Glucocorticoids kill both normal and malignant lymphoid cells. Consequently, steroids of this class remain a principle tool in chemotherapy of both malignant and non-malignant lymphoproliferative diseases. Glucocorticoids also mediate normal immune development and differentiation, and are very important pharmacological and physiological modulators of the immune response. Virtually all individuals in the United States will use glucocorticoids for some purpose during their life. Nevertheless, we do not understand the mechanisms whereby such steroids affect proliferation and death of either normal or transformed lymphoid cells. This proposal presents a cogent hypothesis to explain how glucocorticoids kill malignant T lymphoid cells, and is a continuation of more than 20 years of work in this area. We have determined that glucocorticoids inhibit the expression of cyclin D3. This is a post- transcriptional process in which the degradation of cyclin D3 mRNA increases rapidly upon addition of dexamethasone. Destabilization involves at least two proteins that bind to a defined response element within the 3' untranslated region of cyclin D3 mRNA. Approximately half of the project (Specific Aim 1) deals with isolation and characterization of these two proteins, analysis of their mechanism of action, and identification of other proteins that may participate in glucocorticoid-mediated destabilization of cyclin D3 mRNA. We have determined that inhibition of cyclin D3 expression causes a redistribution of the cyclin-dependent kinase inhibitor, p27Kip1, which dissociates from Cdk4-containing complexes as the abundance of cyclin D3 falls and binds to and inhibits cyclin E/Cdk2 complexes. We have recently discovered that the antiapoptotic protein Bc12 is a substrate for Cdk2. Inhibition of Cdk2 by glucocorticoids and other agents causes net dephosphorylation of Bcl2. We propose that dephosphorylation of Bc12 leads to cell death, perhaps by decreasing the affinity of Bc12 for Bax, favoring the formation of Bax homodimers and cell death. The remaining half of this proposal (Specific Aim 2) deals with testing this hypothesis. We will analyze the phenotype of cells that express dominant negative Cdks and p27, with particular attention to Bc12 phosphorylation and cell death. We will also analyze the phenotype of cells that express Bc12 mutants that cannot be phosphorylated as well as mutants that mimic constitutive phosphorylation. Upon completion of these specific aims. we hope to have, for the first time, a mechanism that accounts for the relationship between glucocorticoid inhibition of Cdk2 activity, cell proliferation, and cell death.
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