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  • DKFZ Publication Database  (6,216)
  • Journal article published  (6,210)
  • Miscellaneous publication  (6)
  • CANCER  (3,234)
  • EXPRESSION  (2,717)
  • PROTEIN  (1,921)
  • GENES  (1,077)
  • ACTIVATION  (967)
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  • DKFZ Publication Database  (6,216)
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  • 1
    Keywords: PROTEIN ; MICE ; ACTIVATION ; PROGRESSION ; inactivation ; NEPHROPATHY ; 2 PARTS ; MANNOSE-BINDING LECTIN ; VASCULAR COMPLICATIONS ; CD59
    Abstract: Coagulation and complement regulators belong to two interactive systems constituting emerging mechanisms of diabetic nephropathy. Thrombomodulin (TM) regulates both coagulation and complement activation, in part through discrete domains. TM's lectin like domain dampens complement activation, while its EGF-like domains independently enhance activation of the anti-coagulant and cytoprotective serine protease protein C (PC). A protective effect of activated PC in diabetic nephropathy is established. We hypothesised that TM controls diabetic nephropathy independent of PC through its lectin-like domain by regulating complement. Diabetic nephropathy was analysed in mice lacking TM's lectin-like domain (TMLeD/LeD) and controls (TMwt/wt). Albuminuria (290 mug/mg vs. 166 mug/mg, p=0.03) and other indices of experimental diabetic nephropathy were aggravated in diabetic TMLeD/LeD mice. Complement deposition (C3 and C5b-9) was markedly increased in glomeruli of diabetic TMLeD/LeD mice. Complement inhibition with enoxaparin ameliorated diabetic nephropathy in TMLeD/LeD mice (e.g. albuminuria 85 mug/mg vs. 290 mug/mg, p〈0.001). In vitro TM's lectin-like domain cell-autonomously prevented glucose-induced complement activation on endothelial cells and - notably - on podocytes. Podocyte injury, which was enhanced in diabetic TMLeD/LeD mice, was reduced following complement inhibition with enoxaparin. The current study identifies a novel mechanism regulating complement activation in diabetic nephropathy. TM's lectin-like domain constrains glucose-induced complement activation on endothelial cells and podocytes and ameliorates albuminuria and glomerular damage in mice.
    Type of Publication: Journal article published
    PubMed ID: 23014597
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  • 2
    Keywords: PROTEINS ; ACTIVATION ; COMPLEXES ; BINDING ; CRYSTAL-STRUCTURE ; sensitivity ; STOICHIOMETRY ; RAFTS ; membrane microdomains ; MULTIVALENT STRUCTURE
    Abstract: The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRbeta chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRbeta chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.
    Type of Publication: Journal article published
    PubMed ID: 23091059
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  • 3
    Keywords: EXPRESSION ; IN-VIVO ; GENE ; transcription ; METABOLISM ; MICE ; NUCLEAR RECEPTORS ; LIVER RECEPTOR HOMOLOG-1 ; LRH-1 ; LIVER-RECEPTOR-HOMOLOG-1
    Abstract: Reverse cholesterol transport (RCT) is an antiatherogenic process in which excessive cholesterol from peripheral tissues is transported to the liver and finally excreted from the body via the bile. The nuclear receptor liver receptor homolog 1 (LRH-1) drives expression of genes regulating RCT, and its activity can be modified by different posttranslational modifications. Here, we show that atherosclerosis-prone mice carrying a mutation that abolishes SUMOylation of LRH-1 on K289R develop less aortic plaques than control littermates when exposed to a high-cholesterol diet. The mechanism underlying this atheroprotection involves an increase in RCT and its associated hepatic genes and is secondary to a compromised interaction of LRH-1 K289R with the corepressor prospero homeobox protein 1 (PROX1). Our study reveals that the SUMOylation status of a single nuclear receptor lysine residue can impact the development of a complex metabolic disease such as atherosclerosis.
    Type of Publication: Journal article published
    PubMed ID: 25176150
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  • 4
    Keywords: EXPRESSION ; TRANSCRIPTION FACTOR ; BINDING ; TARGET ; COLORECTAL-CANCER ; TRANSFORMATION ; OVEREXPRESSION ; cdk4 ; EPITHELIAL-MESENCHYMAL TRANSITION ; CENTROSOME AMPLIFICATION
    Abstract: AP4 represents a c-MYC-inducible bHLH-LZ transcription factor, which displays elevated expression in many types of tumors. We found that serum-starved AP4-deficient mouse embryo fibroblasts (MEFs) were unable to resume proliferation and showed a delayed S-phase entry after restimulation. Furthermore, they accumulated as tetraploid cells due to a cytokinesis defect. In addition, AP4 was required for c-MYC-induced cell cycle re-entry. AP4-deficient MEFs displayed decreased expression of CDK2 (cyclin-dependent kinase 2), which we characterized as a conserved and direct AP4 target. Activation of an AP4 estrogen receptor fusion protein (AP4-ER) enhanced proliferation of human diploid fibroblasts in a CDK2-dependent manner. However, in contrast to c-MYC-ER, AP4-ER activation was not sufficient to induce cell cycle re-entry or apoptosis in serum-starved MEFs. AP4-deficiency was accompanied by increased spontaneous and c-MYC-induced DNA damage in MEFs. Furthermore, c-MYC-induced apoptosis was decreased in AP4-deficient MEFs, suggesting that induction of apoptosis by c-MYC is linked to its ability to activate AP4 and thereby cell cycle progression. Taken together, these results indicate that AP4 is a central mediator and coordinator of cell cycle progression in response to mitogenic signals and c-MYC activation. Therefore, inhibition of AP4 function may represent a therapeutic approach to block tumor cell proliferation.
    Type of Publication: Journal article published
    PubMed ID: 25261373
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  • 5
    Keywords: CANCER ; NETWORKS ; fibroblasts ; cytoskeleton ; DEFORMATION ; EPITHELIAL-MESENCHYMAL TRANSITION ; acrylamide ; VISCOELASTIC PROPERTIES ; CYTOCHALASIN
    Abstract: Mechanical cell properties play an important role in many basic biological functions, including motility, adhesion, proliferation and differentiation. There is a growing body of evidence that the mechanical cell phenotype can be used for detection and, possibly, treatment of various diseases, including cancer. Understanding of pathological mechanisms requires investigation of the relationship between constitutive properties and major structural components of cells, i.e., the nucleus and cytoskeleton. While the contribution of actin und microtubules to cellular rheology has been extensively studied in the past, the role of intermediate filaments has been scarcely investigated up to now. Here, for the first time we compare the effects of drug-induced disruption of actin and vimentin intermediate filaments on mechanical properties of suspended NK cells using high-throughput deformability measurements and computational modeling. Although, molecular mechanisms of actin and vimentin disruption by the applied cytoskeletal drugs, Cytochalasin-D and Withaferin-A, are different, cell softening in both cases can be attributed to reduction of the effective density and stiffness of filament networks. Our experimental data suggest that actin and vimentin deficient cells exhibit, in average, 41% and 20% higher deformability in comparison to untreated control. 3D Finite Element simulation is performed to quantify the contribution of cortical actin and perinuclear vimentin to mechanical phenotype of the whole cell. Our simulation provides quantitative estimates for decreased filament stiffness in drug-treated cells and predicts more than two-fold increase of the strain magnitude in the perinuclear vimentin layer of actin deficient cells relatively to untreated control. Thus, the mechanical function of vimentin becomes particularly essential in motile and proliferating cells that have to dynamically remodel the cortical actin network. These insights add functional cues to frequently observed overexpression of vimentin in diverse types of cancer and underline the role of vimentin targeting drugs, such as Withaferin-A, as a potent cancerostatic supplement.
    Type of Publication: Journal article published
    PubMed ID: 24952458
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  • 6
    Keywords: EXPRESSION ; IN-VIVO ; PATHWAY ; LUNG-CANCER ; ACTIVATION ; RADIATION-THERAPY ; chemotherapy ; SQUAMOUS-CELL CARCINOMA ; CISPLATIN ; tumor microenvironment ; HIF-1 ; GLUCOSE-METABOLISM ; UNFAVORABLE PROGNOSIS ; HUMAN TUMOR XENOGRAFT ; HIF-1-ALPHA ; Local tumor control ; Fractionated radiation ; HIF pathway inhibition ; INDUCIBLE FACTOR-1-ALPHA
    Abstract: BACKGROUND: The transcription factor hypoxia-inducible factor-1 (HIF-1) pathway plays an important role in tumor response to cytotoxic treatments. We investigated the effects of a novel small molecule inhibitor of mitochondrial complex I and hypoxia-induced HIF-1 activity BAY-87-2243, on tumor microenvironment and response of human squamous cell carcinoma (hSCC) to clinically relevant fractionated radiotherapy (RT) with and without concomitant chemotherapy. METHODS: When UT-SCC-5 hSCC xenografts in nude mice reached 6 mm in diameter BAY-87-2243 or carrier was administered before and/or during RT or radiochemotherapy with concomitant cisplatin (RCT). Local tumor control was evaluated 150 days after irradiation and the doses to control 50% of tumors (TCD50) were compared between treatment arms. Tumors were excised at different time points during BAY-87-2243 or carrier treatment for western blot and immunohistological investigations. RESULTS: BAY-87-2243 markedly decreased nuclear HIF-1alpha expression and pimonidazole hypoxic fraction already after 3 days of drug treatment. BAY-87-2243 prior to RT significantly reduced TCD50 from 123 to 100 Gy (p=0.037). Additional BAY-87-2243 application during RT did not decrease TCD50. BAY-87-2243 before and during radiochemotherapy did not improve local tumor control. CONCLUSIONS: Pronounced reduction of tumor hypoxia by application of BAY-87-2243 prior to RT improved local tumor control. The results demonstrate that radiosensitizing effect importantly depends on treatment schedule. The data support further investigations of HIF-1 pathway inhibitors for radiotherapy and of predictive tests to select patients who will benefit from this combined treatment.
    Type of Publication: Journal article published
    PubMed ID: 25234922
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  • 7
    Keywords: APOPTOSIS ; CANCER ; CELLS ; ANTITUMOR-ACTIVITY ; POTENT ; POLO-LIKE KINASE ; SMALL-MOLECULE INHIBITOR ; SELECTIVE INHIBITOR ; POLO-LIKE-KINASE-1 ; BI-2536
    Abstract: We synthesized a series of vanillin-derived compounds and analyzed them in HeLa cells for their effects on the proliferation of cancer cells. The molecules are derivatives of the lead compound SBE13, a potent inhibitor of the inactive conformation of human polo-like kinase 1 (Plk1). Some of the new designs were able to inhibit cancer cell proliferation to a similar extent as the lead structure. Two of the compounds ((({4-[(6-chloropyridin-3-yl)methoxy]-3-methoxyphenyl}methyl)(pyridin-4-ylmethyl) amine) and (({4-[(4-chlorophenyl)methoxy]-3-methoxyphenyl}methyl)(pyridin-4-ylmethyl)amine)) were much stronger in their capacity to reduce HeLa cell proliferation and turned out to potently induce apoptosis and reduce Plk1 kinase activity in vitro.
    Type of Publication: Journal article published
    PubMed ID: 25304894
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  • 8
    Keywords: CANCER ; GENOME ; PATHWAYS ; THERAPY ; GENE ; BREAST-CANCER ; prognosis ; MUTATION ; DATABASE ; MUTATIONS ; SOMATIC MUTATIONS ; tumours ; Type ; LANDSCAPES ; MYELOID-LEUKEMIA GENOME ; PROJECT ; CANCER-THERAPY ; MANAGEMENT ; SUBTYPES ; REPERTOIRE ; SCIENCE ; development
    Abstract: The International Cancer Genome Consortium (ICGC) was launched to coordinate large-scale cancer genome studies in tumours from 50 different cancer types and/or subtypes that are of clinical and societal importance across the globe. Systematic studies of more than 25,000 cancer genomes at the genomic, epigenomic and transcriptomic levels will reveal the repertoire of oncogenic mutations, uncover traces of the mutagenic influences, define clinically relevant subtypes for prognosis and therapeutic management, and enable the development of new cancer therapies
    Type of Publication: Miscellaneous publication
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  • 9
    Keywords: microarrays ; gene expression ; GENE ; EXPRESSION
    Type of Publication: Miscellaneous publication
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  • 10
    Keywords: KINASE INHIBITOR ; PROTEIN-KINASE ; PROTEIN ; COMPLEX ; COMPLEXES ; INHIBITOR ; KINASE
    Type of Publication: Miscellaneous publication
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  • 11
    Keywords: FIELDS ; cancer research ; FIELD ; CANCER
    Type of Publication: Miscellaneous publication
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  • 12
    Keywords: MUTANT P53 ; ANTAGONISTS ; MDM2 ; ACTIVATION ; CELLS
    Type of Publication: Miscellaneous publication
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  • 13
    Keywords: RECEPTOR ; ANGIOGENESIS ; APOPTOSIS ; CANCER ; CANCER CELLS ; CELLS ; ENDOTHELIAL-CELLS ; EXPRESSION ; FLK-1/KDR ; GROWTH ; GROWTH-FACTOR ; IN-VITRO ; INHIBITOR ; INVASION ; IONIZING-RADIATION ; IRRADIATION ; proliferation ; PROTECTION ; radiotherapy ; SURVIVAL ; tumor ; TUMOR-CELLS
    Abstract: In recent decades, radiation research has concentrated primarily on the cancer cell compartment. Much less is known about the effect of ionizing radiation on the endothelial cell compartment and the complex interaction between tumor cells and their microenvironment. Here we report that ionizing radiation is a potent antiangiogenic agent that inhibits endothelial cell survival, proliferation, tube formation and invasion. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor were able to reduce the radiosensitivity of endothelial cells. Yet, it is also found that radiation induces angiogenic factor production by tumor cells that can be abrogated by the addition of antiangiogenic agents. Receptor tyrosine kinase inhibitors of Flk-1/KDR/VEGFR2, FGFR1 and PDGFRbeta, SU5416, and SU6668 enhanced the antiangiogenic effects of direct radiation of the endothelial cells. In a coculture system of PC3 prostate cancer cells and endothelial cells, isolated irradiation of the PC3 cells enhanced endothelial cell invasiveness through a Matrigel matrix, which was inhibited by SU5416 and SU6668. Furthermore, ionizing radiation up-regulated VEGF and basic fibroblast growth factor in PC3 cells and VEGFR2 in endothelial cells. Together these findings suggest a radiation-inducible protective role for tumor cells in the support of their associated vasculature that may be down- regulated by coadministration of angiogenesis inhibitors., These results rationalize concurrent administration of angiogenesis inhibitors and radiotherapy in cancer treatment
    Type of Publication: Journal article published
    PubMed ID: 12839971
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  • 14
    Keywords: RECEPTOR ; SPECTRA ; ANGIOGENESIS ; CANCER ; GROWTH ; GROWTH-FACTOR ; IN-VITRO ; INHIBITOR ; proliferation ; SURVIVAL ; tumor ; ADVANCED SOLID TUMORS ; AGENTS ; ANGIOSTATIN ; BLOOD ; carcinoma ; CELL ; CELL LUNG-CANCER ; CELL-PROLIFERATION ; CLINICAL-TRIAL ; COMBINATION ; DOPPLER ; ENDOTHELIAL GROWTH-FACTOR ; evaluation ; FACTOR RECEPTOR ; Germany ; human ; IN-VIVO ; INHIBITION ; KINASE ; LUNG ; MICROSCOPY ; MICROVESSEL DENSITY ; MODEL ; MODELS ; neoplasms ; PATHWAY ; PATHWAYS ; PERFUSION ; PHASE-I ; PROSTATE ; RECOMBINANT HUMAN ENDOSTATIN ; THERAPY ; TOXICITY ; tumor growth ; TYROSINE KINASE ; VITRO ; VIVO
