Lipopolysaccharide (LPS),

Progression of glioma is associated with local degenerative processes which are attributed to the activity of gelatinases. As glioma cells are candidate for secretion of these enzymes, we have studied in vitro the potential of cytokines (interleukin-1alpha (IL-1), tumor necrosis factor-alpha (TNFalpha) and transforming growth factor-beta (TGFbeta2)) to regulate the expression of gelatinase A and B (Gels A and B, respectively) in two glioma cells of human (A172) and rat origin (C6). We showed that IL-1 and TNFalpha both induced gene expression and protein secretion of Gel B in both cell lines, as revealed by RT-PCR and gelatin zymography, respectively. In C6 cells, TNFalpha had no effect on Gel A constitutive expression while IL-1 increased its production, but only at high doses. We have also demonstrated that TGFbeta2 inhibited both IL-1- or TNFalpha-induced gene expression and Gel B production in a dose-dependent manner but had no effect on Gel A secretion. The effect of TGFbeta2 on Gel B secretion was reversed by phorbol myristate acetate (PMA). Taken together, these data suggest that IL-1, TNFalpha and TGFbeta2 tightly regulate Gel B secretion in glioma cells, an enzyme which is believed to play an important role in the local invasion of brain tissue by tumor cells.

Gelatinase B is a member of the metalloproteinase family of enzymes that degrade the extracellular matrix under normal and pathological conditions, including autoimmune diseases and tumor cell dissemination. In the present work, we describe a simple and reliable method that allows qualitative and quantitative measurements of a specific enzymatic reaction (mediated here by gelatinase B) by flow cytometry using fluorochrome-labeled substrate coated on polystyrene microspheres. Using this approach, proteolytic degradation can be monitored by the decrease of the fluorescence emitted by the microspheres following incubation with the enzyme. In most of the experiments, the signal-to-noise ratio between autofluorescent microspheres and those coated with the fluorescein isothiocyanate (FITC)-labeled substrate was near 500. This ratio allows one to measure accurately the enzymatic activity in the presence of chemical or biological inhibitors. FITC labeling and passive adsorption of substrates on the solid support did not cause significant conformational changes or steric hindrance that would interfere with the proteolytic activity of gelatinase B. This method constitutes a powerful tool for the measurement, on a routine basis, of the net activity resulting from the balance between the gelatinase B activity and the presence of natural inhibitors and for the identification of new metalloproteinase inhibitors that could suppress the excessive proteolytic activity that characterizes many disease processes.