The substances were dissolved in dimethyl sulf oxide. The stock solutions were stored at 20 C. For experimental use the drugs were freshly pre pared from stock solution. Cell lines The Vorinostat order human B ALL cell lines SEM, RS4,11 and the T ALL cell lines Jurkat and MOLT4 were purchased from DSMZ and cultured according Langenselbold, Germany and incubated for up to 72 h with different concentrations of PDA 66. Treated cells were harvested after 4, 24, 48 and 72 h and used for further analyses. Control cells were cultured in medium containing the same concentration of DMSO as the cells treated with the highest dose of PDA 66. Proliferation studies Cell counts were determined by trypan blue staining. Metabolic activity was analyzed by tetrazolium com pound WST 1.

In brief, triplicates of cells were seeded in 96 well plates, treated with different concentrations of PDA 66 and incubated with 15 ul WST 1 for up to 4 h. The mitochondrial dehydrogenases reduce WST 1 to soluble formazan and cause a change of color correlating with the amount of metabolically active cells. Absorbance at 450 nm and a reference wavelength at 620 nm were de termined by an ELISA Reader. The absorbance of culture medium with sup plemented WST 1 in the absence of cells was used as background control. May Gr��nwald Giemsa staining After treatment with 1 uM PDA 66 glass slides were pre pared with 3×104 cells with Cytospin 3 centrifuge. Briefly, slides were incubated 6 min in May Gr��nwald solution, washed with tap water, incubated 20 min in Giemsa solution, and washed in tap water again.

To evaluate morphological changes of the cells slides were analyzed by Nikon Eclipse E 600 light microscope and imaged with NIS Elements soft ware. Analyses of apoptosis and necrosis Apoptosis and necrosis were analyzed by staining the cells with Annexin V FITC and Propidium iodide. Results were assessed by flow cytometry. Briefly, 5×105 cells were harvested and washed twice with PBS. After resuspending the cells in 100 ul of binding buffer 4 ul of Annexin V FITC was added and incubated for 15 min at room temperature, respectively. Following addition of 400 ul binding buffer for a final volume of 500 ul the cells were stained with PI immediately before measure ment. Unstained and single stained cells were included in each experiment as controls. Measurements were per formed using FACSCalibur and data analyzed by CellQuest software.

Cell cycle analysis After treatment cells were harvested and http://www.selleckchem.com/products/PD-0332991.html washed twice in PBS. Cells were fixed with 70% ethanol and incubated with 1 mg ml Ribonuclease A for 30 min at 37 C. After washing the cells twice in PBS, they were stained with PI and DNA content was determined by flow cytometry. Western blot Protein extraction and western blot was performed as de scribed previously.