Biomedical subjects

S Wölfl

Publications and source records attributed to S Wölfl.

At least 19 recordsLinked to original sources

PubMed2002

Molecular characterization of breast cancer cell lines by expression profiling.

PURPOSE: Gene expression patterns provide detailed insights into cellular regulation that reflect minor differences of cellular capacity not accessible by standard descriptions of the cellular phenotype or origin. METHODS: To identify fundamental differences and similarities we analyzed the gene expression patterns of four breast cancer cell lines: MCF-7, SK-BR-3, T-47D, and BT-474. RESULTS: Although only a small subset of genes (597) is represented on the Atlas-cDNA-Array (Clontech) used, clear differences in the expression of a number of genes could be detected. For example, unique high levels of expressions were found for the HLH-protein ID-1 (MCF-7) and the receptor tyrosine kinase erbB2 (SK-BR-3 and T-47D). Most genes analyzed were expressed at comparable levels in all cell lines studied. CONCLUSIONS: For interpretation of the results sets of genes that show similar variation of expression among the cells were grouped together. Furthermore, our analysis allows the assignment of similarity values that lead to a relation profile of the cell lines. How these results correlate with known biological properties of the cell lines is discussed. Additionally, we demonstrate that results obtained by cDNA-Array hybridization for expression of the ErbB receptor family correlate well with competitive RT-PCR, thus confirming the reliability of the cDNA-Array analysis.

Breast Neoplasms↗

PubMed2000

Preparation of DNA and protein micro arrays on glass slides coated with an agarose film.

A thin layered agarose film on microscope slides provides a versatile support for the preparation of arrayed molecular libraries. An activation step leading to the formation of aldehyde groups in the agarose creates reactive sites that allow covalent immobilization of molecules containing amino groups. Arrays of oligonucleotides and PCR products were prepared by tip printing. After hybridization with complementary fluorescence labeled nucleic acid probes strong fluorescence signals of sequence-specific binding to the immobilized probes were detected. The intensity of the fluorescence signals was proportional to the relative amount of immobilized oligonucleotides and to the concentration of the fluorescence labeled probe. We also used the agarose film-coated slides for the preparation of protein arrays. In combination with specific fluorescence labeled antibodies these protein arrays can be used for fluorescence linked immune assays. With this approach different protein tests can be performed in parallel in a single reaction with minimal amounts of the binding reagents.

Carrier Proteins↗

PubMed2000

Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances.

Yeast strains transformed with high copy number plasmids carrying the gene encoding a green fluorescent protein optimised for yeast (yEGFP3) under the control of the RAD54 or RNR2 promoter were used to investigate the activity of potentially DNA-damaging substances. The assays were performed on 96-well microtitre plates in the presence of different concentrations of the test substances. The synthesis of GFP protein was measured through the fluorescence signal and cell growth was monitored by absorption. Here, we demonstrate that this system can be used as a biosensor to assess the genotoxic potential of drugs and other chemical substances. The use of microtitre plates will enable full automation of the system and allows the inclusion of internal reference standards in each assay.

Biosensing Techniques↗

PubMed2000

Inducible cyclooxygenase-2 gene expression in the human thyroid epithelial cell line Nthy-ori3-1.

OBJECTIVE: To investigate whether the genes encoding Cyclooxygenase-1 and -2 are expressed in thyroid epithelial cells, in vitro. MATERIALS AND METHODS: COX-1/-2 gene expression was examined in the thyroid epithelial cell line Nthy-ori3-1 using semi-quantitative RT-PCR and Western blot analysis. ELISAs were employed to assess whole cell COX-enzyme activity, PGE2 and IL-6 formation. RESULTS: In response to IL-1beta and TNF-alpha combined cells of the thyroid epithelial cell line Nthy-ori3-1 secreted marked amounts of PGE2 in a time-dependent fashion. This is attributed to increased levels of COX-2 specific mRNA, increased amounts of COX-2 protein and COX enzyme activity in the absence of detectable COX-1 protein. The inhibition of the induced COX enzyme activity by the selective COX-2 inhibitor NS-398 demonstrated the presence of COX-2 pharmacologically. The expression of the COX-2 gene was also accompanied by a marked induction of IL-6 formation, a well described inflammatory response of thyroid epithelial cells. CONCLUSIONS: Our observation presents first evidence that COX-2 gene expression is inducible in thyroid epithelial cells, in vitro, upon stimulation with the pro-inflammatory cytokines IL-1beta and TNF-alpha. This finding may indicate that thyroid epithelial cells could play an inflammatory controlling role perhaps during auto-immune thyroid diseases.

