Identification of cell surface cathepsin B-like activity on murine melanomas and fibrosarcomas: modulation by butanol extraction.
Cathepsin B (CB) is a lysosomal cysteine protease that may play a role in the activation of extracellular degradative enzymes involved in the destruction of the subendothelial matrix and extravasation of metastatic tumor cells. In this study we have investigated the cell surface expression of a CB-like enzyme on the surface of tumor cell variants expressing both high and low metastatic potentials. Cell surface CB-like activity was demonstrated by incubation of intact viable cells and isolated plasma membranes with the selective chromogenic substrate N-carbobenzoxyvalyllysyllysylarginyl-4-methoxy-beta-naphthylamide. Cell surface CB activity required thiol activation and was blocked by the CB-selective protease inhibitors leupeptin, antipain, and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by inhibitors inactive against CB. Enzymatic activity was significantly reduced when assayed at pH 7 and greater. Although all tumor lines had detectable CB-like activity, we observed a correlation between the expression of cell surface CB-like activity and metastatic phenotype only with isolated plasma membranes, and not with whole cell preparations. Noncytolytic 2% butanol extraction, a technique known to increase the experimental metastatic propensity, also significantly increased cell surface CB-like activity. Incubation of extracted tumor cells with crude butanol extracts prepared from those cells restored the cell surface CB-like activity to that of the unextracted controls, suggesting that the increased enzyme activity observed following extraction may be due to the release of an endogenous cysteine protease inhibitor. These results demonstrate that a CB-like protease is expressed on the surface of several murine tumor cells and that an endogenous inhibitor may play a role in determining experimental metastatic phenotype.
Keren
Z
Z
Department of Immunology, University of Texas M.D. Anderson Hospital and Tumor Institute, Houston 77030.
LeGrue
S J
SJ
D020001:1-Butanol; D000818:Animals; D000440:Butanols / Q000494:pharmacology*; D002401:Cathepsin B / Q000032:analysis* / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D005354:Fibrosarcoma / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008546:Melanoma, Experimental / Q000201:enzymology*; D051379:Mice; D008809:Mice, Inbred C3H; D009362:Neoplasm Metastasis; D011480:Protease Inhibitors / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000440:Butanols; D011480:Protease Inhibitors; D020001:1-Butanol; D002401:Cathepsin B