Down-regulation of Rad51 and decreased homologous recombination in hypoxic cancer cells

Abstract

There is an emerging concept that acquired genetic instability in cancer cells can arise from the dysregulation of critical DNA repair pathways due to cell stresses such as inflammation and hypoxia. Here we report that hypoxia specifically down-regulates the expression of RAD51, a key mediator of homologous recombination in mammalian cells. Decreased levels of Rad51 were observed in multiple cancer cell types during hypoxic exposure and were not associated with the cell cycle profile or with expression of hypoxia-inducible factor. Analyses of RAD51 gene promoter activity, as well as mRNA and protein stability, indicate that the hypoxia-mediated regulation of this gene occurs via transcriptional repression. Decreased expression of Rad51 was also observed to persist in posthypoxic cells for as long as 48 h following reoxygenation. Correspondingly, we found reduced levels of homologous recombination in both hypoxic and posthypoxic cells, suggesting that the hypoxia-associated reduction in Rad51 expression has functional consequences for DNA repair. In addition, hypoxia-mediated down-regulation of Rad51 was confirmed in vivo via immunofluorescent image analysis of experimental tumors in mice. Based on these findings, we propose a novel mechanism of genetic instability in the tumor microenvironment mediated by hypoxia-induced suppression of the homologous recombination pathway in cancer cells. The aberrant regulation of Rad51 expression may also create heterogeneity in the DNA damage response among cells within tumors, with implications for the response to cancer therapies.

Figures

FIG. 1.

Transcriptome response to hypoxia after 24-h exposure of MCF-7 cells to 0.5% O2. (A) Histogram analysis of the approximately 48,000 transcripts (16,000 unique transcripts for each of three biological replicates) detected by the GenCompass microarray, based on H/N expression ratios (log2). (Inset) Percentages of genes up-regulated and down-regulated at two-, four-, and sixfold thresholds. (B) Selection of genes previously identified as regulated by hypoxia which were detected by the GenCompass microarray. Accession numbers are given for reference. H/N ratios are averaged from three independent experiments. NDRG1, N-myc downstream-regulated gene 1. (C) GenCompass H/N expression ratios of selected DNA repair genes, with the respective pathways listed. H/N ratios are averaged from two experiments. BER, base excision repair; MMR, mismatch repair. ERCC1, excision repair cross-complementing group 1; APEX2, apurinic/apyrimidinic endonuclease/redox factor 2; PMS2, postmeiotic segregation increased 2; RAD51, RAD51 homolog.

FIG. 2.

Decreased levels of Rad51 protein in cell lines in response to hypoxia or the Fe2+ chelator DFX, and determination of Rad51 protein stability in the presence of DFX. (A) Western blot analyses were performed to determine the expression of the HR-associated protein Rad51 in MCF-7 cells after exposure to normoxia (lanes N), hypoxia (0.5 or 0.01% O2) (lanes H), or DFX (250 μM). The time for which the cells were maintained under each condition (24 or 48 h) is shown. Expression of HIF-1α and Glut1 is shown for comparison, to verify that physiologically relevant levels of hypoxia were present in the treated cells. Note that Glut1 appears as several isoforms which are detected by our antibody. Tubulin expression is also presented to confirm equal sample loading. (B) Western blot analyses of Rad51 protein expression in A549, HeLa, SW480, and A431 cells after a 48-h exposure to normoxia or hypoxia (0.01% O2). Tubulin expression is presented to confirm equal loading of samples for HeLa and SW480 cells, while β-actin was used as a loading control for A549 and A431 cells. (C) Average reduction in Rad51 protein levels after a 48-h exposure to hypoxia (0.01% O2), as determined by densitometry analysis of Western blots generated from duplicate (RKO, A431, SW480, and PC3 cells) or triplicate (MCF-7, A549, and HeLa cells) hypoxia experiments. The tissue of origin for each cell line is shown. Reductions are expressed as H/N ratios normalized to the expression of β-actin and tubulin, and standard errors for each ratio are given. (D) To assess Rad51 protein stability, MCF-7 cells were either exposed to DFX (250 μM) or left untreated for 24 h, followed by coincubation with CHX (10 μg/ml) to block new protein synthesis. (Upper panels) Cells were harvested at the indicated times after addition of CHX, and Rad51 protein expression was determined by Western blotting. Expression of HIF-1α is shown to confirm both the induction of chemical hypoxia and successful abolition of new protein synthesis. In addition, tubulin protein levels were unchanged and served as standards to confirm equal loading of cellular protein samples. (Lower panel) Analysis of Rad51 protein expression at each time point after addition of CHX in cells exposed to DFX or left untreated, quantified as described in the legend to panel C. Each data point is the percentage of Rad51 protein remaining (after normalization to tubulin levels) in either DFX-treated or untreated cells at the indicated time.

