TISSUE EXTRACTION PROCEDURES FOR INVESTIGATION OF UROKINASE PLASMINOGEN-ACTIVATOR (UPA) AND ITS INHIBITORS PAI-1 AND PAI-2 IN HUMAN BREAST CARCINOMAS

Urokinase type plasminogen activator (uPA) and its inhibitors, plasmin ogen activator inhibitor type I (PAI-1) and type II (PAI-2), are suppo sed to be involved in the expression of the invasive and metastatic ph enotype of cancer cells. However, clinical investigations on the progn ostic significance of their levels in tumor tissue are difficult to re alize because of the absence of a convenient method of measurement of these parameters. The aim of the present investigation was to set up a method allowing the measurement of these enzymes and of sex steroid r eceptor status in appropriate subcellular fraction(s) in conditions ea sily reproducible in routine. We found that a tissue homogenate prepar ed according to the method recommended [5] for current measurement of sex steroid receptors is appropriate far further distinct preparations . One aliquot is used for cytosol preparation; another can be treated by 2% Triton X-100 (vol/vol) and provide an extract containing the tot ality of uPA and PAI-1. The advantage of this procedure is that approp riate subcellular fractions can be derived from a unique homogenizatio n step. Total uPA and PAI-1 are measured in a Triton extract with good performance as compared to previous investigations [4]. PAI-2 is meas ured in the same cytosol fraction used for sex steroid receptors and o ther parameters. Because of its simplicity and its high reliability, t his method could be a useful tool in the investigation of uPA family p roteases and analysis of their prognostic significance in early breast tumors.