Genomically Incorporated 5-Fluorouracil that Escapes UNG-Initiated Base Excision Repair Blocks DNA Replication and Activates Homologous Recombination.
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 δ 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.
Department of Molecular Pharmacology and Experimental Therapeutics (A.M.H., C.J.H., P.M.J., C.A.B., J.M.W., L.M.K.) and Division of Oncology Research (C.J.H., J.M.W., L.M.K), Department of Radiation Oncology (L.M.K.), Mayo Clinic, Rochester, Minnesota; and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York (X.W., M.Y.L.).
Amelia M
AM
Huehls
Department of Molecular Pharmacology and Experimental Therapeutics (A.M.H., C.J.H., P.M.J., C.A.B., J.M.W., L.M.K.) and Division of Oncology Research (C.J.H., J.M.W., L.M.K), Department of Radiation Oncology (L.M.K.), Mayo Clinic, Rochester, Minnesota; and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York (X.W., M.Y.L.).
Catherine J
CJ
Huntoon
Department of Molecular Pharmacology and Experimental Therapeutics (A.M.H., C.J.H., P.M.J., C.A.B., J.M.W., L.M.K.) and Division of Oncology Research (C.J.H., J.M.W., L.M.K), Department of Radiation Oncology (L.M.K.), Mayo Clinic, Rochester, Minnesota; and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York (X.W., M.Y.L.).
Poorval M
PM
Joshi
Department of Molecular Pharmacology and Experimental Therapeutics (A.M.H., C.J.H., P.M.J., C.A.B., J.M.W., L.M.K.) and Division of Oncology Research (C.J.H., J.M.W., L.M.K), Department of Radiation Oncology (L.M.K.), Mayo Clinic, Rochester, Minnesota; and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York (X.W., M.Y.L.).
Carly A
CA
Baehr
Department of Molecular Pharmacology and Experimental Therapeutics (A.M.H., C.J.H., P.M.J., C.A.B., J.M.W., L.M.K.) and Division of Oncology Research (C.J.H., J.M.W., L.M.K), Department of Radiation Oncology (L.M.K.), Mayo Clinic, Rochester, Minnesota; and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York (X.W., M.Y.L.).
Jill M
JM
Wagner
Department of Molecular Pharmacology and Experimental Therapeutics (A.M.H., C.J.H., P.M.J., C.A.B., J.M.W., L.M.K.) and Division of Oncology Research (C.J.H., J.M.W., L.M.K), Department of Radiation Oncology (L.M.K.), Mayo Clinic, Rochester, Minnesota; and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York (X.W., M.Y.L.).
Xiaoxiao
X
Wang
Department of Molecular Pharmacology and Experimental Therapeutics (A.M.H., C.J.H., P.M.J., C.A.B., J.M.W., L.M.K.) and Division of Oncology Research (C.J.H., J.M.W., L.M.K), Department of Radiation Oncology (L.M.K.), Mayo Clinic, Rochester, Minnesota; and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York (X.W., M.Y.L.).
Marietta Y
MY
Lee
Department of Molecular Pharmacology and Experimental Therapeutics (A.M.H., C.J.H., P.M.J., C.A.B., J.M.W., L.M.K.) and Division of Oncology Research (C.J.H., J.M.W., L.M.K), Department of Radiation Oncology (L.M.K.), Mayo Clinic, Rochester, Minnesota; and Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York (X.W., M.Y.L.) karnitz.larry@mayo.edu.
Larry M
LM
Karnitz
D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D005472:Fluorouracil / Q000378:metabolism* / Q000494:pharmacology; D019073:HT29 Cells; D059765:Homologous Recombination / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism*
D051981:Uracil-DNA Glycosidase; D005472:Fluorouracil