Combination of adenovirus-mediated thymidine kinase gene therapy with cytotoxic chemotherapy in bladder cancer in vitro.

We evaluated efficacy, toxicity and potential synergism of adenoviral-mediated thymidine kinase (tk)- ganciclovir (GCV) gene therapy in combination with 4 cytotoxic chemotherapeutic agents (doxorubicin, cisplatin, mitomycin C, and methotrexate) in 3 human bladder cancer cell lines. Cell lines were exposed to (1) 10 different concentrations of adenovirus expressing tk plus GCV; (2) 8 different concentrations of either doxorubicin, methotrexate, mitomycin C or cisplatin; or (3) combination treatment consisting of either low-, medium- or high-dose tk-GCV gene therapy plus 8 different concentrations of a single chemotherapeutic agent. Cell survival was determined using a MTT-based cell proliferation-assay. For most combinations, adding chemotherapy to tk-GCV gene therapy did not result in any therapeutic benefit. In some scenarios, we observed modest improvement with combinations of high-dose tk-GCV gene therapy and high-dose standard chemotherapy over tk-GCV monotherapy. Low concentrations of methotrexate enhanced the antitumor effects of low- and medium-dose tk-GCV gene therapy. Low level negative interference between tk-GCV gene therapy and chemotherapy occurred in some combinations but was overall negligible. In general, adding chemotherapy to tk-GCV gene therapy did not demonstrate significant therapeutic benefit in vitro. High doses of chemotherapeutic agents should be used in combination with tk-GCV gene therapy in order to take advantage of the occasional instance where modest improvement occurred with combination therapy. Additional studies exploring the role of methotrexate in enhancing the tk-GCV system are required. Investigation of other, potentially more synergistic chemotherapeutic agents in combination with tk-GCV is warranted.

Adenovirus-mediated suicide gene therapy for bladder cancer: comparison of the cytomegalovirus- and Rous sarcoma virus-promoter.

BACKGROUND: To compare efficacy and toxicity of the human cytomegalovirus-immediate-early (CMV) promoter and the Rous-sarcoma-virus (RSV) promoter to express thymidine kinase (tk) for adenovirus-mediated suicide gene therapy of experimental bladder cancer in vivo and in vitro. MATERIALS AND METHODS: In vitro: 3 human (5637, RT-4 and TCC-SUP) and one murine (MBT-2) bladder cancer cell line were exposed to ADV/RSV-tk or ADV/CMV-tk vectors and cell survival was determined. In vivo: Subcutaneous tumors were established and adenovirus vectors were injected 10 days later. RESULTS: In vitro: ADV/CMV-tk was up to 4 times more potent in terms of cell killing than ADV/RSV-tk. In vivo: ADV/CMV-tk had a three-fold higher antitumor potency per viral particle as compared to ADV/RSV-tk. Higher doses of ADV/CMV-tk caused treatment-associated hepatotoxicity. CONCLUSIONS: Our results confirm the efficacy of adenovirus-mediated tk suicide gene therapy in the treatment of experimental bladder cancer. Dose-related toxicity was greater with the use of ADV/CMV-tk, but lower doses achieved the same efficacy as ADV/RSV-tk.

In vivo adenovirus-mediated suicide gene therapy of orthotopic bladder cancer.

These studies were undertaken to determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase (ADV/RSV-tk) and the prodrug ganciclovir (GCV) in an orthotopic murine bladder cancer model. We utilized a replication-defective adenoviral construct containing the beta-galactosidase gene as a control and the herpes simplex virus thymidine kinase gene as the therapeutic vector under the transcription control of the Rous sarcoma virus long terminal repeat promoter. Intravesically created, orthotopic bladder tumors were established in syngeneic C3H/He female mice. India ink injection and beta-galactosidase studies were performed to determine if transurethral administration, direct tumor injection, or the combination was the most efficient route of virus administration. Optimal dosing of ADV/RSV-tk was determined by direct tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate but similar controlled experiments. Growth curve studies demonstrated reliable tumor formation by 14 days. Direct transvesical tumor injection resulted in the best distribution and intratumor gene expression as measured by X-gal staining. Dose-ranging experiments demonstrated an optimal viral dose of 5 x 10(8) plaque-forming units and a greater than twofold reduction in tumor growth for the animals treated with ADV/RSV-tk compared to controls. Efficacy studies demonstrated a greater than threefold reduction in tumor growth. No clinical or gross pathologic toxicity was detected. Long-term survival results suggested a survival benefit for the treatment animals compared to controls. We conclude that ADV/RSV-tk in combination with GCV provides effective therapy for orthotopic murine bladder cancer by significantly inhibiting tumor growth with limited toxicity to the host. These data provide further support for testing this suicide gene therapy strategy in human Phase I trials.

