The DNA damage mark pH2AX differentiates the cytotoxic effects of small molecule HDAC inhibitors in ovarian cancer cells

Cancer Biology & Therapy 12:6, 484-493; September 15, 2011; © 2011 Landes Bioscience

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*Correspondence to: Dineo Khabele; Email: [email protected]: 02/25/11; Revised: 05/31/11; Accepted: 06/07/11DOI: 10.4161/cbt.12.6.15956

Introduction

The majority of high grade epithelial ovarian cancers harbor alter-ations in DNA damage response and repair molecules, including TP53 and BRCA mutations.1-3 A promising therapeutic approach for these high grade tumors is to inhibit DNA repair with small molecule compounds and promote DNA damage and cell death by synthetic lethality.3 Histone deacetylase inhibitors (HDACi) could be used to target high grade ovarian tumors in a similar manner since these agents have shown pre-clinical efficacy in ovarian cancer cells alone and combined with DNA damaging drugs.4-8 Studies have shown HDACi contribute to DNA dam-age-induced cell death,5,6,9 through changes in gene transcrip-tion, impaired responses to DNA damage, altered DNA repair

high grade epithelial ovarian cancers are relatively sensitive to DNa damaging platinum-based chemotherapy, suggesting that the dependencies of ovarian tumors on DNa damage response pathways can be harnessed for therapeutic purposes. Our goal was to determine if the DNa damage mark gamma-h2aX phosphorylation (ph2aX) could be used to identify suitable cytotoxic histone deacetylase inhibitors (hDaCi) for ovarian cancer treatment. Nineteen chemically diverse hDaCi compounds were tested in 7 ovarian cancer cell lines. Fluorescent, biochemical and cell-based assays were performed to assess DNa damage by induction of ph2aX and to measure cell viability and apoptosis. The relationships between ph2aX and the cellular effects of cell viability and apoptosis were calculated. Selected hDaCi were tested in combination with cisplatin and other DNa damaging agents to determine if the hDaCi improved upon the effects of the DNa damaging agents. The hDaCi compounds induced differing levels of ph2aX expression. high levels of ph2aX in hDaCi-treated ovarian cancer cells were tightly associated with decreased cell viability and increased apoptosis. Consequently, a ketone-based hDaCi was chosen and found to enhance the effects of cisplatin, even in ovarian cancer cells with extreme resistance to DNa damaging drugs. In conclusion, a fluorescent-based assay for ph2aX can be used to determine cellular responses to hDaCi in vitro and may be a useful tool to identify potentially more effective hDaCi for the treatment of ovarian cancer. In addition, these results lend support to the inclusion of ketone-derived hDaCi compounds for future development.

The DNA damage mark pH2AX differentiates the cytotoxic effects of small molecule HDAC

inhibitors in ovarian cancer cellsandrew J. Wilson,1 edward holson,2 Florence Wagner,2 Yan-Ling Zhang,2 Daniel M. Fass,2 Stephen J. haggarty,2,3

Srividya Bhaskara,4 Scott W. hiebert,4,5 Stuart L. Schreiber6 and Dineo Khabele1,5,*

1Department of Obstetrics and Gynecology; Division of Gynecologic Oncology; Vanderbilt University School of Medicine; Nashville, TN USa; 4Department of Biochemistry; 5Vanderbilt-Ingram Cancer Center; Vanderbilt University School of Medicine; Nashville, TN USa; 2Stanley Center for psychiatric Research; Broad Institute; Cambridge, Ma USa;

3Center for human Genetic Research; Massachusetts General hospital; harvard Medical School; Boston, Ma USa; 6Chemical Biology program; Broad Institute; howard hughes Medical Institute; Chemistry & Chemical Biology; harvard University; Cambridge, Ma USa

Keywords: histone deacetylase inhibitors (HDACi), pH2AX, ovarian cancer

Abbreviations: Ac, acetylation; CL, cleaved; DMSO, dimethyl sulfoxide; MS-275, entinostat; pH2AX, gamma-H2AX phosphorylation; HDACs, histone deacetylases; HDACi, histone deacetylase inhibitors; IC

