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: 3164492
• 负责人: JAMES P QUIGLEY
• 金额: $ 12.86万
• 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 1987-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3164492
• 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)

在过去的一年里，我们成功地获得了一种单克隆抗体， 识别劳斯肉瘤病毒的纤溶酶原激活物（PA） 转化鸡胚成纤维细胞（RSVCEF）。 述抗体 选择它的能力，不仅结合PA，而且还抑制其 催化活性 它是一种高度特异性的抗体， 结合或抑制培养物中存在的人尿激酶或PA活性 但在1 -10 μ g/ml浓度下， 由RSVCEF的培养物释放。 的抗催化活性。 抗体使我们现在可以直接测试PA在行为中的作用。 RSVCEF的培养物。 我们最近开发了一种降解的生化测定法， RSVCEF的侵入行为。 该试验涉及降解的 当RSVCEF培养时，正常CEF产生细胞外基质（ECM） 置于放射性标记的ECM上。 该分析系统允许我们测试 参与特定的蛋白酶，也需要细胞-ECM contact. 我们计划测试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能与PA结合但不与PA结合的单克隆抗体。 抑制其活性，这将作为有用的对照。 除了使用抗体测试PA在细胞中的作用外， 行为，我们现在已经将抗体连接到固体支持物上， 作为免疫亲和配体用于纯化PA。 与标准 免疫亲和柱已经产生了 PA的均匀制备。 这种方法将为我们提供 足够量的PA（0.1 - 1.0 mg），迄今为止还没有， 用生物化学的方法来描述酶的特征，并用它来寻找其他的酶。 相关基质。 我们的初步研究表明，PA（在 不存在纤溶酶原）可以切割鸡的均质制品 细胞纤连蛋白 这些研究对于确定 酶在细胞行为的某些方面的确切作用，包括 生长停滞和细胞迁移。 我们最近建立了一个肿瘤细胞转移试验使用鸡 胚胎 该系统包括将人类肿瘤细胞植入肿瘤细胞。 绒毛尿囊膜（CAM）的胚胎，并随后检测 胚胎外周器官中的人类细胞。 有大量 间接证据表明蛋白水解酶在肿瘤细胞中作用 侵袭和转移涉及肿瘤细胞酶和宿主 内切酶 已经获得了抗人PA的抗催化抗体 （尿激酶），现在有了鸡PA的抗催化抗体，我们将 现在能够检查和解剖出肿瘤细胞各自的作用， PA与宿主细胞PA在鸡胚转移系统中的作用。 （一）