title: ATR-Chk1 signaling pathway and homologous recombinational repair protect cells from 5-fluorouracil cytotoxicity.
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.
authors.affiliation: Department of Molecular Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Fujinaka
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Matsuoka
authors.forename: Makoto
authors.initials: M
authors.lastname: Iimori
authors.forename: Munkhbold
authors.initials: M
authors.lastname: Tuul
authors.forename: Ryo
authors.initials: R
authors.lastname: Sakasai
authors.forename: Keiji
authors.initials: K
authors.lastname: Yoshinaga
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Saeki
authors.forename: Masaru
authors.initials: M
authors.lastname: Morita
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kakeji
authors.forename: David A
authors.initials: DA
authors.lastname: Gillespie
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Minoru
authors.initials: M
authors.lastname: Takata
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kitao
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Maehara
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D002645:Chickens; D019610:Cytoprotection / Q000187:drug effects*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism; D005472:Fluorouracil / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism; D016393:Lymphoma, B-Cell / Q000201:enzymology / Q000473:pathology; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D059767:Recombinational DNA Repair / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects*; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors
chemical_list: D018797:Cell Cycle Proteins; D052236:Fanconi Anemia Complementation Group D2 Protein; D006657:Histones; C069930:rad9 protein; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine; D005472:Fluorouracil