Journal of Neuro-Oncology

, Volume 39, Issue 3, pp 205–216

The importance of cell density in the interpretation of growth factor effects on collagenase IV activity release and extracellular matrix production from C6 astrocytoma cells

  • Masashi Tamaki
  • Warren McDonald
  • Rolando F. Del Maestro


We have examined the influence of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) on the release of collagenase type IV activity and the production of extracellular matrix (ECM) molecules using C6 astrocytoma cells in monolayer culture. Collagenase type IV activity was significantly increased in a dose dependent manner in the low cell density group by treatment with FGF-2 and VEGF but significantly decreased in a dose dependent fashion in the high cell density group. These results were corroborated using Western blot technique with an antibody to gelatinase A. Addition of exogenous laminin and fibronectin to the media decreased collagenase type IV activity in a dose dependent fashion with the minimum concentration of 0.1 μg/ml. Laminin and fibronectin reached a concentration of 0.1 μg/ml in only the high cell density group after treatment with the growth factors tested. These findings indicate that C6 astrocytoma cells appear to have two regulatory mechanisms for collagenase type IV activity which are dependent on cell density. In a low cell density, C6 astrocytoma cells respond to the dominant effect of FGF-2 and VEGF by increasing the release of collagenase IV activity. In a high cell density collagenase type IV activity is decreased due to it's down regulation by released ECM molecules in response to FGF-2 and VEGF. These regulatory mechanisms may be crucial to the understanding of the coordination of tumor-associated angiogenesis by malignant glial cells.

C6 astrocytoma cell density collagenase IV basic fibroblast growth factor vascular endothelial growth factor extracellular matrix molecules 


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  1. 1.
    Park JE, Keller GA, Ferrara N: The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithclial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell 4: 1317–1326, 1993Google Scholar
  2. 2.
    Thorgeirsson UP, Lindsay CK, Cottam DW, Gomez DE: Tumor invasion, proteolysis, and angiogenesis. J Neuro-Oncol 18: 89–103, 1994Google Scholar
  3. 3.
    Folkman J, Klagsbrun M, Sasse J, Wadzinski M, Ingber D, Vlodavsky I: A heparin-binding angiogenic protein — basic fibroblast growth factor — is stored within basement membrane. Am J Pathol 130: 393–400, 1988Google Scholar
  4. 4.
    Edwards DR, Murphy G, Reynolds JJ, Whitham SE, Docherty AJP, Angel P, Heath JK: Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 6: 1899–1904, 1987Google Scholar
  5. 5.
    Cornelius LA, Nehring LC, Roby JD, Parks WC, Welgus HG: Human dermal microvascular endothelial cells produce matrix metalloproteinases in response to angiogenic factors and migration. J Invest Dermatol 105: 170–176, 1995Google Scholar
  6. 6.
    Moscatelli D, Presta M, Rifkin DB: Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration. Proc Natl Acad Sci USA 83: 2091–2095, 1986Google Scholar
  7. 7.
    Moscatelli D, Rifkin DB: Membrane and matrix localization of proteinases: a common theme in tumor cell invasion and angiogenesis. Biochim Biophys Acta 948: 67–85, 1988Google Scholar
  8. 8.
    Unemori EN, Ferrara N, Bauer EA, Amento EP: Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells. J Cell Physiol 153: 557–562, 1992Google Scholar
  9. 9.
    Ingber D: Extracellular matrix and cell shape: potential control points for inhibition of angiogenesis. J Cell Biochem 47: 236–241, 1991Google Scholar
  10. 10.
    Risau W, Lemmon V: Changes in the vascular extracellular matrix during embryonic vasculogenesis and angiogenesis. Dev Biol 125: 441–450, 1988Google Scholar
  11. 11.
    Bowersox JC, Sorgente N: Chemotaxis of aortic endothelial cells in response to fibronectin. Cancer Res 42: 2547–2551, 1982Google Scholar
  12. 12.
    Jaye M, McConathy E, Drohan W, Tong B, Deuel T, Maciag T: Modulation of the sis gene transcript during endothelial cell differentiation in vitro. Science 228: 882–885, 1985Google Scholar
  13. 13.
    Kubota Y, Kleinman HK, Martin GR, Lawley TJ: Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol 107: 1589–1598, 1988Google Scholar
  14. 14.
    Grant DS, Tashiro K, Segui-Real B, Yamada Y, Martin GR, Kleinman HK: Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vivo. Cell 58: 933–943, 1989Google Scholar
  15. 15.
    Del Maestro RF, Vaithilingam IS, McDonald W: Degradation of collagen type IV by C6 astrocytoma cells. J Neurooncol 24: 75–81, 1995Google Scholar
  16. 16.
    Tamaki M, McDonald W, Del Maestro RF: Release of collagen type IV degrading activity from C6 astrocytoma cells and cell density. J Neurosurg 84: 1013–1019, 1996Google Scholar
  17. 17.
    Vaithilingam IS, McDonald W, Stroude EC, Cook RA, Del Maestro RF: Proteolytic activity during the growth of C6 astrocytoma in the murine spheroid implantation model. Can J Neurol Sci 19: 17–22, 1992Google Scholar
  18. 18.
    Vaithilingam IS, McDonald W, Brown NK, Stroude E, Cook RA, Del Maestro RF: Serum proteolytic activity during the growth of C6 astrocytoma. J Neurosurg 77: 595–600, 1992Google Scholar
  19. 19.
    Vaithilingam IS, McDonald W, Malott DW, Del Maestro RF: An extracellular proteasome-like structure from C6 astrocytoma cells with serine collagenase type IV activity and metallo-dependent activity on α-casein and β-insulin. J Biol Chem 270: 4588–4593, 1995Google Scholar
  20. 20.
    Brown PD, Levy AT, Margulies IMK, Liotta LA, Stetler-Stevenson WG: Independent expression and cellular processing of Mr 72000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines. Cancer Res 50: 6184–6191, 1990Google Scholar
  21. 21.
    Vaithilingam IS, Stroude EC, McDonald W, Del Maestro RF: General protease and collagenase (IV) activity in C6 astrocytoma cells, C6 spheroids and implanted C6 spheroids. J Neurooncol 10: 203–212, 1991Google Scholar
  22. 22.
    Stetler-Stevenson WG, Krutzsch HC, Wacher MP, Margulies IMK, Liotta LA: The activation of human type IV collagenase proenzyme. J Biol Chem 264: 1353–1356, 1989Google Scholar
  23. 23.
    Koochekpour S, Merzak A, Pilkington GJ: Growth factors and gangliosides stimulate laminin production by human glioma cells in vitro. Neurosci Lett 186: 53–56, 1995Google Scholar
  24. 24.
    Del Maestro RF, Vaithilingam IS, McDonald W, Weiss JB: Elevated levels of a collagenase type IV activating factor in C6 astrocytoma cells. J Cell Biochem Suppl 18D: 142, 1994Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Masashi Tamaki
    • 1
  • Warren McDonald
    • 1
  • Rolando F. Del Maestro
    • 1
  1. 1.Brain Research Laboratories, Experimental Research Unit, Division of Neurosurgery, London Health Sciences CenterUniversity of Western Ontario, LondonOntarioCanada

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