Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line.
Binding of urokinase-type plasminogen activator (u-PA) to its receptor has been shown not only to focus proteolytic activity to the cell surface but also to exert a mitogenic effect on the human epidermal tumor cell line CCL 20.2. This report shows that u-PA is an autocrine mitogen in the human melanoma cell line GUBSB and that inhibition of receptor-bound u-PA by specific anti-u-PA antibodies causes a significant suppression of cell proliferation in this system. The GUBSB cell line secretes 70-80% of the u-PA in its active form and expresses high-affinity u-PA receptors with a Kd of 5.2 x 10(-10) M and 2.8 x 10(4) binding sites per cell. Approximately 70% of the u-PA receptors on these cells are occupied by endogenously secreted u-PA. Addition of the monoclonal anti-u-PA antibody MPW5UK (10 nM), directed against the active site of u-PA, twice daily to the cell cultures resulted in a significant decrease of [3H]thymidine incorporation by the tumor cells, whereas a 10 times higher concentration of the monoclonal antibody MPW4UK, which does not inhibit plasminogen activator activity of u-PA, was necessary to achieve the same effect. In addition, diisopropyl fluorophosphate-inactivated u-PA, in a concentration 50-fold higher than the concentration necessary to saturate the u-PA receptor (250 pM), decreased [3H]thymidine incorporation similarly to the specific antibody, proving that active u-PA is required for the mitogenic effect. Inhibition of endogenous u-PA production by cycloheximide reduced [3H]thymidine incorporation significantly; after addition of exogenous u-PA, [3H]thymidine incorporation increased again in the cycloheximide-treated cells. Therefore, inhibition of receptor-bound u-PA might represent a tool not only to inactivate cell-bound proteolytic activity, necessary for invasion, but also to exert a specific antiproliferative effect on certain tumor cells.
Kirchheimer
J C
JC
Department of Medical Physiology, University of Vienna, Austria.
Wojta
J
J
Christ
G
G
Binder
B R
BR
D000911:Antibodies, Monoclonal; D002455:Cell Division; D002460:Cell Line; D004261:DNA Replication; D004792:Enzyme Precursors / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008545:Melanoma / Q000201:enzymology; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors / Q000276:immunology / Q000502:physiology*; D015849:Plasminogen Inactivators; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000201:enzymology*; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000276:immunology / Q000502:physiology*
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D015849:Plasminogen Inactivators; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator