Urokinase and the Urokinase Receptor: Association With In Vitro Invasiveness of Human Bladder Cancer Cell Lines

Abstract

The plasminogen activators urokinase and tissue plasminogen activator are enzymes that degrade proteins in tissue basement membranes and the extracellular matrix (a biomolecular complex surrounding individual cells in tissues that serves as a barrier between the cells and the vascular and lymph systems). The action of these enzymes allows tumor cells to escape their local environment and metastasize. Plasminogen activator activity can be influenced by the urokinase receptor, which is expressed on the surface of cells, and by the plasminogen activator inhibitors 1 and 2. Because bladder tumors differ in their propensity to invade local areas and distant sites, we studied the expression of both plasminogen activators, the two plasminogen activator inhibitors, and the urokinase receptor in four human bladder cancer cell lines (RT4, 253J, EJ, and T24) to see if there was an association between the expression of these proteins and tumor cell invasiveness in vitro. The expression of urokinase, tissue plasminogen activator, and the two inhibitors was measured by enzyme-linked immunosorbent assays of serum-free, cell-conditioned media (i.e., culture fluids). Cell surface expression of the urokinase receptor was assayed by flow cytometry, using an anti-receptor monoclonal antibody (Mab3936). The invasive capacity of untreated cells and of cells exposed to exogenous, high-molecular-weight urokinase was analyzed by use of Matrigel invasion chambers. The four bladder cancer cell lines demonstrated differential expression of both plasminogen activators and both inhibitors; three of the cell lines (T24, EJ, and 253J) expressed the urokinase receptor. The four cell lines differed in their invasive potential in vitro. Neither expression of tissue plasminogen activator nor production of the inhibitors appeared to influence Matrigel invasion. EJ cells and 253J cells produced the highest levels of urokinase and demonstrated the greatest propensity for invasion; T24 cells, which produced only small amounts of urokinase, exhibited a low invasive potential. Pretreatment of T24 cells with exogenous high-molecular-weight urokinase markedly increased their invasiveness. Similar pretreatment of EJ and 253J cells increased their invasiveness as well. RT4 cells, which lacked urokinase receptor expression but produced moderate amounts of urokinase, were not invasive and did not become so after exposure to exogenous high-molecular-weight urokinase. Binding of Mab3936 to urokinase receptors inhibited Matrigel invasion. To our knowledge, this is the first study demonstrating that bladder tumor cells express the urokinase receptor and that both receptor expression and urokinase expression are required for bladder tumor cell invasion in vitro.