THE HUMAN GLUCOCORTICOID RECEPTOR, ITS GENE AND ACTIONS

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The long-term objectives of this project are to understand the workings of glucocorticoids and the glucocorticoid receptor in human leukemic cells. In the last four years and two months, we have accomplished most of the major aims originally proposed; we expect to complete the significant ones remaining in the next ten months. In addition, we have obtained new results extending beyond the original aims. To date we have, as originally proposed, successfully collaborated in the cloning and sequencing of the human glucocorticoid receptor (hGR) cDNA; obtained and used both mono- and polyclonal antibodies to the hGR; discovered an RFLP in a family with Primary Cortisol Resistance; further defined the nature of the receptor defects in clones of glucocorticoid-resistant human leukemia CEM cells; shown that cortivazol's two binding sites in CEM C7 cells are both on hGR; shown that hGR+/lysis- CEM cells complement hGR- mutants; found that of the oncogenes tested, only c-myc is acutely, profoundly down-regulated by glucocorticoid, showing that this process requires both the GR and "lysis function." In extensions of the original aims, we have also: overexpressed hGR in the baculovirus system and found that this hGR is extremely potent in stimulating glucocorticoid response element-driven, cell free transcription; shown that hGR is induced by dexamethasone in a line of myeloma cells; begun to map hGR for its ability to kill transfected leukemic cells, with and without ligand; and sequenced about 5000 bp of the 5' flank of the hGR, identifying a previously undescribed first exon and several transcription start sites. Based on our results, we propose to carry out new experiments designed to explain glucocorticoid receptor action at the gene level, to define the topology of the receptor responsible for cell kill, to test the hypothesis that the c-myc gene is a critical site of action for glucocorticoids as they evoke a lethal response in leukemic cells, and to study the regulation of the receptor itself. We will characterize further the overexpressed hGr and use the purified protein to study GR biochemistry. In cell-free systems we want to study both up- and down-regulation of certain genes transcribed by RNA polymerase I or II under the control of glucocorticoid receptors, using our overexpressed hGR to drive the in vitro reactions. We will determine the level(s) at which myc is regulated by glucocorticoids in CEM cells. We will test in several ways the hypothesis that c-myc is a critical regulated step in the lethal response of CEM cells to glucocorticoids. We will map in detail the hGR for the regions necessary for cell kill, seeking the fragment that can efficiently bring about leukemic cell death in the absence of steroid. Finally, we will determine the region of the hGR promoter/enhancer responsible for the inducibility of the hGR itself in CEM cells and will find what transcription factors bind and interact to bring about that response.