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  • GENE  (4)
  • PROTEIN  (3)
  • 1
    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 proteins in human hepatocytes. This is exemplified in hepatocellular carcinomas where SLC21A6 and SLC21A8 were differentially synthesized and showed an irregular staining pattern. Both transport proteins have not been detected in human hepatoma HepG2 cells. In routine paraffin sections, 10 of 12 hepatocellular carcinomas were focally positive with antibody mMDQ. In contrast, cholangiocarcinomas and liver metastases of colorectal and pancreatic adenocarcinoma were negative without exception. This suggests the usefulness of SLC21A6/SLC21A8 within a panel of tumor markers for hepatocellular carcinomas. Moreover, both antibodies should be useful in studies on the expression and localization of two important uptake transporters of human hepatocytes under physiologic and pathophysiologic conditions
    Type of Publication: Journal article published
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  • 2
    Keywords: CELLS ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; IN-VITRO ; proliferation ; Germany ; MODEL ; THERAPY ; VITRO ; GENE ; PROTEIN ; TISSUE ; ACTIVATION ; MECHANISM ; MARKER ; ANTIGEN ; mechanisms ; ALPHA ; MEMBRANE ; LINE ; MARKERS ; EXTRACELLULAR-MATRIX ; BETA ; EPITHELIAL-CELLS ; PHENOTYPE ; vimentin ; adenocarcinoma ; GROWTH-FACTOR-BETA ; chronic pancreatitis ; LONG-TERM CULTURE ; FACTOR-BETA ; molecular ; MATRIX ; fibrosis ; pancreas ; RE ; immortalization ; basement membrane ; BASEMENT-MEMBRANE ; collagen ; HUMAN-FIBROBLASTS ; TRANSFECTION ; TGF-BETA ; VITAMIN-A ; DESMIN ; ACIDIC PROTEIN ; MOLECULAR-MECHANISMS ; DUCT CELLS ; TGF beta ; ABILITY ; PLUS ; CYCLE ARREST ; HEPATIC-FIBROSIS ; LIVER FIBROSIS ; pancreatic stellate cells ; TRANSFORMING GROWTH-FACTOR-BETA-1
    Abstract: Tissue fibrosis is one of the characteristics of chronic pancreatitis and pancreatic adenocarcinoma. Activated pancreatic stellate cells (PSC) play a central role in this process. However, analysis of the molecular mechanisms leading to PSC activation is hampered by the lack of an established human PSC line. To overcome this problem, we immortalized and characterized primary human PSC. The cells were isolated by the outgrowth method and were immortalized by transfection with SV40 large T antigen and human telomerase ( hTERT). Primary human PSC served as controls. An immortalized line, RLT-PSC, was analyzed for the expression of stellate cell markers. Moreover, the effects of transforming growth factor beta 1(TGF beta 1) or platelet-derived growth factor stimulation and of cultivation on basement membrane components or N-acetylcysteine (NAC) treatment on gene and protein expression and proliferation were analyzed. Immortal RLT-PSC cells retained the phenotype of activated PSC proven by the expression of alpha-smooth muscle actin (alpha SMA), vimentin, desmin and glial fibrillary acidic protein (GFAP). TGF beta 1 treatment upregulated the expression of alpha SMA, collagen type I (Col I), fibronectin and TGF beta 1. Incubation of RLT-PSC cells and primary human activated PSC on Matrigel plus NAC treatment resulted in a deactivated phenotype as evidenced by a decrease of aSMA, connective tissue growth factor and Col I expression and by a decreased proliferation of the cells. Moreover, this treatment restored the ability of the cells to store vitamin A in cytoplasmic vesicles. In conclusion, we have established an immortal pancreatic stellate cell line, without changing the characteristic phenotype. Importantly, we were able to demonstrate that besides soluble factors, the matrix surrounding PSC plays a pivotal role in the maintenance of the activation process of PSC. Cultivation of activated PSC on a reconstituted basement membrane plus treatment with NAC was able to deactivate the cells, thus pointing to the possibility of an antifibrosis therapy in chronic pancreatitis
    Type of Publication: Journal article published
    PubMed ID: 16127427
