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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Xrcc3 Induces Cisplatin Resistance by Stimulation of Rad51-Related Recombinational Repair, S-Phase Checkpoint Activation, and Reduced Apoptosis

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.

	Abstract

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Eukaryotic cells respond to DNA damage by activation of DNA repair, cell cycle arrest, and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signaling other cellular responses. The Rad51-guided homologous recombination repair system plays an important role in the recognition and repair of DNA interstrand crosslinks (ICLs), and cells deficient in this repair pathway become hypersensitive to ICL-inducing agents such as cisplatin and melphalan. We investigated the possible role of the Rad51-paralog protein Xrcc3 in drug resistance. Xrcc3 overexpression in MCF-7 cells resulted in 1) a 2- to 6-fold resistance to cisplatin/melphalan, 2) a 2-fold increase in drug-induced Rad51 foci, 3) an increased cisplatin-induced S-phase arrest, 4) decreased cisplatin-induced apoptosis, and 5) increased cisplatin-induced DNA synthesis arrest. Interestingly, Xrcc3 overexpression did not alter the doubling time or cell cycle progression in the absence of DNA damage. Furthermore, Xrcc3 overexpression is associated with increased Rad51C protein levels consistent with the known interaction of these two proteins. Our results demonstrate that Xrcc3 is an important factor in DNA cross-linking drug resistance in human tumor cells and suggest that the response of the homologous recombinational repair machinery and cell cycle checkpoints to DNA cross-linking agents is intertwined.

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.

	Materials and Methods

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Cell Culture and Stable Transfection. MCF-7 cells were maintained as described (Batist et al., 1989). 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).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Xrcc3 constitutive overexpression was determined by Western blot analysis in 50 µg of protein extracts from MOCK cells and OVER cells (A). The cells were treated with cisplatin or melphalan 24 h after seeding. Cisplatin (dashed lines) and MLN (solid line) resistance determined by the SRB assay correlated with Xrcc3 protein levels (B). The y-axis represents the IC50 for the drugs, and the x-axis represents Xrcc3 protein levels. The open, gray, and black circles represent, respectively, the results obtained with MOCK, OVER1, and OVER2 cells, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 2. The resistance to CDDP (A) and MLN (B) was determined using the SRB assay 7 days after treatment. MOCK cells (open circles) and two clones of Xrcc3-overexpressing cells OVER1 (gray circles) and OVER2 (black circles) cells were treated with cisplatin or melphalan 24 h after seeding. The IC50 values in the tables (µM) represent the mean of three independent experiments ± 95% confidence intervals. The bottom panel is a representative survival assay used to obtain by interpolation the IC50 values.

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).

View larger version (23K): [in this window] [in a new window]   Fig. 3. MOCK and OVER cells were treated with CDDP and stained for Annexin V to assess induction of apoptosis. The y-axis indicates the percentage of Annexin V positive cells at a given time (x-axis) 0 to 36 h after 20 µM CDDP treatment. The percentage of MOCK and OVER cells are represented by open and black bars, respectively. The results represent the mean value of two independent experiments ± 95% confidence intervals. The asterisks , *, 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).

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.

	Results

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Xrcc3 Mediates CDDP and MLN Resistance. Two Xrcc3-transfected MCF-7 clones, OVER1 and OVER2, overexpress Xrcc3 by 2- and 4-fold, respectively, when compared with mock-transfected cells (MOCK) (Fig. 1A). The resistance of CDDP and MLN in these cells correlates with Xrcc3 protein levels (r = 0.95) (Fig. 1B). The Xrcc3-overexpressing clones displayed resistance to CDDP (OVER1, 3.4-fold; and OVER2, 4.4-fold) and MLN (OVER1, 2.5-fold; and OVER2, 4-fold) when compared with the MOCK cells (Fig. 2, A and B). The results suggest that Xrcc3 is an important factor in DNA cross-linking agent cytotoxicity since Xrcc3 protein expression correlates with increasing CDDP/MLN resistance. We wanted to determine next whether the differences in drug resistance between MOCK and OVER2 cells (OVER) are due to a difference in CDDP-induced programmed cell death. We assessed differences in Annexin V expression between MOCK and OVER cells after CDDP treatment. We used a 1-h 20 µM CDDP exposure, and we assessed differences in CDDP-induced apoptosis in a two-doubling time period (48 h). In this period of time, the IC₅₀ determined by SRB (7 days after treatment) did not induce detectable apoptosis. We stained MOCK and OVER cells for Annexin V 0 to 36 h after CDDP treatment (Fig. 3). Our results show that CDDP induces some apoptosis in MOCK and OVER cells at 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.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Sister cultures of MOCK (open) and OVER (solid) cells were used to determine the effect of Xrcc3 overexpression on HRR as assessed by Rad51 foci determination after cisplatin treatment at 0 or 10 µM. Rad51 foci density (A). Rad51 density was expressed as (B) percentage of control per field (y-axis) versus time after treatment (x-axis) or (C) percentage of control per nucleus (y-axis) 12 h after cisplatin treatment. The values represents the mean of three independent experiments ± 95% confidence intervals; * and ** indicates significant difference p = 0.012 and 0.19 x 10–9, respectively.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Sister cultures of MOCK (open) and OVER (solid) cells were used to determine the effect of Xrcc3 overexpression on HRR as assessed by quantification of SCEs. SCEs per cell were quantified 36 h after treatment with 0.5 or 2.5 µM cisplatin (x-axis). The results are expressed as percentage of control (0 µM cisplatin; y-axis) (A) or per cell (B) of the mean value of SCEs per cell determined in 25 cells ± 95% confidence intervals; *, **, and indicates significant difference, p = 0.003 and p = 0.00043 and 2.6 x 10–6, respectively.

