Improving HSV -TK /GCV gene therapy through pharmacological modulation of the bystander effect.

Expression of the herpes simplex virus type 1 thymidine kinase (HSV-TK) in tumor cells confers sensitivity to the antiviral drug ganciclovir (GCV). Phosphorylation of GCV by HSV-TK to its monophosphate derivative, and further phosphorylation by endogenous enzymes to the cytotoxic triphosphate form allows for competition with endogenous dGTP for incorporation into DNA, an event correlated with cytotoxicity. Due to the inadequacy of current gene transfer techniques, this form of therapy relies heavily upon and benefits from a phenomenon known as the bystander effect. This dissertation examines the bystander effect and the factors involved in its modulation. Bystander cytotoxicity, or the ability of HSV-TK-expressing cells to cause GCV-mediated cytotoxicity in neighboring non-expressing cells, has been attributed to the transfer of phosphorylated GCV metabolites through gap junctions. However, with bystander cytotoxicity exhibited by cells with negligible gap junction intercellular communication (GJIC), we examined the role gap junctions have on bystander cytotoxicity. Through use of a double dye flow cytometry technique, HeLa cells, previously demonstrated not to have GJIC, exhibited low, yet detectable levels of communication. Further examination demonstrated that lack of bystander effect was not due to inability to transfer phosphorylated GCV metabolites, but low-level transfer along with a rapid half-life of GCV triphosphate accounted for the lack of cytotoxicity. In HeLa cells, the presence of HSV-TK-expressing cells does not elicit bystander cytotoxicity despite transfer of phosphorylated GCV metabolites in amounts sufficient to decrease cytotoxicity to HSV-TK-expressing cells. We hypothesized that introduction of a mechanism based inhibitor designed to decrease endogenous dGTP pools would induce a bystander effect. Not only did the combination of hydroxyurea (HU), a ribonucleotide reductase inhibitor, and GCV result in the induction of bystander effect, but it increased cytotoxicity of HSV-TK-expressing cells to levels exhibited by cultures in which all cells express the transgene. The SW480 cell line exhibited only additive bystander cytotoxicity when incubated with HU and GCV due to an inability to sustain a decrease in dGTP. Further studies demonstrated that HU inhibited ribonucleotide reductase sufficiently, but dGTP was repleted presumably through salvage pathways. These studies suggest that ribonucleotide reductase inhibitors are more effective at enhancing cytotoxicity with HSV-TK/GCV than GJIC modulation and may lead to improved clinical efficacy.