ATR–Chk1 signaling pathway and homologous recombinational repair protect cells from 5-fluorouracil cytotoxicity
Highlights
► DT40 mutants of Rad54, BRCA2, Rad9 and Rad17 exhibited 5-FU hypersensitivity. ► 5-FU induced γH2AX foci, but not double-strand breaks, in wild type cells. ► 5-FU-induced γH2AX foci colocalized with Rad51. ► Double-strand breaks and apoptosis were induced by 5-FU in Rad9 or Rad17 mutants. ► Chk1 inhibitor potentiated 5-FU cytotoxicity wild-type as well as these mutants.
Introduction
5-Fluorouracil (5-FU) is a fluoropyrimidine-type compound with marked anti-tumor effects [1]. For many years, 5-FU has been an important chemotherapeutic drug used in first-line treatment of a range of human cancers, particularly colorectal carcinoma [2]. Intracellularly, 5-FU is converted to several active metabolites, including fluorouridine triphosphate (FUTP), fluorodeoxyuridine triphosphate (FdUTP) and fluorodeoxyuridine monophosphate (FdUMP). These active metabolites compromise global RNA metabolism by incorporation of FUTP into RNA and DNA metabolism by FdUMP-mediated inhibition of thymidylate synthase (TS) and incorporation of FdUTP into DNA [2]. Inhibition of TS is achieved by the formation of a ternary covalent complex consisting of TS-FdUMP-5,10-methylenetetrahydrofolate. Once this complex is formed, cells are unable to synthesize dTMP from dUMP, and the cellular dUTP level increases at the expense of dTTP. The resulting dUTP/dTTP imbalance causes massive misincorporation of dUTP or FdUTP, particularly during DNA replication.
Although DNA damage is considered to be one of the main triggers of the tumor cell killing effects of 5-FU [3], [4], it is not fully understood how misincorporated dUTP or FdUTP are processed and contribute to cytotoxicity. Misincorporated FdUTP or dUTP are recognized, excised from DNA, and undergo base excision repair (BER) or mismatch repair (MMR) [5]. DNA strand breaks are generated as byproducts of the repair processes. Homologous recombinational repair (HRR) has been proposed to contribute to the repair of 5-FU-induced DNA damage, including double-strand breaks (DSBs) [3], but no direct evidence for this process has been obtained in vertebrate cells. In addition, 5-FU activates Chk1 kinase, the main mediator of the activation of cell cycle checkpoints and DNA repair in response to genotoxic stress [6], which is achieved via phosphorylation at Ser317 and Ser345 by ataxia telangiectasia-mutated and Rad3-related kinase (ATR) [7]. The critical role of Chk1 in 5-FU resistance has been reported in human cancer cell lines [8], the chicken DT40 cell line [9] and a mouse xenograft model [10]. Importantly, Chk1 elimination by siRNA in HeLa cells abolishes the S-phase checkpoint induced by 5-FU. This allows DNA synthesis to continue, leading to excessive accumulation of DNA DSBs, and ultimately potentiates the efficacy of 5-FU [8].
ATR is mainly activated in the replication protein A (RPA)-coated single-strand DNA (ssDNA) region by anchoring ATR interacting protein (ATRIP) [11]. Rad9 and Rad17 are essential for the activation of ATR and Chk1 induced by RPA-coated ssDNA [7]. Rad9 is a component of the Rad9-Hus1-Rad1 (9-1-1) complex, a PCNA-like clamp that is loaded at the boundary of dsDNA and ssDNA [12]. The 9-1-1 complex is required for the recruitment of TopBP1 [13], which activates the ATR–ATRIP complex [14]. Rad17 forms a complex with replication factor C (RFC) 2-5 and loads the 9-1-1 clamp to sites of DNA damage [15]. ATR is also activated by direct binding to the MutSα complex, which binds to O6-methylguanine (O6-meG):dTTP adducts generated by SN1-type alkylating agents, such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) [16], [17], [18]. MMR factors, but not RPA or Rad17, are required for ATR-mediated SMC1 phosphorylation by MNNG [18]. ATR and MMR factors, but not RPA, are recruited to O6-meG:dTTP adducts in vitro [16], [17]. A MMR-mediated cell cycle checkpoint response to fluorodeoxyuridine has also been reported [19], but the contribution of this response to cellular tolerance to 5-FU is not clear.
To elucidate the mechanisms responsible for the activation of the DNA damage response by 5-FU, we determined 5-FU sensitivity and cellular responses in chicken B lymphoma DT40 cells and in gene knockout strains as model systems. We found that mutants of HRR factors (Rad54 and BRCA2) and of factors involved in the ATR–Chk1 signaling pathway (Rad9 and Rad17) exhibited significantly increased sensitivity to 5-FU. Consistently, 5-FU induced nuclear foci of phosphorylated histone H2AX (γH2AX), which colocalized with HRR factor Rad51, but not with DNA DSBs. From the phenotypic analysis of these mutant cells and cells whose Chk1 activity was inhibited by UCN-01, we revealed the critical role of the ATR–Chk1 signaling pathway and HRR for protecting cells from 5-FU cytotoxicity.
Section snippets
Cell culture and chemicals
Chicken DT40 and their mutant cells (rad9, rad17 [20], rad54 [21], ku70 [22], brca2ΔCTD [23] and fancd2 [24]) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% chicken serum, 2 mM l-glutamine, 50 mM 2-mercaptoethanol, penicillin and streptomycin in a 5% CO2 incubator at 39.5 °C. The rad54 and ku70 strains were kindly provided by Dr. Shunichi Takeda. 5-FU and UCN-01 were purchased from Sigma (St. Louis, MO).
Western blotting and antibodies
We used antibodies against Chk1 (G-4, Santa Cruz
5-FU induces γH2AX nuclear foci and Chk1-dependent DNA damage responses in chicken B lymphoma cells DT40
5-FU activates Chk1 by ATR-mediated phosphorylation of Ser345 within the C-terminal domain of Chk1, and induces S-phase arrest in HeLa cells and DT40 cells [8], [9]. In HeLa cells, 5-FU also induces γH2AX, a marker of DNA damage including DSBs, although Chk1 Ser345 phosphorylation occurs before this step [8], suggesting that DNA damage is introduced after activation of the S-phase checkpoint.
When DT40 cells were exposed to increasing doses of 5-FU for 16 h, the number of cells with γH2AX nuclear
Discussion
5-FU and its prodrugs have been, and will probably continue to be, the mainstay first-line treatment for major solid tumors. Although 5-FU metabolites affect DNA, RNA and de novo nucleotide synthesis, cytotoxicity associated with DNA damage has been a major focus of research, and BER and MMR are involved in these events [19], [34], [35], [36], [37]. In addition, the Chk1-mediated cell cycle checkpoint also contributes to 5-FU tolerance [8], [9]. From the clonogenic survival assay using DT40
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
We thank Dr. Shunichi Takeda for providing mutant DT40 cells, Dr. Kenshi Komatsu for providing antibodies, Ms. Naoko Katakura, Mariko Shimokawa and Akiko Seo for expert technical assistance, Ms. Satoko Hamatake for secretarial assistance and the Research Support Center, Graduate School of Medical Sciences, Kyushu University for technical support. This work was supported in part by Grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan (H.K. and Y.M.).
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