Induction of functionally active CD95 ligand by doxorubicin or OKT3.
(A) Induction of apoptosis by doxorubicin-treated H9 cells in J16 or CEM target cells is inhibited in J16/FADD-DN and in CD95-resistant CEM target cells. CD3+ H9 cells were treated with 0.1 μg/mL doxorubicin for 6 hours, washed, and cocultured for 24 hours with calcein-stained J16, J16/FADD-DN, CEM, or CD95-resistant CEM (CApoR) cells. Apoptosis in calcein-stained target cells was determined by FSC/SSC analysis. (B) Induction of apoptosis by OKT3-treated H9 cells in J16 or CEM target cells is inhibited in J16/FADD-DN and in CD95-resistant CEM target cells. CD3+ H9 cells were incubated for 6 hours in 75 cm2flasks precoated with 100 μg/mL OKT3, washed, and cocultured with calcein-stained J16, J16/FADD-DN, CEM, or CD95-resistant CEM (CApoR) cells. Apoptosis in calcein-stained target cells was determined by FSC/SSC analysis. (C) Induction of apoptosis by doxorubicin or OKT3-treated H9 cells in J16 target cells is inhibited by Fab anti-CD95 antibody fragments or by anti-CD95L antibody. CD3+ H9 cells were treated for 6 hours with 0.1 μg/mL doxorubicin or were incubated for 6 hours in 75 cm2 flasks precoated with 100 μg/mL OKT3, washed, and cocultured with calcein-stained J16 cells in the presence or absence of 50 μg/mL Fab anti-CD95 antibody fragments or 100 μg/mL Nok-1 antibody. Apoptosis in calcein-stained target cells was determined by FSC/SSC analysis. (D) Lack of apoptosis by doxorubicin-treated Nalm6 cells in J16 or CEM target cells. Nalm6 cells were treated for 6 hours with 0.1 μg/mL doxorubicin, washed, and cocultured with calcein-stained J16, J16/FADD-DN, CEM, or CD95-resistant CEM (CApoR) cells. Apoptosis in calcein-stained target cells was determined by FSC/SSC analysis.