    Abstract: The multifaceted nature of the angiogenic process in malignant neoplasms suggests that protocols that combine antiangiogenic agents may be more effective than single-agent therapies. However it is unclear which combination of agents would be most efficacious and will have the highest degree of synergistic activity while maintaining low overall toxicity. Here we investigate the concept of combining a "direct" angiogenesis inhibitor (endostatin) with an "indirect" antiangiogenic compound [SU5416, a vascular endothelial growth factor receptor 2 (VEGFR2) receptor tyrosine kinase (RTK) inhibitor]. These angiogenic agents were more effective in combination than when used alone in vitro (endothelial cell proliferation, survival, migration/invasion, and tube formation tests) and in vivo. The combination of SU5416 and low-dose endostatin further reduced tumor growth versus monotherapy in human prostate (M), lung (A459), and glioma (U87) xenograft models, and reduced functional microvessel density, tumor microcirculation, and blood perfusion as detected by intravital microscopy and contrast-enhanced Doppler ultrasound. One plausible explanation for the efficacious combination could be that, whereas SU5416 specifically inhibits vascular endothelial growth factor signaling, low-dose endostatin is able to inhibit a broader spectrum of diverse angiogenic pathways directly in the endothelium. The direct antiangiogenic agent might be able to suppress alternative angiogenic pathways up-regulated by the tumor in response to the indirect, specific pathway inhibition. For future clinical evaluation of the concept, a variety of agents with similar mechanistic properties could be tested
    Type of Publication: Journal article published
    PubMed ID: 14695206
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  • 15
    Keywords: measurement ; CANCER ; proliferation ; Germany ; LUNG ; THERAPY ; ALGORITHM ; ALGORITHMS ; ANATOMICAL MODEL ; AUTOMATIC DETECTION ; BAYESIAN-ANALYSIS ; cancer screening ; chest ; CLASSIFICATION ; COMMON ; computed tomography (CT),image processing ; computers,diagnostic aid ; computers,neural networks ; CT ; DENSITY ; DIAGNOSIS ; EMPHYSEMA ; FOLLOW-UP ; follow-up studies ; GENERATION ; GROUND-GLASS OPACITIES ; HIGH-RESOLUTION CT ; IMAGES ; imaging ; INFORMATION ; lung cancer ; LUNG-CANCER ; MASK ; MULTIPLE NEURAL-NETWORKS ; NETWORK ; NETWORKS ; neural networks ; QUANTIFICATION ; screening ; segmentation ; SOLITARY PULMONARY NODULES ; SPIRAL CT ; SUPPORT ; SYSTEM ; SYSTEMS ; thorax ; TOMOGRAPHY IMAGES ; TOOL ; TOTAL LUNG CAPACITY ; VENTILATION ; VISUALIZATION ; VOLUME
    Abstract: The proliferation of digital data sets and the increasing amount of images, e.g. through the use of multislice spiral CT or multiple follow-up examinations in the context of new therapies, are ideal prerequisites for computer-aided diagnosis (CAD) in chest radiology. Multiple studies have described the applications and advantages of computer assistance in performing different diagnostic tasks. More powerful computers will enable the introduction of these systems into the clinical routine and could provide an enormous increase in morphological and functional information. The commercial introduction of tools for detection and visualization of pulmonary nodules has already begun. This is one of the most widely-reported applications in view of the ongoing studies on lung cancer screening. The next generation of tools will improve the diagnosis of emphysema through detection, quantification and classification. Many more uses are being developed, for instance the detection and classification of infiltrates, volume measurements or functional pulmonary imaging (e.g. dynamic ventilation CT or (3)Helium-MRI). Grossly simplified, most systems use a three level structure consisting of segmentation/feature extraction, classification of extracted features and an output unit. The output can be mere visualization through color-coding, volume measurements or calculated probabilities. The output supports the radiologist in establishing his findings and preparing differential and final diagnoses as well as providing quantitative data for follow-up studies. Different techniques are used for segmentation of lung areas as the basis for a variety of applications. Some commonly-used techniques for this and other tasks are density masks and threshold-based algorithms. Data processing is predominantly carried out with Bayesian classifiers or neural networks. This article describes the current status of research and provides insight into the common schemes and capabilities of the systems. It focuses particularly on common topics such as segmentation, volume measurement, detection of pulmonary nodules, quantification of emphysema and analysis of ground glass opacities
    Type of Publication: Journal article published
    PubMed ID: 14610697
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  • 16
    Keywords: CANCER ; Germany ; LUNG ; lung cancer ; LUNG-CANCER ; SYSTEM ; CANCER MORTALITY ; COHORT ; cohort studies ; cohort study ; cohort-studies ; DEATH ; DISEASE ; DISEASES ; DNA adducts ; EPIDEMIOLOGY ; EXPOSURE ; HEPATOCELLULAR-CARCINOMA ; HISTORY ; incidence ; iron foundry ; larynx ; liver ; LONG-TERM ; missing death certificates ; MORTALITY ; mouth ; NEW-YORK ; occupation ; PHARYNX ; POPULATION ; PRIMARY LIVER-CANCER ; RISK ; RISKS ; SITE ; SITES ; WORKERS
    Abstract: Background Observations of an increased incidence of cancers of the upper aero-digestive tract (pharynx, esophagus, larynx, lung) among workers of local German foundries gave rise to concern about a potentially elevated occupational risk of those cancer sites. The purpose of the study was to examine whether occupational exposure in iron foundries increases the risk of cancer. Methods A historical cohort study of 17,708 male German production workers in 37 iron foundries who were first employed in 1950-1985 with a minimum employment period of 1 year was initiated. Employment and occupational histories were collected. Mortality was compared with that of the German general population during 1950-1993 using a new method for computing the SMR when not all causes of death are available (called SMR*). Results Mortality from all causes was elevated to SMR = 115.4 (95% confidence interval (CI) = 111.9-119.1), as was for total cancer (SMR* = 123.8, CI = 102.1-152.6), especially cancers of the lung (SMR* = 163.9, CI = 123.9-223.0) and liver (SMR* = 322.5, CI = 149.5-844.8), and diseases of the respiratory system (SMR* = 147.6, CI = 100.4-221.5). Non- significant elevations of mortality were also found for cancers of the mouth and pharynx (SMR* = 153.5, CI = 82.3-359.8) and larynx (SMR* = 173.1, CI = 85.5-550.5). Mortality from various causes of death was higher among workers with shorter exposure periods than among long-term employees. The elevated mortality persisted for years and decades after termination of employment. Conclusions The results provide further evidence for an increased risk of lung cancer and possibly other cancers of the upper aero-digestive tract among foundry workers. Special attention should be paid to the strongly increased mortality from liver cancer and the mortality pattern among employees having terminated work. (C) 2003 Wiley-Liss, Inc
    Type of Publication: Journal article published
    PubMed ID: 12594777
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  • 17
    Keywords: CELLS ; EXPRESSION ; INHIBITOR ; tumor ; IN-VIVO ; GENE ; TUMORS ; ACCUMULATION ; CATECHOLAMINES ; gene therapy ; human norepinephrine transporter ; I-123 MIBG ; LINES ; METAIODOBENZYLGUANIDINE UPTAKE ; MIBG uptake ; MICE ; NEUROBLASTOMA-CELLS ; NORADRENALINE TRANSPORTER ; NUCLEAR-MEDICINE ; radiation ; RELEASE ; STORAGE ; TIME ; TRANSDUCTION