50, inhibitory concentration at which

50% of cellular proliferation is reduced; IF, immunofluorescence; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly (ADP-ribose) polymerase; FK228, romidepsin; SAHA, vorinostat

and accumulation of reactive oxygen species.10,11 Selective inhibi-tion of class I HDAC isoenzymes with small molecules and gene silencing suppresses ovarian cancer cell viability.4,7 Furthermore, recent publications demonstrate that the class I HDAC 3 is important for genome maintenance and efficient DNA damage response and repair.12-14 Thus, several lines of evidence support the use of HDACi for targeting DNA damage response pathways in the treatment of ovarian cancer.

The HDACi compounds vorinostat (SAHA)15 and romidep-sin (FK228)16 have been approved recently for the treatment of cutaneous T-cell lymphoma. Unfortunately, in vivo responses in solid tumors including ovarian cancers have been disappoint-ing17,18 and HDACi appear to be more effective when used in combination with other drugs.10,11 Variations in responses to

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at 24 h after acute genotoxic injury is also associated with irrepa-rable DNA damage and ultimate cell death,19 suggesting pH2AX could serve as a surrogate mark of response to cytotoxic ther-apy. Recent studies have shown pH2AX is induced by HDACi in tumors, including ovarian cancer cells.5,6,9 However, it is not clear whether HDACi cellular responses can be distinguished by persistent activation of pH2AX. In this report we show that robust and sustained pH2AX induction can be a potential sur-rogate mark of response to HDACi compounds in ovarian can-cer cells with molecular defects in DNA response pathways (all with TP53 mutations).23,24 The differences in response appear to be influenced by chemical class, potency of HDAC isoenzymes inhibition and cell type. This approach also led to the selection of a ketone-derived HDACi that enhanced the effects of DNA dam-aging chemotherapy drugs even in highly chemotherapy-resistant ovarian cancer cells.

Results

Robust induction of pH2AX is tightly associated with the cytotoxic effects of HDACi in ovarian cancer cells. We evalu-ated pH2AX expression by fluorescent assay in SKOV-3 ovar-ian cancer cells (Fig. 1), because IF analysis is emerging as a

HDACi compounds are likely due to differences in: chemical class (e.g., hydroxamic acid or depsipeptide); HDAC isoform selectivity (e.g., HDAC1 vs. HDAC6 inhibition); non-histone effects (e.g., tubulin acetylation); pharmacokinetics and pharma-codynamics; and type of chemotherapy drug combination (e.g., DNA damaging agents). Responses to HDACi drugs also appear to be dependent on cell and tumor type. It is well-known that normal cells are less responsive to HDACi compared with cancer cells.7,9,18 However, it is not known which HDACi are best suited for treating susceptible high grade ovarian cancers. Thus, identi-fying appropriate HDACi for combination with DNA damaging agents could be highly relevant as a “synthetic lethal” approach. To determine if the cytotoxic effects of a diverse set of HDACi compounds could be distinguished in ovarian cancer cells, we used a fluorescent assay for activation of phosphorylated gamma-H2AX (pH2AX), a well-established mark of DNA damage.19-21

Characteristic pH2AX foci mark sites of double-strand DNA breaks and subsequently recruit multiple proteins involved in DNA damage response and repair.19-21 At the completion of proper DNA repair, pH2AX is typically de-activated. In genomi-cally unstable cells with dysfunctional DNA damage responses, pH2AX remains activated and cells continue to replicate with-out proper DNA repair.22 Prolonged activation of pH2AX foci

Figure 1. Differential induction of ph2aX by chemically diverse hDaCi compounds in SKOV-3 ovarian cancer cells. (a) Representative images (40x) of IF staining for ph2aX after 24 h exposure of cell treated with Saha to demonstrate how foci ph2aX were quantified. (B) The percentage distribution of nuclei displaying 1, 2–5 or greater than 5 ph2aX foci was quantified. Values are mean + Se for three independent fields of 100 cells each. (C) Represen-tative images (40x) of high (Saha and BRD7914) and low (BRD5051) ph2aX induction.

