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: 6239910
• 负责人: AGNES A DAY
• 金额: $ 4.83万
• 依托单位: HOWARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6239910
• 关键词: DNA footprinting; binding proteins; biomarker; cell transformation; decorin; extracellular matrix proteins; gel mobility shift assay; gene expression; genetic transcription; metastasis; neoplastic cell; neoplastic transformation; northern blottings; nucleic acid sequence; polymerase chain reaction; protein biosynthesis; protein structure; restriction fragment length polymorphism; site directed mutagenesis; southern blotting; western blottings

Transformation of normal cells to malignant tumors is a multi-step process which results from genetic alterations and environmental insults. Most malignant cells possess the ability to metastasize via the detachment of cells form the primary tumor, intravasation, extravasation and establishment of secondary foci in distal organs. Extracellular matrices (ECM) and basement membranes are composed of a variety of proteins that surround tissues and organs and present impermeable barriers to active cell mobility. The metastatic process involves interactions between wandering malignant cells and macromolecule of basement membranes and extracellular matrices, whereby the cells attach to the matrix and gain egress via the increased synthesis of proteolytic enzymes. Various investigations have shown altered regulation of the synthesis of ECM proteins in various malignant cells. The hypothesis of this proposal is that altered transcriptional regulation of ECM protein synthesis is directly or indirectly correlated with the transformed state, which in turn, enhances metastasis. In this proposal, we propose a systematic approach to exploring this hypothesis. A battery of paired cell lines and tumor specimens (normal cells, polyps, primary tumors and metastatic samples) from various organs will be analyzed by Northern, Southern and slot blot techniques for the increased/decreased transcription of genes encoding osteonectin, decorin, type I collagen, fibronectin, and various oncogenes. The housekeeping protein, B-actin, will serve as a control in these experiments. Nuclear extractions, DNA footprinting, gel shift mobility and site directed mutagenesis assays will be performed to determine the molecular mechanism(s) of the altered transcription. Data from these studies may provide a mechanism by which to determine the probability of metastasis at a specific stage of tumor growth, and provide molecular markers to determine if micro-metastasis has already occurred.

正常细胞转化为恶性肿瘤是多步骤 遗传改变和环境侮辱引起的过程。 大多数恶性细胞具有通过 细胞脱离形成原发性肿瘤，静脉内外渗出 并在远端器官中建立二次焦点。 细胞外 矩阵（ECM）和基底膜由多种 围绕组织和器官并表现出不可渗透的蛋白质 主动细胞迁移率的障碍。 转移过程涉及 流浪的恶性细胞与大分子之间的相互作用 地下膜和细胞外矩阵，细胞附着 到矩阵并通过增加的合成而获得出口 蛋白水解酶。 各种调查显示发生了变化 调节各种恶性细胞中ECM蛋白的合成。 该提议的假设是转录变化 ECM蛋白合成的调节是直接或间接相关的 随着转化状态又增强了转移。 在这个 提案，我们提出了一种系统的方法来探索这一假设。 一组配对的细胞系和肿瘤标本（正常细胞，息肉， 来自各种器官的原发性肿瘤和转移样品） 由北部，南部和老虎机印迹技术分析 编码骨蛋白的基因的转录增加/减少 I型胶原蛋白，纤连蛋白和各种癌基因。 管家 蛋白质B-肌动蛋白将在这些实验中作为对照。 核 萃取，DNA足迹，凝胶移动性和定向位置 将进行诱变测定以确定分子 改变转录的机理。 这些研究的数据可能 提供一种确定转移概率的机制 在肿瘤生长的特定阶段，并提供分子标记 确定是否已经发生了微米。