DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the roles of a novel protein C53 in regulation of cell cycle progression and DNA damage response, and to explore potential utilization of this novel protein as a novel therapeutic target. Regulation of cell cycle and cellular response to DNA damage is critical for genome stability, normal tissue homeostasis, tumorigenesis and cancer treatment. Any defects in cell cycle regulation may lead to genome instability and oncogenesis. Cells utilize the so-called checkpoint mechanisms to ensure accurate transmission of genetic material. The G2/M checkpoint regulates mitotic entry and activation of cyclin- dependent kinase 1 (Cdk1), a driving force for cell division. DNA damage usually evokes such checkpoints through activating a network of molecules including protein kinases ATM/ATR and their targets checkpoint kinases Chk1 and Chk2. By phosphorylating downstream effectors such as p53 and Cdc25 phosphatases, Chk1/Chk2 spread damage signal and elicit either cell cycle arrest or apoptotic cell death. We have recently identified Cdk5 activator p35 binding protein C53 as a novel regulator of cell cycle and DNA damage response. Some p53 deficient tumor cells treated with DNA damaging agents such as etoposide, a potent inhibitor of DNA topoisomerase II, undergo prolonged G2 arrest, and followed by apoptotic cell death. Our study demonstrated that depletion of endogenous C53 leads to defective cell cycle progression and resistance to apoptosis induced by etoposide and ionizing irradiation. Interestingly, C53 overexpression alone induces uneven chromatin condensation (UCC) in the absence of DNA damage. Moreover, C53 overexpression sensitizes cancer cells to various DNA damage agents via impairing DNA damage-induced checkpoint response, including activation of checkpoint kinases, but does not affect ATM/ATR activity. We found that C53 depletion enhances DNA damage-induced checkpoint activation. Intriguingly, we found that C53 interacts with both Chk1 and Chk2, and is able to inhibit their enzymatic activity. Based upon our preliminary study, we hypothesize that C53 is a novel negative regulator of checkpoint kinases via inhibiting their activation and kinase activity. By counteracting checkpoint kinases, C53 acts as a promoter of Cdk1 activation and mitotic entry during normal cell cycle and DNA damage response. To test our hypothesis, we will Aim 1: To elucidate the mechanism of C53-mediated regulation of checkpoint kinase activation. We will determine how C53 affects activation/inactivation of Chk1/Chk2 in DNA damage response. Aim 2: To dissect the C53-Chk interaction. By using deletion and site-specific mutations, we will determine which domains of C53 and Chk1/Chk2 are responsible for their interaction, and the C53 activity to inhibit Chk1/Chk2 kinase activity. Aim 3: To investigate the effect of caspase-mediated cleavage of C53 on mitotic cell death. This study will provide insight on a novel regulatory mechanism for cell cycle control and checkpoint response. It will lay a foundation for exploitation of this protein as a novel therapeutic target in cancer treatment. PUBLIC HEALTH RELEVANCE: Regulation of cell cycle is critical for normal tissue homeostasis, animal development, and the pathogenesis of human diseases such as cancer. Cell cycle progression is a coordinately regulated process that includes accurate replication of DNA during the S phase, correct segregation of chromosomes during mitosis (M phase) and final separation of daughter cells in cytokinesis. Cells utilize the cell cycle checkpoints to ensure the completion of critical event of a particular phase before the next phase is initiated. The checkpoints are crucial for maintaining DNA integrity in normal cells, and defects in the checkpoints may cause genomic instability and ultimately tumorigenesis. In response to DNA damage, cells use similar checkpoint mechanisms to halt cell cycle for DNA repair, or trigger cell death if the damage is beyond repair. We have recently identified a novel protein C53 that plays important roles in these fundamental processes. Further study of this protein will provide insight into a novel regulatory mechanism for cell cycle and cell death. It will lay a foundation for further exploitation of this novel protein as a potential therapeutic target in fighting diseases such as cancer.
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