Vorinostat Induces DNA Damage in AML Cells 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. doi:10.1371/journal.pone.0020987.g005 Vorinostat induces DNA damage in AML patient blasts and reduces their viability To extend our findings, leukemic blasts from AML patients were cultured and treated with vorinostat ex vivo, and DNA damage was assessed using the COMET assay. Vorinostat caused a significant increase in tail moment, beginning 12 h post vorinostat treatment (Figure 5A). The increase in DNA damage correlated with a decrease in cell viability (Figure 5B) and an increase in caspase-3/7 activity (Figure 5C), measured at a later time point (72 h). Blasts from an additional AML patient were assayed for caspase-3/7 activity in response to vorinostat and/or NAC. In these blasts, vorinostat caused a significant increase caspase-3/7 activity and pre- treatment with NAC abolished this increase (Figure S2). These data confirm that, as in our AML cell line models, treatment of AML patient blasts with vorinostat results in an early induction of DNA damage followed at later time points by a decrease in cell viability and apoptosis. In addition, they also suggest vorinostat-induced ROS as an important contributor to vorinostat-induced apoptosis and possibly DNA damage. Importantly, vorinostat did not induce DNA damage in PBMCs collected from healthy volunteers, as measured by alkaline comet assay (Figure S3A). Normal PBMCs also demonstrated a reduced sensitivity to vorinostat-induced loss of cell viability and apoptosis (Figure S3B, and C). Discussion HDACs are overexpressed in many tumor types and have been shown to play an important role in mediating aberrant epigenetic reprogramming, silencing tumor suppressor genes and favoring PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e20987