FTY720-induced toxicity in CLL cells is independent on caspase activation. (A) Pancaspase inhibitor z-VAD-fmk failed to rescue CLL B cells from FTY720-induced cell death. Purified CD19⁺ cells from patients with CLL (10⁶ cells/mL media) were incubated with 10 μM FTY720 in the presence or absence of z-VAD-fmk (150 μM) for 24 hours. 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 the 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 = 3; *P = .001 when compared with FARA [2F-Adenine Arabinoside or fludarabine]-treated group; **P = .998 when compared with FTY720-treated group). (B) FTY720 failed to induce caspase-3, caspase-8, or PARP processing. Purified CD19⁺ cells from patients with CLL (10⁶ cells/mL media) were incubated with DMSO (None) or 10 μM FTY720 for 24 and 48 hours. Western blot analysis of the lysates from each of the conditions were assessed for processed and unprocessed PARP, caspase-3, and caspase-8 as described in “Western blotting.” The bold arrow indicates the unprocessed form of each of the proteins; the dashed arrows indicate cleaved products. Untreated and UV-treated Jurkat cell lysates were used as positive controls. (C) FTY720 induced activation of PARP processing in the Ramos but not the Raji cell line. Ramos or Raji cells (10⁵/mL media) were incubated with DMSO and 150 μM z-VAD-fmk with or without 10 μM FTY720 for 24 hours. Western blotting of PARP protein for each of the conditions for both cell lines is shown.