Decitabine (5-aza-2acute;-deoxycytidine, aza-dCyd) is an anticancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 and dUTPase are two "house-cleaning" nucleotidohydrolases involved in the elimination of non-canonical nucleotides. Here we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase thus suggesting their contribution to the cellular response to this anticancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP, an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of aza-dCyd producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyzes the triphosphate form of decitabine with similar kinetic efficiency than its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy
