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Fas Receptor Clustering and Involvement of the Death Receptor Pathway in Rituximab-Mediated Apoptosis with Concomitant Sensitization of Lymphoma B Cells to Fas-Induced Apoptosis¹

Alja J. Stel^*, Bram ten Cate^*, Susan Jacobs^*, Jan Willem Kok, Diana C. J. Spierings, Monica Dondorff^*, Wijnand Helfrich^*, Hanneke C. Kluin-Nelemans, Lou F. M. H. de Leij^*, Sebo Withoff^2, and Bart Jan Kroesen^2,3,*

^* University Medical Center Groningen, Department of Pathology and Laboratory Medicine, Section Medical Biology–Laboratory Tumor Immunology, Groningen, The Netherlands; Department of Membrane Cell Biology and Department of Hematology, University Medical Center Groningen, Groningen, The Netherlands; and Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This research was funded by grants of the research school Groningen University Institute for Drug Exploration, the J.K. de Cock Foundation, and the foundation Sacha Swarttouw-Hijmans.

² S.W. and B.J.K. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Bart Jan Kroesen, University Medical Center Groningen, Department of Pathology and Laboratory Medicine, Section Medical Biology–Laboratory Tumor Immunology, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail address: B.J.Kroesen{at}med.umcg.nl

⁴ Abbreviations used in this paper: XIAP, X-chromosome-linked inhibitor of apoptosis protein; CL, cross-linker (F(ab')₂ of goat anti-human-IgG; DFF, DNA fragmentation factor; DISC, death-inducing signaling complex; FADD, Fas-associated death domain; FasL, Fas ligand; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; TRITC, tetramethylrhodamine.

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