Acadesine induces caspase processing and mitochondrial cytochrome

c release. (A) Z-VAD.fmk protects cells from acadesine-induced apoptosis. B-CLL cells were preincubated for 1 hour with 200 μM Z-VAD.fmk and 0.5 mM acadesine was added for 24 hours. Viability was measured by analysis of phosphatidylserine exposure and PI uptake as described in “Patients, materials, and methods.” Data are shown as the mean value ± SD of 4 patients. (B) Analysis of the processing of caspase-3, -8, and -9. B-CLL cells were treated with 0.2 or 0.5 mM acadesine for 24 hours. Cells were lysed and analyzed by Western blot as described in “Patients, materials, and methods.” The migration positions of the precursor forms of caspase-3 (32 kDa) and caspase-8 (53/51 kDa) and the cleavage products of caspase-8 (43/41 kDa) and caspase-9 (37 kDa) are indicated. (C) Analysis of cytochrome c release. B-CLL cells were treated with 0.5 mM acadesine for 24 hours and mitochondrial and cytosolic extracts were analyzed by Western blot. Cytochrome oxidase subunit II was analyzed as a control for mitochondrial protein loading. C indicates control; A, acadesine.