XRCC1 down-regulation in human cells leads to DNA-damaging agent hypersensitivity, elevated sister chromatid exchange, and reduced survival of BRCA2 mutant cells.
Previous studies using rodent cells indicate that a deficiency in XRCC1 results in reduced single-strand break repair, increased sensitivity to DNA-damaging agents, and elevated levels of sister chromatid exchange (SCE). Epidemiological studies have suggested an association of certain human XRCC1 polymorphisms with genetic instability and cancer susceptibility. However, investigations on the molecular functions of XRCC1 in human cells are limited. To determine the contributions of this nonenzymatic scaffold protein, we suppressed XRCC1 levels in several human cell lines using small interfering RNA (siRNA) technology. We report that XRCC1 down-regulation in HeLa cells leads to a concomitant decrease in the DNA ligase 3 protein level and an impaired nick ligation capacity. In addition, depletion of XRCC1 resulted in a significantly increased sensitivity to the alkylating agent methyl methanesulfonate and the thymidine base analog 5-hydroxymethyl-2'-deoxyuridine, a slightly increased sensitivity to ethyl methanesulfonate and 1,3-bis(2-chloroethyl)-1-nitrosourea, and no change in the response to camptothecin. We also discovered that a 70-80% reduction in XRCC1 protein leads to an elevated level of SCE in both HeLa cells and normal human fibroblasts, but does not affect chromosome aberrations in the diploid fibroblasts. Last, XRCC1 siRNA transfection led to an approximately 40% decrease in the survival of BRCA2-deficient cells, supporting a model whereby the accumulation of unrepaired SSBs leads to the accumulation of cytotoxic DNA double strand breaks following replication fork collapse in cells defective in homologous recombination.
Fan
Jinshui
J
Laboratory of Molecular Gerontology, National Institute on Aging, Baltimore, Maryland, USA.
Wilson
Paul F
PF
Wong
Heng-Kuan
HK
Urbin
Salustra S
SS
Thompson
Larry H
LH
Wilson
David M
DM
D000818:Animals; D024682:BRCA2 Protein / Q000378:metabolism*; D016466:CHO Cells; D002457:Cell Extracts; D002470:Cell Survival / Q000187:drug effects; D043171:Chromosomal Instability / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics*; D015536:Down-Regulation; D006367:HeLa Cells; D006801:Humans; D008741:Methyl Methanesulfonate / Q000633:toxicity; D015162:Micronucleus Tests; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation / Q000235:genetics*; D009369:Neoplasms / Q000473:pathology; D034741:RNA, Small Interfering / Q000378:metabolism; D012854:Sister Chromatid Exchange / Q000187:drug effects*; D014162:Transfection; D000076105:X-ray Repair Cross Complementing Protein 1
D024682:BRCA2 Protein; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D009153:Mutagens; D034741:RNA, Small Interfering; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D008741:Methyl Methanesulfonate