Resveratrol causes Cdc2-tyr15 phosphorylation via ATM/ATRChk1/2Cdc25C pathway as a central mechanism for S phase arrest in human ovarian carcinoma Ovcar-3 cells
Alpna Tyagi 1,
Rana P. Singh 1,
Chapla Agarwal 1, 2,
Sunitha Siriwardana 2, 3,
Robert A. Sclafani 2, 3 and
Rajesh Agarwal 1, 2, *
1 Department of Pharmaceutical Sciences, School of Pharmacy, 2 University of Colorado Cancer Center and 3 Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA
* To whom correspondence should be addressed. Tel: +1 303 315 1381; Fax: +1 303 315 6281; Email: Rajesh.Agarwal{at}UCHSC.edu
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Abstract
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Resveratrol is one of the most extensively studied cancer chemopreventive agents; however, its mechanisms of action are not completely understood. Here, we observed that resveratrol induces S phase arrest via Tyr15 phosphorylation of Cdc2 in human ovarian carcinoma Ovcar-3 cells. Overexpression of Cdc2AF, a mutant resistant to Thr14 and Tyr15 phosphorylation, ablated resveratrol-induced S phase arrest. Further upstream, we observed that resveratrol causes phosphorylation of cell division cycle 25C (Cdc25C) tyrosine phosphatase via the activation of checkpoint kinases Chk1 and Chk2, which in turn were activated via ATM (ataxia telangiectasia mutated)/ATR (ataxia telangiectasia-Rad3-related) kinase in response to DNA damage, as resveratrol also increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM/ATR in response to DNA damage. The involvement of these molecules in resveratrol-induced S phase was also supported by the studies showing that addition of ATM/ATR inhibitor caffeine reverses resveratrol-caused activation of ATM/ATRChk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X, and S phase arrest. In additional studies assessing whether observed effects of resveratrol are specific to Ovcar-3 cells, we observed that it also induces S phase arrest and H2A.X (Ser139) phosphorylation in other ovarian cancer cell lines PA-1 and SKOV-3, albeit at different levels; whereas, resveratrol showed only marginal S phase arrest in normal human foreskin fibroblasts with undetectable level of phospho-H2A.X (Ser139). These findings for the first time identify that resveratrol causes Cdc2-tyr15 phosphorylation via ATM/ATRChk1/2Cdc25C pathway as a central mechanism for DNA damage and S phase arrest selectively in ovarian cancer cells, and provide a rationale for the potential efficacy of ATM/ATR agonists in the prevention and intervention of cancer.
Abbreviations: ATM, ataxia telangiectasia-mutated; ATR, ataxia telangiectasia-Rad3-related; Cdc25C, cell division cycle 25C; Chk, checkpoint kinase; H2A.X, histone 2AX; GFP, green fluorescent protein; tTA, tetracycline transactivator; FACS, fluorescence activated cell sorting; HFF, human foreskin fibroblast; PBS, phosphate-buffered saline.
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Introduction
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Ovarian cancer remains one of the most lethal gynecological cancers; the high mortality rate makes this malignancy a major health concern. Statistical predictions for 2004 show an estimated 25, 580 new cases of ovarian cancer with 16, 090 deaths in the USA alone (1). Prevention and therapeutic intervention by using dietary agents are newer dimensions in cancer management (2). Administration of phytochemicals prevents initiation, and promotion and progression events associated with carcinogenesis, and may be a direct way to reduce cancer mortality and morbidity (2). Trans (t)-resveratrol (3,4,5-trihydoxystilbene) is present naturally in grapes, other fruits and a variety of medicinal plants (3). Because of its high concentration in grape skin, a significant amount of resveratrol is present in wines, especially red wines (4). Resveratrol has been suggested to be partially responsible for the beneficial effects of red wines against coronary heart disease (4). Cancer preventive and anticancer activities of resveratrol have also been observed, where it prevents skin tumorigenesis in a mouse model (5), and inhibits growth and induces apoptosis as well as S phase cell cycle arrest in various human cancer cell lines (69). The high doses (upto 20 mg/kg/day) of oral trans-resveratrol to rats, which are estimated to be 1000 times of the amount consumed by a person drinking one glass of red wine a day, do not show any adverse health effects (10). Despite encouraging cancer preventive and therapeutic effects (reviewed extensively in ref. 11), the central mechanism responsible for resveratrol efficacy is not fully known.
Several recent studies have shown that resveratrol induces DNA damage in many human cancer cell lines (8,12) and that it is capable of binding to DNA and cleave or damage DNA in a Cu2+ dependent pathway (13). In eukaryotes, the checkpoint pathways initiated by DNA damage consists of several protein kinases, including phosphoinositide kinase homologs ataxia telangiectasia mutated (ATM) kinase, ataxia telangiectasia-Rad3-related (ATR) kinase, Mec1 and Rad3, and the protein kinases Chk2 (also known as Rad53 and Cds1 in yeast), Chk1 and Dun1 (14). In mammals, in response to DNA damage, ATM controls cell cycle arrest in G1 and G2 and also prevents ongoing DNA synthesis. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases known as Chk1 and Chk2 (1517). Though Chk1 and Chk2 are structurally distinct, they have a functionally related kinase domain that phosphorylates an overlapping pool of cellular substrates (18). In mammalian cells, Chk1 and Chk2 have apparently evolved to channel DNA damage signals from ATR and ATM, respectively, which is based on the reports that Chk1 is activated by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (19,20). In recent years, it has been suggested that ATM activation could be used to prevent cancer development (21). In vitro, Chk2 phosphorylated cell division cycle 25C (Cdc25C) on serine 216, a site known to be involved in negative regulation of Cdc25C; this is the same site phosphorylated by the protein kinase Chk1 suggesting that in response to DNA damage and DNA replication stress Chk1 and Chk2 can phosphorylate Cdc25C tyrosine phosphatase, an activator of the cyclin-dependent kinase Cdc2 (22). Phosphorylation of Cdc25C on Ser216 interferes with Cdc25C's ability to promote mitotic entry (22). Cdc25C on Ser216 is phosphorylated throughout inter-phase and is dephosphorylated directly before mitotic entry.
In order to assess the preventive and therapeutic potential of resveratrol in human ovarian cancer, in the present study, we assessed antiproliferative and cytotoxic effects of resveratrol and associated molecular mechanisms employing human ovarian carcinoma Ovcar-3 cells. Our results provide first evidence for the activation of ATM/ATR checkpoint signaling as a central mechanism of resveratrol-induced S phase arrest and growth inhibition in Ovcar-3 cells.
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Materials and methods
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Cell lines and reagents
Human ovarian cancer cell lines Ovcar-3, PA-1 and SKOV-3 were from American Type Culture Collection (Manassas, VA) and normal HFF cells were kindly provided by Dr James DeGregori (UCHSC, Denver, CO). Ovcar-3 cells were cultured in RPMI 1640 containing 20% fetal bovine serum (FBS) under standard culture conditions (37°C, 95% humidified air and 5% CO2). PA-1, SKOV-3 and HFF cells were cultured in Eagle's Minimal Essential medium, McCoy'S 5A and DMEM, respectively, containing 10% FBS under standard culture conditions. Resveratrol used in the present study was from Sigma-Aldrich Chemical Company (St Louis, MO). The primary antibody for ATM was from Oncogene (San Diego, CA) and for ATR was from Novus (Littleton, CO). Primary antibodies to phospho-Cdc2 (Tyr15), phospho-Cdc25C (Ser216) and phospho-Chk1/2, total Chk1 and secondary anti-rabbit antibody were from Cell Signaling (Beverly, MA). Antibodies for Chk2, cyclin B1, Cdc2 and Cdc25C were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho H2A.X (Ser139) was from Trevigen (Gaithersburg, MD). Antibody for ß-actin was from Sigma. Secondary anti-mouse antibody and ECL detection system were from Amersham (Arlington Heights, IL). Cell proliferation ELISA BrdU kit was from Roche Diagnostics (Indianapolis, IN).
Cell culture and treatments
Ovcar-3 cells were cultured in RPMI 1640 medium containing 20% FBS and 1% penicillinstreptomycin under standard culture conditions. At 60% confluency, cultures were treated with desired doses of resveratrol (10, 30 and 50 µM, final concentrations in medium) dissolved in dimethyl sulfoxide (DMSO) or DMSO alone for different time points (2472 h) for cell growth and cell cycle analysis. For lysates preparation, cells were treated with resveratrol at 30 or 50 µM dose for desired time. In caffeineresveratrol combination studies, cells were treated with caffeine (10 mM) 15 min prior to resveratrol treatment. In PI3 Kinase inhibitor study, cells were treated with wortmannin (50 µM) 2 h prior to resveratrol treatment. Cell lysates were prepared in non-denaturing lysis buffer (10 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 1 mM EGTA, 0.3 mM phenyl methyl sulfonyl fluoride, 0.2 mM sodium orthovanadate, 0.5% NP-40, 5 U/ml aprotinin). For lysate preparation, medium was aspirated and cells were washed two times with ice-cold phosphate-buffered saline (PBS) followed by incubation in lysis buffer for 10 min on ice. Then cells were scraped and kept on ice for 30 min, and finally cell lysates were cleared by centrifugation at 4°C for 30 min at 14 000 r.p.m. (18,000x g) Protein concentrations in lysates were determined using Bio-Rad DC protein assay kit (Bio-Rad laboratories, Hercules, CA) by the Lowry method.
Cell growth and DNA synthesis assays
Ovcar-3 cells were plated at 5000 cells/cm2 in 60 mm plates under standard culture conditions. After 24 h, cells were fed with fresh medium and treated with DMSO alone or different concentrations of resveratrol (10, 30 and 50 µM). After 24, 48 and 72 h of treatments, cells were trypsinized, collected and counted using a hemocytometer. Trypan blue dye exclusion was used to determine cell viability. DNA synthesis was assessed by BrdU incorporation employing colorimetric ELISA. Briefly, cells were cultured in 96-well plates at 37°C for 24 h. Cells were treated with DMSO alone or resveratrol in DMSO for 24 and 48 h. After these treatments, BrdU was added and subsequently incubated for another 2 h at 37°C. Thereafter, cells were fixed and incubated with anti-BrdU antibody followed by the addition of substrate. The reaction product was quantified by measuring absorbance at 370 nm using a scanning multi-well spectrophotometer (ELISA reader).
Flow cytometry for cell cycle analysis
Ovcar-3, PA-1, SKOV-3 and HFF cells at 60% confluency were treated with either DMSO alone or various doses of resveratrol (10, 30 and 50 µM) for 24 and 48 h; thereafter, medium was aspirated, cells quickly washed two times with cold PBS and trypsinized, and cell pellets were collected. Approximately 0.5 x 106 cells in 0.5 ml of saponinpropidium iodide (PI) solution (0.3% saponin, 25 µg/ml PI, 0.1 mM EDTA and 10 µg/ml RNase in PBS) were incubated at 4°C for 24 h in the dark. Cell cycle distribution was then analyzed by flow cytometry using the FACS analysis core service of the University of Colorado Cancer Center (Denver, CO).
Immunoblot analysis
Total cell lysates were denatured with 2xsample buffer. Samples were subjected to SDSPAGE on 4, 12 or 16% Trisglycine gels and separated proteins were transferred onto membranes by western blotting. Membranes were blocked with blocking buffer for 1 h at room temperature and, as desired, probed with primary antibody against desired molecule overnight at 4°C followed by peroxidase-conjugated appropriate secondary antibody for 1 h at room temperature and ECL detection.
Cdc2AF transfection assay
Recombinant adenovirus Cdc2AF was a generous gift from David Morgan at UCSF (23,24). Cdc2AF is a Cdc2 mutant that cannot be phosphorylated at inhibitory sites (Tyr15 and Thr14). Ovcar-3 cells were co-infected with a recombinant adenovirus tetracycline transactivator (tTA) plus the desired Cdc2AF virus as described (23,24), each at a multiplicity of infection (MOI) of 510 plaque-forming units per cell for 1 h and then treated with desired dose of resveratrol followed by cell cycle and western blot analyses. Control adeno green fluorescent protein (GFP) virus and the procedures for using recombinant adenoviruses have been described by our laboratory (25,26).
Immunocytochemical staining for phospho-H2A.X (Ser139)
Ovcar-3 cells were seeded on 4-well chamber slides and the next day treated with DMSO (control) or 50 µM resveratrol and/or caffeine 15 min (10 mM) prior to resveratrol. At the end of desired treatment times, cells were fixed with methanol at 20°C for 10 min and washed twice with ice-cold PBS. Cells were gradually rehydrated with PBS and then incubated with 10% BSA in PBS for 30 min at room temperature. Cells were rinsed twice with PBS and incubated with primary rabbit polyclonal anti-phospho H2A.X (Ser139) antibody in PBS with 3% BSA at 4°C overnight. Cells were then rinsed in PBS with 3% BSA six times and incubated with goat Alexa 488-conjugated anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR) for 1 h, and were then rinsed in PBS with 3% BSA six times and observed and photographed under inverted Nikon TE-300 microscope with an epifluorescent attachment equipped with a Princeton Instrument Micromax camera, at 488 nm fluorescence excitation and 520 nm fluorescence emission. Images are acquired with the Image Pro-plus software (Media Cybernetics, Silver Spring, MD) at 200x magnification.
Statistical analysis
Statistical significance of differences between control and treated samples were calculated using Student's t-test (SigmaStat 2.03). P values of <0.05 were considered significant. The gel and cell cycle analysis data in all cases are representative of at least 24 independent studies with reproducible results.
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Results
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Effect of resveratrol on cell viability, DNA synthesis and cell cycle progression
In the studies assessing resveratrol efficacy in Ovcar-3 cells, it caused strong cell growth inhibition (Figure 1a) and cell death induction (Figure 1b) in both a dose- and a time-dependent manner. It also inhibited DNA synthesis (Figure 1c). Based on these results, we next assessed whether the growth inhibitory effect of resveratrol is accompanied by its effect on cell cycle progression. Resveratrol showed 4867% cells in S phase after 2448 h of treatment as compared with controls, in which only 26% of cells were in S phase (Figure 2a and b). The increase in S phase cells by resveratrol was accompanied by a decrease in both G1 and G2/M cell populations (Figure 2a and b).

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Fig. 1. Effect of resveratrol on cell viability and DNA synthesis in Ovcar-3 cells. Resveratrol exhibits growth inhibition in human ovarian carcinoma Ovcar-3 cells. (a and b) Cells were plated in 60 mm dishes (5000 cells/cm2) and treated with DMSO (control) or different concentrations of resveratrol for 24, 48 and 72 h. At the end of treatments cells were harvested and counted using trypan blue as detailed in Materials and methods. (c) For DNA synthesis, cells were grown in 96-well plates followed by treatment with resveratrol. Cells were then incubated with BrdU for 23 h followed by incubation with anti-BrdU antibody and substrate. BrdU incorporation was measured by using ELISA reader. Data are presented as mean ± SE of triplicate samples. $, P < 0.05; #, P < 0.01; and *, P < 0.001 as compared with control. Res, resveratrol.