    Abstract: The transport of MIBG by the human norepinephrine transporter (hNET) seems to be the critical step in the treatment of MIBG- concentrating tumors. Therefore, we investigated whether the accumulation of MIBG may be induced by retroviral transect on of the hNET gene in Morris hepatoma cells. Methods: A bicistronic retroviral vector for the transfer of the hNET coding sequence and the hygromycin resistance gene was generated. Morris hepatoma cells (MH3924A) were infected with the respective retroviral particles, and hNET-expressing cell lines MHhNEThyg1 to MHhNEThyg9 were obtained through hygromycin selection. The uptake of H-3-norepinephrine or I-131-MIBG and the efflux of I-131-MIBG were determined in transfected and wild-type cells. In addition, the I-131-MIBG distribution was monitored in nude mice and rats bearing wild-type and hNET- expressing hepatomas. Results: hNET-expressing hepatoma cell lines accumulated up to 36 times more norepinephrine than did wild-type cells and 8 times more than did hNET-expressing neuroblastoma cell line SK-N-SH. The addition of nisoxetine, a selective inhibitor of noradrenaline uptake, inhibited norepinephrine uptake. Maximal I-131-MIBG accumulation was observed 2 h after incubation and was followed by 43% efflux within 4 h after the I-131-MIBG-containing medium had been removed. In vivo experiments performed with nude mice bearing both hNET-expressing and wild-type tumors showed a 10-fold- higher accumulation of I-131-MIBG in transfected tumors than in wild-type tumors. The ex vivo calculations revealed doses of 605 and 75 mGy in hNET-expressing and wild-type tumor tissues, respectively. Conclusion: Transduction of the hNET gene enables Morris hepatoma cells to accumulate norepinephrine and MIBG. However, the retention of MIBG is brief; therefore, the absorbed dose of radiation in vivo is not expected to be therapeutically effective
    Type of Publication: Journal article published
    PubMed ID: 12791828
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  • 18
    Keywords: CELLS ; EXPRESSION ; CELL ; Germany ; PATHWAY ; PATHWAYS ; ACETATE-NONUTILIZING MUTANTS ; CDNA ; CDNA CLONES ; CLONES ; CLONING ; CRASSA ; DISTINCT ; DNA-microarray analysis,transcriptional profiling,correspondence analysis,acetate metabolism,Neurosp ; ENZYMES ; FILAMENTOUS FUNGUS ; GENE ; GENE-EXPRESSION ; GENES ; GENOME ; GENOME SEQUENCE ; HYBRIDIZATION ; microarray ; NMT1 ; PROTEIN ; PROTEINS ; RNA ; SACCHAROMYCES-CEREVISIAE ; SAMPLE ; SAMPLES ; transcription
    Abstract: Nutrient-dependent variations in transcript levels of the filamentous fungus Neurospora crassa were studied on a microarray containing some 4700 cDNAs. Cells were grown in minimal and acetate medium. The isolated RNA was analyzed in comparison to the results obtained upon the hybridization of samples prepared from the RNA of cells grown in full medium. Altogether, 160 cDNA clones exhibited significant variations, falling into five distinct subgroups of very similar transcription profiles. This is indicative of the occurrence of a high degree of co-regulation of genes in N. crassa. Especially the regulation of the expression of proteins involved in metabolic pathways was found to be strongly regulated at the RNA level. (C) 2003 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 14599890
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  • 19
    Keywords: CELLS ; tumor ; CELL ; Germany ; MICROSCOPY ; DIAGNOSIS ; NEW-YORK ; PROTEIN ; PROTEINS ; ADHESION MOLECULES ; cell line ; COMPONENTS ; CONSTITUTIVE TRANSMEMBRANE GLYCOPROTEIN ; CULTURED-CELLS ; DESMOCOLLIN ; desmoplakin ; desmosome ; DIFFERENTIATION ; EPITHELIA ; HUMAN PLAKOGLOBIN ; INTERMEDIATE-SIZED FILAMENTS ; meninges,meningioma,desmosome,desmocollin 3 ; meningioma ; MOLECULAR CHARACTERIZATION ; MOLECULES ; MONOCLONAL-ANTIBODY ; MR-165000 DESMOGLEIN ; NON-EPIDERMAL DESMOSOMES ; PLAQUE PROTEIN ; SUBDURAL SPACE ; TISSUE ; TUMORS
    Abstract: Intercellular junctions morphologically identical to epithelial desmosomes are known structures in meningiomas and arachnoidal tissue. Desmoplakin as one of the desmosomal plaque components has proven to be a reliable marker for diagnosis of meningeal tumors. Here we demonstrate by immunofluorescence microscopy, immunoblot and reverse transcription-PCR reactions that cells of arachnoidal tissue, of diverse meningioma subtypes and of a meningioma-derived cell line contain the full complement of the typical desmosomal proteins desmoplakin (DP), plakophilin 2 (PP2), desmocollin 2 (Dsc2) and desmoglein 2 (Dsg2). Consequently, all these molecules are suitable for diagnostic applications of meningioma tumors. In addition to these constitutive desmosomal components, representative for single-layered (simple) epithelia, the dural border cells of the arachnoid and about 60% of the meningiomas tested were positive for desmocollin 3 (Dsc3), a protein in epithelia taken as an indicator for differentiation
    Type of Publication: Journal article published
    PubMed ID: 12845453
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  • 20
    Keywords: EXPRESSION ; FACTOR RECEPTOR ; ACID SPHINGOMYELINASE ; DEATH DOMAIN ; ENDOTOXIN-SHOCK ; NF-KAPPA-B ; NITRIC-OXIDE SYNTHASE ; SIGNAL- TRANSDUCTION ; STRESS-INDUCED APOPTOSIS ; TNF-RECEPTOR ; TUMOR-NECROSIS-FACTOR
    Abstract: Stimulation of macrophages by lipopolysaccharide (LPS) leads to the synthesis of proinflammatory cytokines and nitric oxide (NO) and, as a consequence, to endotoxic shock. We provide evidence that LPS stimulates the activity of a membrane- associated neutral sphingomyelinase (nSMase) and that this activity is mandatory for the liberation of nuclear factor- kappaB (NFkappaB) and the induction of inducible NO-synthase (iNOS). With the aid of a newly developed, selective inhibitor of nSMase, C11AG, we could distinguish between nSMase-dependent and -independent LPS-induced signals. C11AG blocked LPS- stimulated sphingomyelin degradation and NFkappaB activation without interfering with p42 tyrosine phosphorylation. Concomitantly, the expression of iNOS was found to be reduced both in mononuclear cells and in murine endotoxemia. Therefore, specific Inhibitors of nSMase may define a new class of antiinflammatory substances
    Type of Publication: Journal article published
    PubMed ID: 12866359
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  • 21
    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; IN-VITRO ; proliferation ; IN-VIVO ; MODEL ; DISEASE ; DISTINCT ; MICE ; ACTIVATED MACROPHAGES ; ACTIVATION ; COMPLEX ; CRESCENTIC GLOMERULONEPHRITIS ; INJURIES ; LIGAND ; MESANGIAL CELLS ; MONOCYTE ARREST ; NEPHRITIS ; NITRIC-OXIDE ; RANTES ; RESPONSES
    Abstract: The chemokine CC chemokine ligand (CCL)5/RANTES as well as its respective receptor CCR5 mediate leukocyte infiltration during inflammation and are up-regulated early during the course of glomerulonephritis (GN). We tested the effects of the two CCL5/RANTES blocking analogs, Met-RANTES and amino-oxypentane- RANTES, on the course of horse apoferritin (RAF)induced GN. HAF-injected control mice had proliferative GN with mesangial immune complex deposits of IgG and HAF. Daily i.p. injections of Met-RANTES or amino-oxypentane-RANTES markedly reduced glomerular cell proliferation and glomerular macrophage infiltration, which is usually associated with less glomerular injury and proteinuria in RAF-GN. Surprisingly, however, RAF-GN mice treated with both analogs showed worse disease with mesangiolysis, capillary obstruction, and nephrotic range albuminuria. These findings were associated with an enhancing effect of the CCL5/RANTES analogs on the macrophage activation state, characterized by a distinct morphology and increased inducible NO synthetase expression in vitro and in vivo, but a reduced uptake of apoptotic cells in vivo. The Immoral response and the Th1/Th2 balance in HAF-GN and mesangial cell proliferation in vitro were not affected by the CCL5/RANTES analogs. We conclude that, despite blocking local leukocyte recruitment, chemokine analogs can aggravate some specific disease models, most likely due to interactions with systemic immune reactions, including the removal of apoptotic cells and inducible NO synthetase expression
