GLUCOCORTICOID REGULATION OF THYMOCYTE APOPTOSIS

细胞凋亡是程序性细胞死亡的一种形式，其特征在于： 一些独特的形态学和生化特征。 不成熟 胸腺细胞对胸腺增生诱导剂特别敏感， 通过细胞凋亡对糖皮质激素产生独特的反应。 以来 糖皮质激素反应由糖皮质激素受体介导， 基因表达的依赖性改变，我们提出， 糖皮质激素诱导的胸腺细胞凋亡是特异性 基因激活为了更好地理解 糖皮质激素调节胸腺细胞凋亡，我们有两条路线 调查。首先，我们分离出一组缺乏免疫力的 （Apt-）WEHI 7.2小鼠胸腺瘤细胞系通过化学诱变和 在含有合成糖皮质激素的培养基中选择 地塞米松。对这些Apt-突变体的表征表明， 它们表达正常水平的全功能糖皮质激素受体， 对几种诱导骨质疏松症的药物具有交叉抗性，属于 至少有三个遗传互补群。第二，我们使用 差减cDNA克隆和mRNA差异显示分离 快速诱导的糖皮质激素调节的转录物。这些研究 导致了Dag-8和GIG-10的特征，这两个基因是 在类固醇治疗的2小时内诱导。我们建议延长这些 细胞凋亡的分子遗传学研究，追求三个具体目标; 1)分离功能上与Apt互补的人类基因组序列， 在突变体中含有早期、中期和晚期缺陷的表型 步骤，2）表征细胞凋亡途径的集合， 差异表达序列标签（DEST），以前从 地塞米松处理的WEHI 7.2细胞，以确定它们中的任何一个是否 基于细胞中的表达模式识别潜在的“凋亡基因”。 Apt-细胞系和3）研究Dag-8、GIG-10和Apt-细胞系的作用。 选择的DEST，在使用四环素调节WEHI 7.2凋亡中， 调控表达系统和反义寡核苷酸。的 这些研究的总体目标是识别和表征 主要的类固醇调节基因和凋亡效应基因， 糖皮质激素诱导的胸腺细胞凋亡所需的。

Apoptosis is a form of programmed cell death which is characterized by a number of distinct morphological and biochemical features. Immature thymocytes are especially sensitive to apoptosis-inducing agents and uniquely respond to glucocorticoids by undergoing apoptosis. Since glucocorticoid responses are mediated by glucocorticoid receptor- dependent alterations in gene expression, we have proposed that glucocorticoid-induced thymocyte apoptosis is the result of specific gene activation. To better understand the molecular basis of glucocorticoid-regulated thymocyte apoptosis, we have pursued two lines of investigation. First, we have isolated a panel of apoptotic-deficient (Apt-) WEHI 7.2 mouse thymoma cell lines by chemical mutagenesis and selection in media containing the synthetic glucocorticoid dexamethasone. Characterization of these Apt- mutants has revealed that they express normal levels of fully functional glucocorticoid receptor, are cross-resistant to several apoptosis-inducing agents, and belong to at least three genetic complementation groups. Second, we have used subtraction cDNA cloning and mRNA differential display to isolate rapidly-induced glucocorticoid-regulated transcripts. These studies have led to the characterization of Dag-8 and GIG-10, two genes that are induced within 2 hours of steroid-treatment. We propose to extend these molecular genetic studies of apoptosis by pursuing three specific aims; 1) Isolate human genomic sequences that functionally complement the Apt- phenotype in mutants containing defects in early, intermediate and late steps in the apoptotic pathway, 2) Characterize a collection of differentially expressed sequence tags (DESTs) previously isolated from dexamethasone-treated WEHI 7.2 cells, to determine if any of them identify potential "apoptotic genes" based on expression patterns in the Apt- cell lines and 3) Investigate the role of Dag-8, GIG-10 and selected DESTs, in modulating WEHI 7.2 apoptosis using a tetracycline- regulated expression system and antisense oligodeoxynucleotides. The overall goal of these studies is to identify and characterize the primary steroid-regulated genes and the apoptotic effector genes, both of which are required for glucocorticoid-induced thymocyte apoptosis.