ROLES OF MICROFILAMENT PROTEINS AND PLASTIN IN NEOPLASIA

微丝蛋白和塑蛋白在肿瘤中的作用

• 批准号: 3172553
• 负责人: JOHN C LEAVITT
• 金额: $ 17.61万
• 依托单位: CALIFORNIA INSTITUTE FOR MEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3172553
• 关键词: RNA splicing; Retroviridae; Retroviridae disease; actins; athymic mouse; bacteriophage lambda; cytoskeleton; fibroblasts; gene expression; genetic library; genetic manipulation; genetic recombination; genetic transcription; genetic translation; hamsters; human tissue; microinjections; molecular cloning; mutant; neoplasm /cancer genetics; neoplastic transformation; nucleic acid sequence; plasmids; protein sequence; structural genes; tissue /cell culture; transfection; transforming virus

We are examining the relationship of beta-actin gene mutations to abnormal cytoarchitecture, elevated growth potential, and neoplastic transformation of human fibroblasts. This is being accomplished in the following steps: (1)\cloned genes for mutant and wildtype beta-actins have been isolated from gene libraries of HUT-14 and HUT-14T cells; these actin genes have been selected by recombination of the actin gene clone in lambda DNA with a specially constructed plasmid PiVX containing a sub-cloned portion of beta-actin cDNA homologous to the 3' untranslated region of the gene; (2)\mutant and wildtype genes were distinguished by DNA sequencing and restriction site polymorphisms at codons 36, 83, and 244; we have established that each of these mutations occurred in separate sequential steps during derivation of the highly tumorigenic cell line HUT-14T from a diploid human fibroblast culture; (3) each of these mutant actin genes and additional mutant actin genes have been inserted into a PSV-Z neoplasmid and incorporated into normal human and rodent fibroblasts via gene transfer; (4) after gene transfer these exogenous actin genes are abundantly expressed such that often the exogenous mutant actin protein is the most abundant cellular protein; (5) upon incorporation and expression of mutant beta-actin genes in diploid human fibroblasts profound morphologic transformation occurs; and (6)\cultures treated with mutant actin genes are being assessed for alterations in cytoarchitecture and transformation by monitoring mutant beta-actin synthesis, escape from senescence, focus formation, anchorage independence, aneuploidy, protein profiles in two-dimensional gels, protease expression, and tumorigenesis in athymic mice. This approach will ultimately test the involvement of actin mutations in abnormal cytoskeletal structure and function and expose their relationship to neoplastic transformation and tumorigenicity. In the course of this study we discovered that normal rodent fibroblasts synthesize an abundant alpha-actin isoform not found in normal human fibroblasts. This alpha-actin is dramatically downregulated following transformation of rodent cells in all instances tested thus far. We are attempting to clone a cDNA for this rodent cell transformation-sensitive actin so that we can identify the actin isoform and examine its role in rodent cell transformation. (L)

我们正在研究β-肌动蛋白基因突变与异常的关系 细胞结构、生长潜力升高和肿瘤转化 人类成纤维细胞。 这是通过以下步骤完成的： (1)\已分离出突变型和野生型β-肌动蛋白的克隆基因 来自 HUT-14 和 HUT-14T 细胞的基因库；这些肌动蛋白基因有 通过将 lambda DNA 中的肌动蛋白基因克隆与 专门构建的质粒 PiVX 含有亚克隆部分 β-肌动蛋白 cDNA 与该基因的 3' 非翻译区同源； (2)通过DNA测序区分突变型和野生型基因 密码子 36、83 和 244 处的限制性位点多态性；我们有 确定这些突变中的每一个都发生在单独的顺序中 从高致瘤性细胞系 HUT-14T 衍生的步骤 二倍体人成纤维细胞培养物； (3) 这些突变肌动蛋白基因中的每一个和 额外的突变肌动蛋白基因已被插入 PSV-Z 肿瘤中 并通过基因整合到正常人类和啮齿动物成纤维细胞中 转移; (4) 基因转移后，这些外源肌动蛋白基因被 大量表达，使得外源突变肌动蛋白经常被 最丰富的细胞蛋白质； (5) 成立及表达时 二倍体人成纤维细胞中突变β-肌动蛋白基因的影响意义深远 发生形态转变； 和 (6)\用突变体处理的培养物 正在评估肌动蛋白基因的细胞结构变化和 通过监测突变β-肌动蛋白合成进行转化，逃避 衰老，病灶形成，锚定独立性，非整倍性，蛋白质 二维凝胶中的概况、蛋白酶表达和肿瘤发生 无胸腺小鼠。 这种方法最终将测试肌动蛋白的参与 异常细胞骨架结构和功能的突变并暴露其 与肿瘤转化和致瘤性的关系。在课程中 在这项研究中，我们发现正常啮齿动物成纤维细胞合成 正常人成纤维细胞中未发现丰富的α-肌动蛋白亚型。 这 α-肌动蛋白在转化后急剧下调 迄今为止测试的所有情况下的啮齿动物细胞。 我们正在尝试克隆 这种啮齿动物细胞转化敏感肌动蛋白的 cDNA，这样我们就可以 鉴定肌动蛋白亚型并检查其在啮齿动物细胞中的作用 转变。 (大)