Cancer Research Meeting Calendar Advances in Breast Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aloyz, R.
Right arrow Articles by Panasci, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aloyz, R.
Right arrow Articles by Panasci, L.
[Cancer Research 62, 5457-5462, October 1, 2002]
© 2002 American Association for Cancer Research

Experimental Therapeutics

Regulation of Cisplatin Resistance and Homologous Recombinational Repair by the TFIIH Subunit XPD1

Raquel Aloyz2, Zhi-Yuan Xu2, Vanessa Bello, Josée 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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have recently completed screening of the National Cancer Institute human tumor cell line panel and demonstrated that among four nucleotide 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 overexpression 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 resistance and accelerated removal of interstrand cross-links.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Resistance to chemotherapeutic agents is a major impediment to the successful treatment of various human cancers. Therefore, the elucidation 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 mechanisms, 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 NER4 (6) . It has been reported that XPD mutations that impair NER activity result in minimal 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 transcription (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. Mutations 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 overexpression on bifunctional alkylating drug resistance vis à vis HRR.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Stable Transfection.
Cells were maintained in McCoy’s 5A medium supplemented with 10% fetal bovine serum, containing 10 µg/ml gentamicin, in a humidified 5% CO2 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 µg/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 referred to as XPD cells) and SK-MG-4 mock-transfected cells (hereafter referred 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 µM) 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 µg/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 KH2PO4, 10 mM EDTA, 0.1 µg/ml N-sarcosyl NaCl, and 20 µg/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 µg/ml ethidium bromide in 50 mM KH2PO4 (pH 12.1) by heating it to 100°C (fn = 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 proportion of total cross-linked DNA (%CT) after cisplatin treatment, was calculated as 100 x (f50 µM-f0 µM/1-f0 µM).

Rad51 Foci Density Determination.
Sister XPD and MOCK cell cultures were seeded at 5 x 103 cells in complete medium onto glass coverslips and allowed to adhere for 24 h. Cells were then treated with cisplatin, washed with PBS, and fixed for 15 min in PBS containing 4% paraformaldehyde, 0.25% glutaraldehyde, and 0.2% Triton X-100, permeabilized for 3 min in 0.5% 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 {alpha}-hRad51 antibody (1:100 dilution), from Santa Cruz and a FITC-conjugated {alpha}-rabbit immunoglobulin (1:50 dilution) from Santa Cruz. The DNA was counterstained by incubation with propidium iodide 2 µg/ml for 5 min, washed with PBS, and mounted in DABCO mounting medium (Sigma-Aldrich). Staining was analyzed by confocal 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 µM). One h after cisplatin addition, fresh medium containing 0.04 µg BrdUrd/ml (Boehringer Mannheim) was added to the cultures for 44 h (two doubling times). During the final 5 h of culture, mitotic cells were arrested in metaphase with 0.01 µg/ml Colcemid (Life Technologies, Inc.). Metaphase preparation was done by standard cytogenetic procedures. 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 x 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 {alpha}-hRad51 antibody (1:100 dilution; Santa Cruz) or {alpha}-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 {alpha}-rabbit FITC (Santa Cruz) and {alpha}-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 µM 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 protease inhibitors mixture (Roche), and 1 mM sodium vanadate]. The Western blot analysis was performed as described in a previous report (10) . Briefly, 50 µg of proteins were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was then blocked and subsequently probed with specific antibodies, {alpha}-TFIIHp80 (XPD; 1:500 dilution; Santa Cruz), {alpha}-hRad51 (1:2,000 dilution; Santa Cruz), antitubulin (1:10,000 dilution; Medicorp), and {alpha}-Xrcc3 (1:1,000 dilution; a kind gift of Dr. P. Sung, Department of Molecular Biology, Howard Hughes Medical Institute, San Antonio, TX). Proteins were detected with horseradish peroxidase-{alpha}-mouse or horseradish peroxidase-{alpha}-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 µg of proteins from nuclear extracts were precleared with protein A-Sepharose or protein G-Agarose by incubation for 2 h at 4°C. The proteins present in the supernatant (1 mg/ml in lysis buffer containing proteases inhibitors) were immunoprecipitated overnight at 4°C with {alpha}-XPD (a kind gift from Dr. J. M. Egly) or {alpha}-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.