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with activating apoptosis and reducing cell viability as expected (p < 0.05). However, the strongest associations were between pH2AX induction, reduced cell viability and activated apoptosis (Fig. 3B). Evidence of apoptosis was also validated biochemically by protein gel blot determination of PARP cleavage (CL-PARP) (Fig. 3C).

In order to characterize the effects of the panel HDACi on non-histone targets, we evaluated the induction of a direct transcriptional target of HDACi, the cyclin-dependent kinase inhibitor, p21 (Fig. 3D).7,26,27 The induction of p21 induc-tion was associated with decreased cell viability (r2 = 0.68, p < 0.0001), and apoptosis by CL-PARP (r2 = 0.73, p < 0.0001) and CL-caspase-3 (r2 = 0.73, p < 0.0001). However, acetylated tubulin, another established non-histone target of HDACi associ-ated with HDAC6 inhibition (Fig. 3D),28 was not associated with cell viability (r2 = 0.04, p = 0.40) or apoptosis by CL-PARP (r2 = 0.12, p = 0.17) and CL-caspase-3 (r2 = 0.12, p = 0.16). BRD8148, a potent inducer of acetylated tubulin, had no effect on cell via-bility or apoptosis and did not activate pH2AX. In contrast, the class I HDACi apicidin, which has weak class II activity and thus does not acetylate tubulin in vitro, strongly induced pH2AX, sup-pressed cell viability and activated apoptosis (Fig. 3).

To test whether high inducers of pH2AX suppressed cell viability in other cells, we treated six additional ovarian cancer

method of choice for detecting pH2AX-associated DNA dam-age.20 Activation of pH2AX was measured by relative intensity to controls and percent distribution of pH2AX foci. Low levels of endogenous pH2AX expression were observed, even in untreated ovarian cancer cells. However, SAHA, FK228, BRD7914, apici-din and BRD0526 induced significantly higher levels of pH2AX, i.e., >20% of cells with more than five nuclear foci.

Large-scale cell viability assays were performed to assess the effects of HDACi treatment at screening concentrations of 10 μM (except for FK228 at 10 nM) compared with controls (Fig. 2A). SAHA, FK228, BRD7914, apicidin and BRD0526 inhibited cell viability by greater than 50% after 72 h of treatment. The same five compounds at the doses above showed significant activation of apoptosis (>20%) by immunofluorescence (IF) staining for cleaved caspase-3 (CL-caspase-3) at the 24 h time point, which was prior to the decrease in cell viability (Fig. 2B and C).

The relative effect of each HDACi on the induction of pH2AX was confirmed by protein gel blot analysis at the screening con-centrations described (Fig. 3A). All of the HDACi compounds increased pan H3 and pan H4 acetylation to some extent, vali-dating the functional efficacy of HDAC inhibition in a cell-based system. The global histone acetylation marks pan H3ac and pan H4ac, which are the most commonly used markers of response to HDACi in clinical trials,25 were positively associated

Figure 2. Distinct effects of hDaCi compounds on cell viability and apoptosis in SKOV-3 ovarian cancer cells. (a) effects of the hDaCi on cell viability were determined by MTT assays after 72 h of treatment. each dose was replicated six times. Values are mean + Se for three independent experiments. (B) apoptosis was detected by immunofluorescence (IF) evaluation of caspase-3 activation in response to 24 h of hDaCi treatment. The percentage of cells displaying cleaved caspase-3 (CL-caspase-3) was quantified (20x). Values are mean + Se for three independent fields of 100 cells each. (C) Repre-sentative fields (40x) demonstrating IF staining for CL-caspase-3 (green) in control and Saha-treated cells. The nuclei are stained with DapI (blue).

