The mitochondrial pathway is involved in differentiation-associated caspase activation.

(A) Mitochondrial membrane depolarization was studied by flow cytometry in parental (U937) and Bcl-2–overexpressing (U937/Bcl-2) cells either left untreated (control) or treated with 20 nM TPA for 12 hours. Lonidamine (LND, 200 μM, 48 hours) was used as a positive control. Mitochondrial depolarization was suggested by reproducible changes in the curve shape of cytosolic green monomers (FL1). (B) Western blot analysis of cytochrome c in the mitochondrial (Mit) and cytosolic (S100) fractions obtained from U937 and U937/Bcl-2 cells exposed for indicated times to 20 nM TPA. (C) Flow cytometry analysis of caspase-3 active fragments (white curves indicate untreated cells; gray curves, TPA-treated cells). (D) Western blot analysis of caspase-3 in cells treated for 24 hours with 20 nM TPA. Numbers on the right indicate molecular weight in kilodaltons; *indicates cleavage products. (E) DEVDase activity measured in lysates from U937 cells preincubated or not for 1 hour with the indicated Abs (antagonistic anti-Fas ZB4, 500 nM; TNF inh.: anti–TNF-α [10 μg/mL] plus antagonistic anti–TNF-R1 [25 μg/mL]) and then either left untreated (Co) or treated with either TPA (20 nM for 24 hours) or agonistic anti-Fas Ab (CH11: 50 ng/mL plus CHX 0.8 μg/mL for 12 hours) or rhTNF-α (25 ng/mL plus CHX 0.8 μg/mL for 5 hours). (A-D) One representative of 3 independent experiments. (E) Mean ± SD of 3 experiments in triplicate.