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  • 1
    Keywords: CELLS ; EXPRESSION ; carcinoma ; Germany ; LUNG ; PATHWAY ; DEATH ; PROTEIN ; PROTEINS ; TUMOR-NECROSIS-FACTOR ; MECHANISM ; TRANSCRIPTION FACTOR ; DOMAIN ; CONTRAST ; mechanisms ; SEQUENCE ; VARIANTS ; MOLECULE ; NUCLEI ; MALIGNANCIES ; PATTERNS ; CARCINOMA CELLS ; CELL-DEATH ; DAMAGE ; LOCALIZATION ; MITOCHONDRIA ; LENGTH ; targeting ; SINGLE ; MALIGNANCY ; HUMAN LUNG ; cell death ; DEPENDENT APOPTOSIS ; HEPATOCYTE APOPTOSIS ; HUMAN-PROSTATE ; LYSOSOMAL PATHWAY ; MATURE ; SUBCELLULAR-DISTRIBUTION
    Abstract: Background: Splicing variants of human cathepsinB primary transcripts ( CB(- 2,3)) result in an expression product product which lacks the signal peptide and parts of the propeptide. This naturally truncated Delta(51)CB is thus unable to follow the regular CB processing and sorting pathway. It is addressed to the mitochondria through an activated N-terminal mitochondrial targeting signal instead. Although Delta(51)CB is supposed to be devoid of the typical CB enzymatic activity, it might play a role in malignancies and trigger cell death/apoptosis independent from the function of the regular enzyme. Cytoplasmic presence of the mature CB might occur as a result of lysosomal damage. Results: We investigated such "aberrant" proteins by artificial CB-GFP chimeras covering various sequence parts in respect to their enzymatic activity, their localization in different cell types, and the effects on the cell viability. Unlike the entire full length CB form, the artificial single chain form was not processed and did not reveal typical enzymatic CB activity during transient overexpression in large cell lung carcinoma cells.. Delta(51)CB was found predominantly in mitochondria. In contrast, the shorter artificial CB constructs localized in the cytoplasm, inside the cell nucleus, and in the midbodies of dividing cells. Bleaching experiments revealed both mobile and immobile fractions of these constructs in the nucleus. Nuclear accumulation of artificially truncated CB variants led to disintegration of nuclei, followed by cell death. Conclusion: We propose that cell death associated with CB is not necessarily triggered by its regular enzymatic activity but alternatively by a yet unknown activity profile of truncated CB. Cytoplasmic CB might be able to enter the cell nucleus. According to a mutational analysis, the part of CB that mediates its nuclear import is a signal patch within its heavy chain domain. The results suggest that besides the N-terminal signal peptide also other CB domains contain patterns which are responsible for a differentiated targeting of the molecule, e. g. to the mitochondria, to the nucleus, or to vesicles. We propose a hierarchy of targeting signals depending on their strength and availability. This implies other possible transport mechanisms besides the usual trafficking via the mannose-6-pathway
    Type of Publication: Journal article published
    PubMed ID: 15807897
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  • 2
    Keywords: CANCER ; CELLS ; SURVIVAL ; carcinoma ; CELL ; LUNG ; INFORMATION ; lung cancer ; LUNG-CANCER ; GENE ; PROTEIN ; TISSUE ; PATIENT ; MACROPHAGES ; MARKER ; prognosis ; CONTRAST ; LYMPH-NODES ; STAGE ; PROGRESSION ; HIGH-RISK ; LOCALIZATION ; HEAD ; HIGH-LEVEL ; non-small cell lung cancer ; CATHEPSIN-B ; STEFIN-A ; TUMOR TISSUE ; LEVEL ; IMMUNOHISTOCHEMICAL ANALYSIS ; NSCLC ; CYSTEINE PROTEASE ; RELEVANCE ; macrophage ; PREDICTOR ; inflammatory cells ; biological markers ; cystatin ; cathepsin ; CYSTEINE PROTEINASE-INHIBITOR ; ENDOGENOUS INHIBITORS ; PROTEASE INHIBITORS ; stefin
