ROLES OF MICROFILAMENT PROTEINS IN NEOPLASIA/METASTASIS

微丝蛋白在肿瘤/转移中的作用

• 批准号: 3172545
• 负责人: JOHN C LEAVITT
• 金额: $ 17.46万
• 依托单位: PALO ALTO MEDICAL FOUNDATION RES INST
• 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 1994-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3172545
• 关键词: actins; athymic mouse; cytoskeletal proteins; fibroblasts; gene expression; gene induction /repression; genetic manipulation; genetic markers; genetic promoter element; genetic regulatory element; genetic transcription; hematopoietic stem cells; human tissue; laboratory rabbit; metastasis; microfilaments; molecular cloning; neoplasm /cancer genetics; neoplastic transformation; northern blottings; nucleic acid probes; protein structure; restriction mapping; tissue /cell culture; transfection; transposon /insertion element

The goal of this program has been to identify, isolate, characterize, and study the effects of expression of human genes that, in a fundamental way, govern the manifestation of the tumorigenic phenotype of human cells. For this work the use of a comparative human cell system is emphasized consisting of diploid/normal fibroblasts, and immortalized/nontumorigenic fibroblasts and immortalized/tumorigenic fibroblasts. This comparative approach has demonstrated that there are significant differences between the normal and transformed fibroblast strains in a limited set of abundant proteins which have been identified as microfilament structural proteins - actins, tropomyosins, and plastins. A mutant beta-actin gene was discovered, cloned, and characterized which, when expressed in transfected cells at high levels, led to tumorigenic conversion of immortalized/nontumorigenic human fibroblast cells. Significant changes in tropomyosin expression were also shown to occur accompanying tumorigenic conversion of human fibroblasts, and the transformation-sensitive Tm3 isoform was cloned and characterized. Finally, plastin was discovered and identified as a family of calcium-binding phosphoproteins whose function is to bundle actin filaments at the edge of the cell. The genes for this family of proteins have been partially cloned and characterized, and it has been established that the L-plastin isoform is specifically expressed in hematopoietic cells and induced in a divergent variety of human tumor- derived cells. The results of this program indicate that disruption of normal microfilament function may be a unifying and fundamental change for all cellular forms of human cancer including metastatic cancer. This leads to the hypothesis that changes in the dynamic regulation of the cytoskeletal microfilament system underlie development of the cancerous phenotype of the human cell. To extend this work research will be continued to: (1) complete the characterization of the human L- and T- plastin genes and the comparative characterization of their regulatory domains to determine the mechanism of activation of the L-plastin gene in human neoplasia; (2) construct a hematopoietic cell-specific expression vector using the L-plastin promoter; (3) develop the L-plastin promoter as a reporter gene for human cell neoplastic transformation; (4) map the chromosomal location of the L- and T-plastin genes; (5) examine the effects of induction of L-plastin synthesis in normal cells; (6) determine the mechanism and cell cycle specificity of plastin phosphorylation and its relevance to neoplasia; (7) examine the separate and synergistic effects of L-plastin, tropomyosin, and mutant and wildtype actin expression on tumorigenesis and metastasis; (8) extend the survey of normal human tissues and tumors for expression of plastin isoforms; (9) develop an L-plastin antibody test for nascent human tumors; (10) characterize the expression of plastin isoforms during embryonic development; and (11) determine the usefulness of L-plastin as a marker of pluripotent hematopoietic stem cells.

该计划的目标是识别、隔离、表征和 研究人类基因表达的影响，从根本上来说， 控制人类细胞致瘤表型的表现。 为了 这项工作强调了比较人体细胞系统的使用 由二倍体/正常成纤维细胞和永生化/非致瘤性细胞组成 成纤维细胞和永生化/致瘤成纤维细胞。 这个比较 方法表明，两者之间存在显着差异 一组有限的丰富的正常和转化的成纤维细胞菌株 已被鉴定为微丝结构蛋白的蛋白质 - 肌动蛋白、原肌球蛋白和塑体蛋白。 突变的β-肌动蛋白基因是 发现、克隆和表征，当在转染中表达时 高水平的细胞，导致致瘤性转化 永生化/非致瘤性人成纤维细胞。 重大变化 原肌球蛋白表达也被证明伴随致瘤性发生 人成纤维细胞的转化，以及转化敏感的 Tm3 克隆并表征了同工型。 最终，发现了塑性蛋白并 被鉴定为钙结合磷蛋白家族，其功能是 将肌动蛋白丝束在细胞边缘。 造成这一现象的基因 蛋白质家族已被部分克隆和表征，并且已 已确定 L-plastin 同种型特异性表达于 造血细胞并在不同种类的人类肿瘤中诱导 衍生细胞。 该计划的结果表明， 正常的微丝功能可能是一个统一的根本性变化 人类癌症的所有细胞形式，包括转移性癌症。 这导致 假设动态调节发生变化 细胞骨架微丝系统是癌变发展的基础 人类细胞的表型。 为了扩展这项工作，研究将 继续：（1）完成人类L-和T-的表征 塑性蛋白基因及其调控的比较特征 结构域来确定 L-plastin 基因的激活机制 人类肿瘤； (2)构建造血细胞特异性表达 使用 L-plastin 启动子的载体； (3) 将L-plastin启动子开发为 人类细胞肿瘤转化的报告基因； (4) 绘制地图 L-和T-塑性蛋白基因的染色体位置； (5)检验效果 正常细胞中 L-塑性蛋白合成的诱导； (6) 确定 质体蛋白磷酸化及其机制和细胞周期特异性 与肿瘤的相关性； (7) 检验单独效应和协同效应 L-塑蛋白、原肌球蛋白以及突变型和野生型肌动蛋白的表达 肿瘤发生和转移； (8)扩大对正常人体组织的调查 以及表达塑性蛋白亚型的肿瘤； (9) 开发L-plastin 新生人类肿瘤的抗体测试； (10) 表征表达式 胚胎发育过程中的塑性蛋白亚型； (11) 确定 L-塑性蛋白作为多能造血干标记物的用途 细胞。