Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.23.5 (cathepsin D)
4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts from vascular smooth muscle cells. The antigen was detected on the cell surface and in cathepsin D-positive and acridine orange-accumulating vesicular compartments of fibroblasts. Ultrastructurally, the antigen was revealed in coated pits and in endosomal and lysosomal structures. 1B10 recognized three major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10 immunoreactivity was specific to fibroblasts and smooth muscle differentiated fibroblasts within the context of vascular smooth muscle cells.
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PMID:A fibroblast-associated antigen: characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells. 155 84

In breast cancer cell lines, pro-cathepsin D is synthesized in excess and abnormally processed, resulting in its slower maturation and increased secretion into the culture medium. Since this lysosomal protease is only active at acidic pH, we have searched for acidic compartments other than lysosomes where cathepsin D might be active when MCF7 cells are plated on corneal extracellular matrix. We found large acidic intracellular vesicles (1.5 to 20 microns in diameter) by acridine orange and 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine staining, two fluorescent probes which reveal acidic compartments. These vesicles were actively acidified. They were 2- to 20-fold more abundant in MCF7 breast cancer cells and primary cultures of human breast cancers cells than in primary cultures of normal mammary epithelial cells. In living MCF7 cells, high resolution video-enhanced microscopy showed that these vesicles were mobile and intracellular. Double immunolocalization indicated that they contained mature cathepsin D (but no detectable pro-cathepsin D) and endocytosed extracellular material. This material (dextran, transferrin, and extracellular matrix) and the association with other lysosomal enzymes varied according to the vesicles, suggesting their heterogeneity (large endosomes or phagosomes). We conclude that, in breast cancer cells, cathepsin D may digest intracellularly phagocytosed and/or endocytosed extracellular matrix in large acidic vesicles. We propose that the higher expression of cathepsin D associated with the increased number of large acidic vesicles in breast cancer cells may facilitate digestion of basement membrane and consequently metastasis.
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PMID:Cathepsin D in breast cancer cells can digest extracellular matrix in large acidic vesicles. 239 69

Human breast cancer cells include large acidic vesicles (LAVs) containing endocytosed extracellular matrix (P. Montcourrier et al., Cancer Res., 50, 1990, p. 6045-6054) and mature cathepsin D, a lysosomal protease associated with metastatic risk. We show that metastatic breast cancer MDA-MB231 cells migrating in vitro through a Matrigel gel in a modified Boyden chamber were enriched in LAVs (28% compared to 6% before plating) as visualized by acridine orange fluorescence. This is the first evidence that LAVs are associated with invasiveness of cancer rather than with an apoptotic process.
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PMID:[The presence of large acidic vesicles is correlated with in vitro invasive potential of breast cancer cells]. 840 70

Apoptosis, a form of active cell death, plays a role during normal limb development. The present study was done to test the hypothesis that the teratogen cyclophosphamide, an alkylating agent and commonly used anticancer drug, produces malformations by disturbing the regulation of apoptosis in the limb. The effects of a preactivated analog of cyclophosphamide, 4-hydroperoxycyclophosphamide, on limb development and on apoptosis in the limb were determined in vitro. Cathepsin D is a lysosomal protease which is induced in tissues undergoing destruction by apoptosis. To further examine the process of apoptosis in the limb, the effects of 4-hydroperoxycyclophosphamide exposure on cathepsin D protein concentration and on the immunolocalization of cathepsin D in limb buds were assessed. Limb buds from gestational day 12 mice were excised and cultured in roller bottles in a chemically defined medium for up to 6 days. The addition of 4-hydroperoxycyclophosphamide (1 or 10 micrograms/ml) to the culture medium produced time- and concentration-dependent limb malformations. Electrophoresis of the DNA extracted from both control and treated limbs revealed a DNA fragmentation pattern characteristic of apoptosis. Limbs cultured in the control medium showed a "DNA ladder" only after 72 hours in vitro; however, those in the drug-treated groups showed fragmentation within 12 hours of drug exposure. Acridine orange staining and examination of cell ultrastructure with the electron microscope further confirmed that apoptotic cell death in the interdigital areas was accelerated in drug-exposed limbs. The relative abundance of cathepsin D in limbs exposed to 4-hydroperoxycyclophosphamide for 24 hours was increased compared to control limbs.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction of apoptosis and cathepsin D in limbs exposed in vitro to an activated analog of cyclophosphamide. 853 10

Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.
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PMID:Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis. 864 84

The cytotoxicity of oxidized LDL (oxLDL) to several types of artery wall cells might contribute to atherosclerosis by causing cell death, presumably by both apoptosis and necrosis. After its uptake into macrophage lysosomes by receptor-mediated endocytosis, oxLDL is poorly degraded, resulting in ceroid-containing foam cells. We studied the influence ofoxLDL on lysosomal enzyme activity and, in particular, on lysosomal membrane stability and the modulation of these cellular characteristics by HDL and vitamin E (vit-E). Unexposed cells and cells exposed to acetylated LDL (AcLDL) were used as controls. The lysosomal marker enzymes cathepsin L and N-acetyl-beta-glucosaminidase (NAbetaGase) were biochemically assayed in J-774 cells after fractionation. Lysosomal integrity in living cells was assayed by the acridine orange (AO) relocation test. Cathepsin D was immunocytochemically demonstrated in J-774 cells and human monocyte-derived macrophages. We found that the total activities of NAbetaGase and cathepsin L were significantly decreased, whereas their relative cytosolic activities were enhanced, after oxLDL exposure. Labilization of the lysosomal membranes was further proven by decreased lysosomal AO uptake and relocation to the cytosol of cathepsin D, as estimated by light and electron microscopic immunocytochemistry. HDL and vit-E diminished the cytotoxicity of oxLDL by decreasing the lysosomal damage. The results indicate that endocytosed oxLDL not only partially inactivates lysosomal enzymes but also destabilizes the acidic vacuolar compartment, causing relocation of lysosomal enzymes to the cytosol. Exposure to AcLDL resulted in its uptake with enlargement of the lysosomal apparatus, but the stability of the lysosomal membranes was not changed.
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PMID:Uptake of oxidized LDL by macrophages results in partial lysosomal enzyme inactivation and relocation. 948 81

To test whether the possibly enhanced sensitivity of aged cells to oxidative stress may depend on their content of ceroid/lipofuscin, AG-1518 human fibroblasts with various amounts of the pigment accumulated due to prolonged cultivation under normobaric hyperoxia were exposed to acute oxidative stress (2.5 microM naphthazarin, 15 min) and then returned to standard culture conditions. Twenty-four hours after the naphthazarin treatment, 37% of the cells were still vital, whereas others had undergone oxidative stress-induced apoptosis with ensuing postapoptotic necrosis. The average amount of ceroid/lipofuscin within the surviving cells was only about half of that of the initial population of cells, as measured before the naphthazarin exposure. This finding suggests that ceroid/lipofuscin-rich cells have an increased sensitivity to oxidative stress. The ceroid/lipofuscin quantity strongly positively correlated with the size of the acidic compartment (as evaluated by uptake of the weakly basic lysosomotropic fluorochrome acridine orange) and with its content of the lysosomal protease cathepsin D, as assayed by immunocytochemistry. We hypothesize that the enhanced sensitivity of ceroid/lipofuscin-loaded cells to oxidative stress may be caused by the increased amounts of lysosomal enzymes, known as mediators of oxidative damage, and/or by catalysis of intralysosomal oxidative reactions by lipofuscin-associated iron.
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PMID:Ceroid/lipofuscin-loaded human fibroblasts show increased susceptibility to oxidative stress. 1057 36

