HUMAN GLUCOCORTICOID RECEPTOR, ITS GENE AND ACTIONS

描述(改编自申请人的摘要)：基本原则 这项资助的目的是了解激活的糖皮质激素如何 受体(GR)发挥其主要的生物学作用之一，即细胞凋亡。 恶性淋巴样细胞。在每一个方面都取得了重大进展 之前的具体目标。在淋巴样细胞中，突然下调 原癌基因c-myc的表达被认为是 糖皮质激素诱导的细胞凋亡。CEM C7细胞的新结果表明 糖皮质激素也下调其他重要的调控基因，但 诱导GR和c-jun。从这些事实出发，提出了一种新的机制模型。 糖皮质激素诱导的CEM细胞凋亡已经形成。其中四个 六个新的具体目标将考验这一模式的各个方面。1) 糖皮质激素的转录/转录后性质 对c-myc的调控将由核Run-on和mRNA决定 稳定性实验。启动子调控序列分析：记者 转基因，外加DNA或RNA结合实验将用于 确定控制的机制。2)假设在 Myc细胞凋亡途径缺失导致其他基因表达减少 将对调控基因进行测试。酶活性、蛋白质和信使核糖核酸 糖皮质激素或抗真菌药物降低Myc后的血药浓度 正义寡核苷酸。3)差异显示寻找其他基因 由糖皮质激素和Myc共同调节的基因将被执行，这些基因 进行了克隆和鉴定。4)手机JUN和Fos水平会有所不同 同时观察对Myc和细胞凋亡的影响。 致死功能的GR结构域图显示了DNA结合 域(DBD)是关键的，死亡的动力学不同 取决于DBD：GR上下文。当与类固醇结合相关联时 结构域(SDB)，DBD介导糖皮质激素依赖的细胞凋亡 很长时间的滞后。单独转染DBD或DBD突变体465*引起细胞 死亡的速度更快。两个具体目标追求基础性和实践性 这些发现的后果。系统结构/功能分析 DBD将执行：1.与SDB相关；以及2.作为 独一无二的致命碎片。提供后者的有效方式，在 将寻求开发一种新的基于基因的治疗方法。 重组DBD片段的结构和功能将进行研究。 将寻找蛋白质伙伴，并定义DNA结合位点。

DESCRIPTION (Adapted from the applicant's abstract): The fundamental objective of this grant is to understand how the activated glucocorticoid receptor (GR) exerts one of its major biological actions, apoptosis of malignant lymphoid cells. Significant progress has been made on each of the previous specific aims. In lymphoid cells, the abrupt down-regulation of the protooncogene c-myc has been identified as a key step in glucocorticoid-evoked apoptosis. New results in CEM C7 cells show that glucocorticoids also down-regulate other important regulatory genes, but induce the GR and c-jun. From these facts, a model for the mechanism of glucocorticoid-evoked apoptosis in CEM cells has been formed. Four of the six new specific aims will test aspects of this model. 1) The transcriptional/post-transcriptional nature of the glucocorticoidal regulation over c-myc will be determined by nuclear run-on and mRNA stability experiments. Regulatory sequence analysis in promoter:reporter transfections, plus DNA or RNA binding experiments will be used to determine the mechanisms of the control. 2) The hypothesis that in the apoptotic pathway loss of Myc leads to reduced expression of other regulatory genes will be tested. Enzyme activities, protein, and mRNA levels will be followed after reducing Myc with glucocorticoid or anti sense oligonucleotides. 3) Differential display to find other genes regulated by both glucocorticoids and Myc will be carried out, these genes cloned and characterized. 4) Cellular Jun and Fos levels will be varied while following the effects on Myc and apoptosis. Domain mapping of the GR for lethal function has shown the DNA Binding Domain (DBD) to be critical, with the kinetics of death different depending on the DBD:GR context. When associated with a Steroid Binding Domain (SDB), the DBD mediates glucocorticoid-dependent apoptosis after a long lag. Transfected alone, the DBD or the DBD mutant 465* cause cell death more quickly. Two specific aims pursue the basic and practical consequences of these findings. Systematic structure/function analyses of the DBD will be carried out: 1. In context with the SDB; and 2. As a unique lethal fragment. Effective ways to deliver the latter, in development of a new gene-based therapeutic method will be sought. Recombinant DBD fragments will be studied for structure and function. Protein partners will be sought and DNA binding sites will be defined.