Molecular determinants of glioma cell migration and invasion

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Object. Migration and invasion are important prerequisites for the infiltrative and destructive growth patterns of malignant gliomas. Infiltrative growth prevents complete tumor resection and causes significant neurological morbidity and mortality.

Methods. The authors assessed the expression of matrix metalloproteinases (MMPs) at messenger RNA and protein levels, MMP-2 and MMP-9 activities, and expression levels of a panel of anti- and proapoptotic proteins of the BCL-2 family. They then correlated their findings with αVβ3 integrin expression and the migratory and invasive potentials in 12 human malignant glioma cell lines.

Multiple MMPs were expressed by most cell lines. The levels of MMP-2 and MMP-3 and the activities of MMP-2 and MMP-9 correlated with tumor cell invasion. Migration and invasion were also correlated. Although the expression levels of αVβ3 integrin did not predict migration or invasion, a neutralizing αVβ3 integrin antibody inhibited migration and invasion selectively in cell lines that contained a high level of αVβ3 integrin expression, thus indicating the important role of αVβ3 integrin for migration and invasion in this subset of cell lines. An expression pattern of BCL-2 family proteins that favor resistance to apoptosis was associated with enhanced migration, invasion, and MMP activity. Wild-type p53 cell lines migrated farther than mutant p53 cell lines.

Conclusions. Activities of MMP-2 and MMP-9 are the best predictors of glioma cell invasion. The αVβ3 integrin mediates migration and invasion in a subset of glioma cell lines, but these processes do not depend on αVβ3 integrin expression. Antiapoptotic BCL-2 family protein expression is a predictor of efficient migration and invasion.

Article Information

Address reprint requests to: Wolfgang Wick, M.D., Department of Neurology, University of Tübingen, School of Medicine, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany. email: wolfgang.wick@uni-tuebingen.de.

© AANS, except where prohibited by US copyright law.

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Figures

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    Findings of mmp mRNA and MMP protein expression and MMP-2 and MMP-9 activities in human malignant glioma cell lines. A: Messenger RNA was isolated from all cell lines and subjected to RT-PCR analysis for mmp-2, mmp-3, mmp-7, mmp-9, mmp-12, upa, or β-actin mRNA expression (for details, see Materials and Methods and Table 1). B: Supernatant proteins were analyzed for MMP-2, MMP-3, MMP-7, MT1-MMP, and TIMP-2 content by immunoblot analysis. Predicted molecular sizes are indicated in the parentheses on the right. Equal protein loading was ascertained using Ponceau S staining (data not shown). C: Activities of MMP-2 and MMP-9 were assessed using zymography. The MMP-2 (gelatinase A) activity is detected as a 72-kD band representing the activated form of the zymogen and, in some cell lines, as a 68-kD band, reflecting the fully active enzyme. The MMP-9 (gelatinase B) protein is detected as a 92-kD band corresponding to the activated zymogen. The figure panel shows an inverse photograph of the gelatin zymogram. All experiments were performed at least three times.

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    Bar graphs demonstrating migratory and invasive potentials in human malignant glioma cell lines. The glioma cells were examined for migration (A) and invasion (B) by using chemotaxis chamber assays with uncoated (A) or Matrigel-coated (B) membranes. Data are expressed as mean values ± standard errors of the means in three experiments.

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    Expression of the αVβ3 integrin and its requirement for glioma cell migration. A: Expression analysis for αV and β3 mRNA was performed by RT-PCR with β-actin serving as a control for equal loading. Negative controls consisted of aliquots containing water instead of cDNA. B: The αVβ3 protein levels at the cell surface were quantified by flow cytometry. Compare the bold specific curve with the dotted curve obtained using an isotype-matched control antibody. C: Bar graph depicting the data shown in B after being quantified by calculating the specific fluorescence indexes for each cell line as the ratio of mean fluorescence intensity of cells stained with the specific antibody by the mean fluorescence intensity obtained using the control antibody. D and E: Bar graphs demonstrating tumor cell migration (D) and invasion (E), as determined in Fig. 2, in the absence (black bars) or presence (open bars) of a neutralizing αVβ3 antibody (10 ng/ml). The degree of inhibition mediated by this antibody, compared with that of a control antibody, is expressed as residual migration or invasion in percentages. All experiments were performed at least three times.

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    Expression of BCL-2 family protein in human glioma cell lines. Soluble protein lysates of the glioma cells were examined for levels of BCL-2, BCL-XL, MCL-1, BAX, BAK, and BAD protein expression by using immunoblot analysis.

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