Doxorubicin- or OKT3-induced DISC formation and activation of caspases.

(A) Detection of DISC-bound CD95 ligand. CD3+ H9 cells were treated with 0.1 μg/mL doxorubicin for the time points indicated. CD3+ or CD3-H9 cells were incubated for 6 hours in 75 cm² flasks precoated with 100 μg/mL OKT3. Cells were lysed and immunoprecipitation of CD95 was performed by FADD monoclonal antibody. Proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). CD95-L protein was detected by mouse anti-CD95-L monoclonal antibody and ECL. (B) Doxorubicin- induced CD95 aggregation. CD3+ H9 cells were treated with 0.1 μg/mL doxorubicin for the time points indicated. Cells were lysed, and immunoprecipitation of CD95 was performed by anti-APO-1 IgG3 monoclonal antibody using limiting antibody concentrations (0.5 μg/mL) or excess antibody concentrations (2 μg/mL). Proteins were separated by 12% SDS-PAGE. CD95 protein was detected by mouse anti-CD95 monoclonal antibody and ECL. (C) Doxorubicin, OKT3-, or anti-APO-1–induced DISC formation. CD3+ H9 cells were treated with 0.1 μg/mL doxorubicin for the time points indicated or with 1 μg/mL anti-APO-1 IgG3 monoclonal antibody for 15 minutes. CD3+ or CD3-H9 cells were incubated for 6 hours in 75 cm² flasks precoated with 100 μg/mL OKT3. Cells were lysed, and immunoprecipitation of CD95 was performed by anti-APO-1 IgG3 monoclonal antibody (I.P.) or Western blot analysis was performed (blot). Proteins were separated by 12% SDS-PAGE. FADD and caspase-8 protein were detected by mouse anti-FADD monoclonal antibody or mouse anti-caspase-8 monoclonal antibody and ECL. (D) Dose response of doxorubicin-induced DISC formation. CD3+ H9 cells were treated with 0.03-0.3 μg/mL doxorubicin for 12 hours. Immunoprecipitation of the CD95 DISC was performed as described in Figure 3C. (E) Doxorubicin- or OKT3-induced activation of caspases. CD3+ H9 cells were treated with 0.1 μg/mL doxorubicin for the time points indicated. CD3+ or CD3-H9 cells were incubated for 6 hours in 75 cm² flasks precoated with 100 μg/mL OKT3. Western blot analysis for caspase-8, caspase-3, or PARP protein was performed using mouse anti-caspase-8 monoclonal antibody, mouse anti-caspase-3 monoclonal antibody, or rabbit anti-PARP polyclonal antibody and ECL. Processing of caspase-8, which was detected as a double band corresponding to 2 caspase-8 isoforms (caspase-8/a and caspase-8/b), resulted in p43 and p41 cleavage intermediates derived from caspase-8/a and caspase-8/b, respectively, and the p18 active subunit.