[CANCER RESEARCH 62, 5457–5462, October 1, 2002] Regulation of Cisplatin Resistance and Homologous Recombinational Repair by the TFIIH Subunit XPD 1 Raquel Aloyz, 2 Zhi-Yuan Xu, 2 Vanessa Bello, Jose ´e Bergeron, Fei-Yu Han, Yifei Yan, Areti Malapetsa, Moulay A. Alaoui-Jamali, Alessandra M. V. Duncan, and Lawrence Panasci 3 Lady Davis Institute for Medical Research, Sir Mortimer B. Davis–Jewish General Hospital, Quebec, Canada H3T 1E2 [R. A., Z-Y. X., V. B., J. B., Y. Y., A. M., M. A. A-J., L. P.], and Pathology and Human Genetics, McGill University and Cytogenetics, McGill University Hospital Center, Montreal Children’s Hospital, Montreal, Quebec, Canada H3H 1P3 [F-Y. H., A. M. V. D.] ABSTRACT We have recently completed screening of the National Cancer Institute human tumor cell line panel and demonstrated that among four nucleo- tide excision repair proteins (XPA, XPB, XPD, and ERCC1), only the TFIIH subunit XPD endogenous protein levels correlate with alkylating agent drug resistance. In the present study, we extended this work by investigating the biological consequences of XPD overexpression in the human glioma cell line SK-MG-4. Our results indicate that XPD overex- pression in SK-MG-4 cells leads to cisplatin resistance without affecting the nucleotide excision repair activity or UV light sensitivity of the cell. In contrast, in SK-MG-4 cells treated with cisplatin, XPD overexpression leads to increased Rad51-related homologous recombinational repair, increased sister chromatid exchanges, and accelerated interstrand cross- link removal. Moreover, we present biochemical evidence of an XPD- Rad51 protein interaction, which is modulated by DNA damage. To our knowledge, this is the first description of functional cross-talk between XPD and Rad51, which leads to bifunctional alkylating agent drug resist- ance and accelerated removal of interstrand cross-links. INTRODUCTION Resistance to chemotherapeutic agents is a major impediment to the successful treatment of various human cancers. Therefore, the eluci- dation of the mechanisms involved in drug resistance is a key element in the development of new strategies to overcome this phenomenon and improve treatment outcomes. Up-regulation of DNA repair mech- anisms, which is necessary for maintenance of the genetic stability of the cell (1), has been associated with resistance to alkylating agents, cisplatin analogues, and radiation (2, 3). Several DNA repair genes, including XPB, XPD, XPA, and ERCC-1, have been implicated in the development of anticancer drug resistance in human tumor cells (3, 4). In a recent study, we assessed the levels of the protein products of the above-mentioned genes in the National Cancer Institute panel of 60 human tumor cell lines in relation to the cytotoxicity profile of 170 compounds that constitute the standard agent database. We found a significant correlation between XPD endogenous protein levels and resistance to alkylating agents (5). The XPD helicase, a component of the TFIIH transcription factor, participates in DNA unwinding to allow gene transcription by RNA polymerase II and/or the removal of DNA lesions—induced by a variety of genotoxic agents, including UV light and some anticancer drugs— by NER 4 (6). It has been reported that XPD mutations that impair NER activity result in min- imal or no DNA-cross-linking agent hypersensitivity (7, 8). DNA ICL-inducing agents, such as cisplatin, are thought to exert their cytotoxic effect by preventing efficient DNA replication and tran- scription (3). In mammalian cells, it has been suggested that ICL repair occurs via the activity of the NER endonuclease (ERCC1/XPF) and Rad51-related HRR proteins, including Xrcc2 and Xrcc3. Muta- tions in these four proteins (ERCC-1, XPF, Xrcc2, and Xrcc3) result in extreme hypersensitivity to ICL-inducing agents (9). Moreover, we have