Abstract
2098
Engineering tumor cells to express the herpes simplex virus type 1 thymidine kinase (HSV-TK) allows the normally innocuous antiviral, GCV, to be phosphorylated to its triphosphate, which elicits cytotoxicity following its incorporation into DNA. Previously we have reported that GCV cytotoxicity can be enhanced by the addition of hydroxyurea (HU), a ribonucleotide reductase inhibitor that produces an imbalance in dNTP pools, resulting in additive cytotoxicity in HSV-TK-expressing cells and synergistic cytotoxicity in bystander cells in a wide variety of solid tumor cell lines. Because HU causes an imbalance in dNTP pools and activates the mismatch repair pathway (MMR), this study aimed to determine if MMR affects sensitivity to GCV. In these studies we used two HCT116 colon carcinoma cell lines which are matched for MMR proficiency and deficiency. The parental HCT116 0-1 are MMR deficient due to the mutation of an essential gene for MMR, hMLH1, whereas HCT116 1-2 stably express MLH1 and are MMR proficient. We have generated stably expressing HSV-TK clones of these two cell lines. Cell survival studies performed after a 24 hr incubation with GCV demonstrated that HCT116 0-1TK cells were significantly more sensitive to GCV than HCT116 1-2TK cells (IC50 = 0.1 μM and 0.2 μM, IC90 = 0.7 μM and 1.3 μM, respectively; p<0.05). To verify that the differences in GCV cytotoxicity observed in the HSV-TK clones were not due to selection of clones with variable expression of TK, we treated parental HCT116 0-1 and 1-2 (non-HSV-TK expressing) with high concentrations of GCV, assuming GCV metabolism by endogenous kinases would be similar at the high concentrations in each cell line. The MMR deficient HCT116 0-1 were significantly more sensitive to GCV than the MMR proficient HCT116 1-2 (IC50= 124 μM and 465 μM, respectively; p<0.01) validating the results observed in the HSV-TK expressing cells. To further evaluate the effect of MMR proficiency on GCV cytotoxicity, siRNA was utilized to decrease expression of hMLH1 in HSV-TK expressing U251 human glioblastoma and SW480 human colon carcinoma cells, both of which are considered MMR proficient cell lines. Suppression of hMLH1 resulted in increased sensitivity to GCV in both cell lines. Taken together, these data suggest that MMR may play a role in sensing or repairing GCV incorporation into DNA.
- American Association for Cancer Research