FIG. 3.

Transcriptional repression of the RAD51 gene by hypoxia. (A) Northern blot analyses were performed on total RNA extracted from MCF-7 cells after exposure to normoxia (lanes N), hypoxia (0.01% O2) (lanes H), or DFX (250 μM). The time for which cells were maintained under each condition (24 or 48 h) is given. VEGF expression is shown for comparison, to verify that physiologically relevant levels of hypoxia were present in the treated cells, and expression of 28S rRNA is presented to confirm equal sample loading. (B) Northern blot analysis of RAD51 mRNA expression in A549, SiHa, and RKO cells after a 24- or 48-h exposure to hypoxia (0.01% O2). VEGF expression is shown for comparison, to verify that physiologically relevant levels of hypoxia were present in the treated cells. Expression of 28S rRNA (MCF-7, SiHa, and RKO cells) and β-actin mRNA (A549 cells) is presented to confirm equal sample loading. (C) To assess the stability of RAD51 mRNA, MCF-7 cells were either left untreated or exposed to DFX (250 μM) for 24 h, followed by coincubation with ActD (5 μg/ml) to block transcription. Cells were harvested at the indicated times after the addition of ActD, and RAD51 mRNA expression was determined by Northern blotting. Expression of VEGF is shown to confirm both the induction of chemical hypoxia and successful abolition of transcription. In addition, 28S rRNA levels were unchanged and served as standards to confirm equal sample loading. (D) Analysis of RAD51 mRNA expression at each time point after ActD addition in cells exposed to DFX or left untreated, as determined by phosphorimager analysis of Northern blots. Values are the percentage of RAD51 mRNA remaining in either DFX-treated or untreated cells at each time point, and error bars are based on standard errors calculated from duplicate experiments. (E) Schematic of the 5′-flanking region of the RAD51 gene, with delineation of the promoter fragment used for luciferase reporter gene assays (pGL3-Rad51p). Approximate locations of the core promoter regions, as described in the Eukaryotic Promoter Database (EPD) and as identified by in silico analysis using the Genomatix promoter identification algorithm PromoterInspector, are shown for reference. Bent arrow above exon 2 indicates the ATG translation start codon. (F) To determine the effect of hypoxia on RAD51 gene promoter activity, the pGL3-Rad51p luciferase (firefly) reporter plasmid was transiently transfected into RKO cells 4 h prior to normoxic or hypoxic exposure (for 48 h), immediately followed by measurement of luciferase activity. Firefly luciferase values were normalized to Renilla luciferase activity from a cotransfected pRL-SV40 control vector, and error bars are based on standard errors calculated from duplicate experiments. The activity of the luciferase reporter plasmid 5X-HRE, which contains five HREs tandemly ligated to a human cytomegalovirus minimal promoter, is shown as a control to confirm physiologically relevant levels of hypoxia. The activity of the promoterless luciferase reporter gene construct pGL3-Basic is also shown as a control.

FIG. 4.

Persistent down-regulation of Rad51 expression after hypoxia. (A) Western blot analyses were performed to determine the expression pattern of Rad51 protein in MCF-7 cells cultured under hypoxia for 48 h (0.01% O2) (lane H) and then following reoxygenation (R), as indicated by the timeline shown. Samples were obtained at 24-h intervals posthypoxia (lanes R), with the 72-, 96-, and 120-h time points representing 24, 48, and 72 h following reoxygenation, respectively. As a control to show that Rad51 levels do not change over the same period under normoxia, Rad51 protein expression in cells grown in parallel under consistently normoxic conditions is shown for each time point (lanes N). Expression of HIF-1α and Glut1 is shown for comparison, to verify that physiologically relevant states of hypoxia and reoxygenation were observed in the treated cells. Tubulin expression is also presented to confirm equal sample loading. (B) Northern blot analysis was performed to determine the expression of RAD51 mRNA in MCF-7 cells during the same time course and under the same conditions described for panel A. As indicated by the timeline shown, RAD51 mRNA expression was assessed at 24 and 48 h of hypoxia (lanes H). RNA samples were also analyzed at 24-h intervals posthypoxia (lanes R), with the 72- and 96-h time points representing 24- and 48-h periods of reoxygenation, respectively. As a control to show that RAD51 expression does not change over the same period under normoxia, RAD51 mRNA expression in normoxic cells grown in parallel is shown for each time point (lanes N). VEGF expression is shown for comparison, to verify that physiologically relevant states of hypoxia and reoxygenation were obtained in the treated cells. Expression of 28S rRNA is also presented to confirm equal sample loading.

