ROLE OF METASCHEMATIN PROTEINS IN NEOPLASIA

元组织素蛋白在肿瘤形成中的作用

• 批准号: 3193491
• 负责人: JOHN C LEAVITT
• 金额: $ 6.94万
• 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 1992-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3193491
• 关键词: actins; 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.

在我们试图研究癌症发生机制的过程中，我们 正在使用一种人类成纤维细胞系统，该系统是由一种 将化学致癌物导入4株克隆细胞系，其中3株为 永生但不会致癌，而且是完全 致癌的。注意到了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， 原肌球蛋白异构体调节及对纤溶酶原激活剂的诱导作用 促化生因子的表达。我们之前已经证明过， 突变的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 在二倍体人类中引起有限的肿瘤相关变化 成纤维细胞。在另一项单独但平行的调查中，我们有 人类编码68号L的纤溶酶原蛋白基因的克隆与鉴定 肿瘤人成纤维细胞中诱导的KD磷酸蛋白。这个 纤溶酶原基因已被置于β-肌动蛋白的调控之下。 启动子，并正在将其导入人和啮齿动物 以确定其在细胞转化中的作用。在这 我们计划分离人类的新陈代谢基因， 一种27-28kd的多肽，上调25-30倍 伴随着人成纤维细胞的肿瘤转化。我们 将确定这种蛋白质的氨基酸序列 ITS基因和基因的核苷酸序列。我们会寻找 从转化细胞中分离的基因的结构变化 试图解释其在表达上的提升机制 转化后的细胞。我们将研究启动子和机制。 通过对启动子周围的核苷酸测序来调控它，以及 通过连接报告基因，如大肠杆菌β-半乳糖苷酶或 CAT到启动子及随后调控序列的定位 通过缺失分析。我们将寻找激活的因素 凝胶移位技术在转化成纤维细胞中合成化疗素的研究 实验。我们将在控制下表达转化趋化蛋白基因 并检测其表达的影响 关于细胞生长、细胞结构和致瘤性的研究。最后，我们 将研究异位趋化蛋白、纤溶酶原激活剂和 突变型β-肌动蛋白在肿瘤诱导中的表达 二倍体人成纤维细胞的表型。