ROLE OF GLOBOTRIAOSYL CERAMIDE (CD77) IN SIGNAL TRANSDUCTION

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Daudi cells and other Burkitt's lymphoma-derived cell lines are frequently used as in vitro models of germinal center B lymphocytes because of their phenotypic resemblance with regard to globotriaosyl ceramide (Gb3 or CD77) and surface protein expression. Gb3 has also been identified as the receptor for verotoxins (VT's). The Gb3-binding VT B subunits have been shown to mimic the cloned subunit of the human interferon-a receptor (IFNAR) and the B cell marker CD19 in their amino acid sequences. Experimental evidence indicates that Gb3 and Gb3-binding proteins are essential components of a number of signal transduction pathways in Burkitt's lymphoma and germinal center B lymphocytes including a role for Gb3/IFNAR interaction in IFN-a induced growth inhibition, Gb3/CD19 interaction in homotypic adhesion and Gb3/VT B-subunit interaction in the induction of apoptosis. We propose to investigate the role of Gb3 in these signal transduction pathways using Gb+ and Gb3-deficient Daudi cell lines. AIMS: 1) ADHESION STUDIES The proposed investigations will determine the effect of CD19 ligation on Gb3+ and Gb3-deficient cells treated with inhibitors/activators of known signaling pathways and/or by monitoring levels of known intermediates in such pathways. Homotypic adhesion induced by antibody ligation of surface proteins, phorbol ester and IFN-a will be compared in these cell lines. Restoration of wild-type responses by addition of exogenous Gb3 will be attempted in cases where Gb3- deficient cells are defective in adhesion mechanisms. 2) IFN-SIGNALING STUDIES IFN-induced signal transduction will be compared and contrasted in the Gb3+ and Gb3- cells with regard to changes in mediators such as activation of ISGF3 and the end results of the signaling including growth inhibition, synthesis of interferon-inducible proteins and homotypic adhesion. Cells will be reconstituted with exogenous Gb3 in cases where Gb3-deficient cells are found to be defective in IFN-signaling. 3) APOPTOSIS STUDIES We will determine if Gb3-mediated apoptosis in the mutants can be reconstituted with exogenous Gb3. We will also compare the activity of other inducers of apoptosis in these cell lines including that induced by anti-CD19, CD22 and IgM antibody, or more general mechanisms such as anti-Fas antibody, radiation and cytotoxic T lymphocyte killing. 4) Gb3 AS MESSENGER The potential role of Gb3 degradation products in anti-CD19 mediated adhesion, IFN-signaling and the apoptotic pathways will be investigated by monitoring changes in the levels of Gb3, ceramide, sphingosine and related sphingolipid compounds.