Vorinostat Induces DNA Damage in AML Cells Figure 2. Vorinostat causes cells to arrest in the G2-M phase of cell cycle and arrested cells have accrued DNA double strand breaks. A Cells were double stained with BrdU and PI, and analyzed by flow cytometry. Graphs show quantification of cells in each phase of the cell cycle: cells arrested in G1 phase (BrdU negative; 2N DNA content), in S phase (BrdU positive) and in G2-M phase (BrdU negative; 4N DNA content). B Western blot analysis of cyclin-E expression in NB4 cells treated with vehicle or 1.5 mM vorinostat. b-Actin was used as a loading control. C Cells were double stained with an antibody against P-Ser10-H3 and PI, and analyzed by flow cytometry. For DNA content, 1 = Sub-Go, 2 = G1, 3 = S and 4 = G2-M. Dot plots are representative of data collected and show an increase in mitotic cells positive for P-Ser10-H3, and an accumulation of cells in G2-M. Graphs show that the ratio of P-Ser10-H3 positive cells to cells with 4N DNA content (G2-M) does not change in response to vorinostat. D Cells were double stained for cH2AX and PI. Dot plots are representative of data collected and show an increase in cH2AX positive cells that is most significantly increased in G2-M. The graphs demonstrate an increase in the percentage of cH2AX positive cells amongst the cells arrested in the G2-M cell cycle phase. Asterisks indicate a significant difference and a non-significant difference is indicated as ‘‘n.s’’ (** ,0.01 *** ,0.001). doi:10.1371/journal.pone.0020987.g002 cells beginning at 24 h (Figure 3A). As opposed to the induction of cell cycle arrest, which was observed after 6 h–12 h of vorinostat treatment, no significant cell death until at least 18 h post-treatment (data not shown). We next assessed caspase- dependency of cell death using PI staining and a caspase-3/7 activity assay. Vorinostat induced caspase-3/7 activity in NB4 and U937 cells after 24 h of treatment, consistent with the appearance of cell death (Figure 3B). Inhibition of caspase activity by the pan-caspase inhibitor Z-VAD-FMK, suppressed vorino- stat-induced cell death (Figure 3C). Thus, DNA damage and G2- M arrest precedes vorinostat-induced apoptosis and this apoptosis is largely caspase-driven. Figure 3. Vorinostat induces apoptosis in AML cells following the induction of DNA damage and cell cycle arrest. NB4 and U937 cells were treated with vorinostat and assayed for apoptosis at the indicated time points. A Cells were stained with PI and analyzed by flow cytometry. Apoptosis was quantified as the percentage of cells with DNA content below that of the Go-G1 peak (Sub-Go). B Cells were counted and then assayed for caspase-3/7 activity using a luciferase-based assay, where reactive light units (RLU) are normalized to number of cells. C Cells were pre- treated with 50 mM Z-VAD-FMK, a pan-caspase inhibitor for 1 h and then treated with vorinostat for 48 h. Apoptosis was then quantified as percentage of cells with Sub-Go DNA content. doi:10.1371/journal.pone.0020987.g003 PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20987