Cyclin E/Cdk2 is a critical regulator of the G1/S phase cell cycle progression that is deregulated in many types of neoplasms. In hematopoietic tumor cells, genotoxic stress causes a dramatic decrease in Cyclin E levels, coinciding with the timely appearance of its proteolytic fragment, p18-Cyclin E. Overexpression of p18-Cyclin E in a variety of cell types induces apoptosis. We have identified Ku70, a critical component of the Non-Homologous End Joining (NHEJ) DNA repair as a specific interacting partner of p18-Cyclin E. Cells stably expressing p18-Cyclin E at non-toxic levels are more sensitive to DNA-damaging agents. Neutral comet assays showed inefficient repair in the presence of p18-Cyclin E following irradiation. Moreover, presence of p18-Cyclin E caused impairment of plasmid end-ligation and reduced colony formation as compared to cell lysates containing wild-type Cyclin E. DNA pull-down assays showed that the assembly of Ku70/Ku80 and DNA-PKcs is not affected by p18-Cyclin E. However, the recruitment of XRCC4, Ligase IV, and the recently identified accessory factor XLF was greatly impaired. It appears that p18-Cyclin E profoundly affects NHEJ which is dependent on its interaction with Ku70 and probably caused by interference with the recruitment of the XLF/XRCC4/Ligase IV heterocomplex to the sites of double strand breaks. We have reported earlier that p18-Cyclin E induces cell death by associating with Ku70 in the cytosol, causing dissociation of Bax from Ku70 and its activation. However, p18-Cyclin E has a much shorter half-life than Cyclin E due to effective ubiquitination and degradation by the proteasome. Interestingly, the SCF-Fbw7 complex is implicated in this ubiquitination process only to a limited extent, with a novel pathway involving Ku70 and Hdm2 being more effective in p18-Cyclin E turnover. Blocking its degradation using specific proteasome inhibitors can stabilize p18-Cyclin E and prolong its association with Ku70, thereby enhancing its apoptotic effect. By preventing its degradation, p18-Cyclin E may become an effective therapeutic target in various hematopoietic malignancies. Interaction of Ku70 with p18-Cyclin E and Bax may be unique in providing the molecular switch between cell cycle control, DNA repair, and activation of apoptosis following genotoxic stress.
|Commitee:||Blank, James, Fraizer, Gail, Marcinkiewicz, Jennifer, Piontkivska, Olena|
|School:||Kent State University|
|School Location:||United States -- Ohio|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Keywords:||Apoptosis, Cell cycle, Cyclin e, Dna repair, Ku70, Proteasome|
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