In Vitro Repair Assay, NER Activity.
The 2959-bp plasmid pSK (Stratagene) 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 DNA were isolated and purified. The pSK plasmid was then treated with cisplatin (0.5 µg cisplatin per 100 µg DNA) as described previously (15) . Nuclear extracts were also prepared according to a previous protocol (16) . Each reaction mixture contained 300 ng of damaged or untreated pSK plasmid, 40 µg of cell extract in reaction buffer containing 45 mM HEPES-KOH (pH 7.8), 7.4 mM MgCl2, 0.9 mM DTT, 0.4 mM EDTA, 2 mM ATP, 20 µM of each dGTP, dTTP and dCTP, 4 µM dATP, 40 mM phosphocreatine, 2.5 µg creatine phosphokinase, 3.4% glycerol, 18 µg BSA, and 4 µCi [{alpha}-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 µg/ml ethidium bromide. The gel was then fixed for 15 min in 15% methanol and 10% acetic acid, dried on Whatman filter paper, and exposed for autoradiography. For data presentation, autoradiographs were scanned and processed with the Adobe Photoshop software. Experiments were repeated three times and the mean ± SE determined.

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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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)Citation . XPD overexpression resulted in a 2–4-fold increase in cisplatin resistance (Fig. 2, A and B)Citation . 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 ± 1.3 and 23 ± 1.5 h, respectively).



View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. XPD constitutive overexpression was determined by Western blot analysis in 50 µg of protein extracts from MOCK cells, XPD cells, and SKMG-4 cells.

 


View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. The resistance to cisplatin-mediated DNA damage of MOCK ({square}) and XPD ({blacksquare}) cells was assessed by (A) sulforhodamine B assay 7 days after cisplatin treatment and by (B) MTT assay 48 h after cisplatin treatment. A, Y-axis (log scale) represents the percentage of surviving cells after treatment with cisplatin as compared with the untreated cells (control). Each value was calculated as (absorbance-treated cells/absorbance-untreated cells) x 100. The LD50 (50% of control), was calculated by interpolation using a linear regression between log (% of control) versus cisplatin concentration. B, Y-axis represents the percentage of cells after treatment with respect to the untreated (control). Each value was calculated as (absorbance-treated cells/absorbance-untreated cells) x 100. The results are expressed as the mean value of three independent experiments; bars, ±SE. * indicates significant differences between MOCK and XPD cells as determined by paired Student t test for means (P < 0.05).

 
XPD Overexpression Increases ICL Repair Rate and Melphalan Resistance without Affecting in Vitro NER Activity or UV Light Sensitivity.
Because XPD is an essential component of the NER-DNA repair pathway, we wanted initially to determine whether cisplatin resistance was related to proficient NER activity in XPD cells. Our results demonstrate that XPD overexpression did not alter in vitro NER activity (Fig. 3)Citation . 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)Citation . We then examined the kinetics of interstrand DNA cross-link formation and removal after cisplatin treatment. The results demonstrate that the kinetics of ICL formation were not affected by XPD overexpression; however, the rate of ICL removal was significantly accelerated by 3-fold (Fig. 5)Citation .



View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. The in vitro NER activity was determined in nuclear extracts of MOCK ({square}) and XPD ({blacksquare}) cell cultures. Y-axis represents the ratio between the {gamma}-P32 incorporation using as a substrate a cisplatin-treated plasmid and the {gamma}-P32 incorporation using as a substrate an untreated plasmid (top panel). The values represent the mean of three independent experiments; bars, ±SE. The bottom panel is a picture of a representative autoradiograph.

 


View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. The sensitivity to melphalan- (A) and UV- (B) mediated DNA damage of MOCK ({square}) and XPD ({blacksquare}) 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) x100]. The results are expressed as the mean value of three independent experiments; bars, ±SE. * indicate significant differences between MOCK and XPD. Cell survival as determined by paired Student t test for means (P < 0.05).

 


View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. MOCK ({square}) and XPD ({blacksquare}) cell cultures were treated with 50 µM cisplatin for 1 h and maintained in fresh complete medium for 4, 8, or 24 h (X-axis, Recovery time). The 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 (n = 6); bars, ±SE. * indicates a significant difference between MOCK ({circ}) and XPD ({bullet}) cells in the %CT (Student t test P < 0.05).

