PROTEASE INHIBITOR EFFECTS IN EPITHELIAL TRANSFORMATION

蛋白水解酶抑制剂已被证明具有广泛的 包括减少肿瘤形成在内的抑制转化作用 以及细胞培养系统中的转化受阻。在以前的工作中 我们的实验室已经描述了这一过程的特定阶段性质 致癌病毒对人表皮角质形成细胞的转化 SV40。这项提议的总体目标是使用病毒转化的 以上皮细胞为模型系统研究丹参的作用机制 抗肿瘤中的蛋白水解酶抑制剂。在这篇文章中描述的研究 提案分为两个部分。在第一部分中，我们将 表征了蛋白酶抑制剂对转化的影响。 使用四种参数的病毒转化的上皮细胞 生长和分化是转化的阶段特异性标志 在体外：a)克隆生长，b)生长依赖于血清和生长 因子，c)锚定独立生长，d)密度依赖调节 关于生长和分化的。这些研究将确定以下方面 增长控制和转型过程中的关键阶段 是蛋白酶抑制转化作用的靶点 抑制剂。在第二部分中，我们将研究基因的作用 表达作为蛋白酶抑制剂作用的分子机制。 这些研究包括三个部分：1)DNA和RNA印迹 杂交分析确定蛋白水解酶抑制剂是否治疗 可以防止两种类型的SV40诱导的myc和ras的变化 原癌基因：a)结构多态和b)改变水平 转录，2)免疫化学分析和RNA印迹杂交 确定蛋白水解酶抑制剂治疗是否可以逆转 与转化相关的简单上皮/胎儿角蛋白基因的诱导 和/或阻断一种新鉴定的细胞骨架基因的表达 转化标记；3)转化相关基因的分离 C DNA文库中表达被阻断或 由蛋白水解酶抑制剂诱导。

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.