    Type of Publication: Journal article published
    PubMed ID: 12759447
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  • 22
    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; IN-VITRO ; Germany ; human ; DISEASE ; DISEASES ; MICE ; ACTIVATION ; COMPLEX ; MESANGIAL CELLS ; ARTHRITIS ; CELL ACTIVATION ; COMPLEXES ; CUTTING EDGE ; DNA ; IFN-GAMMA ; INFECTION ; kidney ; MACROPHAGES ; MECHANISM ; MESSENGER-RNA ; MESSENGER-RNA EXPRESSION ; MOTIFS ; murine ; OLIGODEOXYNUCLEOTIDES ; RECEPTOR EXPRESSION ; SERA ; TH1 RESPONSES ; TRIGGER
    Abstract: Immune complex glomerulonephritis (GN) often deteriorates during infection with viruses and bacteria that, in contrast to mammals, have DNA that contains many unmethylated CpG motifs. Balb/c mice with horse apoferritin-induced GN (HAF-GN) were treated with either saline, CpG-oligodeoxynucleotides (ODN), or control GpC-ODN. Only CpG-ODN exacerbated HAF-GN with an increase of glomerular macrophages, which was associated with massive albuminuria and increased renal MCP-1/CCL2, RANTES/CCL5, CCR1, CCR2, and CCR5 mRNA expression. CpG-ODN induced a Th1 response as indicated by serum anti-HAF IgG(2a) titers, mesangial IgG(2a) deposits, and splenocyte IFN-gamma secretion. Messenger RNA for the CpG-DNA receptor Toll-like reeptor 9 (TLR9) was present in kidneys with HAF-GN but not in normal kidneys. The source of TLR9 mRNA in HAF-GN could. be infiltrating macrophages or intrinsic renal cells, e.g., mesangial cells; but, in vitro, only murine J774 macrophages expressed TLR9. In J774 cells, CpG-ODN induced the chemokines MCP-1/CCL2 and RANTES/CCL5 and the chemokine receptors CCR1 and CCR5. It is concluded that CpG-DNA can aggravate preexisting GN via a shift toward a Th1 response but also by a novel pathway involving TLR9-mediated chemokine and chemokine receptor expression by macrophages, which may contribute to the enhanced glomerular macrophage recruitment and activation. This mechanism may be relevant during infection-triggered exacerbation of human immune-complex GN and other immune- mediated diseases in general
    Type of Publication: Journal article published
    PubMed ID: 12538732
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  • 23
    Keywords: MODEL ; PROTEIN ; FAMILY ; PROTEIN FAMILIES ; PROTEIN FAMILY ; DOMAIN ; PAIRS ; recombination
    Abstract: There is a limited repertoire of domain families in nature that are duplicated and combined in different ways to form the set of proteins in a genome. Most proteins in both prokaryote and eukaryote genomes consist of two or more domains, and we show that the family size distribution of multi-domain protein families follows a power law like that of individual families. Most domain pairs occur in four to six different domain architectures: in isolation and in combinations with different partners. We showed previously that within the set of all pairwise domain combinations, most small and medium-sized families are observed in combination with one or two other families, while a few large families are very versatile and combine with many different partners. Though this may appear to be a stochastic pattern, in which large families have more combination partners by virtue of their size, we establish here that all the domain families with more than three members in genomes are duplicated more frequently than would be expected by chance considering their number of neighbouring domains. This duplication of domain pairs is statistically significant for between one and three quarters of all families with seven or more members. For the majority of pairwise domain combinations, there is no known three-dimensional structure of the two domains together, and we term these novel combinations. Novel domain combinations are interesting and important targets for structural elucidation, as the geometry and interaction between the domains will help understand the function and evolution of multi-domain proteins. Of particular interest are those combinations that occur in the largest number of multi-domain proteins, and several of these frequent novel combinations contain DNA-binding domains.
    Type of Publication: Journal article published
    PubMed ID: 14649290
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  • 24
    Keywords: CANCER ; CELLS ; EXPRESSION ; IN-VIVO ; LUNG-CANCER ; DNA adducts ; RISK ; GENE ; LINES ; ACTIVATION ; DNA ; 3-aminobenzanthrone ; 3-nitrobenzanthrone ; AIR ; CARCINOGENESIS ; CYP1A2 ; CYTO-TOXIC METABOLITES ; DIESEL EXHAUST ; DNA ADDUCT FORMATION ; ENVIRONMENTAL CONTAMINANT 3-NITROBENZANTHRONE ; GENETIC POLYMORPHISMS ; HETEROCYCLIC AMINES ; HETEROLOGOUS EXPRESSION ; HUMAN CYTOSOLIC SULFOTRANSFERASES ; IONS ; metabolic activation ; NAT : SULT ; nitro-PAH ; P-32- postlabeling ; PHENOL SULFOTRANSFERASES ; POSTLABELING ANALYSIS
    Abstract: 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and ambient air pollution. 3-Aminobenzanthrone (3-ABA), 3- acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3- aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites. Recently we found that 3-NBA and its metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) form the same DNA adducts in vivo in rats. In order to investigate whether human cytochrome P450 (CYP) enzymes (i.e., CYPIA2), human N,O- acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA and its metabolites we developed a panel of Chinese hamster V79MZ-hIA2 derived cell lines expressing human CYPIA2 in conjunction with human NATI, NAT2, SULTIAI or SULTIA2, respectively. Cells were treated with 0.01, 0.1 or I muM 3-NBA, or its metabolites (3- ABA, 3-Ac-ABA and N-Ac-N-OH-ABA). Using both enrichment versions of the P-32-postlabeling assay, nuclease P I digestion and butanol extraction, essentially 4 major and 2 minor DNA adducts were detected in the appropriate cell lines with all 4 compounds. The major ones were identical to those detected in rat tissue; the adducts lack an N-acetyl group. Human CYPIA2 was required for the metabolic activation of 3-ABA and 3-Ac-ABA (probably via N-oxidation) and enhanced the activity of 3-NBA (probably via nitroreduction). The lack of acetylated adducts suggests N-deacetylation of 3-Ac-ABA and N-Ac-N-OH-ABA. Thus, N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be a common intermediate for the formation of the electrophilic arylnitrenium ions capable of reacting with DNA. Human NAT I and NAT2 as well as human SULTIAI and SULTIA2 strongly contributed to the high genotoxicity of 3-NBA and its metabolites. Moreover, N,O-acetyltransfer reactions catalyzed by human NATs leading to the corresponding N-acetoxyester may be important in the bioactivation of N-Ac-N-OH-ABA. As human exposure to 3-NBA is likely to occur primarily via the respiratory tract, expression of CYPs, NATs and SULTs in respiratory tissues may contribute significantly and specifically to the metabolic activation of 3-NBA and its metabolites. Consequently, polymorphisms in these genes could be important determinants of lung cancer risk from 3-NBA
    Type of Publication: Journal article published
    PubMed ID: 12740904
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  • 25
    Keywords: EXPRESSION ; INHIBITOR ; Germany ; KINASE ; GENE ; GENES ; PROTEIN ; SACCHAROMYCES-CEREVISIAE ; transcription ; COMPONENTS ; COMPLEX ; COMPLEXES ; cell cycle ; CELL-CYCLE ; CYCLE ; protein kinase ; PROTEIN-KINASE ; IDENTIFICATION ; gene expression ; protein kinase CK2 ; Saccharomyces cerevisiae ; SUBUNIT ; YEAST ; BUDDING YEAST ; DISRUPTION ; EXPRESSION ANALYSIS ; HOLOENZYME ; PHO pathway
