title: Methotrexate-mediated inhibition of RAD51 expression and homologous recombination in cancer cells.
abstract: BACKGROUND
  Methotrexate is an inhibitor of folic acid metabolism. Homologous recombination is one of the most important ways to repair double-stranded breaks in DNA and influence the radio- and chemosensitivity of tumor cells. But the relationship between methotrexate and homologous recombination repair has not been elucidated.
  
  
  METHODS
  Induction of double-strand breaks by methotrexate in HOS cells is assessed by the neutral comet assay. Inhibition of subnuclear repair foci by methotrexate is measured by immunofluorescence. Western blot and quantitative real-time PCR are conducted to detect whether methotrexate affects the expression level of genes involved in homologous recombination. In addition, we used a pCMV3xnls-I-SceI construct to determine whether methotrexate directly inhibits the process of homologous recombinational repair in cells, and the sensitivity to methotrexate in the Ku80-deficient cells is detected using clonogenic survival assays.
  
  
  RESULTS
  The result showed that methotrexate can regulate the repair of DNA double-strand breaks after radiation exposure, and methotrexate inhibition caused the complete inhibition of subnuclear repair foci in response to ionizing radiation. Mechanistic investigation revealed that methotrexate led to a significant reduction in the transcription of RAD51 genes. Treatment with methotrexate resulted in a decreased ability to perform homology-directed repair of I-SceI-induced chromosome breaks. In addition, enhancement of cell death was observed in Ku mutant cells compared to wild-type cells.
  
  
  CONCLUSIONS
  These results demonstrate that methotrexate can affect homologous recombination repair of DNA double-strand breaks by controlling the expression of homologous recombination-related genes and suppressing the proper assembly of homologous recombination-directed subnuclear foci.
authors.affiliation: Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin Key Laboratory of Molecular Nuclear Medicine, Tianjin, China.
authors.forename: Li-Qing
authors.initials: LQ
authors.lastname: Du
authors.forename: Xiao-Qing
authors.initials: XQ
authors.lastname: Du
authors.forename: Jian-Qiang
authors.initials: JQ
authors.lastname: Bai
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Qing-Shan
authors.initials: QS
authors.lastname: Yang
authors.forename: Xiao-Chun
authors.initials: XC
authors.lastname: Wang
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.forename: Fei-Yue
authors.initials: FY
authors.lastname: Fan
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D045744:Cell Line, Tumor; D059766:DNA End-Joining Repair / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D024522:Genes, BRCA2 / Q000187:drug effects; D059765:Homologous Recombination / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051135:Rad51 Recombinase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics; D059767:Recombinational DNA Repair / Q000187:drug effects / Q000235:genetics
chemical_list: D000964:Antimetabolites, Antineoplastic; C497194:RAD52 protein, human; D034741:RNA, Small Interfering; D051721:Rad52 DNA Repair and Recombination Protein; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D008727:Methotrexate