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Fig. 2. Resveratrol induces S phase arrest in the cell cycle progression of Ovcar-3 cells. Cells were cultured as described in Materials and methods and treated with either DMSO alone (control) or varying concentrations of resveratrol for (a) 24 and (b) 48 h. At the end of these treatments, cells were collected and incubated with saponinPI solution at 4°C for 24 h in dark and subjected to FACS analysis as detailed in Materials and methods. Data are presented as mean ± SE of triplicate samples. $, P < 0.05; #, P < 0.01; and *, P < 0.001 as compared with control. Res, resveratrol.
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Effect of resveratrol on cell cycle regulatory proteins
Based on an observed S phase arrest in Ovcar-3 cells by resveratrol, we next assessed the levels of cell cycle regulators associated with this effect. Resveratrol treatment (30 and 50 µM for 24 and 48 h) induced protein expression of CDK2, and cyclin A and E (Figure 3). Furthermore, Cdc25C and Cdc2 were inhibited due to phosphorylation on Ser216 and Tyr15, respectively, following resveratrol treatment without any change in total Cdc25C level and only a slight increase in total Cdc2 level (Figure 3). We did not observe any changes in the protein expression of Wee1, but cyclin B1 accumulation was observed (Figure 3). Overall, these results suggested the possible involvement of Cdc25C, Cdc2 and cyclin B1 in resveratrol-induced S phase arrest in Ovcar-3 cells, and therefore, we next investigated the role of Cdc2 phosphorylation in the observed S phase arrest.

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Fig. 3. Effect of resveratrol on cell cycle regulatory molecules in Ovcar-3 cells. Cells were cultured as described in Materials and methods, and treated with either DMSO alone (control) or varying concentrations of resveratrol as labeled in the figure. At the end of the treatments, total cell lysates were prepared and subjected to SDSPAGE followed by western immunoblotting. Membranes were probed with anti-CDK2, cyclin A, cyclin E, cyclin B1, phospho-Cdc25C (Ser216), Cdc25C, phospho-Cdc2 (Tyr15), Cdc2, Wee1 and ß-actin antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection system. Res, resveratrol.
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Resveratrol-induced S phase arrest involves Cdc2 (Tyr15) phosphorylation
Since we observed that cells with resveratrol-dependent S phase arrest were not able to enter mitosis under normal culture conditions, we synchronized the cells by serum starvation for 48 h and released them using medium containing serum without or with resveratrol, and cell cycle analysis was done as a function of time from 0 to 48 h. As shown in Figure 4a and b, control cells entered S phase after 18 h of the release, and passed through G2M, G1 and S phases of the next cell cycle (Figure 4a); however, resveratrol-treated cells arrested and remained in S phase even after 48 h of the treatment (Figure 4b). Resveratrol caused a time-dependent increase in the S phase cell population from 30% after 18 h to 84% after 3648 h of treatment (Figure 4b). These results demonstrate that resveratrol induces an irreversible S phase arrest under both unsynchronized and synchronized conditions. Next we investigated molecular mechanisms underlying this effect by assessing the phosphorylation of Cdc2 (Tyr15) under synchronized conditions. Immunoblot analysis for phospho-Cdc2 (Tyr15) showed an increased accumulation after 18 h of release in control cells (Figure 4c); however, resveratrol treatment caused an increase in phospho-Cdc2 (Tyr15) as early as 12 h, which became robust at 18 h and remained elevated for up to 48 h (Figure 4c). Total Cdc2 protein expression showed a slight increase in resveratrol-treated cells as compared with control (Figure 4c). Together, these results showed that resveratrol-induced S phase arrest involves Cdc2 (Tyr15) phosphorylation, and therefore, we further elucidated the role of this pathway in resveratrol efficacy in Ovcar-3 cells.

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Fig. 4. Resveratrol induces irreversible S arrests and Cdc2 (Tyr15) phosphorylation in Ovcar-3 cells. Cells were synchronized by serum starvation for 48 h and then released using media containing serum (a) with DMSO (control) or (b) 50 µM resveratrol and cell cycle analysis was done as a function of time from 0 to 48 h as indicated in the figure. (c) In similar treatments, cell lysates were prepared and subjected to SDSPAGE followed by western immunoblotting with anti-phospho-Cdc2 (Tyr15), Cdc2 and ß-actin antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection system. Res, resveratrol.
