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

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.