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broadest inhibitory effects on cell viability. In six of the seven cell lines, FK228 reduced cell viability at nanomolar concen-trations, consistent with what we previously published in refer-ence 4 and 7. The hydroxamates, SAHA and BRD0526, and the ketones, apicidin and BRD7914, were effective at reducing cell

cell lines with the panel of HDACi at the screening concentra-tions of 10 μM, (except for FK228, 10 nM) (Fig. 4A). The com-pounds were categorized by percent inhibition of cell viability in quintiles to generate a heat map. Similar to earlier results, the HDACi that induced the highest levels of pH2AX displayed the

Figure 3. Robust expression of ph2aX is strongly associated with reduced cell viability and activation of apoptosis in SKOV-3 ovarian cancer cells. (a) Representative protein gel blots from histone extraction (ph2aX, pan h3ac and pan h4ac) or whole cell lysates (paRp cleavage (CL-paRp), p21 and acetylated tubulin) after 24 h treatment with each hDaCi. equal loading of histone extracts was determined by probing for total histone h3 (histone extracts) or β-actin and α-tubulin (whole cell lysates). each experiment was repeated at least three times. (B) The association between the relative induction of ph2aX and reduced cell viability or activated apoptosis (CL-caspase-3 and CL-paRp) for each hDaCi compound was evaluated with the Spearman’s correlation coefficient (p < 0.05).

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inhibition alone failed to inhibit growth in any of the ovarian cancer cells represented.

A preclinical, ketone-based HDACi cooperates with the DNA damaging agent cisplatin pH2AX induction in ovarian cancer cells. BRD7914 is a ketone HDACi inspired in design

viability at concentrations less than 10 μM. As a class, the ortho- aminoanilides displayed moderate to low growth inhibition across the ovarian cancer cell lines and only MS-275 appeared to outperform other ortho-aminoanilides. BRD8148, which induced high levels of acetylated tubulin, suggesting HDAC6

Figure 4. Cell viability profiles of hDaCi compounds in a panel of ovarian cancer cells. (a) a heat map representing the effects of the 19 hDaCi compounds on cell viability in seven well-characterized ovarian cancer cell lines. Large-scale MTT assays were performed after 72 h hDaCi treatment. except for FK228 (10 nM), compounds were tested at a concentration of 10 μM. each dose was replicated six times. Values are mean + Se for three independent experiments. Quintiles of cell growth inhibition are represented by a different color in the key. hDaCi chemical class is also represented by a different color in the second key. (B) Representatives of major chemical classes of hDaCi are shown: hydroxamates, peptides, ortho-aminoanilides (benzamides) and ketones (*clinically available).

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with high pH2AX induction and suppressed cell viability in all of the ovarian cancer cell lines, including the chemotherapy resis-tant NCI/ADR-RES cells. NCI/ADR-RES ovarian cancer cells are well-known for extreme resistance to multiple chemotherapeutic agents, including cisplatin.23,24,32 Interestingly, with the exception of FK228, NCI/ADR-RES cells were exquisitely sensitive to all high inducers of pH2AX, and inhibited cell viability even at sub-micromolar concentrations (0.05–0.5 μM) (Figs. 5 and 6). Because of its relatively unique structure and efficacy in chemotherapy resistant cells, we chose BRD7914 for combinatory studies with cis-platin and other DNA damaging agents in NCI/ADR-RES cells.

As predicted by its strong induction of pH2AX, BRD7914 (0.05–5 μM) reduced cell viability in NCI/ADR-RES ovarian cancer cells and enhanced the effects of cisplatin (0.15–15 μM), even at low concentrations of both drugs (Fig. 5A). SAHA (0.05–5 μM) was used as a positive control. BRD7914 (at 0.05 μM—approxi-mately 50% of the IC

