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 细胞结构、生长潜能升高和肿瘤转化 人类成纤维细胞。 这项工作将通过以下步骤来完成： (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-14 T细胞的基因文库;这些肌动蛋白基因具有 通过将λ DNA中的肌动蛋白基因克隆与 特别构建的质粒PiVX，其含有 与该基因的3'非翻译区同源的β-肌动蛋白cDNA; (2)通过DNA测序区分突变基因和野生型基因， 限制性位点多态性在密码子36，83和244;我们有 确定了这些突变中的每一个都以不同的顺序发生， 在高度致瘤性细胞系HUT-14 T的衍生过程中， 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种， 另外的突变肌动蛋白基因被插入到PSV-Z新质粒中 并通过基因整合到正常的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 大量表达，使得通常外源突变肌动蛋白被 最丰富的细胞蛋白质;（5）在掺入和表达后 突变的β-肌动蛋白基因在二倍体人成纤维细胞中的意义深远 发生形态转化;和（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的改变， 通过监测突变体β-肌动蛋白的合成， 衰老，病灶形成，锚定独立性，非整倍体，蛋白质 二维凝胶图谱、蛋白酶表达和肿瘤发生 无胸腺小鼠 这种方法将最终测试肌动蛋白参与 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。过程中 在这项研究中，我们发现正常的啮齿类动物成纤维细胞合成一种 在正常人成纤维细胞中没有发现丰富的α-肌动蛋白同种型。 这 α-肌动蛋白是显着下调后转化为 啮齿动物细胞在所有情况下测试到目前为止。 我们试图克隆 这种啮齿动物细胞转化敏感肌动蛋白的cDNA， 鉴定肌动蛋白亚型并检查其在啮齿动物细胞中的作用 转型 （左）

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)