DISSECTION OF PATHWAYS LEADING TO ANTIGEN RECEPTOR-INDUCED AND FAS CD95-INDUCED APOPTOSIS IN HUMAN B-CELLS/

To dissect intracellular pathways involved in B cell Ag receptor (BCR) -mediated and Fas-induced human B cell death, we isolated clones of th e Burkitt lymphoma cell line Ramos with different apoptosis sensitivit ies, Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In cont rast, clones resistant to Fas-mediated apoptosis could still undergo B CR-induced cell death. Based on the functional phenotypes of these clo nes, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of b oth Fas and BCR resulted in cleavage of the IL-1 beta-converting enzym e/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp- aldehyde and poly(ADP-ribose) polymerase, Markedly, qualitative differ ences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage prod ucts of 19/20 and 17 kDa, only the latter cleavage product was found u pon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylk etone blocked both Fas-and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overe xpression of a Fas-associated death domain (FADD) dominant-negative mu tant protein was found to inhibit Fas-induced apoptosis but not BCR-in duced apoptosis, Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to d istinct proteases upstream of caspase 3.