A new human GR gene sequence (hGR 1Ap/e), which is distinct from the previously identified human GR promoter and coding sequences, has been isolated and characterized. The hGR 1Ap/e sequence is approximately 31 kbp upstream of the human GR coding sequence. This sequence (2,056 bp) contains a novel promoter (the hGR 1A promoter; 1,075 bp) and untranslated exon sequence (hGR exon 1A sequence; 981 bp). Alternative splicing produces three different hGR 1A-containing transcripts, 1A1, 1A2, and 1A3. GR transcripts containing exon 1A1, 1A2, 1B, and 1C are expressed at various levels in many cancer cell lines, while the exon 1A3-containing GR transcript is expressed most abundantly in blood cell cancer cell lines. Glucocorticoid hormone treatment causes an up-regulation of exon 1A3-containing GR transcripts in CEM-C7 T-lymphoblast cells and a down-regulation of exon 1A3-containing transcripts in IM-9 B-lymphoma cells. Deoxyribonuclease I footprinting using CEM-C7 cell nuclear extract reveals four footprints in the promoter region and two intraexonic footprints. Much of the basal promoter-activating function is found in the +41/+269 sequence, which contains two deoxyribonuclease I footprints (FP5 and FP6). When this sequence is cloned into the pXP-1 luciferase reporter gene, hormone treatment causes a significant increase in luciferase activity in Jurkat T cells that are cotransfected with a GR expression vector. FP5 is an interferon regulatory factor-binding element, and it contributes significantly to basal transcription rate, but it is not activated by steroid. FP6 resembles a glucocorticoid response element and can bind GRbeta. This novel hGR 1Ap/e sequence may have future applications for the diagnosis, prognosis, and treatment of T-cell leukemia and lymphoma.

We have quantified the basal and glucocorticoid-regulated levels of different transcripts from the human glucocorticoid receptor (GR) gene in the T-cell acute lymphoblastic leukemia cell line, CEM-C7, and in the B lymphoblastoid cell line, IM-9. Highly specific quantitative, reverse transcription-polymerase chain reaction assays measured total GR transcripts, transcripts encoding the isoforms glucocorticoid receptor alpha (GRalpha) and glucocorticoid receptor beta (GRbeta), and transcripts containing different forms of exon 1: 1A1, 1A2, 1A3, 1B, and 1C. GRalpha and GRbeta transcripts are coordinately upregulated in CEM-C7 cells and coordinately downregulated in IM-9 cells by dexamethasone. The concentration of GRalpha mRNA is more than a 1000-fold higher than that for GRbeta mRNA. Transcripts with different exon 1 forms are all upregulated in CEM-C7 cells and all downregulated in IM-9 cells by dexamethasone, but transcripts containing exons 1A1, 1A2, or 1A3 are regulated to a higher degree than transcripts containing exon 1B or exon 1C. However, exon 1B- and exon 1C-containing transcripts are substantially more abundant than exon 1A-containing transcripts, with exon 1A3-containing transcripts more abundant than exon 1A1- or exon 1A2-containing transcripts. Analysis using models for glucocorticoid receptor autoregulation kinetics suggests that the minor 1A3-containing transcript component could be important for GR protein upregulation, and hence apoptosis, in CEM-C7 cells. These studies suggest that GRalpha transcripts containing exons 1A3, 1B, and 1C contribute most to the intracellular level of GR mRNA and may be the most relevant for steroid-mediated apoptosis in T-lymphoblasts.

For the elucidation of the mechanism of steroid hormone receptor activation, the hydrodynamic properties of the unactivated and activated forms of the nonproteolyzed glucocorticoid receptor from the mouse AtT-20 pituitary tumor cell line were determined. The unactivated, molybdate-stabilized receptor has the following properties: sedimentation coefficient = 9 S; Rs = 8.3 nm; Mr = 317 000; f/f0 = 1.70; axial ratio (prolate ellipsoid) = 14. The activated monomeric receptor has a sedimentation coefficient of 3.2 S, a Stokes radius of 6 nm, a molecular weight of 81 000, a frictional ratio of 1.93, and an axial ratio (prolate ellipsoid) of 18. A receptor species of intermediate size was detected when the analysis was performed in buffer containing both 0.3 M KCl and 20mM Na2MoO4. Its characteristics are as follows: sedimentation coefficient = 5 S; Rs = 8.3 nm; Mr = 176 000; f/f0 = 2.06; axial ratio (prolate ellipsoid) = 22. A preliminary study seemed to indicate that this is an activated form of the receptor. On the basis of the molecular weights, it is likely that the unactivated receptor is a tetramer of identical hormone-binding subunits (Mr = 81 000) while the intermediate form is a homodimer. Alternatively, non-hormone-binding components (receptor-binding factors) may be involved in forming the multimeric, nonactivated receptor complex. In either case, the dissociation of a multimeric, nonactivated receptor into subunits appears to be a possible mechanism of receptor activation. Finally, the addition of high concentrations of 1-thioglycerol promoted activation. Thus, sulfhydryl groups may be involved in receptor subunit interaction.

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