ARTICLE |
Correspondence to: Kamiar Moin, Dept. of Pharmacology, Wayne State U. School of Medicine, 540 E. Canfield, Detroit, MI 48201.
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Summary |
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The cystatin superfamily of cysteine protease inhibitors and target cysteine proteases such as cathepsin B have been implicated in malignant progression. The respective cellular/extracellular localization of cystatins and cysteine proteases in tumors may be critical in regulating activity of the enzymes. Confocal microscopy has enabled us to demonstrate the differential localization of cystatins and cathepsin B in an embryonic liver cell line and an invasive hepatoma cell line. In both, stefins A and B were distributed diffusely throughout the cytoplasm, whereas cystatin C was distributed in juxtanuclear vesicles. Stefin A and cystatin C, but not stefin B, were present on the cell surface. Cystatin C was found on the top surfaces of both cell lines, whereas stefin A was found only on the top surface of the embryonic liver cells. Cathepsin B staining was concentrated in perinuclear vesicles in the embryonic liver cells. In the hepatoma cells, staining for cathepsin B was also present in vesicles adjacent to the cell membrane and on localized regions of the bottom surface. Such a disparate distribution of cathepsin B and its endogenous inhibitors may facilitate proteolysis by the hepatoma cells and thereby contribute to their invasive phenotype. (J Histochem Cytochem 46:745751, 1998)
Key Words: cathepsin, cystatin, cysteine protease inhibitor, immunocytochemistry, stefin
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Introduction |
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Papain family cysteine proteases and their endogenous inhibitors have been implicated in malignant progression of human and animal tumors ( in the rat) and B and cystatin C show differential inhibitory activity against cysteine proteases. For example, the Kis for inhibition of one target protease, cathepsin B, are 0.29 nM for cystatin C, 2.4 nM for stefin A, and 19 nM for stefin B (for review see
The subcellular localization of the stefins and cystatins can be inferred from sequence analysis of their cDNAs. Stefins A and B are presumed to be cytoplasmic because the amino acid sequences deduced from their cDNAs do not have signal peptides (
A membrane localization for stefins and cystatins would not be predicted from their cDNA sequences. However, in murine tumors, we have observed an enrichment of cystatin activity in a plasma membrane fraction along with a decrease in overall cystatin activity (
Previous immunohistochemical studies have not shed light on these discrepancies because their primary goal had been to identify tissues positive for specific low molecular mass cystatins rather than to determine subcellular/extracellular localization. Therefore, in the present study, we employed immunofluorescent confocal microscopy to compare the localization of the cystatins and the target cysteine protease cathepsin B in BNL CL.2 murine embryonic liver cells and Hepa cl 9 murine liver tumor cells. This enabled us to assess whether there were any differences in the subcellular and surface distributions of stefins A and B, cystatin C, and cathepsin B. We were particularly interested in differences that might affect the invasive phenotype of Hepa cl 9 cells.
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Materials and Methods |
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Materials
Dulbecco's modified Eagle's medium (DMEM) was purchased from Sigma (St Louis, MO); secondary antibodies (Texas Red-conjugated affinity-purified donkey anti-rabbit or anti-mouse IgGs and fluorescein-conjugated affinity-purified donkey anti-rabbit or anti-mouse IgGs) and normal donkey serum from Jackson ImmunoResearch (West Grove, PA); and the Slow Fade anti-fade reagent from Molecular Probes (Eugene, OR). Polyclonal antibodies to rat epidermis cystatin- and mouse cystatin C were kind gifts from Dr. Kimie Fukuyuma (University of California at San Francisco, CA) and Dr. Magnus Abrahamson (University of Lund, Lund, Sweden), respectively. Monoclonal antibody to human stefin B was a kind gift from Dr. Vito Turk (Jozef Stefan Institute, Ljubljana, Slovenia). The specificities of the antibodies were established by immunoblot analysis (
Cells and Culture Conditions
Hepa cl 9 is a subclone of Hepa cl 7, originally derived from a murine hepatoma (
Immunolocalization
Intracellular Labeling.
Intracellular stefins A and B, cystatin C, and cathepsin B were localized using our published immunocytochemical protocols ( IgG, which is crossreactive with mouse stefin A, rabbit anti-mouse cystatin C IgG, or mouse anti-human liver stefin B IgG) for 2 hr. In controls, either preimmune serum (rabbit or mouse) or PBSsaponin was substituted for the primary antibody. After washing, cells were blocked with normal donkey serum (5% in PBSsaponin) in the presence of the secondary antibody: either Texas Red-conjugated or fluorescein-conjugated affinity-purified donkey anti-rabbit IgG or mouse IgG at 20 µg/ml. After washing, the coverslips were mounted upside down on slides with Slow Fade anti-fade reagent and observed on a Zeiss LSM-310 confocal microscope.
