Holst-Hansen C, Johannessen B, Høyer-Hansen G, Rømer J, Ellis V, Brünner N
The Finsen Laboratory, Copenhagen, Denmark.
Clin Exp Metastasis. 1996 May;14(3):297-307. doi: 10.1007/BF00053903.
In order to invade and spread cancer cells must degrade extracellular matrix proteins. This degradation is catalysed by the concerted action of several enzymes, including the serine protease plasmin. Several experimental studies have shown that inhibition of plasmin formation reduces cancer cell invasion and metastasis, indicating a critical role of this proteolytic pathway in these processes. In order to further study the role of plasmin in cancer progression, we have characterized urokinase-type plasminogen activator (uPA) mediated plasmin formation in three human breast cancer cell lines. Using monoclonal antibodies against uPA and its receptor uPAR, we have investigated the contribution of uPA and uPAR to invasive capacity in an in vitro invasion assay. MDA-MB-231 BAG cells were found to express high protein levels of uPA, uPAR and PAI-1. MDA-MB 435 BAG cells produced low amounts of uPA, PAI-1 and moderate amounts of uPAR, whereas MCF-7 BAG cells showed low levels of uPA, uPAR and PAI-1 protein. In a plasmin generation assay MDA-MB-231 BAG cells were highly active in mediating plasmin formation, which could be abolished by adding either an anticatalytic monoclonal antibody to uPA (clone 5) or an anti-uPAR monoclonal antibody (clone R3), which blocks binding of uPA to uPAR. The two other cell lines lacked the capacity to mediate plasmin formation. In the Matrigel invasion assay the cells showed activity in this order: MCF-7 BAG < MDA-MB-435 BAG < MDA-MB-231 BAG. Testing MDA-MB-231 BAG cells in the Matrigel invasion assay revealed that invasion could be inhibited in a dose-dependent manner either by the clone 5 uPA antibody or by the clone R3 uPAR antibody, suggesting that the cell surface uPA system is actively involved in this invasive process. It is concluded that these three cell lines constitute a valuable model system for in vitro studies of the role of cell surface uPA in cancer cell invasion and has application in the search for novel compounds which inhibit mechanisms involved in uPA-mediated plasmin generation on cancer cells.
为了实现侵袭和扩散，癌细胞必须降解细胞外基质蛋白。这种降解是由几种酶的协同作用催化的，包括丝氨酸蛋白酶纤溶酶。多项实验研究表明，抑制纤溶酶的形成可减少癌细胞的侵袭和转移，这表明该蛋白水解途径在这些过程中起关键作用。为了进一步研究纤溶酶在癌症进展中的作用，我们对三种人乳腺癌细胞系中尿激酶型纤溶酶原激活剂（uPA）介导的纤溶酶形成进行了表征。使用针对uPA及其受体uPAR的单克隆抗体，我们在体外侵袭试验中研究了uPA和uPAR对侵袭能力的贡献。发现MDA-MB-231 BAG细胞表达高水平的uPA、uPAR和PAI-1蛋白。MDA-MB 435 BAG细胞产生少量的uPA、PAI-1和中等量的uPAR，而MCF-7 BAG细胞显示uPA、uPAR和PAI-1蛋白水平较低。在纤溶酶生成试验中，MDA-MB-231 BAG细胞在介导纤溶酶形成方面高度活跃，添加针对uPA的抗催化单克隆抗体（克隆5）或抗uPAR单克隆抗体（克隆R3）可消除这种活性，后者可阻断uPA与uPAR的结合。另外两种细胞系缺乏介导纤溶酶形成的能力。在基质胶侵袭试验中，细胞的活性顺序为：MCF-7 BAG < MDA-MB-435 BAG < MDA-MB-231 BAG。在基质胶侵袭试验中对MDA-MB-231 BAG细胞进行测试发现，克隆5 uPA抗体或克隆R3 uPAR抗体均可剂量依赖性地抑制侵袭，这表明细胞表面uPA系统积极参与了这一侵袭过程。结论是，这三种细胞系构成了一个有价值的模型系统，用于体外研究细胞表面uPA在癌细胞侵袭中的作用，并可应用于寻找抑制癌细胞上uPA介导的纤溶酶生成机制的新型化合物。
