Translational control of p27 in breast cancer cells

乳腺癌细胞中 p27 的翻译控制

• 批准号: 7029647
• 负责人: W KEITH MISKIMINS
• 金额: $ 22.55万
• 依托单位: UNIVERSITY OF SOUTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-07 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7029647
• 关键词: 3T3 cells; DNA footprinting; RNA binding protein; RNA interference; SDS polyacrylamide gel electrophoresis; binding proteins; breast neoplasms; cell cycle proteins; cell differentiation; cell proliferation; clinical research; complementary DNA; cyclin dependent kinase; enzyme inhibitors; genetic translation; messenger RNA; molecular cloning; neoplastic cell; polymerase chain reaction; posttranslational modifications; ribosomes; translation factor; western blottings

DESCRIPTION (provided by applicant): The broad goal of this application is to understand the mechanisms that regulate translation of the CDK inhibitor p27Kip1 (p27) in normal cells and in breast cancer cells, p27 is an essential component of normal cell cycle control and is expressed at elevated levels in quiescent cells and downregulated upon reentry into the cell cycle. There is a very strong correlation between low p27 expression and poor clinical prognosis for numerous types of cancer, including breast cancer, p27 levels are regulated by multiple mechanisms including translation initiation efficiency. Evidence is provided demonstrating that the ability to use the p27 5'-UTR for cap-independent translation strongly correlates with steady state p27 levels in a panel of breast cancer cell lines. A set of factors that bind to the p27 5'-UTR has been identified. Preliminary data show that differences in the binding of these factors to the 5'-UTR corresponds to differences in p27 expression. Based on these findings, a model is presented for the mechanism by which these factors mediate cap-independent translation of p27. The 1st specific aim is intended to test this model. This will involve defining the RNA sequences and structures required for translational initiation at the p27 internal ribosome entry site. The p27 5'-UTR-interacting proteins will be characterized and the mechanism by which they affect translation will be explored. The 2nd aim is to determine if changes in the p27 5'-UTR-interacting proteins play a role in decreased levels of p27 in breast cancer cells. This will involve examining the levels, binding activity, subcellular localization, and post-translational modifications of these proteins. These experiments will be performed using the same panel of breast cancer cells that have already been characterized for p27 levels and the ability to use the p27 5'-UTR for cap-independent translation. The same experiments will be performed using normal fibroblasts that are either quiescent or proliferating. These experiments are expected to lead to an understanding of how these proteins function in normal cells and whether alterations in this process contribute to the loss of p27 expression that is observed in some tumor cells.

描述（由申请人提供）：本应用的广泛目标是了解正常细胞和乳腺癌细胞中CDK抑制剂P27KIP1（P27）的转化的机制，p27是正常细胞周期控制的重要组成部分，并且在静脉细胞中升高并在重新定位后下调的水平上表达了正常细胞周期的重要组成部分。低p27表达与多种类型的癌症（包括乳腺癌，p27水平）的临床预后较差之间存在非常强的相关性，包括多种机制，包括翻译起始效率。提供的证据表明，使用p27 5'-UTR进行盖胶囊不依赖性翻译的能力与乳腺癌细胞系列的稳态P27水平密切相关。已经确定了与P27 5'-UTR结合的一组因素。初步数据表明，这些因子与5'-UTR的结合差异对应于p27表达的差异。基于这些发现，为这些因素介导了p27的帽单位翻译的机制提出了一个模型。第一个特定目的旨在测试该模型。这将涉及定义P27内部核糖体进入位点翻译起始所需的RNA序列和结构。将表征P27 5'-UTR相互作用的蛋白质，并将探索它们影响翻译的机制。第二个目的是确定P27 5'-UTR相互作用蛋白的变化是否在乳腺癌细胞中P27水平降低中起作用。这将涉及检查这些蛋白质的水平，结合活性，亚细胞定位和翻译后修饰。这些实验将使用已经针对P27水平进行表征的相同乳腺癌细胞以及使用P27 5'-UTR进行帽非依赖性翻译的能力进行。将使用静止或增殖的正常成纤维细胞进行相同的实验。这些实验有望导致对这些蛋白质在正常细胞中的功能以及此过程中的改变是否有助于在某些肿瘤细胞中观察到的P27表达的丧失。