Blotting, Western↗

PubMed2000

Bone morphogenetic protein 2 (BMP-2) induces sequential changes of Id gene expression in the breast cancer cell line MCF-7.

Bone morphogenetic proteins (BMPs) are involved in the development of various organs including the mammary gland. They are well-regulated and act in a time-, concentration- and cell-type-specific manner. We found that BMP-2 is expressed in primary breast tumor tissue samples and in breast cancer cell lines. Hybridization of labeled cDNA, obtained from the breast cancer cell line MCF-7, against the Atlas human cDNA expression array revealed differential gene expression depending on BMP-2 treatment. The most prominent changes were observed for the helix-loop-helix proteins Id-1, Id-2 and Id-3. Id-1 expression had increased severalfold after 4 h and was even higher after 24 h. Id-2 and Id-3 were more strongly induced after 4 h and showed no further significant change after 24 h. Analysis of cell-cycle distribution revealed a marked increase of the sub-G1 phase after 48 h in serum-deprived cells. In the presence of BMP-2 no change was observed over 48 h indicating that BMP-2 does not induce apoptosis. In addition, expression of caspase-3 was reduced in BMP-2-treated cells after 24 h. In summary, our results clearly indicate that BMP-2 is a susceptibility factor keeping the cells ready for the integration of various other signals for cell progression.

Bone Morphogenetic Protein 2↗

PubMed2000

Aptamers.

Explore the source record for details and available documents.

DNA, Single-Stranded↗

PubMed1999

UV-Laser induced protein/DNA crosslinking reveals sequence variations of DNA elements bound by c-Jun in vivo.

Many proteins involved in the modulation of gene expression exert their function through direct interaction with DNA. The sequence specificity of these interactions provides the basis for many regulatory mechanisms. The sites that are utilized by a transcription factor are usually analyzed using in vitro binding studies. To detect true in vivo binding sites we developed a method, presented here, that allows construction of recognition element DNA (reDNA) libraries which represent in vivo binding sites plus flanking sequences. reDNA libraries can be constructed for any well-characterized transcription factor. Here we used this method for an in vivo study of genomic DNA elements that interact with the transcription factor c-Jun in rat cerebellum.

Animals↗

PubMed1999

Std1 and Mth1 proteins interact with the glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae.

The Std1 protein modulates the expression of glucose-regulated genes, but its exact molecular role in this process is unclear. A two-hybrid screen for Std1-interacting proteins identified the hydrophilic C-terminal domains of the glucose sensors, Snf3 and Rgt2. The homologue of Std1, Mth1, behaves differently from Std1 in this assay by interacting with Snf3 but not Rgt2. Genetic interactions between STD1, MTH1, SNF3, and RGT2 suggest that the glucose signaling is mediated, at least in part, through interactions of the products of these four genes. Mutations in MTH1 can suppress the raffinose growth defect of a snf3 mutant as well as the glucose fermentation defect present in cells lacking both glucose sensors (snf3 rgt2). Genetic suppression by mutations in MTH1 is likely to be due to the increased and unregulated expression of hexose transporter genes. In media lacking glucose or with low levels of glucose, the hexose transporter genes are subject to repression by a mechanism that requires the Std1 and Mth1 proteins. An additional mechanism for glucose sensing must exist since a strain lacking all four genes (snf3 rgt2 std1 mth1) is still able to regulate SUC2 gene expression in response to changes in glucose concentration. Finally, studies with green fluorescent protein fusions indicate that Std1 is localized to the cell periphery and the cell nucleus, supporting the idea that it may transduce signals from the plasma membrane to the nucleus.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Inducible binding of cyclic adenosine 3',5'-monophosphate (cAMP)-responsive element binding protein (CREB) to a cAMP-responsive promoter in vivo.

