Effects of rolipram and forskolin on CLL cells.

The PDE4 inhibitor rolipram, but not the adenylate cyclase activator forskolin, augments caspase-3–like activity and induces caspase-3 and PARP cleavage in CLL cells. (A) CLL cells were cultured for 24 or 48 hours, as indicated (24 hours for middle panel), with media (control; CT), 10 μM rolipram (R), 40 μM forskolin (F), or both (R/F). (Top) Whole-cell lysates were immunoblotted with a caspase-3–specific antibody. Full-length procaspase-3 and the caspase-3 cleavage product p18 are indicated by arrows. Similar cleavage of caspase-3 was detected in rolipram/forskolin-treated leukemic cells from 7 of 8 CLL patients. (Middle) Cells were assessed for caspase-3–like activity with the substrate Ac-DEVD-pNA by means of a colorimetric assay. The standard error of the mean (SEM) of triplicate samples is shown. Similar activation of caspase-3–like activity was detected in 7 of 8 CLL patients. (Bottom) Whole-cell lysates were immunoblotted with a PARP-specific antibody. The 85-kDa cleavage product is indicated by an arrow. Similar rolipram/forskolin-induced PARP cleavage was observed in 7 of 8 CLL patients. (B) CLL cells were cultured for the indicated time period in 10 μM rolipram and 40 μM forskolin. The “0” time point refers to culture for 18 hours in media with vehicle control alone. (Top) Whole-cell lysates were immunoblotted for caspase-3. (Middle) Cells were assayed for caspase-3–like activity. The SEM of triplicate samples is shown. (Bottom) Whole-cell lysates were immunoblotted with a PARP-specific antibody. (C) CLL cells were treated with media alone (0) or 40 μM forskolin in combination with the indicated concentration of rolipram, followed by an assay for caspase-3–like activity.