Biomedical subjects

Anja-Katrin Bosserhoff

Publications and source records attributed to Anja-Katrin Bosserhoff.

At least 19 recordsLinked to original sources

PubMed2006

A novel drug-eluting stent coated with an integrin-binding cyclic Arg-Gly-Asp peptide inhibits neointimal hyperplasia by recruiting endothelial progenitor cells.

OBJECTIVES: Novel stents loaded with an integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) were analyzed for their potential to limit coronary neointima formation and to accelerate endothelialization by attracting endothelial progenitor cells (EPCs). BACKGROUND: Re-endothelialization is important for healing after arterial injury. METHODS: Effects of cRGD on EPC number, recruitment in flow, and invasion were analyzed in vitro. A durable polymer coating containing 67 microg cRGD per stent was developed for Guidant Tetra stents. Twelve cRGD-loaded polymer, 12 unloaded polymer, and 12 bare metal stents were deployed in porcine coronary arteries. Quantification of cRGD in peri-stent tissue was established by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Histomorphometry and immunostaining were performed after 4 and 12 weeks. Recruitment of labeled porcine EPCs was assessed 7 days after intracoronary infusion. RESULTS: The cRGD clearly supported the outgrowth, recruitment, and migration of EPCs in vitro. At 4 weeks, there was no difference for mean neointimal area and percent area stenosis in the cRGD-loaded, polymer, or bare metal stent group. At 12 weeks, neointimal area (2.2 +/- 0.3 mm2) and percent area stenosis (33 +/- 5%) were significantly reduced compared with polymer stents (3.8 +/- 0.4 mm2, 54 +/- 6%; p = 0.010) or bare metal stents (3.8 +/- 0.3 mm2, 53 +/- 3%; p < 0.001). The HPLC/MS confirmed cRGD tissue levels of 1 to 3 mug/stent at 4 weeks, whereas cRGD was not detectable at 12 weeks. Staining for CD34 and scanning electron microscopy indicated enhanced endothelial coverage on cRGD-loaded stents at 4 weeks associated with a significant increase in the early recruitment of infused EPCs. CONCLUSIONS: Stent coating with cRGD may be useful for reducing in-stent restenosis by accelerating endothelialization.

Animals↗

PubMed2006

Reduced expression of beta-catenin inhibitor Chibby in colon carcinoma cell lines.

AIM: To analyse the Chibby expression and its function in colon carcinoma cell lines and colorectal carcinoma (CRC). METHODS: Chibby expression levels were investigated by quantitative RT-PCR in a panel of seven different colon carcinoma cell lines. By sequencing, we analysed mutational status of Chibby. To test whether Chibby exhibited effects on beta-catenin signalling in colon carcinoma cells, we transfected SW480 cells with Chibby expression plasmid and, subsequently, analysed activity of beta-catenin and tested for alterations in cellular phenotype. In addition, we examined Chibby mRNA levels in samples of colorectal carcinomas and adjacent normal tissues by using quantitative RT-PCR and hybridised gene chips with samples from CRC and normal tissues. RESULTS: Chibby mRNA expression was strongly down-regulated in colon carcinoma cell lines in comparison to normal colon epithelial cells and no mutation in any of the examined colon carcinoma cell lines was found. Further, we could show that Chibby inhibited beta-catenin activity in TOPflash assays when over-expressed in SW480 cells. Proliferation and invasion assays with Chibby transfected SW480 cells did not reveal profound differences compared to control cells. In contrast to these in vitro data, quantitative RT-PCR analyses of Chibby mRNA levels in CRC tumor samples did not show significant differences to specimens in adjacent non-cancerous tissue. Consistent with these findings, gene chips analysing tissue samples of tumors and corresponding normal tissue did not show altered Chibby expression. CONCLUSION: Altered Chibby expression might be observed in vitro in different colon carcinoma cell lines. However, this finding could not be confirmed in vitro in CRC tumors, indicating that Chibby is not likely to promote CRC tumor development or progression. As Chibby is an important inhibitor of beta-catenin signalling, our data implicate that the usability of colon carcinoma cell lines for in vitro studies analysing the Wnt/beta-catenin pathway in colorectal carcinoma needs extensive verification.

