Biomedical subjects

Ina Poser

Publications and source records attributed to Ina Poser.

14 recordsLinked to original sources

PubMed2005

RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells.

RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.

Alternative Splicing↗

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

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

Snail-regulated genes in malignant melanoma.

The demonstration that zinc-finger transcriptional repressors can control E-cadherin expression in epithelial cells has provided a new avenue of research in the field of epithelial-mesenchymal transition (EMT). One of these zinc-finger molecules is the transcription factor Snail, which controls gastrulation and neural crest EMT in different species. Additionally, Snail is involved in the development of malignant melanoma where a dramatic change in E-cadherin expression is an important early step for melanoma progression. For this study, a human cancer cDNA array was used which includes genes involved in cancer development and progression. Using the array we compared the gene expression pattern of the melanoma cell line Mel Im with a Mel Im cell clone stable transfected with antisense (as) SNAIL cDNA. We validated the significant differences of the expression of genes on mRNA level. Primarily, we observed changes in the expression of genes involved in EMT. Quantitative real-time polymerase chain reaction showed a down-regulation of MMP-2, EMMPRIN, SPARC, TIMP-1, t-PA, RhoA and Notch4 expression and a re-induction of E-cadherin expression in the as Snail cell clones. Furthermore, we measured the expression of integrin beta3, NM23b and RhoB. Additionally, we investigated whether the selected genes are influenced only through Snail or if E-cadherin can influence the expression of these genes. In summary, all examined genes which are influenced through Snail have a regulatory function in EMT processes as does Snail itself. The Snail target gene E-cadherin has no regulatory function with respect to the selected genes.

Cadherins↗

PubMed2005

Inhibition of melanoma inhibitory activity (MIA) expression in melanoma cells leads to molecular and phenotypic changes.

The secreted protein melanoma inhibitory activity (MIA) is highly expressed in malignant melanoma but not in melanocytes and is associated with tumor progression in vivo. Here, we further investigated the functional role of MIA by inhibiting MIA expression of the human melanoma cell line HMB2 via stable antisense MIA cDNA transfection, and subsequent analysis of the cell clones. MIA-deficient cell clones showed several changes in cell morphology and growth pattern. In monolayer and three-dimensional culture enhanced cell-cell contacts were formed. Furthermore, a re-induction of pigment synthesis in comparison with the amelanotic parental cell line HMB2 was observed. Molecular analyses revealed a re-expression of tyrosinase-related protein 1 (Trp-1) and tyrosinase in the MIA-deficient cell clones necessary for melanin synthesis. In accordance, re-expression of MIA in the MIA-deficient melanoma cell clones resulted in downregulation of Trp-1. To identify the molecular mechanisms of MIA regulating pigmentation, MITF and PAX3, two positive regulators of Trp-1 and tyrosinase transcription, and PIAS3, a negative regulator of MITF activity, were analyzed. Only in MIA-deficient cells, expression of PAX3 mRNA and MITF protein was found. In contrast, strong expression of PIAS3 was detected in HMB2 but not in the MIA-deficient cells. To our knowledge this is the first report demonstrating a correlation between MIA expression and pigmentation and morphology of melanocytic cells.

Acetylation↗

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

An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

Cell Division↗

PubMed2004

Functional role of MIA in melanocytes and early development of melanoma.

The protein MIA (melanoma inhibitory activity) is highly expressed in malignant melanomas but not in melanocytes. Furthermore, expression of MIA correlates with tumor progression in vivo. Here, MIA-dependent changes of gene expression after long-term inhibition of MIA expression in the human melanoma cell line HMB2 were investigated. Primarily, we observed characteristic changes in cell morphology, and also found re-established cell-cell contacts in MIA-deficient cell clones grown in monolayer culture. Real-time reverse transcription-polymerase chain reaction (RT-PCR) showed a downregulation of N-cadherin expression and a reinduction of E-cadherin expression in the MIA-deficient cell clones. Further, both cancer cDNA array and protein arrays verified a marked downregulation of several other melanoma-associated genes (e.g. membrane-type 1 matrix metalloproteinase tissue-type plasminogen activator integrin beta3, secreted protein acidic and rich in cysteins and fibronectin) in the MIA-deficient melanoma cells, confirmed by real-time RT-PCR and Western blotting. As all these molecules are associated with migration, the effect of MIA on migration of human primary melanocytes was analysed. In the presence of MIA, we observed enhanced migratory ability of melanocytic cells, induction of melanoma-associated genes as well as inhibition of apoptosis due to anoikis. These results suggest that expression of MIA promotes melanoma progression by inducing further melanoma-associated genes.

Cell Adhesion Molecules↗

PubMed2004

Modulation of genotoxic effects in asbestos-exposed primary human mesothelial cells by radical scavengers, metal chelators and a glutathione precursor.

The genotoxicity of asbestos fibers is generally mediated by reactive oxygen species (ROS) and by insufficient antioxidant protection. To further elucidate which radicals are involved in asbestos-mediated genotoxicity and to which extent, we have carried out experiments with the metal chelators deferoxamine (DEF) and phytic acid (PA), and with the radical scavengers superoxide dismutase (SOD), dimethylthiourea (DMTU) and the glutathione precursor Nacystelyn trade mark (NAL). We investigated the influence of these compounds on the potency of crocidolite, an amphibole asbestos fiber with a high iron content (27%), and chrysotile, a serpentine asbestos fiber with a low iron content (2%), to induce micronuclei (MN) in human mesothelial cells (HMC) after an exposure time of 24-72 h. Our results show that the number of crocidolite-induced MN is significantly reduced after pretreatment of fibers with PA and DEF. This effect was not observed with chrysotile. In contrast, simultaneous treatment of cells with asbestos and the OH*scavenging DMTU or the O2- -scavenging SOD significantly decreased the number of MN induced by chrysotile and crocidolite. In particular, DMTU almost completely suppressed micronucleus induction by both fiber types. A similar effect was observed in the presence of the H(2)O(2)-scavenging NAL after chrysotile treatment of HMC. By means of kinetochore analysis, it could be shown that the number of clastogenic events is decreased after PA and DEF pretreatment of fibers as well as after application of the above-mentioned scavengers. Our results show that chrysotile asbestos induces an increased release of H(2)O(2) in contrast to crocidolite. Also, the iron content of the fiber plays an important role in radical formation, but nevertheless, chrysotile produces oxy radicals to a similar extent as crocidolite, probably by phagocytosis-mediated oxidative bursting.

Acetylcysteine↗

PubMed2004

Ultraviolet radiation induces release of MIA: a new mechanism for UVR-induced progression of melanoma.

MIA is a potent melanoma detachment factor that interferes with cellular adherence by binding to fibronectin and laminin, blocking their interaction with alpha4beta1 and alpha5beta1 integrins. The direct correlation between serum MIA levels of patients with progressing melanomas and tumor load supports a role for MIA as a melanoma progression factor. The goal of this study was to determine the effect of ultraviolet radiation (UVR), activating ras mutation, or loss of p53 function on MIA expression and release from melanoma cells. We previously showed that transfection of a mutant constitutively active ras into the melanoma cell line, WM35, induces a phenotypic change from radial to vertical growth, exhibiting increased proliferation and migration. Here, we report that MIA release was elevated in a ras-transfected cell line. In addition, loss of functional p53, using a dominant negative construct, substantially lowered the level of MIA release compared to control. UVR stimulated release of MIA into the extracellular compartment in both the control and ras-transfected cell lines. In addition, MIA mRNA was increased following UVR in all cell lines tested. By inducing either apoptosis or necrosis, we were able to confirm that MIA protein is not released from cells due to cell death alone. We have identified a transcriptional effect of UVR on MIA expression and have shown that release of MIA protein is dependent upon functional p53. We propose that UVR-induced production and release of MIA may promote the detachment of radial and vertical growth phase melanomas from basement membrane or matrix proteins, serving as a unique progression mechanism for melanoma.

