despite inducing expression of p21 (data not shown), a powerful inducer of the G1-phase arrest. Consistent with this observation, p21 expression does not coincide with a degradation of cyclin-E (Figure 2B). A number of studies have reported that vorinostat induces ROS in leukemic cells [18,42] and other malignant cell types [43,44]. To expand on these findings, we found that vorinostat induced 8- oxo-G, a marker of oxidative DNA damage (Figure 1D). It should be noted that the increase in vorinostat-induced 8-oxo-G was statistically significant in U937 but not NB4 cells. 8-oxo-G represents a single oxidative lesion and vorinostat may induce other oxidative lesions like 2,6-diamino-4-hydroxy-5-formamido- pyrimidine, 3-methyladenine and/or 7-methyladenine to a greater extent in NB4 cells. The ability of vorinostat to induce such oxidative lesions will be assessed in future studies. The persistence of such damaged bases can lead to increasingly cytotoxic lesions such as DSBs [45]. Indeed, pre-treatment with NAC greatly diminished vorinostat-induced cH2AX, resulting in both a reduced G2-M phase arrest and apoptosis (Figure 4). However, the reduction in apoptosis by NAC is modest and highlights the possibly pleiotrophic mechanisms of vorinostat-induced DNA damage and apoptosis. Indeed, HDACi have been demonstrated to activate both death receptor (extrinsic) and mitochondrial (intrinsic) apoptotic pathways. HDACi have been shown to up- regulate death ligands TRAIL, TRAIL-R2, Fas, Fas-L and TNFa of the extrinsic pathway [3]. HDACi have also been shown to induce pro-apoptotic genes (i.e. Bmf, Bim, Bax, Puma, Noxa) and down-regulate pro-survival genes (i.e. survivin, XIAP) favoring initiation of the intrinsic apoptotic program [3]. Finally, we investigated DNA damage in primary leukemic blasts. Consistent with our findings in cell lines, vorinostat-induced DNA damage could be measured at an early time point (Figure 5A). The induction of DNA damage by vorinostat was also consistent with a reduction in cell viability and an increase in caspase-3/7 activity (Figure 5B, 5C). It is interesting to note that while patient-derived leukemia blasts cultured ex vivo do not cycle, DNA damage is still induced in response to vorinostat. It is possible that in the context of this model, vorinostat-induced DNA damage is replication-independent and transcription-dependent, as previously demonstrated by Conti et al. [24]. In the clinic, the results of vorinostat monotherapy have been mixed [46,47]. Our data demonstrate that DNA damage is a relevant mechanism of vorinostat-induced cytotoxicity in AML cells. We propose the assessment of DNA damage in patients treated with vorinostat to ascertain whether the induction of DNA damage correlates with response. Our findings suggest that AML patients unable to mount a G1-phase arrest to vorinostat may respond favorably. Indeed, future directions will be aimed at understanding the mechanisms by which vorinostat bypass the G1-phase arrest in our cell line models. The existence of numerous mechanisms by which vorinostat promotes genomic instability suggests the combination of vorinostat with other DNA damaging agents, or possibly G1-arrest inhibitors. Indeed, vorinostat along with other HDACi have been demonstrated to enhance sensitivity to ionizing radiation and DNA damaging drugs such as mitomycin C, cisplatin, bleomycin, doxorubicin, etoposide, 5-fluorouracil, Ara-C and topotecan [48,49]. Alternatively, the many mecha- nisms of vorinostat may complicate more precisely targeting a desired cellular function, thereby lessening its therapeutic efficacy. References Vorinostat Induces DNA Damage in AML Cells For example, the vorinostat target HDAC3 has been meticulously demonstrated in knockout and knockdown experiments to be vital to DSB repair [24,50]. Thus, the development of more specific HDACi that target a single HDAC class or isoform may be a promising approach to improve efficacy of this novel class of anti- cancer agents. Supporting Information Figure S1 Vorinostat does not alter intracellular GSH, and antioxidants reduce vorinostat-induced G2-M arrest and apoptosis. A GSH levels were quantified after a 4 h treatment with vorinostat in NB4 and U937 cells. BSO inhibits GSH synthesis and was used as a control. B NB4 and U937 cells were pre-treated for 1 h with NAC followed by vorinostat treatment for 18 h. Afterwards, the cells were stained with propidium iodide to measure DNA content. C NB4 and U937 cells were pre-treated for 1 h with the antioxidant L-GSH or the SOD mimetric MnTMPyP for 1 h followed by vorinostat treatment for 24 h. Cells were then stained with propidium iodide to quantify DNA fragmentation. Asterisks indicate a significant difference (*** ,0.001). (TIF) Figure S2 The antioxidant NAC protects against vorino- stat-induced caspase-3/7 activity. Blasts from an AML patient were treated with vorinostat and/or NAC for 72 h at which point caspase-3/7 activity was quantified. Asterisks indicate a significant difference and a non-significant difference is indicated as ‘‘n.s’’. (*** ,0.001). (TIF) Figure S3 Normal PBMCs are less sensitive to vorino- stat-induced DNA damage and apoptosis. PBMCs were collected from healthy volunteers and treated with vorinostat. 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). B 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). C Cells were treated with vorinostat for 72 h and assayed for viability using a luciferase based assay. D Caspase-3/7 activity (normalized to cell number) was assessed after 72 h as a marker of apoptosis. Asterisks indicate a significant difference and a non-significant difference is indicated as ‘‘n.s’’. (*** ,0.001). (TIF) Figure S4 Vorinostat has no effect on DNA repair protein expression. Protein levels for the DNA repair proteins Ku70, RAD51 and NBS1 were assayed by western blot. b-Actin was used as a loading control. (TIF) Author Contributions Conceived and designed the experiments: LAP DD-R FP WHM. Performed the experiments: LAP DD-R FP HR SS. Analyzed the data: LAP DD-R FP HR SS WHM. Contributed reagents/materials/analysis tools: WHM. Wrote the paper: LAP DD-R FP WHM. 1. Martens JH, Stunnenberg HG (2010) The molecular signature of oncofusion proteins in acute myeloid leukemia. FEBS Lett 584: 2662–2669. 2. Pan LN, Lu J, Huang B (2007) HDAC inhibitors: a potential new category of anti-tumor agents. Cell Mol Immunol 4: 337–343. PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e20987