The long term goal of the proposed research is to understand the action of adrenal steroid hormones (glucocorticoids) on lymphocytes and the immune system in general. We will continue to investigate in detail the glucocorticoid induced cellular mechanisms which ultimately lead to lymphocyte death and thereby suppressed immune function. The thymic lymphocyte of the adrenalectomized rat will be the focus of our attention. The cells are well suited for these investigations because they are abundant, uniform in cell cycle stage. uniform in state of differentiation, responsive to glucocorticoids in vitro and in vivo and a well studied model for adrenal steroid action. Three seminal observations concerning glucocorticoid induced lymphocytolysis have been made during the past granting period. They are: 1) Glucocorticoid induced lymphocyte cell death is preceeded by DNA degradation; 2) Glucocorticoid induced DNA degradation is not random, implying specificity; 3) A nuclear glucocorticoid """"""""induced"""""""" nuclease activity has been identified. Based on these observations, we wish to propose the following hypothesis: Glucocorticoids induce or activate a deoxyribonuclease which selectively alters the integrity of genomic DNA and causes the cells to die. To test this hypothesis, we propose the following Specific Aims: 1) To evaluate whether glucocorticoid induced """"""""breaks"""""""" in DNA occur in transcribed or non-transcribed genomic sequences; 2) To purify to homogeneity the glucocorticoid """"""""induced"""""""" nuclease and test its specificity in vitro; 3) To clone the glucocorticoid """"""""induced"""""""" nuclease gene and evaluate its role in glucocorticoid induced cell death by transfection experiments; 4) Lastly, to evaluate via selective cell sorting the development of glucocorticoid resistance during the differentiation of rat thymic lymphocytes to mature T cells. Together, these studies should test the proposed hypothesis and provide data on the molecular basis for glucocorticoid induced lymphoid cell death.
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