Carrier Proteins↗

PubMed2006

Regulation of integrin activity by MIA.

MIA (melanoma inhibitory activity) has been identified as a small protein secreted from malignant melanoma cells, which interacts with extracellular matrix proteins including fibronectin. Here, we show that MIA negatively regulates the activity of the mitogen-activated protein kinase pathway in malignant melanoma. Using far Western blotting and co-immunoprecipitation we searched for MIA-binding cell surface proteins. We found that MIA interacts with integrin alpha4beta1 and alpha5beta1, leading to down-regulation of integrin activity and reduction of mitogen-activated protein kinase signaling. These findings also suggest that MIA may play a role in tumor progression and the spread of malignant melanomas via mediating detachment of cells from extracellular matrix molecules by modulating integrin activity. Inhibiting MIA functions in vivo may therefore provide a novel therapeutic strategy for metastatic melanoma disease.

Animals↗

PubMed2006

Influence of melanoma inhibitory activity on transforming growth factor-beta signaling in malignant melanoma.

Melanoma cells escape transforming growth factor-beta (TGFbeta)-mediated growth inhibition by expressing the Smad (mothers against decapentaplegic homolog, Drosophila) inhibitors Ski and Sno. Here, we demonstrate that melanoma inhibitory activity (MIA) influences the expression of these inhibitors. A Smad responsive reporter construct was activated after TGFbeta1 treatment in the MIA-deficient cell clones but not in the parental cell line. According to this finding, the TGFbeta target genes JunB and Id-1 showed a strong induction of expression. Additional analyses revealed that Ski and Sno, repressors of TGFbeta/SMAD signaling, are not expressed in the MIA-deficient cells but in the parental cell line HMB2 and the mock control. Further investigation showed that Ski and Sno expression might be regulated via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling cascade. Treatment of HMB2 cells with a MEK inhibitor revealed a reduction of Ski and Sno expression, which leads to the conclusion that, in our melanoma cell model, Ski and Sno expression is regulated via MAPK/ERK signaling.

Blotting, Western↗

PubMed2006

Multiparameter immunofluorescence on paraffin-embedded tissue sections.

Immunohistochemical techniques have gained increasing importance in diagnostics and research. While formalin-fixed, paraffin-embedded human tissue retains excellent morphology, the detection of antigens by immunofluorescence in its sections and especially the demonstration of multiple simultaneous antibodies have limitations. Double immunofluorescence labeling of routinely processed paraffin sections has been described previously. The signal intensity observed after triple labeling has been reported to be significantly inferior to that obtained by application of double fluorochromes. The authors show multicolor labeling of three and four primary antibodies in routinely processed paraffin-embedded tissue sections using a standardized immunofluorescence technique. In addition, procedures to reduce background staining and to avoid nonspecific double staining are described.

Antibodies↗

PubMed2006

Detailed analysis of MIA protein by mutagenesis.

MIA (melanoma inhibitory activity) has been identified as a small protein secreted by malignant melanoma cells that interacts with extracellular matrix proteins including fibronectin. These findings suggest that MIA may play a role in tumor progression and the spread of malignant melanomas by mediating detachment of cells from extracellular matrix molecules. Here, we present a detailed study on functionally important MIA domains. Using site-directed mutagenesis, amino acids important for MIA structure and/or function were determined. Amino acids conserved in SH3 domains were shown to be important for structural integrity. In addition, amino acid residues necessary for MIA function were identified. Interestingly, not all of them are conserved with respect to other members of the MIA protein family. In summary, our results lead to a better understanding of MIA function. Regulating MIA functions in vivo may provide a novel therapeutic strategy for metastatic melanoma disease.