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).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Cell cycle progression was determined as described under Materials and Methods in sister cultures of MOCK (open) and OVER (solid) cells. The DNA content of untreated (left panel) or treated cells, 6 h (middle panel) and 12 h (right panel) after 20 µM cisplatin treatment was determined by FACS analysis after DNA labeling with propidium iodide. The values represent the mean of three independent experiments ± 95% confidence intervals. Asterisk indicates significant difference, p = 0.024 (A). Representative plots obtained by FACS analysis are reproduced in B.

View larger version (14K): [in this window] [in a new window]   Fig. 7. The effect of Xrcc3 overexpression on cisplatin-induced DNA synthesis arrest was determined as described under Materials and Methods in sister cultures of MOCK (open) and OVER (solid) cells. Subconfluent cultures were plated in the presence of [14C]thymidine. Forty-eight hours later (70–80% confluence), the cells were treated for 1 h with 0 or 10 µM cisplatin. The DNA synthesis progression was assessed by a 1-h [3H]thymidine chase (x-axis; 0, 2, 6, 12, and 24 h after treatment). The values represent the mean of three independent experiments ± 95% confidence intervals; the asterisk indicates significant differences: *, p =0.0096, **, p = 0.0074, and ***, p = 0.0012.

View larger version (29K): [in this window] [in a new window]   Fig. 8. The effect of Xrcc3 in signaling proteins involved in cell cycle arrest and apoptosis was determined as described under Materials and Methods in protein extracts of MOCK and OVER cells by Western blot at different times after 20 µM CDDP treatment (A). Changes in p53 and p21 protein levels relative to extracellular signal-regulated kinase 2 and Chk2 phosphorylation status (y-axis) after CDDP treatment (0–48 h; x-axis) are represented in the right panel (B). The results are a representative experiment of three independent determinations. Cell cycle progression was analyzed 36 h after treatment as described in Fig. 6 (C). The profiles are a representative experiment of two independent determinations.

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.

View larger version (23K): [in this window] [in a new window]   Fig. 9. Protein extracts from MOCK cells (open symbols) and OVER cells (solid symbols) were separated by 12% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane and probed sequentially with specific antibodies against Xrcc3, Rad51B, and Rad51C (A, left panel). The signals in each lane were analyzed using the Scion Image software (A, right panel; Scion Corporation, Frederick, MD). The Xrcc3 optical density (x-axis) correlates with Rad51C optical density (circles) but not with Rad51B optical density (squares). The linear regression analysis between the optical densities was obtained using the Microsoft Excel Statistical Tool Pack (B; Microsoft, Redmond, WA). The results indicate that there is a significant linear correlation (p = 0.036, r = 0.902) between Xrcc3 and Rad51C expression.

	Discussion

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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.

	Footnotes

 
Supported by a National Cancer Institute Cancer Research Society (Toronto, ON, Canada) grant and a generous private donation from the Helen and Nicky Rosenbloom Lang Scholarship to L.P., a U.S. Army grant and a Lymphoma Foundation of Canada fellowship to R.A., and a Leukemia Lymphoma Fund of Canada Fellowship to Z.-Y.X.

doi:10.1124/jpet.105.084053.

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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