CONTROL OF GENE EXPRESSION--A ROLE FOR PROTO-ONCOGENES

已经努力开发了一种体外系统来研究 细胞的多步致癌转化。许多细胞系都有 最近通过DNA介导的大鼠REF52细胞转染法分离到一株 克隆的病毒和细胞癌基因。表达SV40T/t的线条 抗原或激活的ras癌基因(T24H-ras1)腺病毒-5早期 A区基因(Ad5E1A)被完全转化，而表达 只有T24H-ras1基因或Ad5E1a基因不是。 来自每个品系的大量克隆已经被描述为关于 形态、生长速度、血清依赖性、致瘤性和表达 2D-Gel上可见p21ras蛋白产物。初步结果表明 正常细胞p21基因的表达会因为 T24H-ras1基因的表达，以及含有较高水平的 大量的T24H-ras 1基因产物的致瘤性低于细胞 含有较低数量的基因产物。 这项研究将通过以下方式进一步表征这些细胞系 细胞周期的计算机分析双向凝胶电泳法 特定的细胞群体，按DNA含量的函数分类。通过 研究处于细胞周期代表性阶段的细胞，我们将 确定：(1)蛋白质合成和周转的详细模式 在整个细胞周期中；(2)每个细胞系对刺激的反应 血清剥夺后的生长因子；以及(3)发生的变化 在体内肿瘤形成过程中的每一条线。这些实验将展示如何 基因表达的基础水平受T24H-ras1存在的影响 或E1a基因，或两者兼而有之，它们将显示正常对血清的反应 对纯化后的生长促进因子和抑制因子分别进行改变 排队。 我们还将继续研究推出这些措施的效果。 将克隆的基因显微注射入正常的REF52细胞。 使用显微注射细胞的2D凝胶分析的初步研究证实 T24 H-ras 1基因表达对检测稳态水平的影响 细胞ras基因。(S)

An effort has been made to develop an in vitro system for the study of multistep oncogenic transformation of cells. A number of cell lines have been recently isolated by DNA-mediated transfection of the rat cell REF52 with cloned viral and cellular oncogenes. Lines expressing the SV40 T/t antigens or the activated ras oncogene (T24 H-ras 1) + adenovirus-5 early region 1A gene (Ad5E1A) are fully transformed, whereas lines expressing only the T24 H-ras 1 gene or the Ad5E1A gene are not. Numerous clones from each line have been characterized with respect to morphology, growth rate, serum dependence, oncogenicity, and expression of the p21 ras protein products as seen on 2D-gels. Initial results indicate that expression of the normal cellular p21 genes is altered as a result of expression of the T24 H-ras 1 gene, and that cells containing higher amounts of the T24 H-ras 1 gene products are less tumorigenic than cells containing lower amounts of the gene products. This research will further characterize these cell lines by computer-analyzed two-dimensional gel electrophoresis of cell cycle specific populations of cells, sorted as a function of DNA content. By studying cells at representative stages of the cell cycle we will determine: (1) detailed patterns of protein synthesis and turnover throughout the cell cycle; (2) the responses of each line to stimulation of growth factors after serum-deprivation; and (3) the changes that occur in each line during in vivo tumorigenesis. These experiments will show how basal levels of gene expression are affected by the presence of T24 H-ras 1 or E1A genes, or both, and they will show how the normal responses to serum and to purified growth stimulatory and inhibitory factors are altered each line. We will also continue studying the effect of introducing each of these genes into normal REF52 cells by microinjection of the cloned gene. Initial studies using 2D gel analysis of microinjected cells confirm the effect of T24 H-ras 1 gene expression on the detected steady-state levels of the cellular ras genes. (S)