GLUCOCORTICOID ACTION IN SYNCHRONIZED CELLS

这项建议的目标是研究 同步培养的HeLa S3细胞在细胞周期中的糖皮质激素作用 我们将测试这一独特的假设，即生理变化在 糖皮质激素受体蛋白可导致类固醇反应和 人类细胞周期的抗性阶段。此前的调查显示 显示存在“电荷”修饰的未蛋白水解糖皮质激素 地塞米松不能诱导细胞周期阶段的受体 HeLa S3细胞中的碱性磷酸酶(AP)。这种受体不是核受体 在37℃下在整个细胞内移位，或在体外25℃加温激活。 我们建议进一步刻画其物理性质 地塞米松-甲磺酸亲和力调节糖皮质激素受体 细胞周期和确定受体修饰的分子基础 以及研究这一修改的规则。基于 我们的文献和我们自己的研究可能涉及到修改 受体的磷酸化/去磷酸化。这种可能性将是 用高纯度亲和标记受体进行体外探索 细胞周期中的整个细胞。此外，单抗将会 被提升为亲和标记糖皮质激素的各种修饰形式 受体，并用作唯一的探针来建立受体 结构/功能关系。这一目标将在1)之前实现 用单抗筛选反应性和非反应性细胞 碱性磷酸酶在细胞分选机上的应用及其物理特性 受体，2)微量注射针对修饰受体的单抗 和正常受体形成HeLa S3细胞和评估碱性 响应地塞米松挑战的磷酸酶的产生。这些 研究应该提供必要的生化信息，说明 糖皮质激素受体是一种可修饰的蛋白质，这种表观遗传学 机制可以解释细胞周期中类固醇抵抗的原因。一个 对这一假设的彻底检验和概念的理解 发达的应该提供足够的生化基础来准确地 预测人类肿瘤和其他疾病的类固醇反应状态 涉及肾上腺轴的疾病。

The objectives of this proposal are to examine the mechanism of glucocorticoid action during the cell cycle in synchronized HeLa S3 cells. We will test the unique hypothesis that physiological modifications in the glucocorticoid receptor proteins can result in steroid responsive and resistant phases of the human cell cycle. Previous investigations have shown the presence of a "charge" modified unproteolyzed glucocorticoid receptor during cell cycle stages when dexamethasone does not induce alkaline phosphatase (AP) in HeLa S3 cells. This receptor does not nuclear translocate at 37C in whole cells or activate by warming at 25C in vitro. We propose to characterize further the physical properties of dexamethasone-mesylate affinity laveled glucocorticoid receptors during the cell cycle and determine the molecular basis for modification in receptor as well as study the regulation of this modification. Based on the literaturwe and our own studies the modification may likely involve phosphorylation/dephosphorylation of receptor. This possibility will be explored in vitro with highly purified affinity labeled receptors and in whole cells during the cell cycle. Furthermore monoclonal antibodies will be raised to the various modified forms of affinity labeled glucocrticoid receptors and used as unique probes to establish receptor structure/function relationships. This goal will be achieved by 1) selecting responsive and nonresponsive cells with monoclonal antibodies to alkaline phosphatase on a cell sorter and physically characterizing their receptors, 2) microinjecting monoclonal antibodies to the modified receptor and normal receptor forms into HeLa S3 cells and assessing alkaline phosphatase production in response to a dexamethasone challenge. These studies should provide needed biochemical information on whether the glucocorticoid receptor is a modifiable protein and whether such epigenetic mechanisms can account for steroid resistance during the cell cycle. A thorough testing of this hypothesis and an understanding of the concepts developed should provide an adequate biochemical basis to accurately predict the state of steroid responsiveness in human neoplasia and other diseases involving the adrenal axis.