Effect of rolipram/forskolin on PP2A_C- and PP2A_C- associated phosphatase activity in CLL cells.

Rolipram/forskolin treatment of CLL cells induces augmented levels of PP2A_C- and PP2A_C-associated phosphatase activity. (A) CLL cells were cultured for 18 hours in media (CT), 5 nM okadaic acid (OA), 10 μM rolipram, and 40 μM forskolin (R/F), or the combination of okadaic acid, rolipram, and forskolin (R/F + OA). PP2A_C was immunoprecipitated from whole-cell lysates, then assayed for phosphatase activity by means of the substrate KRpTIRR and a colorimetric phosphatase assay. The SEM of triplicate samples is shown. Similar results were obtained in 2 CLL patients. (B) (Left) CLL cells from a representative patient were cultured for 18 hours in media (0) or 40 μM forskolin combined with the indicated concentration of rolipram, followed by immunoprecipitation of PP2A_C and phosphatase assay as for panel A. The inset shows the mean ± SEM increase in PP2A activity relative to control (CT) in leukemic cells from 5 CLL patients treated for 18 hours with 10 μM rolipram and 40 μM forskolin (RF). ** = P < .01. (Right) CLL cells from a representative patient were cultured for 18 hours in media (CT) or 200 μM 8-Br-cAMP (Br) or 200 μM 6-Bnz-cAMP (Bz) as indicated, followed by immunoprecipitation of PP2A_C and phosphatase assay as for panel A. (C) CLL cells from a representative patient cultured as for panel B were immunoblotted for PP2A_C. The graph in panel C represents the mean ± SEM of densitometric analyses of immunoblots from 3 patients. * = P < .05. ** = P < .01. (D) CLL cells were cultured in media (0) or 10 μM rolipram and 40 μM forskolin for the indicated number of hours, followed by immunoblotting for PP2A_C. The “0” time point refers to culture for 18 hours in media with vehicle control alone. Similar results were obtained in 2 CLL patients. For panels C-D, loading and transfer were assessed by subsequent immunoblotting for tubulin, as shown.