Article pubs.acs.org/crt
Unpredicted Downregulation of RAD51 Suggests Genome Instability Induced by Tetrachlorobenzoquinone Xiufang Song, Qiong Shi, Zixuan Liu, Yawen Wang, Yuxin Wang, Erqun Song, and Yang Song* Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, People’s Republic of China ABSTRACT: We previously demonstrated that halogenated quinone induces DNA double strand breaks (DSBs) in a ROS-dependent manner, which coordinates with downstream repair cascade including nonhomologous end joining, base excision repair, and nucleotide excision repair. However, these error-prone processes may cause the potential risk of genome instability, and current has no information on how faithful repair route, such as homologous recombination (HR), was affected. RAD51 is a key protein in the HR pathway of DSBs repair. Here, we found that tetrachlorobenzoquinone (TCBQ) causes a time-dependent reverse U-shape biphasic trend of RAD51 expression. An increase in the early stage and a following decrease of RAD51 expression were found in both 12.5 and 25 μM TCBQ groups, wherein higher concentration faced a faster response. The upregulated RAD51 in the early phase suggested the attempting to repair TCBQinduced DNA damage; however, the downregulation of RAD51 in the late phase implicated that the rescue probably be abandoned with severe DNA damage. This phenomenon is a general toxic manner of TCBQ regardless of cell type. Surprisingly, TCBQ showed minimum effect on RAD51 mRNA (or protein) synthesis as well as RAD51 degradation. Specific inhibition of RAD51 by siRNA amplified TCBQ-induced DNA damage and cytotoxicity, while cells with enhanced RAD51 expression resisted TCBQ-induced toxicity. The modulation of RAD51 is correlated with p53 level, which suggests p53 has a role in TCBQ-induced RAD51 clearance. Together, our data suggested that TCBQ increases genome instability and cell death through a unique mechanism of inducing DNA damage and inhibiting DNA repair.
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linked with the development of carcinogenesis.8 Fortunately, cells rapidly respond to DNA damage by stimulating intricate signaling pathways, referred to as DNA damage response (DDR). Bernstein et al. summarized five major DNA repair pathways, namely, homologous recombination (HR), nonhomologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER), and mismatch repair (MMR).9 Among these signaling pathways, sensor proteins are responsible for the recognition of DNA lesions, and then transducers convey the damage signal to downstream effectors. However, these signal molecules not only orchestrate DNA repair and pro-survival, but also cell cycle arrest and cell death. On the occasion of irreversible DNA damage, a switching mechanism that allows the signal shift from DNA repair to apoptosis appears to prevent unrepaired damage-caused carcinogenesis and is responsible for keeping faithful genome transmission. DSBs are the most severe form of DNA damage. Two major repair pathways for DSBs are NHEJ and HR, which are homology-independent and homology-dependent recombination, respectively.10 NHEJ is an error-prone process because it does not require the sequence homologies during the ligation of damaged DNA. On the contrary, HR takes advantage of large sequence homologies to repair DSBs. NHEJ and HR are
INTRODUCTION Tetrachlorobenzoquinone (TCBQ) is the major genotoxic and carcinogenic quinoid metabolite of a widely used wood preservative and biocide pentachlorophenol (PCP). Long-term exposure of PCP increased the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine, in the liver of mice.1 Analysis of hepatic DNA adducts in rats exposed to PCP indicated the formation of TCBQ−DNA conjugates,2 which were further confirmed by the direct characterization of a deoxyguanosine adduct of TCBQ.3 Moreover, TCBQ forms a mixture of hemoglobin and albumin adducts in rat blood (both in vitro and in vivo studies), which also confirmed the nucleophilic feature of TCBQ.4 The increases in abasic sites and level of 8oxodeoxyguanosine upon TCBQ treatment supported that these damages are also derived from ROS damage.5 3-(2′-Deoxy-β-Derythro-pentofuranosyl)-pyrimido[1,2-α]-purin-10(3H)-one (M1G-dR) is a biomarker of DNA damage from primary ROS insults to the DNA backbone.6 Jeong et al. demonstrated that TCBQ promoted the level of M1G-dR,7 which implied hydroxyl radical generated from TCBQ plays an important role in the formation of DNA lesions and DNA strand breaks.5 Alkylation DNA damage or ROS-derived DNA damage resulted in single strand breaks (SSBs), double strand breaks (DSBs), DNA cross-linking, apurinic/apyrimidinic sites, purine/ pyrimidine bases, or deoxyribose backbone modifications. If these damages have not been repaired or removed before replication, they might result in chromosome instability, which © XXXX American Chemical Society
Received: October 6, 2016 Published: November 8, 2016 A
DOI: 10.1021/acs.chemrestox.6b00369 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology
