Cdk-2 Independent role of cyclin E in Cell survival

Cdk-2 细胞周期蛋白 E 在细胞存活中的独立作用

• 批准号: 8197137
• 负责人: Alexandru Almasan
• 金额: $ 31.55万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-23 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197137
• 关键词: Affect; Apoptosis; Apoptotic; Binding; Biological Markers; C-terminal; Caspase; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Cycle Regulation; Cell Proliferation; Cell Survival; Cells; Characteristics; Chronic Lymphocytic Leukemia; Cleaved cell; Clinical; Clinical Treatment; Competitive Binding; Cyclin E; Cytosol; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA-PKcs; Data; Ectopic Expression; Epithelial; Fibroblasts; G22P1 gene; Generations; Genetic Models; Genotoxic Stress; Hematopoietic; Hematopoietic Neoplasms; Human; Individual; Investigation; Ionizing radiation; Knock-out; Knowledge; Ligase; Lymphocyte; Mammalian Cell; Mediating; Mitochondria; Modeling; Molecular; Mutagens; Nonhomologous DNA End Joining; Nuclear; Patients; Phosphotransferases; Play; Process; Proteasome Inhibition; Proteasome Inhibitor; Proteins; Proteolysis; Radiation; Radiation Tolerance; Radiation therapy; Regulation; Repair Complex; Resistance; Role; Signal Transduction; Site; Small Interfering RNA; Testing; Therapeutic; Tumor Cell Line; Ubiquitination; caspase-3; cell growth regulation; chemotherapy; cytochrome c; established cell line; human CCNE1 protein; knock-down; multicatalytic endopeptidase complex; mutant; neoplastic cell; novel; prevent; repaired; response

Cyclin E1 (CycE) represents an essential regulator of the mammalian cell cycle and DNA replication. Our studies have recently uncovered its new role in the cell survival of hematopoietic tumor cells. We found that CycE is cleaved proteolytically during apoptosis induced by typical genotoxic agents such as ionizing radiation. This cleavage is dramatic as it affects most cellular CycE and is characteristic to all human tumor hematopoietic cells we have examined, both established cell lines or primary, freshly isolated from human patients. This cleavage is important for apoptosis as expression of a cleavage-resistant CycE mutant blocks this process. CycE cleavage generates p18CycE, which is unable to bind Cdk2 or other known interactors of CycE. Our hypothesis, supported by preliminary data, is that CycE function is mediated by its interaction with Ku70, which we have identified as a novel p18CycE-binding partner. By binding to Ku70, p18CycE1 displaces Bax, leading to its activation. Indeed, p18CycE genesis coincides with Bax activation and loss of mitochondrial functions and the ensuing apoptosis is dependent on both Bax and Ku70, since it is prevented in cells rendered deficient in these proteins by knockout or siRNA-mediated knock-down. As Ku70 is a critical component of the nonhomologous end joining (NHEJ) DNA repair, our preliminary data indicate that the CycE fragment also affects the response to DNA damage and its repair. We will determine the mechanism by which p18CycE regulates apoptosis and DNA repair, in addition to inactivating CycE function. These studies are focused on a molecule that is unique in regulating all three responses to genotoxic stress: cell cycle control, DNA repair, and apoptosis. Our objective is to understand the regulation of CycE and derivative p18CycE following genotoxic stress and their impact on cells with differential radiation sensitivity. We seek to determine: 1) how the Ku70 interaction with p18CycE regulates cell survival, 2) the regulation and role of p18CycE1, 3) the therapeutic potential of p18CycE1. These investigations will establish the contribution to apoptosis of CycE1 independent of Cdk2. Our studies will increase our understanding of the mechanism by which CycE may provide a molecular switch by coordinating key responses to genotoxic stress, that include cell cycle control, DNA repair, and apoptosis that govern the radio- or chemotherapy-induced signals in clinical therapy.

细胞周期蛋白E1（CycE）是哺乳动物细胞周期的重要调节因子， DNA复制。我们的研究最近发现了它在细胞存活中的新作用， 造血肿瘤细胞我们发现，CycE是裂解蛋白水解过程中， 典型的遗传毒性剂如电离辐射诱导的细胞凋亡。该裂解 是戏剧性的，因为它影响大多数细胞CycE，是所有人类肿瘤的特征 我们已经检查了造血细胞，无论是建立的细胞系或原代，新鲜 从人类病人身上分离出来的。这种切割对于细胞凋亡是重要的，因为 抗切割的CycE突变体阻断了这一过程。CycE切割产生 p18CycE，其不能结合Cdk2或其他已知的CycE相互作用物。我们 初步数据支持的假设是，CycE功能是由其 与Ku70的相互作用，我们已经确定为一种新的p18CycE结合伴侣。通过 p18CycE1与Ku70结合，置换Bax，导致其活化。事实上，p18CycE 发生与Bax激活和线粒体功能丧失一致， 随后的凋亡依赖于Bax和Ku70，因为它在细胞中被阻止， 通过敲除或siRNA介导的敲除使这些蛋白质缺陷。作为 Ku70是非同源末端连接（NHEJ）DNA修复的关键组分，我们的研究表明， 初步数据表明，CycE片段也影响对DNA的反应， 损坏及其修复。我们将确定p18CycE调节 细胞凋亡和DNA修复，除了失活CycE功能。这些研究 专注于一种独特的调节基因毒性的所有三种反应的分子， 应激：细胞周期控制、DNA修复和凋亡。我们的目标是了解 基因毒性应激后CycE及其衍生物p18 CycE的调节及其影响 对辐射敏感性不同的细胞。我们试图确定：1）Ku70如何 与p18CycE的相互作用调节细胞存活，2）p18CycE1的调节和作用， 3)p18CycE1的治疗潜力。这些调查将确定 CycE1对细胞凋亡的贡献独立于Cdk2。我们的研究将增加我们的 理解CycE通过以下方式提供分子开关的机制： 协调对遗传毒性应激的关键反应，包括细胞周期控制，DNA 修复和细胞凋亡，在临床上控制放射或化疗诱导的信号 疗法