    Abstract: The budding yeast Saccharomyces cerevisiae encounters phosphate starvation by the transcription-regulated PHO pathway. We find that genetic perturbation of protein kinase CK2, a conserved tetrameric Ser/Thr phosphotransferase with links to cell cycle and transcription, affects expression of PHO pathway genes in a subunit- and isoform-specific manner. Remarkably, the genes encoding phosphate supplying phosphatases and transporters are significantly repressed, while the genes encoding components of the central pathway regulator complex, a cyclin-dependent kinase (CDK), a cyclin, and a CDK inhibitor, remain unaltered. Thus, perturbation of CK2 uncouples the executive part of the PHO pathway from its cyclin-CDK control complex. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies
    Type of Publication: Journal article published
    PubMed ID: 12606059
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  • 26
    Keywords: EXPRESSION ; screening ; DISTINCT ; GENE ; GENES ; GENOME ; DNA ; FAMILY ; ASSOCIATION ; autistic disorder,susceptibility gene,chromosome 2,mutation screening,association ; CANDIDATE GENE ; chromosome ; DISORDER ; DLX GENES ; FREQUENCY ; GENOMEWIDE SCREEN ; GENOMIC SCREEN ; GLUTAMIC-ACID DECARBOXYLASE ; LINKAGE ; polymorphism ; POLYMORPHISMS ; SIGNAL ; single nucleotide polymorphism ; SUSCEPTIBILITY ; SUSCEPTIBILITY GENES ; SUSCEPTIBILITY LOCUS ; VARIANTS
    Abstract: The results from several genome scans indicate that chromosome 2q21-q33 is likely to contain an autism susceptibility locus. We studied the potential contribution of nine positional and functional candidate genes: TBR-1; GAD1; DLX1; DLX2; cAMP-GEFII; CHN1; ATF2; HOXD1 and NEUROD1. Screening these genes for DNA variants and association analysis using intragenic single nucleotide polymorphisms did not provide evidence for a major role in the aetiology of autism. Four rare nonsynonymous variants were identified, however, in the cAMP-GEFII gene. These variants were present in five families, where they segregate with the autistic phenotype, and were not observed in control individuals. The significance of these variants is unclear, as their low frequency in IMGSAC families does not account for the relatively strong linkage signal at the 2q locus. Further studies are needed to clarify the contribution of cAMP-GEFII gene variants to autism susceptibility
    Type of Publication: Journal article published
    PubMed ID: 14593429
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  • 27
    Keywords: CANCER ; PROTECTION ; MODEL ; DISEASE ; EPIDEMIOLOGY ; HISTORY ; RISK ; GENE ; GENES ; SAMPLE ; FAMILY ; RISK-FACTORS ; SUSCEPTIBILITY ; BREAST ; breast cancer ; BREAST-CANCER ; AGE ; BRCA1 ; case-only design ; family history ; gene carrier probability ; LINKAGE ANALYSIS ; mixture logistic model ; ovarian cancer ; OVARIAN-CANCER ; population and sibling controls ; WOMEN
    Abstract: Background The effect of environmental/lifestyle factors on breast cancer risk may be modified by genetic predisposition. Methods In a population-based case-control-family study performed in Germany including 706 cases by age 50 years, 1381 population, and 252 sister controls, we investigated main effects for environmental/lifestyle factors and genetic susceptibility and gene-environment interaction (G x E). Different surrogate measures for genetic predisposition using pedigree information were used: first-degree family history of breast or ovarian cancer; and gene carrier probability using a genetic model based on rare dominant genes. Possible G x E interaction was studied by (1) logistic regression using cases and population controls including an interaction term; (2) comparing results using sister controls and population controls; (3) case-only analysis with logistic regression and (4) a mixture logistic model. Results Familial predisposition showed the strongest main effect and the estimated gene carrier probability gave the best fit. High parity and longer duration of breastfeeding reduced breast cancer risk significantly, a history of abortions increased risk and age at menarche showed no significant effect. We found significant G x E interaction between parity and genetic susceptibility using different surrogate measures. In women most likely to have a high genetic susceptibility, high parity was less protective. Later age at menarche was protective in women with a positive family history. No evidence for G x E interaction was found for breastfeeding and abortion. Conclusions These findings corroborate results from other studies and provide further evidence that the magnitude of protection from parity is reduced in women most likely to have a genetic risk in spite of the limitations of using surrogate genetic measures
    Type of Publication: Journal article published
    PubMed ID: 12690006
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  • 28
    Keywords: RECEPTOR ; EXPRESSION ; GROWTH ; IN-VIVO ; PATHWAYS ; PROTEIN ; SACCHAROMYCES-CEREVISIAE ; COMPLEX ; COMPLEXES ; BINDING ; IDENTIFICATION ; Saccharomyces cerevisiae ; YEAST ; lifestyle ; BETA ; CONSERVATION ; EGG EXTRACTS ; GTPASE RAN ; IMPORTIN-ALPHA ; LOCALIZATION ; MESSENGER-RNA EXPORT ; NUCLEAR EXPORT RECEPTOR ; NUCLEUS ; TRANSPORT FACTOR ; XENOPUS
    Abstract: The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1, 2) and nuclear envelope formation (3, 4) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins. In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins. At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding. Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth. Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle
    Type of Publication: Journal article published
    PubMed ID: 12578832
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  • 29
    Keywords: CANCER ; CELLS ; IRRADIATION ; carcinoma ; CELL ; Germany ; THERAPY ; DEATH ; EXPOSURE ; radiation ; DNA ; REDUCTION ; SUFFICIENT ; FLOW ; treatment ; PARTICLES ; CARCINOMA CELLS ; CELL-DEATH ; CERVIX ; DAMAGE ; FRANCE ; BEAM ; bioshuttle ; BORON ; boron neutron capture therapy (BNCT) ; boronophenylalanine (BPA) ; CAPTURE THERAPY ; CARCINOMA-CELLS ; CELLULAR UPTAKE ; DELIVERY ; DNA-DAMAGE ; drug delivery ; LET-effects ; nuclear transport
    Abstract: Boron neutron capture therapy (BNCT) is an experimental treatment modality which depends on a sufficient cellular uptake of Boron (B-10) followed by an exposure to a thermal neutron beam from a. nuclear reactor. High energetic particles (He-4 and Li-7) are. created during the neutron capture reaction and produce DNA damages, which lead to cell killing. Regarding BNCT, the short radiation range of He- and Li-particles is decisive for the distribution of B-10. Until now, BNCT has been lacking for therapeutically effective concentrations of B-10. Twenty-four hours after the combined use of our 'Bioshuttle'-p-borono-phenylalanine(10)-constructs ('Bioshuttle'-p-BPA(10)) and neutron-irradiation, an obvious reduction of the radiation-resistant HeLa-S cells could be observed. No cells were alive 72 h after the incubation with 'Bioshuttle'-p-BPA(10) followed by neutron irradiation. A post-mitotic cell death could be assumed based on flow cytometrical data. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 12832130
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  • 30
    Keywords: CANCER ; CELLS ; INHIBITOR ; INVASION ; tumor ; CELL ; COMBINATION ; Germany ; INHIBITION ; KINASE ; PATHWAY ; THERAPY ; DISEASE ; DISEASES ; SITE ; PROTEIN ; COMPLEX ; COMPLEXES ; MECHANISM ; DOMAIN ; BINDING ; PHOSPHORYLATION ; protein kinase ; PROTEIN-KINASE ; treatment ; CATALYTIC SUBUNIT ; inactivation ; FIBER ; ADHESION ; ATP ; CELL-ADHESION ; crystal structure ; CRYSTAL-STRUCTURE ; MUSCLE ; PKA ; PROTEIN-KINASES
