Vorinostat has previously been shown to induce reactive oxygen species, growth arrest and apoptosis in leukemia cells and leukemia mouse models [19,20,21,22]. However, the ability of vorinostat to induce DNA damage in leukemia cells is yet to be reported. In this study, we investigated the mechanisms by which vorinostat- induced DNA damage affects cell growth and apoptosis. We show for the first time that at low, clinically achievable doses, vorinostat induces a variety of DNA damage in leukemia cell lines and AML patient-derived blasts cultured ex vivo. These data demonstrate DNA damage to be a contributor to vorinostat-mediated growth arrest and apoptosis mechanisms. Materials and Methods Ethics Statement Patient samples obtained from the Quebec Leukemia Cell Bank were collected in accordance with the ethical requirements and regulations of the Quebec Leukemia Cell Bank Ethics Committee. Ethical approval for this study was obtained from the Jewish General Hospital and the McGill Institutional Review Board. All patient samples obtained, collected and used were done so in accordance to the ethical requirements and regulations of the Jewish General Hospital and the McGill Institutional Review Board. Informed written consent was obtained from all partici- pants involved. Cell lines, patient cells and reagents Vorinostat was obtained courtesy of Merck (Whitehouse Station, NJ, USA) and dissolved in DMSO. Z-VAD-FMK was purchased from Promega. N-acetyl cysteine (NAC) was purchased from Sigma and dissolved in DMEM. NB4 and U937 cells were obtained from ATCC and cultured in RPMI supplemented with 10% fetal bovine serum (FBS) at 37uC with 5% CO2. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and blasts from patient FG04290 (AML type M3) and 281010 (AML inv(3)) were isolated from blood samples obtained with patient informed consent. Briefly, blood was centrifuged and the serum discarded. Cells were then resuspended in HBSS, layered over Ficoll and centrifuged. Blasts that remained suspended in the Ficoll were collected for cell culture and treatment. Cryopreserved blasts from patients 03-H070 and 07-H020 (type M3) were obtained from the Quebec Leukemia Cell Bank (Maisonneuve- Rosemont Hospital). COMET assay COMET assays was performed under alkaline or neutral conditions as previously described [23]. Briefly, cells were imbedded in low melting point agarose and layered onto glass slides (Trevigen). Slides were incubated in lysis buffer overnight at 4uC. Slides were then washed and incubated in alkaline buffer (pH.13) for 30 minutes. The COMET slides were then subjected to electrophoresis at 20 V and 240 mA for 30 minutes. After- wards, the slides were washed in distilled water and fixed using 70% ethanol. Finally, cells were stained with ethidium bromide (1 mg6mL21). Images were randomly collected for analysis of at least 100 nuclei in duplicate experiments using a fluorescent microscope (Leica). Comets were scored using CometScore (TriTek) for tail moment (TM) and tail length (TL). TM and TL are presented in arbitrary units (a.u). Western analysis Whole cell extracts were prepared from pelleted cells lysed in RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0). Western Vorinostat Induces DNA Damage in AML Cells blot was performed on extracts to detect protein levels of cH2AXser139, NBS1 (Cell Signaling), Ku70, cyclin-E and RAD51 (Santa Cruz Biotechnology). b-Actin was purchased from Sigma and confirmed equal protein loading. BrdU-PI staining BrdU was added to cells in culture for 15 minutes, collected, washed twice in warm PBS, and fixed in ice cold 70% ethanol. Fixed cells were then washed twice in PBS and once in PBS with 0.1% BSA. Finally, cells were incubated with BrdU-FITC conjugated antibody, washed and counterstained with propidium iodide. Samples were analyzed using a FACSCalibur flow cytometry (BD Bioscience) and FCS Express v3.0 (De Novo Software). Ten thousand events were collected per sample. Quantification of cH2AXser139 and P-Ser10-H3 by flow cytometry Cells were treated and fixed in 1% paraformaldehyde. Afterwards, cells were permeabilized by 1% Triton dissolved in PBS. Permeabilized cells were incubated with a FITC-conjugated antibody directed against cH2AXser139 or P-Ser10-H3 at room temperature. Cells were concomitantly stained with propidium iodide to assess DNA content. Fluorescence was measured by flow cytometer and analyzed using FCS Express v3.0. Ten thousand events were recorded per sample. Propidium iodide staining Cells were seeded at 26105cells6mL21 and treated as described. Subsequently, cells were collected and washed in supplemented PBS (5% FBS/0.01 M NaN3) at 4uC, pelleted, and resuspended in a hypotonic fluorochrome solution (50 mg?ml21propidium iodide, 0.1% sodium citrate, 0.1% Triton X-100). Fluorescence was detected by flow cytometry and analysis was performed using FCS Express v3.0. Ten thousand events were recorded per sample and allowed for investigation of cell cycle and apoptosis. Cells undergoing DNA fragmentation and apoptosis were defined as events with fluorescence weaker than the Go-G1 cell cycle peak (Sub-Go). ROS Detection Cells were treated with vorinostat for 6 h and 18 h. Cells were then incubated with 25 mg/ml 29-79-dichlorofluorescin diacetate (DCF-DA) for 30 minutes at 37uC for the detection of intracellular peroxides (mainly H2O2). Cells were collected and washed in PBS. Fluorescence was measured by flow cytometry and analyzed using FCS Express v3.0. Cell viability and caspase-3/7 assay Cell viability was assayed using CellTiter-GloH Luminescent Cell Viability Assay (Promega). This assay aims to quantify the amount of ATP present as this correlates with the number of metabolically viable cells in culture. Briefly, cells treated with vorinostat were incubated in a cell lysis solution containing a luciferin derivative, Ultra-GloTM Recombinant Luciferase and Mg2+ for 10 minutes at room temperature. The solution lyses the cells and converts the luciferin derivative into a luminescent signal proportional to the amount of ATP present. Caspase-3/7 activity was assayed using Caspase-Glo 3/7H Assay (Promega). Briefly, cells treated with vorinostat were incubated in a cell lysis solution containing a luciferase substrate derivative, Ultra-GloTM Recom- binant Luciferase and Mg2+ for 1 hour at room temperature. The luciferase substrate derivative is specifically cleaved by active caspase-3/7 resulting in the conversion of the substrate into a PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20987