ATR-Chk1 signaling pathway and homologous recombinational repair protect cells from 5-fluorouracil cytotoxicity

Yoshihiko Fujinaka, Kazuaki Matsuoka, Makoto Iimori, Munkhbold Tuul, Ryo Sakasai, Keiji Yoshinaga, Hiroshi Saeki, Masaru Morita, Yoshihiro Kakeji, David A. Gillespie, Ken ichi Yamamoto, Minoru Takata, Hiroyuki Kitao, Yoshihiko Maehara

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Abstract

5-Fluorouracil (5-FU) has long been a mainstay antimetabolite chemotherapeutic drug for the treatment of major solid tumors, particularly colorectal cancer. 5-FU is processed intracellularly to yield active metabolites that compromise RNA and DNA metabolism. However, the mechanisms responsible for its cytotoxicity are not fully understood. From the phenotypic analysis of mutant chicken B lymphoma DT40 cells, we found that homologous recombinational repair (HRR), involving Rad54 and BRCA2, and the ATR-Chk1 signaling pathway, involving Rad9 and Rad17, significantly contribute to 5-FU tolerance. 5-FU induced γH2AX nuclear foci, which were colocalized with the key HRR factor Rad51, but not with DNA double-strand breaks (DSBs), in a dose-dependent manner as cells accumulated in the S phase. Inhibition of Chk1 kinase by UCN-01 increased 5-FU-induced γH2AX and enhanced 5-FU cytotoxicity not only in wild-type cells but also in Rad54- or BRCA2-deficient cells, suggesting that HRR and Chk1 kinase have non-overlapping roles in 5-FU tolerance. 5-FU-induced Chk1 phosphorylation was significantly impaired in Rad9- or Rad17-deficient cells, and severe γH2AX nuclear foci and DSBs were formed, which was followed by apoptosis. Finally, inhibition of Chk1 kinase by UCN-01 increased 5-FU-induced γH2AX nuclear foci and enhanced 5-FU cytotoxicity in Rad9- or Rad17-deficient cells. These results suggest that Rad9- and Rad17-independent activation of the ATR-Chk1 signaling pathway also significantly contributes to 5-FU tolerance.

Original languageEnglish
Pages (from-to)247-258
Number of pages12
JournalDNA Repair
Volume11
Issue number3
DOIs
Publication statusPublished - Mar 1 2012

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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