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(PP250) The interaction of the proteolytic fragment of Cyclin E with Ku70 enhances apoptosis by inhibiting DNA repair.
Plesca, Dragos*,1, 2, Mazumder, Suparna1, Almasan, Alex1, 2, 3, 1 Department of Cancer Biology, Cleveland, OH, USA2 School of Biomedical Sciences, Kent, OH, USA3 Department of Radiation Oncology, Cleveland, OH
ABSTRACT- Cyclin E (CycE)/Cdk2 is a critical regulator of the cell cycle progression from G1 to S phase in mammalian cells and has been found to be deregulated in many types of neoplasms. However, the influence of CycE upon apoptosis has not been determined. We found that in hematopoietic cells, genotoxic stress leads to an increase in the expression of CycE followed by a decrease of its levels coinciding with the time-dependent appearance of its proteolytic cleavage-dependent fragment, p18-CycE (Mazumder et. al; MCB 2002). The interaction with Cdk2, as well as the associated kinase activity were lost following caspase-mediated CycE cleavage. Overexpression of p18-CycE induces apoptosis. Since p18-CycE no longer interacts with Cdk2, we have investigated its involvement in amplification of apoptosis through novel interacting partners. By yeast-two-hybrid screening, mass spectrometry analyses, and co-immunoprecipitation assays we have identified Ku70 and KUB3, components of the Non-Homologous End Joining (NHEJ) machinery, as two interacting partners of p18-CycE. We have also found that p18-CycE affects the cytosolic to nuclear translocation of the Ku70-Ku80 complex. Two in vitro NHEJ assays, end-ligation and plasmid reactivation assays showed that cellular extracts containing p18-CycE reduced the DNA ligation ability considerably as compared to extracts containing CycE. Current studies are examining the effect of p18-CycE on V(D)J recombination using a transfection based assay with the expression constructs of the Recombination Activating Genes 1 and 2 and the pJH299 extrachromosomal substrate in MEFs. In addition to its role in DNA repair, Ku70 regulates apoptosis by sequestering Bax, which is released following p18-CycE binding. Interaction of Ku70 with p18-CycE and Bax may link cell cycle control, DNA repair, and activation of apoptosis following genotoxic stress as well as provide mechanistic insights into the choice between cell death and cell survival depending on the cell type and nature of the genotoxic challenge.
Key words: Cyclin E, Non Homologous End Joining
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