    Abstract: Protein kinases require strict inactivation to prevent spurious cellular signaling; overactivity can cause cancer or other diseases and necessitates selective inhibition for therapy. Rho-kinase is involved in such processes as tumor invasion, cell adhesion, smooth muscle contraction, and formation of focal adhesion fibers, as revealed using inhibitor Y-27632. Another Rho-kinase inhibitor, HA-1077 or Fasudil, is currently used in the treatment of cerebral vasospasm; the related nanomolar inhibitor H-1152P improves on its selectivity and potency. We have determined the crystal structures of HA-1077, H-1152P, and Y-27632 in complexes with protein kinase A (PKA) as a surrogate kinase to analyze Rho-kinase inhibitor binding properties. Features conserved between PKA and Rho-kinase are involved in the key binding interactions, while a combination of residues at the ATP binding pocket that are unique to Rho-kinase may explain the inhibitors' Rho-kinase selectivity. Further, a second H-1152P binding site potentially points toward PKA regulatory domain interaction modulators
    Type of Publication: Journal article published
    PubMed ID: 14656443
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  • 31
    Keywords: PEPTIDE ; RECEPTOR ; CELLS ; EXPRESSION ; Germany ; PROTEIN ; DRUG ; MOLECULES ; LINES ; MICE ; COMPLEX ; murine ; primary ; ANTIGEN ; ANTIGENS ; BIOSYNTHESIS ; LYMPHOCYTES ; antigen presentation ; B-CELLS ; CLASS-II MOLECULES ; EXCHANGE ; H2-O ; HLA-DM ; HLA-DO ; IA MOLECULES ; INVARIANT CHAIN ; MHC MOLECULES ; MONOCLONAL- ANTIBODY ; PEPTIDE REPERTOIRE ; PEPTIDES ; T- CELLS
    Abstract: Peptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory molecule H2-M, which helps to exchange the invariant chain-derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2-O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2-M and selectively expressed in B cells. Using stable H2-O ribozyme-antisense transfectants, H2-O overexpressing murine B cell lines, and H2-O-transgenic mice, we investigated the effects of H2-O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levels of H2-O in the antigenpresenting B cells. Thus, increased H2-O expression downmodulated, whereas reduced H2-O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2-O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from A(b) molecules were very similar in cells expressing different H2-O levels. The intracellular location of H2-O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2-O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2-O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor
    Type of Publication: Journal article published
    PubMed ID: 12645938
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  • 32
    Keywords: ANGIOGENESIS ; CANCER ; GROWTH-FACTOR ; tumor ; carcinoma ; MODEL ; neoplasms ; THERAPY ; MICE ; magnetic resonance imaging ; BREAST ; PROGRESSION ; MR-ANGIOGRAPHY ; blood pool contrast agents ; contrast enhanced MRA ; GADOMER-17 ; POOL CONTRAST AGENTS
    Abstract: Purpose: To evaluate high-resolution three-dimensional MR angiography (MRA) for the visualization and morphologic characterization of intratumoral vasculature. Materials and Methods: Two subcutaneous rodent tumor models (human skin carcinoma HaCaT-ras-A-5RT3 grown in nude mice and rat prostate carcinoma R3327-AT1 grown in Copenhagen rats) were examined with a clinical 1.5 T MR-system. For MRA a dedicated high- resolution three-dimensional gradient echo pulse sequence with a voxel size of 166 x 206 x 320 mum(3) was performed after injection of Gadomer-17. The image analysis included a correlation of intratumoral vessels with histology. Signal intensity measurements were performed in the vena cava, the tumor underlying muscle, and in various regions of the tumor. Signal-to-noise-ratios (SNR) and contrast-to-noise-ratios (CNR) were calculated from this measurement. Results: High-resolution MRA allowed a clear distinction of intratumoral blood vessels. The mouse tumor model tended to be strongly vascularized with several intratumoral blood vessels clearly displayed by MRA. When correlated with histology, these intratumoral blood vessels had a size in the range of 300 to 400 mum. In contrast, rat tumors had only sparse capillary intratumoral blood vessels that could only be demonstrated by histology. In both tumor models, dilated blood vessels were observed in the subcutaneous tissue near the tumor. In general, areas with a strong contrast enhancement correlated with viable, well vascularized tumor regions, whereas non-enhancing tumor areas correlated with tumor necrosis or hypoxic areas. Conclusion: High-resolution three-dimensional MRA allows the visualization of intratumoral vasculature in rodent models. With minimal hardware and software modifications, high-resolution MRA could be performed on a clinical 1.5 T MRI scanner. Morphologic characterization of intratumoral blood vessels could add important insights into the process of tumor angiogenesis
    Type of Publication: Journal article published
    PubMed ID: 12815640
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  • 33
    Keywords: EXPRESSION ; tumor ; carcinoma ; CELL ; Germany ; TYROSINE KINASE ; screening ; SITE ; SITES ; DISTINCT ; microarray ; PROTEIN ; TISSUE ; TUMORS ; primary ; GROWTH-FACTOR RECEPTOR ; FREQUENCY ; FREQUENCIES ; STAGE ; PROGRESSION ; immunohistochemistry ; ABERRATIONS ; HEAD ; ONCOPROTEIN ; CARCINOMAS ; NECK ; squamous cell carcinoma ; GREECE ; gene amplification ; head and neck ; laryngeal carcinoma ; OROPHARYNGEAL ; C-MYC ; CANCER PATIENTS ; CYCLIN D1 OVEREXPRESSION ; cytogenetic aberration ; head and neck squamous cell carcinoma (HNSCC) ; immunohistochemistry (IHC) ; MICROARRAY ANALYSIS ; oncoprotein overexpression ; OVEREXPRESSION ; POOR-PROGNOSIS ; tissue microarray (TMA) ; tumor classification
    Abstract: Background: Tissue microarray (TMA) analysis is a high-throughput approach that allows the screening of large tumor collectives for cytogenetic aberrations. In this study, a TMA of a large collection of clinically well-defined primary squamous cell carcinomas of the head and neck (HNSCC) was used to determine the expression of several oncoproteins. Materials and Methods: A TMA containing 547 primary HNSCC was used for the analysis of cyclinD1, c-myc, erbb1 and erbb2 expression by immunohistochemistry (IHC). Results: CyclinD1 and c-myc were overexpressed at higher frequencies in primary pharyngeal and laryngeal carcinomas compared with primary oral carcinomas (p 〈 0.001 and p 〈 0.001), while erbb1 and erbb2 overexpression was associated with oral site (p 〈 0.001 and p = 0.04, respectively). Furthermore, cyclinD1 overexpression correlated with stage IV primary carcinomas (p = 0.04). Conclusion: HNSCC is a heterogenous group of tumors, which, depending on anatomic sites and clinical stage, shows variable expressions of the oncoproteins described. This indicates a specific pathogenic role of these oncoproteins in different subtypes of HNSCC and may have therapeutic implications
    Type of Publication: Journal article published
    PubMed ID: 14666705
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  • 34
    Keywords: EXPRESSION ; Germany ; QUANTIFICATION ; TIME ; PATIENT ; treatment ; ALPHA ; ASSAY ; DECREASE ; POLYMERASE-CHAIN-REACTION ; INTERFERON
    Abstract: We developed a real-time quantitative polymerase chain reaction-based assay for quantification of PRV-1 mRNA. We found that the expression of PRV-1 in granulocytes of patients with polycythemia vera (PV) who were pretreated with phlebotomy or hydroxyurea was significantly higher than that in normal controls. Surprisingly, in PV patients who had received interferon-alpha (IFN) for five or more months no significant PRV-1 upregulation was found. Observation of four PV patients treated with IFN over six months revealed a uniform time- dependent decrease of initially upregulated PRV-1
    Type of Publication: Journal article published
    PubMed ID: 12651277
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  • 35
    Keywords: OPTIMIZATION ; PEPTIDE ; SPECTRA ; CELLS ; EXPRESSION ; INHIBITOR ; Germany ; PROTEIN ; PROTEINS ; MOLECULES ; MICE ; ACTIVATION ; COMPLEX ; COMPLEXES ; murine ; MEMBER ; MHC ; LYMPHOCYTES ; antigen presentation ; PEPTIDES ; STABILITY ; MHC class I ; DEGRADATION ; SUBUNITS ; TRANSLOCATION ; antigen processing ; CELL-LINE .220 ; HISTOCOMPATIBILITY COMPLEX ; LOADING COMPLEX ; MUTANT MICE ; NEWLY SYNTHESIZED PROTEINS
    Abstract: Tapasin is a member of the MHC class I loading complex where it bridges the TAP peptide transporter to class I molecules. The main role of tapasin is assumed to be the facilitation of peptide loading and optimization of the peptide cargo. Here, we describe another important function for tapasin. In tapasin- deficient (Tpn(-/-)) mice the absence of tapasin was found to have a dramatic effect on the stability of the TAP1/TAP2 heterodimeric peptide transporter. Steady-state expression of TAP protein was reduced more than 100-fold from about 3 x 10(4) TAP molecules per wild-type splenocyte to about 1 x 10(2) TAP per Tpn(-/-) splenocyte. Thus, a major function of murine tapasin appears to be the stabilization of TAR The low amount of TAP molecules in Tpn(-/-) lymphocytes is likely to contribute to the severe impairment of MHC class I expression. Surprisingly, activation of Tpn(-/-) lymphocytes yielded strongly enhanced class I expression comparable to wild-type levels, although TAP expression remained low and in the magnitude of several hundred molecules per cell. The high level of class I on activated Tpn(-/-) cells depended on peptides generated by the proteasome as indicated by blockade with the proteasome-specific inhibitor lactacystin. Lymphocyte activation induced an increase in ubiquitinated proteins that are cleaved into peptides by the proteasome. These findings suggest that in the presence of a large peptide pool in the cytosol, a small number of TAP transporters is sufficient to translocate enough peptides for high class I expression. However, these class I molecules were less stable than those of wild-type cells, indicating that tapasin is not only required for stabilization of TAP but also for optimization of the spectrum of bound peptides
    Type of Publication: Journal article published
    PubMed ID: 12594855
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  • 36
    Keywords: CELLS ; EXPRESSION ; CELL ; Germany ; human ; SYSTEM ; DISTINCT ; PROTEIN ; EPITHELIA ; MOLECULES ; TISSUE ; TISSUES ; SKIN ; GLYCOPROTEIN ; ELEMENTS ; SURFACE ; LOCALIZATION ; GLANDS ; SEGMENTS ; calnexin ; ESTABLISHMENT ; MUCINS ; salivary gland ; sebaceous gland ; SEBACEOUS GLANDS
    Abstract: Calnexin (Cnx) has been characterized as a membrane-bound protein that transiently interacts in a unique chaperone system with newly synthesized glycoproteins in order to allow the establishment of their proper tertiary and, in most cases, quarternary structures. The aim of the study was to identify and to locate the expression of Cnx in the three major salivary glands of humans by different methods. Strong expression of Cnx protein and mRNA were generally found in serous salivary secretory units. With regard to mucous secretory units, expression of Cnx was only detectable at a low level in mucous acinar cells of sublingual glands, but not of submandibular glands. Expression of Cnx was always preserved in the surface epithelium of intralobar and interlobular duct segments. In addition, expression of Cnx was detected in sebaceous glands of parotid tissues, with a distribution pattern resembling that seen in sebaceous glands of the normal skin. In conclusion, production of saliva is associated with the expression of Cnx. Synthesis of molecules in mucous secretory units is not necessarily associated with a strong Cnx expression, whereas synthesis in serous secretory units apparently is. The tissue- specific Cnx expression is also paralleled by the observation that the secretions produced by the major salivary glands differ in their composition and amount
    Type of Publication: Journal article published
    PubMed ID: 12507291
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  • 37
    Keywords: APOPTOSIS ; CANCER ; CANCER CELLS ; CELLS ; EXPRESSION ; INHIBITOR ; tumor ; TUMOR-CELLS ; CELL ; Germany ; human ; IN-VIVO ; INHIBITION ; DEATH ; GENE ; GENE-EXPRESSION ; RNA ; TUMORS ; ACTIVATION ; PROTEIN FAMILY ; INDUCTION ; SUSCEPTIBILITY ; TARGET ; gene expression ; resistance ; HUMAN-TUMORS ; STIMULI ; CANCER-CELLS ; DELIVERY ; MAMMALIAN-CELLS ; SMALL INTERFERING RNAS ; doxorubicin ; HUMAN-TUMOR-CELLS ; IAP PROTEINS ; POSITIVE CANCER-CELLS ; RNA interference,inhibitors of apoptosis,chemotherapy,HeLa,melanoma ; STRATEGIES
    Abstract: Increased resistance to apoptosis is a hallmark of many tumor cells. The functional inhibition of specific antiapoptotic factors may provide a rational basis for the development of novel therapeutic strategies. We investigated here whether the RNA interference (RNAi) technology could be used to increase the apoptotic susceptibility of cancer cells. As a molecular target, we chose the antiapoptotic livin (ML-IAP, KIAP) gene, which is expressed in a subset of human tumors. We identified vector-borne small interfering (si)RNAs, which could efficiently block endogenous livin gene expression. Silencing of livin was associated with caspase-3 activation and a strongly increased apoptotic rate in response to different proapoptotic stimuli, such as doxorubicin, UV-irradiation, or TNFalpha. The effects were specific for Livin-expressing tumor cells. Our results (i) provide direct evidence that the intracellular interference with livin gene expression resensitizes human tumor cells to apoptosis, (ii) define the livin gene as a promising molecular target for therapeutic inhibition, and (iii) show that the livin gene is susceptible to efficient and specific silencing by the siRNA technology
    Type of Publication: Journal article published
    PubMed ID: 14614456
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    Keywords: CELLS ; EXPRESSION ; tumor ; BLOOD ; carcinoma ; human ; MICROSCOPY ; liver ; PROTEIN ; PROTEINS ; TUMORS ; FAMILY ; RAT ; hepatocytes ; MEMBER ; MEMBERS ; antibodies ; MOUSE ; IDENTIFICATION ; RAT-LIVER ; MEMBRANE ; metastases ; CONJUGATE ; LOCALIZATION ; EPITHELIAL-CELLS ; HEPATOCYTE CANALICULAR ISOFORM ; METASTATIC CARCINOMAS ; MULTIDRUG-RESISTANCE PROTEIN ; POLYPEPTIDE OATP2
    Abstract: Transport proteins mediating the selective uptake of organic anions into human hepatocytes include the organic anion transporters SLC21A6 (also termed OATP2, OATP-C, or LST-1) and SLC21A8 (OATP8). Both transporters are localized to the basolateral membrane of human hepatocytes. Because of the importance of these transporters for hepatobiliary elimination, including the removal of bilirubin and its conjugates from the blood circulation, we have generated monoclonal antibodies for studies on the expression and localization of these transport proteins. We describe two antibodies, designated monoclonal antibody MDQ (mMDQ) and monoclonal antibody ESL (mESL), directed against the amino terminus and the carboxyl terminus of human SLC21A6, respectively. Both antibodies have been characterized by immunoblot analysis, immunoprecipitation, and immunofluorescence microscopy. While mESL reacted specifically with SLC21A6, mMDQ detects both SLC21A6 and SLC21A8. Neither of the two antibodies reacted with other human, or with dog, rat, or mouse liver SLC21A family members. Antibody mMDQ may be used for the simultaneous detection of SLC21A6 and SLC21A8 in immunoblotting because of its immunoreactivity with both molecules and because of the different molecular masses of both glycosylated p