Glioma Cells Transduced with anEscherichia coliCD/HSV-1 TK Fusion Gene Exhibit Enhanced Metabolic Suicide and Radiosensitivity

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Abstract
To ascertain whether concomitant expression of Escherichia coli cytosine deaminase (CD) and herpes simplex virus type-1 thymidine kinase (HSV-1 TK) could mediate greater levels of cytotoxicity beyond that observed with either suicide gene alone, 9L gliosarcoma cells were transduced with a retrovirus encoding a CD/HSV-1 TK fusion gene. The resultant CD/HSV-1 TK fusion protein (CDglyTK) was found to be bifunctional via CD and HSV-1 TK enzymatic assays, and conferred upon cells prodrug sensitivities equivalent to or better than that observed for each enzyme independently (ganciclovir [GCV] and bromovinyldeoxyuridine [BVdU] for HSV-1 TK and 5-fluorocytosine [5-FC] for CD). Simultaneous treatment of CDglyTK-expressing cells with prodrugs specific for HSV-1 TK and CD (GCV/5-FC or BVdU/5-FC) resulted in slight synergistic toxicity, two- to three-fold greater than that expected if the cytotoxic effects of each prodrug were purely additive. More importantly, co-treatment with HSV-1 TK- and CD-specific prodrugs was found to increase greatly the radiosensitivity of CDglyTK-expressing cells. Sensitivity enhancement ratios of 2.44 (GCV/5-FC) and 3.90 (BVdU/5-FC) were achieved. The results suggest that double suicide gene therapy, using a bifunctional CD/HSV-1 TK fusion gene, coupled with radiotherapy may provide a highly efficient means of selectively treating cancer. Herplex simplex virus type-1 thymidine kinase (HSV-1 TK) and Escherichia coli cytosine deaminase (CD) are two of the best-characterized suicide genes. Cells genetically modified to express these genes acquire sensitivity to normally innocuous prodrugs because these nonmammalian enzymes convert the nontoxic prodrug into a cytotoxic metabolite. Both HSV-1 TK and CD suicide gene therapy have been employed independently to eliminate neoplastic cells both in vitro and in vivo. As a means of achieving significantly greater levels of targeted cytotoxicity, HSV-1 TK and CD were co-expressed as a single polypeptide within the same cell. The CD/HSV-1 TK fusion protein was found to be bifunctional, sensitizing transduced 9L gliosarcoma cells to both CD and HSV-1 TK-specific prodrugs. Co-administration of both classes of prodrugs achieved mild synergistic cytotoxicity. More importantly, double suicide gene therapy significantly radiosensitized tumor cells, resulting in even greater cytotoxicity.