REGULATION OF ECM PROTEIN BIOSYNTHESIS IN NORMAL, MALIGNANT AND METASTATIC CELLS

正常细胞、恶性细胞和转移细胞中 ECM 蛋白质生物合成的调节

• 批准号: 6316634
• 负责人: AGNES A DAY
• 金额: $ 8.82万
• 依托单位: HOWARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6316634
• 关键词: binding proteins; biomarker; decorin; enzyme activity; extracellular matrix proteins; gene expression; genetic regulation; genetic transcription; human tissue; membrane structure; metalloendopeptidases; metastasis; neoplastic cell; neoplastic process; northern blottings; nucleic acid sequence; polymerase chain reaction; protein biosynthesis; protein degradation; protein structure; restriction fragment length polymorphism; single strand conformation polymorphism

It is known that metastasis results from increased metalloprotease production/activity by cancerous cells resulting in the breaching of the extracellular matrix (ECM) and basement membranes, which serve as barriers to ingress and egress of metastatic cells through the circulatory and lymphatic systems. Few studies have been done to ascertain the regulatory and synthetic status of the connective tissue proteins (CTP) during tumor progression. The goal of this research is to determine what other regulatory events occur in cancerous cells which further facilitate the process of metastasis. Specifically, this research will explore the possibility that down-regulation of ECM protein synthesis is an integral component in the loss of structural integrity of the basement membrane and ECM, thus facilitating metastasis. Our previous studies demonstrated altered expression of decorin, fibronectin, osteonectin and type I collagen when transcriptional profiles were compared among breast colon and skin (normal, benign, primary cancerous and metastatic) cell lines and clinical samples. Additionally, restriction fragment length polymorphism (RFLP) analyses have revealed mutationally altered genes (decorin, fibronectin and osteonectin) in two metastatic cell lines (melanoma and breast). The specific aims of this proposal are: (1) perform a systematic analysis of normal, benign, primary cancerous and metastatic cell lines and clinical samples for further correlations of decreased ECM synthesis and increased metastatic potential, (2) develop a metastatic profile of decreased ECM production and increased metalloprotease activity in clinical derived tissues, (3) determine the structural and functional domains of decorin and osteonectin which demonstrate RFLP polymorphisms via single strand conformational polymorphism (SSCP) analysis, RT-PCR cloning and sequence analysis and (4) determine regulatory regions of selected ECM genes and their binding proteins which are involved in the observed altered regulation. Completion of the above specific aims should provide a means of determining the metastatic capability of a primary tumor in situ.

众所周知，金属蛋白酶增加导致转移 癌细胞的生产/活性导致破坏 细胞外基质（ECM）和基底膜，它们充当障碍物 通过循环系统和 淋巴系统。很少有研究来确定调节性 肿瘤期间结缔组织蛋白（CTP）的合成状态 进展。这项研究的目的是确定其他什么 调节事件发生在癌细胞中，进一步促进 转移过程。具体来说，这项研究将探索 ECM蛋白质合成下调的可能性是积分 损失基底膜结构完整性的组成部分 ECM，从而促进转移。我们以前的研究表明 Decorin，纤连蛋白，骨蛋白丝蛋白和I型的表达改变了 比较乳腺结肠的转录曲线时的胶原蛋白 和皮肤（正常，良性，原发性癌和转移性）细胞系，以及 临床样品。另外，限制片段长度多态性 （RFLP）分析显示突变改变的基因（decorin，，，， 两种转移细胞系（黑色素瘤和 胸部）。该提案的具体目的是：（1）执行系统 分析正常，良性，原发性癌细胞和转移性细胞系 和临床样品，以进一步的ECM合成降低 并提高转移性​​电位，（2）发展 降低ECM生产并增加了金属蛋白酶活性 临床衍生组织，（3）确定结构和功能 Decorin和ostolettin的域，这些领域表现出RFLP多态性 通过单链构象多态性（SSCP）分析，RT-PCR 克隆和序列分析以及（4）确定调节区域 选定的ECM基因及其结合蛋白参与 观察到的调节改变。上述特定目标的完成应 提供确定主要能力的转移能力的方法 原位肿瘤。