time courses. Solvent control group was cultured in medium containing 0.1% DMSO. Cell Viability. In brief, cells were seeded in 96-well culture plates at 104 cells/well. Cells were permitted to adhere overnight at 37 °C and incubated with TCBQ. CCK-8 solution (10 μL of kit reagent) was added to each well, and the plates were maintained at 37 °C for 3 h. Then the optical density (OD) value was read at 450 nm using a microplate reader (BioTek ELX800, Vermont). Experiments were conducted in triplicate, and results were expressed as a percentages of DMSO controls. Single Cell Gel Electrophoresis (SCGE) Assay. SCGE assay, also known as alkaline comet assay (pH ≥ 13), was used to measure DNA damage following exposure to TCBQ. The frosted glass microscope slide was precoated with a layer of normal-melting agarose (0.8% in PBS) and baked at 37 °C. After exposure to TCBQ for indicated times, MDA-MB-231 cells were collected. Fifty-microliter aliquots of cell suspensions (106 cells/mL) were mixed with 50 μL of low-melting agarose (0.8% in PBS), immediately pipetted onto a frosted glass microscope slide coated with 100 μL of normal-melting agarose, and covered with a coverslip for 40 min at 4 °C. Then the coverslip was gently removed and slide was covered with 100 μL of low-melting agarose, and agarose allowed to solidify for 20 min at 4 °C. After which, the coverslip was removed, and slides were incubated in cold lysis buffer (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% DMSO, with NaOH used to adjust pH to 10.0) in darkness at 4 °C for 1 h. The slide was washed twice with cold PBS and then placed in a cold fresh alkaline solution (1 mM Na2EDTA and 300 mM NaOH, PH ≥ 13) at room temperature for 30 min to allowed DNA denaturation. Thereafter, electrophoresis was performed at 25 V and 300 mA for 30 min. After electrophoresis, the slide was neutralized twice with 0.4 M Tris buffer (pH = 7.5) at 4 °C for 15 min. The slide was washed twice in distilled water and stained with 10 μg/mL of ethidium bromide for 10 min, and an image was taken by fluorescent microscope (OLYMPUS IX71). Comets were coded, and at least 100 randomly selected cells were analyzed per sample with Comet Assay Software Project (CASP, Wroclaw University, Poland), and Olive tail moment (OTM) was recorded to describe DNA damage to cells. Three independent experiments were conducted. Protein Extraction and Quantification. Approximately 106 cells were plated, and after different treatments, cells were washed twice with cold PBS and lysed with RIPA lysis buffer (Dingguo Biotechnology Co., Ltd. Beijing, China) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and proteinase inhibitor cocktail. The lysate was incubated on ice for 30 min and homogenized with an ultrasonicator for 5 min. The cell lysate was collected by centrifugation at 10 000g for 10 min. The supernatant was collected, and the concentration of protein was determined by Bradford Protein Assay Kit. Whole cell protein lysate was solubilized in loading buffer. For nuclear and cytosolic proteins, they were extracted by a nuclear/cytosol fractionation kit (Sangon Biotech Co., Ltd. Shanghai, China) according to the manufacturer’s instructions. The whole or cytosolic and nuclear fractions were stored at −20 °C, respectively. Western Blotting Analysis. Cell extracts were resolved by electrophoresis on standard 12.5% or 15% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. The resulting filters were blocked with 5% BSA or 5% nonfat dried milk at 37 °C for 1.5 h, and membranes were washed with TBST three times and incubated with the appropriately diluted primary antibodies at 37 °C for 3 h or at 4 °C overnight. The membranes were washed and further incubated with the secondary goat antirabbit IgG-HRP-conjugated antibody (1:3000 dilution) for 2 h, followed by visualization using the HRP substrate DAB system or ECL system. The relative densities of protein expression were quantitated by ImageJ software (National Institutes of Health, Maryland, USA). Protein levels were standardized by comparison with β-actin in different cell substrates. RNA Extraction and Reverse Transcription Quantitative PCR (RT-qPCR). The MDA-MB-231 cells were seeded in 100 mm Petri dishes for 24 h and then treated with TCBQ (or DMSO as a control) for desired time. Total RNA was extracted with a total RNA Purification Kit (BioTeke, Beijing, China) as described in the manufacturer’s manual.
considered competitive, and they can also cooperate with each other.11 Interestingly, NHEJ and HR take over sequentially in the early and late in S-phase, correspondingly.12 Our previous study illustrated that polychlorinated biphenyl quinone activates NHEJ, BER, and NER signalings.13 Nevertheless, whether quinone compound has implication in HR signaling has not been detected. Mammalian RAD51 protein is a strand transferase, which represents an essential component of HR repair pathway.14 RAD51 assembles into helical polymers that bind to damaged DNA that facilitates the annealing to a complementary homologous strand on the intact chromatid. After homologous strand invasion to form a D-loop, RAD51 is then removed from DNA to allow the DNA synthesis to progress across the break site. Understanding the molecular basis of DNA repair pathway is important for gaining insight into TCBQ-induced toxicity. The aim of the current study is to reveal the possible molecular mechanisms of TCBQ-induced DDR and provide information on the regulation of “survival” and “death” fate of TCBQ-exposed cells. The effect of TCBQ on RAD51 expression and alternatively, the protective role of RAD51 on TCBQ-induced DNA integrity insult were studied. The putative role of p53 signaling pathway on TCBQ-caused RAD51 downregulation was also discussed.
