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）：本申请的主要目标是了解正常细胞和乳腺癌细胞中调节 CDK 抑制剂 p27Kip1 (p27) 翻译的机制，p27 是正常细胞周期控制的重要组成部分，并且是在静止细胞中表达水平升高，并在重新进入细胞周期时下调。对于包括乳腺癌在内的多种类型的癌症，p27 表达低与临床预后不良之间存在非常强的相关性，p27 水平受到包括翻译起始效率在内的多种机制的调节。有证据表明，使用 p27 5'-UTR 进行帽独立翻译的能力与一组乳腺癌细胞系中的稳态 p27 水平密切相关。已鉴定出一组与 p27 5'-UTR 结合的因子。初步数据表明，这些因子与 5'-UTR 结合的差异对应于 p27 表达的差异。基于这些发现，提出了一个模型来解释这些因素介导 p27 的帽独立翻译的机制。第一个具体目标是测试该模型。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 相互作用蛋白，并探索它们影响翻译的机制。第二个目标是确定 p27 5'-UTR 相互作用蛋白的变化是否在乳腺癌细胞中 p27 水平降低中发挥作用。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。这些实验将使用相同的乳腺癌细胞组进行，这些细胞已经表征了 p27 水平，并且能够使用 p27 5'-UTR 进行帽独立翻译。将使用静止或增殖的正常成纤维细胞进行相同的实验。这些实验预计将有助于了解这些蛋白质在正常细胞中如何发挥作用，以及该过程的改变是否会导致在某些肿瘤细胞中观察到的 p27 表达丧失。