CERAMIDE AS AN INTRA-CELLULAR MEDIATOR

这个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，通过这种途径，维生素D3和 肿瘤坏死因子-α被证明能激活一种中性的鞘磷脂酶，这种酶能裂解 神经鞘磷脂转化为神经酰胺和磷酸胆碱。神经酰胺能够 抑制细胞生长，诱导分化。根据我们最近的调查 发现神经酰胺下调c-myc，下面的假设将 有待研究：神经酰胺是肿瘤坏死因子-α的细胞内介质 下调c-myc活性，抑制细胞增殖，以及 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的调控。计划的目标包括a)演示 神经酰胺的产生对肿瘤坏死因子-α的反应 定量酶分析；b)建立神经酰胺对C-反应的影响。 使用Northern Blot分析MYC下调；以及c)确认 神经酰胺下调c-myc的机制与神经酰胺类似。 肿瘤坏死因子-α放线菌素D和核连续检测。2.划定 神经酰胺的作用机制。因为初步工作表明神经酰胺 激活蛋白磷酸酶，目标包括确定a) 2D研究神经酰胺对内源蛋白质磷酸化的影响 电泳法；b)神经酰胺激活蛋白质的能力 磷酸酶；c)神经酰胺类似物的结构-功能活性 激活磷酸酶和下调c-myc；以及d) 磷酸酶抑制剂对神经酰胺和肿瘤坏死因子-α作用的影响。希望是这样的 这些研究将定义一种新的信号通路，可能揭示 重要的治疗靶点，调节细胞增殖和 差异化。

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.