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MATERIALS AND METHODS
Chemicals and Reagents. TCBQ (CAS number: 118−75−2) was purchased from Aladdin Reagent Database Inc. (Shanghai, China). A stock solution of TCBQ (50 mM) was prepared in DMSO before use. RAD51, p53, Lamin B, cleaved caspase-3, and Alexa Fluor 488-labeled goat antirabbit IgG (H+L) antibodies were purchased from Proteintech Group Co. Ltd. (Wuhan, China). β-Actin, p-p53, and HRP-conjugated goat antirabbit IgG (H+L) antibodies were supplied by Sangon Biotech Co., Ltd. (Shanghai, China). Antibodies against phospho-histone H2A.X (Ser139) (γ-H2AX) were purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Ubiquitin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Pifithrin-α (a p53 inhibitor), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), EasyBlot ECL kit, and protein (A+G) agarose beads were obtained from Beyotime Institute of Biotechnology (Nanjing, China). The proteasome inhibitor MG132 was obtained from Selleck Chemicals (Shanghai, China). Cycloheximide (CHX) and actinomycin D (ActD) were obtained from YuanYe Biotechnology Co, Ltd. (Shanghai, China). Cell counting kit-8 (CCK-8) was purchased from Genview (Shanghai, China). Ethidium bromide, agarose (normal and low melting point), Bradford Protein Assay Kit, and RT-qPCR amplification primers of RAD51 and β-actin were supplied by Dingguo Biotechnology Co., Ltd. (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Keygen Biotech (Nanjing, China). RAD51, p53, and control siRNA, lipofectamine 2000, and siRNA-mate transfection reagents were purchased from GenePharma Co. Ltd. (Shanghai, China). pEGFP-N1-RAD51 plasmid was constructed by Zhongding Biological (Nanjing, China). All other chemicals were of the highest grade commercially available and used without further purification. Cell Culture and Treatment. The breast carcinoma MDA-MB-231 and rat pheochromocytoma PC12 cell lines were purchased from Nanjing KeyGEN Biotech. Co. Ltd. (Nanjing, China). Human liver hepatocellular carcinoma HepG2, human lung cell carcinoma A549, and human breast carcinoma MCF-7 cell lines were purchased from the Third Military Medical University (Chongqing, China). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co. Ltd.) and antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere of 5% CO2. Cells were exposed to TCBQ at concentrations between 0 and 50 μM in serum-free media for desired B
DOI: 10.1021/acs.chemrestox.6b00369 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Figure 1. TCBQ induced the loss of cell viability, DNA damage, and apoptosis in MDA-MB-231 cells. (A) The effect of TCBQ on MDA-MB-231 cell viability. The cell viability was performed using CCK-8 assay after the exposure to TCBQ (0−50 μM) for indicated times. Results are displayed as the means ± SD of the three independent experiments. ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 indicate significant difference from cells exposed to control group. (B) DNA damage induced by TCBQ was assessed by the comet assay. Representative images were shown. Each figure represents the typical comet tails of the 150 observed cells. Data represent the mean values ± SD obtained in three independent experiments. ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001 indicate significant difference from cells exposed to control group. (C) Western blotting analysis of the levels of γ-H2AX and cleaved caspase-3 after treatment with TCBQ. β-actin was used as a loading control. Immunoprecipitation (IP) Assay. For IP, whole cell lysates were precleared with Protein (A+G) Agarose beads at 4 °C for 1 h. The cell lysate was collected by centrifugation at 450 g for 5 min. Then 2 mg lysates obtained were incubated with approximately 3 μg of anti-RAD51 or anti-p53 antibody and mixed with 100 μL of Protein (A+G) agarose beads overnight at 4 °C. Then IP solutions were collected by centrifuging at 450g for 5 min, and supernatant was separated. Then the pellets were washed five times with PBS. The complexes from each sample were eluted with loading buffer, heated at boiling water for 8 min, and subsequently analyzed by Western blotting. Small RNA (siRNA) Interference. The MDA-MB-231 cells were seeded in 100 mm Petri dishes and transfected at 30−50% confluence with 50 nM p53 or RAD51 siRNA using siRNA-mate transfection reagent according to the manufacturer’s instructions. The sense-strand sequences of siRNA duplexes used were as follows: p53 siRNA sense, 5′GGAAGACUCCAGUGGUAAUTT-3′, and antisense, 5′AUUACCACUGGAGUCUUCCAG-3′, RAD51 siRNA sense, 5′GAGCUUGACAAACUACUACUUTT-3′, and antisense, 5′AAGUAGUUUGUCAAGCUCTT-3′, control siRNA, 5′UUCUCCGAACGUGUCACGUTT-3′, and antisense, 5′ACGUGACACGUUCGGAGAATT-3′. After 12 h transfection, fresh medium was added to each cell, and cells were cultured for another 24 h. The cells were treated with TCBQ for 6 h and then were collected for further examination. Plasmid Transfections. For RAD51 expression vector, cDNA was cloned into the expression vector pEGFP-N1. Transfections were done in MDA-MB-231 cells using the Lipo2000 transfection reagent according to the manufacturer’s instructions. The GFP-positive cells were sorted and analyzed for GFP-tagged RAD51 by Western blotting with anti-RDA51. After 24 h of transfection, cells were used for further experiments. Statistical Analysis. For each protocol, at least three independent experiments and results were expressed as mean ± standard deviation (SD). The statistical significance of the differences was evaluated by a
RNA was reversely transcribed into cDNA (Roche, Switzerland) to perform RT-qPCR analysis using LightCycler 96 instrument protocol with Faststart Essential DNA Green Master (Roche, Switzerland). The following oligonucleotide primer pairs were used to construct the probes: 5′-AGGTGAAGGAAAGGCCATGTAC-3′, and reverse, 5′CATATGCTACATTATCCAGGACATCA-3′ were used to amplify human RAD51, and the primers forward, 5′-TCCTCCCTGGAGAAGAGCTAC-3′, and reverse, 5′-TCCTGCTTGCTGATCCACAT3′ were used to amplify human β-actin as a housekeeping gene. These primers were designed based on the human gene sequences. Three independent replicates were analyzed per sample, and the relative gene expression normalized to the internal control gene β-actin was obtained by the 2−ΔΔCt method. Immunofluorescence Staining. TCBQ-induced RAD51 expressions were measured by immunofluorescence analysis. Briefly, cells were treated with TCBQ for indicated concentrations and periods. Cells were washed briefly with cold PBS and were fixed in 4% paraformaldehyde with 3% sucrose for 20 min at room temperature and then blocked in 5% BSA blocking buffer at room temperature for 1 h. Cells were incubated with respective primary antibody (1:500 dilution for RAD51 detection) for 2 h at room temperature. After three times washing with PBS, they were developed with Alexa Fluor 488-labeled goat antirabbit IgG (H+L) antibody (1:250 dilution). Finally, the sections were counterstained with DAPI (1:250 dilution) for 10 min and analyzed under fluorescence microscope (OLYMPUS IX71). Protein and mRNA Stability Analyses. Cells were plated in 60 mm diameter dishes and allowed to attach for 24 h, followed by a 12 h incubation in the presence or absence of TCBQ. CHX (10 μg/mL), as an inhibitor of protein synthesis, or ActD (5 μg/mL), as an inhibitor of transcription, was then added to the cell culture. Cells were harvested at the indicated times after the addition of inhibitor. RAD51 protein and mRNA levels were determined by Western blotting and RT-qPCR, respectively. RAD51 protein or mRNA were deduced from the regression line based on protein or mRNA degradation plots. C
DOI: 10.1021/acs.chemrestox.6b00369 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Figure 2. TCBQ regulated the expression of RAD51 in MDA-MB-231 cells. (A) Western blotting analysis was performed to determine the expression of the protein RAD51 in MDA-MB-231 cells after exposure to TCBQ. β-actin expression was also presented to confirm equal sample loading. (B) RTqPCR analysis was performed on total RNA extracted from MDA-MB-231 cells after exposure to TCBQ. (C) MDA-MB-231 cells were treated with 25 μM TCBQ for indicated times, and RAD51 nuclear and cytoplasmic protein expressions were assessed by Western blotting. Fold change in protein expression over untreated control normalized to either Lamin B or β-actin for nuclear and cytoplasmic fraction, respectively. (D) Effect of TCBQ on RAD51 foci performed with immunofluorescence analysis. Cells were treated with 25 μM TCBQ for indicated times and were stained with anti-RAD51 antibody or DAPI. The merged images of cells showed RAD51 foci.
