Activation of mismatch repair protects cells against ganciclovir cytotoxicity through decreasing errors in DNA replication

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4038 Suicide gene therapy with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) is notable for producing multi-log cytotoxicity in a unique pattern of delayed cell death. This cytotoxicity can be enhanced by the addition of hydroxyurea (HU), a ribonucleotide reductase inhibitor that decreases dNTP pools, which combined with GCV produces additive or synergistic cytotoxicity in many tumor cell types. Because the HU-mediated imbalance in dNTP pools activates mismatch repair (MMR), we evaluated the role of MMR in GCV + HU cytotoxicity. Using isogenic HCT116TK (HSV-TK-expressing) colon carcinoma cells that are deficient or proficient in MMR, cell survival studies demonstrated greater GCV sensitivity in the MMR- compared to the MMR+ cells, primarily at high concentrations. The combination of GCV and HU exhibited additive cytotoxicity in MMR- cells but resulted in antagonism in the MMR+ cells. To understand the molecular basis for the sparing of GCV cytotoxicity in MMR+ cells, we measured the nature and frequency of mutations following exposure to GCV and/or HU using a shuttle vector mutation assay. There was no significant difference in the basal mutation frequency of untreated cells between the HCT116TK MMR+ and MMR- cell lines (0.062 ± 0.032% and 0.116 ± 0.0049%, respectively; p=0.116). A 24 hr incubation with GCV resulted in similar mutation frequencies in both the MMR+ and MMR- cell lines (0.16 ± 0.018% and 0.16 ± 0.014, respectively) that were not different from controls. Treatment with HU resulted in a significant increase in mutation frequency in the MMR- cells (0.307 ± 0.016%; p=0.0004), but not in the MMR+ cells (0.0320 ± 0.017%; p=0.495) compared to untreated controls. Interestingly, the predominance of a unique transversion mutation (C->A) was observed following incubation with GCV alone in both cell lines, whereas HU induced single base substitutions in which no single mutation predominated. The combined treatment with GCV and HU produced a further increase in mutation frequency in the MMR- cell line (0.379 ± 0.030%) and an increase in HU-associated mutations. However, in the MMR+ cell line, the GCV + HU resulted in a decrease in the mutation frequency (0.0784 ± 0.042 %), although the unique C->A mutation still predominated. These data demonstrated that the unique GCV-associated mutation was neither detected nor repaired by MMR. In addition, the data suggest that the additive cytotoxicity with GCV and HU in the MMR- cells may be due to an increase in errors in DNA replication. However, in the MMR+ cell line, the addition of HU activates MMR which decreases replication errors, resulting in antagonistic cytotoxicity. Taken together, these results demonstrate that activation of MMR protects cells against GCV cytotoxicity.