    Abstract: Cystatins regulate tumour-associated cysteine proteases, however, their role in tumour progression is not clear yet. To assess their relevance in the progression of nonsmall cell lung cancer (NSCLC) the protein level, cysteine protease activity (CPI) and localization of type I (stefins A and B) and type H (C, E/M and F) cystatins were defined in tumours and control lung counterparts from 165 patients. The medians of CPI activity, stefins A and B were significantly greater in tumour than in lung tissue (2.1-fold, 1.7-fold, 1.2-fold, respectively, all p 〈 0.001). The median levels of cystatin C and cystatin E/M were lower in tumour tissue (0.9-fold, p=0.06; 0.6-fold, p 〈 0.01). In all the samples the levels of cystatin F were below the detection limit. Immunohistochemical analysis revealed the presence of all cystatins in tumour cells and infiltrated inflammatory cells such as macrophages and neutrophils. In univariate survival analysis patients with high levels of stefin A, stefin B and CPI activity exhibited a better survival probability (p=0.05, p=0.05, p 〈 0.01, respectively). In contrast, cystatins C and E/M provided no prognostic information. In multivariate analysis the most powerful predictor of survival was the pTNM stage (p 〈 0.0001; RR 3.5), followed by stefin A, stefin B and CPI activity (all p=0.03; RR 1.5). Our results suggest that only stefins A and B, i.e. type I cystatins, are up-regulated in lung tumours and thus able to counteract harmful tumour-associated proteolytic activity. As biological markers they may add independent prognostic information for better assessment of low- and high-risk patients with NSCLC
    Type of Publication: Journal article published
    PubMed ID: 16969475
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  • 3
    Abstract: Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.
    Type of Publication: Journal article published
    PubMed ID: 12000759
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  • 4
    Keywords: CANCER ; EXPRESSION ; tumor ; carcinoma ; Germany ; human ; LUNG ; GENE ; GENE-EXPRESSION ; HYBRIDIZATION ; PROTEIN ; MONOCLONAL-ANTIBODY ; TISSUE ; TUMORS ; kidney ; TISSUES ; BREAST-CANCER ; antibodies ; antibody ; STAGE ; IN-SITU ; PROGRESSION ; ARRAYS ; metastases ; HETEROZYGOSITY ; REGION ; MONOCLONAL-ANTIBODIES ; CARCINOMAS ; NORMAL TISSUE ; HISTONE ACETYLTRANSFERASE ; ovarian carcinoma ; METHYLATION ; MITOSIS ; DEFICIENCY ; ONCOLOGY ; pancreas ; RE ; PATTERN ; ARRAY ; mRNA ; MALIGNANT PROGRESSION ; biomarker ; monoclonal antibodies ; EPITHELIAL TUMORS ; USA ; LOSSES ; MAMMARY-CARCINOMA ; SET ; NOV ; cDNA array ; NUCLEAR-MEMBRANE ; epigenetic regulation ; hMOF ; RECESSIVE CEREBELLAR-ATAXIA ; SYNE-1
    Abstract: In a study on gene deregulation in ovarian carcinoma we found a mRNA ceding for a 350 kDa protein, Drop1, to be downregulated 20- to 180-fold in the majority of ovarian and mammary carcinomas. The mRNA is encoded by a set of exons in the 5' region of the SYNE1 gene. Immunohistochemical staining for Drop1 protein by a specific monoclonal antibody corresponds to the pattern seen for the mRNA. cDNA arrays of matched pairs of tumor and normal tissue and in situ hybridizations confirmed the drastic loss of Drop1 mRNA as a common feature in uterus, cervix, kidney, lung, thyroid and pancreas carcinomas, already at early tumor stages and in all metastases. Two-hybrid studies suggest a role of this deficiency in the malignant progression of epithelial tumors. (c) 2008 Wiley-Liss, Inc
    Type of Publication: Journal article published
    PubMed ID: 18709643
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  • 5
    Keywords: brain ; RECEPTOR ; CELLS ; EXPRESSION ; IN-VITRO ; CELL ; Germany ; DEATH ; POPULATION ; TISSUE ; MACROPHAGES ; MARKER ; RECOGNITION ; MOUSE ; prevention ; PHAGOCYTOSIS ; MARKERS ; DAMAGE ; NETHERLANDS ; CLEARANCE ; CENTRAL-NERVOUS-SYSTEM ; RECEPTORS ; microglia ; MULTIPLE-SCLEROSIS ; mannose receptor ; SUBPOPULATION ; RE ; BRAIN-TUMORS ; GLIOMA ; GLIOMA-CELLS ; FUNCTIONAL-CHARACTERIZATION ; cell death ; APOPTOTIC CELLS ; immunology ; STRAIN
