QUANTIFICATION OF INTRACELLULAR CATHEPSIN ACTIVITIES IN HUMAN LUNG-TUMOR CELL-LINES BY FLOW-CYTOMETRY

The cysteine proteases cathepsin B and cathepsin L are very likely inv olved in invasive processes of normal and malignant cells, they become relevant for a number of diseases and are possibly prognostic markers for the outcome of human lung cancer. Therefore, we have determined a ctivities of these related enzymes in cells and in cell extracts of hu man lung carcinoma cell lines of different cathepsin composition by fl ow cytometry and by spectrophotometry, respectively. To this end we ap plied the synthethic dipeptidyl substrates benzoxycarbonyl-arginyl-arg inine- and benzoxycarbonyl-phenyl-arginine- coupled to 4-methoxy-beta- naphthylamide, aminomethyl-coumarine or rhodamine R110, The apparent e nzymatic activities were differentially defined by protease inhibitors , particularly E-64 and CA-074. Independent of the dipeptidyl-composit ion more than 99 per cent of the apparent activity was due to cathepsi n B when 4-methoxy-beta-naphthylamide or aminomethylcoumarine were the leaving groups, The 4-methoxy-beta-naphthylamide precipitate used for detection of cell associated activities revealed a wide spectrum of e xcitation to fluorescence thwarting the application of other possible fluorescent tags. Therefore, its application is restricted to uniparam etric fluorescence investigations. Both dipeptidylgroups coupled to rh odamine R110 were promiscuous: only 25 to 30% of the apparent activity were due to cathepsin B; the predominant activity came from cathepsin L, irrespective whether intracellular or activities of cellular extra cts were analyzed, However, rhodamine R110-coupled substrates open the way for multiparametric fluorescent analysis of cathepsins B and L co ntaining cells if appropriate inhibitors for specification of the enzy matic activities are additionally applied, In very contrast to 4-metho xy-beta-naphthylamide, which causes irreparable damage to the cells, t he rhodamine substrates permit studies with living cells and live cell sorting.