Substratum acidification and proteinase activation by murine B16F10 melanoma cultures.
Murine B16F10 melanoma cells, cultured within 0.7% agarose gels containing the fluorescent proteinase substrate acetamidofluorescein-BSA, catalyze the hydrolysis of the substrate in the region immediately surrounding the cell. Fluorescence ratio measurements on hydrolyzed substrate correlate with an average pH of 5.5 +/- 0.2 in the adjacent substratum region. Enzymatic activity within the gel is partially inhibited by leupeptin, pepstatin, phenylmethylsulfonyl fluoride. EDTA and by anti-human cathepsin B, suggesting potential roles for thiol-, aspartic- and metalloproteinases. The time-course of fluorescence intensity, correlated with substratum pH measurements, suggest that substrate hydrolysis is catalyzed by enzymes with pH optima of < 5.5. Invasion by these cells through thin barriers of reconstituted basement membrane gel (Matrigel) is totally blocked by the thiol proteinase inhibitor, leupeptin. It is suggested that secreted or cell-surface acid proteinase enzymes, activated by the cell-mediated local hyperacidity, are involved in substrate hydrolysis and that these enzymes may be important in invasiveness by this cell-line.
Young
P R
PR
Department of Chemistry, University of Illinois, Chicago 60680.
Spevacek
S M
SM
D000818:Animals; D002401:Cathepsin B / Q000032:analysis; D002460:Cell Line / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D005452:Fluoresceins; D005782:Gels; D006863:Hydrogen-Ion Concentration; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000201:enzymology
D005452:Fluoresceins; D005782:Gels; D007976:Leupeptins; D012710:Serum Albumin, Bovine; C059221:acetamidofluorescein; D010450:Endopeptidases; D002401:Cathepsin B; C032854:leupeptin