Hormonal regulation of extracellular plasminogen activators and Mr approximately 54,000 plasminogen activator inhibitor in human neoplastic cell lines, studied with monoclonal antibodies

We have studied the regulation by glucocorticoids and dibutyryl cAMP of the amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and a Mr approximately 54000 plasminogen activator inhibitor accumulated in serum-free conditioned culture fluid by a human fibrosarcoma, a human glioblastoma and a human melanoma cell line (HT-1080, UCT/gl-1 and Bowes). For the quantitation of u-PA and t-PA, we used sandwich-type ELISA with a combination of polyclonal and monoclonal antibodies. For an estimation of variations in the amount of the inhibitor, we used sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by Coomassie blue staining of conditioned culture fluid proteins, the inhibitor protein band being identified by its selective removal by passage of the conditioned culture fluids through a column with monoclonal antibodies against the inhibitor. The modulation of the 3 proteins by the hormonal agents varied greatly between the cell lines. The proteins were independently regulated, in the sense that the hormonal agents did not concomitantly change their levels in the direction expected either to increase or decrease total extracellular plasminogen activator activity. In conditioned culture fluids containing both t-PA and inhibitor, the two were present in the medium as a Mr approximately 120 000 complex. In contrast, no u-PA inhibitor complexes were found in conditioned culture fluid from any of the cell lines; this is likely to be due to the occurrence of u-PA in the culture fluid in the one-chain proenzyme form, which, unlike active u-PA, does not react with the inhibitor. These findings illustrate the complexity of the regulation of extracellular plasminogen activator activity, and imply that the presumed functional diversity of u-PA and t-PA may be related to their independent regulation.