Resistance to CD95-mediated apoptosis through constitutive c-FLIP expression in a non-Hodgkin's lymphoma B cell line

CD95 (Fas/Apo-1) is a transmembrane molecule that induces apoptosis and pla ys a central role in the regulation of the immune response. The present stu dy describes two new 8 lymphoid cell lines, B593 and BR97, derived from non -Hodgkin's lymphoma, which differ in susceptibility to CD95-mediated apopto sis. While B593 cells are sensitive to CD95-mediated apoptosis, BR97 cells are completely resistant. Activation of caspase-8 and caspase-3 proteases p lays an important role in the CD95 signalling pathway. CD95 stimulation ind uced caspase-8 and caspase-3 activation in B593, but not in BR97 cells. How ever, activation of both caspase-8 and caspase-3 was achieved in BR97 cells treated with staurosporine. Furthermore, protein synthesis inhibition by c ycloheximide restored sensitivity to CD95-mediated apoptosis and allowed ac tivation of both caspase-8 and caspase-3 in BR97 cells. These results indic ate that, in BR97 cells, both caspases are functional and suggest that CD95 -apoptosis resistance may result from the presence of inhibitory factor(s). Constitutive high level expression of the apoptotic inhibitor c-FLIP was o bserved in the CD95-resistant BR97 cell line compared to 8593. Moreover, do wnregulation of c-FLIP expression level by protein synthesis inhibition str ictly correlated with restored sensitivity to CD95-mediated apoptosis in BR 97 cells. Furthermore, we demonstrate that c-FLIP is recruited to the CD95 DISC in BR97 cells together with caspase-8 and FADD. The data presented in this study strongly suggests that, in a B-NHL-derived cell line, resistance to CD95-mediated apoptosis results from endogenous high level expression o f apoptotic inhibitor c-FLIP.