Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only <11% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cellsurface plasmin formation was inhibited by an anticatalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted a2-macroglobulin (a2M), as shown by a2M-specific mRNA in Northern blotting and detection of a2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their a2M content, and immunoabsorption of a2M removed the inhibitory activity.These studies suggest that t-PA can bind to the surface of melanoma cells and generate surfacebound plasmin. Because t-PA and cell-bound plasmin are unaffected by a2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.
In previous studies we showed that human sarcoma and melanoma cell lines synthesize and secrete into culture medium a glycoprotein, migrating in urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 140,000. It is not detected in cultures of the corresponding normal cells. Conditioned medium of the melanoma cell line HMB-2, producing among the cell lines tested the largest amounts of this glycoprotein, has now been used as a source for purification of the protein. NH2-terminal amino-acid sequence determination of the purified glycoprotein showed that it is identical to human alpha 2-macroglobulin (alpha 2M). Rabbit antibodies raised against the glycoprotein specifically reacted in immunoblotting and immunodiffusion tests with alpha 2M present in human plasma. Likewise, these antibodies immunoprecipitated from the conditioned media of 35S-methionine-labelled melanoma and osteosarcoma cell lines the protein which had a molecular weight corresponding to alpha 2M. alpha 2M was also synthesized and secreted by 2 strains of fetal lung fibroblasts but not by fetal skin fibroblasts or adult skin fibroblasts autologous to the osteosarcoma cell line.
alpha 2-Macroglobulin (alpha 2M) is known as an inhibitor of various proteinases and to bind several of the growth factors. We previously demonstrated that clonal variation exists in the production of alpha 2M in a human melanoma and that this variation may be associated with growth stimulation. We have now analyzed six human melanoma cell lines for the simultaneous expression of TGF-alpha, TGF-beta, PDGF-A chain, PDGF-B chain, and tumor-associated alpha 2M. In Northern blot analysis TGF-alpha was detected in four of the cell lines, TGF-beta in all, PDGF-A chain in three, and PDGF-B chain in none of the cell lines. alpha 2M, detected by immunoblotting, varied significantly between the different melanoma cell lines and only one cell line was found to be negative. Evaluation of growth-promoting activity in conditioned media suggested that alpha 2-macroglobulin, secreted by these tumor cell lines, is a significant modulator of melanoma cell growth.
We and others have previously shown that human melanoma cell lines in culture synthesize alpha-2-macroglobulin (alpha 2M). We have now studied melanomas from 30 patients for the presence of alpha 2M using the peroxidase anti-peroxidase technique on histologic sections from paraffin-embedded tissues and primary antibody raised against tumor-associated alpha 2M in rabbits. alpha 2M was detected in 10 of the 30 melanomas studied. In all but 2 cases the presence of alpha 2M was restricted to solitary tumor cells or to solitary foci of tumor tissue. In one case of melanoma almost all tumor cells were positive for alpha 2M, while in the others between 20% and 50% of tumor cells were positive. In all but one of the melanomas, the positivity was characteristic of epithelioid or large-cell type or was confined to this component in melanomas with more than one cell type. In 4 positive cases, differences in the extent of alpha 2M-containing tumor tissue were observed between primary tumor and metastases or metastases from different localizations, with equivocal trend. Clinical follow-up of the melanoma patients suggested that alpha 2M-positively tends to correlate with an unfavorable prognosis.
We have shown (Bizik et al., Cell Regul 1:895-905, 1990) that tPA can activate plasminogen on the surface of human melanoma cells in the presence of alpha 2-macroglobulin (alpha 2M) secretion. In the present study, we investigated the binding of tPA on the surface of Bowes melanoma cells, selected since they lacked production of PAI-1 and alpha 2M. Elution of tPA from the cell layers indicated that polylysine (5 micrograms/ml) and tranexamic acid (10 mM), an analog of lysine, were the most efficient agents for disrupting the interaction between tPA and cell surface component(s). Using a panel of monoclonal antibodies against individual domains of tPA revealed that an antibody directed to the kringle-2 domain of tPA interfered most significantly with cell-surface plasmin generation. As tPA is a glycoprotein, interactions between the tPA sugar moieties and cell surface were also tested by the use of a series of monosaccharides. N-acetyl-D-glucosamine (100 mM) was the most potent sugar to release tPA from melanoma cells, but the results indicated that the oligosaccharides of tPA play only a supportive role in the binding of tPA to the cell surface. Quantitative comparison of the cell surface localized tPA, which was eluted by tranexamic acid, with the total cellular tPA showed that cell surface bound tPA could represent up to 10%. We conclude that tPA interacts with the melanoma cell surface in a similar manner as has been described for binding of tPA to fibrin and to the putative endothelial cell surface receptor.
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