SAHA down-regulates Bcr-Abl levels and induces apoptosis of K562 and LAMA-84 cells.

(A) Cells were treated with the indicated concentrations of SAHA for 48 hours. Following this treatment, the percentage of annexin V–stained apoptotic cells was determined by flow cytometry. (B) Western blot analyses of acetylated (Ac) histone 3, histone H3, p21, and p27 in the cell lysates from K562 and LAMA-84 cells, following treatment with the indicated concentrations of SAHA for 48 hours. The levels of β-actin served as the loading control. (C) Western blot analyses of Bcr-Abl, Bcl-x_L, and phospho(p)-AKT in the cell lysates from K562 and LAMA-84 cells, following treatment with the indicated concentrations of SAHA for 48 hours. Alternatively, immunoprecipitates with anti-Abl antibody were probed and immunoblotted with anti-phosphotyrosine (p-tyr) antibody. (D) Treatment with SAHA reduces bcr-abl mRNA in K562 cells. Samples of 15 μg total cellular RNA were isolated from K562 cells treated with SAHA (2 μM) for the indicated exposure intervals and analyzed by Northern blotting. A 1000-bp probe from the b3-a2 junction sequence of bcr-abl was used as a probe, which detected a 615-Kb transcript. The same membrane was stripped and rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. (E) Cotreatment with z-VAD-fmk (25 μM) PARP cleavage activity of caspases without affecting the SAHA-mediated decline in Bcr-Abl levels. K562 cells were exposed to SAHA and/or z-VAD-fmk for 48 hours. Following this exposure, immunoblot analyses of Bcr-Abl and PARP, and the cleavage product, were performed on the cell lysates. The levels of β-actin served as the loading control.