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  • 3
    Keywords: CELLS ; EXPRESSION ; SYSTEM ; DEATH ; CLONING ; GENE ; GENES ; transcription ; MICE ; PATIENT ; SERA ; TRANSCRIPTION FACTOR ; animals ; MOUSE ; DNA-BINDING ; BETA ; EPITHELIAL-CELLS ; AP-2-GAMMA ; DROSOPHILA HOMOLOG ; FACTOR AP-2 ; KIDNEY-FUNCTION ; LINEAGES ; MOUSE EMBRYOGENESIS ; PARATHYROID- HORMONE
    Abstract: Inactivation of the transcription factor AP-2beta in a genetically mixed C57BU6/129S1 mouse strain resulted in perinatal lethality as a consequence of massively enhanced apoptotic death of renal epithelial cells (Genes Dev 1997;11:19381948). Recently, we observed that the phenotype is modulated by genetic background because AP-2beta mutant mice, backcrossed onto 129P2 background, survive approximately 2 weeks after birth, allowing for a detailed analysis of kidney function. Here we show that kidneys reveal Varying amounts of cysts derived from all tubular structures (proximal and distal tubuli, collecting ducts). However, all mice died irrespective of the degree of cyst formation. Serum analysis of AP-2beta mutant animals revealed defective tubular secretory function and ion homeostasis including severe hypocalcemia, hyperphosphatemia, and hyperuremia. Because hormonal calcium regulation was not impaired, the mice developed secondary renal hyperparathyroidism as typically observed in patients with terminal renal failure. We further demonstrate that molecular defects in the collecting duct system lead to insufficient water retention and urinary concentration. In summary, our studies reveal essential, nonredundant roles of AP-2beta in renal tubular functions
    Type of Publication: Journal article published
    PubMed ID: 12695560
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  • 4
    Keywords: CANCER ; CELLS ; EXPRESSION ; GENE ; GENE-EXPRESSION ; MESSENGER-RNA ; DOWN-REGULATION ; human papillomavirus ; HUMAN-PAPILLOMAVIRUS ; intraepithelial neoplasia ; E7 ONCOPROTEIN ; INTERFERON-ALPHA ; GAMMA ; LASER CAPTURE MICRODISSECTION ; CYTOKINE PRODUCTION ; cervical tissue ; interferon-kappa ; quantitative real-time polymerase chain reaction
    Abstract: Interferons (IFNs) are expressed by many cell types and play a pivotal role in the generation of immune responses against viral infections. IFN-kappa, a novel type I IFN, displays a tight tropism for keratinocytes and specific lymphoid populations and exhibits functional similarities with other type I IFNs. The human papillomavirus (HPV), the etiological agent for cervical cancer, infects keratinocytes of the uterine cervix and has been shown to directly inhibit the IFN pathway. We evaluated IFN-kappa, -beta, and -gamma gene expression in HPV-negative normal and HPV-positive pre-malignant and malignant ex vivo cervical tissue covering the entire spectrum of cervical disease. Quantitative real-time polymerase chain reaction and methods previously optimized for detecting low-expressing genes in cervical tissue were used. In contrast to IFN-beta and -gamma, IFN-kappa mRNA prevalence and levels were unexpectedly higher in diseased compared with normal whole cervical tissue with highest levels observed in invasive carcinoma tissue. Strikingly, laser capture microdissection revealed an absence of IFN-kappa mRNA in diseased epithelium, whereas stromal IFN-kappa was found exclusively in diseased tissue. IFN-gamma and IFN-beta were likewise found to be upregulated in diseased cervical stroma. Immunofluorescence supports the involvement of monocytes and dendritic cells in the stromal induction of IFNs in diseased tissue. Further, using three-dimensional raft cultures in which the viral life cycle can be mimicked, human keratinocytes transfected with full-length HPV16 displayed a significant decrease in IFN-kappa mRNA compared with non-transfected human keratinocytes. Altogether, these findings show that IFN-kappa is down-regulated in cervical keratinocytes harboring HPV, which may be a contributing factor in the progression of a cervical lesion.
    Type of Publication: Journal article published
    PubMed ID: 20479716
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  • 5
    Keywords: RECEPTOR ; APOPTOSIS ; CELLS ; EXPRESSION ; IN-VITRO ; IN-VIVO ; PROTEIN ; DEATH DOMAIN ; NF-KAPPA-B ; LIGAND ; FAMILY ; MEMBER ; IDENTIFICATION ; INTERFERON ; real-time PCR ; CYTOTOXIC LIGAND TRAIL ; KILLER/DR5
    Abstract: TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis by cross-linking of the two TRAIL receptors that contain a death domain, TRAIL-R1 and TRAIL-R2. TRAIL-R3 and TRAIL-R4 are receptors that do not transmit an apoptotic signal. Our aim was to determine the expression of TRAIL and its receptors in normal pancreas and chronic pancreatitis. We applied real-time PCR, immunohisto(cyto)chemistry, and nick-end labeling of apoptoses. In normal pancreas, a minor subset of acinar cells coexpressed TRAIL-R2 and TRAIL-R4, whereas ductular epithelium and interstitial fibroblast-like cells (FLC) expressed TRAIL- R4. TRAIL-R1 and TRAIL-R3 were not detected in normal pancreas. In chronic pancreatitis, the exocrine epithelium strongly expressed TRAIL-R1, -R2, -R4, and, to a lesser extent, TRAIL- R3. Islets focally neoexpressed TRAIL-R1 and -R2 and intensely expressed TRAIL-R4. Changes in TRAIL receptor expression were most pronounced in areas of inflammatory infiltration and active fibrosis. In normal pancreas, expression of TRAIL was low on the mRNA level and undetectable on the protein level. In chronic pancreatitis, FLC in areas of active fibrosis expressed TRAIL. In addition, apoptoses were most numerous in these areas. We show that these FLC are pancreatic stellate cells. Pancreatic stellate cells express TRAIL in vivo and in vitro, and TRAIL expression is enhanced by IFN-gamma. Our findings indicate that the TRAIL/TRAIL receptor system is likely to be involved in chronic pancreatitis and suggest that pancreatic stellate cells may directly contribute to acinar regression by inducing apoptosis of parenchymal cells in a TRAIL-dependent manner
    Type of Publication: Journal article published
    PubMed ID: 12808117
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  • 6
    Keywords: EXPRESSION ; carcinoma ; Germany ; GENE ; GENE-EXPRESSION ; GENES ; HYBRIDIZATION ; microarray ; transcription ; ACCURACY ; validation ; PATIENT ; IN-SITU ; gene expression ; microarrays ; ARRAYS ; PCR ; PROBES ; HEAD ; NECK ; squamous cell carcinoma ; expression profiling ; SINGLE ; SUBSET ; head and neck cancer ; OLIGONUCLEOTIDE ; ARRAY ; CARCINOMA PATIENTS ; PROFILES ; ADJUSTMENT ; DNA-MICROARRAY ; CELL-CARCINOMA ; RESOURCES ; SIGNATURE ; EXPRESSION PROFILES ; comparative study ; head and neck squamous cell carcinoma ; LOCUSLINK ; NCBI ; oligonucleotide microarray ; oligonucleotide probes ; reproducibility of results ; statistics and numerical data
    Abstract: The comparison of gene expression measurements obtained with different technical approaches is of substantial interest in order to clarify whether interplatform differences may conceal biologically significant information. To address this concern, we analyzed gene expression in a set of head and neck squamous cell carcinoma patients, using both spotted oligonucleotide microarrays made from a large collection of 70-mer probes and commercial arrays produced by in situ synthesis of sets of multiple 25-mer oligonucleotides per gene. Expression measurements were compared for 4425 genes represented on both platforms, which revealed strong correlations between the corresponding data sets. Of note, a global tendency towards smaller absolute ratios was observed when using the 70-mer probes. Real-time quantitative reverse transcription PCR measurements were conducted to verify expression ratios for a subset of genes and achieved good agreement regarding both array platforms. In conclusion, similar profiles of relative gene expression were obtained using arrays of either single 70-mer or multiple short 25-mer oligonucleotide probes per gene. Although qualitative assessments of the expression of individual genes have to be made with caution, our results indicate that the comparison of gene expression profiles generated on these platforms will help to discover disease-related gene signatures in general
    Type of Publication: Journal article published
    PubMed ID: 16205657
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