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Cdc2 (Tyr15) phosphorylation is essential for resveratrol-induced S phase arrest
Cell cycle entry into mitotic phase is initiated by the dephosphorylation of two inhibitory residues Tyr15 and Thr14 of Cdc2 in G2 phase followed by activation of Cdc2cyclin B1 complex (15). Consistent with this, transient overexpression of Cdc2AF, a dominant mutant form of Cdc2 in which Tyr15 and Thr14 are changed to phenylalanine and alanine, respectively, has been shown to promote mitotic events in mammalian cells (24). Accordingly, to further define the role of Cdc2 (Tyr15) phosphorylation in resveratrol-induced S phase arrest, Ovcar-3 cells were co-infected with a recombinant adenovirus for Cdc2AF under Tet control (23,24) and a recombinant adenovirus expressing tTA. Overexpression of Cdc2AF significantly decreased resveratrol-induced S phase arrest from 61 to 41% (P < 0.001) after 24 h of treatment (Figure 5a). Under the same conditions, cell lysates were analyzed for phospho-Cdc2 (Tyr15) and total Cdc2 levels. Cdc2AF overexpression abolished basal as well as resveratrol-induced Cdc2 (Tyr15) phosphorylation (Figure 5b), consistent with the observed reversal in resveratrol-induced S phase arrest. Cells infected with only tTA or GFP control viruses did not affect resveratrol-induced S phase arrest and Cdc2 (Tyr15) phosphorylation (Figure 5). Together, these results clearly suggest that resveratrol induced phosphorylation of Cdc2 (Tyr15) is an essential event for S phase arrest in Ovcar-3 cells.

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Fig. 5. Resveratrol-induced S phase arrest is dependent on Cdc2 (Tyr15) phosphorylation. Cells were cultured and co-infected with recombinant adenovirus construct of Cdc2AF under Tet control and/or a recombinant adenovirus expressing tTA or GFP and treated with resveratrol (50 µM) for 24 h. Different treatments are as labeled in the figure. At the end of these treatments, (a) cells were harvested and analyzed for cell cycle distribution as detailed in Materials and methods, or (b) total cell lysates were prepared and subjected to SDSPAGE followed by western immunoblotting for phospho-Cdc2 (Tyr15), Cdc2 and ß-actin. Cdc2AF protein is larger than endogenous wild-type Cdc2 protein because of the HA tag (23). Cell cycle distribution data are presented as mean ± SE of triplicate samples. *, P < 0.001 as compared with control, GFP, and Tet treatments; , P < 0.001 as compared with Res treatment (pair-wise multiple comparisons by Bonferroni t-test). Res, resveratrol.
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Resveratrol activates ATM/ATR kinases
Based on the results described above and earlier reports, we hypothesized that resveratrol induces DNA damage that is sensed by members of phosphoinositide 3-kinase-related kinases (ATM and ATR) for the early signal transmission through the cell cycle checkpoint (27,28). ATM and ATR are nuclear kinases recently identified as being activated in response to DNA damage/genotoxic stress in eukaryotic cells (16,27,29). Resveratrol treatment (50 µM) of cells for 48 h resulted in a strong increase in the levels of both ATM and ATR proteins (Figure 6a), which was abrogated when cells were pre-treated with 10 mM caffeine (Figure 6a), a known inhibitor of ATM/ATR kinases (30). Exposure of cells to UVC (used as a positive control), a known activator of ATM/ATR, also showed a strong increase in the levels of both these kinases (Figure 6a).

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Fig. 6. Resveratrol increases ATM, ATR and Chk1/2 protein expression, and activates ATM/ATR-dependent phosphorylation of Chk1/2. Cells were cultured as described in Materials and methods, and treated with DMSO alone (control) or resveratrol (50 µM) and/or caffeine (10 mM) (an inhibitor of ATM/ATR) for 48 h. Caffeine was added 15 min before resveratrol. At the end of these treatments, total cell lysates were prepared and subjected to SDSPAGE followed by western immunoblotting. Membranes were probed with (a) anti-ATM and ATR, (b) anti-phospho-Chk1 (Ser296), Chk1, phospho-Chk2 (Thr387, Thr68 and Ser19), Chk2 and ß-actin antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection. Res, resveratrol.
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Resveratrol activates cell cycle checkpoint kinases through ATM/ATR signaling
Since exposure of cells to resveratrol resulted in an increase in ATM/ATR kinases, which are known to activate Chk1 and Chk2 (31), we next examined the phosphorylation of Chk1 and Chk2. Resveratrol treatment (50 µM for 48 h) resulted in an increase in phosphorylation of Chk1 (Ser296) as well as its total protein level (Figure 6b). Similarly, resveratrol strongly induced the phosphorylation of Chk2 at Thr68, Thr387 and Ser19 sites together with a moderate increase in total Chk2 protein level (Figure 6b). We also used caffeine pre-treatment, which is reported to inhibit both ATM and ATR (30), to examine whether Chk1 and Chk2 phosphorylation in response to resveratrol is ATM/ATR dependent (Figure 6a). Caffeine pre-treatment of cells blocked the resveratrol-induced activation/serine-threonine phosphorylation of both Chk1 and Chk2 kinases (Figure 6b). Overall, these results suggest that resveratrol causes activation of Chk1 and Chk2 in an ATM/ATR-dependent manner.
Resveratrol causes phosphorylation of Cdc25C and Cdc2, accumulation of cyclin B1, and S phase arrest via ATM/ATR-dependent Chk1/Chk2 pathway
Chk1 and Chk2 kinases are known to phosphorylate Cdc25C phosphatase at Ser216 in response to DNA damage (31). Negative regulation of Cdc25C by phosphorylation at Ser216 is an important regulatory mechanism used by cells to block mitotic entry under normal cell cycle progression or after DNA damage. The findings discussed above suggested that resveratrol-induced ATM/ATR checkpoint signaling followed by Chk1/Chk2 activation possibly results in phosphorylation of the Cdc25C phosphatase at Ser216 and associated negative regulation, which in turn produces accumulation of inactive phosphorylated Cdc2 at Thr14 and Tyr15. Thus, the cell cycle progression is prevented by inhibition of the mitotic CDK. Consistent with this hypothesis, we observed that resveratrol very strongly induces the phosphorylation of Cdc25C at Ser216 (Figure 7a), which was inhibited by caffeine pre-treatment of cells (Figure 7a), clearly suggesting the role of ATM/ATR-mediated Chk1/2 kinase pathway for inhibitory phosphorylation of Cdc25C (Ser216). As phospho-Cdc25C (Ser216) is impaired for dephosphorylation of Cdc2 (Tyr15), we observed a strong increase in phospho-Cdc2 (Tyr15) levels following resveratrol treatment, which was also totally inhibited by caffeine (Figure 7a), clearly suggesting the upstream involvement of ATM/ATRChk1/2Cdc25C pathway in this effect. On the other hand, nuclear Cdc2 remains phosphorylated in the absence of Cdc25C, causing a G2 phase arrest. During S and G2 phases Cdc2cyclin B complexes are kept in the inactive state through Tyr15 phosphorylation of Cdc2 by Wee1/Mik1/Myt1 tyrosine kinases (32,33). Elevated levels of Tyr15-phosphorylated Cdc2 are associated with S phase arrest after DNA damage in several systems (34,35). We did not find any change in the protein level of Wee1 (data not shown) following resveratrol treatment; however, there was a strong accumulation of the Cdc2 regulatory subunit cyclin B1, which was also completely reversed by caffeine pre-treatment (Figure 7a). This observation is in accord with a report showing accumulation of cyclin B1 in S or G2 phase arrest following DNA damage (23).

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Fig. 7. Resveratrol causes S phase arrest via ATM/ATR-dependent phosphorylation of Cdc25C (Ser216) and Cdc2 (Tyr15), and cyclin B1 accumulation. Ovcar-3 cells were cultured as described in Materials and methods and treated with either DMSO (control) or 50 µM resveratrol and/or caffeine (10 mM) for 48 h. Caffeine was added 15 min before resveratrol, or in another experiment wortmannin was added 2 h before resveratrol. At the end of these treatments, (a) cells were harvested and cell lysates were prepared and subjected to SDSPAGE followed by western immunoblotting for phospho-Cdc25C (Ser216), phospho-Cdc2 (Tyr15), Cdc2 and ß-actin. (b and c) In similar treatments, cells were harvested and analyzed for cell cycle distribution as detailed in Materials and methods. Data are presented as mean ± SE of triplicate samples. $, P < 0.05; #, P < 0.01; and *, P < 0.001 as compared with control. Res, resveratrol; wort, wortmannin.
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Next, we examined the biological relevance of these molecular alterations following caffeine and/or resveratrol treatments. Consistent with the above results, resveratrol induced a strong S phase arrest, which was reversed almost to control levels by caffeine (10 mM) pre-treatment (Figure 7b). We further confirmed this observation using wortmannin, a PI3K inhibitor that also inhibits PI3K-related kinases ATM and ATR. Similar to caffeine, wortmannin (50 µM) pre-treatment of cells almost completely reversed resveratrol-induced S phase arrest in Ovcar-3 cells (Figure 7c). Together, these results suggested that resveratrol-induced S phase arrest involves ATM/ATR checkpoint signaling.
Resveratrol induces Ser139 phosphorylation of H2A.X
H2A.X is a variant form of histone H2A that gets phosphorylated directly by ATM at serine 139, marking an early event in response to DNA damage (36). H2A.X is also known to play a critical role in the retention of DNA repair factors at DNA damaged sites. Resveratrol (50 µM) treatment of cells for 6 h showed increased serine 139 phosphorylation of histone H2A.X, as judged by immunocytochemistry using anti-phospho-histone H2A.X (Ser139) antibody, providing evidence that resveratrol activates ATM/ATR kinases following DNA damage in Ovcar-3 cells (Figure 8a). This is further supported by the observation that caffeine pre-treatment almost completely blocked resveratrol-induced serine 139 phosphorylation of H2A.X (Figure 8a). As a positive control, cells that were exposed to UVC for 3 h showed a strong serine 139 phosphorylation of H2A.X (Figure 8a). Similar results were also observed for H2A.X (Ser139) in western immunoblotting (Figure 8b) after identical treatment of Ovcar-3 cells with resveratrol without or with caffeine pre-treatment. Together, these findings suggest that resveratrol causes DNA damage leading to ATM/ATR activation followed by serine 139 phosphorylation of H2A.X, which is also known as an early marker of apoptosis induction.

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Fig. 8. Resveratrol induces H2A.X (Ser139) phosphorylation. (a) Ovcar-3 cells were treated with DMSO (control), or 50 µM dose of resveratrol without or with caffeine (10 mM) pre-treatment for 6 h, or exposed to UVC for 3 h followed by immunocytochemical staining for phospho-H2A.X (Ser139) using specific antibody as detailed in Materials and methods. Microscope images were taken under inverted Nikon TE-300 microscope with an epifluorescent attachment equipped with Princeton Instrument Micromax camera at 200x magnification using the Image Pro-plus software. Green fluorescence represents staining for phospho-H2A.X (Ser139). (b) Cells were cultured as described in Materials and methods, and treated with DMSO alone (control), or resveratrol (50 µM) without or with caffeine (10 mM) for 6 h. Caffeine was added 15 min before resveratrol treatment. At the end of these treatments, total cell lysates were prepared and subjected to SDSPAGE followed by western immunoblotting. Membranes were probed with anti-H2A.X (Ser139) and ß-actin antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection. Res, resveratrol; UVC, ultraviolet C. See online Supplementary material for a colour version of this figure.
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Resveratrol selectively induces S phase arrest and Ser139 phosphorylation of H2A.X in ovarian cancer cells but not normal cells
As we observed in the above results that resveratrol induces strong S phase arrest and phosphorylation of H2A.X at Ser139 in Ovcar-3 cells, additional studies were also performed to assess whether observed effects of resveratrol are specific to Ovcar-3 human ovarian cancer cells. Treatment of normal human foreskin fibroblast (HFF) and ovarian cancer PA-1 and SKOV-3 cells for 24 h with resveratrol at 10, 30 or 50 µM dose resulted in only a marginal increase (statistically not significant) in S phase population in HFF cells (Figure 9a). However, 24 h of resveratrol treatment showed a strong S phase arrest (5077% S phase population) in PA-1 cells as compared with DMSO control (43% S phase population, Figure 9b). The observed increase in S phase cell population was at the expense of both G1 as well as G2M phase cell populations (Figure 9b). Similar to Ovcar-3 and PA-1 cells, resveratrol also caused a strong S phase arrest at 10 and 30 µM doses in SKOV-3 ovarian cancer cells as compared with DMSO control; however, 50 µM dose of resveratrol showed lesser S phase population to its lower doses, and the cells started shifting towards G1 phase population (Figure 9c).

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Fig. 9. Resveratrol selectively induces S phase arrest and phosphorylation of H2A.X at Ser139 in ovarian cancer PA-1 and SKOV-3 cells, but not in normal HFF. Cells were cultured as described in Materials and methods and treated with either DMSO alone (control) or varying concentrations of resveratrol for 24 h. At the end of these treatments, cells were collected and incubated with saponinPI solution at 4°C for 24 h in the dark and subjected to FACS analysis as detailed in Materials and methods. The percentage of cell cycle distribution in HFF, PA-1 and SKOV-3 cells are shown in a, b and c, respectively. Data are presented as mean ± SE of triplicate samples. $, P < 0.05; #, P < 0.01; and *, P < 0.001 as compared with control. (d) Cells were cultured as described in Materials and methods, and treated with DMSO alone (control) or resveratrol (50 µM) for 24 h. At the end of these treatments, total cell lysates were prepared and subjected to SDSPAGE followed by western immunoblotting. Membranes were probed with anti-H2A.X (Ser139) and ß-actin antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection. Res, resveratrol.
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In the other studies assessing whether selective S phase arrest by resveratrol in ovarian cancer cells, but not in normal HFF, is also associated with the phosphorylation of H2A.X at Ser139, we observed that indeed that is the case. Following resveratrol treatment (50 µM dose for 24 h) of different cells, western immunoblot analysis showed a lack of phospho-H2A.X (Ser139) band in normal HFF cells, a strong band in Ovcar-3 and PA-1 cells, and a weak band in case of SKOV-3 cells (Figure 9d).
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Discussion
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Extensive data are now accumulating showing that dietary constituents can strongly influence the potential for disease outcome (37,38). A vast variety of naturally occurring substances have been shown to protect against experimental carcinogenesis and an increasing amount of evidence suggests that certain phytochemicals, particularly those included in our daily diet, have marked cancer chemopreventive properties (39,40). Resveratrol is a stilbene with a relatively broad distribution in plants and is present in various human foods, including red wines, peanuts and mulberries (41). It is an antioxidant and has been shown to inhibit various stages of tumor development (5,11). Evidence is provided that resveratrol is capable of binding to DNA and causing DNA damage (13). Our present study indicated that resveratrol induces S phase arrest and phosphorylation of H2A.X at Ser139 selectively in various ovarian cancer cells (Ovcar-3, PA-1 and SKOV-3), and that one of the plausible mechanisms accounting for the chemopreventive activity of resveratrol in Ovcar-3 cells occurs through ATM/ATR-dependent DNA damage and S phase arrest.