exposure to TCBQ (0−50 μM) for indicated times. TCBQ exposure caused concentration and time-dependent loss of cell viabilities. TCBQ dosage regimen in the following investigation was used based on cell viability results; therefore, concentrations of TCBQ up to 25 μM were used in the subsequent experiments to avoid artifact result from cell death. TCBQ-induced DNA DSBs were then assessed by two different methods. When cells were treated with 25 μM TCBQ for different times and subjected to SCGE, significant increases in
one-way ANOVA. Differences in measured variables between experimental and control groups were assessed using an unpaired student’s t test. P < 0.05 was considered statistically significant.
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RESULTS AND DISCUSSION TCBQ Induces the Loss of Cell Viability, DNA Damage, and Apoptosis in MDA-MB-231 Cells. The cytotoxic effect of TCBQ toward MDA-MB-231 cells was represented in Figure 1, panel A. Cell viability was determined by CCK-8 assay after D
DOI: 10.1021/acs.chemrestox.6b00369 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology
Figure 3. TCBQ regulated the expression of RAD51 protein in different cells. Hepatocellular carcinoma cell HepG2, human lung cancer cell A549, human breast carcinoma cell MCF-7, and rat pheochromocytoma PC12 cells were treated with TCBQ, and the levels of RAD51 in TCBQ-treated lysates were determined by Western blotting. β-actin was used as the loading control. Shown here is the representative blot from at least three independent experiments.
Figure 4. Influence of TCBQ on RAD51 protein and mRNA stabilities in MDA-MB-231 cells. (A) Western blotting showed that TCBQ leads to RAD51 downregulation and is independent of ubiquitin (Ub)-proteasome system. Cells were treated with DMSO or TCBQ (25 μM) in the presence or absence of MG132 (10 μM) for 6 h, followed by IP and Western blotting. (B) MDA-MB-231 cells were either exposed to TCBQ (12.5 μM) or DMSO for 12 h, followed by coincubation with CHX (10 μg/mL) to block new protein synthesis. RAD51 and β-actin protein expressions were determined by Western blotting. Quantification of RAD51 protein expression at each time point after addition of CHX in cells exposed to TCBQ or DMSO. Each data point is the percentage of RAD51 protein remaining (after normalization to β-actin) in either TCBQ-treated or control cells at the indicated time. (C) MDAMB-231 cells were either exposed to TCBQ (12.5 μM) or DMSO for 12 h, followed by coincubation with ActD (5 μg/mL) to block new mRNA synthesis. RAD51 mRNA expression was determined by RT-qPCR. In addition, β-actin mRNA levels were served as standards to confirm equal sample loading. RAD51 mRNA expression at each time point after ActD addition in cells exposed to TCBQ or DMSO as determined by phosphorimager analysis of RT-qPCR. Values are the percentages of RAD51 mRNA remaining in either TCBQ-treated or control cells at each time point.
dependent manner (∗, p < 0.05; ∗∗, p < 0.01; and ∗∗, p < 0.01 at 6.25, 12.5, and 25 μM, respectively), Figure 1, panel B. Phosphorylation of H2A.X at Ser 139 indicated cellular
OTM were observed (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗, p < 0.001 at 3, 6, 12, and 24 h, respectively). Meanwhile, TCBQ caused dramatic increases in OTM in a concentrationE
DOI: 10.1021/acs.chemrestox.6b00369 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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induced DNA damage through the upregulation of RAD51 at the first place; however, the decay of RAD51 suggested that the rescue probably was abolished. TCBQ Regulates the Expression of RAD51 in Different Cell Lines. Given such regulation in RAD51 protein expression after TCBQ treatment, we sought to determine whether this phenomenon exclusively occurred in MDA-MB-231 cell line. To address this issue, human liver hepatocellular carcinoma cell HepG2, human lung cancer cell A549, human breast carcinoma cell MCF-7, and rat pheochromocytoma PC12 cells were treated with TCBQ, followed by Western blotting analysis. We found that RAD51 protein levels were similarly regulated by TCBQ in these different cell lines, Figure 3. These results suggested that TCBQ-regulated RAD51 is not restricted to MDA-MB-231 cells, but a general manner of TCBQ on the regulation of RAD51 expression. Influence of TCBQ on RAD51 Ubiquitination and Protein/mRNA Synthesis in MDA-MB-231 Cells. We examined the possibility of post-translational control of RAD51 by inquiring whether TCBQ promotes RAD51 proteasome-mediated degradation. As shown in Figure 4, panel A, MG132, rather than TCBQ, increased the basal ubiquitin level in MDA-MB-231 cells (left panel), which allowed us to detect ubiquitinated RAD51. Indeed, after IP with anti-RAD51 antibody, an unchanged ubiquitination of RAD51 was detected in MDA-MB-231 cells cotreated with TCBQ and MG132, compared with MG132 treatment alone (right panel, lane 3 vs 4). These results suggested that TCBQ leads to RAD51 downregulation is independent of ubiquitin-proteasome system. To examine whether TCBQ affects RAD51 at the translational level, CHX was treated to block protein biosynthesis. As shown in Figure 4, panel B, the RAD51 protein levels in MDA-MB-231 cells were decreased over time with CHX treatment, which suggested that maintaining of certain level of RAD51 requires synthesis of new protein. Cotreatment with TCBQ did not affect the slope of RAD51 protein degradation, which is consistent with the result from Figure 4, panel A, that is, TCBQ shows no significant effect on RAD51 protein level. Next, we determined whether TCBQ affected RAD51 at the transcriptional level. MDA-MB-231 cells were exposed to TCBQ, followed by coincubation with ActD to block mRNA synthesis. Cells were then harvested, and RAD51 mRNA expression was determined by RT-qPCR. As shown in Figure 4, panel C, RAD51 mRNA was gradually downregulated by ActD; however, TCBQ further