The precise molecular mechanisms involved in the cancer therapeutics of HDAC inhibitors may depend highly on the cellular context or the genetic lesion and epigenetic background of the cancer. For targeted or cus tomized www.selleckchem.com/products/nutlin-3a.html cancer therapy, it is essential to understand the distinct mechanisms of apoptotic cell death induced by HDAC inhibitors. Our study demonstrates that although the primary target of HDAC inhibitors may be transcrip tion, it is the cellular environment, or the ability of cells to maintain their survival protein networks that determines their fate, to die or to survive in response to the treatment. Methods Cell culture and reagents The cells were maintained in Dulbeccos Modified Eagle Medium supplemented with 10% fetal bovine serum at 37 C with 5% CO2.

Valproic acid and sodium butyrate were purchased from Sigma. Antibodies against Akt, phosphor Akt and caspase3 were obtained from Cell Signalling. Flow cytometry analysis Following exposure to valproic acid or sodium butyrate for 8, 16 or 24 hours, cells were detached from tissue culture dishes by trypsinization, combined with floating cells and fixed in 70% ethanol for 30 min utes at 20 C. After washing with phosphate buffered saline, the cells were incubated with 50 g ml of RNase A and 50 g ml of propidium iodide for 30 minutes at 37 C. DNA contents of the cells were then profiled by flu orescence activated cell analyzer to determine the distribution of cells in different phases of the cell cycle. Quantitative real time RT PCR Total RNA was isolated using RNeasy Mini Kit and reverse transcribed using random primers and Super script II.

Serial dilution of the cDNA was used to determine the appropriate concentration required for the real time PCR amplification which was carried out by using TaqMan reporter assay with a 7500 Fast Real Time PCR System. Gene specific primers and TaqMan probes used for the amplification were ordered from Applied Biosystems. Quantification of mRNA levels was performed by using 18S rRNA as an internal control. Protein extraction and Western blot analysis Following various treatments, cells were washed and har vested. The cell pellets were suspended and incubated for 30 minutes at 4 C in whole cell extraction buffer consist ing of 10% glycerol, 50 mM Tris HCl, 400 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P 40.

The lysates were then cen trifuged at 14,000 g for 10 minutes at 4 C. Protein con centrations were determined by Bradford assay using bovine serum albumin as standard. PerkinElmer Life Sciences ECL system AV-951 was used for the detection and Scion Image software was used to quantify the Western blots. Caspase activity assay Following various treatments, cells were assayed for cas pase activities by using fluorescent assay kits and a microplate fluorometer.