Cdk-2 Independent role of cyclin E in Cell survival

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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.