Tumor-associated cysteine proteinase activities in human melanoma cells and fibroblasts of different origin.
Specific catalytic activities of cysteine proteinases including cathepsins B (EC 3.4.22.1) and L (EC 3.4.22.15) in human melanoma cell lines SK-MEL-28, SK-MEL-30, MEL-HO and in fibroblasts of different origin are reported. Cell line-specific pH profiles of these cysteine proteinases were determined fluorometrically with benzyloxycarbonyl-phenylalanyl-arginine-amidomethylcoumarine (Z-Phe-Arg-AMC) under saturated conditions. Single activities of cathepsins B and L were inactivated by urea and by benzyloxycarbonyl-phenylalanyl-phenylalanine-diazomethylketone (Z-Phe-Phe-CHN2) in order to describe the activities of these enzymes separately. The melanoma cell line MEL-HO, which originated from a primary lesion, showed highest activity of an unknown cysteine proteinase. This enzyme is not inactivated by urea and Z-Phe-Phe-CHN2 and has a Michaelis constant (K(M) value) of approximately 1 mM. The specific characteristics suggest that it is a tumor-associated cathepsin B. In addition, high invasive subpopulations of SK-MEL-28 and SK-MEL-30 cell lines isolated by an invasion assay showed higher proteinase activities than the low invasive subpopulations. Furthermore, in fibroblasts originating from melanoma tissue cysteine proteinase activities were increased compared to normal skin fibroblasts. In conclusion, these results indicate that these cysteine proteinases shown here are tumor-associated proteinases, possibly facilitating invasion and dissemination of melanoma cells.
Mayer
P
P
Department of Dermatology, University of Tübingen, Germany.
Schmid
H
H
Schaber
B
B
Fierlbeck
G
G
D002460:Cell Line; D003374:Coumarins; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003978:Diazomethane / Q000031:analogs & derivatives; D004151:Dipeptides; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008545:Melanoma / Q000201:enzymology* / Q000473:pathology; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D009363:Neoplasm Proteins / Q000378:metabolism*; D012867:Skin / Q000166:cytology; D012878:Skin Neoplasms / Q000201:enzymology / Q000473:pathology; D014407:Tumor Cells, Cultured; D014508:Urea / Q000494:pharmacology
D003374:Coumarins; D004151:Dipeptides; D009363:Neoplasm Proteins; D003978:Diazomethane; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; C031947:benzyloxycarbonylphenylalanylphenylalanine diazomethyl ketone; D014508:Urea; D003546:Cysteine Endopeptidases