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  • 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
    Abstract: CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, is consistently expressed by tumor cells of anaplastic large-cell lymphoma (ALCL). CD30 stimulation induces massive caspase-dependent cell death of ALCL cells in case of canonical NFkappaB inhibition or proteasome inhibition. However, CD30, a TNFR lacking a death domain (DD), is unable to recruit a death inducing complex containing TRADD (TNFR1-associated DD-protein) or FADD (FAS-associated DD-domain protein) together with the receptor-interacting protein 1 (RIP1) and caspase-8. Thus, the mechanism explaining CD30-induced cell death of lymphocytes remains obscure. Here, we demonstrate that blockage of RIP1 by siRNA or pharmacological inhibition of RIP1 by Necrostatin-1 almost completely prevented CD30-induced cell death. In addition, we revealed CD30-induced accumulation of RIP1 at the cytoplasma membrane of NFkappaB-inhibited ALCL cells by confocal laser scanning microscopy. Finally, primary ALCL cases can be subdivided into two groups based on the presence or absence of RIP1 as revealed by immunohistology. Taken together, our study identified RIP1 as a crucial mediator of CD30-induced cell death that bears features of apoptosis as well as necroptosis. RIP1 expression in ALCL tumor cells might eligible for the therapeutic application of CD30 antibodies in combination with NFkappaB/proteasome inhibitors that should result in CD30-induced cell death.
    Type of Publication: Journal article published
    PubMed ID: 23545938
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  • 4
    Abstract: We previously showed that histone deacetylase inhibitor (HDACi) and 5-azacytidine (AZA) treatment selectively induced cell death of esophageal cancer cells. The mechanisms of cancer selectivity, however, remained unclear. Here we examined whether the cancer selectivity of HDACi/AZA treatment is mediated by the thioredoxin (Trx) system and reactive oxygen species (ROS) in esophageal cancer cells. For this, we first analyzed human tissue specimens of 37 esophageal cancer patients by immunohistochemistry for Trx, Trx-interacting protein (TXNIP) and Trx reductase (TXNRD). This revealed a loss or at least reduction of nuclear Trx in esophageal cancer cells, compared with normal epithelial cells (P〈0.001). Although no differences were observed for TXNIP, TXNRD was more frequently expressed in cancer cells (P〈0.001). In the two main histotypes of esophageal squamous cell carcinomas (ESCCs, n=19) and esophageal adenomcarcinomas (EAC, n=16), similar Trx, TXNIP and TXNRD expression patterns were observed. Also in vitro, nuclear Trx was only detectable in non-neoplastic Het-1A cells, but not in OE21/ESCC or OE33/EAC cell lines. Moreover, the two cancer cell lines showed an increased Trx activity, being significant for OE21 (P=0.0237). After treatment with HDACi and/or AZA, ROS were exclusively increased in both cancer cell lines (P=0.048-0.017), with parallel decrease of Trx activity. This was variably accompanied by increased TXNIP levels upon AZA, MS-275 or MS-275/AZA treatment for 6 or 24 h in OE21, but not in Het-1A or OE33 cells. In summary, this study evaluated Trx and its associated proteins TXNIP and TXNRD for the first time in esophageal cancers. The analyses revealed an altered subcellular localization of Trx and strong upregulation of TXNRD in esophageal cancer cells. Moreover, HDACi and AZA disrupted Trx function and induced accumulation of ROS with subsequent apoptosis in esophageal cancer cells exclusively. Trx function is hence an important cellular mediator conferring non-neoplastic cell resistance for HDACi and/or AZA.
    Type of Publication: Journal article published
    PubMed ID: 26692290
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  • 5
    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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  • 6
    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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  • 7
    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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  • 8
    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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  • 9
    Abstract: CD24 is a small, highly glycosylated cell surface protein that is linked to the membrane through a glycosyl-phosphatidylinositol anchor. It is overexpressed in many human carcinomas and its expression is linked to bad prognosis. Lately, lack or low expression of CD24 was used to identify tumor stem cells resulting in conflicting data on the usefulness of this marker. In many immunohistochemical studies, the mAb SN3b was used but the epitope and specificity of this antibody have never been thoroughly investigated. In other studies based mainly on cytofluorographic analysis, the mAb ML-5 was applied. In this study, we compared the epitope of mAb SN3b to the CD24 mAbs SWA-11 and ML-5 that both bind to the core protein of CD24. Using tissue microarrays and affinity-purified CD24 glycoforms, we observed only a partial overlap of SN3b and SWA11 reactivity. The mAb SN3b recognizes sialic acid most likely on O-linked glycans that can occur independently of the CD24 protein backbone. The SN3b epitope was not related to common sialylated cancer-associated glycan structures. Both SN3b epitope positive or negative CD24 glycoforms supported the binding of P-selectin and Siglec-5. In breast cancer, the SN3b reactivity was associated with bad prognosis, whereas SWA11 was not. In renal cell cancer, the SN3b epitope was completely absent but SWA11 reactivity was a prognostic factor. Our results shed new light on the tumorbiological role of CD24 and resolve discrepancies in the literature related to the use of different CD24 mAbs.
    Type of Publication: Journal article published
    PubMed ID: 20351695
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  • 10
    Abstract: It has been suggested that the behavior and function of Paneth cells in metaplasia are different from those found in normal intestinal mucosa. In this study, we investigated whether calnexin, a protein involved in secretory pathways, might be associated with differentiation and function of Paneth cells in normal small intestine, in complete intestinal metaplasia of the stomach, and in Paneth cell-rich adenomas. Differentiation and function of Paneth cells was monitored by Ki67, lysozyme, and morphologic features. Using a newly established monoclonal antibody, we found that calnexin is regularly synthesized by Paneth cells of normal small intestine. In these cells, the staining intensity of calnexin was inversely correlated with their content of secretory granules (lysozyme). In contrast, Paneth cells of intestinal metaplasia and Paneth cell-rich adenomas showed a reduced immunostaining of both calnexin and lysozyme. Moreover, these Paneth cells synthesized the proliferation marker Ki67, a phenomenon that was never observed in Paneth cells of normal small intestine. In vitro experiments using CaCo2 cells showed that the expression of calnexin is not directly affected by the induction of mitosis. In conclusion, calnexin probably reflects the status of Paneth cell differentiation and function. The results do not necessarily indicate that calnexin has a function in Paneth cell proliferation.
    Type of Publication: Journal article published
    PubMed ID: 12480915
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