TRANSFORMATION-RESISTANT REVERTANTS

致癌作用涉及多个独立的体细胞突变， 原癌基因和肿瘤抑制基因。 这种突变会导致 去调节控制细胞的信号转导级联 生长和分化。 虽然个别功能 某些癌基因和肿瘤抑制基因是已知的， 关于信号的组成或调节知之甚少 他们控制的道路。 分子遗传学方法，即;的 癌基因转化细胞的回复突变体分析，已被选择 为了深入了解这些通路中的异常， 肿瘤生长 研究重点是两个独立的 来自大鼠成纤维细胞的回复突变细胞系， 过度产生蛋白激酶C并被ras癌基因转化。 与其转化的亲本系相反，这些回复突变细胞 品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，并对 由几种不同的癌基因引起的再转化。 两种回复突变细胞 株系也表现出金属硫蛋白（MT）基因表达的缺陷 对不同刺激的反应。 这些数据表明，占主导地位的- 每个回复突变体中的作用突变基因可以驱动合成， 一组特定基因（包括MT）阻遏物的活性 还没有被鉴定出的基因， 转型 这项建议的具体目标是： 1.）为了分离出导致突变的显性突变基因， 在两个回复突变体细胞系中的每一个中的回复突变体表型。 这将 可以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， 插入突变基因的克隆。 2.）分析MT基因负调控的机制 通过使用报告基因， 在各种MT基因启动子元件控制下的构建体， 然后进行电泳迁移率变动测定和对 与目的启动子元件相互作用的蛋白质。 3.）第三章分离除了表达受到抑制的MT以外的基因 通过cDNA文库的差异筛选， 从对照和回复突变体细胞系构建。 此类基因 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.