Amino Acid Sequence↗

PubMed2006

Multiple colon carcinomas in a patient with Cowden syndrome.

Cowden syndrome is a non-adenomatous gastrointestinal polyposis syndrome with inactivation of PTEN, a dual-phosphatase tumor suppressor gene. Patients with loss of wildtype PTEN expression from one allele carry an increased risk of malignant breast, thyroid and brain tumors. However, the risk of malignant transformation in gastrointestinal polyps is still unclear. In this study, we describe a kindred with Cowden syndrome and identify a heterozygous germline mutation causing truncation of the PTEN tumor suppressor. The index patient was a 56 year-old woman having multiple facial papules, acral keratosis, oral papillomatosis, multiple benign breast and thyroid tumors and gastrointestinal polyposis. Progression to invasive adenocarcinoma occured in two pre-existing hamartomatous polyps. Analysis of one of the carcinomas revealed somatic inactivation of the wildtype PTEN allele by exon-skipping. This case demonstrates that gastrointestinal hamartomas in Cowden syndrome patients can progress to invasive adenocarcinomas and should therefore be carefully monitored.

Base Sequence↗

PubMed2005

Tenascin-C protein is induced by transforming growth factor-beta1 but does not correlate with time to tumor progression in high-grade gliomas.

BACKGROUND: Tenascin-C is an extracellular matrix protein known to correlate with prognosis in patients with glioblastoma, probably by stimulation of invasion and neoangiogenesis. Transforming Growth Factor-beta1 (TGF-beta1) plays an important role in the biology of high-grade gliomas, partly by regulating invasion of these tumors into parenchyma. This study was designed to evaluate if TGF-beta1 induces the expression and deposition of Tenascin-C in the extracellular matrix of high-grade gliomas which may be pivotal for the invasion of these tumors into healthy parenchyma. METHODS: A series of 20 high-grade gliomas was stained immunohistochemically with Tenascin-C- and TGF-beta1- specific antibodies. Expression levels of both proteins were evaluated and correlated with each other, time to progression and molecular and morphological markers of invasion. A quantitative PCR assay was performed evaluating the induction of Tenascin-C mRNA by treatment with TGF-beta1 in vitro. RESULTS: Tenascin-C was expressed in 18 of 19 (95%) evaluable tumors, whereas 14 of 20 tumors (70%) expressed TGF-beta1 in a significant percentage of cells. Treatment with TGF-beta1 did induce the expression of Tenascin-C at the mRNA and protein level in vitro. The expression of Tenascin-C and TGF-beta1 did neighter statistically correlate with each other nor with time to progression. CONCLUSION: In our series, Tenascin-C and TGF-beta1 were expressed in the vast majority of high-grade gliomas. We could not detect a correlation of one of the proteins with time to progression. Nevertheless, we describe induction of Tenascin-C by TGF-beta1, possibly providing a mechanism for the invasion of high-grade gliomas into healthy parenchyma.

Adult↗

PubMed2005

Promoter-hypermethylation is causing functional relevant downregulation of methylthioadenosine phosphorylase (MTAP) expression in hepatocellular carcinoma.

The methylthioadenosine phosphorylase (MTAP) gene is localized in the chromosomal region 9p21. Here, frequently homozygous deletions occur in several kinds of cancer associated with the loss of tumour suppressor genes as p16 and p15. The aim of this study was to analyse MTAP expression in hepatocellular carcinoma (HCC) and to get an insight into the regulation and functional role of MTAP in hepatocancerogenesis. Compared with primary human hepatocytes MTAP expression was markedly downregulated in three different HCC cell lines as determined by real-time PCR and western blotting. This was not due to genomic losses or mutations but to promoter-hypermethylation. Reduced MTAP-expression was confirmed in vivo in HCC compared with non-cancerous liver tissue on both mRNA and protein levels. To study the functional relevance of the downregulated MTAP expression in HCC, MTAP expression was re-induced in HCC cell lines by stable transfection. In these MTAP re-expressing cell clones the invasive potential was strongly reduced, whereas no effects on cell proliferation were observed in comparison with mock transfected cell clones. Furthermore, in MTAP re-expressing cells interferon (IFN)-alpha and IFN-gamma induced a significantly stronger inhibition of cell proliferation than in mock transfected cells. In conclusion, our results suggest a functional role of MTAP inactivation in HCC development and invasiveness. Furthermore, in the light of a recent report revealing an association between MTAP activity and IFN sensitivity, our findings may have clinical significance for therapeutic strategies.