50) and cis-

platin (3 μM) increased pH2AX intensity and number of foci compared with each agent alone (Fig. 5B). The effects of SAHA, BRD7914 alone (both 0.05 μM) or in combination with cisplatin (3 μM) were validated with pro-tein gel blots for pH2AX induc-tion and CL-PARP (Fig. 5C). Furthermore, the ketone BRD7914 enhanced the effects of two mechanistically distinct DNA damaging agents, doxo-rubicin (0.005–0.5 μM) and etoposide (0.2–20 μM) in NCI/ADR-RES cells (Figs. 6A and B). As a comparison, two HDACi (BRD6929 and BRD5298) that

did not induce high pH2AX levels did not activate apoptosis alone or in combination with cisplatin (Figs. S1 and S2A). Neither of these HDACi, alone or with cisplatin, increased pH2AX to the same extent as BRD7914 or significantly induced CL-PARP in

by apicidin.29 Apicidin is a potent HDACi in vitro, but has not been developed clinically, because of poor pharmacokinetics and pharmacodynamics.29,30 BRD7914 and similar derivatives have shown some efficacy in vitro and in vivo and appear to have a bet-ter metabolic profile than SAHA.30,31 BRD7914 was associated

Figure 5. a ketone hDaCi compound is enhances the effects of cisplatin, even in cisplatin-resistant NCI/aDR-Res ovarian cancer cells. (a) effects on cell viability determined by MTT assays after 72 h treatment with Saha and ketone (BRD7914) alone or in simultaneous treatment with cisplatin at the indicated concentra-tions (in μM). (B) a Combination Index (CI) of hDaCi and cisplatin treatment was derived from experiments described in (a) at different fractional effects. (C) IF analysis of the effects of a 24 h exposure to Saha or BRD7914 alone (at 0.05 μM—approximately 50% of the IC50) or in combination with cisplatin (3 μM) on ph2aX (green) in NCI/aDR-ReS cells. DapI-stained nuclei are in blue. (D) protein gel Blot analysis of the effects of Saha, BRD7914 alone (both 0.05 μM) or in combination with cisplatin (3 μM) on ph2aX and paRp cleavage (CL-paRp), in NCI/aDR-ReS cells following 24 h treatment. equal loading for histone and whole cell extracts was confirmed by probing for total histone h3 and actin, respectively.

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that target DNA damage response and repair pathways to induce synthetic lethality as a treatment strategy.3 That chromatin modulating HDACi cause DNA damage via direct and indirect mechanisms preferentially in tumor cells, suggest these com-pounds could be used similarly to treat ovarian cancers.10,11 In this study, we have expanded upon previous results demonstrating the use of HDACi to enhance DNA damage and increase the cyto-toxicity of DNA damaging agents in susceptible ovarian cancer

the presence of cisplatin (Fig. S2A). Similar effects were observed in SKOV-3 ovarian cancer cells (Figs. 6C, D and S2B, C).

Discussion

High grade ovarian cancers are characterized by defective DNA repair processes associated with TP53 and BRCA mutations.1-3 Therefore, there is strong rationale for designing compounds

Figure 6. The effects of Saha and BRD7914 alone and in combination with other DNa damaging agents, doxorubicin and etoposide in ovarian cancer cells. Cell viability was determined by MTT assays in NCI/aDR-ReS cells following 72 h treatment of (a) doxorubicin or (B) etoposide alone or in concur-rent combination with the hDaCi, Saha or BRD7914. SKOV-3 cells were similarly treated with (C) doxorubicin and (D) etoposide with or without each hDaCi. Values shown are mean + Se of three independent experiments.

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resistant gene transporter36 and NCI/ADR-RES cells overexpress ABCB1.24 Neither of the clinically available ortho-aminoanilides (MS-275 or CI-994) exhibited significant potency in our system. Despite robust induction of pan H3ac and pan H4ac, this class of compounds did not significantly activate pH2AX by 24 h and exerted at best modest effects on apoptosis and cell viability. A recent clinical trial showed pH2AX induction did not accurately reflect the clinical efficacy of MS-275.37 In that study, activation of pH2AX occurred several days after treatment with MS-275, suggesting induction of pH2AX could be a result of rather than a contributor to tumor cell death. A potential explanation for this observation is the known slow on/slow off kinetics of some ortho-aminoanilides38 and suggests a different mechanism of action than other HDACi compounds. We are investigating whether this effect extends to other types of HDACi. Nevertheless, our finding that a preclinical ketone-based HDACi with a distinct chemical structure from currently available clinical compounds enhances the effects of cisplatin with concurrent treatment, par-ticularly in drug resistant cells, is pertinent for future develop-ment of this chemical class of HDACi.