    Abstract: Microglia phagocytic activity for apoptotic glioma cells is hardly analysed inspite of its relevance to tissue damage prevention. We provide evidence for a phosphatidyl serine-independent clearance of mouse glioma cells at an advanced stage of death, suggesting microglia recognition of late apoptotic markers. Dying cells were immediately cleared or stayed for hours in that stage before engulfment occurred. This phagocytic activity was restricted to a microglia subset representing 30 to 70% of the population according to the used strain. Expression of receptors involved in late apoptotic markers recognition therefore seems confined to a subpopulation of microglia and to be strain-dependent. (C) 2008 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 18495256
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  • 6
    Keywords: CELLS ; IN-VITRO ; IN-VIVO ; MICROSCOPY ; PROTEIN ; PROTEINS ; ACTIVATION ; DOMAIN ; C-JUN ; CATALYTIC SUBUNIT ; SUBUNIT ; LOCALIZATION ; NUCLEUS ; CRYSTAL-STRUCTURE ; LIVING CELLS ; CALCIUM-DEPENDENT PROTEASE ; DISSOCIATION ; MYOFIBRILLAR PROTEIN-TURNOVER ; NEUTRAL PROTEASE ; NUCLEAR-LOCALIZATION ; RAT CALPAIN ; THIOL PROTEASE
    Abstract: Ubiquitously expressed calpains are Ca2+-dependent, intracellular cysteine proteases comprising a large catalytic subunit (domains DI-DIV) and a noncovalently bound small regulatory subunit (domains DV and DVI). It is unclear whether Ca2+-induced calpain activation is followed by subunit dissociation or not. Here, we have applied advanced fluorescence microscopy techniques to study calpain subunit interactions in living cells using recombinant calpain subunits or domains fused to enhanced cyan and enhanced yellow fluorescent reporter proteins. All of the overexpressed variants of the catalytic subunit (DI-IV, DI-III, and DI-IIb) were active and Ca2+-dependent. The intact large subunit, but not its truncated variants, associates with the small subunit under resting and ionomycin-activated conditions. All of the variants were localized in cytoplasm and nuclei, except DI-IIb, which accumulates in the nucleus and in nucleoli as shown by microscopy and cell fractionation. Localization studies with mutated and chimeric variants indicate that nuclear targeting of the DI-IIb variant is conferred by the two N-terminal helices of DI. Only those variants that contain DIII migrated to membranes upon the addition of ionomycin, suggesting that DIII is essential for membrane targeting. We propose that intracellular localization and in particular membrane targeting of activated calpain, but not dissociation of its intact subunits, contribute to regulate its proteolytic activity in vivo
    Type of Publication: Journal article published
    PubMed ID: 12591934
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  • 7
    Keywords: APOPTOSIS ; CELLS ; EXPRESSION ; AGENTS ; CELL ; PATHWAY ; DISEASE ; DISEASES ; PROTEIN ; CULTURED-CELLS ; ACCUMULATION ; RELEASE ; GLYCOPROTEIN ; ISOFORM ; MOUSE ; UP-REGULATION ; PLASMA ; MEMBRANE ; PLASMA-MEMBRANE ; CELL-SURFACE ; protease ; cysteine ; ageing ; CATHEPSIN-B ; CONFORMATIONAL-CHANGES ; CASPASE ; CONFORMATIONAL-CHANGE ; CREUTZFELDT-JAKOB-DISEASE ; INHIBITORS ; PRION PROTEIN ; PRP ACCUMULATION
    Abstract: Prion diseases are characterized by the accumulation of an abnormal, proteinase K-resistant isoform of the prion protein, PrPSc, which is generated by a post-translational conversion of the protease-sensitive normal cell-surface glycoprotein PrPc involving major conformational changes. The conversion is thought to occur at the plasma membrane or along the endocytic pathway towards the lysosome. PrPSc aggregates have been found to accumulate in secondary lysosomes. In our study, the activities of two major lysosomal cysteine proteases, cathepsins B and L, were found to be significantly increased in scrapie-infected Neuro2a cells compared with uninfected cells using biochemical and cytochemical methods. We hypothesize that lysosomal proteases may be involved in a 'second autocatalytic loop' of PrPSc formation, acting in concert with the well-known autocatalytic enhancement of PrP conversion in the presence of PrPSc
