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 resistance 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

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 obtained 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 (f50 ␮M-F0 ␮M/1-F0 ␮M). Rad51 Foci Density Determination. Sister XPD and MOCK cell cultures Nuclear extracts were also prepared according to a previous protocol (16). were seeded at 5 ϫ 103 cells in complete medium onto glass coverslips and Each reaction mixture contained 300 ng of damaged or untreated pSK plasmid, allowed to adhere for 24 h. Cells were then treated with cisplatin, washed with 40 ␮g of cell extract in reaction buffer containing 45 mM HEPES-KOH (pH ␮ PBS, and fixed for 15 min in PBS containing 4% paraformaldehyde, 0.25% 7.8), 7.4 mM MgCl2, 0.9 mM DTT, 0.4 mM EDTA, 2 mM ATP, 20 M of each glutaraldehyde, and 0.2% Triton X-100, permeabilized for 3 min in 0.5% dGTP, dTTP and dCTP, 4 ␮M dATP, 40 mM phosphocreatine, 2.5 ␮g creatine Triton X-100 in PBS, and then washed with PBS. Rad51 foci density was determined as described previously with minor modifications (10, 11). Briefly, Rad51 protein was detected using a specific ␣-hRad51 antibody (1:100 dilution), from Santa Cruz and a FITC-conjugated ␣-rabbit immunoglobulin (1:50 dilution) from Santa Cruz. The DNA was counterstained by incubation with propidium iodide 2 ␮g/ml for 5 min, washed with PBS, and mounted in DABCO mounting medium (Sigma-Aldrich). Staining was analyzed by con- focal microscopy. Rad51 foci are defined as the yellow staining resulting from colocalization of Rad51 labeling (green) and DNA staining (red). Rad51 foci density was calculated as the ratio between yellow intensity staining and yellow surface staining using the Northern Eclipse software as described (10). SCE. Twenty-four h after seeding, XPD and MOCK cells were treated with cisplatin (0, 0.25, or 0.50 ␮M). One h after cisplatin addition, fresh medium containing 0.04 ␮g BrdUrd/ml (Boehringer Mannheim) was added to the cultures for 44 h (two doubling times). During the final5hofculture, mitotic cells were arrested in metaphase with 0.01 ␮g/ml Colcemid (Life Technolo- gies, Inc.). Metaphase preparation was done by standard cytogenetic proce- dures. Differential sister chromatid staining was achieved by the fluorescence- plus-Giemsa method (13). Enumeration of SCEs was done without knowledge of treatment in 10 well-spread second-division metaphases for each culture. Immunocytochemistry Assays. XPD and MOCK cells were seeded in eight-well chamber slides at 4 ϫ 103cells/well and treated 24 h later with cisplatin. Cells were fixed as mentioned above followed by blocking in 1% rat serum. Cells were then incubated overnight at 4°C with ␣-hRad51 antibody (1:100 dilution; Santa Cruz) or ␣-XPD antibody (1:200 dilution; a kind gift from Dr. J. M. Egly (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France) in blocking solution followed by a 2-h incubation with the secondary antibodies ␣-rabbit FITC (Santa Cruz) and ␣-mouse Cy3 (Jackson Labs), respectively. The slides were mounted using Sigma mounting medium and analyzed by one-dimensional microscopy. The images were captured using a Zeiss microscope, entered into a computer, and merged using the Northern Eclipse software. Western Blot Analysis. XPD and MOCK cells were cultured in 100-mm Petri dishes in complete medium and treated 24 h later with 25 ␮M cisplatin. The cells were harvested 12 h after treatment and lysed at 4°C in lysis buffer [20 mM Tris (pH 8), 135 mM NaCl, 1% NP40, 10% glycerol containing a Fig. 2. The resistance to cisplatin-mediated DNA damage of MOCK (Ⅺ) and XPD (f) protease inhibitors mixture (Roche), and 1 mM sodium vanadate]. The Western cells was assessed by (A) sulforhodamine B assay 7 days after cisplatin treatment and by blot analysis was performed as described in a previous report (10). Briefly, 50 (B) MTT assay 48 h after cisplatin treatment. A, Y-axis (log scale) represents the ␮g of proteins were separated by 10% SDS-PAGE and transferred onto a percentage of surviving cells after treatment with cisplatin as compared with the untreated cells (control). Each value was calculated as (absorbance-treated cells/absorbance- nitrocellulose membrane. The membrane was then blocked and subsequently ϫ untreated cells) 100. The LD50 (50% of control), was calculated by interpolation using probed with specific antibodies, ␣-TFIIHp80 (XPD; 1:500 dilution; Santa a linear regression between log (% of control) versus cisplatin concentration. B, Y-axis Cruz), ␣-hRad51 (1:2,000 dilution; Santa Cruz), antitubulin (1:10,000 dilution; represents the percentage of cells after treatment with respect to the untreated (control). ϫ Medicorp), and ␣-Xrcc3 (1:1,000 dilution; a kind gift of Dr. P. Sung, Depart- Each value was calculated as (absorbance-treated cells/absorbance-untreated cells) 100. The results are expressed as the mean value of three independent experiments; bars, ϮSE. indicates significant differences between MOCK and XPD cells as determined by paired ء ,ment of Molecular Biology, Howard Hughes Medical Institute, San Antonio TX). Proteins were detected with horseradish peroxidase-␣-mouse or horse- Student t test for means (P Ͻ 0.05). 5458

