Regulation of Cisplatin Resistance and Homologous Recombinational Repair by the TFIIH Subunit XPD1
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[CANCER RESEARCH 62, 5457–5462, October 1, 2002] Regulation of Cisplatin Resistance and Homologous Recombinational Repair by the TFIIH Subunit XPD1 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 Panasci3 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 imal or no DNA-cross-linking agent hypersensitivity (7, 8). DNA ICL-inducing agents, such as cisplatin, are thought to exert their We have recently completed screening of the National Cancer Institute cytotoxic effect by preventing efficient DNA replication and tran- human tumor cell line panel and demonstrated that among four nucleo- scription (3). In mammalian cells, it has been suggested that ICL tide excision repair proteins (XPA, XPB, XPD, and ERCC1), only the TFIIH subunit XPD endogenous protein levels correlate with alkylating repair occurs via the activity of the NER endonuclease (ERCC1/XPF) agent drug resistance. In the present study, we extended this work by and Rad51-related HRR proteins, including Xrcc2 and Xrcc3. Muta- investigating the biological consequences of XPD overexpression in the tions in these four proteins (ERCC-1, XPF, Xrcc2, and Xrcc3) result human glioma cell line SK-MG-4. Our results indicate that XPD overex- in extreme hypersensitivity to ICL-inducing agents (9). Moreover, we pression in SK-MG-4 cells leads to cisplatin resistance without affecting have demonstrated recently that increased HRR, as seen by increased the nucleotide excision repair activity or UV light sensitivity of the cell. In Rad51 nuclear foci density, correlates with melphalan/cisplatin drug contrast, in SK-MG-4 cells treated with cisplatin, XPD overexpression resistance in a variety of human tumor cell lines (10). leads to increased Rad51-related homologous recombinational repair, In the present study, we investigate the effect of XPD overexpres- increased sister chromatid exchanges, and accelerated interstrand cross- sion on bifunctional alkylating drug resistance vis a`vis HRR. 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 MATERIALS AND METHODS XPD and Rad51, which leads to bifunctional alkylating agent drug resist- ance and accelerated removal of interstrand cross-links. 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% CO2 atmosphere. The XPD open reading frame INTRODUCTION sequence (a kind gift from Dr. L. Thompson, Biology and Biotechnology Resistance to chemotherapeutic agents is a major impediment to the Research Program, Lawrence Livermore National Laboratory, Livermore, CA) was subcloned into the pcDNA3.1 expression vector (Invitrogen), amplified, successful treatment of various human cancers. Therefore, the eluci- and stably transfected into the glioma cell line SK-MG-4 (Dr. Caincross, dation of the mechanisms involved in drug resistance is a key element University of Western Ontario, Ontario, Canada) using the Effectine reagent in the development of new strategies to overcome this phenomenon (Qiagen) following the manufacturer’s instructions. The transfected cells were and improve treatment outcomes. Up-regulation of DNA repair mech- maintained in medium for 48 h, trypsinized, and serially diluted. Single clones anisms, which is necessary for maintenance of the genetic stability of were amplified for 3 weeks in medium containing 600 g/ml G418. Mock- the cell (1), has been associated with resistance to alkylating agents, transfected SK-MG-4 cells were obtained by transfection of the empty cisplatin analogues, and radiation (2, 3). Several DNA repair genes, pcDNA3.1 expression vector. including XPB, XPD, XPA, and ERCC-1, have been implicated in the Cell Survival Assay. SK-MG-4 cells overexpressing XPD (hereafter re- development of anticancer drug resistance in human tumor cells (3, 4). ferred to as XPD cells) and SK-MG-4 mock-transfected cells (hereafter re- In a recent study, we assessed the levels of the protein products of the 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 above-mentioned genes in the National Cancer Institute panel of 60 (Jewish General Hospital, Montreal, Quebec, Canada), melphalan (Sigma- human tumor cell lines in relation to the cytotoxicity profile