Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line.

Abstract

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 104 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 antiu-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 fluorophosphateinactivated 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.Malignant cells contain and secrete plasminogen activators (PAs; EC 3.4.21.31), trypsin-like serine proteases, to a much higher extent than their respective normal counterparts (1-3). Immunological characterization has shown that most malignant cells produce urokinase-type PA (u-PA) rather than tissue-type PA (t-PA) (2). u-PA contains not only an activesite domain consistent with its serine protease character and involved in the activation ofplasminogen but also a "kringle" domain, found in several other seine proteases, and a growth-factor domain homologous to the epidermal growth factor (4).

Urokinase-type plasminogen activator induces proliferation in breast cancer cells

“…5). Kirchheimer and coworkers have found similar results in human melanoma cell line GUBSB using monoclonal anti-uPA antibody (MPW5UK) which blocks binding of uPA to uPAR (22).…”

Oestradiol regulation of the components of the plasminogen-plasmin system in MDA-MB-231 human breast cancer cells stably expressing the oestrogen receptor

“…Conversely, the inhibition of endogenously produced uPA by human malignant melanoma cells impairs cell proliferation (Kirchheimer et aL 1989). It is believed that the mitogenic and growth factor activity of uPA occurs throgh the amnotermnal fragment (AIT) of uPA, containing the growth factor domain, which shares sucntures homologous to the EGF, the TGF-a and the Krinkle domain.…”

Role of multidomain structure of urokinase in regulation of growth and remodeling of vessels

“…Митогенная активность урокиназы наб-людалась на многих типах клеток, в том чис-ле на человеческих эпидермальных клетках, нормальных и злокачественных клетках почки и клетках меланомы [96,97]. недавние иссле-дования показали, что пролиферация раковых клеток человека зависит от взаимодействия комплекса uPa-uPar с интегринами, что ве-дет к активации p38 maPK [98].…”