The nucleotidohydrolases DCTPP 1 and dUTPase are involved in the cellular response to the DNA-demethylating drug decitabine

Decitabine (5-aza-2’-deoxycytidine, aza-dCyd) is a potent DNA-demethylating and genotoxic drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukemia. 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 aza-dCyd 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-dCMP, an alternative cytotoxic mechanism for decitabine involving 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 nucleosides triphosphate 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 decitabineinduced global DNA demethylation. We propose that DCTPP1 and dUTPase nucleotidohydrolases are key factors in decitabine mode of action with potential value as predictive markers of the clinical response or as enzymatic targets to improve decitabine-based chemotherapy.