Plasminogen activator mediated degradation of subendothelial extracellular matrix by human squamous carcinoma cell...

Extracellular matrix (ECM) produced by bovine corneal endothelial cells was used to investigate the role of the plasminogen activator/plasmin system in the degradation of ECM by human squamous cell carcinoma (SqCCs) and human foreskin epidermal cells (HFEC). SqCCs caused an 8- to 34-fold greater solubilization of 3H-glucosamine-labeled ECM than HFEC. This action in SqCCs was dependent upon the presence of acid-treated serum, indicating that tumor-associated proteinases were sensitive to the inhibitory action of acid-labile proteinase inhibitors present in the serum. SqCC mediated digestion of radiolabeled ECM was decreased by 14- to 55-fold in plasminogen depleted serum, and the addition of 100 micrograms/mL of purified human plasminogen resulted in up to a 30-fold increase in the degradation of the ECM. Inhibitors of this proteinase system and murine monoclonal antibodies (MAb) specific for human urokinase plasminogen activator (uPA) decreased the SqCC mediated digestion of radiolabeled ECM in a concentration dependent manner. SqCCs exhibited 10- to 30-fold higher extracellular uPA levels than HFEC, as assayed by substrate hydrolysis, zymography, micro-ELISA, western analysis, and northern analysis. These findings reflect the differential ability of these cell types to degrade the ECM. In addition, immuno-cross-reactive plasminogen activator inhibitor type I (PAI type 1) and type II (PAI type 2) were identified in cell-free conditioned medium produced by both tumor cells and normal epidermal cells, using a micro-ELISA assay. Indirect immunofluorescence flow cytometry, employing MAbs directed against uPA, detected the presence and localization of uPA on the SqCC cell surface. These findings were specific for uPA, since cell surface associated tissue plasminogen activator was not detected in these cell types under analogous conditions. In addition, partially purified SqCC plasma membrane preparations exhibited 2- to 10-fold higher uPA-like activity than HFEC, as determined by zymography. The findings support the concept that the plasminogen activator system is important in the breakdown of ECM by SqCCs and suggest that regulatory mechanisms involved in this proteolytic system may be important targets for chemotherapeutic intervention to limit tumor cell invasion and metastasis.