In conclusion, induction of pH2AX is highly associated with the in vitro efficacy of select HDACi, alone and in combination with DNA damaging agents in ovarian cancer cells, including chemotherapy-resistant cells and provides additional support for the use of HDACi in ovarian cancer therapy. It is to be acknowl-edged that in vitro testing is not adequate for clinical applica-tion. However, these results may have clinical implications. First, consideration of HDACi chemical class, selectivity of HDAC isoenzymes and molecular features of the cells are critical for selecting HDACi for treating ovarian cancer cells. Second, the unexpected finding that pH2AX correlates more tightly with in vitro cytotoxicity than established HDACi clinical biomarkers, pan H3ac and pan H4ac, suggests that pH2AX could be a more relevant surrogate marker of response in the clinical investigation of HDACi. Finally, small molecule ketone-based HDACi should be considered as viable therapeutic options and warrant further development.

Materials and Methods

Cell culture. Ovarian cancer cell lines SKOV-3 and OVCAR-3 (American Type Culture Collection, Manassas, VA) and IGROV-1, OVCAR-4, OVCAR-5, OVCAR-8 and NCI/ADR-RES (National Cancer Institute, Bethesda, MD) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin, as previously described in reference 7. The seven well-characterized cell lines23,24 were used within 6 mo of receipt from the cell line banks and tested negative for mycoplasma. Cells were treated with HDACi com-pounds (below); DNA-damaging agents, cisplatin, doxorubicin and etoposide (Sigma Chemical Co., St. Louis, MO); and 0.01% dimethyl sulfoxide, DMSO (Sigma) in media as a control.

HDACi compounds. The panel of 19 HDACi compounds was selected by representative chemical class, current clinical use, preclinical novelty, knowledge of biochemical inhibition of HDAC proteins and availability. The HDACi represented

cells. Small molecule HDACi from distinct chemical classes were examined to determine the relative effects of the compounds on DNA damage, cell viability and apoptosis in ovarian cancer cells (all with TP53 mutations),23,24 to identify relevant HDACi for combination with cisplatin.

Robust induction of pH2AX, an established mark of DNA damage, was measured in ovarian cancer cells treated with hydroxamate (SAHA, BRD0526), ketone (apicidin, BRD7914) and depsipeptide (FK228) HDACi, with a more modest induc-tion in cells treated with ortho-aminoanilides (MS-275 and CI-994). Importantly, induction of pH2AX was tightly associ-ated with marked anti-tumor effects (reduced cell viability and increased apoptosis), consistent with observations that prolonged activation and persistence of pH2AX foci are associated with irreparable DNA damage and cytotoxicity.19,20 Through this process, we selected a preclinical ketone-based HDACi, which enhanced the effects of cisplatin, even in highly drug-resistant ovarian cancer cells.

There are multiple mechanisms by which HDACi cause DNA damage-induced apoptosis, including alterations in gene transcription and modulation of DNA damage response and repair proteins.10,11 Of ongoing investigation is whether selec-tivity toward specific class I (1–3 and 8) or class II (4 and 6) HDAC isoforms impacts the therapeutic efficacy of HDACi compounds.33 Here, we provide indirect evidence that targeting class I HDACs contributes to the mechanism of HDACi acti-vated DNA damage-induced cell death. First, FK228 and apici-din exhibited the strongest cytotoxic effects in our system and are known to be relatively class I HDAC-selective.34 Neither FK228 nor apicidin appeared to inhibit the class II HDAC 6, confirmed by lack of acetylated tubulin, the HDAC6 target, at the observed cytotoxic concentrations. Second, BRD8148, an HDAC6 selec-tive inhibitor, which is equipotent with SAHA toward HDAC6 in biochemical enzymatic assays (unpublished data) and induced tubulin acetylation similarly to SAHA in this study, demon-strated little to no effect across the cell lines examined. These findings are consistent with our previous work linking inhibi-tion of class I HDACs to the potent suppression of cell viability in ovarian cancer cells4,7 and the more recent studies showing that prolonged inhibition of the class I HDAC 3, disrupts proper DNA damage response and genome maintenance.12-14,35 It is to be acknowledged that alternative mechanisms of DNA damage, apoptosis and cell growth suppression through inhibition of the class II HDAC 6 have been described in prostate and breast can-cer cell lines.9 These discrepant results are consistent with other data showing that cellular responses to HDACi appear to be cell-type dependent.9-11,18

The development of new HDACi for ovarian cancer therapy is driven by the clinical limitations of current HDACi com-pounds. Despite successes in hematologic malignancies, the hydroxamate SAHA is rapidly metabolized10,11 and shows lim-ited clinical activity in ovarian cancer.17,18 The cyclic depsipep-tide FK228 has the most potent antitumor activity in the ovarian cancer cells tested, with the exception of multi-drug resistant NCI/ADR-RES cells. This observation could be explained by the fact that FK228 is known to induce ABCB1, the multi-drug

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5. Ozaki K, Kishikawa F, Tanaka M, Sakamoto T, Tanimura S, Kohno M. Histone deacetylase inhibitors enhance the chemosensitivity of tumor cells with cross-resistance to a wide range of DNA-damaging drugs. Cancer Sci 2008; 99:376-84; PMID: 18201278; DOI: 10.1111/j.1349-7006.2007.00669.x.

6. Yang YT, Balch C, Kulp SK, Mand MR, Nephew KP, Chen CS. A rationally designed histone deacety-lase inhibitor with distinct antitumor activity against ovarian cancer. Neoplasia 2009; 11:552-63; PMID: 19484144.

3. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 2009; 361:123-34; PMID: 19553641; DOI: 10.1056/NEJMoa0900212.

4. Son DS, Wilson AJ, Parl AK, Khabele D. The effects of the histone deacetylase inhibitor romidepsin (FK228) are enhanced by aspirin (ASA) in COX-1 positive ovar-ian cancer cells through augmentation of p21. Cancer Biol Ther 2010; 9:928-35; PMID: 20404564; DOI: 10.4161/cbt.9.11.11873.

References1. Bast RC Jr, Hennessy B, Mills GB. The biology of

ovarian cancer: new opportunities for translation. Nat Rev Cancer 2009; 9:415-28; PMID: 19461667; DOI: 10.1038/nrc2644.

2. Landen CN Jr, Birrer MJ, Sood AK. Early events in the pathogenesis of epithelial ovarian cancer. J Clin Oncol 2008; 26:995-1005; PMID: 18195328; DOI: 10.1200/JCO.2006.07.9970.

measuring the percentage of viable treated cells compared with controls.

Protein gel blotting. Whole cell protein isolation, protein gel blotting and signal detection were performed as described in ref-erence 7 and 26. For histone extraction, cell pellets were resus-pended in an extraction buffer: PBS containing 0.5% Triton X100 (v/v) (Sigma), 2 mM phenylmethylsulfonyl fluoride (Sigma) and 1:100 dilutions of protease and phosphatase inhibitor cocktails (Sigma). Cells were lysed on ice for 10 min and centrifuged for 10 min at 2,000 rpm at 4°C. The pellet was resuspended in 0.2 N hydrochloric acid (Sigma) supplemented with protease and phosphatase inhibitors and acid extracted overnight at 4°C. The samples were centrifuged for 10 min at 2,000 rpm at 4°C and the supernatant was collected for further analysis.

