[PubMed] [Google Scholar]Donze O, bbas-Terki T, Picard D. with potential implications in the treatment with histone deacetylase inhibitors. a, c, e) and total eIF2 (b, d, f). (B) eIF2S/S and eIF2A/A MEFs were treated with DMSO (con) or 10 M of vorinostat (SAHA) for the indicated time periods. Protein components (50 g) were subjected to western blot analysis for phosphorylated eIF2 (a) and total eIF2 (b). Representative blots are demonstrated. The percentage of the phosphorylated protein to total normalized to its control is definitely indicated. Quantification of the bands was performed by densitometry using the Scion Image software. Multiple eIF2 kinases are responsible for the induction of eIF2 phosphorylation upon treatment with vorinostat Next we wished to determine which of the eIF2 kinases is responsible Deoxycorticosterone for mediating eIF2 phosphorylation in response to vorinostat. To this end, we treated MEFs deficient in each of the four eIF2 kinases together with their isogenic wildtype MEFs with the chemotherapeutic agent and examined eIF2 phosphorylation. Consistent with the previous findings, we recognized an induction of eIF2 phosphorylation in all MEFs examined. However, even though the induction of eIF2 phosphorylation was reduced the knockouts (KO) of PERK, GCN2 and HRI compared to their isogenic wildtype cells (WT), it was not completely abolished in any of them, suggesting that vorinostat can activate more than one of the eIF2kinases (Number ?(Figure2A).2A). The redundancy of the eIF2 kinases was further confirmed by the use of double knock-outs of GCN2 and PERK(DKO) where the upregulation of eIF2 phosphorylation was only partially diminished in the absence of the two kinases (Number ?(Number2B),2B), further indicating that the induction observed, is a combinatorial event involving multiple kinases. Open in a separate window Number 2. Multiple eIF2 kinases are responsible for the induction of eIF2 phosphorylation upon treatment with vorinostat.(A) The indicated MEFs were treated with DMSO (con) or 10 M vorinostat for the indicated time periods. Protein components (50 g) were subjected to western blot analysis for phosphorylated eIF2 (a, c, e, g) and total eIF2 (b, d, f, h). (B) GCN2 -/- PERK -/- MEFs (DKO) were treated together with Deoxycorticosterone their isogenic control (WT) with DMSO (con) or 10 M vorinostat (SAHA) for the indicated time periods. Protein components (50 g) were subjected to western blot analysis for phosphorylated eIF2 (a) and total eIF2 (b). Representative blots are demonstrated. The percentage of the phosphorylated protein to total normalized to its control is definitely indicated. Quantification of the bands was performed by densitometry using the Scion Image software. eIF2 phosphorylation protects against vorinostat-induced cell death It is founded in the literature that eIF2 phosphorylation can play both cytoprotective or proapoptotic tasks depending on the type and period of stress [10;20]. Here, we wished to investigate the effect of eIF2 phosphorylation in respect to cell fate upon treatment with vorinostat. To this end, we treated eIF2S/S and eIF2/ MEFs with this drug and measured the cell death index by FACS analysis using Rabbit Polyclonal to OR2L5 propidium iodide (PI) staining. Our data display that eIF2/ MEFs are more sensitive to this drug than eIF2S/S MEFs, indicating that eIF2 phosphorylation protects against Deoxycorticosterone vorinostat-induced cell death (Number ?(Figure3A).3A). In order to confirm the FACS analysis data we examined the levels of cleaved caspase 3, a downstream effector of apoptosis. We observed high levels of cleaved caspase 3 in the treated eIF2/ MEFs, in contrast to the treated eIF2S/S MEFs where cleaved caspase 3 was almost not detectable (Number ?(Figure3B).3B). To extend our observations to human being cells, we treated HepG2 cells with vorinostat together with a derivative of salubrinal [21], sal003, a compound that raises phosphorylation of eIF2 by obstructing its dephosphorylation. Treatment with both providers decreased the cell death index in the co-treated cells compared to the cells treated only with the HDACi (Number ?(Number3C),3C), further validating that eIF2 phosphorylation protects from your apoptotic effects of the drug not only in mouse but also in human being cells. Open in a separate window Number 3. Phosphorylation of eIF2 protects against vorinostat-induced cell death.(A) eIF2S/S and eIF2A/A MEFs were treated with DMSO (con) or 10 M vorinostat (SAHA) for 48h and 72h and were subjected to FACS analysis after propidium iodide staining. Cell death is represented from the percentage (%) of cells in SubG1. Histograms symbolize the mean.