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, plasminogen activator inhibitor type I (PAI-1) and type II (PAI-2), are supposed to be involved in the expression of the invasive and metastatic phenotype of cancer cells. However, clinical investigations on the prognostic significance of their levels in tumor tissue are difficult to realize 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 receptor status in appropriate subcellular fraction(s) in conditions easily reproducible in routine. We found that a tissue homogenate prepared according to the method recommended [5] for current measurement of sex steroid receptors is appropriate for 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 totality of uPA and PAI-1. The advantage of this procedure is that appropriate subcellular fractions can be derived from a unique homogenization step. Total uPA and PAI-1 are measured in a Triton extract with good performance as compared to previous investigations [4]. PAI-2 is measured in the same cytosol fraction used for sex steroid receptors and other parameters. Because of its simplicity and its high reliability, this method could be a useful tool in the investigation of uPA family proteases and analysis of their prognostic significance in early breast tumors.