THE HUMAN GLUCOCORTICOID RECEPTOR, ITS GENE AND ACTIONS

本项目的长期目标是了解 糖皮质激素和糖皮质激素受体在人类白血病细胞。 在过去的四年零两个月里，我们已经完成了大部分的工作。 最初提出的主要目标;我们期望完成重要目标 剩下的十个月。此外，我们还获得了新的结果 超越了最初的目标。到目前为止，我们已经，作为最初的 提出，成功地合作克隆和测序的 人糖皮质激素受体（hGR）cDNA;获得并使用单-和 hGR的多克隆抗体;在一个家族中发现了RFLP， 原发性皮质醇抵抗;进一步定义了受体的性质 糖皮质激素抗性人白血病CEM细胞克隆缺陷; 结果表明，cortivazol在CEM C7细胞中的两个结合位点均在hGR上; 显示hGR+/裂解- CEM细胞补充hGR-突变体;发现 癌基因测试，只有c-myc是急性，深刻下调 糖皮质激素，表明这一过程需要GR和“溶解” 功能“在最初目标的延伸中，我们还： hGR在杆状病毒系统中的作用，并发现这种hGR是非常有效的 在刺激糖皮质激素反应元件驱动，无细胞 转录;表明hGR是由地塞米松诱导的，在一个细胞系中， 骨髓瘤细胞;开始绘制hGR杀死转染细胞的能力 白血病细胞，有和没有配体;和测序约5000 bp的 hGR的5'侧翼，鉴定先前未描述的第一外显子， 多个转录起始位点。 根据我们的研究结果，我们建议进行新的实验， 在基因水平上解释糖皮质激素受体的作用， 负责细胞杀伤的受体的拓扑结构，以检验假设 c-myc基因是糖皮质激素的关键作用位点， 它们在白血病细胞中引起致死反应， 受体本身。我们将进一步表征过表达的hGr 并利用纯化的蛋白进行GR生物化学研究。在无细胞系统中 我们想研究某些基因转录的上调和下调， 通过糖皮质激素受体控制下的RNA聚合酶I或II， 使用我们的过表达hGR来驱动体外反应。我们将 确定CEM中糖皮质激素调节myc的水平 细胞我们将用几种方法来检验c-myc是一种关键的 在CEM细胞对糖皮质激素的致死反应中的调节步骤。我们 将详细绘制细胞杀伤所需区域的hGR， 能有效导致白血病细胞死亡的片段 没有类固醇。最后，我们将确定hGR的区域 启动子/增强子负责CEM中hGR本身的诱导 细胞，并将找到转录因子结合和相互作用， 关于这个反应。

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.