Carcinoma, Hepatocellular↗

PubMed2005

Enhanced migration of tissue inhibitor of metalloproteinase overexpressing hepatoma cells is attributed to gelatinases: relevance to intracellular signaling pathways.

AIM: To study the effect of gelatinases (especially MMP-9) on migration of tissue inhibitor of metalloproteinase (TIMP-1) overexpressing hepatoma cells. METHODS: Wild type HepG2 cells, cells stably transfected with TIMP-1 and TIMP-1 antagonist (MMP-9-H401A, a catalytically inactive matrix metalloproteinase (MMP) which still binds and neutralizes TIMP-1) were incubated in Boyden chambers either with or without Galardin (a synthetic inhibitor of MMP-1, -2, -3, -8, -9) or a specific inhibitor of gelatinases. RESULTS: Compared to wild type HepG2 cells, the cells overexpressing TIMP-1 showed 115% migration (P<0.05) and the cells overexpressing MMP-9-H401A showed 62% migration (P<0.01). Galardin reduced cell migration dose dependently in all cases. The gelatinase inhibitor reduced migration in TIMP-1 overexpressing cells predominantly. Furthermore, we examined intracellular signal transduction pathways of TIMP-1-dependent HepG2 cells. TIMP-1 deactivates cell signaling pathways of MMP-2 and MMP-9 involving p38 mitogen-activated protein kinase. Specific blockade of the ERK pathway suppresses gelatinase expression either in the presence or absence of TIMP-1. CONCLUSION: Overexpressing functional TIMP-1- enhanced migration of HepG2-TIMP-1 cells depends on enhanced MMP-activity, especially MMP-9.

Carcinoma, Hepatocellular↗

PubMed2005

Bone morphogenic proteins are overexpressed in malignant melanoma and promote cell invasion and migration.

Malignant melanoma cells are known to have altered expression of growth factors compared with normal human melanocytes. These changes probably favor tumor growth and progression and influence the tumor environment. The induction of transforming growth factor beta1 (TGF-beta1), TGF-beta2, and TGF-beta3 expression in malignant melanoma has been reported before, whereas the expression of related bone morphogenic protein (BMP) molecules has not been analyzed in melanomas until now. Here, we show that BMP4 and BMP7 are up-regulated in nine melanoma cell lines, whereas BMP2 is overexpressed in only two of the analyzed cell lines. Immunohistochemistry of primary and metastatic melanoma also shows increased BMP4 and BMP7 expression compared with nevi. Promoter studies reveal that expression is controlled at the transcriptional level. The transcription factor Ets-1 was identified as a positive regulator for BMP4 expression. In order to determine the functional relevance of BMP expression in malignant melanoma, chordin-expressing cell clones and antisense BMP4 cell clones were generated. The clones in which BMP4 activity and expression are reduced show no changes in proliferation or in attachment-independent growth when compared with controls. However, a strong reduction of migratory and invasive properties was observed in these cells, suggesting that BMP4 promotes melanoma cell invasion and migration and therefore has an important role in the progression of malignant melanoma.

Animals↗

PubMed2005

Integrin signaling in malignant melanoma.