decreased RAD51 mRNA level upon ActD. This may explain that the holistic lower level of remaining RAD51 protein with TCBQ cotreatment, compared with CHX alone group (DMSO), Figure 4, panel B. These data indicated that TCBQ-induced regulates RAD51 mRNA rather than protein stability, which accounted for the decrease of RAD51. TCBQ Downregulates RAD51 in a p53-Dependent Manner. The tumor suppressor protein p53 participates in the five major DNA repair pathways.22,23 However, unrepairable DNA damage may lead to p53-dependent HR protein inhibition24 through transcriptional-independent25 and -dependent26 mechanisms. Mdm2 (the negative regulator of p53) and p53 feedback loop orchestrated the response of p53 to DNA damage of which determined the “survival” and “death” fate of cell.23,27,28 We therefore clarify whether the p53 signaling was involved in the downregulation of RAD51. TCBQ upregulated p53 and p-p53 in both dose-and time-dependent manners, Figure 5, panel A. Pretreatment with pifithrin-α caused a strong
responses to genotoxic stress; thus, it functions as a marker of DNA DSBs. Cells were treated TCBQ within a range of 6.25−25 μM for 6 h, and γ-H2AX expressions were detected by Western blotting assay, and results showed TCBQ increased γ-H2AX expression in both time- and dose-dependent manners, Figure 1, panel C. Furthermore, we examined the effect of TCBQ on the activation of apoptotic signaling. TCBQ significantly increased the cleavage of caspase-3 in both time- and dose-dependent manners, Figure 1, panel C. In summary, these data indicated that TCBQ acts as a genotoxic agent, which induces DNA damage and apoptosis in MAD-MB-231 cells. Besides TCBQ, our group and other groups demonstrated the genotoxic potential of (halogenated) benzoquinone against mammalian cells, mostly through oxidative stress- and free radical-related manners.13,15,16 TCBQ Regulates the Levels of RAD51 Protein and mRNA in MDA-MB-231 Cells. Our previous study demonstrated that p53 activation is an early response to DSBs and activates cell cycle checkpoint, DNA repair, and apoptosis.13 Then p53 triggers various transduction cascades to different downstream effector molecules, among them, the principal step of gene conversion is performed by RAD51.10 RAD51 is a key machinery in the HR pathway of DSBs repair, which might control gene conversion17 and sister chromatid exchange.18 Therefore, we explored the effect of RAD51 on TCBQ-induced DSBs in MDA-MB-231 cells. MDA-MB-231 cells were treated with TCBQ for indicated time and concentration, followed by RAD51 Western blotting analysis for its protein expression. We found that the exposure of TCBQ for 6 h slightly decreased the level of RAD51, Figure 2, panel A. However, a two-phase change of RAD51 was found in our time-course study. As shown, an increase in the early stage and a following decrease of RAD51 expression was found in both 12.5 and 25 μM TCBQ groups, wherein higher concentration faced a faster response. The expression of RAD51 reached the summit at 1 h after 25 μM TCBQ treatment. We also explored the effect of TCBQ on the RAD51 expression at the mRNA level by RT-qPCR analysis, and similar trends were found compare with RAD51 protein expressions, Figure 2, panel B. A previous study revealed that endogenous RAD51 is primarily located in cytoplasm but relocated to the nucleus upon DNA damage.19 We next investigated whether TCBQ has effect on the cellular distribution of RAD51. MDA-MB-231 cells were incubated with 25 μM TCBQ for indicated times. Cell fractionation was performed to assess RAD51 protein expression in the nucleus and cytoplasm. As shown in Figure 2, panel C, TCBQ decreased RAD51 expression in cytoplasmic fraction in a time-dependent manner. Interestingly, nuclear RAD51 increased at 1 h and decreased afterward. Notably, breast cancer susceptibility gene 2 (BRCA2) protein is accused of relocating RAD51 to the nucleus.20 The formation of RAD51 foci was also evaluated in MDA-MB-231 cells, Figure 2, panel D. In the control group, RAD51 was mainly located in the cytoplasm, while the fraction of nucleus with RAD51 foci and the number of foci per nuclei increased within 3 h of TCBQ treatment, which showed that repair had taken place within this time interval. Afterward, the number of foci per nucleus and the fraction of cells with RAD51 foci decreased. The export of RAD51 in the late phase suggested that DNA repair was impaired, which might lead to TCBQ-mediated cell death. Similar findings have been reported that strigolactone (a plant hormone) downregulates RAD51 through ubiquitination and proteasomal degradation and prevents RAD51 colocalize with γ-H2AX foci.21 Taken together, these results indicated cell attempt to repair TCBQF
DOI: 10.1021/acs.chemrestox.6b00369 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Figure 5. Involvement of p53 signaling pathway in TCBQ-induced downregulation of RAD51. (A) TCBQ induces dose- and time-dependent p53 and the phosphorylated p53 expression. MDA-MB-231 cells were treated with TCBQ. (B) MDA-MB-231 cells were pretreated with pifithrin-α for 1 h before treatment with TCBQ for another 6 h. (C) MDA-MB-231 cells were pretreated with pifithrin-α for 1 h before TCBQ treatment (25 μM). The cell viability was determined by CCK-8 assay. Data from three independent experiments are expressed as means ± SD. MDA-MB-231 cells transiently transfected with p53 siRNA were treated with or without 25 μM TCBQ for 6 h. (D) Western blotting analysis for the levels of p53, p-p53, RAD51, cleaved caspase-3, and γ-H2AX protein. (E) The RAD51 foci were determined by immunofluorescence staining. (F) DNA damage was assessed by comet assay. (G) Cells were treated with 25 μM TCBQ for indicated times. Equal amounts of the cell lysates were immunoprecipitated with an antiRAD51 antibody and then blotted with p53 or RAD51 antibody. (H) Cell viability was measured by CCK-8 assay. Each sample was prepared in triplicate.
