5-Fluorouracil (5-FU) and its metabolite 5-fluorodeoxyuridine (FdUrd, floxuridine) are chemotherapy agents that are converted to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP). FdUMP inhibits thymidylate synthase and causes the accumulation of uracil in the genome, whereas FdUTP is incorporated by DNA polymerases as 5-FU in the genome; however, it remains unclear how either genomically incorporated U or 5-FU contributes to killing. We show that depletion of the uracil DNA glycosylase (UNG) sensitizes tumor cells to FdUrd. Furthermore, we show that UNG depletion does not sensitize cells to the thymidylate synthase inhibitor (raltitrexed), which induces uracil but not 5-FU accumulation, thus indicating that genomically incorporated 5-FU plays a major role in the antineoplastic effects of FdUrd. We also show that 5-FU metabolites do not block the first round of DNA synthesis but instead arrest cells at the G1/S border when cells again attempt replication and activate homologous recombination (HR). This arrest is not due to 5-FU lesions blocking DNA polymerase d but instead depends, in part, on the thymine DNA glycosylase. Consistent with the activation of HR repair, disruption of HR sensitized cells to FdUrd, especially when UNG was disabled. These results show that 5-FU lesions that escape UNG repair activate HR, which promotes cell survival.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health [Grants GM72474, R01-GM031973, and R01-ES014737], the Mayo Clinic Ovarian Cancer SPORE [P50 CA136393], a grant from the Fred C. and Katherine Andersen Foundation, and a Mayo Clinic Eagles Pilot Grant. Authorship Contributions Participated in research design: Huehls, Huntoon, Joshi, Baehr, Wagner, Wang, Lee, Karnitz. Conducted experiments: Huehls, Huntoon, Joshi, Baehr, Wagner, Wang, Karnitz. Contributed new reagents or analytic tools: Huehls, Huntoon, Joshi, Baehr, Wagner, Wang, Karnitz. Performed data analysis: Huehls, Huntoon, Joshi, Baehr, Wagner, Wang, Lee, Karnitz. Wrote or contributed to the writing of the manuscript: Huehls, Karnitz.