The overall goal of the proposed research is to understand the mechanism(s) by which glucocorticoids act to suppress the function of the immune system. In particular, we will focus our studies elucidating the molecular events that are involved in the regulation of thymocyte cell death by glucocorticoids. Studies undertaken in several laboratories including our own have revealed that glucocorticoid regulated lymphocytolysis occurs by receptor dependent mechanisms which activate inherent programmed call death processes commonly referred to as """"""""apoptosis"""""""". The biological hallmark of all apoptosis forms of cell death in profound internucleosomal fragmentation of genomic DNA which occurs prior to any detectable loss in call viability. Accordingly, the nuclease responsible for this DNA degradation is a key component to the apoptotic process. During the previous granting period, we have succeeded in the identification, purification and determination of a partial amino acid sequence for a """"""""unique"""""""" nuclease which has inherent internucleosomal DNA cleavage activity. In this research proposal, we propose to clone the gene for this nuclease and use the cloned gene to directly test the hypothesis that internucleosomal DNA fragmentation is the cause of cell death. The availability of the cloned nuclease gene will permit us to address previously unapproachable questions pertaining to the mode of regulation of apoptosis by glucocorticoids. Additionally, we will define the molecular basis for tissue and call type specific apoptosis. To test this hypothesis and address the ensuing issues, we propose the following specific aims: (I) To clone and sequence the glucocorticoid regulated nuclease from rat thymocytes. (II) To verify the authenticity of the cloned nuclease and determine its role in apoptotic forms of cell death. (III) To define the tissue specific mechanisms that regulate nuclease gene expression and nuclease activity in both apoptotic and non-apoptotic cells. The knowledge gained from these investigations should define the precise cellular mechanisms which activate the physiological process of apoptosis and provide key insights into the mechanisms of immunosuppression by glucocorticoids.
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