IDENTIFICATION OF CELL CYCLE SPECIFIC PROTEINS AFFECTED

受影响的细胞周期特异性蛋白质的鉴定

• 批准号: 3180572
• 负责人: B FRANZA
• 金额: $ 12.87万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-30 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3180572
• 关键词: cell differentiation; cell growth regulation; complementary DNA; cytogenetics; flow cytometry; gel electrophoresis; gene expression; gene mutation; genetic manipulation; genetic transcription; growth factor; growth inhibitors; growth media; immunoprecipitation; microinjections; molecular cloning; morphology; mouse sarcoma virus; neoplastic growth; neoplastic transformation; oncogenes; proteolysis; radiotracer; simian virus 40; tissue /cell culture; viral carcinogenesis

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)

已经做出努力来开发用于以下研究的体外系统： 细胞的多步致癌转化。 许多细胞系具有 最近通过DNA介导转染大鼠细胞REF 52分离到 病毒和细胞癌基因的克隆。 表达SV 40 T/t的细胞系 抗原或激活的ras癌基因（T24 H-ras 1）+腺病毒-5早期 区域1A基因（Ad 5E 1A）被完全转化，而表达 只有T24 H-ras 1基因或Ad 5E 1A基因不表达。 来自每个品系的许多克隆已经在以下方面表征： 形态学、生长速率、血清依赖性、致癌性和 p21 ras蛋白产物，如在2D-凝胶上所见。 初步结果表明 正常细胞p21基因的表达被改变， T24 H-ras 1基因的表达，以及含有较高表达的细胞， 大量的T24 H-ras 1基因产物的致瘤性低于细胞 基因产物含量较低。 这项研究将进一步表征这些细胞系， 细胞周期计算机分析双向凝胶电泳 特定的细胞群，根据DNA含量进行分类。通过 研究细胞周期的代表性阶段，我们将 确定：（1）蛋白质合成和周转的详细模式 （2）各细胞系对刺激的反应; 血清剥夺后的生长因子;和（3）发生的变化， 在体内肿瘤发生过程中的每一个线。 这些实验将展示 基因表达的基础水平受T24 H-ras 1的存在的影响 或E1 A基因，或两者兼而有之，他们将显示如何正常反应， 以及纯化的生长刺激和抑制因子各自改变 线 我们亦会继续研究每项措施的效果 通过显微注射克隆的基因将基因导入正常REF 52细胞。 使用显微注射细胞的2D凝胶分析的初步研究证实了 T24 H-ras 1基因表达对检测到的稳态水平的影响 细胞ras基因。 （S）