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Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter.

Beliën AT, Paganetti PA, Schwab ME - J. Cell Biol. (1999)

Bottom Line: Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors.Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property.These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.

View Article: PubMed Central - PubMed

Affiliation: Brain Research Institute, University of Zurich and Swiss Federal Institute of Technology, 8057 Zurich, Switzerland. belien@hifo.unizh.ch

ABSTRACT
Invasive glioma cells migrate preferentially along central nervous system (CNS) white matter fiber tracts irrespective of the fact that CNS myelin contains proteins that inhibit cell migration and neurite outgrowth. Previous work has demonstrated that to migrate on a myelin substrate and to overcome its inhibitory effect, rat C6 and human glioblastoma cells require a membrane-bound metalloproteolytic activity (C6-MP) which shares several biochemical and pharmacological characteristics with MT1-MMP. We show now that MT1-MMP is expressed on the surface of rat C6 glioblastoma cells and is coenriched with C6-MP activity. Immunodepletion of C6-MP activity is achieved with an anti-MT1-MMP antibody. These data suggest that MT1-MMP and the C6-MP are closely related or identical. When mouse 3T3 fibroblasts were transfected with MT1-MMP they acquired the ability to spread and migrate on the nonpermissive myelin substrate and to infiltrate into adult rat optic nerve explants. MT1-MMP-transfected fibroblasts and C6 glioma cells were able to digest bNI-220, one of the most potent CNS myelin inhibitory proteins. Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors. Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property. These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.

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(a) Optic nerve explant infiltration assay. Adult rat optic nerves were  placed underneath a teflon ring sealed to a  culture dish with silicon grease. Hoechst  dye-prelabeled C6 cells, MT1-MMP–transfected fibroblasts, or mock-transfected fibroblasts (100,000 cells) were placed in the  middle chamber, in contact with the tips of  the nerves (a). After 7 d Hoechst dye-labeled  cells are observed infiltrating the optic  nerve explants in longitudinal sections.  MT1-MMP–transfected fibroblasts (b) but  not mock-transfected fibroblasts (c) infiltrated the optic nerves. Enlargements show  a high density of MT1-MMP–transfected fibroblasts at a distance of 1.6 mm. Very few  mock-transfected fibroblasts were found at  this depth. C6 cells infiltrated the optic  nerve to the same extent as the MT1-MMP– transfected fibroblasts (d). Bar, 0.3 mm.
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Figure 9: (a) Optic nerve explant infiltration assay. Adult rat optic nerves were placed underneath a teflon ring sealed to a culture dish with silicon grease. Hoechst dye-prelabeled C6 cells, MT1-MMP–transfected fibroblasts, or mock-transfected fibroblasts (100,000 cells) were placed in the middle chamber, in contact with the tips of the nerves (a). After 7 d Hoechst dye-labeled cells are observed infiltrating the optic nerve explants in longitudinal sections. MT1-MMP–transfected fibroblasts (b) but not mock-transfected fibroblasts (c) infiltrated the optic nerves. Enlargements show a high density of MT1-MMP–transfected fibroblasts at a distance of 1.6 mm. Very few mock-transfected fibroblasts were found at this depth. C6 cells infiltrated the optic nerve to the same extent as the MT1-MMP– transfected fibroblasts (d). Bar, 0.3 mm.

Mentions: Optic nerve explants in chamber cultures were prepared as described by Schwab and Thoenen (1985). In brief, the optic nerves were rapidly dissected from 3–4-mo-old Lewis rats and then trimmed. Subsequently, the nerves were X-irradiated at 5,000 g for 7 min to reduce glial cell proliferation and placed under a teflon ring sealed to a culture dish with silicon grease. Three chambers in contact only via the explants were obtained in this way (see Fig. 9 a). C6 glioblastoma cells, MT1-MMP–transfected, or mock-transfected fibroblasts were prelabeled with Hoechst-dye for 1.5–3 h (50 μl Hoechst-dye/10 ml DME/FCS), washed three times with DME/ FCS, and then 100,000 cells were placed into the middle chamber. Cultures were incubated for 7 d. Medium was exchanged every other day. At the end of the experiment the optic nerves were fixed for 48 h in 4% PFA containing 5% sucrose, washed, removed from the cultures, flat mounted in Tissue Tek (Sakura), and then frozen at −40°C. 20-μm longitudinal sections were serially cut on a cryostate. Labeled infiltrated cells were counted under a fluorescences microscope (Zeiss Axiophot) starting from the tip of the nerve exposed to the middle, cell-containing chamber. The cells were counted at distances of 0.4–0.8, 0.8–1.2, and 1.2–1.8 mm from the middle chamber stump. Three sections were counted for each distance, and the cell numbers were extrapolated for the thickness of the nerve. 12 nerves infiltrated by MT1-MMP–transfected fibroblasts, 12 nerves with mock-transfected fibroblasts, and eight nerves with C6 cells were evaluated.


Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter.

