Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6 glioma.
OBJECTIVE
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.


METHODS
Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the beta-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-deltaE1 (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, 1x10(5) cells were stereotactically injected into the right caudate-putamen of female Wistar rat brains. At 3 days after implantation, 1x10(8) plaque-forming units of ad-CD/TK or ad-deltaE1 (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.


RESULTS
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 microg/ml or GCV at concentrations greater than 0.3 microg/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.


CONCLUSIONS
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.
Chang
J W
JW
Department of Neurosurgery, Brain Research Institute, Yonsei University, College of Medicine, Seoul, Korea.
Lee
H
H
Kim
E
E
Lee
Y
Y
Chung
S S
SS
Kim
J H
JH
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000963:Antimetabolites / Q000627:therapeutic use; D000998:Antiviral Agents / Q000627:therapeutic use; D019677:Artificial Gene Fusion; D001932:Brain Neoplasms / Q000378:metabolism / Q000628:therapy*; D003131:Combined Modality Therapy; D043525:Cytosine Deaminase; D005240:Feasibility Studies; D005260:Female; D005437:Flucytosine / Q000627:therapeutic use; D015774:Ganciclovir / Q000627:therapeutic use; D005910:Glioma / Q000378:metabolism / Q000628:therapy*; D009368:Neoplasm Transplantation; D009700:Nucleoside Deaminases / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics*; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D000963:Antimetabolites; D000998:Antiviral Agents; D011993:Recombinant Fusion Proteins; D005437:Flucytosine; D013937:Thymidine Kinase; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D015774:Ganciclovir