synchronously increased with RAD51 upregulation. Moreover, p53 siRNA treatment increased cell viability in TCBQ-treated cells (Figure 5H), which is consistently with our previous finding on a similar structure quinone.13 Overall, these results indicated that p53 signaling pathway was essential for the regulation of RAD51 expression upon TCBQ stimulation. Previous reports showed that the depletion of RAD51 signaling is associated with increased extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling.32−34 However, our result illustrated moderate effect of c-Jun Nterminal kinase (JNK), ERK, or p38 MAPK on TCBQ-mediated RAD51 suppression (data not shown). RAD51 Level Correlated with the Cytotoxic Effect of TCBQ. To further study the contribution of RAD51 expression on TCBQ-induced cytotoxicity, RAD51 expression was first modulated by RAD51 and was knocked down using specific siRNA. As shown in Figure 6, panel A, transfection with RAD51 siRNA significantly suppressed RAD51 protein levels in 12 and 24 h. However, RAD51 siRNA did not affect TCBQ-induced p53 activation (Figure 6B), which suggested p53 function as the upstream molecule of RAD51. RAD51 depletion increased γH2AX and cleaved caspase-3 protein levels. To determine the effect of RAD51 overexpression on the protection of TCBQ-
inhibitory effect on TCBQ-elicited RAD51 protein level, Figure 5, panel B. Moreover, CCK-8 analysis indicated that pharmacological inhibition of p53 clearly restored the cell viability in MDAMB-231 cells treated with TCBQ, Figure 5, panel C. Arias-Lopez et al. demonstrated that p53 transcriptional downregulates RAD51 gene and requires specific binding to DNA.26 In parallel, HR repair is more effective in p53 mutant cell than p53 wild type cell due to the lack of RAD51 suppression.29−31 The knockdown of p53 significantly reversed the downregulation of RAD51 and the upregulation of γ-H2AX and cleaved caspase-3 in TCBQtreated cells, Figure 5, panel D. We also found that p53 knockdown reverses TCBQ-induced formation of RAD51 foci in MDA-MB-231 cells, as shown in Figure 5, panel E. SCGE assay demonstrated that p53 siRNA significantly decreased OTM in transfected groups compared with corresponding sham siRNA groups (∗∗, p < 0.01), Figure 5, panel F. p53 has been found to interact with RAD51 and inhibit its activity during DNA repair.26 Therefore, it is interesting to investigate how TCBQ interferes with the binding of RAD51 with p53. IP experiment showed that p53 bound to RAD51 after TCBQ treatment. Interestingly, this interaction was augmented for the next 3 h; then p53/RAD51 interaction decreased, Figure 5, panel G. Although p53 negatively regulates RAD51, it seems that p53/RAD51 interaction G
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Figure 6. RAD51 level correlated with the cytotoxic effect of TCBQ. (A) Western blotting analysis performed after treatment of MDA-MB-231 cells with control or RAD51 siRNA. (B) MDA-MB-231 cells transiently transfected with RAD51 siRNA were treated with or without 25 μM TCBQ for 6 h. The levels of p53, p-p53, cleaved caspase-3, and γ-H2AX protein were determined by Western blotting. (C) Western blotting analysis of RAD51 protein that were transfected with the respective parental controls or pEGFP-N1-RAD51. (D) Western blotting analysis for the levels of p53, p-p53, RAD51, cleaved caspase-3, and γ-H2AX protein. (E) DNA damage was assessed by comet assay. ∗∗, p < 0.01 or #, p < 0.05 indicate significant difference from cells exposed to TCBQ. (F) Cell viability was measured by CCK-8 assay. ∗, p < 0.05 or #, p < 0.05 indicate significant difference from cells exposed to TCBQ.
in downregulation of RAD51 by TCBQ in MAD-MB-231 cells. Our results have potentially significant implications for understanding genome surveillance and protection mechanisms provided by p53 in response to TCBQ exposure.
induced cytotoxicity, a pEGFP-N1-RAD51 vector was transfected into MDA-MB-231 cells. Western blotting showed an increase in total RAD51 protein in overexpressing MDA-MB231 cells compared with the respective control, Figure 6, panel C. RAD51 overexpression did not interfere with TCBQ-induced p53 activation; however, the expressions of γ-H2AX and cleaved caspase-3 were decreased, Figure 6, panel D. Consistently, RAD51 overexpression corresponded to a decrease in OTM (∗∗, p < 0.01). On the contrary, transfection of RAD51 siRNA significantly enhanced TCBQ-induced DNA damage. A statistically significant increase in OTM was observed in MDA-MB-231 cells transfected RAD51 siRNA (#, p < 0.05), Figure 6, panel E. In addition, cells containing RAD51 overexpression were more resistant to TCBQ than cells containing the respective parental controls (∗, p < 0.05); suppression of RAD51 by corresponding siRNA significantly enhanced the cytotoxicity caused by TCBQ (#, p < 0.05), Figure 6, panel F. This result is compatible with previous reports that RAD51-deficient leads to embryonic lethality in mice35 and cell death in the chicken DT40 lymphoma B-cell line.36 Thus, it appears that the RAD51 plays an important role in the resistance to TCBQ-induced cytotoxicity.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected];
[email protected]. Phone: +86-23-68251503. Fax: +86-23-68251225. ORCID
Yang Song: 0000-0001-7716-9216 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding
This work is supported by National Natural Science Foundation of China (21622704, 21575118, and 21477098) and Fundamental Research Funds for the Central Universities (XDJK2015A017 and XDJK2016A004).
