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E2F-1响应于遗传毒性应激，介导并介导凋亡。 但是，p53和E2F-1如何介导细胞凋亡的机制尚不清楚。我们发现p53和E2F-1共同调节了ECK的表达，ECK是上皮细胞受体蛋白 - 酪氨酸激酶的表达，并且ECK在氧化应激后介导了凋亡。 在p53诱导系统中，ECK在激活p53时大大上调。氧化应激以p53依赖性方式增加了ECK的表达。 我们已经克隆了人类ECK基因的启动子区域，并从ECK的转录启动位点确定了700 bp内的两个潜在的p53结合位点。我们表明，p53极大地诱导了包含荧光素酶基因上游的荧光素酶报告基因的荧光素酶活性。 有趣的是，ECK启动子中还有一个E2F结合位点。 E2F-1与含有潜在的E2F-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。 我们进一步证明了E2F-1的异位表达提高了ECK蛋白的水平。 最后，ECK的过表达大大增加了细胞对细胞凋亡的敏感性，并抑制了软琼脂中的菌落形成。 我们的发现表明，p53和E2F-1是ECK的转录调节剂，ECK是p53和E2F-1途径中的细胞死亡受体。 这是p53和E2F-1在信号凋亡中具有共同靶基因的第一个证据。 在此赠款应用中，我们提出了一系列的实验方法，将ECK分类为p53-Downstream基因家族的新成员，并且是P53和E2F-1的双重目标。 我们将确定这两个基本肿瘤抑制因子是否以及如何调节ECK。 我们将证明ECK通过细胞凋亡及其在肿瘤发生中的潜在作用在信号氧化细胞死亡中的重要性。 成功实现我们的特定目标将为p53和E2F-1如何合作调节蛋白质受体，该蛋白受体受到损伤信号并介导细胞死亡。 ECK作为细胞死亡受体的表征可能会揭示基因治疗的新靶标或导致癌症治疗中有效的化学治疗策略的发展。