 
XPD Overexpression Mediates Increases in Both Rad51 Nuclear Foci Density and SCE after Cisplatin Treatment.
The next step was to evaluate whether another DNA repair pathway known to be involved in ICL repair, HRR, was affected by XPD overexpression. Rad51 is an essential component of the HRR pathway. Multienzymatic complexes, which include Rad51, localize in the cell nucleus after DNA damage. These complexes are visualized immunocytochemically as Rad51 nuclear foci and are believed to represent active DNA repair. The quantification of Rad51 foci density induced 24 h after cisplatin treatment in MOCK cells and XPD cells was determined. XPD overexpression resulted in a 1.8-fold increased rate of HRR as evidenced by an increase in cisplatin-induced Rad51 foci density in XPD cells (Fig. 6A)Citation . To additionally assess the effect of XPD overexpression in cisplatin-induced HRR, we analyzed the cisplatin-induced SCEs in both cell lines. In agreement with the Rad51 foci density results mentioned above, XPD cells showed a 1.5-fold increase in the number of SCEs per cell when compared with MOCK cells after treatment (Fig. 6B)Citation .



View larger version (20K):
[in this window]
[in a new window]
[Download PPT slide]
  		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 µM concentration (left panel). A, Rad51 foci density was calculated as described; Y-axis values represents the mean of two independent experiments (right panel); bars, ±SE. B, SCEs/cell (Y-axis) were determined 36 h after treatment with 0, 0.25, or 0.5 µM cisplatin (X-axis). The results represent the mean value of SCE determined in 10 cells; bars, ±SE. * indicates a significant differences by Student t test (P < 0.05) between MOCK ({square}) and XPD ({blacksquare}) cells.

 
XPD and Rad51 Proteins Coimmunoprecipitate in MOCK Cells and XPD Cells.
Recent reports suggest that the different DNA repair pathways are not independent. It is possible that XPD and Rad51 interact as part of a multienzymatic complex involved in ICL repair. Immunochemical analysis of XPD and MOCK cells revealed that XPD and Rad51 colocalize constitutively in the nucleus of these cells (Fig. 7A)Citation . Colocalized staining increases after cisplatin treatment of XPD and MOCK cells (Fig. 7B)Citation . We additionally investigated the possible association between Rad51 and XPD. Immunoprecipitation analysis of nuclear extracts from XPD and MOCK cells confirmed the immunocytochemical results, demonstrating that XPD and Rad51 interact constitutively (Fig. 8A)Citation . This association is increased after cisplatin treatment in both XPD and MOCK cells, suggesting that it is a physiological response to cisplatin-mediated DNA damage (Fig. 8B)Citation . Although basal Rad51 protein levels are not affected by XPD overexpression, after cisplatin treatment, Rad51 protein levels are specifically increased in XPD cells (Fig. 8C)Citation .



View larger version (48K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. XPD and Rad51 proteins localization was determined by immunocytochemistry 24 h after 25.0 µM 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.

 


View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. Sister cultures were maintained in complete medium with (+) or without (-) 25 µM cisplatin and collected 16 h later. Rad51-XPD interaction was tested by cross-coimmunoprecipitation of nuclear enriched extracts. A, Rad51 immunoprecipitation and (B) XPD immunoprecipitation. C, XPD and Rad51 protein levels were tested in whole cell lysates by Western blot. The blots were reprobed with an XRCC3 and tubulin antibody to asses equal protein loading.

 
XPD Overexpression Increases the Percentage of Cells Arrested in S Phase after Cisplatin Treatment.
FACS analysis confirmed our results that XPD overexpression did not affect the cell cycle profile in the absence of DNA damage (Fig. 9A)Citation . However, after cisplatin treatment, the percentage of cells arrested in S phase was higher in XPD cells compared with MOCK cells. Also, the percentage of XPD cells in G2/M and apoptotic phase were lower than that in MOCK cells (Fig. 9B)Citation .



View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 9. Changes in cell cycle progression of MOCK ({circ}) and XPD ({bullet}) cells before (A) and after treatment with 25 µM (B) cisplatin were addressed by FACS. The values correspond to the percentage obtained in a representative experiment (n = 3).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
XPD overexpression results in cisplatin drug resistance, and is associated with accelerated ICL removal and increased HRR. We 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, 20, 21, 22, 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). However, 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 both part of a large complex with other factors. XPD is known to interact with XPB, other TFIIH components, p53, and other proteins (27 , 28) . We detected both XPB (TFIIH89 subunit) and BRCA1 proteins in Rad51 and XPD immunoprecipitate (data not shown), in agreement with recent reports suggesting that HRR requires the assembly of multienzymatic complexes.