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CONCLUSION In summary, our study shows that RAD51 plays a vital role in TCBQ-induced DNA damage and apoptosis. p53 was involved
Notes
The authors declare no competing financial interest. H
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Hepatocellular Carcinoma HepG2 Cells. Chem. Res. Toxicol. 28, 2160−2169. (14) Wan, L., Han, J., Liu, T., Dong, S., Xie, F., Chen, H., and Huang, J. (2013) Scaffolding protein SPIDR/KIAA0146 connects the Bloom syndrome helicase with homologous recombination repair. Proc. Natl. Acad. Sci. U. S. A. 110, 10646−10651. (15) Lai, Y., Lu, M., Gao, X., Wu, H., and Cai, Z. (2011) New evidence for toxicity of polybrominated diphenyl ethers: DNA adduct formation from quinone metabolites. Environ. Sci. Technol. 45, 10720−10727. (16) Xiao, W., Sarsour, E. H., Wagner, B. A., Doskey, C. M., Buettner, G. R., Domann, F. E., and Goswami, P. C. (2016) Succinate dehydrogenase activity regulates PCB3-quinone-induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes. Arch. Toxicol. 90, 319−332. (17) Lambert, S., and Lopez, B. S. (2000) Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms. EMBO J. 19, 3090−3099. (18) Lambert, S., and Lopez, B. S. (2001) Role of RAD51 in sisterchromatid exchanges in mammalian cells. Oncogene 20, 6627−6631. (19) Magwood, A. C., Mundia, M. M., and Baker, M. D. (2012) High levels of wild-type BRCA2 suppress homologous recombination. J. Mol. Biol. 421, 38−53. (20) Marmorstein, L. Y., Ouchi, T., and Aaronson, S. A. (1998) The BRCA2 gene product functionally interacts with p53 and RAD51. Proc. Natl. Acad. Sci. U. S. A. 95, 13869−13874. (21) Croglio, M. P., Haake, J. M., Ryan, C. P., Wang, V. S., Lapier, J., Schlarbaum, J. P., Dayani, Y., Artuso, E., Prandi, C., Koltai, H., Agama, K., Pommier, Y., Chen, Y., Tricoli, L., LaRocque, J. R., Albanese, C., and Yarden, R. I. (2016) Analogs of the novel phytohormone, strigolactone, trigger apoptosis and synergize with PARP inhibitors by inducing DNA damage and inhibiting DNA repair. Oncotarget 7, 13984−14001. (22) Sengupta, S., and Harris, C. C. (2005) p53: traffic cop at the crossroads of DNA repair and recombination. Nat. Rev. Mol. Cell Biol. 6, 44−55. (23) Meek, D. W. (2004) The p53 response to DNA damage. DNA Repair 3, 1049−1056. (24) Linke, S. P., Sengupta, S., Khabie, N., Jeffries, B. A., Buchhop, S., Miska, S., Henning, W., Pedeux, R., Wang, X. W., Hofseth, L. J., Yang, Q., Garfield, S. H., Sturzbecher, H. W., and Harris, C. C. (2003) p53 interacts with hRAD51 and hRAD54, and directly modulates homologous recombination. Cancer Res. 63, 2596−2605. (25) Bertrand, P., Saintigny, Y., and Lopez, B. S. (2004) p53’s double life: transactivation-independent repression of homologous recombination. Trends Genet. 20, 235−243. (26) Arias-Lopez, C., Lazaro-Trueba, I., Kerr, P., Lord, C. J., Dexter, T., Iravani, M., Ashworth, A., and Silva, A. (2006) p53 modulates homologous recombination by transcriptional regulation of the RAD51 gene. EMBO Rep. 7, 219−224. (27) Ma, L., Wagner, J., Rice, J. J., Hu, W., Levine, A. J., and Stolovitzky, G. A. (2005) A plausible model for the digital response of p53 to DNA damage. Proc. Natl. Acad. Sci. U. S. A. 102, 14266−14271. (28) Kohn, K. W., and Pommier, Y. (2005) Molecular interaction map of the p53 and Mdm2 logic elements, which control the Off-On switch of p53 in response to DNA damage. Biochem. Biophys. Res. Commun. 331, 816−827. (29) Wu, M., Wang, X., McGregor, N., Pienta, K. J., and Zhang, J. (2014) Dynamic regulation of Rad51 by E2F1 and p53 in prostate cancer cells upon drug-induced DNA damage under hypoxia. Mol. Pharmacol. 85, 866−876. (30) Ambrosini, G., Seelman, S. L., Qin, L. X., and Schwartz, G. K. (2008) The cyclin-dependent kinase inhibitor flavopiridol potentiates the effects of topoisomerase I poisons by suppressing Rad51 expression in a p53-dependent manner. Cancer Res. 68, 2312−2320. (31) Orre, L. M., Stenerlow, B., Dhar, S., Larsson, R., Lewensohn, R., and Lehtio, J. (2006) p53 is involved in clearance of ionizing radiationinduced RAD51 foci in a human colon cancer cell line. Biochem. Biophys. Res. Commun. 342, 1211−1217. (32) Wiegmans, A. P., Miranda, M., Wen, S. W., Al-Ejeh, F., and Moeller, A. (2016) RAD51 inhibition in triple negative breast cancer