Beliën AT, Paganetti PA, Schwab ME - J. Cell Biol. (1999)

(a) Optic nerve explant infiltration assay. Adult rat optic nerves were  placed underneath a teflon ring sealed to a  culture dish with silicon grease. Hoechst  dye-prelabeled C6 cells, MT1-MMP–transfected fibroblasts, or mock-transfected fibroblasts (100,000 cells) were placed in the  middle chamber, in contact with the tips of  the nerves (a). After 7 d Hoechst dye-labeled  cells are observed infiltrating the optic  nerve explants in longitudinal sections.  MT1-MMP–transfected fibroblasts (b) but  not mock-transfected fibroblasts (c) infiltrated the optic nerves. Enlargements show  a high density of MT1-MMP–transfected fibroblasts at a distance of 1.6 mm. Very few  mock-transfected fibroblasts were found at  this depth. C6 cells infiltrated the optic  nerve to the same extent as the MT1-MMP– transfected fibroblasts (d). Bar, 0.3 mm.
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Related In: Results  -  Collection

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Figure 9: (a) Optic nerve explant infiltration assay. Adult rat optic nerves were placed underneath a teflon ring sealed to a culture dish with silicon grease. Hoechst dye-prelabeled C6 cells, MT1-MMP–transfected fibroblasts, or mock-transfected fibroblasts (100,000 cells) were placed in the middle chamber, in contact with the tips of the nerves (a). After 7 d Hoechst dye-labeled cells are observed infiltrating the optic nerve explants in longitudinal sections. MT1-MMP–transfected fibroblasts (b) but not mock-transfected fibroblasts (c) infiltrated the optic nerves. Enlargements show a high density of MT1-MMP–transfected fibroblasts at a distance of 1.6 mm. Very few mock-transfected fibroblasts were found at this depth. C6 cells infiltrated the optic nerve to the same extent as the MT1-MMP– transfected fibroblasts (d). Bar, 0.3 mm.
Mentions: Optic nerve explants in chamber cultures were prepared as described by Schwab and Thoenen (1985). In brief, the optic nerves were rapidly dissected from 3–4-mo-old Lewis rats and then trimmed. Subsequently, the nerves were X-irradiated at 5,000 g for 7 min to reduce glial cell proliferation and placed under a teflon ring sealed to a culture dish with silicon grease. Three chambers in contact only via the explants were obtained in this way (see Fig. 9 a). C6 glioblastoma cells, MT1-MMP–transfected, or mock-transfected fibroblasts were prelabeled with Hoechst-dye for 1.5–3 h (50 μl Hoechst-dye/10 ml DME/FCS), washed three times with DME/ FCS, and then 100,000 cells were placed into the middle chamber. Cultures were incubated for 7 d. Medium was exchanged every other day. At the end of the experiment the optic nerves were fixed for 48 h in 4% PFA containing 5% sucrose, washed, removed from the cultures, flat mounted in Tissue Tek (Sakura), and then frozen at −40°C. 20-μm longitudinal sections were serially cut on a cryostate. Labeled infiltrated cells were counted under a fluorescences microscope (Zeiss Axiophot) starting from the tip of the nerve exposed to the middle, cell-containing chamber. The cells were counted at distances of 0.4–0.8, 0.8–1.2, and 1.2–1.8 mm from the middle chamber stump. Three sections were counted for each distance, and the cell numbers were extrapolated for the thickness of the nerve. 12 nerves infiltrated by MT1-MMP–transfected fibroblasts, 12 nerves with mock-transfected fibroblasts, and eight nerves with C6 cells were evaluated.

Bottom Line: Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors.Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property.These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.

View Article: PubMed Central - PubMed

Affiliation: Brain Research Institute, University of Zurich and Swiss Federal Institute of Technology, 8057 Zurich, Switzerland. belien@hifo.unizh.ch

ABSTRACT
Invasive glioma cells migrate preferentially along central nervous system (CNS) white matter fiber tracts irrespective of the fact that CNS myelin contains proteins that inhibit cell migration and neurite outgrowth. Previous work has demonstrated that to migrate on a myelin substrate and to overcome its inhibitory effect, rat C6 and human glioblastoma cells require a membrane-bound metalloproteolytic activity (C6-MP) which shares several biochemical and pharmacological characteristics with MT1-MMP. We show now that MT1-MMP is expressed on the surface of rat C6 glioblastoma cells and is coenriched with C6-MP activity. Immunodepletion of C6-MP activity is achieved with an anti-MT1-MMP antibody. These data suggest that MT1-MMP and the C6-MP are closely related or identical. When mouse 3T3 fibroblasts were transfected with MT1-MMP they acquired the ability to spread and migrate on the nonpermissive myelin substrate and to infiltrate into adult rat optic nerve explants. MT1-MMP-transfected fibroblasts and C6 glioma cells were able to digest bNI-220, one of the most potent CNS myelin inhibitory proteins. Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors. Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property. These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.

Show MeSH
Related in: MedlinePlus