TRANSFORMATION-RESISTANT REVERTANTS

癌变涉及多个独立的体细胞突变 原癌基因和抑癌基因。这种突变可能会导致 对控制细胞的信号转导级联的放松管制 生长和分化。尽管的各个功能 某些癌基因和肿瘤抑制基因是已知的较详细的， 对信令的组成或调节知之甚少 它们控制的路径。分子遗传学方法，即 癌基因转化细胞的返回体分析已选定 为了洞察这种通路中导致 肿瘤生长。拟议的研究集中在两个独立的 从大鼠成纤维细胞衍生的回复细胞系 过度产生蛋白激酶C，并由ras癌基因转化。 与它们转化的亲本不同，这些回复细胞 线条在软琼脂中不会形成菌落。此外，他们还压制 体细胞杂交种的转化表型，并对 由几个不同的癌基因进行的再转化。两种回变细胞 金属硫蛋白(MT)基因的表达也存在缺陷 对不同的刺激做出反应。这些数据表明，主要- 每个回复突变体中的作用突变基因可能驱动合成或 包括MTS在内的一组特定基因的抑制子的活性 目前还没有确定在基因中起关键作用的基因 转型。 这项建议的具体目标是： 1)分离致病的显性突变基因(S) 两个回变细胞系中每一个的回变表型。这将是 是通过对这些细胞系进行插入突变来实现的 专门化逆转录病毒，然后选择重组体和 插入突变基因的克隆。 2.)MT基因负性调控机制的分析 利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， 随后进行了电泳迁移率变化分析和对 与目的启动子元件(S)相互作用的蛋白质。 3.)分离除MTS外的表达被抑制的基因 用差异筛选法筛选回变株中的cDNA文库 由对照和突变细胞系构建而成。这样的基因 代表转化表型的潜在介体。

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.