FTY720-induced toxicity in CLL cells is dependent on activation of PP2a. (A) FTY720-induced PP2A activity in CD19⁺ B cells from patients with CLL. Purified B-lymphocytes from patients with CLL (10⁶ cells/mL media) were incubated with DMSO or 10 μM FTY720 for 0, 1, 2, 3, or 15 hours. The PP2A activity in the cell lysates were measured as described in “Methods.” The left panel shows the time kinetics of a representative experiment. The right panel shows summary of PP2A activity at 4 hours in 5 independent samples in response to DMSO, 10 μM FTY720, or 1,9 di-deoxy-forskolin. (B) FTY720-induced PP2a activity in CD19⁺ B cells is inhibited by okadaic acid. Purified B-lymphocytes from patients with CLL (10⁶ cells/mL media) were pretreated with media or okadaic acid (5 nM) for 2 hours, followed by incubation with DMSO or 10 μM FTY720 for the indicated time periods. The PP2a activity in the cell lysates were measured by a nonradioactive assay as described in “PP2a activity (nonradioactive assay)” (n = 4; *P < .001 when compared with FTY720-treated group). (C) FTY720-induced PP2a activity in CD19⁺ B cells is inhibited by okadaic acid. Purified B-lymphocytes from patients with CLL (10⁶ cells/mL media) were pretreated with media or okadaic acid (5 nM) for 2 hours, followed by incubation with DMSO or 10 μM FTY720 for the indicated time periods. The PP2a activity in the cell lysates was measured using a radioactive assay as described in “PP2a activity (radioactive assay).” (D) FTY720-induced dephosphorylation of ERK1/2 in CD19⁺ B cells is inhibited by okadaic acid. Purified B-lymphocytes from patients with CLL (10⁶ cells/mL media) were pretreated with media or okadaic acid (5 nM) for 2 hours, followed by incubation with DMSO or 10 μM FTY720 for 3 hours. The ERK1/2 phosphorylation status was analyzed in cell lysates obtained under indicated treatment conditions using anti–phospho-ERK1/2 antibody. The levels of total ERK1/2 were analyzed in each lane by reprobing the blot with anti-ERK1/2 antibody. (E) FTY720-induced cellular toxicity is partially rescued by okadaic acid. Purified B-lymphocytes from patients with CLL (10⁶ cells/mL media) were pretreated with media or okadaic acid (5 nM) for 2 hours, followed by incubation with DMSO or 10 μM FTY720. The cells were stained with Annexin V–FITC and PI as described in “Analysis of cell viability and apoptosis.” The cells were analyzed by flow cytometry, and data were collected under list mode. The data shown represent the percentage of Annexin V⁻/PI⁻ viable cells plus or minus SD that are normalized to media control (n = 6; *P = .028 when comparing FTY720-treated vs OA plus FTY720-treated groups).