ROLES OF MICROFILAMENT PROTEINS AND PLASTIN IN NEOPLASIA

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、高生长潜能和肿瘤转化 人类成纤维细胞。这是通过以下步骤完成的： (1)克隆了突变型和野生型β-肌动蛋白基因 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 含有亚克隆部分的特制表达载体pIVX 与该基因3‘端非翻译区同源的β-肌动蛋白基因； (2)对突变型和野生型基因进行DNA测序和比较 密码子36、83和244的限制酶切位点多态；我们有 确定了这些突变中的每一个都是以不同的顺序发生的 高致瘤性细胞系HUT-14T诱导过程中的步骤 二倍体人成纤维细胞培养；(3)这些突变的肌动蛋白基因和 其他突变的肌动蛋白基因已插入到PSV-Z新质粒中 并通过基因导入正常人和啮齿动物成纤维细胞 转移；(4)基因转移后这些外源肌动蛋白基因 大量表达，因此通常外源突变肌动蛋白是 最丰富的细胞蛋白；(5)掺入和表达 二倍体人成纤维细胞中突变的β-肌动蛋白基因的研究 发生形态转化；(6)用突变体处理的培养物 肌动蛋白基因正在评估细胞结构和细胞结构的变化 通过监测突变的β-肌动蛋白合成进行转化，从 衰老，病灶形成，锚定独立性，非整倍体，蛋白质 二维凝胶中的分布、蛋白酶表达与肿瘤发生 无性系小鼠。这一方法最终将测试肌动蛋白的参与。 细胞骨架结构和功能异常的突变并揭示其 与肿瘤转化和致瘤性的关系。在课程中 在这项研究中，我们发现正常的啮齿动物成纤维细胞合成了一个 在正常的人成纤维细胞中没有发现丰富的α-肌动蛋白亚型。这 α-肌动蛋白在转化为 到目前为止测试过的所有实例中的啮齿动物细胞。我们正试图克隆 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研究其在啮齿动物细胞中的作用 转型。(L)

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)