APO866-induced cell death is independent of caspase activation, and is associated with depolarization of mitochondrial membrane. Caspase inhibitors failed to prevent APO866-induced cell death in ML-2 cells (A), Jurkat cells (B), or primary AML cells (C). Cells were exposed to APO866 (10 nM) for 4 days in the presence or absence of zVAD-fmk (100 μM), zDEVD-fmk, zIETD-fmk, or zLEHD-fmk (50 μM). Controls consisted of untreated cells similarly analyzed (A-C). Control of the activity of the pan-caspase inhibitor (D) included wild-type Jurkat cells incubated for 4 days with 100 ng/ml MegaFasL alone (“MegaFasL”) or with zVAD (100 μM, “zVAD-fmk + MegaFasL”). ML-2 (E), Namalwa (F), and Jurkat (G) cells were incubated without or with various concentrations of APO866 for 96 hours. Mitochondrial potential was measured using JC-1 staining red versus green fluorescence as described in “Methods.” Data are derived from at least 3 independent experiments.