Effect of rolipram/forskolin on mitochondrial depolarization and accumulation of cytosolic cytochrome c in CLL cells.
Rolipram/forskolin treatment of CLL cells induces mitochondrial depolarization and accumulation of cytosolic cytochrome c. (A) CLL cells were treated for the indicated number of hours with 10 μM rolipram and 40 μM forskolin. Whole-cell lysates were then separated into cytosolic (cytosol) and membrane-rich (heavy-membrane) fractions, followed by immunoblotting for cytochrome c. Total cytochrome c, shown at the bottom, represents a sample in which equivalent portions of cytosolic and membrane fractions were combined. Similar rolipram/forskolin-induced augmentation of cytosolic cytochrome c was seen in leukemic cells from 7 of 8 CLL patients. (B) CLL cells were treated for 18 hours with either media alone (0) or forskolin (40 μM) combined with the indicated concentration of rolipram. Whole-cell lysates were subfractionated and immunoblotted as for panel A. (C) CLL cells were treated for the indicated period of time with 10 μM rolipram and 40 μM forskolin. The “0” time point refers to culture for 18 hours in media with vehicle control alone. Mitochondrial membrane potential was then assessed by means of a DiOC6(3) flow cytometry assay where reduced immunofluorescence reflects mitochondrial depolarization.
