Vorinostat Induces DNA Damage in AML Cells Figure 1. Vorinostat induces different types of DNA damage in NB4 and U937 cells. NB4 and U937 cells were treated with 1.5 mM and 2.0 mM vorinostat, respectively, for the indicated time points. A Alkaline COMET assay to measure DNA double strand breaks, single-strand breaks and alkalile-labile sites. At least 100 nuclei were randomly selected and quantified for DNA damage, represented as an increase in Tail Moment (a.u) and Tail Length (a.u). Representative images of COMET tails obtained are presented in the lower panel. B Neutral COMET assay to measure DNA double strand breaks. DNA double strand breaks are represented as Tail Moment (a.u) and Tail Length (a.u). C Time-dependent induction of cH2AX in response to vorinostat as measured by western blot. b-Actin was used as a loading control. D Induction of the oxidative DNA lesion 8-oxoguanine in response to vorinostat was quantified by flow cytometry using a FITC-conjugated antibody. Asterisks show significant difference and a non- significant difference is indicated as ‘‘n.s’’. (** ,0.01 *** ,0.001). doi:10.1371/journal.pone.0020987.g001 luminescent signal (RLU). Luminescence was measured using a FLUOstar OPTIMA microplate reader (BMGLabtech). The luminescent signal was then normalized to cell number and is reported as RLU/cell. Glutathione Quantification Glutathione (GSH) was quantified using GSH-Glo Glutathione Assay (Promega). Briefly, cells were counted and washed with PBS. For each condition, 10 000 cells were plated. Cells were then incubated in a solution containing a luciferin derivative and Glutathione S-Transferase for 30 min at room temperature. In the presence of GSH, the luciferin derivative is converted to luciferin. The cells were then incubated with luciferase enzyme, which initiates a luminescence signal directly proportional to the luciferin previously generated. Luminescence was measured using a FLUOstar OPTIMA microplate reader. Serial dilution of a GSH standard solution was included in order to generate a standard curve, facilitating the conversion of RLU to GSH concentration. Cells treated with buthionine sulphoximine (BSO) were also included as a control. Statistics Significance was determined by ANOVA followed by Newman- Keuls post-tests using Prism version 4.0 (GraphPad). Results Vorinostat induces a variety of DNA damage in AML cells Previously, vorinostat has been shown to induce DNA double strand breaks (DSBs) [24]. To investigate the possibility of other types of DNA damage, we performed a comprehensive analysis of vorinostat-induced DNA damage. Two AML cell lines, NB4 and U937, were treated with equally cytotoxic and clinically achievable concentrations of vorinostat before being subjected to single-cell gel electrophoresis (COMET assay). The COMET assay was performed under alkaline conditions, as this permits the detection of a broad spectrum of DNA lesions including DSBs, DNA single- strand breaks and alkalile-labile sites. Treatment with vorinostat caused a significant increase in COMET tail moment and tail length (Figure 1A). To specifically assess vorinostat-induced DSBs, the COMET assay was also performed under neutral conditions, which makes the assay more specific to DSBs but still allows for the detection of cross-links. Indeed, a significant increase in tail moment and tail length was observed and increased in a time- dependent manner (Figure 1B). To verify induction of DSBs, phosphorylation of H2AX was measured by western blot. The phosphorylated form of H2AX (cH2AX) localizes to DSBs within minutes of their formation and is therefore a sensitive marker for this lesion [25]. Vorinostat induced cH2AX after 12 h of treatment in both NB4 and U937 cells (Figure 1C). It should be noted that an increase in cH2AX could be observed after 6 h of vorinostat when blots were over-exposed (data not shown). Also, a number of studies have shown that vorinostat induces ROS. Therefore, we assayed for oxidative DNA damage by staining cells with a fluorescent antibody that recognizes 8-oxoguanine (8- oxo-G) and quantified its induction by flow cytometry. This DNA adduct is a sensitive marker of oxidative stress. We detected an early and time-dependent increase of 8-oxo-G in both NB4 and U937 cells (Figure 1D). Collectively, these data demonstrate that vorinostat is able to induce a variety of DNA damage including DSBs and oxidative DNA lesions. Vorinostat increases DNA replication and induces a G2-M arrest followed by apoptosis DNA damage is often accompanied by arrest in cell cycle, and HDACi have been shown to cause a G2-M and/or G1 arrest depending on the malignancy/model and HDACi concentration used. To determine if vorinostat-induced DNA damage resulted in a cell cycle arrest, we performed a double-stain with BrdU (incorporates into replicating cells) and PI (provides a measure of DNA content). The BrdU-PI stain allows for gating and accurate calculation of the percentage of cells in each phase of the cell cycle and discriminates late S-phase from G2 cells and early S-phase cells from G1, as opposed to only staining with PI. We observed a substantial increase in BrdU negative cells with 4N DNA content (G2-M phase) after 6 h–12 h of vorinostat with a decrease in BrdU negative cells with 2N DNA content (G1 phase) (Figure 2A). This demonstrates that vorinostat induces a G2-M phase arrest consistent with the appearance of DNA damage. We detected a slight increase in replicating cells (BrdU positive cells), which is supported by an increase in cyclin-E as detected by western blot (Figure 2B). To further characterize the G2-M arrest, a double-stain with histone H3 phosphorylated at serine 10 (P- Ser10-H3), a marker of mitosis, and PI was performed and quantified by flow cytometry. Vorinostat was able to induce P- Ser10-H3 and, as previously shown, caused an accumulation of cells with a G2-M DNA content (Figure 2C). However, there was no change in the ratio of P-Ser10-H3 positive cells to cells arresting in G2-M. This suggests that the cell cycle arrest is not specific to the G2-phase or mitosis. Taxol was used as a positive control, as it stabilizes microtubules, causing cells to arrest in mitosis resulting in a significant increase in the percentage of P- Ser10-H3 positive cells within the G2-M population [26]. Next, to determine if the cells arresting in G2-M have damaged DNA, a double-stain with cH2AX and PI was performed and quantified by flow cytometry. The cH2AX-PI stain demonstrated that vorinostat induces a significant increase of cH2AX positive cells arresting in G2-M versus vehicle treated control (Figure 2D). This suggests that cells with DSBs eventually arrest and accumulate in the G2-M phase. Importantly, the cH2AX positive cells were not predominantly found in the Sub-Go population. This indicates that cH2AX is not induced solely as a consequence of apoptosis. These data suggest that vorinostat causes cells to exit the G1 phase of the cell cycle and that cycling cells from G1 eventually arrest in the G2-M phase, likely due to an accumulation of DNA damage. To assay for cell death, NB4 and U937 cells were treated with vorinostat and subsequently stained with PI in order to quantify fragmented DNA by flow cytometry. Vorinostat induced cell death in a dose- and time-dependent manner in both NB4 and U937 PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20987