FIG. 5.

Decreased Rad51 expression is not associated with the cell cycle profile. (A) Quantitative assessment of cell cycle profiles of four cell lines exposed to hypoxia (0.01% O2) or normoxia for 48 h. Calculated proportions are expressed as percentages based on triplicate hypoxia experiments using flow cytometric analysis of PI-stained cells and histogram analysis software. (B) Fold changes in both the percentage of cells in the G1 phase and the Rad51 protein level in hypoxia compared to normoxia, as calculated from panel A and Fig. 2C, respectively. (C) Semiquantitative RT-PCR analysis of RAD51 mRNA expression in isolated G1- and S-phase populations of normoxic and hypoxic cells. Equal numbers of cell cycle-specific populations were obtained by DNA staining of cells with the vital fluorochrome Hoechst 33342, followed by flow cytometric analysis and cell sorting. Unsorted cells processed in parallel are also shown for reference (lanes 1 and 2). VEGF expression is shown for comparison, to verify physiologically relevant states of hypoxia in normoxic and hypoxic cell populations, and the expression of β-actin is presented to confirm equal sample loading. (D) Cell cycle profiles of A549 cells exposed to either normoxia or hypoxia for 48 h and of A549 cells reoxygenated immediately following hypoxia for the indicated times, shown as histograms based on PI staining for DNA content. Approximate ranges of G1-, S-, and G2/M-phase populations are shown for reference. (Inset) Rad51 protein expression in the A549 cells at the corresponding time points. (E) S-phase proliferation in normoxic A549 cells or in A549 cells reoxygenated for the indicated times, as assessed by BrdU incorporation. Dotted lines represent the threshold for positive BrdU incorporation, based on cells assayed in parallel without BrdU incubation at each time point. Quantitative assessments of cell cycle profiles at each time point are shown in each panel and were calculated as described in the legend to panel A. The percentage of total cells in each sample that incorporated BrdU is given in parentheses.

FIG. 6.

Decreased Rad51 expression is not associated with HIF expression. (A) Western blot analyses were performed to determine the expression of Rad51 protein in log-phase 786-0 cells expressing a wild-type (+VHL) or mutant (−VHL) VHL gene. Expression of VHL and Glut1 is shown for comparison to verify the status of these cells. Tubulin expression is also presented to confirm equal sample loading. (B) Western blot analysis of Rad51 protein expression in 786-0 cells expressing either wild-type or mutant VHL following exposure to hypoxia (0.01% O2) for 48 h. (C) Western blot analysis of Rad51 and HIF-1α expression levels in HeLa cells 48 h after transfection with the HIF-1α expression vector pCEP4-HIF-1α.

FIG. 7.

Decreased HR in hypoxic and posthypoxic cells. (A) Schematic of the shuttle vector plasmid and donor DNA fragment used to assess HR. Plasmid pSupFG1/G144C contains a supFG1 gene with an inactivating G:C-to-C:G point mutation at position 144. The donor is a homologous DNA fragment containing a portion of the wild-type supFG1 gene. Abbreviations: ampr, ampicillin resistance gene; WT, wild type. (B) Recombination frequencies in MCF-7 cells cotransfected with the shuttle vector and donor fragment, followed by culture either under normoxic conditions alone (72 h) or under hypoxia (0.01% O2) for 48 h, immediately followed by reoxygenation and 24 h of normoxia (Hypoxia + post-hypoxia). The total number of blue colonies/ total number of colonies in each sample is given in parentheses. Error bars are based on standard errors calculated from duplicate experiments. (C) Recombination frequencies in MCF-7 cells cotransfected with the shuttle vector and donor fragment under normoxic conditions immediately following a 48-h exposure either to normoxia or to hypoxia (0.01% O2) (Post-hypoxia).

FIG. 8.

Inverse association between hypoxia marker staining and Rad51 expression in cervical and prostate cancer xenografts. Shown are immunofluorescence analyses of staining with the hypoxia marker EF5 (red) and Rad51 expression (green) in tumor xenografts derived from Me180 cervical cancer cells (A), PC3 prostate cancer cells (B), and SiHa6 cervical cancer cells (C). Individual EF5, Rad51, and DAPI staining (blue), and Rad51-EF5 merged staining (yellow), are shown in panel A. Arrowheads in panel A indicate substantially decreased Rad51 expression within a region of strong EF5 staining.