Cell Line, Tumor↗

PubMed2003

Upregulation of HMG1 leads to melanoma inhibitory activity expression in malignant melanoma cells and contributes to their malignancy phenotype.

Malignant transformation of melanocytes to melanoma cells closely parallels activation of melanoma inhibitory activity (MIA) expression. We have previously shown that upregulation of MIA occurs on a transcriptional level and involves the highly conserved region (HCR) promoter element. We further observed that the HCR element interacts with the melanoma-associated transcription factor (MATF) and thereby confers strong promoter activation. In this study we identify the peptide sequence of MATF and show that it is identical with the transcription factor HMG1. HMG1 was upregulated in malignant melanoma cells and further activated by hypophosphorylation. Stable antisense-HMG1 expression in melanoma cells led to the reduction of MIA promoter activity and protein expression, indicating that HMG1 is a potent regulator of MIA expression. Interestingly, chromatin immunoprecipitation and electrophoretic mobility shift experiments indicated that HMG1 and the NF-kappa B family member p65 both interact and bind to the HCR promoter element. In summary, our study proves HMG1 and p65 to be important factors in MIA regulation and melanoma progression.

Amino Acid Sequence↗

PubMed2002

Down-regulation of COOH-terminal binding protein expression in malignant melanomas leads to induction of MIA expression.

Malignant transformation of melanocytes to melanoma cells closely parallels activation of MIA expression and involves a promoter region that we referred to previously as a HCR (highly conserved region). The HCR element interacts with the melanoma-associated transcription factor and confers strong activation of the promoter. Furthermore, mutation and deletion studies described in this study revealed that the permissive site for cell-specific promoter activity was located directly 5' to the HCR region. Changes in the DNA sequence 5' adjacent to the melanoma-associated transcription factor binding site led to an MIA promoter activity in benign melanocytes and nonmelanocytic cells that usually do not express MIA. Detailed analysis revealed binding of T-cell factor family transcription factors to the repressor element. Because this family is known to interact with COOH-terminal binding protein, we explored the role of COOH-terminal binding protein 1(CtBP1) in silencing MIA gene expression. By reporter gene analysis, we determined a strict negative regulation of MIA promoter activity in melanoma cells by CtBP1. Furthermore, we observed strong expression of CtBP1 in primary melanocytes but a loss of wild-type CtBP1 expression in malignant melanoma in vitro and in vivo. Therefore, we speculate that CtBP1 has an important negative role in MIA regulation, and loss of CtBP1 is implicated in melanoma progression.

Alcohol Oxidoreductases↗

PubMed2002

Interphase fish analysis of cell cycle genes in asbestos-treated human mesothelial cells (HMC), SV40-transformed HMC (MeT-5A) and mesothelioma cells (COLO).

The epidemiologic association between asbestos exposure and human malignant mesothelioma is well established. However, the molecular mechanisms linking asbestos exposure of humans and the subsequent mesothelioma formation is not well understood. The most frequent genetic changes found so far in human malignant mesothelioma (HMM) are deletions and point mutations in the tumor suppressor genes p16INK4a and NF2. Whereas homozygous deletions appear to be the predominant mechanism leading to p16/CDKN2A inactivation, inactivating point mutations coupled with allelic loss mainly occur at the NF2 locus. In the present study, asbestos-treated human mesothelial cells (HMC), SV40-transformed human mesothelial cells (MeT-5A) and a human mesothelioma cell line (COLO) were investigated for genetic changes of cell cycle genes (cyclin D1, p16INK4a, RB1, CDK2) using multicolor fluorescence in situ hybridization (mFISH) in interphase cells. The results show that cyclin D1 is unaffected in all investigated cells. The p16INK4a gene locus was shown to be mutated in COLO cells but not in HMC. After labeling of CDK2 and RB1, hemizygous loss of one allele of each gene was observed in asbestos-treated HMC whereas gene amplification of these genes was detectable in MeT-5A and COLO cells. Our data indicate that disarrangement of the RB1 dependent pathway seems to be involved in mesothelioma formation.

Asbestos↗