XPD overexpression does not affect cell cycle progression under basal conditions (in the absence of DNA damage). After cisplatin treatment, the percentage of cells in S phase is higher in XPD cells than in MOCK cells. Moreover, after cisplatin treatment, the percentage of cells in G2/M and apoptotic phase is lower in XPD cells than in MOCK cells. These results suggest that XPD-overexpressing cells are arrested in S phase, whereas mock-transfected cells undergo mitosis and cell death. Other researchers have shown that Rad51 foci formation in response to DNA damage takes place in postreplicative DNA during S phase concomitantly with the SCE process (23 , 29) . Our data lead us to hypothesize that a higher percentage of XPD cells compared with MOCK cells is undergoing HRR. This may be because XPD overexpression may alter the intra-S phase checkpoint induced after cisplatin treatment. Although our study was carried out in nonsynchronized cells, the increase in Rad51 protein levels after cisplatin treatment is in agreement with the higher percentage of XPD cells found in S phase after cisplatin treatment. This is in agreement with a previous report showing nuclear Rad51 protein level changes throughout the cell cycle, with a maximal expression in the S phase (30) .

In summary, our results support the hypothesis that the mechanism of DNA cross-linking agent resistance mediated by XPD involves an increase in homologous recombination after ICLs inducing agent treatment. To our knowledge, this is the first example of an NER protein, XPD, exhibiting a modulatory effect on HRR and DNA cross-linking agent resistance.


   FOOTNOTES
 
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. Back

2 These authors contributed equally to this work. Back

3 To whom reprint requests should be addressed, at the Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Côte Ste. Catherine, Montreal, Quebec, Canada H3T 1E2. Phone: (514) 340-8248; Fax: (514) 340-8302. Back

