Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites.
Virus-transformed fibroblasts show an increased production of proteases as well as loss of extracellular adhesive proteins. To determine whether these transformation-associated events are related, we investigated the capacity of Rous sarcoma virus-transformed cells (embryonic chick fibroblasts and mouse BALB/c 3T3) to degrade fibronectin by using a novel cross-linked protein substratum: fluorescence-labeled or radiolabeled fibronectin covalently linked to the surface of a fixed gelatin film. In serum-containing medium, the coupled fibronectin was not released when incubated without cells, and only a small amount was released when incubated with nontransformed cells. However, when transformed cells were seeded on the radiolabeled fibronectin-coupled substratum, there was a threefold increase in the time-dependent release of radioactivity into the medium. The released material was characterized as peptides with molecular sizes of less than 30,000 daltons. Correspondingly, growth of transformed cells on the rhodamine-fibronectin substratum resulted in the appearance of discrete negative fluorescent spots beneath the cells and along their migratory paths, whereas a uniform fluorescent carpet was detected with nontransformed cells. The release of radioactivity was partially inhibited by protease inhibitors, including alpha 2-macroglobulin, leupeptin, and benzamidine, but the negative fluorescent spots appeared unaffected by any of these inhibitors. However, both the release of radiolabeled peptides and the appearance of fluorescence-negative spots were inhibited by 1,10-phenanthroline at concentrations that did not affect cellular attachment and protein synthesis, thus supporting a role for proteases in localized degradation of fibronectin substratum. These fluorescence-negative spots coincided with sites of fibronectin disappearance as judged by indirect labeling with antibodies to cellular fibronectin. In addition, immunofluorescent analyses showed a correlation between vinculin localization and the negative fibronectin spots found under transformed cells, indicating that degradation occurs at cell substratum contact sites. These results can be correlated with other transformation-associated phenotypic changes, and are discussed in terms of the invasion of tumor cells into the extracellular matrix.
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts; D005353:Fibronectins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005780:Gelatin; D006651:Histocytochemistry; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology
D005353:Fibronectins; D011480:Protease Inhibitors; D005780:Gelatin; D010447:Peptide Hydrolases