title: Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis.
abstract: Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.
authors.affiliation: University Medical Center Groningen, Department of Pathology and Laboratory Medicine, Section Medical Biology-Laboratory Tumor Immunology, Groningen, The Netherlands.
authors.forename: Alja J
authors.initials: AJ
authors.lastname: Stel
authors.forename: Bram
authors.initials: B
authors.lastname: Ten Cate
authors.forename: Susan
authors.initials: S
authors.lastname: Jacobs
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: Kok
authors.forename: Diana C J
authors.initials: DC
authors.lastname: Spierings
authors.forename: Monica
authors.initials: M
authors.lastname: Dondorff
authors.forename: Wijnand
authors.initials: W
authors.lastname: Helfrich
authors.forename: Hanneke C
authors.initials: HC
authors.lastname: Kluin-Nelemans
authors.forename: Lou F M H
authors.initials: LF
authors.lastname: de Leij
authors.forename: Sebo
authors.initials: S
authors.lastname: Withoff
authors.forename: Bart Jan
authors.initials: BJ
authors.lastname: Kroesen
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology* / Q000627:therapeutic use; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20 / Q000276:immunology; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects* / Q000276:immunology; D002051:Burkitt Lymphoma / Q000188:drug therapy / Q000276:immunology*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000235:genetics / Q000276:immunology; D053181:Caspase 8 / Q000276:immunology; D045744:Cell Line, Tumor; D053222:Fas Ligand Protein / Q000276:immunology*; D053200:Fas-Associated Death Domain Protein / Q000276:immunology; D006801:Humans; D021962:Membrane Microdomains / Q000276:immunology; D021381:Protein Transport / Q000187:drug effects / Q000276:immunology; D011940:Receptor Aggregation / Q000187:drug effects* / Q000276:immunology; D000069283:Rituximab; D019014:fas Receptor / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000970:Antineoplastic Agents; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C505533:FADD protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D019014:fas Receptor; D000069283:Rituximab; C505530:CASP8 protein, human; D053181:Caspase 8