Oxidative stress is a primary pathogenesis in the brain, which is particularly vulnerable to oxidative stress. Maintenance of astrocyte functions under oxidative stress is essential to prevent neuronal injuries and to recover neuronal functions in various pathologic conditions. Imidazoline drugs have affinities for imidazoline receptors, which are highly distributed in the brain, and have been shown to be neuroprotective. This study presented the protective effects of several imidazoline drugs against oxidative cytotoxicity, in primary cultures of astrocytes. Imidazoline drugs, such as idazoxan, guanabenz, guanfacine, BU224, and RS-45041-190, showed protective effects against naphthazarin-induced oxidative cytotoxicity, as evidenced by LDH release and Hoechst 33342/propidium iodide staining. The imidazoline drugs stabilized lysosomes and inhibited naphthazarin-induced lysosomal destabilization, as evidenced by acridine orange relocation. Guanabenz inhibited, the leakage of lysosomal cathepsin D to cytosol, the decreased mitochondrial potential, and the release of mitochondrial cytochrome c, which were induced by naphthazarin. The lysosomal destabilization by oxidative stress and other apoptotic signals and subsequent cathepsin D leakage to the cytosol can induce apoptotic changes of mitochondria and eventually cell death. Therefore, lysosomal stabilization by imidazoline drugs may be ascribed to their protective effects against oxidative cytotoxicity.
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PMID:Imidazoline drugs stabilize lysosomes and inhibit oxidative cytotoxicity in astrocytes. 1186 79

Blockade of the mitochondrial permeability transition pore (mPTP) by cyclosporin A (CsA) inhibits apoptosis in various cell types. However, use of CsA in humans is associated with damage to the arterial endothelium. We evaluated whether inhibition of the apoptotic machinery by CsA promotes other forms of cell death in arterial endothelial cells (EC). Exposure of human umbilical artery EC (HUAEC) to clinically relevant concentrations of CsA for up to 24 h was associated with a significant increase in necrotic features. We detected inhibition of apoptosis and a significant increase in necrosis in HUAEC exposed concomitantly to CsA and mitomycin C, a proapoptotic DNA damaging agent. We found that CsA-induced cell death is independent of caspase activation, p53 induction, and calcineurin inhibition. However, bongkrekic acid, another mPTP blocker, also increased necrosis in HUAEC. Dihydroethidium and acridine orange staining revealed increased intracellular production of reactive oxygen species (ROS) followed by lysosomal damage in HUAEC exposed to CsA. Hydroxyl radical and superoxide scavengers and inhibition of cathepsin D activity significantly attenuated CsA-induced EC death. These results suggest that inhibition of the apoptotic machinery by CsA in arterial EC favors development of a necrotic form of cell death regulated by ROS and secondary lysosomal damage.
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PMID:Blockade of the apoptotic machinery by cyclosporin A redirects cell death toward necrosis in arterial endothelial cells: regulation by reactive oxygen species and cathepsin D. 1251 15

Past studies in our laboratory have shown that silica (-quartz) particle exposure of a mouse alveolar macrophage cell line (MH-S) elicits mitochondrial depolarization and caspase 3 and 9 activation, contributing to apoptosis. However, cellular pathways leading to these outcomes have not been extensively investigated. Initial studies revealed that silica exposure elicits lysosomal permeability after 1 h, as evidenced by leakage of FITC-conjugated dextran and acridine orange. We next evaluated a role for the lysosomal acidic compartment in apoptosis. Cells pretreated with the lysosomotropic weak base ammonium chloride, to increase lysosomal pH, showed decreased caspase activation and apoptotic DNA fragmentation. MH-S cells pretreated with pepstatin A, an inhibitor of lysosomal cathepsin D, showed decreased caspase 9 and 3 activation as well as a decreased percentage of cells that became apoptotic. DNA fragmentation and caspase 9 and 3 activation were also decreased in cells pretreated with despiramine, an inhibitor of lysosomal acidic sphingomyelinase. Silica pretreated with aluminum lactate (to blunt surface active sites) reduced caspase activation and apoptosis. Although aluminum lactate-treated silica still induced lysosomal permeability (by FITC-dextran leakage), one measure of lysosome integrity and function suggested a reduction in the extent and/or nature of lysosomal injury (by acridine orange retention). A role for reactive oxygen species (ROS) was investigated to explore another pathway for silica-induced apoptosis in addition to lysosomal enzymes; however, no role for ROS was apparent. Thus, following silica exposure, lysosomal injury precedes apoptosis, and the apoptotic signaling pathway includes cathepsin D and acidic sphingomyelinase.
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PMID:Silica-induced apoptosis in mouse alveolar macrophages is initiated by lysosomal enzyme activity. 1505 7


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