ROLE OF METASCHEMATIN PROTEINS IN NEOPLASIA

元组织素蛋白在肿瘤形成中的作用

• 批准号: 3193492
• 负责人: JOHN C LEAVITT
• 金额: $ 6.52万
• 依托单位: CALIFORNIA INSTITUTE FOR MEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-05-10 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3193492
• 关键词: actins; antisense nucleic acid; athymic mouse; chemical carcinogenesis; clone cells; complementary DNA; fibroblasts; gel electrophoresis; gene deletion mutation; gene expression; genetic promoter element; human tissue; molecular cloning; neoplasm /cancer genetics; oncoproteins; phosphoproteins; protein sequence; reporter genes; transposon /insertion element

In our attempt to investigate the mechanism of carcinogenesis, we are using a human fibroblast system which was transformed by a chemical carcinogen into 4 clonal cell lines, 3 of which were immortalized but not tumorigenic, and one which was fully tumorigenic. Changes in expression of several proteins were noted in the cell lines such as a mutation in the beta-actin gene, tropomyosin isoform modulation, and the induction of plastin and metaschematin expression. We have previously shown that high expression of the mutant beta-actin gene promotes stable tumorigenic conversion of immortalized HuT-12 fibroblasts, and elicits limited neoplasia-related changes in diploid human fibroblasts. In a separate but parallel investigation, we have cloned and characterized the human gene encoding l-plastin, a 68 kd phosphoprotein induced In neoplastic human fibroblasts. The plastin gene has been put under the control of the beta-actin promoter, and is being transfected into human and rodent fibroblasts to ascertain its role in cell transformation. In this research we propose to isolate the human gene for metaschematin, a 27-28 kd polypeptide that is up-regulated 25 to 30 fold accompanying neoplastic transformation of human fibroblasts. We will determine the amino acid sequence for this protein from the nucleotide sequence of its cDNA and gene. We will look for structural changes in the gene isolated from the transformed cell to try to explain the mechanism of its elevation in expression in the transformed cell. We will examine the promoter and mechanism of its regulation by nucleotide sequencing around the promoter, and by linking a reporter gene such as E.coli beta-galactosidase or CAT to the promoter and subsequent mapping of regulatory sequences by deletion analysis. We will look for factors which activate metaschematin synthesis in transformed fibroblasts using gel shift experiments. We will express the metaschematin gene under control of the beta-actin promoter and examine the effect of its expression on cell growth, cytoarchitecture, and tumorigenicity. Finally, we will study the synergistic effect of metaschematin, plastin, and mutant beta-actin expression on the induction of the neoplastic phenotype in diploid human fibroblasts.

在我们尝试研究致癌机制时，我们 正在使用人类成纤维细胞系统，该系统是由 化学致癌物进入4个克隆细胞系，其中3个是 永生化但不致瘤，并且是完全 致瘤性。 注意到几种蛋白质表达的变化 在细胞系中，例如β-肌动蛋白基因的突变， 原肌球蛋白亚型调节，以及塑性蛋白和 元架构蛋白表达。 我们之前已经表明，高 突变β-肌动蛋白基因的表达促进稳定 永生化 HuT-12 成纤维细胞的致瘤转化，以及 在二倍体人类中引起有限的肿瘤相关变化 成纤维细胞。 在一项单独但平行的调查中，我们有 第 68 章 kd 磷蛋白在肿瘤性人成纤维细胞中诱导。 这 塑蛋白基因已被置于β-肌动蛋白的控制之下 启动子，并正在转染到人类和啮齿动物体内 成纤维细胞以确定其在细胞转化中的作用。 在这个 我们建议在研究中分离出metaschematin的人类基因， 上调 25 至 30 倍的 27-28 kd 多肽 伴随人成纤维细胞的肿瘤转化。 我们 将确定该蛋白质的氨基酸序列 其cDNA和基因的核苷酸序列。 我们会寻找 从转化细胞中分离出的基因的结构变化 试图解释其表达升高的机制 转化的细胞。 我们将研究启动子和机制 通过启动子周围的核苷酸测序对其进行调节，以及 通过连接报告基因，例如大肠杆菌β-半乳糖苷酶或 CAT 至启动子以及随后的调控序列图谱 通过删除分析。 我们将寻找激活因素 使用凝胶位移在转化的成纤维细胞中合成metschematin 实验。 我们将在控制下表达metaschematin基因 β-肌动蛋白启动子并检查其表达的效果 细胞生长、细胞结构和致瘤性。 最后，我们 将研究metaschematin、plastin 和 突变型β-肌动蛋白表达对肿瘤诱导的影响 二倍体人成纤维细胞的表型。