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)

在过去的一年中，我们已经成功获得了单克隆抗体 识别Rous肉瘤病毒的纤溶酶原激活剂（PA） 转化的雏鸡胚胎成纤维细胞（RSVCEF）。 抗体已经 选择其能力不仅与PA结合，还可以抑制其 催化活性。 它是一种高度特异的抗体 结合或抑制人类尿激酶或培养物中存在的PA活性 人类肿瘤细胞，但在pa活性（在L-10 mu g/ml时） 由RSVCEF文化发行。 抗催化活性的 抗体使我们现在可以直接测试PA在行为中的作用 RSVCEF文化。 我们最近开发了一种降解和 RSVCEF的侵入性行为。 该测定涉及降解 RSVCEF培养时，正常CEF产生的细胞外基质（ECM） 放置在放射标记的ECM上。 测定系统允许我们测试 特定蛋白酶的参与以及细胞ECM的需求 接触。 我们计划通过通过 在存在或不存在抗体的情况下进行降解研究。 我们计划分离与PA结合但不结合的单克隆抗体 抑制其活性，这将作为有用的控制。 除了使用抗体测试PA在细胞中的作用 行为，我们现在将抗体与固体支持联系起来，并正在使用它 作为用于净化PA的免疫亲属配体。 与标准同时 色谱程序免疫亲和力柱产生了 PA的均匀准备。 这种方法将为我们提供 足够数量的PA（0.1至1.0 mg），迄今为止无法使用， 以生物化学来表征酶，并使用它来搜索其他 相关底物。 我们的初步研究表明PA（在 缺乏纤溶酶原）可以裂解鸡的均匀制剂 细胞纤连蛋白。 这些研究将在定义 酶在细胞行为的某些方面的确切作用 生长停滞和细胞迁移。 我们最近使用雏鸡建立了肿瘤细胞转移测定法 胚胎。 该系统涉及将人类肿瘤细胞植入 胚胎的绒毛膜膜（CAM），然后检测 胚胎外围器官中的人类细胞。 有很大的机构 间接证据表明蛋白水解酶在肿瘤细胞中的作用 侵袭和转移涉及肿瘤细胞酶和宿主 酶。 采购了对人PA的抗催化抗体 （尿蛋白酶）现在具有抗小鸡PA的抗催化抗体，我们将 现在能够检查并剖析肿瘤细胞的各自作用 PA和宿主细胞PA在雏鸡胚胎转移系统中。 （一个）