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  • 1
    ISSN: 1432-0878
    Keywords: Key words: Tumor cells ; Enterocytic differentiation ; Lysosomal cathepsins ; Leupeptin ; HT-29 colon carcinoma cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Enterocyte-like differentiated HT-29 colon carcinoma cells were shown to contain far higher intracellular levels of activity of lysosomal cathepsins B, D, and L than their undifferentiated counterparts. In the latter, inhibition of lysosomal functions by leupeptin or ammonium chloride led to a marked increase in the cell-associated activity of the three cathepsins. High levels of pro-cathepsins B, D, and L were found in the culture media of both HT-29 cell populations. Ammonium chloride and chloroquine, which are known to impair the mannose-6-phosphate-dependent trafficking of lysosomal-targeted proteins, did not increase the secretion of the three cathepsins in either undifferentiated or differentiated cultures of HT-29 cells. Analyses by cell fractionation revealed heterogeneities with regard to the density and the content of lysosomal cathepsins between the two cell populations. Leupeptin induced the accumulation of mature lysosomal cathepsins B and L in light density organelles in undifferentiated HT-29 cells. Altogether, these data demonstrate that (1) the expression and subcellular distribution of cathepsins B, D, and L in HT-29 cells are influenced by their state of enterocytic differentiation, (2) the segregation of lysosomal cathepsins is largely inefficient in this tumor cell line and does not increase upon differentiation, and (3) the mannose-6-phosphate-receptor-dependent pathway plays a minor role in the sorting of the three cathepsins, both in undifferentiated and enterocytic-differentiated HT-29 cells.
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  • 2
    ISSN: 1432-0878
    Keywords: Key words Procathepsin D ; Lysosomes ; Endocytosis ; Baby hamster kidney (BHK) cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  BHK cells transfected with human cathepsin D (CD) cDNA normally segregate the autologous hamster cathepsin D while secreting a large proportion of the human proenzyme. In the present work, we have utilized these transfectants to examine to what extent the mannose-6-phosphate-dependent pathway for lysosomal enzyme segregation contributes to the differential sorting of human and hamster CD. We report that, in recipient control BHK cells, the rate of mannose-6-phosphate-dependent endocytosis of human procathepsin D secreted by transfected BHK cells is lower than that of hamster procathepsin D and much lower than that of human arylsulphatase A. The missorted human enzyme bears phosphorylated oligosaccharides and most of its phosphate residues are “uncovered”, like the autologous enzyme. Thus, despite both the Golgi-associated modifications of oligosaccharides, i.e. the phosphorylation of mannose and the uncovering of mannose-6-phosphate residues, which proceed on human and hamster procathepsin D with comparable efficiency, only the latter is accurately packaged into lysosomes. Ammonium chloride partially affects the lysosomal targeting of cathepsin D in control BHK cells, whereas in transfected cells, this drug strongly inhibits the maturation of human procathepsin D and slightly enhances its secretion. These data indicate that: (1) over-expression of a lysosomal protein does not saturate the Golgi-associated reactions leading to the synthesis of mannose-6-phosphate; (2) a portion of cathepsin D is targeted independently of mannose-6-phosphate receptors in the transfected BHK cells; and (3) whichever mechanism for lysosomal delivery of autologous procathepsin D is involved, this is not saturated by the high rate of expression of human cathepsin D.
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  • 3
    ISSN: 0263-6484
    Keywords: Lysosomal hydrolases ; regenerating hepatocytes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cathepsins B and D, β-galactosidase, and acid phosphatase activities were found to be decreased in the regenerating rat liver, the reduction being maximal around the peak of hepatocyte mitoses (30 h). To investigate whether these changes could be heterogeneously distributed among hepatic cells, total cell populations from control or two-thirds hepatectomized rat livers were dissociated by the collagenase perfusion technique and analysed by different procedures. Isopycnic centrifugation in a Metrizamide gradient satisfactorily resolved hepatocytes and non-parenchymal cells from control animals but was not adequate when applied to 30-h regenerating liver cells. Colchicine treatment of the hepatectomized animals, resulted in substantial accumulation of phase M-hepatocytes. Subpopulations considerably enriched in fast-sedimenting phase M-cells were obtained by sedimentation at 1 g of the total liver cell population, and subsequently analysed by isopycnic equilibration. Phase M-hepatocytes were shown to have markedly reduced levels of β-galactosidase, acid phosphatase, and cathepsin B activities in comparison, not only with control hepatocytes, but also with those parenchymal cells which were not metaphase-arrested in the same regenerating livers. Therefore, in partially-hepatectomized rats, hepatocytes progressing up to metaphase in the first mitotic cycle exhibited a selective depletion of lysosomal enzyme activities. The mechanism(s) underlying this change remain(s) presently unknown.
    Additional Material: 4 Ill.
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