GLUCOCORTICOID INHIBITION OF PLASMINOGEN ACTIVATOR

纤溶酶原激活物(Pas)是一种高度特异的蛋白水解酶。 专用于失活的酶原纤溶酶原，当被PAS切割时， 形成活性丝氨酸蛋白酶纤溶酶。PA，这是由两者表示的 正常和恶性细胞类型，通常受类固醇和 多肽激素。已有研究表明，PA的表达可能与 对细胞生长和/或分化状态的调节。我们有 证实LICR-LON-HMy2人骨髓瘤淋巴母细胞(HMy2) 细胞)分泌PA尿激酶(UK)。这些细胞的生长受到抑制 糖皮质激素，其胞外UK活性几乎是 在接触糖皮质激素后消除。这种对UK的抑制是 与糖皮质激素对UK或UK的抑制剂诱导无关 激活的纤溶酶。我们建议研究糖皮质激素的作用机制。 抑制HMy2 UK。我们将确定1)是否禁止 胞外UK涉及UK的生物合成和/或分泌减少；2) UK编码的mRNA的稳态水平是否下降 参与；3)UK基因转录是否降低；以及4) UK基因转录减少是否是 糖皮质激素或是否由糖皮质激素诱导 转录因子。分子生物学的方法将是 通过使用人UK cDNA质粒Phuk-8促进，该质粒已被 由F.Blasi博士(目前在NIH工作)提供给我们。适当的应急措施 讨论了所有可能结果的计划。中概述的实验 这项提案将确立使用糖皮质激素的有效性 在未来的研究中抑制HMy2 UK，以检查通过什么机制 类固醇受体关闭了构成基因的转录。这样的未来 研究将与目前使用的许多系统进行对比 研究类固醇对特定基因的诱导作用。总之，这项工作将 为一系列重要的长期目标奠定基础：1) 糖皮质激素对构成基因抑制作用机制的研究 转录；2)研究糖皮质激素之间的潜在关系 UK的抑制、细胞生长的抑制和类固醇的发展 骨髓瘤淋巴母细胞的耐药性；以及3)潜在的 糖皮质激素反应性的重要临床预测指标 骨髓瘤患者。

Plasminogen activators (PAs) are specialized proteases which are highly specific for the inactive zymogen plasminogen, which, upon cleavage by PAs, forms the active serine protease plasmin. PAs, which are expressed by both normal and malignant cell types, are frequently regulated by steroid and peptide hormones. It has been suggested that PA expression may be related to regulation of cell growth and/or differentiation state. We have demonstrated that LICR-LON-HMy2 human myeloma lymphoblast cells (HMy2 cells) secrete the PA urokinase (UK). These cells are growth inhibited by glucocorticoids, and their extracellular UK activity is virtually eliminated following exposure to glucocorticoids. This inhibition of UK is unrelated to glucocorticoid induction of inhibitors to either UK or activated plasmin. We propose to examine the mechanisms of glucocorticoid inhibition of HMy2 UK. We will establish 1) whether inhibition of extracellular UK involves decreased biosynthesis and/or secretion of UK; 2) whether decreases in steady-state levels of mRNA coding for UK are involved; 3) whether decreased transcription of the UK gene occurs; and 4) whether decreased UK gene transcription is a primary effect of glucocorticoids or whether it is mediated by glucocorticoid-induced transcriptional factors. The molecular biology approaches will be facilitated by the use of the human UK cDNA plasmid pHUK-8, which has been supplied to us by Dr. F. Blasi (currently at NIH). Appropriate contingency plans for all possible outcomes are discussed. The experiments outlined in this proposal will establish the validity of using glucocorticoid inhibition of HMy2 UK in future studies to examine mechanisms by which steroid-receptors turn off constitutive gene transcription. Such future studies would contrast with the many systems currently being used to examine steroid induction of specific genes. In summary, this work will provide the groundwork for a number of significant long-range goals: 1) examining the mechanisms of glucocorticoid inhibition of constitutive gene transcription; 2) studying potential relationships between glucocorticoid inhibition of UK, inhibition of cell growth, and development of steroid resistance in myeloma lymphoblasts; and 3) development of a potentially important clinical assay for prediction of glucocorticoid responsiveness in myeloma patients.