PROTEASE INHIBITOR EFFECTS IN EPITHELIAL TRANSFORMATION

蛋白酶抑制剂对上皮转化的影响

• 批准号: 6271638
• 负责人: TADMIRI Rangachar VENKATESH
• 金额: $ 3.01万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271638
• 关键词: biomarker; cell differentiation; cell growth regulation; epithelium; gene expression; genetic library; genetic transcription; neoplastic transformation; nucleic acid sequence; oncogenic virus; protease inhibitor; protooncogene; restriction mapping; southern blotting; western blottings

Protease inhibitors have been shown to possess a broad range of transformation-suppressing effects including reduction of tumor formation and blockage of transformation in cell culture systems. In previous work our laboratory has described the stage-specific nature of the process of transformation of human epidermal keratinocytes by the oncogenic virus, SV40. The overall goal of this proposal is to use the viral transformed epithelial cells as a model system to study the mechanism of action of protease inhibitors in antioncogenesis. The studies described in this proposal are divided into two parts. In the first part we will characterize the effects of protease inhibitors on transformation in the viral transformed epithelial cells using four types of parameters of growth and differentiation as stage-specific markers of transformation in vitro: a) clonal growth, b) growth dependence on serum and growth factors, c) anchorage independent growth, d) density dependent regulation of growth and differentiation. These studies will determine aspects of growth control and critical stages in the transformation process which are targets of the transformation suppressing effects of protease inhibitors. In the second part we will investigate the role of gene expression as a molecular mechanism of action of protease inhibitors. These studies will have three components: 1) DNA and RNA blot hybridization analyses to determine whether protease inhibitor treatment can prevent two types of SV40-induced changes in the myc and ras protooncogenes: a) structural polymorphisms and b) altered levels of transcription, 2) Immunochemical analysis and RNA blot hybridization to determine whether protease inhibitor treatment can reverse the transformation-related induction of simple epithelial/fetal keratin genes and/or block the expression of a newly characterized cytoskeletal cDNA marker of transformation and, 3) Isolation of transformation-related sequences from cDNA libraries whose expression is either blocked or induced by protease inhibitors.

蛋白酶抑制剂已被证明具有广泛的 转化抑制作用，包括减少肿瘤形成 和细胞培养系统中转化的阻断。 在以前的工作中 我们的实验室已经描述了这一过程的阶段特异性， 致癌病毒对人表皮角质形成细胞的转化， SV40 这项提案的总体目标是利用病毒转化的 上皮细胞作为模型系统来研究的作用机制， 蛋白酶抑制剂的抗癌作用。 本文中描述的研究 建议分为两部分。 在第一部分，我们将 表征蛋白酶抑制剂对转化的影响， 使用四种参数的病毒转化上皮细胞 生长和分化作为阶段特异性转化标记 体外：a）克隆生长，B）生长对血清和生长依赖性 因素，c）锚定独立生长，d）密度依赖性调节 生长和分化。 这些研究将决定 增长控制和转型过程中的关键阶段， 是蛋白酶转化抑制作用的靶点 抑制剂的 在第二部分中，我们将探讨基因的作用， 表达作为蛋白酶抑制剂的分子作用机制。 这些研究将有三个组成部分：1）DNA和RNA印迹 杂交分析以确定蛋白酶抑制剂处理是否 可以阻止SV 40诱导的myc和ras两种类型的变化 原癌基因：a）结构多态性和B） 转录，2）免疫化学分析和RNA印迹杂交， 确定蛋白酶抑制剂治疗是否可以逆转 简单上皮/胎儿角蛋白基因的转化相关诱导 和/或阻断新表征的细胞骨架cDNA的表达 转化标记，3）分离转化相关的 来自cDNA文库的序列，其表达被阻断或 由蛋白酶抑制剂诱导。