ABBREVIATIONS ActD, actinomycin D; BER, base excision repair; BRCA2, breast cancer susceptibility gene 2; CCK-8, cell counting kit-8; CHX, cycloheximide; DAPI, 4′, 6-diamidino-2-phenylindole dihydrochloride; DDR, DNA damage response; DMEM, Dulbecco’s modified Eagle’s medium; DSBs, double strand breaks; ERK, extracellular signal-regulated protein kinase; γ-H2AX, phosphohistone H2A.X; HR, homologous recombination; JNK, c-Jun Nterminal kinase; MAPK, mitogen-activated protein kinase; M1GdR, 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purin-10(3H)-one; MMR, mismatch repair; NER, nucleotide excision repair; NHEJ, nonhomologous end joining; OD, optical density; OTM, olive tail moment; PCP, pentachlorophenol; RTqPCR, reverse transcription quantitative polymerase chain reaction; SCGE, single cell gel electrophoresis; siRNA, small RNA; SSBs, single-strand breaks; TCBQ, tetrachlorobenzoquinone
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REFERENCES
(1) Dahlhaus, M., Almstadt, E., and Appel, K. E. (1994) The pentachlorophenol metabolite tetrachloro-p-hydroquinone induces the formation of 8-hydroxy-2-deoxyguanosine in liver DNA of male B6C3F1 mice. Toxicol. Lett. 74, 265−274. (2) Lin, P. H., La, D. K., Upton, P. B., and Swenberg, J. A. (2002) Analysis of DNA adducts in rats exposed to pentachlorophenol. Carcinogenesis 23, 365−369. (3) Nguyen, T. N., Bertagnolli, A. D., Villalta, P. W., Buhlmann, P., and Sturla, S. J. (2005) Characterization of a deoxyguanosine adduct of tetrachlorobenzoquinone: dichlorobenzoquinone-1,N2-etheno-2′-deoxyguanosine. Chem. Res. Toxicol. 18, 1770−1776. (4) Waidyanatha, S., McDonald, T. A., Lin, P. H., and Rappaport, S. M. (1994) Measurement of hemoglobin and albumin adducts of tetrachlorobenzoquinone. Chem. Res. Toxicol. 7, 463−468. (5) Lin, P. H., Nakamura, J., Yamaguchi, S., Upton, P. B., La, D. K., and Swenberg, J. A. (2001) Oxidative damage and direct adducts in calf thymus DNA induced by the pentachlorophenol metabolites, tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone. Carcinogenesis 22, 627−634. (6) Dedon, P. C., Plastaras, J. P., Rouzer, C. A., and Marnett, L. J. (1998) Indirect mutagenesis by oxidative DNA damage: formation of the pyrimidopurinone adduct of deoxyguanosine by base propenal. Proc. Natl. Acad. Sci. U. S. A. 95, 11113−11116. (7) Jeong, Y. C., and Swenberg, J. A. (2005) Formation of M1G-dR from endogenous and exogenous ROS-inducing chemicals. Free Radical Biol. Med. 39, 1021−1029. (8) Roos, W. P., Thomas, A. D., and Kaina, B. (2015) DNA damage and the balance between survival and death in cancer biology. Nat. Rev. Cancer 16, 20−33. (9) Bernstein, C., Bernstein, H., Payne, C. M., and Garewal, H. (2002) DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis. Mutat. Res., Rev. Mutat. Res. 511, 145−178. (10) Daboussi, F., Dumay, A., Delacote, F., and Lopez, B. S. (2002) DNA double-strand break repair signalling: the case of RAD51 posttranslational regulation. Cell. Signalling 14, 969−975. (11) Richardson, C., and Jasin, M. (2000) Coupled homologous and nonhomologous repair of a double-strand break preserves genomic integrity in mammalian cells. Mol. Cell. Biol. 20, 9068−9075. (12) Saintigny, Y., Delacote, F., Vares, G., Petitot, F., Lambert, S., Averbeck, D., and Lopez, B. S. (2001) Characterization of homologous recombination induced by replication inhibition in mammalian cells. EMBO J. 20, 3861−3870. (13) Song, X., Li, L., Shi, Q., Lehmler, H. J., Fu, J., Su, C., Xia, X., Song, E., and Song, Y. (2015) Polychlorinated Biphenyl Quinone Metabolite Promotes p53-Dependent DNA Damage Checkpoint Activation, SPhase Cycle Arrest and Extrinsic Apoptosis in Human Liver I
DOI: 10.1021/acs.chemrestox.6b00369 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology cells is challenged by compensatory survival signaling and requires rational combination therapy. Oncotarget, DOI: 10.18632/oncotarget.11065. (33) Ko, J. C., Ciou, S. C., Jhan, J. Y., Cheng, C. M., Su, Y. J., Chuang, S. M., Lin, S. T., Chang, C. C., and Lin, Y. W. (2009) Roles of MKK1/2ERK1/2 and phosphoinositide 3-kinase-AKT signaling pathways in erlotinib-induced Rad51 suppression and cytotoxicity in human nonsmall cell lung cancer cells. Mol. Cancer Res. 7, 1378−1389. (34) Chen, R. S., Jhan, J. Y., Su, Y. J., Lee, W. T., Cheng, C. M., Ciou, S. C., Lin, S. T., Chuang, S. M., Ko, J. C., and Lin, Y. W. (2009) Emodin enhances gefitinib-induced cytotoxicity via Rad51 downregulation and ERK1/2 inactivation. Exp. Cell Res. 315, 2658−2672. (35) Tsuzuki, T., Fujii, Y., Sakumi, K., Tominaga, Y., Nakao, K., Sekiguchi, M., Matsushiro, A., Yoshimura, Y., and Morita, T. (1996) Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. U. S. A. 93, 6236−6240. (36) Sonoda, E., Sasaki, M. S., Buerstedde, J. M., Bezzubova, O., Shinohara, A., Ogawa, H., Takata, M., Yamaguchi-Iwai, Y., and Takeda, S. (1998) Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J. 17, 598−608.
J
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