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 the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In contrast, clones resistant to Fas-mediated apoptosis could still undergo BCR-induced cell death. Based on the functional phenotypes of these clones, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of both Fas and BCR resulted in cleavage of the IL-1beta-converting enzyme/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp-aldehyde and poly(ADP-ribose) polymerase. Markedly, qualitative differences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage products of 19/20 and 17 kDa, only the latter cleavage product was found upon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylketone blocked both Fas- and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overexpression of a Fas-associated death domain (FADD) dominant-negative mutant protein was found to inhibit Fas-induced apoptosis but not BCR-induced apoptosis. Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to distinct proteases upstream of caspase 3.
Lens
S M
SM
Department of Clinical Viro-Immunology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam. clbkvi@xs4all.nl
den Drijver
B F
BF
Pötgens
A J
AJ
Tesselaar
K
K
van Oers
M H
MH
van Lier
R A
RA
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis; D001402:B-Lymphocytes / Q000502:physiology*; D002352:Carrier Proteins / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein; D005434:Flow Cytometry; D006801:Humans; D008970:Molecular Weight; D009842:Oligopeptides / Q000494:pharmacology; D010641:Phenotype; D011947:Receptors, Antigen, B-Cell / Q000502:physiology*; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D009842:Oligopeptides; D011947:Receptors, Antigen, B-Cell; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D019014:fas Receptor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases