Caspase activation by proteasome inhibitors. (A) Cleavage of the caspase substrate, PARP, by treatment with proteasome inhibitor. Cells were treated with either 10 μmol/L methylprednisolone, 25 μmol/L zAPFcmk, or various doses of MG132. PARP was detected by immunoblotting. Intact (p116) and fragmented (p85) forms of PARP are indicated by arrows. (B) Effect of MG132 on DEVDase activity. Cells were treated in the absence or presence of 10 μmol/L MG132 for 8 hours, and hydrolysis of the caspase substrate DEVD-AMC was measured in a spectrofluorimeter. Results are from two experiments with independent patient isolates. Cells treated with 10 μmol/L BDcmk, a caspase inhibitor, did not show DEVDase activity more than 50 U above baseline levels. DNA fragmentation from these patients was measured in parallel. These patients are not included in Table 1. Patient no. 1: control = 18.0, MG132 = 96.9; patient no. 2: control = 1.4, MG132 = 32.5. (C) Activation of caspase-3 by proteasome inhibitor. Cells were treated with either 25 μmol/L zAPFcmk, 10 μmol/L MG132, or 10 μmol/L methylprednisolone for 16 hours and procaspase-3 was detected by immunoblotting. Results of one experiment representative of three replicates with independent patient isolates.