CERAMIDE AND RAD INCLUDED APOPTOSIS OF CNS GLIA

神经酰胺和 RAD 包括中枢神经系统胶质细胞凋亡

• 批准号: 2871991
• 负责人: Louis A Pena
• 金额: $ 2.06万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-13 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2871991
• 关键词: apoptosis; astrocytes; biological signal transduction; cell line; cell type; ceramides; enzyme activity; gene expression; genetically modified animals; glia; histochemistry /cytochemistry; laboratory mouse; laboratory rat; oligodendroglia; phosphodiesterases; radiation; radiation sensitivity; reporter genes; second messengers; sphingomyelin phosphodiesterase; tissue /cell culture; transfection

DESCRIPTION (Applicant's Description): The objective of this project is to study the role of radiation-induced ceramide generation via sphingomyelinase (ASMase) in the induction of apoptosis in CNS glial cells, with emphasis on oligodendrocytes (OLs). A novel signal transduction system has recently been character in which the lipid second messenger, ceramide, is linked to the stress activated kinase (SAPK/JNK) cascade, resulting in cell death by apoptosis. Each of the CNS glial cell types, the myelin-forming OLs and astrocytes, varies in its sensitivity to ionizing radiation and to exogenous ceramide in inducing apoptosis. These differences create distinct, opposing clinical relevancies. OLs are radiosensitive and undergo apoptosis following radiation-induced ceramide generation via ASMase. The CNS is a dose limiting tissue for therapeutic irradiation, partly because of radiation-induced cell death of OLs, and as hypothesized in this project, partly because of loss of clonogenic viability of OL progenitors. Thus the issue is ultimately to protect OLs. In contrast astrocytes are radioresistant, show no radiation-induced ceramide generation, and show no ceramide-dependent apoptosis. The majority of gliomas are astrocytic in character, and are known for a high degree of radioresistance. Thus the issue is ultimately to sensitize astrocytes. To investigate the role of radiation-induced ceramide generation via ASMase in contributing to the mechanisms of radioresistance/sensitivity, glial cell lines derived from an ASMase-deficient transgenic mouse (ASM-KO) will be generated. Primary glial cultures will be immortalized by transduction with SV40 large T antigen. In the ASM-KO vs. wild type mouse in vivo, and in these new ASMasc(+/-) cell lines and in existing glial cell lines in vitro, the kinetics and magnitude of ASMase activation, ceramide generation, SAPK/JNK activation, and apoptosis will be characterized by specific biochemical assays, in response to irradiation. Agents which inhibit specific points in the ceramide-SAPK/JNK pathway will be tested in vitro for OL lines, and stimulatory agents for astrocyte lines. To validate these concepts for OLs in vivo, ASMase(+/-) OL lines will be transfected with reporter genes and transplanted intracranially in the nude rat. After irradiation, the level of apoptosis and clonogenic survival of transplants will be assessed by histochemical techniques. Useful agents identified by in vitro screening will be tested in this in vivo model for protection of OLs from radiation-induced cell death.

描述（申请人的描述）：该项目的目的是 研究辐射诱导的神经酰胺通过鞘磷脂酶的作用 （ASMase）在中枢神经系统胶质细胞中凋亡的诱导，重点 少突胶质细胞（OLS）。 一个新型的信号转导系统最近有 是脂质第二信使Ceramide与之相关的性格 应力激活激酶（SAPK/JNK）级联反应，导致细胞死亡 凋亡。 每种CNS神经胶质细胞类型，形成髓磷脂的OL和 星形胶质细胞，其对电离辐射的敏感性以及对外源性的敏感性 神经酰胺诱导凋亡。 这些差异造成了独特的反对 临床相关性。 OLS是放射素敏感的，凋亡 辐射诱导的神经酰胺通过ASMase产生。 中枢神经系统是 剂量限制组织以进行治疗辐射，部分是由于 辐射引起的OL的细胞死亡，并在该项目中提到的那样， 部分原因是OL祖细胞的克隆生存力丧失。 因此 问题最终是为了保护OLS。 相比之下，星形胶质细胞是 放射耐药物，没有辐射引起的神经酰胺的产生，并显示不显示 神经酰胺依赖性凋亡。 大多数神经胶质瘤是星形胶质细胞 性格，以高度放射线而闻名。 因此 问题最终是为了使星形胶质细胞敏感。 通过ASMase研究辐射诱导的神经酰胺的生成的作用 在有助于放射性/灵敏度的机理中，神经胶质细胞 源自ASMase缺陷的转基因小鼠（ASM-KO）的线将是 生成。 一级神经胶质培养物将通过转导而永生 SV40大T抗原。 在ASM-KO与野生型鼠标中，在体内和 这些新的ASMASC（+/-）细胞系以及在体外现有的神经胶质细胞系中， ASMase激活的动力学和大小，神经酰胺的产生， SAPK/JNK激活和凋亡将以特定为特征 生化测定，以响应辐照。 抑制的药物 神经酰胺-SAPK/JNK途径中的特定点将在体外测试 OL线和星形胶质细胞线的刺激剂。 验证这些 OLS在体内的概念，ASMase（+/-）OL线将被转染 报告基因并在裸鼠内颅内移植。 后 辐照，移植物的细胞凋亡水平和移植的克隆生存水平 将通过组织化学技术评估。 有用的代理由 体外筛查将在该体内模型中进行测试，以保护 来自辐射引起的细胞死亡的OL。