ORIGINAL PAPERS Hypersensitivity of Brca1-deficient MEF to the DNA interstrand crosslinking agent mitomycin C is associated with defect in homologous recombination repair and aberrant S-phase arrest Jeanho Yun 1,2 , Qing Zhong 3 , Jong-Young Kwak 1,2 and Wen-Hwa Lee* ,3 1 Medical Research Center for Cancer Molecular Therapy, College of Medicine, Dong-A University, Busan 602-714, South Korea; 2 Department of Biochemistry, College of Medicine, Dong-A University, Busan 602-714, South Korea; 3 Department of Biological Chemistry, School of Medicine, University of California, 124 Sprague Hall, 839 Medical Science Court, Irvine, CA 92697, USA Hypersensitivity of Brca1-deficient cells to interstrand crosslinking (ICL) agents such as cisplatin and mitomycin C (MMC) implicates an important role for Brca1 in cellular response to the ICL DNA damage repair. However, the detailed mechanism of how Brca1 is involved in the ICL response remains unclear. In this study, we analysed the cellular response to MMC treatment using isogenic mouse embryonic fibroblasts (MEFs) including wild type, p53 À/À and p53 À/À Brca1 À/À . Marked hyper- sensitivity of p53 À/À Brca1 À/À MEFs to MMC was found, and the reconstitution of Brca1 expression in these cells restored resistance to MMC. Upon MMC treatment, wild-type MEF was temporarily arrested at G2/M phase but subsequently resumed a normal cell cycle progression. In contrast, Brca1-deficient MEF exhibited a marked time-dependent accumulation of cells arrested at S phase and a prolonged increase in the G2/M population, followed by extensive cell deaths. Importantly, DNA damage-induced Rad51 foci were not formed in these cells, suggesting a defect in homologous recombination. Such defects are fully rescued by reconstitution of Brca1 expression in Brca1-deficient MEF, suggesting that Brca1 directly plays an essential role in ICL repair, which depends on homologous recombination during S phase. Oncogene (2005) 24, 4009–4016. doi:10.1038/sj.onc.1208575 Published online 21 March 2005 Keywords: Brca1; mitomycin C; DNA interstrand cross- link (ICL) repair; Rad51; S phase Introduction DNA interstrand crosslinks (ICLs) are among the most toxic of all the DNA lesions. ICL prevents DNA strand separation, blocks essential cellular processes such as DNA replication, transcription and recombination, and eventually induces cell cycle arrest, chromosomal rearrangement and cell death. ICL agents such as cisplatin, mitomycin C (MMC) and nitrogen mustard are widely used as potent anticancer therapeutics (Metzler, 1986); however, the molecular detail of ICL repair is still not well understood in higher mammalian cells. Although the detailed sequential events remain to be determined, previous studies suggest that nucleotide excision repair (NER) and homologous recombination (HR) are involved in ICL repair (reviewed in Dronkert and Kanaar, 2001). In contrast to procaryotes, cumulative evidence suggests that ICLs elicit double-stranded breaks (DSBs) in eucaryotes (Dardalhon and Averbeck, 1995; De Silva et al., 2000; McHugh et al., 2000). DSBs are produced as repair intermediate following treatment with ICL agents (De Silva et al., 2000) and can also be generated near the sites of ICL during S phase of the cell cycle when encountered by replication fork (Akkari et al., 2000). Thus, it has been proposed that in mammalian cells, the repair of ICL proceeds via the formation of a DSB and followed by DSB repair via HR (Rothfuss and Grompe, 2004). Consistent with this model, cells deficient in Rad51 paralog or other proteins involved in HR-mediated DSB repair are sensitive to DNA damage induced by ICL agents (De Silva et al., 2000; Dronkert and Kanaar, 2001). Rad51, a mammalian homolog of the bacterial protein RecA, is essential for repair of DNA DSB by HR (Sonoda et al., 1998). The importance of understanding ICL repair is also highlighted by the hypersensitivity to ICL agents among Fanconi anemia (FA) patients (Sasaki and Tonomura, 1973) and in cells lacking breast cancer tumor suppres- sor genes, BRCA1 and BRCA2 (Bhattacharyya et al., 2000; Yu et al., 2000; Moynahan et al., 2001). Interestingly, recent studies suggest interactions between FA gene products and BRCA1 and BRCA2 (Garcia- Higuera et al., 2001; D’Andrea and Grompe, 2003). Following DNA damage, FANCD2 protein colocalizes in nuclear foci with BRCA1 and Rad51 in Sphase (Taniguchi et al., 2002). These studies indicate the involvement of the FA/BRCA pathway in HR-mediated repair of DNA damage induced by ICL agents. Substantial evidence exists to support a role for BRCA1 in mediating the cellular response to DNA lesions, especially, DNA DSBs (reviewed in Zheng et al., Received 8 December 2004; revised 8 January 2005; accepted 26 January 2005; published online 21 March 2005 *Correspondence: W-H Lee; E-mail: whlee@uci.edu Oncogene (2005) 24, 4009–4016 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc