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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montréal, 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, Montréal, Quebec, Canada (F.-Y.H.)
Received January 21, 2005; accepted April 18, 2005.
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Abstract |
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; McHugh et al., 2001; West, 2003). The HRR process requires the assembly of multienzymatic complexes visualized 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 attenuated Rad51 focus formation after exposure to ionizing radiation (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 deficiency results in milder sensitivity to DNA cross-linking agents (2-fold) when compared with previous results in deficient hamster cells. Moreover, Xrcc3 deficiency resulted in increased endoreduplication.
Rad51C is involved in at least two complexes, one containing Rad51B-Rad51C-Rad51D-Xrcc2 and another containing Rad51C-Xrcc3 (Masson et al., 2001). Rad51C and Xrcc3 may be involved in Holliday junction resolution, whereas other Rad51 paralog members may be involved in branch migration processes (Liu et al., 2004).
One of the biological responses to DNA damage is to slow progression through S-phase as a consequence of activating a checkpoint. For example, following exposure to IR, cells activate the ATM kinase, which initiates a generalized response that includes the S-phase checkpoint pathway to delay DNA replication and allows the repair of DNA. In response to double strand break induction, ATM triggers two parallel cascades that cooperate to inhibit DNA replication, resulting in the S-phase checkpoint. The two parallel cascades involve ATM-dependent phosphorylation of Chk2 and Nbs1, respectively. The DNA damage-activated kinases Chk1 and Chk2 are also implicated in regulation of G2 checkpoints (Falck et al., 2002). p53 has been shown as well to be an important component of G1 and G2 arrest in response to DNA damage. The p53-responsive p21 protein inhibits cyclin-dependent kinases that drive cell cycle progression. p53-dependent p21 induction is thought to mediate G1 and G2 arrest in response to DNA. Although the IR-induced S-phase checkpoint summarized above has been extensively described, little is known about the molecular mechanisms involved in the S-phase checkpoint elicited in mammalian cells by DNA cross-linking agents. Enhanced DNA repair of ICLs produced by DNA cross-linking agents has been associated with resistance to these agents (Batist et al., 1989; Torres-Garcia et al., 1989; Spanswick et al., 2002). Moreover, increased Xrcc3 protein levels in cell lines and clinical samples correlated with DNA cross-linking agent resistance (Wang et al., 2001; Bello et al., 2002).
In the present study we investigate the consequences of the Rad51-related paralog, Xrcc3 overexpression in terms of cell cycle progression, Rad51-related homologous recombinational repair, and cell survival after cisplatin treatment in the breast cancer cell line MCF-7. Our results demonstrate that Xrcc3 mediates cisplatin resistance by a Rad51-dependent mechanism and suggest that crosstalk between HRR and cell cycle checkpoints may exist.
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Materials and Methods |
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). The Xrcc3 open reading frame sequence was subcloned into the pcDNA3.0 expression vector (Invitrogen, Carlsbad, CA), amplified, and stably transfected into the human breast cancer cell line MCF-7 using the Effectine reagent (QIAGEN, Valencia, CA) following the manufacturer's instructions. The transfected cells were maintained in medium for 36 h, trypsinized, and serially diluted. Single clones were amplified for 3 weeks in medium containing 600 µg/ml G418 (geneticin). Mock-transfected MCF-7 cells were obtained by transfection of the empty pcDNA3.0 expression vector.
Cell Survival Assay. The cells were seeded in 96-well plates until 50% confluent and then treated with cisplatin (CDDP; 0–100 µM) (Mayne, Montreal, QC, Canada) or melphalan (MLN; 0–100 µM) (Sigma-Aldrich, St. Louis, MO). Survival was assessed 7 days after treatment using the SRB colorimetric assay described previously (Batist et al., 1989; Aloyz et al., 2002; Bello et al., 2002). The IC50 (concentration of drug) that results in 50% of control was calculated as previously described (Batist et al., 1989; Aloyz et al., 2002; Bello et al., 2002). The IC50 values represent the mean and the 95% confidence intervals (CIs) of three independent experiments.
Annexin V Assay. Subconfluent cultures were treated with CDDP (0 or 20 µM), and the induction of apoptosis was determined as described before (Aloyz et al., 2004) using the Annexin V-EGF (BD Biosciences Clontech, Palo Alto, CA). Briefly, 0 to 36 h after treatment, floating and adherent cells were harvested, washed with phosphate-buffered saline, fixed, and stained following the manufacturer instructions. 7-Amino actinomycin D (BD Pharmingen, San Diego, CA) was used to discriminate apoptosis from necrosis. The cells were immediately subjected to bivariate analysis using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The percentage of Annexin V cells represents the mean value and the 95% CIs of two independent experiments.
FACS Analysis. Subconfluent cultures were treated with CDDP (0 or 20 µM) for 1 h, and the DNA content was determined by FACS analysis using propidium iodide as described before (Aloyz et al., 2002). Cell cycle analysis was performed using an EPICS XL-MCL fluorescent-activated cell sorter (Beckman Coulter, Fullerton, CA). The percentages represent the mean value and the 95% CIs of two independent experiments.
Rad51 Foci Density Determination. Rad51 foci density was determined as described previously with minor modifications (Wang et al., 2001; Aloyz et al., 2002). At various time points after 1-h exposure to 20 µM CDDP treatment, the cells were washed with phosphate-buffered saline, fixed, and stained with a specific Rad51 rabbit antibody (H-92; Santa Cruz Biotechnology, Santa Cruz, CA). A fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin (Santa Cruz Biotechnology) was used as secondary antibody, and the nuclei were counterstained with propidium iodide (Sigma-Aldrich).
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Sister Chromatid Exchanges (SCEs). Sister chromatid exchanges/cell were determined after treatment with cisplatin (0, 0.5, or 2.5 µM) as described before (Aloyz et al., 2002). Twenty-four hours after seeding, OVER and MOCK cells were treated with cisplatin. One hour after cisplatin treatment, fresh medium containing 0.04 µg of bromodeoxyuridine per milliliter (Roche Diagnostics, Indianapolis, IN) 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 (Invitrogen). Metaphase preparation was done by standard cytogenetic procedures. Differential sister chromatid staining was achieved by the fluorescence-plus-Giemsa method (Aloyz et al., 2002). Enumeration of SCEs was done without knowledge of treatment in 10-well spread second-division metaphases for each culture.
Western Blot Analysis. Western blot analysis was performed using specific antibodies to 
-Xrcc3 (1/1000 dilution; a kind gift of Dr. P. Sung, Howard Hughes Medical Institute, Department of Molecular Biology, Chevy Chase, MD), p53, p21 (1/2000 dilution; Lab Vision, Fremont, CA), phosphorylated Chk2 (Cell Signaling Technology Inc., Beverly, MA), extracellular signal-regulated kinase 2 (Santa Cruz), -tubulin (1/10,000 dilution; Medicorp, Montreal, QC, Canada), Rad51C (1/1000 dilution; 7898; Abcam Ltd, Cambridge, UK), and Rad51B (1/500 dilution; a kind gift of Dr. Jean-Ives Masson, Laval University, Laval, QC, Canada) as described (Wang et al., 2001; Aloyz et al., 2002).
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). Statistical Analysis. The results are expressed as the mean values ± 95% confidence intervals. Differences between mean values were assessed using the two-tailed, paired t test for means.
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Results |
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18 h after treatment. Significantly increased CDDP-induced apoptosis was observed in MOCK cells when compared with OVER cells 18, 24, and 36 h after CDDP. The majority of OVER cells were not apoptotic at 36 h.
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). Nevertheless, whereas Rad51 foci occur during the S-phase of the cell cycle, the SCEs are the reflection of postreplicational repair associated with crossing-over between sister duplexes (Sonoda et al., 1999). Because Xrcc3-null cells are hypersensitive to cisplatin and display deficient spontaneous or induced Rad51 foci and SCEs, we investigated the effect of Xrcc3 expression levels on Rad51 foci formation and SCEs before and after 10 µM CDDP treatment (Bishop et al., 1998; West, 2003). Rad51 foci nuclear density was similar in OVER and MOCK cells in basal conditions (data not shown). However, 12 h after treatment, when significant CDDP-induced apoptosis is not observed, drug-induced Rad51 foci density (Fig. 4), expressed either as density by field (Fig. 4B) or by nucleus (Fig. 4C), was increased by 2-fold in OVER cells with respect to MOCK cells. In contrast, there was no real difference in the percentage of increase of SCEs/cell induced by CDDP between OVER and MOCK cells (Fig. 5, A and B). However, surprisingly, Xrcc3 overexpression resulted in a 2-fold decrease in SCEs in the absence of induced DNA damage (Fig. 5, A and C).
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Xrcc3 Overexpression Alters the CDDP-Induced S-Phase Checkpoint. Xrcc3 overexpression did not affect the doubling time of MCF-7 cells (22 h), suggesting that although Xrcc3 expression is required for normal cell division, Xrcc3 overexpression does not alter this process in a Rad51-proficient background (Tebbs et al., 1995). Similarly, there were no significant differences between OVER and MOCK cells in the FACS profile in basal conditions (Fig. 6, left panel), suggesting that cell cycle progression is not affected by Xrcc3 overexpression in the absence of induced DNA damage. Early after CDDP treatment, there were no significant differences in cell cycle progression between MOCK and OVER cells. Six hours after CDDP treatment, MOCK and OVER cells undergo S-phase arrest (Fig. 6, middle panel). However, 12 h after treatment, a significantly higher percentage of OVER cells was arrested in S-phase when compared with MOCK cells (Fig. 6, right panel). Since S-phase checkpoint induction involves DNA synthesis arrest to allow DNA repair before the cells undergo mitosis (Cliby et al., 1998), CDDP-induced DNA synthesis arrest in OVER and MOCK cells after CDDP treatment was examined. There was no significant difference in DNA synthesis rate in the absence of CDDP treatment between the cell lines (data not shown). CDDP induced a transient DNA synthesis arrest (2–40 h) in both cell lines. However, the DNA synthesis arrest induced by CDDP was more pronounced in OVER than in MOCK cells (1.3- to 1.5-fold) (Fig. 7). Moreover, in keeping with the role of Chk2 in S-phase arrest, CDDP-induced Chk2 phosphorylation was more sustained in OVER cells (6–24 h) than in MOCK cells (6–12 h) (Fig. 8, A and B). At a later time point (36 h), when Chk2 phosphorylation was decreased in both cell lines to basal levels, the percentage of OVER cells in S-phase was higher than in MOCK cells (Fig. 8C).
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p53 Protein Levels Correlates with Increased CDDP-Induced Apoptosis in Mock Cells. We did not find differences in the inductions of p53 and p21 between MOCK and OVER in p53 protein levels 6 to 24 h after CDDP treatment (Fig. 8, A and B). However, 48 h after treatment, p53 levels were close to basal values in OVER cells, whereas in MOCK cells p53 levels remained elevated (Fig. 8, A and B).
Xrcc3 Protein Levels Correlate with Rad51C Protein Levels. Overexpression of Xrcc3 alters Rad51C protein levels. Increasing Xrcc3 protein levels correlate with Rad51C protein levels in the MOCK and OVER cell lines (Fig. 9), whereas Rad51B protein levels are not affected. This is consistent with the known protein-protein interaction of Xrcc3 and Rad51C (Masson et al., 2001). The increased Rad51C protein levels associated with overexpression of Xrcc3 are possibly secondary to altered protein stability.
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Discussion |
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). Our results demonstrate that Xrcc3 overexpression in MCF-7 cells results in increased survival after cisplatin treatment associated with enhanced homologous recombinational DNA repair and consequently decreased cisplatin-induced apoptosis. These results are consistent with previous reports suggesting that in primary human tumor cells and human tumor epithelial cell lines, increased HRR is associated with interstrand cross-linking agent drug resistance (Batist et al., 1989; Torres-Garcia et al., 1989; Christodoulopoulos et al., 1999; Slupianek et al., 2001; Wang et al., 2001; Bello et al., 2002; Spanswick et al., 2002). However, Xrcc3 overexpression in a lymphoblastoid cell line did not increase mitomycin C resistance in preliminary results (Wiese et al., 2002). The divergent results may be due to a difference in the origin of the cell lines.
The increased percentage of Xrcc3-overexpressing cells arrested in S-phase 12 h after cisplatin treatment may be the reflection of increased HRR. Alternatively and/or additionally, it is also possible that the Xrcc3-mediated S-phase arrest after cisplatin treatment reflects the activation of a checkpoint response that alters Rad51-related HRR. The observation that CDDP-induced DNA synthesis arrest and Chk2 phosphorylation is more sustained in Xrcc3-overexpressing cells than in mock-transfected cells suggests that Rad51 HRR and S-phase checkpoint activation are intertwined. Our biochemical results are consistent with a scenario in which cisplatin-induced S-phase arrest in MCF-7 cells would be mediated by Chk2 phosphorylation. The sustained Chk2 phosphorylation observed in Xrcc3-overexpressing cells suggests that Xrcc3 is affecting the S-phase checkpoint pathway activated by ATM/ATR signaling pathways. Moreover, it has been reported that Xrcc3 may be involved in cell cycle progression since Xrcc3 modulates replication fork progression on cisplatin-damaged chromosomes via its role in homologous recombination (Henry-Mowatt et al., 2003).
In MCF-7 cells, cisplatin-induced apoptosis is mediated by the induction of the tumor suppressor gene p53 (Lee et al., 1999). It has been shown that in breast cancer cell lines, cisplatin-induces S-phase arrest is independent of p53 status. Accordingly, Xrcc3 overexpression did not alter p53 or p21 protein levels up to 24 h after CDDP treatment. The late differences in p53 protein levels, 48 h following CDDP treatment, when the surviving cells are recovering from DNA synthesis arrest, may be secondary to a higher number of Xrcc3-overexpressing cells entering mitosis after a successful DNA repair, whereas mock-transfected cells undergo apoptosis. This hypothesis is sustained by the DNA-content profiles and the percentage of apoptotic cells observed 36 h after treatment.
Since HHR takes place during S/G2M phases of the cell cycle, it is possible that the prolonged/increased S-phase checkpoint observed in Xrcc3-overexpressing cells is the reflection of increased cisplatin-induced DNA repair. This hypothesis is sustained by the fact that Xrcc3-overexpressing cells displayed increased Rad51 foci formation and survival after cisplatin treatment when compared with mock-transfected cells. Furthermore, we have not demonstrated that Xrcc3 is necessary for activation of the S-phase checkpoint in response to cisplatin. Also, it is possible that the effect on S-phase checkpoint after cisplatin treatment may be due in part to differences in the stress response to DNA damage in Xrcc3-overexpressing cells. Whether or not Xrcc3 is required for the S-phase checkpoint is not known. This question could be further assessed using specific Xrcc3-siRNA inhibitors.
Increased Xrcc3 protein levels correlate specifically with an increased Rad51C protein level, suggesting that that Xrcc3 overexpression stabilizes Rad51C, presumably due to increased formation of the stabilizing Rad51C-Xrcc3 heterodimer (Masson et al., 2001). For example, it has been reported that a depletion of Rad51C protein in human cells causes a sharp reduction of Xrcc3 protein levels (Lio et al., 2004). Thus, Xrcc3 may indirectly modulate the HRR process via its interaction with Rad51C. Previously, a modest increment in Rad51C was seen with Xrcc3 overexpression (Wiese et al., 2002). Moreover, it has been shown that disruption of Xrcc3-Rad51C and Rad51B-Rad51C interaction using a peptide corresponding to the amino acids 14 to 25 of RAD51C-sensitized hamster cells to cisplatin and reduced cisplatin-induced Rad51 foci (Connell et al., 2004).
The differences observed in the effect of Xrcc3 expression levels on Rad51 foci formation (occurring during S-phase) and SCEs (postreplicative) may imply that these HRR-related processes involve different subpathways in which Xrcc3 plays different roles. In conclusion, our findings have clinical and biological implications. Xrcc3 may be useful as a marker of prognosis in the efficacy of DNA cross-linking agents in the treatment of human tumors. From the biological point of view, the results suggest the existence of crosstalk between HRR and the S-phase checkpoint machinery.
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Footnotes |
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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, confidence interval; FACS, fluorescence-activated cell sorting; SCE, sister chromatid exchange(s); MOCK, mock-transfected cells; OVER, Xrcc3-overexpressing cells.
Address correspondence to: Dr. Lawrence Panasci, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote Ste Catherine Road, Montreal, QC, Canada H3T 1E2. E-mail: lpanasci{at}hotmail.com
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, *, and ** indicate significant differences between MOCK and OVER cells. The respective p values are 0.042, 0.0016, and 0.045 (A). The bottom panel is the analysis of a representative FACS analysis of Annexin V positive cells (B).
