CERAMIDE AS AN INTRA-CELLULAR MEDIATOR

该项目的长期目标是了解分子 细胞分化和增殖的机制。 赞助商的实验室 最近发现了一种新的信号通路，维生素D3和 TNF-α被证明可以激活中性鞘磷脂酶， 神经酰胺和磷酸胆碱。 神经酰胺能够 抑制细胞生长并诱导分化。 根据我们最近的 发现神经酰胺下调c-myc，以下假设将 神经酰胺是TNF-α的细胞内介质 下调c-myc、抑制细胞增殖的活性，和 诱导分化。 因此，该项目的具体目标是： 1. 确定神经酰胺作为第二信使的生理作用 参与c-myc调控。 计划目标包括：（a）展示 神经酰胺的产生响应于TNF-α使用敏感的和 定量酶测定; B）确定神经酰胺对c- 使用北方印迹分析的myc下调;和c）证实 神经酰胺对c-myc下调的机制类似于 TNF-α使用放线菌素D和核运行试验。 2. 划定 神经酰胺的作用机制。 由于初步研究表明神经酰胺 激活蛋白磷酸酶，目的包括确定a） 神经酰胺对内源性蛋白磷酸化的影响 电泳; B）神经酰胺激活蛋白质的能力 c）神经酰胺类似物在磷酸酶中的结构-功能活性; 激活磷酸酶和下调c-myc;和d） 磷酸酶抑制剂对神经酰胺和TNF-α的作用。 希望 这些研究将定义一种新的信号通路， 调节细胞增殖的重要治疗靶点， 分化

The long term goals of this project are to understand the molecular mechanisms of cell differentiation and proliferation. The sponsor's lab has recently identified a novel signalling pathway whereby Vitamin D3 and TNF-alpha were shown to activate a neutral sphingomyelinase which cleaves sphingomyelin into Ceramide and cholinephosphate. Ceramide is capable of inhibiting cell growth and inducing differentiation. Based on our recent finding that Ceramide down regulates c-myc, the following hypothesis will be investigated: Ceramide is an intracellular mediator of TNF-alpha's activity in down-regulating c-myc, inhibiting cell proliferation, and inducing differentiation. The specific aims of this project are therefore: 1. To define the physiologic role of Ceramide as a second messenger involved in c-myc regulation. Planned objectives include a) demonstrating Ceramide generation in response to TNF-alpha using a sensitive and quantitative enzymatic assay; b) establishing the effects of Ceramide on c- myc down-regulation using Northern Blot analysis; and c) confirming that the mechanism of Ceramide on c-myc down-regulation is analogous to that of TNF-alpha using Actinomycin D and nuclear run--on assays. 2. To delineate Ceramide's mechanism of action. Since preliminary work suggests Ceramide activates a protein phosphatase, the objectives include determining a) the effects of Ceramide on endogenous protein phosphorylation using 2D electrophoresis; b) the ability of Ceramide to activate a protein phosphatase; c) structure-function activity of Ceramide analogs in activating phosphatase and in down-regulating c-myc; and d) the effects of phosphatase inhibitors on Ceramide's and TNF-alpha's actions. It is hoped that these studies will define a novel signaling pathway that may disclose important therapeutic targets for modulation of cell proliferation and differentiation.