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Cathepsins B and L are catabolic lysosomal enzymes but are likely candidates for extracellular proteolysis in normal and malignant processes. The signal mediator 12(S)-HETE selectively triggers a shot-gun release of cathepsin B. We have therefore investigated the intracellular distribution of cathepsins in unstimulated and 12(S)-HETE-stimulated tumor cells. Cathepsins B and L have only limited colocalization, which is found in the regions of synthesis and sorting (endoplasmic reticulum, Golgi, trans Golgi network). Treatment by 12(S)-HETE scatters cathepsin B but not cathepsin L and proform of cathepsin B. Colocalization with both mannose 6-phosphate receptors is very limited for both cathepsins. But extensive colocalization of cathepsin B and the endosomal/lysosomal marker CD63 (LIMP-I) documents the main fraction of the enzyme in these compartments. The supposed non-lysosomal fraction of cathepsin B is very likely the secretable material which follows a regulated secretory pathway. Storage and regulated secretion in tumor cells support extracellular proteolysis as a means in invasion which may lead to metastasis. But the mechanisms by which cells might acquire and eventually apply this means is still unknown.

Cathepsins B and L play roles in intracellular and extracellular proteolysis in normal and malignant processes. A directed extracellular proteolysis by regulated secretion could facilitate the process of invasion. We have therefore investigated the effect of the physiological signal mediator 12(S)-hydroxy-eicosatetraenoic acid on the release of cathepsins B and L in normal and malignant human lung cells. Quantitative determinations of cathepsin activities were done by flow cytometry and spectrofluorometry using synthetic dipeptidyl substrates coupled to fluorogens. Most interestingly, a difference in the secretion of cathepsins B and L was found: only release of active cathepsin B was detected. The effect was specific for 12(S)-hydroxy-eicosatetraenoic acid, 12(R)-hydroxy-eicosatetraenoic acid, and 5(S)-hydroxy-eicosatetraenoic acid were ineffective. The response was immediate but a substantial amount of nonreleasable activity remained cell bound. Alveolar macrophages, Wi-38 fibroblasts, and tumor cells derived from large cell carcinomas and adenocarcinomas were sensitive to 12(S)-hydroxy-eicosatetraenoic acid, but cells from undifferentiated squamous cell carcinomas were not. Sensitivity did not parallel malignancy but more likely the degree of differentiation of cells. The investigated tumor cell lines showed no detectable endogenous 12-lipoxy-genase activity to synthesize 12(S)-hydroxy-eicosatetraenoic acid from arachidonate; therefore, we assume a paracrine mechanism for 12(S)-hydroxy-eicosatetraenoic acid action. Protein kinase C alpha, a key enzyme involved in 12(S)-hydroxy-eicosatetraenoic acid-elicited responses, was expressed in all sensitive tumor cells, but insignificantly in a sensitive normal cell line and an insensitive tumor cell line. From our experiments we propose two separate intracellular pools of active cathepsin B: an unreleasable, lysosomal fraction and a fraction available for regulated secretion. Different processing and sorting mechanisms may be responsible for the generation of these cathepsin B-fractions in these pools.

The cysteine proteases cathepsin B and cathepsin L are very likely involved 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 activities of these related enzymes in cells and in cell extracts of human lung carcinoma cell lines of different cathepsin composition by flow cytometry and by spectrophotometry, respectively. To this end we applied the synthetic dipeptidyl substrates benzoxycarbonyl-arginyl-arginine- and benzoxycarbonyl-phenyl-arginine- coupled to 4-methoxy-beta-naphthylamide, aminomethyl-coumarine or rhodamine R110. The apparent enzymatic activities were differentially defined by protease inhibitors, particularly E-64 and CA-074. Independent of the dipeptidyl-composition more than 99 per cent of the apparent activity was due to cathepsin 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 excitation to fluorescence thwarting the application of other possible fluorescent tags. Therefore, its application is restricted to uniparametric fluorescence investigations. Both dipeptidylgroups coupled to rhodamine 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 extracts were analyzed. However, rhodamine R110-coupled substrates open the way for multiparametric fluorescent analysis of cathepsins B and L containing cells if appropriate inhibitors for specification of the enzymatic activities are additionally applied. In very contrast to 4-methoxy-beta-naphthylamide, which causes irreparable damage to the cells, the rhodamine substrates permit studies with living cells and live cell sorting.