Transfer of cDNA for the herpes simplex virus thymidine kinase HSV-TK) into tumor cells allows activation of ganciclovir (GCV) to a cytotoxic nucleotide. This approach is novel in that, not only are cells that express HSV-TK killed by treatment with GCV, but neighboring, non-HSV-TK-expressing cells are also killed. This phenomenon, called the bystander effect, has produced some complete regressions of tumors in animals and is now in clinical trials. Recent studies in the applicant's laboratory have shown that GCV induced a greater than 4-log cell kill in glioblastoma and colon carcinoma cell lines compared to only a 1 - 2 log cell kill induced by other HSV-TK substrates such as thymine arabinoside (araT). The applicant has recently observed that another HSV-TK substrate, D- carbocyclic-2'-deoxyguanosine (CdG), induces multi-log kill similar to that observed with GCV. The data suggest that GCV is unique in that it weakly inhibits DNA synthesis, allowing cells to progress through the cell cycle when GCV nucleotide levels are high, resulting in massive apoptotic cell death. Furthermore, we demonstrate that hydroxyurea enhances cytotoxicity with GCV additively in HSV-TK cells and synergistically in bystander cells. Although other investigators have implicated gap junctional intercellular communication (GJIC) as mediating bystander killing with GCV, our studies show that cells deficient in GJIC also exhibit significant bystander killing in the presence of HSV-TK cells and GCV. Because of the potential of this approach for significant antitumor activity, the studies proposed here will elucidate the mechanism of multi-log cytotoxicity and bystander killing for GCV.
In Specific Aim 1, the mechanism of cytotoxicity will be determined for GCV and CdG and compared to the less toxic araT using cells which all express HSV-TK. These experiments will focus on the uptake and metabolism of GCV, and characterize its effects on DNA synthesis and cell cycle progression.
Specific Aim 2 will evaluate the mechanism of cytotoxicity in bystander cells and elucidate the novel mechanism of GCV nucleotide transfer in GJIC-deficient cells.
Specific Aim 3 will elucidate the mechanism by which hydroxyurea enhances GCV cytotoxicity and evaluate its therapeutic potential in a nude mouse human tumor model.
Specific Aim 4 will evaluate the prolonged apoptotic response observed with GCV and its inhibition by bcl-2 or bcl-xl. These studies will define the critical determinants of the cytotoxic and bystander effects with GCV, and the results will form the basis for improved clinical implementation of this novel approach to cancer chemotherapy.
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