title: SMUG1 but not UNG DNA glycosylase contributes to the cellular response to recovery from 5-fluorouracil induced replication stress.
abstract: 5-Fluorouracil (5-FU) is a widely utilized cancer chemotherapeutic that causes DNA damage via two mechanisms. Its active metabolite inhibits thymidylate synthase, which deprives cells of TTP and causes the introduction of uracil in DNA. Also, 5-FU is directly incorporated into DNA. Both uracil and 5-FU in DNA are recognized by uracil-DNA glycosylases (UDGs), which initiate base excision repair. UNG and SMUG1 are the two human UDGs most likely to combat the genomic incorporation of uracil and 5-FU during replication. In this study, we examined the roles of UNG and SMUG1 in the initial cellular response to 5-FU and compared continuous exposure to a 24h exposure followed by incubation in drug-free media, which mimics what occurs clinically. Loss of UNG did not alter cellular sensitivity to 5-FU in two human cell lines, despite its predominant biochemical activity for uracil and 5-FU in DNA. Loss of SMUG1 corresponded with >2-fold increase in sensitivity to 5-FU, but only with a 24h treatment followed by recovery. There was no difference between SMUG1 proficient and depleted cells following continuous exposure. We observed that 5-FU treatment induced an enhanced S-phase arrest and CHK1 activation plus an increase in the formation of strand breaks and alkali-labile sites in all sublines. However, SMUG1-depleted cells showed a prolonged S-phase arrest, a transient increase in DNA double-strand breaks following 5-FU treatment and an altered phosphorylation of CHK1 following removal of drug. Collectively, the results suggest that SMUG1 has a role in the resumption of replication following 5-FU treatment.
authors.affiliation: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, United States.
authors.forename: Pratik
authors.initials: P
authors.lastname: Nagaria
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine & University of Pittsburgh Cancer Institute, Hillman Cancer Center, United States.
authors.forename: David
authors.initials: D
authors.lastname: Svilar
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine & University of Pittsburgh Cancer Institute, Hillman Cancer Center, United States.
authors.forename: Ashley R
authors.initials: AR
authors.lastname: Brown
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine & University of Pittsburgh Cancer Institute, Hillman Cancer Center, United States.
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Wang
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine & University of Pittsburgh Cancer Institute, Hillman Cancer Center, United States; Department of Human Genetics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15213, United States.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Sobol
authors.affiliation: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, United States. Electronic address: Wyatt@cop.sc.edu.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Wyatt
mesh_terms: D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects* / Q000235:genetics; D005472:Fluorouracil / Q000494:pharmacology*; D055785:Gene Knockdown Techniques; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000187:drug effects / Q000235:genetics; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D014498:Uracil; D004247:DNA; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C498095:SMUG1 protein, human; D051981:Uracil-DNA Glycosidase; D005472:Fluorouracil