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ROLES OF MICROFILAMENT PROTEINS IN NEOPLASIA/METASTASIS

微丝蛋白在肿瘤/转移中的作用

基本信息

批准号：
3172555
负责人：
JOHN C LEAVITT
金额：
$ 18.49万
依托单位：
PALO ALTO MEDICAL FOUNDATION RES INST
依托单位国家：
美国
项目类别：
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

项目摘要

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、分离、表征和研究人类基因表达的影响，从根本上说，控制人细胞的致瘤表型的表现。为这项工作强调了比较人类细胞系统的使用由二倍体/正常成纤维细胞和永生化/非致瘤性成纤维细胞和永生化/致瘤性成纤维细胞。本次比较方法表明，正常和转化的成纤维细胞株在有限的一组丰富的已被鉴定为微丝结构蛋白的蛋白质- 肌动蛋白、原肌球蛋白和质体蛋白。一个突变的β-肌动蛋白基因，发现，克隆和表征，当在转染的高水平的细胞，导致肿瘤转化，永生化/非致瘤性人成纤维细胞。显著变化原肌球蛋白的表达也显示出伴随着肿瘤的发生，人成纤维细胞的转化，以及转化敏感性Tm 3 克隆并表征同种型。最后，发现了Plastin，被鉴定为钙结合磷蛋白家族，其功能是将肌动蛋白纤维束在细胞边缘。这方面的基因蛋白质家族已部分克隆和表征，已经确定L-塑性蛋白同种型特异性表达于造血细胞和诱导在不同种类的人类肿瘤- 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。这导致这一假设的变化，在动态调节的细胞骨架微丝系统是肿瘤发生的基础人类细胞的表型。为了扩大这项工作的研究将是继续：（1）完成人L-和T-的表征 plastin基因及其调控的比较特性结构域，以确定L-plastin基因的激活机制，人肿瘤;（2）构建造血细胞特异性表达（3）开发L-plastin启动子，人细胞肿瘤转化的报告基因;（4）定位染色体定位的L-和T-plastin基因;（5）检查的影响，诱导正常细胞中L-纤溶酶原蛋白合成;（6）确定纤维蛋白原磷酸化的机制和细胞周期特异性与肿瘤形成的相关性;（7）检查以下因素的单独和协同作用： L-plastin，tropomyosin，和突变型和野生型肌动蛋白表达，肿瘤的发生和转移;（8）扩大对正常人体组织的调查和肿瘤的表达;（9）开发一种L-plastin 新生人类肿瘤的抗体测试;（10）表征胚胎发育过程中的质体异构体;和（11）确定 L-plastin作为多能造血干细胞标志物的用途细胞