Our present study identifies resveratrol activity in Ovcar-3 cells via ATM/ATR checkpoint signaling-dependent S phase arrest. We also showed that ATM/ATR activation by resveratrol induces phosphorylation of Chk1 (Ser296) and Chk2 (Ser387, Ser19 and Thr68) followed by that of Cdc25C (Ser216) and Cdc2 (Tyr15), and cyclin B1 accumulation. Inhibition of ATM and ATR kinases by their specific inhibitor caffeine abrogated the phosphorylation of Chk1 and Chk2 as well as Cdc25C and Cdc2 (Tyr15). Inhibition of ATM and ATR kinases using caffeine/wortmannin treatment also reversed resveratrol-induced S phase arrest. Further, our data from the dominant Cdc2AF mutant experiment show that Cdc2 (Tyr15) phosphorylation is an essential event in resveratrol-induced S phase arrest in Ovcar-3 cells. Many DNA damaging agents, especially those that generate DNA double-strand breaks, are known to activate ATM kinase and induce ATM-dependent apoptosis (14). Histone H2A.X, one of the target proteins of ATM, is phosphorylated by activated ATM at serine 139 (31). In the present study, we have also demonstrated that resveratrol selectively induces serine 139 phosphorylation of H2A.X in various ovarian cancer cell lines including Ovcar-3, PA-1 and SKOV-3, which is inhibited by caffeine, suggesting the involvement of ATM/ATR in resveratrol-induced DNA damage signaling.
The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2 (42). Although Chk1 and Chk2 are structurally distinct they have functionally related kinases that phosphorylate an overlapping pool of cellular substrates (18). In mammalian cells, evidence has been presented that Chk1 and Chk2 have apparently evolved to channel DNA damage signals from ATM and ATR, respectively. This is based on reports that Chk1 appears to be activated by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions are activated primarily through ATM in response to IR (20,22). The activation of Chk2 in response to DNA damage requires phosphorylation at threonine 68 (43,44). Accordingly, our data is consistent with other emerging information on the phosphorylation of Chk1 and Chk2 by ATR and ATM, respectively. Resveratrol treatment induced pChk1 (Ser296) and pChk2 (Thr68, Ser387 and Ser19), which could possibly involve the activation of ATR and ATM kinases. By using caffeine, a known inhibitor of ATR and ATM, we observed that resveratrol-induced phosphorylation of Chk1 and Chk2 was abrogated. In western blot analysis, we also observed that resveratrol showed more effect on ATMChk2 as compared with ATRChk1 in Ovcar-3 cells.
Phosphorylation of Cdc25C on serine 216 by Chk1 or Chk2 creates a binding site for 14-3-3 proteins and results in export to and retention in the cytoplasm (45,46). Nuclear Cdc2 remains phosphorylated in the absence of Cdc25C and the cells remain arrested in the G2 phase. During S and G2 phase the Cdc2cyclinB complex is kept in the inactive state through Tyr15 phosphorylation of Cdc2 by Wee1/Mik1/Myt1 tyrosine kinases (32,33). During the G2/M transition Cdc2 is rapidly converted into the active form by Tyr15 dephosphorylation catalyzed by the Cdc25 tyrosine phosphatase (47,48). Elevated levels of Cdc2 (Tyr15) phosphorylation are found to be associated with S phase arrest after DNA damage in many cell culture systems (20,35). Consistent with these reports, our study shows that resveratrol induces phosphorylation of Cdc25C (Ser216) through Chk1 and Chk2 kinases and remains inactive. Further downstream, Cdc2 (Tyr15) did not get dephosphorylated by Cdc25C; therefore, due to lack of active Cdc2cyclin B1 complex, cells were not able to move through the mitotic phase. In the present study, we have confirmed that even in the synchronized condition cells accumulate in the S phase up to 48 h of resveratrol treatment. One possible reason could be that resveratrol-induced DNA damage is not getting repaired leaving the cells in S phase, which is later removed by the cytotoxic effect of the compound. Our further study supports this assumption as we observed that caffeine pre-treatment to resveratrol showed increase in G1 phase cell population as compared with S phase. These results suggest that resveratrol induces irreparable DNA damage through ATM/ATR-dependent signaling causing S phase arrest in Ovcar-3 cells. The results of our study are also in accord with another naturally occurring cancer preventive agent sulforaphane. In a recent study it has been shown that sulforaphane-induced G2M arrest in prostate cancer cells was associated with a rapid and sustained phosphorylation of Cdc25C at Ser216 via activation of Chk2 (49).
In summary, this study provides a mechanistic basis for resveratrol efficacy selectively in ovarian cancer cells, but not in normal HFF. Our data indicate that resveratrol efficacy in Ovcar-3 cells is via DNA damage and S phase arrest involving ATM/ATRChk1/2Cdc25CCdc2 pathway. We suggest that ATM/ATR could be a novel target for phytochemicals including resveratrol for cancer prevention and treatment.
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Supplementary material
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Supplementary material can be found at http://carcin.oxfordjournals.org/
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Acknowledgments
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This work was supported by USPHS grant CA64514, and the Flow Cytometry Core of CU Cancer Center is supported by Core Grant CA046934. The authors thank James Maller, PhD, for critical reading of the manuscript.Conflict of Interest Statement: None declared.
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Received May 2, 2005;
revised June 8, 2005;
accepted June 15, 2005.