Figure 2.

Activation of pro-MMP-2 in cocultures of astrocytes and U251N glioma cells. A Gelatin zymogram of pro- and active MMP-2 is displayed. Recombinant human (rH) MMP-2 was used as a standard for both pro- and active MMP-2, which were ∼72 and 65 kDa, respectively. Although the conditioned medium of astrocytes contained high levels of pro-MMP-2 and negligible active MMP-2, U251N glioma cells secreted very low but detectable levels of both forms. In the coculture of astrocytes with U251N cells, pro-MMP-2 remained high; significantly, a substantial amount of active MMP-2 was generated. This result has been replicated in >20 experiments in which each test condition was always performed in duplicate (duplicate lanes per condition are displayed for this and other zymograms). B, The amount of active MMP-2 present in the conditioned medium of cells is documented by degradation of fluorescent substrate. In correspondence with the zymography data, increased MMP-2 activity was generated in U251N glioma–astrocyte coculture. The media histogram refers to feeding medium that had not been exposed to cells. Values shown are the mean±SEM of triplicate samples from a single experiment. This result has been reproduced in two other experiments; ***p < 0.001 compared against U251N glioma, one-way ANOVA with Bonferroni post hoc test. C, Shown is the GFAP status of cells, whereby the U251N cells (left panel) were found to contain GFAP intermediate filament protein (green stain) as do the fetal astrocytes (right panel); in the middle panel the primary anti-GFAP antibody has been omitted from the staining of U251N cells. All cells were counterlabeled with Hoechst dye to illuminate their nuclei. The presence of GFAP immunoreactivity in the U251 line used here indicates that the results of this manuscript are not attributable to the loss of GFAP expression, which can confer to glioma cells a more invasive phenotype (Rutka and Smith, 1993).