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In vitro degradation of radiolabelled, intact basement membrane mediated by cellular plasminogen activator
Environmental Carcinogenesis Group, Laboratory of Pulmonary Function and Toxicology, National Institute of Environmental Health Sciences Research Triangle Park, NC 27709, USA
1To Whom reprint request should be addressed
A simple and unique procedure for the isolation of intact basement membrane from Syrian hamster lung was developed. The method involved an initial 24 h extraction of minced lungs with 0.3 M acetic acid followed by treatment with N-lauroyl sarcosine, an anionic detergent. The tissue was washed several times in 0.85% NaCl and subjected to deoxyribonuclease treatment followed by washings with 0.85% NaCl and distilled water. The residue was shown to be basement membrane by electron microscopy and by amino acid analysis. The basement membrane was radiolabelled by reductive alkylation and had a specific activity of 3–5 x 105 c.p.m./mg and 80–90% of the radiolabel was present in the glycoprotein component of the basement membrane. The abilities of malignant fibrosarcoma cell lines to degrade the [3H]basement membrane was examined. A simple assay to measure degradation of [3H]basement membrane was developed based on the solubilization of the insoluble material after degradation. When added to growing tumor cells in the presence of growth medium and serum, the [3H]basement membrane was solubilized extensively. The reaction was linear for 24 h at which time up to 90% of the labelled material had been released. In contrast, <5% of the label was solubilized in medium plus serum alone or in the presence of normal Syrian hamster embryo cells. A preneoplastic cell line was also capable of degrading the [3H]basement membrane. The solubilization of the [3H]basement membrane was primarily due to degradation of the glycoproteins of the basement membrane as shown by Sephadex G-200 gel chromatography. The abilities of the tumor cells to degrade the [3H]basement membrane correlated with their fibrinolytic activity and inhibitors of plasmin inhibited the reaction. Furthermore, the activity of the cells in this assay was dependent upon the presence of plasminogen in the medium. No degradation of [3H]basement membrane was observed if plasminogen depleted serum was employed, but complete degradation was accomplished if purified plasminogen was added to the medium with plasminogen-depleted serum. These results indicate a role for plasminogen activator in the pathogenesis of invasive tumor cells.
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