Urokinase plasminogen activator and gelatinases are associated with membrane vesicles shed by human HT1080 fibrosarcoma cells

Membrane vesicles are shed by tumor cells both in vivo and in vitro, Although their functions are not well understood, it has been proposed that they may play multiple roles in tumor progression, We characterized membrane vesicles from human HT1080 fibrosarcoma cell cultures for the presence of proteinases involved in tumor invasion, By gelatin zymography and Western blotting, these Vesicles showed major bands corresponding to the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2) and to the MMP-9.tissue inhibitor of metalloproteinase 1 complex, Both gelatinases appeared to be associated with the vesicle membrane, HT1080 cell vesicles also showed a strong, plasminogen-dependent fibrinolytic activity in I-125 fibrin assays; this activity was associated with urokinase plasminogen activator, as shown by casein zymography and Western blotting, Urokinase was bound to its high affinity receptor on the vesicle membrane, Addition of plasminogen resulted in activation of the progelatinases associated with the vesicles, indicating a role of the urokinase-plasmin system in MMP-2 and MMP-9 activation, We propose that vesicles shed by tumor cells may provide a large membrane surface for the activation of membrane-associated proteinases involved in extracellular matrix degradation and tissue invasion.