Increased invasive capacity of connexin43-overexpressing malignant glioma cells

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Object. Malignant glioma cells, similar to astrocytes, express connexin43 (Cx43) universally but at widely varied levels. Data from previous studies have demonstrated that malignant glioma cells form functional gap junction channels among themselves as well as with astrocytes and that such a communication has the potential to modulate the phenotypic characteristics of astrocytes. Recently, gap junctions have been demonstrated to play a role in the invasive phenotype of malignant gliomas. In this study, the authors have further investigated the motility and invasion ability of Cx43-overexpressing and Cx43-deficient malignant glioma cells.

Methods. Using a standard invasion system of a Matrigel transwell invasion chamber, the authors found that the number of Cx43-transfected C6 glioma cells (C6-Cx43 cells) migrating through the Matrigel-coated membrane was similar to that of mock-transfected control cells (C6-mock cells) during the first 24 hours, but increased significantly thereafter. When these cells were cocultured with astrocytes, the number of invading C6-Cx43 cells was more than threefold greater than the number of invading C6-mock cells. Results of an in vitro cell motility assay also demonstrated that C6-Cx43 cells were more motile and scatter-active than C6-mock cells. Furthermore, zymographic analysis of MMPs, an important determinant in glioma invasion, demonstrated that the amounts of MMP-2 and MMP-9 in culture medium collected from C6-Cx43 cells were orders of magnitude higher than those from C6-mock cells. In addition, BB-94, a synthetic MMP inhibitor, significantly inhibited C6-Cx43 cell invasion.

Conclusions. The overexpression of gap junction proteins in glioma cells and the intercellular communication between tumor and nontumor glia cells may play important roles in the facilitation of glioma cell invasion.

Article Information

Address reprint requests to: William T. Couldwell, M.D., Ph.D., Department of Neurosurgery, University of Utah, 30 North 1900 East, Suite 3B409, Salt Lake City, Utah, 84132. email: william.couldwell@hsc.utah.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    Overexpression of Cx43 and Cx32 in transfectants and actin cytoskeleton organization. Upper: Immunocytochemical study results demonstrating that C6-Cx43 and C6-Cx32 cells (A and C) strongly express gap junction proteins, whereas C6-mock cells (B) do not express immunoreactive plaques (green). Texas Red-phalloidin staining of stress fibers was combined with outlining of cell—cell boundaries and extended uninterrupted from cell to cell (red). Lower: Dye transfer assay results demonstrating that functional gap junction coupling formed in cocultures of rat astrocytes with C6-Cx43 (D) but not with C6-mock (E) or C6-Cx32 (F) cells. C6-Cx43 cells = connexin43-transfected C6 glioma cells; C6-Cx32 cells = connexin32-transfected C6 glioma cells; C6-mock cells = vector control—transfected C6 glioma cells.

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    Invasion of connexin- and mock-transfected C6 glioma cells, Part 1. Left: Both C6-Cx43 and C6-mock cells were prelabeled with membrane dyes and seeded on top of Matrigel-coated chambers. After a 24-hour incubation, the cells that had invaded the Matrigel-coated transwell inserts (pore size 8 µm) were quantified with the aid of confocal microscopy. The C6-Cx43 cells migrated through the Matrigel-coated membrane at a migration rate similar to that of the mock-transfected C6 cells (C6-Mock)/a and c. When they were mixed with astrocytes, however, the C6-Cx43 cells exhibited a migration rate higher than that of C6-mock cells (b and d). Right: Bar graph showing the statistical results. Data shown are representative of three independent experiments and data are presented as the means ± standard error of the mean (SEM); **p < 0.01.

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    Invasion of connexin- and mock-transfected C6 glioma cells, Part 2. Upper: Using a cell invasion assay kit, C6-Cx43, C6-Cx32, or C6-mock cells were seeded on the top of ECM-coated chambers with or without rat astrocytes. After a 48-hour incubation, the migrated cells were detected using light microscopy. The C6-Cx43 cells (a and b), but not the C6-Cx32 (c and d) and C6-mock (e and f) cells, exhibited an increased invasion rate when they were mixed with astrocytes that express Cx43. Lower: Bar graph (mean ± standard deviation [SD], n = 12) showing the representative of two independent experiments (**p < 0.01).

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    Results of gelatin zymographic analysis of MMP-2 and MMP-9 in conditioned media from mock-, Cx43, and Cx32-transfected C6 glioma cells. Upper: Gelatin zymograph demonstrating MMP levels. Equal amounts of protein were electrophoresed on SDS—polyacrylamide gel electrophoresis gels containing gelatin as a substrate to detect enzyme activity. Samples were added in quadruplicate: conditioned media from C6-Cx43 cells (lanes 1–4), C6-mock cells (lanes 5–8), and C6-Cx32 cells (lanes 9–12). Lower: Bar graph demonstrating results of zymographic analysis. Data are representative of two separate assay sets (relative activity; mean ± SD, n = 4).

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    Bar graph demonstrating the invasive profile of Cx43-transfected cells correlated with metalloproteinase activation. The C6-Cx43 cells were placed in Matrigel transwell invasion chambers that had been preseeded with or without rat astrocytes and treated with the MMP inhibitor BB-94. After incubation for 24 hours, the C6-Cx43 cells invading through the Matrigel or the confluent monolayer of astrocytes plus Matrigel were counted. Note that the percentages of invading C6-Cx43 cells were significantly reduced by BB-94. Data shown are representative of two separate assay sets and are presented as the means ± SEM (**p < 0.01).

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    Cell growth and motility of Cx43- and mock-transfected C6 glioma cells. To exclude the possibility that any increase in the migrating cell numbers revealed on the radial dish assay was not simply caused by a difference in proliferation potential of the Cx43-transfected cells, both cell growth and motility were tested. Upper: Graph demonstrating that C6-Cx43 cells exhibit a similar or lower proliferation ability than C6-mock cells, as evidenced by counting cell numbers. Lower: Radial dish assay shows an increased motility in C6-Cx43 cells. The C6-Cx43 cells or C6-mock cells were plated in the center of a 100-mm-diameter round Petri dish at 2 × 104 cells in 200 µl of medium, as described in Materials and Methods. When a circular zone of plated cells in the center of the Petri dish was established, the cells were washed and incubated for 7 days. The photograph is representative of two separate assays performed in triplicate for each group. The C6-Cx43 cells exhibited greater motility and clonogenicity after they migrated to a new location.

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