Blockage of the urokinase receptor on the cell surface: Construction and characterization of a hybrid protein consisting of the N‐terminal fragment of human urokinase and human albumin

Abstract

Receptor-bound urokinase is likely to be a crucial determinant in both tumor invasion and angiogenesis. We report here that a yeast-derived genetic conjugate between human serum albumin and the 1-135 N-terminal residues of urokinase (u-PA) competitively inhibits the binding of exogenous and endogenous u-PA to its cell-anchored receptor (u-PAR). This hybrid molecule (ATF-HSA) also inhibits in vitro pro-urokinasedependent plasminogen activation in the presence of u-PAR bearing cells. These effects are probably responsible for the observed in vitro inhibition of tumor cell invasion in a reconstituted basement membrane extract (Matrigel).

Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice

“…Invasion assays were carried out in a 12-well Transwell unit (Costar, Cambridge, MA, USA) as described. 11 Briefly, polycarbonate filters of 12 m pore size were coated with 160 g Matrigel (Becton Dickinson France, le Pont-deChaix, France) and dried. The lower chambers of the Transwell units were filled with human fibroblast-conditioned medium containing 10 ng/ml EGF, and the upper chambers were seeded with 3 × 10 5 infected cells.…”

“…Among them, the amino-terminal fragment of urokinase (ATF) is of particular interest because it can also exert a direct effect on the tumor cells and inhibit metastasis in experimental models. [9][10][11] Urokinase and its high affinity receptor, a GPI-anchored membrane protein (CD87), are believed to be critical elements in tumor biology because they control cell motility, tissue remodeling and the bioavailability of angiogenic factors. For example, uPA/uPAR complex formation at the cell surface is required for efficient activation of plasmin, a wide range protease that can degrade components of the basement membrane and the extracellular matrix (eg vitronectin, laminin, fibronectin), a prerequisite for tumor cell invasion.…”

Blockage of urokinase receptor reduces in vitro the motility and the deformability of endothelial cells

“…Apart from specifically displacing urokinase from its cell surface receptor, the hybrid molecule could also inhibit pro-urokinase-dependent plasminogen activation in the presence of u-PAR-bearing cells as well as in vitro tumor cell invasiveness [15]. In this study, we addressed the effect of the blockage of u-PAR by ATF-HSA or anti-u-PAR antibodies on the migration of endothelial cells and compared its effect with that of aprotinin, a potent inhibitor of plasmin.…”

A Novel Type of Bifunctional Inhibitor Directed against Proteolytic Activity and Receptor/Ligand Interaction

“…A fusion protein harboring the amino-terminal part of uPA (aa 1Ϫ135) and human serum albumin (ATF-HSA hybrid) significantly reduced the invasive capacity of a breast cancer cell line in vitro (27). Similarly, a uPA-(1-137)/IgG chimera suppressed the metastatic capacity of human PC3 prostate carcinoma cells in nude mice (28).…”

“…Hybrid molecules harboring different parts of the two serpins, ␣ 1 -proteinase inhibitor and thyroxine-binding globulin, inhibited human leukocyte elastase and bound to thyroxine with high affinity (31). Nevertheless, all of the chimeric proteins discussed here have been constructed by fusion of independent domains of different proteins (27,30,(32)(33)(34) or by fusion of modules of structurally highly conserved proteins based on their modular architecture (31). In contrast, the presented cystatin variants have been generated, in a novel approach, by replacing 11 aa of the parent cystatin sequence with 13 aa of uPA, resulting in a biologically bifunctional active protein.…”