Chemotherapeutic agents induce processing of caspase-8 in the absence of CD95 signaling. (A) Jurkat cells (1 × 106) were treated with different concentrations of either etoposide (50 μg/mL lane 4; 5 μg/mL lane 3; 0.5 μg/mL lane 2; diluent control lane 1) for 6 hours or anti-CD95 (1,000 ng/mL lane 8; 50 ng/mL lane 7; 2.5 ng/mL lane 6; diluent control lane 5) for 3 hours. Cellular proteins were separated by SDS-PAGE and processing of procaspase-8 was detected by immunoblotting with anti-caspase-8. Open arrowheads (◃) indicate the two different isoforms of procaspase-8 (caspase-8/a and caspase-8/b) that are cleaved into the intermediate forms p43 and p41 (◂) and finally processed to the active p18 subunit (←). Ig light chain of stimulatory anti-CD95 antibody is indicated with an asterisk. (B) Jurkat or Jurkat-R cells (1 × 106) were treated for the indicated time with anti-CD95 (1 μg/mL), doxorubicin (Doxo; 1 μg/mL), or (C) with daunorubicin (Dauno; 5 μg/mL), etoposide (Etopo; 20 μg/mL), or mitomycin C (Mito; 25 μg/mL). Cellular proteins were immunoblotted with anti-caspase-8 as described in (A). Only a section of the immunoblots indicating the cleaved intermediate forms (p43 and p41) of caspase-8a and caspase-8b is shown.
