title: Inhibition of uracil DNA glycosylase sensitizes cancer cells to 5-fluorodeoxyuridine through replication fork collapse-induced DNA damage.
abstract: 5-fluorodeoxyuridine (5-FdU, floxuridine) is active against multiple cancers through the inhibition of thymidylate synthase, which consequently introduces uracil and 5-FU incorporation into the genome. Uracil DNA glycosylase (UDG) is one of the main enzymes responsible for the removal of uracil and 5-FU. However, how exactly UDG mediates cellular sensitivity to 5-FdU, and if so whether it is through its ability to remove uracil and 5-FU have not been well characterized. In this study, we report that UDG depletion led to incorporation of uracil and 5-FU in DNA following 5-FdU treatment and significantly enhanced 5-FdU's cytotoxicity in cancer cell lines. Co-treatment, but not post-treatment with thymidine prevented cell death of UDG depleted cells by 5-FdU, indicating that the enhanced cytotoxicity is due to the retention of uracil and 5-FU in genomic DNA in the absence of UDG. Furthermore, UDG depleted cells were arrested at late G1 and early S phase by 5-FdU, followed by accumulation of sub-G1 population indicating cell death. Mechanistically, 5-FdU dramatically reduced DNA replication speed in UDG depleted cells. UDG depletion also greatly enhanced DNA damage as shown by γH2AX foci formation. Notably, the increased γH2AX foci formation was not suppressed by caspase inhibitor treatment, suggesting that DNA damage precedes cell death induced by 5-FdU. Together, these data provide novel mechanistic insights into the roles of UDG in DNA replication, damage repair, and cell death in response to 5-FdU and suggest that UDG is a target for improving the anticancer effect of this agent.
authors: Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA.
authors: Yan
authors: Y
authors: Yan
authors: Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA.
authors: Xiangzi
authors: X
authors: Han
authors: Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
authors: Yulan
authors: Y
authors: Qing
authors: Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Chemistry, and Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA.
authors: Allison G
authors: AG
authors: Condie
authors: Department of Chemistry, Cleveland State University, Cleveland, OH, USA.
authors: Shashank
authors: S
authors: Gorityala
authors: Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
authors: Shuming
authors: S
authors: Yang
authors: Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.|Department of Chemistry, Cleveland State University, Cleveland, OH, USA.
authors: Yan
authors: Y
authors: Xu
authors: Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA.
authors: Youwei
authors: Y
authors: Zhang
authors: Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA.
authors: Stanton L
authors: SL
authors: Gerson
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D004249:DNA Damage; D004261:DNA Replication; D004351:Drug Resistance; D005467:Floxuridine / Q000494:pharmacology / Q000627:therapeutic use*; D006657:Histones / Q000378:metabolism; D006801:Humans; D009369:Neoplasms / Q000188:drug therapy*; D034741:RNA, Small Interfering / Q000235:genetics; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; C468783:H2AX protein, human; D006657:Histones; D034741:RNA, Small Interfering; D005467:Floxuridine; D014498:Uracil; D051981:Uracil-DNA Glycosidase
keywords: 5-fluorodeoxyuridine; base excision repair; colon cancer; double strand breaks; uracil DNA glycosylase