Vorinostat Induces DNA Damage in AML Cells 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. doi:10.1371/journal.pone.0020987.g006 transformation [2,27]. HDAC activity can be targeted using small molecule HDACi. Currently, two HDACi (vorinostat and romidepsin) have garnered FDA approval, both for the treatment of CTCL [28,29]. One manner by which HDACi have been proposed to induce cell death is through the induction of DNA damage and/or genomic instability. In this work, we have performed a careful analysis of a clinically relevant HDAC inhibitor, vorinostat, on DNA damage, cell growth and apoptosis. For the first time, we show that vorinostat induces DNA damage followed by a G2-M arrest (Figure 6). This DNA damage correlates with increased apoptosis in leukemic cell lines (Figure 6) and, importantly, increased apoptosis in primary leukemic blasts. Vorinostat is FDA approved for the treatment of CTCL, where it is administered at a dose of 400 mg once daily [29]. In CTCL patients this regimen resulted in a maximum concentration 1.260.53 mM. The same regimen in patients with advanced solid malignancies resulted in a maximum concentration of 1.8160.70 mM [30]. Recently, maximum concentrations of 4.8062.8 mM vorinostat have been achieved in patients using a pulse dose approach [31]. The doses used in this study to induce DNA damage and apoptosis in AML cells are within the clinically achievable range. At lower doses vorinostat does not induce apoptosis but rather growth arrest and differentiation [20]. We speculate that the DNA damage inflicted at the higher vorinostat doses is a sufficiently traumatic event to trigger apoptosis, and as such is an important factor determining whether the cells will growth arrest or die. Consistent with a previous report [16], we find DNA double strand breaks (DSBs) to be induced by vorinostat as early as 6 h (by neutral COMET assay) coinciding with the induction of cH2AX (Figure 1). The induction and persistence of DSBs we observe may be due to a number of mechanisms. For example, HDACi have previously been shown to induce DSBs or inhibit their repair by down-regulating expression of DNA repair proteins including NBS1, BRAC1/2, Ku70, Ku80 and RAD51 [15,32,33]. However, we were unable to detect any reduction in Ku70, RAD51 or NBS1 protein levels in vorinostat treated NB4 or U937 cells (Figure S4). This leads us to hypothesize that the DSBs may be due altered repair protein function [14] or modulation of the DNA damage ‘‘histone-code’’ [34,35,36]. It is worth noting that malignant cells have been shown to be more sensitive to HDACi induced DNA damage and apoptosis than non-malignant cells [37,38], an observation that we confirmed in vorinostat-treated leukemic blasts, compared to normal PBMCs (Figure S3). Of interest is a recent study where vorinostat treatment in MCF-7 breast cancer cells induced DSBs that were found to co- localize with replication factories and correlated with a slight decrease in DNA replication and a significant reduction in replication fork velocity [24]. Using similar methods (BrdU incorporation) we show the opposite observation that vorinostat increases DNA replication in NB4 and U937 cells. It is tempting to speculate that in our cell lines, the increase in DNA replication results in a greater amount of DNA damage compared to in MCF- 7 cells, where vorinostat-induced damage results in the arrest of replication forks. In addition, MCF-7 cells arrest both in G1 and G2-M in response to vorinostat, possibly limiting the effect of vorinostat-induced DNA damage and cell death. Indeed, in these cells, higher doses of vorinostat (4–10 mM) are generally required to slow DNA replication, induce cell cycle arrest or apoptosis and even then the apoptosis observed is relatively modest [24,39]. Interestingly, inhibiting the G1-phase arrest has been demonstrat- ed to sensitize MCF-7 cell to HDAC inhibitor-induced apoptosis [40]. In contrast, NB4 and U937 cells are unable to mount a G1- phase arrest in response to vorinostat, possibly leading to a greater accumulation of DNA damage and more cell death. This is supported by the significant increase in cH2AX positive cells arresting in G2-M in response to vorinostat (Figure 2). It is also of general interest to mention that while capable of a G1-arrest [41], U937 cells do not arrest at the G1-phase in response to vorinostat, PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e20987