    Type of Publication: Journal article published
    PubMed ID: 12867662
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  • 8
    Keywords: RECEPTOR ; CANCER ; CELLS ; INHIBITOR ; SURVIVAL ; carcinoma ; CELL ; Germany ; LUNG ; INFORMATION ; lung cancer ; LUNG-CANCER ; SYSTEM ; SYSTEMS ; COHORT ; POPULATION ; PROTEIN ; TISSUE ; TIME ; PATIENT ; primary ; prognosis ; tumour ; ASSAY ; metastases ; PROGNOSTIC-FACTORS ; PARAMETERS ; MULTIVARIATE ; UROKINASE RECEPTOR ; CARCINOMAS ; PROGNOSTIC FACTORS ; PROTEIN LEVELS ; squamous cell carcinoma ; PARENCHYMA ; RECEPTORS ; POOR-PROGNOSIS ; protease ; PROGNOSTIC FACTOR ; non-small cell lung cancer ; CATHEPSIN-B ; STEFIN-A ; INHIBITORS ; CELL CARCINOMA ; ELISA ; FRACTION ; overall survival ; PH ; CARCINOMA PATIENTS ; carcinoid ; PROGNOSTIC-FACTOR ; ACTIVATOR INHIBITOR ; cathepsin B (cath B) ; CLINICAL-RELEVANCE ; CYSTEINE PROTEASE INHIBITORS ; nonsmall cell lung cancer (NSCLC) ; plasminogenactivator-inhibitor (PAI-1) ; plasminoggenactivator-receptor (uPAR) ; PROTEINASE-INHIBITORS ; PULMONARY ADENOCARCINOMA ; TISSUE-FACTOR ; TUMOR TISSUE
    Abstract: To evaluate the possible role of cysteine proteases and serine proteases.. as well as their respective inhibitors and receptors, as new prognostic factors in NSCLC, we examined, for the first time, 10 biological parameters related to three proteolytic systems within a homogeneous collective of 147 cases of NSCLC. Activities (cath B-AT, cath B-A7.5) and protein levels of cath B-C, cath L-C, uPA, PAI-1, uPAR [measured by three different assays uPAR (ADI), uPAR (HD13), uPAR (IIIF10)] and TF were measured in homogenates of lung tumour tissue and corresponding non-malignant lung parenchyma. Total cath B activity (cath BAT) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath B-A7.5), were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The concentrations of cath B-C, cath L-C, uPA, PAI-1, uPAR and TF were determined by ELISAs. uPAR was determined using three different ELISA formats. The median levels of cath B-AT (5.1-fold), cath BA7.5 (2.5-fold), cath BC, (8.5-fold), cath L-C (6.6-fold), uPA (6.5-fold), PAI-1 (4.2-fold),x uPAR (ADI) (2.2-fold), uPAR (HD13) (4.0-fold) and uPAR (IIIF10)(2.6-fold) were higher in tumour tissue compared to the lung parenchyma. Cath B-AT, cath B-A7.5 and cath B-C in primary tumours correlated with lymph node metastases. Regarding histologies, the concentration of PAI-1 seems to be associated with the histological cell types of NSCLC. We found the highest values of PAI-1 in large cell carcinoma 〉 SCC, AC 〉 carcinoid and lowest values in metastases of primary tumours of other organs. Only PAI-1 was significantly increased in poorly-differentiated cells (G3) compared to well- and moderately- differentiated cells (G1/G2). PAI-1 significantly correlated with cath B-AT, and cath B-A7.5 with uPAR (ADI), uPAR (HD13), uPAR (IIIF10) with uPA, and only weakly with TF, but not with cath B-C, and cath L-C Significant cot-relations with overall survival in the total population of NSCLC patients were observed in univariate analysis for cath B-AT, cath B-C, PAI-1, uPAR (ADI), uPAR (HD13), and uPAR (IIIF10). Cath L-C was not significantly associated with poor prognosis. Regarding the histological tumour type, only in patients with squamous cell carcinomas did cath B-A7.5 and PAI-1 remain significant prognostic factors. In multivariate survival analysis only two proteolytic factors, PAI-1 and uPAR (III10F), stayed significant. In conclusion, among 10 biological parameters evaluated within the same cohort of patients, only PAI-1, uPAR (ADI), uPAR (HD13), uPAR (IIIF10), cath B-AT and cath B-C are prognostic factors for overall survival of NSCLC patients. Moreover, PAI-1 and uPAR (IIIF10) add independent prognostic information with regard to established clinical and histomorphological factors in NSCLC
