title: Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed.
abstract: Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG(+/+) and UNG(-/-) cells. We report that despite identical growth rates and DSB repair capacities, UNG(-/-) cells accumulated significantly greater uracil and DSBs compared with UNG(+/+) cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG(+/+) cells. Furthermore, DSBs in UNG(+/+) and UNG(-/-) cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG(-/-) cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG(+/+) and UNG(-/-) cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG(-/-) but not UNG(+/+) cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG(-/-) cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death.
authors: Department of Pathology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
authors: L D
authors: LD
authors: Weeks
authors: Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
authors: G E
authors: GE
authors: Zentner
authors: 1] Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA [2] Case Comprehensive Cancer Center, Case Western Reserve University, 2103 Cornell Road, Cleveland, OH 44106, USA.
authors: P C
authors: PC
authors: Scacheri
authors: 1] Department of Pathology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA [2] Case Comprehensive Cancer Center, Case Western Reserve University, 2103 Cornell Road, Cleveland, OH 44106, USA [3] Department of Medicine, Division of Hematology/Oncology, Case Western Reserve University School of Medicine, 2103 Cornell Road, Cleveland, OH 44106, USA.
authors: S L
authors: SL
authors: Gerson
mesh_terms: D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004260:DNA Repair; D005971:Glutamates / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D009369:Neoplasms / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D000068437:Pemetrexed; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase / Q000172:deficiency* / Q000235:genetics
chemical_list: D005971:Glutamates; D000068437:Pemetrexed; D014498:Uracil; D006147:Guanine; D051981:Uracil-DNA Glycosidase