Cdk-2 Independent role of cyclin E in Cell survival

描述(由申请人提供)： 细胞周期蛋白E(Cyclin E1，CycE)是哺乳动物细胞周期和DNA复制的重要调节因子。我们的研究最近发现了它在造血肿瘤细胞存活中的新作用。我们发现，在典型的遗传毒性物质如电离辐射诱导的细胞凋亡过程中，CycE被蛋白降解。这种分裂是戏剧性的，因为它影响到大多数细胞周期蛋白E，并且是我们所检查的所有人类肿瘤造血细胞的特征，无论是已建立的细胞系还是从人类患者中分离的原代细胞。这种切割对细胞凋亡很重要，因为抗切割的CycE突变体的表达阻止了这一过程。CycE裂解产生p18CycE，p18CycE不能结合CDK2或其他已知的CycE的相互作用因子。我们的假设得到了初步数据的支持，即CycE的功能是通过它与Ku70的相互作用来调节的，我们已经确定Ku70是一个新的p18CycE结合伙伴。通过与Ku70结合，p18CycE1取代Bax，导致其激活。事实上，p18CycE的发生与Bax的激活和线粒体功能的丧失是一致的，随后的凋亡依赖于Bax和Ku70，因为在缺乏这些蛋白质的细胞中，通过基因敲除或siRNA介导的敲除而阻止了这一过程。由于Ku70是非同源末端连接(NHEJ)DNA修复的关键成分，我们的初步数据表明，CycE片段也影响对DNA损伤及其修复的反应。我们将确定p18CycE除了失活CycE功能外，还调节细胞凋亡和DNA修复的机制。这些研究集中在一种独特的分子上，该分子调节对遗传毒性应激的所有三种反应：细胞周期控制、DNA修复和细胞凋亡。我们的目的是了解CycE及其衍生物p18CycE在遗传毒性应激后的调节及其对具有差异辐射敏感性的细胞的影响。我们试图确定：1)Ku70与p18CycE的相互作用如何调节细胞存活，2)p18CycE1的调节和作用，3)p18CycE1的治疗潜力。这些研究将建立独立于CDK2的CycE1在细胞凋亡中的作用。我们的研究将增加我们对CycE可能通过协调对遗传毒性应激的关键反应而提供分子开关的机制的理解，这些反应包括细胞周期控制、DNA修复和细胞凋亡，这些因素控制着临床治疗中放射或化疗诱导的信号。 公共卫生相关性： CycE1到p18CycE1的转换是独一无二的，因为它影响到对遗传毒性应激的所有关键反应：细胞增殖、凋亡和DNA修复。此外，在正常人的淋巴细胞、人上皮细胞或成纤维细胞中，CycE1不能转化为p18CycE1。在细胞周期和DNA复制调控功能缺失的情况下，对p18CycE1功能的研究将有助于我们理解p18CycE1在细胞凋亡和DNA修复中所起的新作用。此外，这些研究提供了一种独特的方法来揭示有关密切联系、协调调节以及可能调节造血细胞对DNA损伤疗法反应的分子开关的基础知识，这些疗法涉及细胞周期控制、DNA修复和细胞凋亡。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Conversion of CycE1 to p18CycE1 is unique as it impacts on all key responses to genotoxic stress: cell proliferation, apoptosis, and DNA repair. Moreover, CycE1 is not converted to p18CycE1 in lymphocytes from normal individuals or in human epithelial or fibroblast cells. These investigations of p18CycE1 function in the absence of its Cdk2-dependent cell cycle and DNA replication regulatory function will contribute to our understanding of the novel role it plays, through p18CycE1, in apoptosis and DNA repair. In addition, these studies provide a unique approach to uncover fundamental knowledge on the intimate connections, coordinate regulation, and a possible molecular switch regulating the response of hematopoietic cells to DNA-damaging therapeutics that involve cell cycle control, DNA repair, and apoptosis.