demonstrated recently that increased HRR, as seen by increased Rad51 nuclear foci density, correlates with melphalan/cisplatin drug resistance in a variety of human tumor cell lines (10). In the present study, we investigate the effect of XPD overexpres- sion on bifunctional alkylating drug resistance vis a ` vis HRR. MATERIALS AND METHODS Cell Culture and Stable Transfection. Cells were maintained in McCoy’s 5A medium supplemented with 10% fetal bovine serum, containing 10 g/ml gentamicin, in a humidified 5% CO 2 atmosphere. The XPD open reading frame sequence (a kind gift from Dr. L. Thompson, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA) was subcloned into the pcDNA3.1 expression vector (Invitrogen), amplified, and stably transfected into the glioma cell line SK-MG-4 (Dr. Caincross, University of Western Ontario, Ontario, Canada) using the Effectine reagent (Qiagen) following the manufacturer’s instructions. The transfected cells were maintained in medium for 48 h, trypsinized, and serially diluted. Single clones were amplified for 3 weeks in medium containing 600 g/ml G418. Mock- transfected SK-MG-4 cells were obtained by transfection of the empty pcDNA3.1 expression vector. Cell Survival Assay. SK-MG-4 cells overexpressing XPD (hereafter re- ferred to as XPD cells) and SK-MG-4 mock-transfected cells (hereafter re- ferred as MOCK cells) were maintained in McCoy’s 5A complete medium and seeded in six-well plates until 70% confluent and then treated with cisplatin (Jewish General Hospital, Montreal, Quebec, Canada), melphalan (Sigma- Aldrich), or UV light. Survival was assessed 48 h and 7 days after treatment using the MTT and sulforhodamine B assay, respectively (Sigma-Aldrich), as described previously (10, 11). FACS Analysis. XPD and MOCK subconfluent cultures were treated with cisplatin (0, 2, or 25 M) for 36 h. Floating and adherent cells were then harvested, fixed with ice-cold 70% ethanol, and the DNA was stained with propidium iodide 5 g/ml for 5 min, washed with PBS, and stored in the dark at 4°C for no longer that 8 h. Cell cycle analysis was performed using a FACS (EPICS XL-MCL; Beckman/Coulter). DNA Cross-linking Assay. The ethidium bromide assay was performed as described in a previous report (12). Briefly, confluent XPD and MOCK cultures were trypsinized and collected in PBS, lysed in lysing buffer (4.0 M NaCl, 50 mM KH 2 PO 4 , 10 mM EDTA, 0.1 g/ml N-sarcosyl NaCl, and 20 g/ml RNase), and incubated at 37°C for 16 h. After a 20-min incubation with 12 IU Heparin at 37°C, the DNA was denatured in the presence of 10 g/ml ethidium bromide in 50 mM KH 2 PO 4 (pH 12.1) by heating it to 100°C (f n = fluorescence after heating/fluorescence before heating). The fraction of nondenatured DNA (f) for each sample was calculated as the ratio between the absorbance (at 580 nm) after and before a 5-min incubation at 100°C. The Received 11/15/01; accepted 7/29/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by a grant from the Leukemia and Lymphoma Society, and a private donation from Helen Rosenbloom Lang. L. P. is the recipient of the Gertrude and Stanley Vineberg Clinical Scientist Award. 2 These authors contributed equally to this work. 3 To whom reprint requests should be addressed, at the Lady Davis Institute for Medical Research, Sir Mortimer B. Davis–Jewish General Hospital, 3755 Co ˆte Ste. Catherine, Montreal, Quebec, Canada H3T 1E2. Phone: (514) 340-8248; Fax: (514) 340- 8302. 4 The abbreviations used are: NER, nucleotide excision repair; ICL, interstrand cross- link; HRR, homologous recombinational repair; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; FACS, fluorescence-activated cell sorter; pSK, pBluescript; SCE, sister chromatid exchange. 5457 American Association for Cancer Research Copyright © 2002 on December 30, 2011 cancerres.aacrjournals.org Downloaded from