Adenovirus-mediated combination suicide and cytokine gene therapy for bladder cancer.

BACKGROUND: The present study tests the hypothesis that adenovirus-mediated transfer of murine IL-2 (ADV/RSV-mIL-2) alone or in combination with HSV-tk + GCV will improve antitumorigenic response in the murine MBT-2 model. MATERIALS AND METHODS: mIL-2 production and toxicity were determined in vitro using an ELISA and a cell proliferation assay. Tumor-bearing animals were randomly assigned into four treatment groups and directly injected with combinations of ADV/RSV-tk and ADV/RSV-mIL-2. In a separate experiment, the above-mentioned groups were followed by two subsequent treatments with ADV/RSV-mIL-2. RESULTS: Transduced MBT-2 cells were able to express mIL-2 in a time and dose dependent fashion. We could not demonstrate any improvement in antitumorigenic response with mIL-2 gene therapy alone or in combination with HSV-tk-suicide gene therapy over HSV-tk suicide gene therapy alone. CONCLUSIONS: Although ADV/RSV-mIL-2 transduced MBT-2 cells were able to produce large amounts of mIL-2 in vitro, we could not demonstrate significant tumor growth inhibition by adding mIL-2 gene therapy to suicide gene therapy. The growth inhibitory effects of sequential suicide and cytokine gene therapy were transient and not superior to single dose suicide and cytokine gene therapy.

Androgen receptor gene mutations do not occur in ovarian cancer.

Genetic alterations have been frequently found in ovarian cancer. There is some indirect evidence indicating that mutation of the steroid receptor genes may play a role in the carcinogenesis of ovarian cancer. Human androgen receptor (hAR) gene mutations have been found in up to 50% of hormone-relapsed prostate cancer. The role of hAR mutation and its association with decreased expression in ovarian cancer has never been elucidated. In this study mutations of hAR gene in 38 human ovarian cancer cell lines with different AR expression pattern were studied using SSCP. No mutation of the hAR gene was found. Mutation of hAR gene is an infrequent event and therefore unlikely to be involved in the development of ovarian cancer. The decreased expression of hAR in advanced ovarian tumor is not due to genetic aberration of hAR. Mutation screening of hAR may not provide any information for risk assessment of developing ovarian cancer.

Gene therapy of metastatic colon carcinoma: regression of multiple hepatic metastases by adenoviral expression of bacterial cytosine deaminase.

Colon carcinoma accounts for 20% of deaths due to malignancies in the Western world. Once metastases occur, therapeutic options are limited, with an approximate 5-year survival of only 5%. To investigate the potential of new gene therapeutic approaches, a hepatic micrometastasis model of colon carcinoma in BALB/c mice was established. Inoculation of syngeneic MCA26 colon carcinoma cells into the spleens of 18- to 20-week-old mice resulted in the formation of multiple hepatic metastases. Selective transduction of developing hepatic metastases was demonstrated using a beta-galactosidase-expressing recombinant adenovirus. Cytosine deaminase (CD) can metabolize 5-fluorocytosine into the chemotherapeutic reagent 5-fluorouracil (5FU). The antitumoral potential of this suicide gene therapy approach was explored by systemic application of a recombinant replication-deficient adenovirus encoding for the bacterial CD gene under the control of the cytomegalovirus promoter (Ad.CMV-CD). Injection into the tail vein of tumor-bearing mice resulted in delayed tumor growth with significant reduction in hepatic metastases. The potential of this experimental approach for possible future clinical applications was evaluated by investigating adenoviral transduction efficiency, 5FU sensitivity, and 5-fluorocytosine-dependent Ad.CMV-CD toxicity in a variety of human colon cancer cell lines. Although the murine cell lines MCA26 and CC36 were highly sensitive to 5FU, the human colon cancer cell lines showed a 1-100 times higher resistance to 5FU. Specific Ad.CMV-CD toxicity correlates with 5FU toxicity. Transduction efficiency in human colon carcinoma cell lines was shown to be 10-1700 times higher compared with murine cell lines, thus compensating for 5FU resistance. In conclusion, suicide gene therapy using CD may be promising as an adjuvant treatment regimen for hepatic micrometastases of human colon carcinoma.

Comparison of long-term survival of cytomegalovirus promotre versus Rous Sarcoma virus promoter-driven thymidine kinase gene therapy in nude mice bearing human ovarian cancer.

