ROLE OF GLOBOTRIAOSYL CERAMIDE (CD77) IN SIGNAL TRANSDUCTION

球三酰神经酰胺 (CD77) 在信号转导中的作用

• 批准号: 6206496
• 负责人: MARK MALONEY
• 金额: $ 20.17万
• 依托单位: SPELMAN COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2000-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6206496
• 关键词: CD antigens; apoptosis; biological signal transduction; cell adhesion; ceramides; clone cells; flow cytometry; gel mobility shift assay; interferon alpha; monoclonal antibody; neoplastic cell culture for noncancer research; northern blottings; receptor binding; receptor expression; tissue /cell culture; western blottings

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.

Daudi细胞和其他Burkitt淋巴瘤衍生的细胞系经常被认为是恶性的。 作为生殖中心B淋巴细胞的体外模型， 关于神经酰胺三己糖基（Gb 3或CD 77）的表型相似性 和表面蛋白表达。 Gb 3也被认为是 Verotoxins（VT） 结合Gb 3的VT B亚基已经被证实是 显示其模拟人干扰素-A受体的克隆亚基 （IFNAR）和B细胞标志物CD 19。 实验证据表明，Gb 3和Gb 3结合蛋白是 许多信号转导途径的重要组成部分， 伯基特淋巴瘤和生殖中心B淋巴细胞，包括 Gb 3/IFNAR在IFN-α诱导的生长抑制中的相互作用，Gb 3/CD 19 同种型粘附中的相互作用和 诱导细胞凋亡。 我们建议研究Gb 3在 这些信号转导通路使用Gb+和Gb 3-缺陷的Daudi细胞 线 目的：1）粘附研究 ⑶ 19连接对用以下处理的Gb 3+和Gb 3-缺陷细胞的影响 已知信号传导途径的抑制剂/激活剂和/或通过监测 这些途径中的已知中间体水平。 同型粘附 通过表面蛋白、佛波酯和IFN-α的抗体连接诱导 将在这些细胞系中进行比较。 野生型应答恢复 通过添加外源性Gb 3，将尝试在Gb 3- 缺陷细胞在粘附机制上有缺陷。 2)干扰素信号 研究IFN诱导的信号转导将进行比较和对比 在Gb 3+和Gb 3-细胞中，关于介质的变化， ISGF 3的激活和信号传导的最终结果，包括生长 干扰素诱导蛋白质和同型 粘连 在以下情况下，细胞将用外源性Gb 3重建： Gb 3缺陷型细胞被发现在IFN信号传导中有缺陷。 第三章 我们将确定Gb 3介导的细胞凋亡是否在肿瘤细胞中起作用。 突变体可以用外源Gb 3重建。 我们还将比较 这些细胞系中的其他凋亡诱导剂的活性，包括 由抗CD 19、CD 22和IgM抗体诱导，或更一般的机制 如抗Fas抗体、辐射和细胞毒性T淋巴细胞杀伤。 4)Gb 3作为信使Gb 3降解产物的潜在作用 抗CD 19介导的粘附、IFN信号传导和凋亡途径将 通过监测Gb 3，神经酰胺， 鞘氨醇和相关鞘脂化合物。