TRANSFORMATION-RESISTANT REVERTANTS

癌发生涉及多个独立的体细胞突变 原癌基因和抑癌基因。 此类突变可导致 放松控制细胞的信号转导级联 生长和分化。 虽然个别职能 某些致癌基因和肿瘤抑制基因已被详细了解， 关于信号传导的组成或调节知之甚少 他们控制的途径。 分子遗传学方法，即；的 已选择对癌基因转化细胞的回复体进行分析 深入了解导致这些路径的畸变 肿瘤生长。 拟议的研究重点关注两个独立的 源自大鼠成纤维细胞的回复回复细胞系 过量产生蛋白激酶 C，并被 ras 癌基因转化。 与它们转化的亲本系相比，这些回复细胞 细胞系在软琼脂中不形成菌落。 此外，它们还抑制 体细胞杂种中的转化表型并且具有抗性 几种不同癌基因的再转化。 两种回复细胞 品系还表现出金属硫蛋白（MT）基因表达缺陷 以应对不同的刺激。 这些数据表明，主要是—— 每个回复体中起作用的突变基因可能驱动合成或 特定基因组（包括 MT）的抑制子的活性 尚未确定的在其中发挥关键作用的基因 转变。 该提案的具体目标是： 1.) 分离导致该现象的显性突变基因 两个回复细胞系中每一个的回复表型。 这将 通过这些细胞系的插入诱变来完成 专门的逆转录病毒，然后选择再转化体和 插入突变基因的克隆。 2.) 分析MT基因负调控机制 通过使用报告基因在回复系中表达 在各种MT基因启动子元件的控制下构建， 随后进行电泳迁移率变动测定和分析 与感兴趣的启动子元件相互作用的蛋白质。 3.) 分离除MT之外的表达被抑制的基因 通过差异筛选 cDNA 文库在回复体中 由对照和回复细胞系构建。 这样的基因 代表转化表型的潜在介质。

Carcinogenesis involves multiple, independent somatic mutations in proto-oncogenes and tumor suppressor genes. Such mutations can lead to deregulation of signal transduction cascades that control cell growth and differentiation. Although the individual functions of certain oncogenes and tumor suppressor genes are known in some detail, little is known about the components or regulation of the signalling pathways they control. A molecular genetic approach, i.e.; the analysis of revertants of oncogene-transformed cells, has been chosen to gain insight into aberrations in such pathways that result in neoplastic growth. The proposed studies focus on two independent revertant cell lines that were derived from rat fibroblasts that overproduce protein kinase C and are transformed by a ras oncogene. In contrast to their transformed parent line, these revertant cell lines do not form colonies in soft agar. Additionally, they suppress the transformed phenotype in somatic cell hybrids and are resistant to retransformation by several different oncogenes. Both revertant cell lines also exhibit defects in expression of metallothionein (MT) genes in response to diverse stimuli. These data suggest that dominantly- acting mutant genes in each revertant may drive the synthesis or activity of a repressor of a specific battery of genes, including MTs and yet to be identified genes that play a critical role in transformation. The specific aims of this proposal are: 1.) To isolate the dominant mutant gene(s) that are responsible for the revertant phenotype in each of the two revertant cell lines. This will be done by insertional mutagenesis of these cell lines with a specialized retrovirus, followed by selection for retransformants and cloning of the insertionally-mutated gene. 2.) To analyze the mechanism of negative regulation of MT gene expression in the revertant lines, through the use of reporter gene constructs under the control of various MT gene promoter elements, followed by electrophoretic mobility shift assays and analysis of the proteins that interact with the promotor element(s) of interest. 3.) To isolate genes in addition to MTs whose expression is inhibited in the revertants by differential screening of cDNA libraries constructed from control and revertant cell lines. Such genes represent potential mediators of the transformed phenotype.