Regulation and Function of ECK in Apoptosis

ECK在细胞凋亡中的调控及作用

• 批准号: 6418332
• 负责人: YUXIN YIN
• 金额: $ 28.09万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-18 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6418332
• 关键词: apoptosis; athymic mouse; breast neoplasms; enzyme activity; enzyme induction /repression; fibroblasts; ionizing radiation; neoplasm /cancer genetics; northern blottings; oxidative stress; p53 gene /protein; polymerase chain reaction; protein kinase; radiation genetics; tissue /cell culture; tumor suppressor genes; tumor suppressor proteins

Transcription factors p53 and E2F-1 coordinate and mediate apoptosis in response to genotoxic stress. However, the mechanism whereby how p53 and E2F-1 mediate apoptosis is unclear. We found that p53 and E2F-1 co-regulate the expression of ECK, an epithelial cell receptor protein-tyrosine kinase and that ECK mediates apoptosis following oxidative stress. In p53 inducible systems, ECK is greatly upregulated upon the activation of p53. The expression of ECK is increased following oxidative stress in a p53-dependent manner. We have cloned the promoter region of human ECK gene and identified two potential p53-binding sites within a 700 bp from the transcriptional initiation site of ECK. We show that p53 greatly induces luciferase activity of the luciferase reporter containing this sequence upstream of the luciferase gene. Interestingly, there is also an E2F-binding site in the ECK promoter. Cotransfection of E2F-1 with the luciferase reporter containing the potential E2F-1 binding site leads to the activation of this promoter reporter. We further demonstrate that the ectopic expression of E2F-1 elevates the levels of ECK protein. Finally, overexpression of ECK greatly increases cellular susceptibility to apoptosis and suppresses colony formation in soft agar. Our findings indicate that p53 and E2F-1 are transcriptional regulators of ECK and that ECK is a cell death receptor in the p53 and E2F-1 pathways. This is the first evidence that p53 and E2F-1 share a common target gene in signaling apoptosis. In this grant application, we propose a series of experimental approaches to classify ECK as a new member of the p53-downstream gene family and a dual target for p53 and E2F-1. We will determine whether and how ECK is regulated by these two fundamental tumor suppressors. We will demonstrate the importance of ECK in signaling oxidative cell death by apoptosis and its potential role in tumorigenesis. Successful achievement of our specific aims will provide evidence for how p53 and E2F-1 cooperatively regulate a protein receptor, which receives damage signals and mediates cell death. Characterization of ECK as a cell death receptor may reveal a new target for gene therapy or lead to the development of effective chemotherapeutical strategies in cancer treatment.

转录因子p53和E2 F-1协调并介导细胞凋亡对遗传毒性应激的响应。 然而，p53和E2 F-1如何介导细胞凋亡的机制尚不清楚。我们发现p53和E2 F-1共同调节ECK的表达，ECK是一种上皮细胞受体蛋白酪氨酸激酶，并且ECK介导氧化应激后的细胞凋亡。 在p53诱导型系统中，ECK在p53激活后大大上调。ECK的表达在氧化应激后以p53依赖的方式增加。 我们克隆了人ECK基因的启动子区，并在ECK转录起始位点的700 bp范围内发现了两个p53结合位点。我们发现，p53极大地诱导荧光素酶报告基因上游含有此序列的荧光素酶活性。 有趣的是，在ECK启动子中也存在E2 F结合位点。 E2 F-1与含有潜在E2 F-1结合位点的荧光素酶报告基因的共转染导致该启动子报告基因的激活。 我们进一步证明E2 F-1的异位表达升高了ECK蛋白的水平。 最后，ECK的过度表达大大增加了细胞对凋亡的敏感性，并抑制软琼脂中的集落形成。 我们的研究结果表明，p53和E2 F-1是ECK的转录调节因子，ECK是p53和E2 F-1途径中的细胞死亡受体。 这是第一个证据表明p53和E2 F-1在细胞凋亡信号转导中共享共同的靶基因。 在本申请中，我们提出了一系列的实验方法，将ECK归类为p53下游基因家族的新成员和p53和E2 F-1的双重靶点。 我们将确定ECK是否以及如何受到这两种基本肿瘤抑制因子的调节。 我们将证明ECK在通过细胞凋亡信号传导氧化性细胞死亡中的重要性及其在肿瘤发生中的潜在作用。 我们特定目标的成功实现将为p53和E2 F-1如何协同调节蛋白受体提供证据，该蛋白受体接收损伤信号并介导细胞死亡。 ECK作为细胞死亡受体的特性可能揭示基因治疗的新靶点或导致癌症治疗中有效的化学治疗策略的发展。