Mitochondrial pathway is activated in ex vivo B-CLL cells, but caspase-9 plays a minor role in their spontaneous apoptosis. (A-B) Time-course analysis of mitochondrial apoptogenic protein release into the cytosol, Bax translocation to the mitochondria, and caspase-9 proteolytic processing during spontaneous B-CLL cell apoptosis. Freshly isolated B-CLL cells were cultured for the indicated times in complete medium (n = 12). The levels of cytochrome c, Smac/DIABLO, Omi/HtrA2, and Bax (A) were analyzed by Western blot in cytosol and mitochondria-enriched fraction (MF) extracts (20 μg). The blots were reprobed with an anti-COX IV mAb to control the purity of cytosolic fraction and the loading of MF, and with an anti–β-actin mAb to control the loading of cytosolic extracts. Caspase-9 proteolytic processing (B) was analyzed by Western blot in 25 μg whole-cell lysates, and protein loading was assessed reprobing the blots with an anti–β-actin mAb. (A-B) Data shown for patients 1 and 2 are representative of 12 patients. (C-D) Effect of pharmacologic caspase-9 inhibition on spontaneous B-CLL cell apoptosis, caspase-8 and -3 proteolytic processing, and PARP degradation. Freshly isolated B-CLL cells were cultured for 24 hours in complete medium with 50μM caspase-9 inhibitor z-LEHD-fmk or 0.005% DMSO as control (n = 7). Apoptosis (C) was evaluated by flow cytometric analysis of hypodiploid nuclei, and results are the mean ± SD of all 7 patients examined. The effect of z-LEHD-fmk on spontaneous B-CLL cell apoptosis is not significant. Caspase-3 and -8 processing and PARP degradation (D) were analyzed by Western blot in 25 μg whole-cell lysates, and protein loading was assessed by reprobing the blots with an anti–β-actin mAb. The data shown for patients 1 and 2 are representative of 7 patients.