PROTEASES IN GROWTH CONTROL AND MALIGNANT TRANSFORMATION

在过去的一年里，我们成功地获得了一种单抗 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)。抗体已经被 选择它不仅是因为它能与PA结合，而且还能抑制它的 催化活性。它是一种高度特异的抗体，因为它不 结合或抑制人尿激酶或存在于培养物中的PA活性 对人肿瘤细胞有抑制作用，但对PA活性有抑制作用(在L-10微克/毫升) 由RSVCEF的培养物释放。它的抗催化活性 抗体使我们现在可以直接测试PA在 RSVCEF的培养。 我们最近开发了一种用于降解的生化分析方法， RSVCEF的入侵行为。该化验涉及到对 RSVCEF培养时正常CEF产生的细胞外基质 被放置在放射性标记的ECM上。化验系统允许我们测试 特异性蛋白水解酶的参与及其对细胞外基质的要求 联系。我们计划测试PA在ECM降解中的直接作用 在我们的抗体存在或不存在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PA的单抗 抑制它的活性，这将是一种有用的控制。 除了使用抗体测试PA在细胞中的作用外 行为，我们现在已经将抗体与固体载体联系起来，并正在使用它 作为一种免疫亲和配体用于纯化PA。与标准同步 层析程序免疫亲和柱产生了一种 PA的均相制备。这种方法论将为我们提供 足够量的PA(0.1至1.0毫克)，迄今无法获得，以 对这种酶进行生化表征，并用它来寻找其他 相关底物。我们的初步研究表明，PA(在 缺乏纤溶酶原)可以裂解均匀的鸡肉制剂 细胞纤维连接蛋白。这些研究将对定义 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。 我们最近用鸡建立了一种肿瘤细胞转移试验。 胚胎。该系统涉及将人类肿瘤细胞植入 胚胎绒毛尿囊膜(CAM)和随后的检测 胚胎外周器官中的人类细胞。有一大堆 间接证据表明蛋白水解酶在肿瘤细胞中的作用 侵袭和转移与肿瘤细胞酶和宿主有关 酵素。获得了抗人PA的抗催化抗体 (尿激酶)，现在有了针对鸡PA的抗催化抗体，我们将 现在能够检查和剖析出肿瘤细胞各自的作用 PA和宿主细胞PA在鸡胚转移系统中的表达。(A)

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)