. Our laboratory centers on the transition events that occur between DNA repair and apoptosis in cells that have accrued irreversible DNA double strand breaks. This proposal focuses on ATM, a major participant to recognizing and initiating DNA double strand break repair, and PP2A, a protein phosphatase that regulates many kinases involved with basic cellular functions. PP2A activity is regulated by ceramide. We hypothesize that ATM function is regulated by PP2A and ceramide. We will show that there is a downregulation of ATM due to ceramide-dependent PP2A dephosphorylation, resulting in deactivating ATM-dependent prosurvival pathways and increasing the apoptotic efforts within the damaged cell. This proposal directly links DNA repair with apoptotic triggers, proposing a mechanisms where cells transition from survival to cell death by decreasing DNA repair efforts and increasing proapoptotic pathways. Describing this ATM-PP2A relationship may provide insight on the mechanisms responsible for radiosensitivity, a phenotype of genetic disorders with abnormal DNA repair, and neurodegeneration, as seen in A-T and other neurodegenerative syndromes. A means to inhibiting these cell death events can potentially decrease these debilitating outcomes. Understanding this mechanism may also present an approach to improving the efficiency of radiation or chemotherapy treatments, such as those used in fighting cancer, by using these pathways to stimulate these apoptotic triggers and create cells less resistant to these treatments. Our model will be normal lymphoblastoid cells treated with bleomycin to induce DNA double strand breaks, which will be analyzed in three specific aims for ATM and PP2A interaction.
Specific Aim 1 will examine the ATM autophosphorylation and activation kinetics in cells with bleomycin-induced DNA double strand breaks. We will achieve this by immunoblotting for autophosphorylation of ATM and phosphorylation of p53 from nuclear lysates of treated normal cells for several timepoints over a 24-hour period.
Specific Aim 2 will observe ATM-PP2A complex formation and how it correlates to ATM activity. Coimmunoprecipitation of ATM and PP2A will demonstrate the direct interaction between the two proteins, resulting in inhibition of ATM function. Treatment of cells with bleomycin and the PP2A inhibitor, okadaic acid, is expected to prolong ATM activation due to PP2A inability to dephosphorylate ATM.
Specific Aim 3 will measure and correlate ceramide levels in bleomycin-treated normal cells to ATM activation and complex formation with PP2A. Artificial increase of ceramide levels with C6-ceramide is expected to induce early ATM-PP2A complex formation, prematurely inhibiting ATM kinase activity. The AREA award will provide financial support to expose undergraduate students to biomedical research and allow them to contribute to the advancement of research in my laboratory. Linking ATM directly to cell death will describe how a cell generates a signal to transition from survival to death. Comprehension of such signals will lead to new information on the cause behind hypersensitivity to DNA damaging agents and neurodegeneration, as well as present possible therapeutic options in treating cancer. ? ? ?
