ROLES OF MICROFILAMENT PROTEINS IN NEOPLASIA/METASTASIS

微丝蛋白在肿瘤/转移中的作用

• 批准号: 3172554
• 负责人: JOHN C LEAVITT
• 金额: $ 17.34万
• 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/3172554
• 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.

该计划的目标一直是识别、隔离、表征和 研究人类基因表达的影响，从根本上说， 管理人类细胞致瘤表型的表现。为 这项工作强调了比较人体细胞系统的使用 由二倍体/正常成纤维细胞组成，永生化/不致瘤 成纤维细胞和永生化/致瘤成纤维细胞。这个比较性 方法已经证明，两者之间存在显著差异 正常和转化的成纤维细胞株在有限的丰富集合中 已被鉴定为微丝结构蛋白的蛋白质- 肌动蛋白、原肌球蛋白和塑料蛋白。一个突变的β-肌动蛋白基因是 发现、克隆并鉴定了当在转基因细胞中表达时 细胞处于高水平，导致肿瘤转化为 永生化/不致瘤的人成纤维细胞。发生了重大变化 原肌球蛋白的表达也显示与肿瘤的发生有关。 人成纤维细胞的转化及对转化敏感的TM3 对异构体进行了克隆和鉴定。最后，发现了纤溶酶原蛋白， 被鉴定为钙结合磷蛋白家族，其功能是 在细胞边缘捆绑肌动蛋白细丝。产生这种现象的基因 蛋白质家族已经被部分克隆和鉴定，而且它已经 已确定L-纤溶酶原异构体在 造血细胞，并在不同种类的人类肿瘤中诱导- 派生的细胞。该计划的结果表明， 正常的微丝功能可能是一个统一的和根本的变化 所有细胞形式的人类癌症，包括转移性癌症。这条线索 对这样一种假设，即动态调节的变化 细胞骨架微丝系统是肿瘤发生发展的基础 人类细胞的表型。为了扩展这项工作，研究将是 继续：(1)完成了对人类L和T-的刻画。 纤溶酶基因及其调控特性的比较研究 确定L-纤溶酶基因激活机制的结构域 人肿瘤；(2)构建造血细胞特异性表达 利用L-纤溶酶启动子将L-纤溶酶启动子发展为 人细胞肿瘤转化的报告基因；(4)定位 L和T-纤溶酶基因的染色体定位；(5)检测其作用 在正常细胞中诱导L-纤溶酶原合成；(6)测定 纤溶酶原激活剂磷酸化的机制和细胞周期特异性 与肿瘤的相关性；(7)检查单独的和协同的影响 L-纤溶酶、原肌球蛋白及突变型和野生型肌动蛋白的表达 肿瘤的发生和转移；(8)扩大对正常人体组织的调查 和肿瘤对纤溶酶亚型的表达；(9)发展L-纤溶酶 对新生人类肿瘤的抗体检测；(10)表征 胚胎发育过程中的纤溶酶同工酶；以及(11)确定 L-纤溶酶作为多能造血干细胞标志物的价值 细胞。