807. Addition of HU to GCV Induces Bystander Cytotoxicity in Co-Cultures of HSVTK-Expressing and Non-Expressing HeLa Cells

Abstract

Expression of the herpes simplex virus type I thymidine kinase (HSVTK) gene in tumor cells sensitizes them to the antiviral agent ganciclovir (GCV). Incubation with GCV results not only in cytotoxicity from cells that express the transgene, but neighboring bystander cells as well. Bystander cytotoxicity has been attributed to the transfer of phosphorylated GCV metabolites from HSVTK-expressing to non-expressing cells through protein channels known as gap junctions. We have previously reported good bystander cytotoxicity in the U251 glioblastoma and SW620 colon carcinoma cell lines, which have 84% and 39% gap junction intercellular communication (GJIC), respectively. However, in the HeLa cell line (23% GJIC), no bystander cytotoxicity resulted from incubating a co-culture of HSVTK-expressing and bystander HeLa cells with GCV. Further analysis revealed that lack of bystander cytotoxicity in these cells was not due to an inability to transfer phosphorylated GCV, but resulted from a low level of transfer along with a rapid half-life of GCVTP in bystander cells (3.1 hrs) when compared to the HSVTK-expressing cells (12.3 hrs). We hypothesized that reduction of the endogenous competitor for GCVTP incorporation into DNA, dGTP, would increase levels of GCV incorporation into DNA resulting in the induction of bystander cytotoxicity. To that end, the effect of hydroxyurea (HU), a ribonucleotide reductase inhibitor, on GCV cytotoxicity in cultures of 100% HSVTK-expressing HeLa cells and co-cultures of HSVTK-expressing and bystander HeLa cells was examined. Isobologram analysis of the interaction between GCV and HU demonstrated an additive-antagonistic effect on 100% cultures of HSVTK-expressing HeLa cells, while exhibiting a synergistic effect on the HSVTK-expressing and bystander cells in a co-culture. To determine the mechanism for the synergistic cytotoxicity demonstrated in each of the populations from the co-culture when incubated with GCV and HU, GCVTP and dNTP pools were quantified and the results were compared to 100% cultures of HSVTK-expressing HeLa cells. The results demonstrated a significant increase in the GCVTP:dGTP values in cultures of 100% HSVTK-expressing cells (>2.5 fold) and in co-cultures of HSVTK-expressing cells (>1.4 fold) and bystander cells (>1.8 fold) at 4 hours. However, at 12 hours the GCVTP:dGTP value for 100% cultures of HSVTK-expressing HeLa cells was at control levels (1.3 0.5 fold) while the values for HSVTK-expressing cells (>2.7 fold) and bystander cells (>4.9 fold) in co-culture were still significantly higher. These data suggest that the prolonged increase in the GCVTP:dGTP value in co-cultures incubated with GCV and HU leads to an increase in the levels of GCVTP incorporated into DNA resulting in synergistic cytotoxicity.