MOLECULAR BIOLOGY OF HORMONE REGULATED CYTOLYSIS

类固醇受体是配体激活的转录因子，与 定位于类固醇靶标调控区的DNA序列 基因。激活的受体既可以诱导转录，也可以抑制转录 取决于发起人的背景和某些 转录因子。尽管我们知道很多关于对 很少有经过充分研究的靶基因，对决定因素了解更少 细胞特异性类固醇调节。因此，根据公认的 模型认为类固醇反应是通过改变细胞内 有限数量的目的基因的表达，我们已经 糖皮质激素诱导细胞死亡机制的探讨 用几种不同的方法诱导两种小鼠淋巴瘤细胞系的(凋亡) 分子遗传学方法。我们对糖皮质激素受体的研究 (GR)结构和功能支持类固醇调节 淋巴细胞凋亡处于转录水平，并导致 旨在分离和鉴定的初步工作 淋巴细胞靶基因。 本提案中描述了三种独立的方法，它们扩展了 我们以前的研究有以下几个目标：1)使用 受体融合和遗传选择来识别氨基酸 类固醇诱导细胞死亡所需的GR氨基末端，2) 为了鉴定我们最近通过以下方式分离的cdna序列 消减杂交技术，并确定它们所起的作用 可能在诱导细胞死亡表型中起作用，以及3)分离和 鉴定表达正常水平的淋巴细胞突变细胞系 野生型GR BUT对类固醇耐药(死亡表型)。同舟共济 这些分析应该有助于更好地理解细胞特异性 类固醇基因网络和潜在地识别所需的关键靶基因 导致淋巴细胞死亡。此外，还进行了一次遗传选择 稳定转GR变异体的细胞对类固醇的耐药性 意义重大，因为它可能允许更彻底地检查 受体功能，如那些需要蛋白质-蛋白质相互作用的功能。

Steroid receptors are ligand-activated transcription factors which bind to DNA sequences located in the regulatory regions of steroid target genes. Activated receptors can both induce and repress transcription depending on the promoter context and availability of certain transcription factors. Although much is known about the regulation of a few well-studied target genes, less is understood about the determinants of cell-specific steroid regulation. Therefore, based on the accepted model that steroid responses are mediated through changes in the expression of a limited number of target genes, we have been investigating the mechanism of glucocorticoid-induced cell death (apoptosis) in two murine lymphoma cell lines using several different molecular genetic approaches. Our studies of glucocorticoid receptor (GR) structure and function support the notion that steroid regulation of lymphocyte apoptosis is at the level of transcription and have led to preliminary work aimed at the isolation and characterization of lymphocyte target genes. Three independent approaches are described in this proposal which extend our earlier studies and have the following objectives: 1) to use receptor fusions and genetic selections to identify amino acids in the GR amino terminus which are required for steroid-induced cell death, 2) to characterize cDNA sequences which we have recently isolated by subtraction hybridization techniques, and to determine what role they may have in eliciting the cell death phenotype, and 3) to isolate and characterize lymphocyte mutant cell lines which express normal levels of wild-type GR but are steroid-resistant (deathless phenotype). Together these analyses should lead to a better understanding of cell-specific steroid gene networks and potentially identify key target genes required for lymphocyte cell death. Moreover, a genetic selection for steroid-resistance among cells stably transfected with GR variants is significant since it may allow for a more thorough examination of receptor functions such as those requiring protein-protein interactions.