Differential regulation of distinct nuclear and mitochondrial isoforms of human dutpase

dUTPase is an essential enzyme which hydrolyzes dUTP to dUMP and pyrophosphate. This reaction is thought to occur primarily to reduce dUTP pools and prevent uracil incorporation into DNA during replication and repair. Chemotherapeutic agents which affect thymidylate metabolism are thought to function by promoting mi si n corporation of uracil into DNA followed by uracil base excision repair-mediated DNA degradation and cell death. Recent studies suggest that elevated levels of dUTPase in certain cancer cell lines cause resistance to thymidylate synthase (TS) inhibition. These studies suggest that levels of dUTPase may be critical in determining resistance to TS inhibition in certain cancers. We have previously identified distinct nuclear (DUT-N) and mitochondria! (DUT-M) isoforms of dUTPase in humans. Here we investigate the patterns of dUTPase isoform expression in cell culture and provide the basis for further investigation of dUTPase expression in normal tissues by immunohistochemistry. Western blot analysis of stimulated peripheral blood lymphocytes reveals that DUT-N protein levels are induced over 1000 fold. This increase in DUT-N protein coincides with increased cellular dUTPase activity and the onset of DNA replication as measured by s#-thymidine incorporation. In contrast, DUT-M protein levels remained constant throughout the cell cycle, suggesting that the expression of these isoforms are goverened by independent mechanisms. Preliminary immunohistochemical localization of dUTPase in normal human tissues reveals a strong correlation between nuclear dUTPase expression and highly proliferative tissue types.