Abstract
Tumor cell adhesion and proteolysis of the extracellular matrix proteins surrounding the cells are tightly linked processes in tumor invasion. In this study, we sought to identify components of the cell surface of a vertical growth phase melanoma cell line, WM1341D, that mediate invasive cellular behavior. We determined by antisense inhibition that melanoma chondroitin sulfate proteoglycan (MCSP) and membrane-type 3 matrix metalloproteinase (MT3-MMP) expressed on WM1341D are required for invasion of type I collagen and degradation of type I gelatin. MT3-MMP co-immunoprecipitated with MCSP in WM1341D melanoma cells cultured on type I collagen or laminin. The association between MT3-MMP and MCSP was largely disrupted by removing chondroitin sulfate glycosaminoglycan (CS) from the cell surface, suggesting CS could mediate the association between the two cell surface core proteins. Recombinant MT3-MMP and MT3-MMP from whole cell lysates of WM1341D cells were specifically eluted from CS- conjugated affinity columns. The results indicate that MT3-MMP possesses the potential to promote melanoma invasion and proteolysis and that the formation of a complex between MT3-MMP and MCSP may be a crucial step in activating these processes.
Highlights
Tumor cell adhesion and proteolysis of the extracellular matrix proteins surrounding the cells are tightly linked processes in tumor invasion
melanoma chondroitin sulfate proteoglycan (MCSP) Mediates Melanoma Invasion into Type I Collagen— vertical growth phase (VGP) melanoma cells aggressively invade the collagen-rich dermis in vivo as a presage to metastasis
We previously reported that cell surface chondroitin sulfate proteoglycans facilitate melanoma invasion into type I collagen gels and/or basement membrane matrices in vitro [4, 5]
Summary
Cell Culture—The vertical growth phase (VGP) human melanoma cell line WM1341D was maintained in RPMI 1640 supplemented with 10% FCS [31,32,33]. Beads were extensively washed with lysis buffer, and bound proteins were released in 50 l of PBS containing 1% SDS for 10 min at room temperature and diluted in 1 ml of 50 mM Tris-HCl (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2, 1 mM NEM, 1 mM PMSF. Transfectants were lysed in 100 mM Tris-HCl (pH7.5) containing 1% Triton X-100, 0.14 M NaCl, 10 mM EDTA, 1 mM NEM, and 1 mM PMSF and incubated with anti-FLAG M2-agarose affinity beads overnight at 4 °C. The beads were washed four times in lysis buffer, bound MT3-MMP was released in SDS sample buffer, and blotted with anti-FLAG and HRP-goat anti-mouse IgG
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