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. Back

Received 11/15/01. Accepted 7/29/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hoeijmakers J. H. Genome maintenance mechanisms for preventing cancer. Nature (Lond.), 411: 366-374, 2001.[Medline]
  2. Panasci L., Paiement J. P., Christodoulopoulos G., Belenkov A., Malapetsa A., Aloyz R. Chlorambucil drug resistance in chronic lymphocytic leukemia: the emerging role of DNA repair. Clin. Cancer Res., 7: 454-461, 2001.[Abstract/Free Full Text]
  3. Chaney S. G., Sancar A. DNA repair: enzymatic mechanisms and relevance to drug response. J. Natl. Cancer Inst., 88: 1346-1360, 1996.[Abstract/Free Full Text]
  4. Dabholkar M., Thornton K., Vionnet J., Bostick-Bruton F., Yu J. J., Reed E. Increased mRNA levels of xeroderma pigmentosum complementation group B (XPB) and Cockayne’s syndrome complementation group B (CSB) without increased mRNA levels of multidrug-resistance gene (MDR1) or metallothionein-II (MT-II) in platinum-resistant human ovarian cancer tissues. Biochem. Pharmacol., 60: 1611-1619, 2000.[Medline]
  5. Xu Z. Y., Chen Z. P., Malapetsa A., Alaoui-Jamali M., Bergeron J., Monks A., Myers T. G., Mohr G., Sausville E. A., Scudiero D. A., Aloyz R., Panasci L. C. DNA repair protein levels vis-à-vis anticancer drug resistance in the human tumor cell lines of the National Cancer Institute drug screening program. Anti-Cancer Drugs, 13: 511-519, 2002.[Medline]
  6. Egly J. M. The 14th Data Lecture. TFIIH: from transcription to clinic. FEBS Lett., 498: 124-128, 2001.[Medline]
  7. Damia G., Imperatori L., Stefanini M., D’Incalci M. Sensitivity of CHO mutant cell lines with specific defects in nucleotide excision repair to different anti-cancer agents. Int. J. Cancer, 66: 779-783, 1996.[Medline]
  8. Hoy C. A., Thompson L. H., Mooney C. L., Salazar E. P. [title] Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents. Cancer Res., 45: 1737-1743, 1985.[Abstract/Free Full Text]
  9. Thompson L. H., Shild D. Homologous recombinational repair of DNA ensures mammalian chromosome stability. Mutat. Res., 477: 131-153, 2001.[Medline]
  10. Wang Z. M., Chen Z. P., Xu Z. Y., Christodoulopoulos G., Bello V., Mohr G., Aloyz R., Panasci L. In vitro evidence for homologous recombinational repair in resistance to melphalan. J. Natl. Cancer. Inst., 93: 1473-1478, 2001.[Abstract/Free Full Text]
  11. Christodoulopoulos G., Malapetsa A., Schipper H., Golub E., Radding C., Panasci L. C. Chlorambucil induction of HsRad51 in B-cell chronic lymphocytic leukemia. Clin. Cancer Res., 5: 2178-2184, 1999.[Abstract/Free Full Text]
  12. De Jong S., Zijlstra J. G., Timmer-Bosscha H., Mulder N. H., Vries E. G. Detection of DNA cross-links in tumor cells with the ethidium bromide fluorescence assay. Int. J. Cancer, 37: 557-561, 1986.[Medline]
  13. Perry P. E., Wolff S. New Giemsa method for the differential staining of sister chromatids. Nature (Lond.), 258: 121-125, 1974.
  14. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acid Res., 11: 1475-1489, 1983.[Abstract/Free Full Text]
  15. Aboussekhra A., Biggerstaff M., Shivji M. K., Vilpo J. A., Moncollin V., Podust V. N., Protic M., Hubscher U., Egly J. M., Wood R. D. Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell, 80: 859-868, 1995.[Medline]
  16. Marinoni J. C., Rossignol M., Egly J. M. Purification of the transcription/repair factor TFIIH and evaluation of its associated activities in vitro. Methods (Orlando), 12: 235-253, 1997.[Medline]
  17. Wood R. D., Biggerstaff M., Shivji M. K. K. Detection and measurement of nucleotide excision repair synthesis by mammalian cells extracts in vitro. Methods: A Companion to Methods in Enzymology, 7: 163-175, 1995.
  18. Vogel U., Dybdahl M., Frentz G., Nexo B. A. DNA repair capacity: inconsistency between effect of over-expression of five NER genes and the correlation to mRNA levels in primary lymphocytes. Mutat. Res., 461: 197-210, 2000.[Medline]
  19. Lim D. S., Hasty P. A mutation in mouse Rad51 results in an early embryonic lethal that is suppressed by a mutation in P53. Mol. Cell. Biol., 16: 7133-7143, 1996.[Abstract]
  20. Thompson L. H., Shild D. The contribution of homologous recombination in preserving genome integrity in mammalian cells. Biochimie, 81: 87-105, 1999.[Medline]
  21. Tsuzuki T., Fujii Y., Sakumi K., Tominaga Y., Nakao K., Sekiguchi M., Matsushiro A., Yoshimura Y., Morita T. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad Sci. USA, 93: 6236-6240, 1996.[Abstract/Free Full Text]
  22. Takata M., Sasaki M. S., Tachiiri S., Fukushima T., Sonoda E., Schild D., Thompson L. H., Takeda S. Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol. Cell. Biol., 21: 2858-2866, 2001.[Abstract/Free Full Text]
  23. Sonoda E., Sasaki M. S., Morrison C., Yamaguchi-Iwai Y., Takata M., Takeda S. Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells. Mol. Cell. Biol., 19: 5166-5169, 1999.[Abstract/Free Full Text]
  24. Vispe S., Cazaux C., Lesca C., Defais M. Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation. Nucleic Acids Res., 26: 2859-2864, 1998.[Abstract/Free Full Text]
  25. Wang X. W., Yeh H., Schaeffer L., Roy R., Moncollin V., Egly J. M., Wang Z., Freidberg E. C., Evans M. K., Taffe B. G. p53 modulation of TFIIH-associated nucleotide excision repair activity. Nat. Genet., 10: 188-195, 1995.[Medline]
  26. Sturzbecher H. W., Donzelmann B., Henning W., Knippchild U., Buchhop S. p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. EMBO J., 15: 1992-2002, 1996.[Medline]
  27. Schultz P., Fribourg S., Poterszman A., Mallouh V., Moras D., Egly J. M. Molecular structure of human TFIIH. Cell, 102: 599-607, 2000.[Medline]
  28. Kohn K. W. Molecular interaction map of the mammalian cell cycle control and DNA repair systems. Mol. Biol. Cell, 10: 2703-2734, 1999.[Abstract/Free Full Text]
  29. Tashiro S., Walter J., Shinohara A., Kamada N., Cremer T. Rad51 accumulation at sites of DNA damage and in postreplicative chromatin. J. Cell. Biol., 150: 283-291, 2000.[Abstract/Free Full Text]
  30. Chen F., Nastasi A., Shen Z., Brenneman M., Crissman H., Chen D. J. Cell cycle-dependent protein expression of mammalian homologs of yeast DNA double-strand break repair genes Rad51 and Rad52. Mutat. Res., 384: 205-211, 1997.[Medline]