of 170 Aldrich), or UV light. Survival was assessed 48 h and 7 days after treatment compounds that constitute the standard agent database. We found a using the MTT and sulforhodamine B assay, respectively (Sigma-Aldrich), as significant correlation between XPD endogenous protein levels and described previously (10, 11). resistance to alkylating agents (5). The XPD helicase, a component of FACS Analysis. XPD and MOCK subconfluent cultures were treated with the TFIIH transcription factor, participates in DNA unwinding to cisplatin (0, 2, or 25 M) for 36 h. Floating and adherent cells were then allow gene transcription by RNA polymerase II and/or the removal of harvested, fixed with ice-cold 70% ethanol, and the DNA was stained with DNA lesions—induced by a variety of genotoxic agents, including propidium iodide 5 g/ml for 5 min, washed with PBS, and stored in the dark UV light and some anticancer drugs—by NER4 (6). It has been at 4°C for no longer that 8 h. Cell cycle analysis was performed using a FACS reported that XPD mutations that impair NER activity result in min- (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 Received 11/15/01; accepted 7/29/02. cultures were trypsinized and collected in PBS, lysed in lysing buffer (4.0 M 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 NaCl, 50 mM KH2PO4,10mM EDTA, 0.1 g/ml N-sarcosyl NaCl, and 20 18 U.S.C. Section 1734 solely to indicate this fact. g/ml RNase), and incubated at 37°C for 16 h. After a 20-min incubation with 1 Supported by a grant from the Leukemia and Lymphoma Society, and a private 12 IU Heparin at 37°C, the DNA was denatured in the presence of 10 g/ml donation from Helen Rosenbloom Lang. L. P. is the recipient of the Gertrude and Stanley ethidium bromide in 50 mM KH2PO4 (pH 12.1) by heating it to 100°C Vineberg Clinical Scientist Award. ϭ 2 These authors contributed equally to this work. (fn fluorescence after heating/fluorescence before heating). The fraction of 3 To whom reprint requests should be addressed, at the Lady Davis Institute for nondenatured DNA (f) for each sample was calculated as the ratio between the Medical Research, Sir Mortimer B. Davis–Jewish General Hospital, 3755 Coˆte Ste. absorbance (at 580 nm) after and before a 5-min incubation at 100°C. The 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- diphenyltetrazolium bromide; FACS, fluorescence-activated cell sorter; pSK, pBluescript; link; HRR, homologous recombinational repair; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- SCE, sister chromatid exchange. 5457 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2002 American Association for Cancer Research. XPD OVEREXPRESSION LEADS TO CISPLATIN RESISTANCE radish peroxidase-␣-rabbit secondary antibodies (Roche) and the enhanced chemiluminescence (Amersham) reagent. Western blots were scanned and analyzed using the Scion Image software. Immunoprecipitation Experiments. Nuclear-enriched fractions were ob- tained from XPD and MOCK cells (14). Five-hundred g of proteins from nuclear extracts were precleared with protein A-Sepharose or protein G- Agarose by incubation for2hat4°C. The proteins present in the supernatant (1 mg/ml in lysis buffer containing proteases inhibitors) were immunoprecipi- tated overnight at 4°C with ␣-XPD (a kind gift from Dr. J. M. Egly) or ␣-Rad51 (Santa Cruz), antibody. Immunoprecipitates were collected after a 2-h incubation with protein G-Agarose or protein A-Sepharose, respectively, washed three times with cold lysis buffer, and separated by 8% SDS-PAGE. Rad51 and XPD proteins were detected as described above. Fig. 1. XPD constitutive overexpression was determined by Western blot analysis in 50 In Vitro Repair Assay, NER Activity. The 2959-bp plasmid pSK (Strat- g of protein extracts from MOCK cells, XPD cells, and SKMG-4 cells. agene) was prepared by alkaline lysis method (Qiagen). Linear, circular, and supercoiled forms of DNA obtained after plasmid preparation were separated on two successive sucrose gradients, and the fractions containing supercoiled proportion of total cross-linked DNA (%CT) after cisplatin treatment, was DNA were isolated and purified. The pSK plasmid was then treated with ϫ cisplatin (0.5 g cisplatin per 100 g DNA) as described previously (15). calculated as 100