The following antibodies were used: pH2AX, anti-phospho H2AX (Ser139), anti-H3ac and anti-H4 ac (Millipore); anti-acetylated α-tubulin, anti-α-tubulin and anti-β-Actin (Sigma); anti-p21 (Thermo Scientific, Fremont, CA); and anti-Poly (ADP-ribose) polymerase (PARP) (Cell Signaling). Densitometry was conducted using Quantity One Version 4.5.2 software (Bio-Rad Laboratories, Hercules, CA). Raw values of protein were expressed relative to corresponding total histone H3 levels, β-Actin or α-tubulin levels.

Statistics. Unless otherwise indicated, values shown were the mean + SE of three independent experiments with p < 0.05 relative to control using the Student’s t-test. The relationships between HDACi induction of pH2AX, p21 and acetylated tubulin to cell viability and apoptosis were calculated using the Spearman’s correlation coefficient, p < 0.05.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

NIH grants: 1K08CA148887-01 (D.K.); 5P30 CA068485; CA091408 5 U54; 1UL1 RR024975; and 5R01DA028301-02 (S.J.H.), Stanley Medical Research Institute. The Gynecologic Cancer Foundation and Gloucester Pharmaceuticals (Celgene Corp.) (D.K.). The Vanderbilt immunohistochem-istry, Translational Pathology Shared Resource and High-Throughput Screening Cores. The Broad Institute Visiting Faculty Program.

Note

Supplemental materials can be found at:www.landesbioscience.com/journals/cbt/article/15956

five chemical classes: hydroxamates, ketones, thiols (depsipep-tides), esters and ortho-aminoanilides (benzamides). The dep-sipeptide romidepsin (FK228)39 was provided by Gloucester Pharmaceuticals (Cambridge, MA) and the ketone apicidin29 was purchased from Fermentek, Ltd. (Jerusalem, Israel). The following HDACi compounds were synthesized at the Broad Institute (Cambridge, MA) utilizing known procedures: the hydroxamates, vorinostat (SAHA)40 and BRD0526;41 the ester BRD5051;41 the ortho-aminoanilides, entinostat (MS-275),42 CI-994,43 BRD6929,44,45 BRD4657,38 BRD6498 and BRD3042)46 and ketone BRD7914.30 The following ortho-aminoanilides are unique to the Broad (BRD5100, BRD5298, BRD8451, BRD9773, BRD1783 and BRD1596)47 and were synthesized according to procedures previously published in ref-erence 34. BRD8148 is a hydroxamate unique to the Broad and has not been previously described. Biochemical assays confirm-ing HDAC inhibition for our panel of HDACi have been previ-ously published in reference 34. All of the HDACi compounds, with the exception of FK228, were tested at an initial concentra-tion of 10 μM, based on well-known methods for compound screening.32 FK228 was used at a 10 nM concentration because of its known potency.4,7,34,39

Immunofluorescence. After treatment with the HDACi compounds and/or cisplatin, ovarian cancer cells were fixed, permeabilized and stained for immunofluorescence (IF) analysis with anti-gamma phospho H2Ax (Ser139) (pH2AX) (Millipore Corp., Billerica, MA) (Millipore) and anti-caspase 3 (Cell Signaling Technology, Inc., Danvers, MA) as described in refer-ence 48. Binding of the primary antibodies was detected with Alexa Flour anti-rabbit IgG 594 or Alexa Flour anti-mouse IgG 488 secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA), as appropriate. For counts of pH2AX foci or activated caspase-3 expression, following drug treatments, three independent cell fields of 100 cells each were examined at 20X. Fluorescence microscopy was performed utilizing a Zeiss Axioplan 2 HAL100 fluorescence microscope (Carl Zeiss MicroImaging Inc.). Images were captured in 24 bit TIFF for-mat with a Zeiss Axiocam HRC camera and visualized utilizing Zeiss Axiovision 3.1 software.

MTT cell viability assays. Cell viability assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) were conducted using established methods,7,26,27 seeding at a density of 1,250 cells/well in 384 well plates (Corning Life Sciences, Lowell, MA). Absorbance was measured at 540 nm using a Spectramax M5 spectrophotometer (Molecular Devices, Sunnyvale, CA). Cell viability/survival was determined by

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