Cell adhesion and migration are essential for embryonic development, tissue regeneration, but also for tumor development. The physical link between the extracellular matrix (ECM) and the actin cytoskeleton is mainly mediated by receptors of the integrin family. Through signals transduced upon integrin ligation to ECM proteins, this family of proteins plays key roles in regulating tumor growth and metastasis as well as tumor angiogenesis. During melanoma development, changes in integrin expression, intracellular control of integrin functions and signals perceived from integrin ligand binding impact upon the ability of tumor cells to interact with their environment and enable melanoma cells to convert from a sessile, stationary to a migratory and invasive phenotype. Antagonists of several integrins are now under evaluation in clinical trials to determine their potential as therapeutics for malignant melanoma and other kinds of cancer.

Cell Adhesion↗

PubMed2005

Strong expression of methylthioadenosine phosphorylase (MTAP) in human colon carcinoma cells is regulated by TCF1/[beta]-catenin.

Methylthioadenosine phosphorylase (MTAP) is known as a ubiquitously expressed house keeping gene important in biochemical salvage processes. The MTAP gene is localized on the human chromosomal region 9p21, a region often deleted in cancer. Recently, several groups including our own have shown that MTAP serves as a tumour suppressor gene. The aim of this study was to analyse the role of MTAP in colon carcinoma and normal colon epithelium and the regulation of gene expression. To examine MTAP RNA and protein expression, we screened six colon carcinoma cell lines and human primary colon epithelial cells by RT-PCR and immunoblotting. MTAP expression was confirmed in vivo by immunohistochemical staining of normal colon tissue compared to adenoma and colon carcinoma. Interestingly, we found strong MTAP mRNA and protein expression by colon carcinoma cell lines but no expression by colonic epithelial cells. To analyse the regulation of MTAP expression, promoter studies were performed and revealed control of MTAP expression by LEF/TCF/beta-catenin. Furthermore, we demonstrated a significant correlation between MTAP protein expression and tumour progression as the intensity of MTAP protein staining increased from normal tissue to carcinoma. In addition, the recently postulated association between MTAP activity and interferon (IFN) sensitivity was confirmed in colon epithelial cells showing only little response to IFN-gamma, in contrast to the carcinoma cell lines. In summary, these data indicate for the first time that MTAP is not expressed in normal human colonic epithelium but is strongly upregulated in colon carcinoma. This finding may be of clinical significance concerning the homeostasis of normal colon epithelium and potential treatment of colon carcinoma.

Adenocarcinoma↗

PubMed2005

Cloning and functional characterization of a new Ski homolog, Fussel-18, specifically expressed in neuronal tissues.

The Sloan Kettering Virus (Ski) family of nuclear oncoproteins represses transforming growth factor-beta (TGF-beta) signaling through inhibition of transcriptional activity of Smad proteins. Here, we report the discovery of a new functional Smad suppressing element on chromosome 18 (Fussel-18). Fussel-18 encodes for a protein of 297 amino acids sharing characteristic structural features, significant homology and similar genomic organization with the homolog Ski family members, Ski and Ski-related novel sequence (Sno). In contrast to Ski and Sno, which are ubiquitously expressed in human tissues, in situ hybridization, RT-PCR, Western blot and immunohistochemistry revealed a highly specific expression pattern for Fussel-18 in neuronal tissues, especially in the cerebellum, the spinal cord and dorsal root ganglia, during both embryogenesis and adult stage. Functionally, we determined interaction of Fussel-18 with Smad 2 and Smad 3 together with an inhibitory activity on TGF-beta signaling. Fussel-18 is the first example of a Smad-binding protein with a highly restricted expression pattern within the nervous system.

Amino Acid Motifs↗

PubMed2005

In situ expression patterns of melanoma inhibitory activity 2 in healthy and diseased livers.

