Figure 1.
Vorinostat induces different types of DNA damage in NB4 and U937 cells.
NB4 and U937 cells were treated with 1.5 µM and 2.0 µM 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 γH2AX in response to vorinostat as measured by western blot. β-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).
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 µM vorinostat. β-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 γH2AX and PI. Dot plots are representative of data collected and show an increase in γH2AX positive cells that is most significantly increased in G2-M. The graphs demonstrate an increase in the percentage of γH2AX 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).
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 µM 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.
Figure 4.
Vorinostat mediated DNA damage and apoptosis is in part due to reactive oxygen species.
A NB4 and U937 cells were treated with vorinostat for 6 h and 18 h and stained with DCF-DA to quantify intracellular peroxides. B NB4 and U937 cells were treated with vorinostat for 6 h, 12 h and 18 h and induction of HO-1 was assessed by western blot. β-Actin was used as a loading control. C U937 cells were pre-treated with 2 mM N-acetyl cysteine (NAC), an antioxidant, followed by 2.0 µM vorinostat for 18 h. Induction of γH2AX was assessed by western blot. β-Actin was used as a loading control. D Cells were stained with PI after 24 h of exposure to vorinostat and/or N-acetyl cysteine, and apoptosis was quantified as percentage of cells with Sub-Go DNA content. Asterisks show significant difference (** <0.01 *** <0.001).
Figure 5.
Vorinostat induces DNA damage and apoptosis at clinically achievable concentrations in AML-patient derived blasts cultured ex vivo.
A Alkaline COMET assay measures DNA double strand breaks, single strand breaks and alkalile-labile sites. DNA damage is represented as an increase in Tail Moment (a.u.) after 12 h exposure to vorinostat. B Cells were assayed for viability 72 h post-vorinostat treatment using a luciferase based assay. C Caspase-3/7 activity (normalized to cell number) was assessed as a marker of apoptosis.
Figure 6.
Treatment of acute myeloid leukemia cells with vorinostat results in an early induction of reactive oxygen species.
The production of reactive oxygen species coincides with the appearance of DNA damage and partially contributes to the DNA damage accumulated. Reactive oxygen species production and accrual of DNA damage is followed by a G2-M cell cycle arrest and finally apoptosis at later time points.