Downstream caspases are present and functional in BL cell lines. (A) Immunoblot analysis for caspase-3 (Yama/CPP32) and caspase-7 (ICE-LAP3). Whole-cell lysates (50 μg) were separated on 15% SDS-PAGE gels under reducing conditions and immobilized on nitrocellulose membranes. Caspase-3 and caspase-7 were detected with anti-CPP32 and anti-ICE-LAP3 p20 antibodies, respectively, followed by development with chemiluminescence. (B) Caspase-3 can be activated in Fas-resistant BL cells. Cells (2 × 106; 5 × 105/mL) were left untreated (control, □) or treated with anti-Fas IgM (▨), C2-ceramide (▪), and staurosporine (▤). Nuclei-free lysates were made from each sample and used in DEVD-pNA cleavage assays. Controls for nonspecific protease activity were obtained by preincubating one set of staurosporine-induced cell lysates with the caspase-3 inhibitor DEVD-fmk (5 μmol/L) for 30 minutes at 37°C before addition of the substrate. Absorbance was read at 405 nm. Caspase activity is expressed in units with 1 unit being the amount of enzyme activity liberating 1 pmol of pNA per minute. (C) Induction of caspase-3 activity in resistant cell lysates by granzyme B. Cleavage assays were performed as in (B) with naı̈ve cell lysates of SKW6.4 and Mutu-BL III cultures. Before performing the cleavage assay, lysates were preincubated for 30 minutes at 37°C in the absence (−, □) or presence (+, ▪) of 5 U of granzyme B (GraB). Reactions performed without cytosolic extract (buffer) showed the failure of granzyme B to independently use DEVD-pNA as a substrate.