UNLABELLED: Recently we identified a novel gene of the MIA gene family, melanoma inhibitory activity 2 (MIA2) and found that MIA2 mRNA is selectively expressed in hepatocytes. Here, we analyzed the in situ expression of MIA2 protein and mRNA in healthy and diseased livers to get first insights into the function of MIA2. METHODS: We analyzed liver tissue of patients with chronic hepatitis C (HepC) infection and hepatocellular carcinoma (HCC) as well as a human multi-tissue array, primary human hepatocytes and the hepatoma cell-lines HepG2, Hep3B and PLC by immunohistochemical staining and quantitative RT-PCR. In addition to MIA2, the expression of alpha-smooth muscle actin (alpha-sma), a marker for activated hepatic stellate cells (HSCs)/myofibroblast, was analyzed. RESULTS: Hepatocytes were confirmed as the exclusive cellular source of MIA2 expression, with a granular, cytoplasmatic staining pattern without enhancement at the cell membrane. In contrast, only low MIA2 expression levels were detected in most HCC and hepatoma cell lines. Only in HCC that contained fibrous stroma or thick hyalinized bundles, adjacent atypical hepatocytes revealed strong staining. In accordance, MIA2 expression was also upregulated in non-tumorous livers of patients with HepC and correlated with the staging of fibrosis. Interestingly, both in HCC and liver tissues of patients with HepC we found a correlation of MIA2 and alpha-sma expression. DISCUSSION: We define for the first time in situ expression patterns of MIA2 in healthy and diseased livers. Our data raise the hypothesis that activation of HSCs/myofibroblasts has influence on MIA2 expression in vivo, consistent with our previous in vitro findings. Since the staining pattern and the protein structure highly suggests that MIA2 is a secreted protein, it may possibly serve as a marker of hepatic fibrosis.

Adult↗

PubMed2005

Melanoma inhibitory activity (MIA): an important molecule in melanoma development and progression.

Cutaneous malignant melanoma is the leading cause of skin cancer death in industrialized countries. Melanoma development and progression are well defined by clinical and histopathological aspects; however, detailed analysis of molecular changes is still ongoing. The protein MIA, which is strongly expressed in melanoma cells but not in melanocytes, is likely to represent a key molecule regulating melanoma progression. Consistent with this, several in vitro and in vivo model systems indicate a direct involvement of MIA in melanoma migration and invasion, with recent studies suggesting a central role for MIA in early melanoma development by regulating important melanoma-related pathways and molecules. The latest developments in MIA research are summarized in this review, which describes recently published data related to the MIA protein structure and function, the role of MIA in melanoma development and progression, and the regulation of MIA expression. Furthermore, newly discovered MIA-homologous genes are discussed.

Animals↗

PubMed2005

Characterization of Sno expression in malignant melanoma.

Normal cells are controlled by several exocrine factors, whereas tumor cells often lose control by antiproliferative stimuli. It is known that melanoma cells produce transforming growth factor (TGF) beta1, 2 and 3, but do not respond with growth inhibition. Recently, the Smad inhibitor Ski was found to play a role in this process. We originally analyzed Ski expression in nine melanoma cell lines, however, only one cell line (SK Mel 28) was positive. As all nine cell lines were unresponsive to TGF-beta, we continued to search for the responsible mechanism. Sequencing of the TGF-beta-receptor II, known to be mutated in other kinds of cancer, did not reveal any mutation. A family member of Ski, the proto-oncogene Sno was strongly expressed in all melanoma cell lines on RNA and protein level, but not in melanocytes. To confirm functional relevance of this observation, we used stable antisense Sno transfection for the generation of cell clones with reduced Sno expression. These cell clones displayed reduced cell proliferation, indicating participation of Sno in the escape of melanoma cells from TGF-beta dependent growth control. Searching for TGF-beta target genes that are under control of Sno interference, Id1 but not antiproliferative genes p21, p15, p57 and p27 was identified in the cell clones after antisense Sno expression. In summary, constitutive Sno expression was identified as an important mechanism to shut off antiproliferative TGF-beta signaling in malignant melanoma.

Humans↗

PubMed2004

Reduction in Raf kinase inhibitor protein expression is associated with increased Ras-extracellular signal-regulated kinase signaling in melanoma cell lines.

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.

Androgen-Binding Protein↗