Statistical Analysis. The ToolPak from Microsoft Excel 97 software was used to perform linear regression analysis. The Student t test of the statistical tests were two-sided.

RESULTS XPD Overexpression Mediates Cisplatin Resistance without Affecting Cell Cycle Progression. To evaluate the effect of XPD overexpression in DNA-damaging agent drug resistance, we stably overexpressed the XPD protein in a glioblastoma cell line, SK-MG-4 (Fig. 1). XPD overexpression resulted in a 2–4-fold increase in cisplatin resistance (Fig. 2, A and B). This increase was not likely because of an effect in the cell cycle progression because the doubling time of MOCK cells and XPD cells was the same (23 Ϯ 1.3 and 23 Ϯ 1.5 h, respectively). XPD Overexpression Increases ICL Repair Rate and Melpha- lan Resistance without Affecting in Vitro NER Activity or UV Light Sensitivity. Because XPD is an essential component of the Fig. 3. The in vitro NER activity was determined in nuclear extracts of MOCK (Ⅺ) and NER-DNA repair pathway, we wanted initially to determine whether XPD (f) cell cultures. Y-axis represents the ratio between the ␥-P32 incorporation using cisplatin resistance was related to proficient NER activity in XPD as a substrate a cisplatin-treated plasmid and the ␥-P32 incorporation using as a substrate an untreated plasmid (top panel). The values represent the mean of three independent cells. Our results demonstrate that XPD overexpression did not alter experiments; bars, ϮSE. The bottom panel is a picture of a representative autoradiograph. in vitro NER activity (Fig. 3). Cisplatin induces a number of different DNA lesions, which are repaired preferentially by different mechanisms. These lesions include monoadducts and intrastrand cross-links, which are repaired by the NER pathway, and ICLs, which are possibly repaired by the sequential activity of the NER endonuclease (ERCC- 1/XPF) followed by HRR. We then wanted to additionally investigate the possible role of XPD as a modulator of the repair of specific DNA lesions. To this end, we tested the sensitivity of XPD cells and MOCK cells to UV light, which DNA-induced damage is repaired by NER and to melphalan, a clinical relevant ICL inducing agent. XPD overexpression resulted in a 2-fold increase in melphalan resistance without affecting UV light sensitivity (Fig. 4, A and B). We then examined

