Combined Antitumor Effects of an Adenoviral Cytosine Deaminase/Thymidine Kinase Fusion Gene in Rat C6 Glioma

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Abstract
In this study, we investigated the feasibility of a double-suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type 1 thymidine kinase (TK) gene and the Escherichia coli cytosine deaminase (CD) gene, via a recombinant adenoviral vector, and ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) treatment, in a rat C6 glioma model. Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the β-galactosidase gene and then staining cells with X-5-bromo-4-chloro-3-indolyl-13-d-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene (ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-ΔE1 (as a control). After the addition of a variety of concentrations of GCV and 5-FC, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. For in vivo antitumor experiments, 1 × 105 cells were stereotactically injected into the right caudate-putamen of female Wistar rat brains. At 3 days after implantation, 1 × 108 plaque-forming units of ad-CD/TK or ad-ΔE1 (as a control) were stereotactically injected into the tumors and GCV (25 mg/kg) and 5-FC (250 mg/kg), alone or in combination, were intraperitoneally administered. Animals were then killed, and tumor volumes were measured by determining the tumor area in every fifth section, using a light microscope. C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity (>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30 μg/ml or GCV at concentrations greater than 0.3 μg/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after single-prodrug treatments, indicating additive effects of the prodrug treatments. In in vivo experiments, the tumor volumes of the rats treated with GCV or 5-FC alone after ad-CD/TK injection (59.1 ± 4.6 and 57.4 ± 7.1 mm3, respectively) were significantly smaller than that of the control rats (157 ± 8.9 mm3, P < 0.05). Furthermore, the tumor volume of the rats treated with GCV and 5-FC in combination was 14.7 ± 1.8 mm3. These results demonstrated the efficient transduction of C6 glioma cells with a recombinant adenovirus and the additive effects of CD/TK fusion gene/GCV/5-FC treatment, compared with single-gene therapy with the TK or CD gene. Therefore, our data suggest that the direct administration of a double-suicide gene/prodrug therapy has great potential in the treatment of brain tumors.