Small interfering RNA-mediated suppression of dUTPase sensitizes cancer cell lines to thymidylate synthase inhibition

Uracil misincorporation into DNA and its associated misrepair have been implicated as contributing components of cytotoxicity resulting from treatment with thymidylate synthase inhibitors. dUTPase, which eliminates dUTP from the DNA biosynthetic pathway, opposes uracil misincorporation; therefore, elevation of this enzyme in cancer cells may contribute to drug resistance. To validate the potential of dUTPase as a target for drug development, we used small interfering RNA directed against this enzyme and determined the effects of decreasing levels of dUTPase on sensitivity to the thymidylate synthase (TS) inhibitor fluorodeoxyuridine (FUdR) in human cancer cell lines. Suppression of dUTPase in SW620 and MCF-7 cells resulted in a significant enhancement in dUTP pool expansion after TS inhibition. This shift in nucleotide pool levels was accompanied by a significant decrease in the FUdR IC(50)(72h) ( approximately 75-fold for SW620 cells and approximately 6-fold for MCF-7 cells), a decline in clonogenic survival, and enhanced DNA double strand break formation. In contrast, depletion of dUTPase in HT29 cells did not substantially affect chemosensitivity or the amount of DNA damage incurred despite a 3-fold increase in dUTP pool expansion. This observation implies that the cytotoxic impact of uracil misincorporation may reach a saturation point in HT29 cells and that a further increase in dUTP levels has no additive effect. Together, these results suggest that uracil misincorporation is a potent determinant of cytotoxicity to TS inhibition and indicate that partial inhibition of dUTPase is a viable therapeutic approach to enhance the efficacy of broadly used chemotherapeutic agents that inhibit TS.