Forodesine triggered caspase-9, -3, and -8 activation and processing of BID protein to its proapoptotic form tBID. (A) Cells from a representative patient with 17p-deleted CLL were treated with forodesine (2 μM) and dGuo (10 and 20 μM) for 10 and 16 hours. Caspase-9, caspase-3, and caspase-8 were analyzed by Western blot in total protein extracts. β-Actin was used as internal loading control. (B) Cells from 2 representative patients with CLL (normal and 17p deleted) were treated with forodesine (2 μM) and dGuo (10-20 μM) for 16 hours, and caspase-8, BID, truncated BID, XIAP, and survivin levels were analyzed by Western blot in total protein extracts. Actin was used as internal loading control. Cell death was assessed by annexin V labeling as described in “Methods.” (C-D) Cells from 4 representative patients with CLL were preincubated for 1 hour with the broad-range caspase inhibitor z-VAD.fmk and treated with forodesine (2 μM) and dGuo (10 and 20 μM) for 16 to 24 hours. Apoptosis induction (percentage of annexin V⁺ cells at 24 hours; C) and loss of ΔΨ_m (DIOC₆ staining at 16 hours; D) were analyzed by flow cytometry.