Surface Labeling. Surface stefins A and B, cystatin C, and cathepsin B were localized on living cells by the following modification of the procedures described above. Incubation with the primary antibody was performed at 4C to prevent endocytosis of antibodies, followed by fixation with 3.7% formaldehyde, saponin was omitted, and only Texas Red-conjugated secondary antibodies were used.
Confocal Image Analysis. For each antigen, 40 consecutive optical sections were obtained utilizing the LSM-310 in confocal mode; individual sections were 0.25 µm and the 40 sections encompassed the entire cell volume. Three-dimensional images and galleries of the optical sections were analyzed to demonstrate the spatial distribution of cell surface cathepsin B and cystatins. The three-dimensional images were assembled from all 40 optical sections utilizing the software VoxelViewUltra from Vital Images (Fairfield, IA). Galleries of confocal images were assembled to encompass the entire cell volume. For cystatin C and stefin A, the galleries contained 25 images of 512 x 768 pixels and, for cathepsin B, 24 images of 512 x 512 pixels.
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Results |
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Intracellular Localization
The staining for stefin A (Figure 1A and Figure 1B) and stefin B (Figure 1C and Figure 1D) was diffuse and was distributed throughout the cytoplasm of both the embryonic liver (Figure 1A and Figure 1C) and hepatoma (Figure 1B and Figure 1D) cells. In the hepatoma cells, the many vesicles presented a negative image because of an apparent concentration of stefins A and B at their membranes (see arrows, Figure 1B and Figure 1D). In contrast, staining for cystatin C was punctate and thus presumably vesicular (Figure 2). The distribution of the vesicles staining for cystatin C was primarily juxtanuclear and was similar in embryonic liver (Figure 2A) and hepatoma (Figure 2B) cells.
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One of the enzymes presumed to be a target of the low molecular mass cystatins is the lysosomal cysteine protease cathepsin B. We had established previously that cathepsin B is localized in perinuclear vesicles in normal cells. In transformed and tumor cells, cathepsin B is distributed both in perinuclear vesicles and in vesicles throughout the cytoplasm, including vesicles at the cell periphery and in cell processes (
Surface Localization
Our previous observations of an enrichment of cystatin activity in a plasma membrane fraction of murine B16 melanomas and Lewis lung carcinomas (
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We had established previously that cathepsin B staining is localized on the bottom surface of transformed breast epithelial cells, breast tumor cells, and glioblastoma cells (
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Discussion |
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Our immunofluorescent studies confirmed a cytoplasmic localization for stefins A and B and a vesicular localization for cystatin C in murine cell lines of liver origin. A surface association of stefin A was observed only in the embryonic liver cells. In contrast, cystatin C was associated with the surface membranes of both cell lines. A cytoplasmic localization for stefin A and B and vesicular localization for cystatin C is consistent with the absence and presence of signal peptides in their respective cDNAs (
In the present study, the staining for stefins A and B in the tumor cells appeared to be concentrated adjacent to vesicular membranes. The pattern of staining suggested that stefins A and B may interact specifically with certain vesicular membranes. There are previous reports of association of cystatins with vesicular membranes/vesicles: cystatin activity with a lysosomal membrane fraction of rabbit liver () with keratohylin granules of rat epidermis (
Association of the cytoplasmic stefin A with vesicular membranes/vesicles intracellularly might explain how stefin A becomes associated with the cell surface. There is precedent for translocation of cytoplasmic proteins to vesicular and cell surface membranes. For example, in chromaffin cells on nicotine stimulation, annexin 2, a cytoplasmic protein, has been shown to be translocated to a membrane fraction containing chromaffin granule membrane and plasma membrane (
We and others have postulated that alterations in the balance between endogenous cystatins and cysteine proteases may contribute to the malignant progression of tumors (
Cathepsin B has been localized to the inner and external aspects of the bottom surface of transformed and tumor cells (
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Footnotes |
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1 Present address: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA.
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Acknowledgments |
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Supported by US Public Health Service grant CA36481. The Confocal Imaging Core is supported in part by US Public Health Service Center grants P30 ES06639 from the National Institutes of Environmental Health Sciences and P30 CA22453 from the National Cancer Institute.
We thank Dr Edith Elliott (University of Natal, South Africa) for critical review of the manuscript and Ms Grace Ziegler for technical assistance.
Received for publication September 24, 1997; accepted January 7, 1998.
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