Fig. 4. The sensitivity to melphalan- (A) and UV- (B) mediated DNA damage of MOCK (Ⅺ) and XPD (f) cells was assessed by MTT assay 48 h after treatment. Y-axis represents the percentage of surviving cells after treatment; this was calculated as the percentage of control [(absorbance-treated cells/absorbance-untreated cells) ϫ100]. The ء .results are expressed as the mean value of three independent experiments; bars, ϮSE indicate significant differences between MOCK and XPD. Cell survival as determined by paired Student t test for means (P Ͻ 0.05). phosphokinase, 3.4% glycerol, 18 ␮g BSA, and 4 ␮Ci [␣-32P]ATP (17). Reactions were carried out at 30°C for 3 h. The plasmid DNA was then purified and linearized with EcoRI and electrophoresed overnight on a 1% agarose gel containing 0.5 ␮g/ml ethidium bromide. The gel was then fixed for Fig. 5. MOCK (Ⅺ) and XPD (f) cell cultures were treated with 50 ␮M cisplatin for 1 h 15 min in 15% methanol and 10% acetic acid, dried on Whatman filter paper, and maintained in fresh complete medium for 4, 8, or 24 h (X-axis, Recovery time). The and exposed for autoradiography. For data presentation, autoradiographs were percentage of total cross-links was determined using the ethidium bromide fluorescence assay (Y-axis, %CT). The values represent the mean value of six independent experiments (indicates a significant difference between MOCK (E) and XPD (F ء .scanned and processed with the Adobe Photoshop software. Experiments were (n ϭ 6); bars, ϮSE repeated three times and the mean Ϯ SE determined. cells in the %CT (Student t test P Ͻ 0.05). 5459

Fig. 6. Rad51 foci were immunostained in sister cultures of MOCK and XPD cells 24 h after cisplatin treatment at 0, 2.0, or 25.0 ␮M concentration (left panel). A, Rad51 foci density was calculated as described; Y- axis values represents the mean of two independent experiments (right panel); bars, ϮSE. B, SCEs/cell (Y- axis) were determined 36 h after treatment with 0, 0.25, or 0.5 ␮M cisplatin (X-axis). The results represent the ء .mean value of SCE determined in 10 cells; bars, ϮSE indicates a significant differences by Student t test (P Ͻ 0.05) between MOCK (Ⅺ) and XPD (f) cells.

the kinetics of interstrand DNA cross-link formation and removal repair. Immunochemical analysis of XPD and MOCK cells revealed after cisplatin treatment. The results demonstrate that the kinetics of that XPD and Rad51 colocalize constitutively in the nucleus of these ICL formation were not affected by XPD overexpression; however, the cells (Fig. 7A). Colocalized staining increases after cisplatin treatment rate of ICL removal was significantly accelerated by 3-fold (Fig. 5). of XPD and MOCK cells (Fig. 7B). We additionally investigated the XPD Overexpression Mediates Increases in Both Rad51 Nu- possible association between Rad51 and XPD. Immunoprecipitation clear Foci Density and SCE after Cisplatin Treatment. The next analysis of nuclear extracts from XPD and MOCK cells confirmed the step was to evaluate whether another DNA repair pathway known to immunocytochemical results, demonstrating that XPD and Rad51 be involved in ICL repair, HRR, was affected by XPD overexpression. interact constitutively (Fig. 8A). This association is increased after Rad51 is an essential component of the HRR pathway. Multienzy- cisplatin treatment in both XPD and MOCK cells, suggesting that it is matic complexes, which include Rad51, localize in the cell nucleus a physiological response to cisplatin-mediated DNA damage (Fig. after DNA damage. These complexes are visualized immunocyto- 8B). Although basal Rad51 protein levels are not affected by XPD chemically as Rad51 nuclear foci and are believed to represent active overexpression, after cisplatin treatment, Rad51 protein levels are DNA repair. The quantification of Rad51 foci density induced 24 h specifically increased in XPD cells (Fig. 8C). after cisplatin treatment in MOCK cells and XPD cells was deter- XPD Overexpression Increases the Percentage of Cells Ar- mined. XPD overexpression resulted in a 1.8-fold increased rate of rested in S Phase after Cisplatin Treatment. FACS analysis con- HRR as evidenced by an increase in cisplatin-induced Rad51 foci firmed our results that XPD overexpression did not affect the cell density in XPD cells (Fig. 6A). To additionally assess the effect of cycle profile in the absence of DNA damage (Fig. 9A). However, after XPD overexpression in cisplatin-induced HRR, we analyzed the cisplatin treatment, the percentage of cells arrested in S phase was cisplatin-induced SCEs in both cell lines. In agreement with the higher in XPD cells compared with MOCK cells. Also, the percentage

