PROTEASES IN GROWTH CONTROL AND MALIGNANT TRANSFORMATION

蛋白酶在生长控制和恶性转化中的作用

• 批准号: 3164491
• 负责人: JAMES P QUIGLEY
• 金额: $ 12.43万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-08-01 至 1987-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3164491
• 关键词: Rous sarcoma virus; antibody formation; cell growth regulation; cell membrane; gel electrophoresis; glucose transport; hybrid cells; hybridomas; neoplasm /cancer invasiveness; neoplastic transformation; peptidases; plasminogen activator; protease inhibitor; radiotracer; temperature sensitive mutant; tissue /cell culture; tumor promoters

In the past year we have been successful in obtaining a monoclonal antibody that recognizes the plasminogen activator (PA) from Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF). The antibody has been selected for its ability not only to bind to PA but also to inhibit its catalytic activity. It is a highly specific antibody in tht it does not bind to or inhibit human urokinase or the PA activity present in cultures of human tumor cells, but does inhbit (at l-10 mu g/ml) the PA activity released by cultures of RSVCEF. The anti-catalytic activity of the antibody allows us now to directly test the role of PA in the behavior of cultures of RSVCEF. We have recently developed a biochemical assay for the degradation and invasive behavior of RSVCEF. The assay involves the degradation of the extracellular matrix (ECM) produced by normal CEF when cultures of RSVCEF are placed on radiolabeled ECM. The assay system allows us to test for the involvement of specific proteases and also for the requirement of cell-ECM contact. We plan to test for the direct role of PA in ECM degradation by conducting degradation studies in the presence or absence of our antibody. We plan to isolate a monoclonal antibody that binds to PA but does not inhibit its activity and this will serve as a useful control. In addition to using the antibody to test for PA's role in cellular behavior, we have now linked the antibody to solid support and are using it as an immunoaffinity ligand for purifying PA. In tandem with standard chromatography procedures the immunoaffinity column has yielded a homogeneous preparation of PA. This methodology will provide us with sufficient quantities of PA (0.1 to 1.0 mg), heretofore not available, to characterize the enzyme biochemically and use it to search for other relevant substrates. Our preliminary studies indicate that PA (in the absence of plasminogen) can cleave a homogeneous preparation of chick cellular fibronectin. These studies will be important in defining the exact role of the enzyme in certain aspects of cellular behavior including growth arrest and cell migration. We have recently established a tumor cell metastasis assay using the chick embryo. The system involves implanting human tumor cells on the chorioallantoic membrane (CAM) of the embryo and subsequently detecting human cells in peripheral organs of the embryo. There is a large body of indirect evidence suggesting a role for proteolytic enzymes in tumor cell invasion and metastasis implicating both tumor cell enzymes and host enzymes. Having procured an anti-catalytic antibody to human PA (urokinase) and now having an anti-catalytic antibody to chick PA, we will now be able to examine and dissect out the respective roles of tumor cell PA and host cell PA in the chick embryo metastasis system. (A)

去年我们成功获得了单克隆抗体 识别劳斯肉瘤病毒的纤溶酶原激活剂 (PA) 转化鸡胚成纤维细胞（RSVCEF）。 该抗体已 选择它不仅能够与 PA 结合，而且能够抑制其 催化活性。 它是一种高度特异性的抗体，因为它不 结合或抑制人尿激酶或培养物中存在的 PA 活性 人类肿瘤细胞，但确实抑制（l-10μg/ml）PA活性 由 RSVCEF 文化发布。 的抗催化活性 抗体现在使我们能够直接测试 PA 在行为中的作用 RSVCEF 文化。 我们最近开发了一种用于降解和 RSVCEF 的侵入行为。 该测定涉及降解 RSVCEF 培养时正常 CEF 产生的细胞外基质 (ECM) 被放置在放射性标记的 ECM 上。 该测定系统使我们能够测试 涉及特定蛋白酶以及细胞 ECM 的要求 接触。 我们计划通过以下方法测试 PA 在 ECM 降解中的直接作用 在存在或不存在我们的抗体的情况下进行降解研究。 我们计划分离出一种与 PA 结合但不结合的单克隆抗体 抑制其活性，这将作为一种有用的控制。 除了使用抗体测试 PA 在细胞中的作用外， 行为，我们现在已将抗体连接到固体支持物上并正在使用它 作为纯化 PA 的免疫亲和配体。 与标准同步 免疫亲和柱的层析程序已产生 PA的均质制备。 该方法将为我们提供 迄今为止还没有足够数量的 PA（0.1 至 1.0 mg），以 对酶进行生化表征并用它来寻找其他酶 相关基材。 我们的初步研究表明，PA（在 缺乏纤溶酶原）可以裂解小鸡的均质制剂 细胞纤连蛋白。 这些研究对于定义 该酶在细胞行为某些方面的确切作用，包括 生长停滞和细胞迁移。 我们最近使用小鸡建立了肿瘤细胞转移测定 胚胎。 该系统涉及将人类肿瘤细胞植入 胚胎绒毛尿囊膜（CAM）并随后检测 胚胎周围器官中的人类细胞。 有一个很大的身体 间接证据表明蛋白水解酶在肿瘤细胞中的作用 涉及肿瘤细胞酶和宿主的侵袭和转移 酶。 获得人PA抗催化抗体 （尿激酶），现在有了针对小鸡 PA 的抗催化抗体，我们将 现在能够检查和剖析肿瘤细胞各自的作用 鸡胚转移系统中的PA和宿主细胞PA。 （一个）