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: 3172556
• 负责人: JOHN C LEAVITT
• 金额: $ 13.43万
• 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 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3172556
• 关键词: 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-肌动蛋白基因突变与异常的关系