Cathepsin B activity in human lung tumor cell lines: ultrastructural localization, pH sensitivity, and inhibitor status at the cellular level.

Abstract

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefii A and B, at the cellular level in human tumor cell lines HS-24, derived from a primary lung tumor (squamous cell), and SB-3, derived from a metastasis (lung adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and hctionally related cathepsin L, but not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefm B was found in both cell types. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized and quantified. Thus, the cellular cathepin B kinetics for the synthetic substrates ZArgArg-4MPNA and Z-Val-Lys-Lys-Arg-4M~NA, its pH dependence and inhibition by E64, stefii A and B, and cystatin C could be determined. From these measurements it appeared that the enzyme exhibited different cleavage rates for these substrates in the different cell types, showed considerable cleavage activity at neutral pH, which was stable under these conditions for extended time periods, and was highly sensi- IntroductionThe cysteine proteinase cathepsin B (cath B: EC 3.4.22.1) is a ubiquitous enzyme. It is involved in lysosomal proteolysis (6) and protein processing (20,26,53,75). After detection and biochemical characterization it was described as a lysosomal enzyme with optimal activity in a slightly acidic (pH 5-6) environment (4). Subsequent investigations also revealed secreted forms ofthe enzyme under physiological (66) and malignant (32,37,44,52) conditions. Recently, interest focused on the appearance of elevated levels of the enzyme in tumors and tumor cells, particularly those derived from metastases or with metastatic character (29,33,57,(60)(61)(62)(63). The ability of the Supported in part by a grant from the Tumor Zcntrum Heidelber Mannheim.'-Correspondence tO: Dr. Eberhard Spiess, Biomedizinische Suukturforschung 0195, Deutsches Krebsforschungszentrum, PO Box 101949, D 69009 Heidelberg, Germany. tive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefim, particularly stefim 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 only pre-mature enzyme forms exist, which are usually not active. The appearance of enzymatic activity at the plasma membrane confiims earlier biochemical and immunofluorescence microscopic investigations. The different sites of localization within the cells make it likely that di&ent forms ofthe enzyme are expmsed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative proasses. (JHistochem Cyrochem 4291 7-929,lW) KEY WORDS: Cathepsin B Stefin; Activity; Tumor cell; Localization; Endoplasmi...

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants

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Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells

“…Among the cysteine proteinases, cathepsin B expression has been particularly well documented in a variety of tumor cell types (Friedrich et al, 1999;Heidtmann et al, 1997) including brain tumors (Demchik et al, 1999;Rempel et al, 1994). It has been suggested that cathepsin B can mediate dissemination of cancer cells by degrading ECM (Spiess et al, 1994) and/or activating other proteinases capable of degrading the matrix (Kobayashi et al, 1993). Buck et al (1992) have shown that cathepsin B can degrade the major components of basement membranes, i.e.…”

Blockade of cathepsin B expression in human glioblastoma cells is associated with suppression of angiogenesis

“…Cathepsin B levels are elevated in malignant tumors, particularly at the invasive edge of the tumor. The increased expression of cathepsin B mRNA has been reported in several human tumors including brain, colon, lung, prostate and esophageal adenocarcinoma (Spiess et al, 1994;Mikkelsen et al, 1995;Iacobuzio-Donahue et al, 1997;Hizel et al, 1998;Hughes et al, 1998). In addition, an increased abundance of cathepsin B transcripts and protein in tumor tissues correlates with clinical invasive features (Iacobuzio-Donahue et al, 1997;Hizel et al, 1998).…”