Several tumor cells secrete significantly increased amounts of the plasminogen activator urokinase, a trypsinlike serine protease, whose biological function in tumor biology is unclear. In this study we report that cells of the human epidermal tumor cell line CCL 20.2 express about 80,000 high-affinity urokinase receptors per cell that bind active as well as diisopropylfluorophosphate-treated high-molecular-weight (HMW) urokinase. Low-molecular-weight (LMW) urokinase is not bound to the receptor. Occupation of these receptors by active HMW urokinase stimulates cell proliferation independently in the presence of plasminogen in the culture medium. LMW urokinase has again no effect on cell proliferation. Calculated on a molar basis, this effect is about 28% of that of epidermal growth factor. Active HMW urokinase might therefore provide an autocrine receptor-mediated growth-promoting mechanism for tumor cells similar to those described for other growth factors.

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Plasma levels of urokinase-type plasminogen activator have been investigated in 80 patients with prostatic carcinoma by means of a radioimmunoassay. A total of 30 patients with disseminated prostatic carcinoma had significantly elevated levels of urokinase-type plasminogen activator, whereas the plasma levels in patients without metastases did not differ from a healthy age matched control group. Sensitivity of elevated urokinase-type plasminogen activator levels in patients with prostatic carcinoma for the presence of metastases was 80 per cent. Therefore, urokinase-type plasminogen activator appears to be a reliable marker for the formation of metastases in prostatic carcinoma.

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).

In this study we report on the effect of urokinase fragments on the proliferation of cells of the human epidermal cell line, CCL 20.2, which expresses high-affinity receptors for urokinase and the growth of which is stimulated by intact active 54-kDa urokinase. The 33-kDa fragment containing only the active-site domain, does not bind to the receptors and does not stimulate cell proliferation, while the 17-kDa fragment, containing only the kringle and the growth-factor domains binds to the receptor but does not stimulate growth of the human epidermal cell line. Growth promotion of this tumor cell line by urokinase is therefore restricted to the complete intact and active urokinase molecule.Activation of plasminogen to plasmin by plasminogen activators may play an important role in the invasion and formation of metastases of tumor cells [l -31. Plasmin is a degradative enzyme capable of digesting the extracellular matrix; it exerts its action either directly on glycoproteins, such as fibronectin or laminin [4,5], or indirectly by activation of latent collagenase [6]. A correlation between cancer and increased plasminogen-activator activity has been found by several groups [3, 71. Significantly elevated levels of plasminogen activator activity in extracts of primary tumors and metastases were found when compared to their normal counterparts [3, 8, 91. Immunological characterization has revealed that most malignant cells produce urokinase-type plasminogen activator (uPA) rather than tissue-type plasminogen activator (tPA) [3, 8, 91. While these data present only indirect support for the involvement of plasminogen activators in malignant processes, more direct evidence can be obtained from the following experiments. Inhibition of plasmin action by tranexamic acid reduced tumor growth of several tumor cell lines implanted into nude mice and inhibited the formation of metastases in rabbits [lo, 111. Moreover, antibodies against urokinase could be shown to inhibit the formation of metastases by a human tumor cell line in an animal model [ 121. We have found previously [ 131 that a human epidermal cell line CCL 20.2, neither containing nor secreting plasminogen activators or plasminogen-activator inhibitors, expresses high-affinity urokinase receptors, similar to those found for several other cell lines [14-161 and that active high-molecular-mass urokinase stimulates proliferation of this cell line. Mitogenic effects of fluid-phase uPA have been previously reported, however, without characterization of urokinase determinants for the observed effects [17]. To prove further that both an intact active site as well as binding of the urokinase molecule to the urokinase receptor are necessary for growth stimulation of the human epidermal tumor cell line, CCL 20.2, we compared, in the present study, the proliferative effect of intact urokinase to that of urokinase fragments. MATERIALS AND METHODS MaterialsTissue-culture cluster plates, 24 flat-bottom wells (Costar, Cambridge, MA); Tween 20, diisopropylfluorophosphate,

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Primary cultures of renal cell carcinomas and of the corresponding normal adjacent kidney tissue from 6 patients were analyzed for the effects of exogenously added urokinase-type plasminogen activator on cell proliferation as compared to the effects of tissue type plasminogen activator, plasmin and dihydrocortisone. Cell proliferation was studied over a period of up to 5 days by measuring 3H-thymidine incorporation as well as cell viability and cell count; conditioned media of the cultures were also analyzed for their plasminogen activator and plasminogen activator inhibitor content. Addition of urokinase stimulated cell proliferation in a time and dose dependent fashion; after 3 days 3H-thymidine incorporation was significantly increased in malignant renal cells (188.3 +/- 28.7%), while it reached in normal renal cells approximately 130% of the 3H-thymidine incorporation of untreated cultures. Tissue-type plasminogen activator had no effect and plasmin decreased cell proliferation slightly while dihydrocortisone inhibited cell proliferation significantly (34.1 +/- 4.9%) in malignant cells. It is concluded that urokinase-type plasminogen activator itself exhibits a mitogenic effect also on primary cultures of renal cell carcinomas.

Urokinase-type (u-PA) and tissue-type plasminogen activator antigen (t-PA) as well as plasminogen activator-inhibitor activity were determined in seminal plasma and lysates of the respective spermatozoas in 67 ejaculate of males in infertile marriage without genito urinary pathology. U-PA was determined by a competition RIA, t-PA by an ELISA and PAI by a spectrophotometric assay. 15 patients showed normozoospermia, 11 azoospermia and 41 oligoasthenoteratozoospermia (OAT-syndrome). In lysates of spermatozoas, significantly higher levels of both plasminogenactivators and PAI were found in patients with OAT syndrome as compared to those exhibiting normozoospermia. Whereas PAI was absent in the seminal plasma of normozoospermic ejaculate, patients with azoospermia (180 +/- 13 mU/ml.) and OAT-syndrome (60 +/- 5 mU/ml.) showed high PAI levels. The similarly high values of t-PA (190.8-227.8 ng./ml.) and u-PA (19.4-32 ng./ml.) in the same compartment confirm their predominantly prostatic origin and seem to have no influence on the quality of the ejaculate.