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 125I 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

Comparative study of T84 and T84SF human colon carcinoma cells: in vitro and in vivo ultrastructural and functional characterization of cell culture and metastasis

To better understand the relationship between tumor heterogeneity, differentiation, and metastasis, suitable experimental models permitting in vitro and in vivo studies are necessary. A new variant cell line (T84SF) exhibiting an altered phenotype was recently selected from a colon cancer cell line (T84) by repetitive plating on TNF-alpha treated human endothelial cells and subsequent selection for adherent cells. The matched pair of cell lines provides a useful system to investigate the extravasation step of the metastatic cascade. Since analysis of morphological differences can be instructive to the understanding of metastatic potential of tumor cells, we compared the ultrastructural and functional phenotype of T84 and T84SF cells in vitro and in vivo. The reported ultrastructural features evidence differences between the two cell lines; selected cells showed a marked pleomorphism of cell size and nuclei, shape, and greater surface complexity. These morphological differences were also coupled with biochemical data showing a distinct tyrosine phosphorylation-based signaling, an altered localization of beta-catenin, MAPK, and AKT activation, as well as an increased expression in T84SF cells of Bcl-X-L, a major regulator of apoptosis. Therefore, these cell lines represent a step forward in the development of appropriate models in vitro and in vivo to investigate colon cancer progression

Effect of T-R conformational change on Sickle Haemoglobin interactions and aggregation

We compare the role of a conformational switch and that of a point mutation in the thermodynamic stability of a protein solution and in the consequent propensity toward aggregation. We study sickle-cell hemoglobin (HbS), the β6 Glu-Val point mutant of adult human hemoglobin (HbA), in its R (CO-liganded) conformation, and compare its aggregation properties to those of both HbS and HbA in their T (unliganded) conformation. Static and dynamic light scattering measurements performed for various hemoglobin concentrations showed critical divergences with mean field exponents as temperature was increased. This allowed determining spinodal data points TS(c) by extrapolation. These points were fitted to theoretical expressions of the TS(c) spinodal line, which delimits the region where the homogeneous solution becomes thermodynamically unstable against demixing in two sets of denser and dilute mesoscopic domains, while remaining still liquid. Fitting provided model-free numerical values of enthalpy and entropy parameters measuring the stability of solutions against demixing, namely, 93.2 kJ/ mol and 314 J/°K-mol, respectively. Aggregation was observed also for R-HbS, but in amorphous form and above physiological temperatures close to the spinodal, consistent with the role played in nucleation by anomalous fluctuations governed by the parameter ε = (T-TS)/TS. Fourier transform infrared (FTIR) and optical spectroscopy showed that aggregation is neither preceded nor followed by denaturation. Transient multiple interprotein contacts occur in the denser liquid domains for R-HbS, T-HbS, and T-HbA The distinct effects of their specific nature and configurations, and those of desolvation on the demising and aggregation thermodynamics, and on the aggregate structure are highlighted

Matrix-degrading proteinases are shed in membrane vesicles by ovarian cancer cells in vivo and in vitro

The in vitro release of matrix-degrading proteinases from breast cancer cells is associated in part with shed membrane vesicles. To determine whether shed vesicles might play a similar role in ovarian cancer cells, we analyzed the shedding phenomenon in vivo and in vitro as well as the enzymatic content of their vesicles. This is the first time that an immunoelectron microscopical analysis revealed membrane vesicles carrying tumor-associated antigen alpha-Folate Receptor (alpha-FR), circulating in biological fluids (ascites and serum) of an ovarian carcinoma patient. These vesicles were trapped in a fiber network with characteristic fibrin periodicity. An ovarian cancer cell line (CABA I) established from ascitic fluid cells of this patient, grew in Matrigel and formed tubular structures suggesting invasive capability. Immunofluorescence analysis demonstrated strong cytoplasmic staining of CABA I cells with anti-matrix metalloproteinase-9 (MMP-9) and anti-urokinase-type plasminogen activator (uPA) antibodies. CABA I cells shed membrane vesicles, which were morphologically similar to those identified in vivo, as determined by electron microscopy. Gelatin zymography of vesicles isolated both in vivo and in vitro revealed major gelatinolytic bands of the MMP family, identified as the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2). By casein-plasminogen zymography we observed high-molecular weight (HMW)-uPA and plasmin bands. Incubation of purified vesicles from CABA I cells with Matrigel led to cleavage of Matrigel components. Taken together, our results point to a possible role of shed vesicles, both in vivo and in vitro, in proteolysis that mediates invasion and spread of ovarian epithelial carcinoma cells