5-chloro-2 '-deosyuridine cytotoxicity results from base excision repair of uracil subsequent to thymidylate synthase inhibition

The lack of a phenotypic alteration of 5-hydroxymethyluracil (hmUra) DNA gl ycosylase (hmUDG) deficient Chinese hamster V79mut1 cells exposed to DNA-da maging agents known to produce hmUra has raised the question whether there might be DNA substrates other than hmUra for hmUDG. Based on the structural similarity between 5-chlorouracil (ClUra) and hmUra and the observations t hat 5-chloro-2'-deoxyuridine (CldUrd) induces base excision repair (BER) ev ents, we asked whether hmUDG or some other DNA BER enzyme is responsible fo r the removal of ClUra from DNA. An in vivo flow cytometry assay with FITC- anti-BrdUrd (which cross-reacts with CldUrd) showed that exogenous CldUrd i s incorporated into DNA. However, both in vivo and in vitro experiments ind icated that ClUra is not excised from DNA by hmUDG or other DNA glycosylase activities. The absence of removal of ClUra by hmUDG raised the question w hether DNA strand breaks occurred subsequent to thymidylate synthase inhibi tion, leading to deoxyuridine incorporation, followed by cleavage of uracil from DNA by uracil DNA glycosylase (UDG). An in vivo thymidylate synthase activity assay in V79 cells demonstrated that CldUrd treatment inhibits thy midylate synthase as effectively as 5-fluoro-2'-deoxyuridine (FdUrd) treatm ent. Uracil, a known UDG inhibitor, partially reverses the cytotoxic effect s of CldUrd on V79 cells, thus confirming that CldUrd induced cytotoxicity is a result of UDG activity. Our results demonstrated that while CldUrd is not directly repaired from DNA, its cytotoxicity is directly due to the UDG removing uracil subsequent to inhibition of thymidylate synthase by CldUMP . (C) 2000 Elsevier Science B.V. All rights reserved.