The cytomegalovirus (CMV) promoter is considered one of the strongest positive regulators leading to expression of higher levels of the thymidine kinase (TK) enzyme than the Rous Sarcoma virus (RSV) promoter in vitro and in vivo. Cell killing efficacy of adenovirus-mediated CMV promoter-driven herpes simplex virus (HSV) TK gene therapy has been found to be 2 to 10 times more effective than RSV driven HSV-TK gene therapy in vitro. In this study the impact of CMV- versus RSV-driven HSV-TK gene therapy on long-term survival of nude mice bearing human ovarian cancer has been evaluated using a prospective randomized experimental design. The experiment was designed to show significance of survival differences from a 50% increase of survived days at a p-value of 0.05 with a power of 80%. All treatment groups showed an increase in median survival compared with control groups. Treatment benefit was ADV/CMV-TK vector dose dependent. At a given viral dose, no significant prolongation of survival was observed comparing CMV- and RSV-driven ADV-TK indicating that simply increasing cell killing efficacy in vitro above a minimal threshold level using a stronger promoter may not lead to prolongation of survival in the HSV-TK/GCV system.

Adenovirus mediated thymidine kinase gene therapy may enhance sensitivity of ovarian cancer cells to chemotherapeutic agents.

The current treatment concept of ovarian cancer consists of radical surgery with subsequent chemotherapy. We have shown that adenovirus (ADV) mediated thymidine kinase (TK) gene transduction of cisplatin-resistant human ovarian cancer xenotransplanted into nude mice followed by ganciclovir (GCV) administration leads to prolongation of survival or cure. In this study the interaction of ADV-TK gene therapy and selected chemotherapeutic agents commonly used for the treatment of ovarian cancer was investigated in three ovarian cancer cell lines with different growth patterns. Toxicity and cell killing efficacy of gene therapy, chemotherapy and their combinations with different concentrations and time intervals were measured by a 3-(4,5- dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) based assay. A slightly increased resistance to gene therapy was observed in cells pretreated with chemotherapy. Removal of the drugs restored the previous susceptibility of the cells to gene therapy. No antagonism was observed with gene therapy followed by chemotherapy. The concomitant application of gene therapy and chemotherapy resulted in a higher rate of cell death than the interval therapy. A dose dependent synergistic interaction was observed only for the combination of gene therapy and the topoisomerase 1 inhibitor topotecan. This synergistic effect was still seen even if the chemotherapeutic agent was added 72 hours later. Our data demonstrate that in addition to its own therapeutic efficacy, ADV-TK based gene therapy may enhance the effect of subsequent chemotherapy while up-front chemotherapy was disadvantageous.

Improvement of gene therapy for ovarian cancer by using acyclovir instead of ganciclovir in adenovirus mediated thymidine kinase gene therapy.

Adenovirus(ADV) mediated thymidine kinase(TK) gene therapy followed by ganciclovir(GCV) administration is widely used in different types of cancer. ACV shares the same mechanism of selective cell killing in ADV/TK positive cells as GCV and can be used at 4.5 times higher doses in patients without significant side effects. An increased dose of TK substrate is associated with improved bystander effect and more efficient cell killing. Toxicity and cell killing efficacy were assessed using a 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide(MTT) based assay in three ovarian cancer cell lines with different proliferation patterns. At the same concentration, equal or higher cell killing efficacy and bystander effect were observed using ACV rather than GCV. 2.5 and 5 times (25 micrograms/ml and 50 micrograms/ml) higher concentrations of ACV always resulted in more effective cell killing than GCV (10 micrograms/ml, P < 0.01). Our data indicate that replacing GCV with ACV in the ADV-TK gene therapy may increase the treatment effect without increasing toxicity.

The efficacy of adenovirus-mediated gene therapy of ovarian cancer is enhanced by using the cytomegalovirus promoter.

The cytomegalovirus(CMV) promoter is considered one of the strongest positive regulators. In this study toxicity, cell killing efficacy and bystander effect of Rous Sarcoma Virus(RSV) driven herpes simplex thymidine kinase(TK) gene therapy was compared with CMV driven TK gene therapy in three ovarian cancer cell lines with different growth patterns using a 3-(4,5-dimethylthiazol)-2,5-diphenyl tetra-zolium bromide (MTT) based assay. ADV/CMV-TK was shown to be 2 to 10 times more effective in tumor cell killing than ADV/RSV-TK. The difference in cell killing efficacy between ADV/CMV-TK and ADV/RSV-TK was dependent on the individual cell line. A CMV promoter dependent eight to ten fold improvement in cell killing efficacy was observed in the relatively slow growing SKOV3 cell line which is not easily transducible, while only a 2 to 4 fold difference was observed in the easily transducible OV-CA-2774 and OV-CA-1225 cell lines. ADV/CMV-TK also showed a stronger bystander effect than ADV/RSV-TK in all three ovarian cancer cell lines. Our data demonstrated that the efficacy of adenovirus-mediated gene therapy of ovarian cancer can be enhanced by using the CMV promoter without increasing toxicity.