ROLE OF GLOBOTRIAOSYL CERAMIDE (CD77) IN SIGNAL TRANSDUCTION

Daudi细胞和其他Burkitt淋巴瘤来源的细胞系经常 生发中心B淋巴细胞的体外模型 球三糖神经酰胺(Gb3或CD77)的表型相似性 和表面蛋白的表达。GB3也被确定为 维罗毒素受体(VT‘s)。结合Gb3的VT B亚基已被 显示为模拟人类干扰素-a受体的克隆亚单位 (IFNAR)和B细胞标记CD19的氨基酸序列。 实验证据表明Gb3和Gb3结合蛋白是 许多信号转导通路的基本组成部分 Burkitt淋巴瘤和生发中心B淋巴细胞包括 Gb3/IFNAR相互作用在干扰素-α诱导的生长抑制中的作用 同型黏附中的相互作用和Gb3/VT B-亚基相互作用 诱导细胞凋亡。我们建议研究Gb3在 这些信号转导通路使用Gb和Gb3缺陷的Daudi细胞 台词。 目标：1)附着力研究拟议的调查将确定 CD19结扎对Gb3和Gb3缺陷细胞的影响 已知信号通路的抑制剂/激活剂和/或通过监测 这些途径中已知中间体的水平。同型粘连 表面蛋白、佛波酯和干扰素-a的抗体结合诱导 将在这些细胞系中进行比较。野生型反应的恢复 通过添加外源Gb3，在Gb3- 缺陷细胞在黏附机制上有缺陷。2)干扰素信号 干扰素诱导的信号转导研究将进行比较和对比 在Gb3和Gb3细胞中，关于介质的变化，如 ISGF3的激活和信号转导的最终结果包括生长 干扰素诱导蛋白的抑制、合成和同型 粘附力。在以下情况下，细胞将与外源Gb3重组 Gb3基因缺陷的细胞在干扰素信号转导方面存在缺陷。3) 细胞凋亡研究我们将确定Gb3介导的细胞凋亡是否 突变体可以与外源Gb3重组。我们还将比较 这些细胞系中其他诱导细胞凋亡的活性，包括 由抗CD19、CD22和IgM抗体或更一般的机制诱导 如抗Fas抗体、放射和细胞毒T淋巴细胞杀伤等。 4)Gb3作为信使Gb3降解产物在 抗CD19介导的黏附、干扰素信号和细胞凋亡途径 通过监测Gb3、神经酰胺、 鞘氨醇和相关的鞘糖脂化合物。

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.