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: 5225959
• 负责人: MARK MALONEY
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5225959
• 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 细胞和其他伯基特淋巴瘤衍生细胞系经常被 用作生发中心 B 淋巴细胞的体外模型，因为它们 与 Globottriosyl ceramide（Gb3 或 CD77）有关的表型相似性 和表面蛋白表达。 Gb3 也被确定为 维罗毒素（VT）受体。 Gb3 结合 VT B 亚基已被 被证明可以模仿人类干扰素α受体的克隆亚基 (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 多种信号转导途径的重要组成部分 伯基特淋巴瘤和生发中心 B 淋巴细胞包括 IFN-a 诱导的生长抑制中的 Gb3/IFNAR 相互作用，Gb3/CD19 同型粘附中的相互作用和 Gb3/VT B 亚基相互作用 诱导细胞凋亡。 我们建议研究 Gb3 在以下方面的作用： 这些信号转导途径使用 Gb+ 和 Gb3 缺陷的 Daudi 细胞 线。 目的： 1) 粘附力研究 拟议的研究将确定 CD19 连接对 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 此类途径中已知中间体的水平。 同型粘附 由表面蛋白、佛波酯和 IFN-a 的抗体连接诱导 将在这些细胞系中进行比较。 野生型反应的恢复 在 Gb3- 的情况下，将尝试添加外源性 Gb3 缺陷细胞的粘附机制有缺陷。 2) 干扰素信号传导 研究将对 IFN 诱导的信号转导进行比较和对比 在 Gb3+ 和 Gb3- 细胞中关于介质的变化，例如 ISGF3 的激活和信号传导的最终结果（包括生长） 干扰素诱导蛋白的抑制、合成和同型 附着力。 在以下情况下，细胞将用外源 Gb3 重建 发现 Gb3 缺陷细胞的 IFN 信号传导存在缺陷。 3） 细胞凋亡研究 我们将确定 Gb3 介导的细胞凋亡是否在 突变体可以用外源 Gb3 重建。 我们还将比较 这些细胞系中其他细胞凋亡诱导剂的活性，包括 由抗 CD19、CD22 和 IgM 抗体或更一般的机制诱导 如抗Fas抗体、放射线和细胞毒性T淋巴细胞杀伤等。 4) Gb3 AS MESSENGER Gb3 降解产物的潜在作用 抗 CD19 介导的粘附、干扰素信号传导和细胞凋亡途径将 通过监测 Gb3、神经酰胺、 鞘氨醇和相关的鞘脂化合物。