This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
Y. Yoshimoto, C. K. Augustine, J. S. Yoo, P. A. Zipfel, M. A. Selim, S. K. Pruitt, H. S. Friedman, F. Ali-Osman, and D. S. Tyler
Defining regional infusion treatment strategies for extremity melanoma: comparative analysis of melphalan and temozolomide as regional chemotherapeutic agents
Mol. Cancer Ther., May 1, 2007; 6(5): 1492 - 1500.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. Zorbas-Seifried, M. A. Jakupec, N. V. Kukushkin, M. Groessl, C. G. Hartinger, O. Semenova, H. Zorbas, V. Yu. Kukushkin, and B. K. Keppler
Reversion of Structure-Activity Relationships of Antitumor Platinum Complexes by Acetoxime but Not Hydroxylamine Ligands
Mol. Pharmacol., January 1, 2007; 71(1): 357 - 365.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
X. Lin and S. B. Howell
DNA mismatch repair and p53 function are major determinants of the rate of development of cisplatin resistance
Mol. Cancer Ther., May 1, 2006; 5(5): 1239 - 1247.
[Abstract] [Full Text] [PDF]

Home page
 Ann OncolHome page
R. de las Penas, M. Sanchez-Ronco, V. Alberola, M. Taron, C. Camps, R. Garcia-Carbonero, B. Massuti, C. Queralt, M. Botia, R. Garcia-Gomez, et al.
Polymorphisms in DNA repair genes modulate survival in cisplatin/gemcitabine-treated non-small-cell lung cancer patients
Ann. Onc., April 1, 2006; 17(4): 668 - 675.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
Z.-Y. Xu, M. Loignon, F.-Y. Han, L. Panasci, and R. Aloyz
Xrcc3 Induces Cisplatin Resistance by Stimulation of Rad51-Related Recombinational Repair, S-Phase Checkpoint Activation, and Reduced Apoptosis
J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 495 - 505.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
T. Okuda, X. Lin, J. Trang, and S. B. Howell
Suppression of hREV1 Expression Reduces the Rate at Which Human Ovarian Carcinoma Cells Acquire Resistance to Cisplatin
Mol. Pharmacol., June 1, 2005; 67(6): 1852 - 1860.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
F. Wu, X. Lin, T. Okuda, and S. B. Howell
DNA Polymerase {zeta} Regulates Cisplatin Cytotoxicity, Mutagenicity, and The Rate of Development of Cisplatin Resistance
Cancer Res., November 1, 2004; 64(21): 8029 - 8035.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
M. Taron, R. Rosell, E. Felip, P. Mendez, J. Souglakos, M. S. Ronco, C. Queralt, J. Majo, J. M. Sanchez, J. J. Sanchez, et al.
BRCA1 mRNA expression levels as an indicator of chemoresistance in lung cancer
Hum. Mol. Genet., October 1, 2004; 13(20): 2443 - 2449.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Yoshida and T. Morita
Control of Radiosensitivity of F9 Mouse Teratocarcinoma Cells by Regulation of Histone H2AX Gene Expression using a Tetracycline Turn-Off System
Cancer Res., June 15, 2004; 64(12): 4131 - 4136.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
R. Jensen and P. M. Glazer
Cell-interdependent cisplatin killing by Ku/DNA-dependent protein kinase signaling transduced through gap junctions
PNAS, April 20, 2004; 101(16): 6134 - 6139.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
X. Wu, W. Fan, S. Xu, and Y. Zhou
Sensitization to the Cytotoxicity of Cisplatin by Transfection with Nucleotide Excision Repair Gene Xeroderma Pigmentosun Group A Antisense RNA in Human Lung Adenocarcinoma Cells
Clin. Cancer Res., December 1, 2003; 9(16): 5874 - 5879.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aloyz, R.
Right arrow Articles by Panasci, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aloyz, R.
Right arrow Articles by Panasci, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online