THE HUMAN GLUCOCORTICOID RECEPTORS, ITS GENE AND ACTIONS

该项目的长期目标是了解 人类白血病细胞中的糖皮质激素和糖皮质激素受体。 在过去的四年零两个月里，我们已经完成了大部分 最初提出的主要目标；我们希望完成重要的目标 在接下来的十个月里。此外，我们还获得了新的结果 超出了最初的目标。到目前为止，我们有，和最初一样 提出并成功合作克隆和测序 人糖皮质激素受体(HGR)基因；获得并使用单链和 HGR的多克隆抗体；在一个患有HGR的家庭中发现了RFLP 初级皮质醇抵抗；进一步确定了受体的性质 糖皮质激素耐药人白血病CEM细胞克隆缺陷； 结果表明，西维拉唑在CEM C7细胞中的两个结合部位均位于HGR上； 显示HGR+/Lysis-CEM细胞补充HGR-突变体；发现 癌基因测试表明，只有c-myc基因被显著下调 糖皮质激素，表明这一过程既需要GR，也需要裂解 在最初目标的延伸中，我们还：过度表达了 在杆状病毒系统中发现这种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；结果表明，HGR是由地塞米松在一系列 骨髓瘤细胞；开始绘制HGR的图谱，以了解其杀死转基因细胞的能力 白血病细胞，有和没有配体；并测定了约5000个碱基的 HGR的5‘侧翼，识别了之前未描述的第一外显子和 几个转录起始点。 基于我们的结果，我们建议进行新的实验，旨在 在基因水平上解释糖皮质激素受体的作用，以定义 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部位 它们在白血病细胞中引起致命反应，并研究其调节 感受器本身。我们将进一步鉴定过度表达的HGR 并用纯化的蛋白进行GR生化研究。在无细胞系统中 我们想要研究转录的某些基因的上调和下调。 由糖皮质激素受体控制下的RNA聚合酶I或II， 使用我们过表达的hGR来驱动体外反应。我们会 确定CEM中糖皮质激素对myc的调节水平(S) 细胞。我们将用几种方法检验c-myc是一个关键的假说。 CEM细胞对糖皮质激素致死反应的调节步骤。我们 将详细绘制细胞杀伤所需区域的HGR图，寻求 能有效地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 在CEM中负责HGR自身诱导性的启动子/增强子 细胞，并将发现什么转录因子结合和相互作用带来 关于这一反应。

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.