    Type of Publication: Journal article published
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  • 9
    Keywords: SPECTRA ; CELLS ; Germany ; MICROSCOPY ; GENE-EXPRESSION ; PROTEIN ; PROTEINS ; tumour ; DYNAMICS ; VARIANTS ; MOLECULE ; SPECTROSCOPY ; STATES ; EXCITED-STATES ; GFP ; PH ; intensity ; MUTANTS ; PROFILES ; excited states ; CORAL ; CROSS-SECTIONS ; two-photon excitation
    Abstract: Two-photon (TP) excitation (820-1150 nm) and emission (280-700 nm) spectra for the fluorescent proteins (FPs) ECFP3, EGFP(3) and EYFP3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one-photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue-shifted and emission red-shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out-of-maximum excitation, common practice in TP excitation microscopy, can be used for routine application
    Type of Publication: Journal article published
    PubMed ID: 15725123
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  • 10
    Keywords: CELLS ; INHIBITOR ; INVASION ; Germany ; human ; DISEASE ; DISEASES ; CLONING ; PROTEIN ; PATIENT ; INFECTION ; primary ; tumour ; IDENTIFICATION ; ESCHERICHIA-COLI ; METASTASIS ; TYPE-2 ; mesothelioma ; OSTEOPOROSIS ; pleural effusion ; CATHEPSIN-B ; cysteine peptidases ; CYSTEINE PROTEINASE-INHIBITORS ; HUMAN SPUTUM ; lung inflammation ; NEUTROPHIL ELASTASE ; peptidase inhibitors ; STEFIN-A
    Abstract: Secretory type 2 cystatins, like cystatins C, E/M and F, are thought to be involved in many pathobiological processes, including vascular amyloidosis, rheumatoid arthritis, Alzheimers disease, osteoporosis, viral and bacterial infections, inflammatory disorders and tumour invasion and metastasis. In order to define the levels of cystatins C, E/M, and F in pleural effusions and to investigate whether these cystatins correlate with diagnostic parameters of pleural and lung diseases, we determined their concentrations in 160 pleural effusions. The median concentration of cystatin C in pleural effusions was 1437 mug/l (95.8 nM), ranging between 18- 3967 mug/l. Cystatin C did neither correlate with malignant nor with benign diseases. The concentration of cystatin E/M was significantly higher in effusions of primary pleural tumours (mesotheliomas) compared to secondary pleural tumours and benign diseases. Furthermore, there was a significant correlation between the concentration of cystatin E/M of mesotheliomas and the pleural fluid tumour cell count and of cystatin C. The median values of cystatin F were significantly increased in parapneumonic/ empyema thoracis pleural effusions and tuberculous pleurisy compared to malignant pleural effusions, respectively. The concentration of cystatin F in benign effusions correlated significantly with diagnostic parameters and inflammation (total protein; lactate dehydrogenase; C-reactive protein). Finally, only in the group of parapneumonic/empyema thoracis was there a significant correlation between cystatin F and the neutrophil count. In conclusion, pleural effusions of different origin contain high levels of cystatin C, perhaps constituting the major part of an inhibitor reservoir. The level of cystatin E/M appears to be significantly associated with primary pleural tumours and cystatin F correlates with inflammatory processes of lung disorders
    Type of Publication: Journal article published
    PubMed ID: 12675521
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