Xrcc3 Induces Cisplatin Resistance by Stimulation of Rad51- Related Recombinational Repair, S-Phase Checkpoint Activation, and Reduced Apoptosis Zhi-Yuan Xu, Martin Loignon, Fei-Yu Han, Lawrence Panasci, and Raquel Aloyz Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montre ´ al, Quebec, Canada (Z.-Y.X., M.L., L.P., R.A.); and Pathology and Human Genetics, McGill University and Cytogenetics, McGill University Hospital Center, Montreal Children’s Hospital, Montre ´ al, Quebec, Canada (F.-Y.H.) Received January 21, 2005; accepted April 18, 2005 ABSTRACT Eukaryotic cells respond to DNA damage by activation of DNA repair, cell cycle arrest, and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signaling other cellular responses. The Rad51-guided homologous recombina- tion repair system plays an important role in the recognition and repair of DNA interstrand crosslinks (ICLs), and cells deficient in this repair pathway become hypersensitive to ICL-inducing agents such as cisplatin and melphalan. We investigated the possible role of the Rad51-paralog protein Xrcc3 in drug resis- tance. Xrcc3 overexpression in MCF-7 cells resulted in 1) a 2- to 6-fold resistance to cisplatin/melphalan, 2) a 2-fold increase in drug-induced Rad51 foci, 3) an increased cisplatin-induced S-phase arrest, 4) decreased cisplatin-induced apoptosis, and 5) increased cisplatin-induced DNA synthesis arrest. Interest- ingly, Xrcc3 overexpression did not alter the doubling time or cell cycle progression in the absence of DNA damage. Further- more, Xrcc3 overexpression is associated with increased Rad51C protein levels consistent with the known interaction of these two proteins. Our results demonstrate that Xrcc3 is an important factor in DNA cross-linking drug resistance in human tumor cells and suggest that the response of the homologous recombinational repair machinery and cell cycle checkpoints to DNA cross-linking agents is intertwined. Cisplatin is one of the most potent antitumor agents known, displaying clinical activity against a wide variety of tumors. Its cytotoxicity is mediated by the induction of DNA intrastrand adducts and DNA interstrand cross-links (ICLs). ICLs are particularly deadly because a single unrepaired ICL impairs DNA replication and translation. The repair of ICLs is thought to involve the combined action of nucleotide exci- sion repair components, which unhooks the ICL, either ini- tially or after strand invasion, along with the action of the homologous recombinational repair (HRR) machinery. In cy- cling mammalian cells, HRR is a major pathway involved in the repair of double strand breaks, which are probably pro- duced during the ICL repair process, whereas nonhomolo- gous end-joining is favored when recombinational substrates are not available (G 1 phase of the cell cycle) (De Silva et al., 2000; McHugh et al., 2001; West, 2003). The HRR process requires the assembly of multienzymatic complexes visual- ized immunocytochemically as Rad51 nuclear foci. These complexes include the Rad51 paralog family members such as Rad51, Rad54, Rad51B, Rad51C, Rad51D, Xrcc2, and Xrcc3 (reviewed in West, 2003). Rad51 paralog-defective cell lines (Rad51B, Rad51C, Rad51D, Xrcc2, and Xrcc3) present similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents, and atten- uated Rad51 focus formation after exposure to ionizing radi- ation (IR) (Liu et al., 1998; Takata et al., 2001). Recently, similar results have been obtained in a human colon cancer cell line in which Xrcc3 was inactivated by gene targeting (Yoshihara et al., 2004). However, in these cells, Xrcc3 defi- ciency results in milder sensitivity to DNA cross-linking agents (2-fold) when compared with previous results in defi- cient hamster cells. Moreover, Xrcc3 deficiency resulted in increased endoreduplication. Supported by a National Cancer Institute Cancer Research Society (Toronto, ON, Canada) grant and a generous private donation from the Helen and Nicky Rosenbloom Lang Scholarship to L.P., a U.S. Army grant and a Lymphoma Foundation of Canada fellowship to R.A., and a Leukemia Lymphoma Fund of Canada Fellowship to Z.-Y.X. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.084053. ABBREVIATIONS: ICL, interstrand crosslink(s); HRR, homologous recombinational repair; IR, ionizing radiation; ATM, ataxia telangiectasia- mutated homolog; ATR, ataxia telangiectasia-mutated and Rad3-related; CDDP, cisplatin; MLN, melphalan; SRB, sulforhodamine B; CI, confi- dence interval; FACS, fluorescence-activated cell sorting; SCE, sister chromatid exchange(s); MOCK, mock-transfected cells; OVER, Xrcc3- overexpressing cells. 0022-3565/05/3142-495–505$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 314, No. 2 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 84053/3039841 JPET 314:495–505, 2005 Printed in U.S.A. 495 at UNIVERSITY OF VIRGINIA Health Sciences Library on December 29, 2011 jpet.aspetjournals.org Downloaded from