian cancer tumors studied (17). Similarly, Dong et al.(18) found that 11% of ovarian cancers did not express p16 and that increased p16 expression correlated with progression and unfavorable prognosis. The mechanism for the decreased p16 expression in these two studies has not been identified; however, it does not appear to be secondary to hypermethylation. Further work needs to be done to delineate the role of p16 in the origination and progression of ovarian cancer.

Our data support the premise that p16 is more effective than p53 or controls in suppressing ovarian cancer cell growth. Other investigators have also demonstrated increased cell cycle arrest, growth inhibition, and apoptosis after adenovirus-mediated transduction of p16 in gliomas and lung, pancreas, liver, and head and neck tumor cell lines (10, 19, 20, 21, 22, 23, 24). Schreiber et al.(10) have compared the efficacy of various cyclin kinase inhibitors (p16, p18, p19, p21, and p27) and found that although they all inhibit growth and increase apoptosis in vitro, only p16 slows tumor progression in vivo. Surprisingly, p16 is also effective in the induction of apoptosis, although the mechanism remains unclear and may be attributable to actions of p16 that are outside the cell cycle regulatory pathway. Other investigators have also proposed that p16 may have other functions aside from cell cycle regulation; for example, p16 was shown to reduce the expression of vascular endothelial growth factor and angiogenesis in vivo while having no effect on cell growth in gliomas (20).

Here we report the first in vivo experiment to evaluate the effect of treating an ovarian cancer nude mouse model with p16 via an adenovirus vector. The results demonstrated that p16 was the only treatment effective in prolonging survival. Similar findings in head and neck cancers and other cell lines show that p16 can significantly decrease the size of established tumors in nude mice (10, 21). Adenovirus-mediated p16 appears to be more effective than p53 or adenovirus alone, although the adenovirus alone did demonstrate some tumoricidal activity. The question of adenoviral vectors having independent action is a chronic issue in gene therapy studies. Unfortunately, it is a question that is difficult to address in a clinical setting because trials with vector alone raise serious ethical considerations. Further work needs to be done in this model to pave the way for future trials in women with recurrent ovarian cancer.

Because p16 and p53 act through separate pathways, it may be beneficial to combine the two constructs to enhance their therapeutic benefit. Our data showed that the p16/p53 combination increased the percentage of cells undergoing apoptosis and inhibited growth more than either tumor suppressor alone. Additional benefit demonstrated in the in vitro assays did not translate into increased survival in the nude mouse model. However, the only other p16/p53 combination study in the literature has reported that the p16/p53 combination can increase apoptosis and that infected cancer cells cannot form tumors in the nude mouse model (25). More work is necessary to further delineate the complex set of cellular events that are initiated by these two tumor suppressors. The present results lend credence to the idea that p16 may be superior to p53 in gene therapy for ovarian cancer.

Combination therapy is gaining popularity in attempts to improve survival in ovarian cancer. Heretofore, most of the effort has been geared toward identifying chemotherapeutic agents that act synergistically to improve outcome. Gene therapy may prove to be a useful adjunctive treatment to complement current therapies. Future work might focus on combining p53 with other agents, perhaps tumor suppressors such as p16 or chemotherapeutic agents such as cisplatin or paclitaxel. Preliminary work with in vitro and in vivo adenovirus-mediated p53 transfection in combination with paclitaxel, cisplatin, doxorubicin, 5-fluorouracil, methotrexate, or etoposide in human head and neck, ovarian, prostate, and breast cancer demonstrated greater anticancer effects than with any of the agents alone (26, 27). In contrast, the work with p16 in combination with chemotherapy has been far less encouraging. Work with bladder cancer cells and human glioma cells has demonstrated that adenovirus-mediated p16 transfer actually results in chemoresistance to several agents, including platinum agents, paclitaxel, topotecan, and carmustine (28, 29, 30).

In conclusion, the adenovirus-mediated transduction of p16 is more efficacious than that of p53 in suppressing growth and increasing survival in the nude mouse model. Although more research is required to characterize the optimal dosages and protocols, adenovirus-mediated p16 gene therapy may soon be a viable treatment option for ovarian cancer patients.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

Supported by a research grant from the Gynecologic Cancer Foundation.

                
3

The abbreviations used are: β-gal, β-galactosidase; CMV, cytomegalovirus; MTT, microtetrazolium; PI, propidium iodide; HSD, honestly significant difference; NED, no evidence of disease.

Fig. 1.

A, expression of p16, p21, p53, and actin controls after infection of 2774 cells with controls and the appropriate adenovirus. Western blot analysis was performed with 50 μg of protein and probing with the antibody of interest. Relative densitometry data compared with controls are listed for each lane. B, expression of RB, bcl-2, bax, and actin controls after infection of 2774 cells with controls and the appropriate adenovirus. Western blot analysis was performed with 50 μg of protein and probing with the antibody of interest. Relative densitometry data compared with controls are listed for each lane.

Fig. 1.

A, expression of p16, p21, p53, and actin controls after infection of 2774 cells with controls and the appropriate adenovirus. Western blot analysis was performed with 50 μg of protein and probing with the antibody of interest. Relative densitometry data compared with controls are listed for each lane. B, expression of RB, bcl-2, bax, and actin controls after infection of 2774 cells with controls and the appropriate adenovirus. Western blot analysis was performed with 50 μg of protein and probing with the antibody of interest. Relative densitometry data compared with controls are listed for each lane.

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Fig. 2.

A, SKOV-3 ip1 cell counting assay that demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average growth curves after infection with adenovirus vectors for nine total experiments. B, 2774 cell counting assay that demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average growth curves after infection with adenovirus vectors for nine total experiments. C, 420 cell counting assay that demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average growth curves after infection with adenovirus vectors for nine total experiments. D, cell counting assay that demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average growth curves for all three ovarian cancer cell lines after infection with adenovirus vectors for nine total experiments.

Fig. 2.

A, SKOV-3 ip1 cell counting assay that demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average growth curves after infection with adenovirus vectors for nine total experiments. B, 2774 cell counting assay that demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average growth curves after infection with adenovirus vectors for nine total experiments. C, 420 cell counting assay that demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average growth curves after infection with adenovirus vectors for nine total experiments. D, cell counting assay that demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average growth curves for all three ovarian cancer cell lines after infection with adenovirus vectors for nine total experiments.

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Fig. 3.

A, SKOV-3 ip1 MTT assay that also demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average MTT after infection with adenovirus vectors for nine total experiments. B, 2774 MTT assay that also demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average MTT after infection with adenovirus vectors for nine total experiments. C, 420 MTT assay that also demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average MTT after infection with adenovirus vectors for nine total experiments. D, pooled data MTT assay that also demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average MTT for all three ovarian cancer cell lines after infection with adenovirus vectors for nine total experiments.

Fig. 3.

A, SKOV-3 ip1 MTT assay that also demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average MTT after infection with adenovirus vectors for nine total experiments. B, 2774 MTT assay that also demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average MTT after infection with adenovirus vectors for nine total experiments. C, 420 MTT assay that also demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average MTT after infection with adenovirus vectors for nine total experiments. D, pooled data MTT assay that also demonstrates decreased growth after infection with adenovirus containing p16 or both p16 and p53. Curves represent average MTT for all three ovarian cancer cell lines after infection with adenovirus vectors for nine total experiments.

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Fig. 4.

A, Kaplan-Meier survival curve for Hey A8-inoculated nude mice after treatment with adenovirus vectors. B, Kaplan-Meier survival curve for SKOV-3 ip1-inoculated nude mice after treatment with adenovirus vectors. C, Kaplan-Meier survival curve for 2774 inoculated nude mice after treatment with adenovirus vectors.

Fig. 4.

A, Kaplan-Meier survival curve for Hey A8-inoculated nude mice after treatment with adenovirus vectors. B, Kaplan-Meier survival curve for SKOV-3 ip1-inoculated nude mice after treatment with adenovirus vectors. C, Kaplan-Meier survival curve for 2774 inoculated nude mice after treatment with adenovirus vectors.

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Table 1

Efficiency of colony formation in soft agarose

Adenovirus% (P)Pooled
SKOV-3 ip1OVCA 4202774
Mock infected 99 51 69 73 
β-gala 88 (0.38) 42 (0.078) 58 (0.241) 63 (0.206) 
p53                  b 54 (0.000) 12 (0.000) 18 (0.000) 28 (0.000) 
p16                  b 0.5 (0.000) 0.2 (0.000) 1 (0.000) 0.6 (0.000) 
p16 andp53b 0.27 (0.000) 0.05 (0.000) 0.05 (0.000) 0.1 (0.000) 
Adenovirus% (P)Pooled
SKOV-3 ip1OVCA 4202774
Mock infected 99 51 69 73 
β-gala 88 (0.38) 42 (0.078) 58 (0.241) 63 (0.206) 
p53                  b 54 (0.000) 12 (0.000) 18 (0.000) 28 (0.000) 
p16                  b 0.5 (0.000) 0.2 (0.000) 1 (0.000) 0.6 (0.000) 
p16 andp53b 0.27 (0.000) 0.05 (0.000) 0.05 (0.000) 0.1 (0.000) 
a

P compared with mock control.

b

P compared with mock and β-gal controls. p16 and p16/p53 were significantly different from p53 in all cell lines (P = 0.000–0.028). p16 did not differ significantly from p16/p53 (P = 1.0).

Table 2

Mean percentage of cells undergoing apoptosis

Adenovirus% (P)Pooled
SKOV-3 ip1OVCA 4202774
Mock 2.4 4.6 1.7 2.9 
β-gala 1.5 (0.978) 5.1 (1.000) 2.9 (0.997) 3.2 (1.000) 
p53                  b 7.0 (0.003–0.020) 34.9 (0.000) 22.9 (0.000) 21.9 (0.000) 
p16                  b 1.9 (0.997–0.999) 28.4 (0.000) 21.3 (0.000) 17.2 (0.000) 
p16 and p53b 3.2 (0.785–0.983) 49.0 (0.000) 34.6 (0.000) 28.9 (0.000) 
Adenovirus% (P)Pooled
SKOV-3 ip1OVCA 4202774
Mock 2.4 4.6 1.7 2.9 
β-gala 1.5 (0.978) 5.1 (1.000) 2.9 (0.997) 3.2 (1.000) 
p53                  b 7.0 (0.003–0.020) 34.9 (0.000) 22.9 (0.000) 21.9 (0.000) 
p16                  b 1.9 (0.997–0.999) 28.4 (0.000) 21.3 (0.000) 17.2 (0.000) 
p16 and p53b 3.2 (0.785–0.983) 49.0 (0.000) 34.6 (0.000) 28.9 (0.000) 
a

P compared with mock control.

b

P compared with mock and β-gal controls. p16 and p53 values were not significantly different except in the SKOV-3 ip1 (P = 0.004). p16 and p16/p53 were significantly different (P = 0.000–0.008) except in SKOV-3 ip1 (P = 0.889). p53 and p16/p53 were significantly different in OVCA 420 and 2774 (P = 0.008–0.011) but not in SKOV-3 ip1 or pooled data (P = 0.06–0.107).

Table 3

Cell cycle analysis: percentage of cells in G1

Adenovirus% (P)Pooled
SKOV-3 ip1OVCA 4202774
Mock 63 53 53 56 
β-gala 55 (0.228) 40 (0.034) 39 (0.005) 44 (0.005) 
p53                  a 77 (0.001) 54 (0.999) 28 (0.000) 54 (0.983) 
p16                  a 73 (0.038) 50 (0.977) 46 (0.433) 53 (0.999) 
p16 and p53a 72 (0.066) 44 (0.256) 41 (0.029) 49 (0.935) 
Adenovirus% (P)Pooled
SKOV-3 ip1OVCA 4202774
Mock 63 53 53 56 
β-gala 55 (0.228) 40 (0.034) 39 (0.005) 44 (0.005) 
p53                  a 77 (0.001) 54 (0.999) 28 (0.000) 54 (0.983) 
p16                  a 73 (0.038) 50 (0.977) 46 (0.433) 53 (0.999) 
p16 and p53a 72 (0.066) 44 (0.256) 41 (0.029) 49 (0.935) 
a

P compared with mock control.

Table 4

Survival data in nude mouse model

AdenovirusSKOV-3 ip1Hey A82774
Mock 104 (0) 38 (0) 43 (0) 
β-gal 128 (20) 37 (0) 89 (20) 
p53 85 (20) 53 (0)b 66 (20) 
p16 143 (40)c 60 (20) 127 (80) 
p16/p53 119 (40) 56 (0) 82 (40) 
AdenovirusSKOV-3 ip1Hey A82774
Mock 104 (0) 38 (0) 43 (0) 
β-gal 128 (20) 37 (0) 89 (20) 
p53 85 (20) 53 (0)b 66 (20) 
p16 143 (40)c 60 (20) 127 (80) 
p16/p53 119 (40) 56 (0) 82 (40) 
a

Mean survival in days (% of NED mice at 150 days).

b

One mouse became paralyzed and was euthanized at 99 days but had NED.

c

One mouse died of unknown causes at 145 days but had NED.

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