GLUCOCORTICOID REGULATION OF THYMOCYTE APOPTOSIS

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Apoptosis is a form of programmed cell death which is characterized by a number of distinct morphological and biochemical features. Immature thymocytes are especially sensitive to apoptosis-inducing agents and uniquely respond to glucocorticoids by undergoing apoptosis. Since glucocorticoid responses are mediated by glucocorticoid receptor- dependent alterations in gene expression, we have proposed that glucocorticoid-induced thymocyte apoptosis is the result of specific gene activation. To better understand the molecular basis of glucocorticoid-regulated thymocyte apoptosis, we have pursued two lines of investigation. First, we have isolated a panel of apoptotic-deficient (Apt-) WEHI 7.2 mouse thymoma cell lines by chemical mutagenesis and selection in media containing the synthetic glucocorticoid dexamethasone. Characterization of these Apt- mutants has revealed that they express normal levels of fully functional glucocorticoid receptor, are cross-resistant to several apoptosis-inducing agents, and belong to at least three genetic complementation groups. Second, we have used subtraction cDNA cloning and mRNA differential display to isolate rapidly-induced glucocorticoid-regulated transcripts. These studies have led to the characterization of Dag-8 and GIG-10, two genes that are induced within 2 hours of steroid-treatment. We propose to extend these molecular genetic studies of apoptosis by pursuing three specific aims; 1) Isolate human genomic sequences that functionally complement the Apt- phenotype in mutants containing defects in early, intermediate and late steps in the apoptotic pathway, 2) Characterize a collection of differentially expressed sequence tags (DESTs) previously isolated from dexamethasone-treated WEHI 7.2 cells, to determine if any of them identify potential "apoptotic genes" based on expression patterns in the Apt- cell lines and 3) Investigate the role of Dag-8, GIG-10 and selected DESTs, in modulating WEHI 7.2 apoptosis using a tetracycline- regulated expression system and antisense oligodeoxynucleotides. The overall goal of these studies is to identify and characterize the primary steroid-regulated genes and the apoptotic effector genes, both of which are required for glucocorticoid-induced thymocyte apoptosis.