Kinetic analysis of plasminogen activator inhibitor type-2: urokinase complex formation and subsequent internalisation by carcinoma cell lines

被引:41
作者
Al-Ejeh, F [1 ]
Croucher, D [1 ]
Ranson, M [1 ]
机构
[1] Univ Wollongong, Dept Biol Sci, Wollongong, NSW 2522, Australia
关键词
cancer cells; endocytosis; inactivation constant (K-I); inactivation rate constant (k(inact)); plasminogen activator inhibitor type-2 (PAI-2); urokinase (uPA);
D O I
10.1016/j.yexcr.2004.03.030
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
The overexpression of urokinase (uPA), which plays a key role in tumour invasion and metastasis, is an established prognostic marker and potential therapeutic target. Plasminogen activator inhibitor type 2 (PAI-2), an efficient and specific inhibitor of uPA, has been shown to selectively deliver potent cytotoxins to tumour cells. However, a direct quantitative analysis of both the inhibition kinetics and subsequent fate of PAI-2 upon interaction with cell-surface uPA has not been previously undertaken. In this study, we analysed specific PAI-2 binding to receptor-bound uPA on human breast and prostate cancer cell lines to directly measure inhibition kinetics. Cell-surface uPA:PAI-2 complex formation, which is reflective of complete uPA inhibition, was found to be very efficient (inactivation constant [K-1] = 60-80 pM, depending on cell line used) and rapid (inactivation rate constant [k(inact)] = 0.32-0.47 min(-1) at 37degreesC, depending on cell line used). To directly quantify and visualise cellular internalisation and localisation, we developed a novel assay based on the use of PAI-2 labelled with Alexa(488) fluorochrome and a polyclonal antibody to quench Alexa(488) fluorescence. The efficient and rapid formation of uPA:PAI-2 complexes was thus shown to be associated with specific and rapid internalisation of PAI-2, which could be localised within endosomes and lysosomes. PAI-2 was subsequently degraded, presumably within lysosomes. This study is the first to provide definitive evidence for uPA/uPAR-mediated PAI-2 endocytosis. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:259 / 271
页数:13
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