A human endothelial cDNA library was screened with a 1.7 kbp human fibroblast mammalian collagenase clone under low stringency conditions in an attempt to isolate cDNA clones of different collagenolytic metalloproteinases. From 500,000 plaques used for primary screening, nine positive clones were plaque purified after 3-4 rounds of screening. Three clones of 1.7, 1.5 and 0.7 kbp were subcloned into pGem 3Z. All three endothelial clones sequenced so far turned out to be identical to the published sequence of the fibroblast mammalian collagenase. The study on metalloproteinase activity during tumor-endothelial cell interaction has just started. The effect of humoral factors from endothelial cells on tumor cells or vice versa were studied by using serum-free conditioned medium from each cell type. Conditioned medium from human endothelial cells inhibited metalloproteinase activity of A2058 cells in a dose-responsive manner. On the other hand, A2058 melanoma cell conditioned medium did not affect the proteolytic activity of the endothelial cells using 0.1, 1, 5, 10, 15 and 25% conditioned medium, but the activity was increased severalfold in the presence of 50% or higher conditioned medium. These data suggest that the basement membrane degradation during tumor-endothelial cell interaction may depend on the balance between metalloproteinase and tumor inhibitor of metalloproteinase (TIMP) expression by the two cell types. Future studies will involve in situ hybridization techniques to examine expression of metalloproteinases and TIMP in cocultures of tumor and endothelial cells.
NIH ExPorter
NIH ExPorter