Rad51 foci density results mentioned above, XPD cells showed a of XPD cells in G2/M and apoptotic phase were lower than that in 1.5-fold increase in the number of SCEs per cell when compared with MOCK cells (Fig. 9B). MOCK cells after treatment (Fig. 6B). XPD and Rad51 Proteins Coimmunoprecipitate in MOCK DISCUSSION Cells and XPD Cells. Recent reports suggest that the different DNA repair pathways are not independent. It is possible that XPD and XPD overexpression results in cisplatin drug resistance, and is Rad51 interact as part of a multienzymatic complex involved in ICL associated with accelerated ICL removal and increased HRR. We 5460

Fig. 7. XPD and Rad51 proteins localization was determined by immunocytochemistry 24 h after 25.0 ␮M cisplatin treatment. The green (Rad51) and red (XPD) labeling were merged electronically, and the yellow areas represent the colocalization of both proteins. A–D, magnifications of the merged nuclear staining.

have herein provided evidence that accelerated ICL repair is involved in XPD-mediated cisplatin resistance by a mechanism independent of NER activity. This is in agreement with previous investigations in which NER activity did not correlate with endogenous or overexpressed XPD protein levels (5, 18). The finding that XPD cells are resistant to melphalan and cisplatin but not to UV light suggests that XPD mediates resistance specifically to bifunctional alkylating agents. Our results imply that XPD can modulate Rad51-related HRR rather than altering NER activity. As Rad51-null mice are not viable because they fail to complete mitotic and meiotic processes, the role of Rad51 in HRR has been assessed in genetically engineered cell lines. These studies have shown that inducible Rad51-null cells are hypersensitive to DNA damage and display defective HRR as visualized by Rad51 foci formation and SCE after DNA damage (19–23). The Rad51-related HRR pathway has been associated with DNA-damage resistance in Rad51-overexpressing cells, in clinical samples from cancer patients, and in epithelial cell lines (10, 11, 24). The tumor suppressor protein p53 interacts with both XPD and Rad51, inhibiting their biological functions (p53 inhibits TFIIH helicase and inhibits both Rad51-directed homologous recombination and HRR; Refs. 25, 26). We found that the interaction between XPD and Rad51 is not likely to be mediated by p53, because both proteins coimmunoprecipitate in SAOS-p53 null cells (data not shown). How- ever, this does not exclude a possible modulatory role of p53 in the Rad51-XPD interaction. It is unknown whether the physical interaction between XPD and Rad51 involves a direct protein-protein interaction or whether they are Fig. 8. Sister cultures were maintained in complete medium with (ϩ) or without (Ϫ) both part of a large complex with other factors. XPD is known to 25 ␮M cisplatin and collected 16 h later. Rad51-XPD interaction was tested by cross- interact with XPB, other TFIIH components, p53, and other proteins coimmunoprecipitation of nuclear enriched extracts. A, Rad51 immunoprecipitation and (B) XPD immunoprecipitation. C, XPD and Rad51 protein levels were tested in whole cell (27, 28). We detected both XPB (TFIIH89 subunit) and BRCA1 lysates by Western blot. The blots were reprobed with an XRCC3 and tubulin antibody to proteins in Rad51 and XPD immunoprecipitate (data not shown), in asses equal protein loading. 5461

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Raquel Aloyz, Zhi-Yuan Xu, Vanessa Bello, et al.

Cancer Res 2002;62:5457-5462.

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