CATHEPSIN-B ACTIVITY IN HUMAN LUNG-TUMOR CELL-LINES - ULTRASTRUCTURAL-LOCALIZATION, PH SENSITIVITY, AND INHIBITOR STATUS AT THE CELLULAR-LEVEL

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefins A and B, at the cellular level in human tumor cell lines HS-24, derived from a primar y lung tumor (squamous cell), and SB-3, derived from a metastasis (lun g adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and functionally related cathepsin L, bu t not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefin B was found in both cell types. Using a specific fluorogenic cy tochemical assay, the intracellular activity of the enzyme was localiz ed and quantified. Thus, the cellular cathepsin B kinetics for the syn thetic substrates Z-Arg-Arg-4M beta NA and Z-Val-Lys-Lys-Arg-4M beta N A, its pH dependence and inhibition by E64, stefins A and B, and cysta tin C could be determined. From these measurements it appeared that th e enzyme exhibited different cleavage rates for these substrates in th e different cell types, showed considerable cleavage activity at neutr al pH, which was stable under these conditions for extended time perio ds, and was highly sensitive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefins, particularly stefin A. By conventional light microscopy, confocal laser scanning microscopy, and electron microscopy the enzymatic activity was localized in lysosomes , as expected, but also in the endoplasmic reticulum, nuclear membrane , and plasma membrane. The endoplasmic reticulum is a site at which on ly pre-mature enzyme forms exist, which are usually not active. The ap pearance of enzymatic activity at the plasma membrane confirms earlier biochemical and immunofluorescence microscopic investigations. The di fferent sites of localization within the cells make it likely that dif ferent forms of the enzyme are expressed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative processes.