In general, DNA-binding factors that activate gene transcription are thought to do so via reversible interaction with DNA. However, most studies, largely performed in vitro, suggest that the transcriptional activator, cAMP response element-binding protein (CREB), is exceptional in that it is constitutively bound to the promoter, where its phosphorylation leads to the recruitment of CREB-binding protein (CBP) to form a CREB/CBP/promoter complex. We have studied how CREB interacts with DNA in vivo to regulate the cAMP-responsive gene encoding human CRH (hCRH). Protein-DNA complexes were cross-linked in cells expressing the endogenous hCRH gene by exposure to a 10 nsec pulse of high-energy UV-laser light, followed by immunoaffinity purification of CREB-DNA complexes. Binding of CREB to a fragment of the hCRH promoter containing a canonical, functional cAMP response element was absent in untreated cells, but was specifically induced after activation of the protein kinase A pathway with forskolin. These data indicate that, in vivo, CREB, like the majority of other DNA-binding transcriptional activators, undergoes signal-mediated promoter interaction.

Binding Sites↗

PubMed1998

L7 protein is a coregulator of vitamin D receptor-retinoid X receptor-mediated transactivation.

The vitamin D receptor (VDR) heterodimerizes with the retinoid X receptor (RXR) and requires additional protein-protein interactions to regulate the expression of target genes. Using the yeast two-hybrid system, we identified the previously described protein L7, that specifically interacted with the VDR in the presence of vitamin D. Deletion analysis indicated, that the N-terminus of L7, which harbours a basic region leucine zipper like domain, mediated interaction with the VDR. Binding assays with purified GST-L7 demonstrated, that L7 specifically pulled down the VDR, that was either expressed in yeast or endogenously contained in the cell line U937. Interestingly, L7 inhibited ligand-dependent VDR-RXR heterodimerization, when constitutively expressed in yeast. We also demonstrate that L7 repressed binding of VDR-RXR heterodimers to a vitamin D response element. Surprisingly, L7 recruited RXR to the same response element in the presence of 9-cis retinoic acid. Ligand-dependent protein-protein interaction in the yeast two-hybrid system confirmed, that binding of L7 also was targeted at the RXR. Our data suggest, that protein L7 is a coregulator of VDR-RXR mediated transactivation of genes, that modulates transcriptional activity by interfering with binding of the receptors to genomic enhancer elements.

Base Sequence↗

PubMed1998

Intramolecular derivatization of 2'-amino-pyrimidine modified RNA with functional groups that is compatible with re-amplification.

To expand the scope of nucleic acid aptamers as a tool for precise molecular recognition, functional groups that are not naturally present in nucleic acid molecules are desired. For in vitro selection these new functional groups must be compatible with the selection process. The present method allows the introduction of succinimide activated side chains at internal amino groups of 2'-amino-pyrimidine-RNA in a combinatorial fashion that is compatible with enzymatic re-amplification.

Base Sequence↗

PubMed1998

Single-read sequence tags of a limited number of genomic DNA fragments provide an inexpensive tool for comparative genome analysis.

Single-read sequences from both ends of 415 3-kb average size genomic DNA fragments of Candida albicans were compared with the complete sequence data of Saccharomyces cerevisiae. Comparison at the protein level, translated DNA against protein sequences, revealed 138 sequence tags with clear similarity to S. cerevisiae proteins or open reading frames. One case of synteny was found for the open reading frames of RAD16 and LYS2, which are adjacent to each other in S. cerevisiae and C. albicans.

Adenosine Triphosphatases↗

PubMed1997

Design of leader sequences that improve the efficiency of the enzymatic synthesis of 2'-amino-pyrimidine RNA for in vitro selection.

The application of nucleic acids obtained by in vitro selection from a large pool of molecules with random sequences in medical diagnosis or therapy requires nucleic acids with enhanced stability in biological fluids. Chemical modifications introduced after selection are likely to alter the structure and the properties of the selected molecules. Therefore, the chemical modifications used must be present throughout the selection. This can be achieved for example by the incorporation of 2'-amino-pyrimidine nucleotides into RNA in the transcription step. Though modified molecules could be transcribed from some generally designed dsDNA templates, the efficiency of transcription and reverse transcription and reverse transcription was very low making this strategy too inefficient. Templates and primers with varying amounts of pyrimidines in the constant flanking region of the RNA molecule were designed and their efficiency in transcription and reverse transcription tested. The obtained 2'-amino-pyrimidine RNA molecules showed enhanced stability in serum and RNAse cocktails. Here we present optimized leader sequences flanking the random core-sequence and reaction conditions that allow the reliable utilization of this modification in in vitro selection.

Base Sequence↗

PubMed1997

Identification of processes that influence negative supercoiling in the human c-myc gene.

DNA elements with sequences suitable for Z-DNA formation are found frequently at various positions in chromatin. Z-DNA formation in these sequences depends largely on the level of local negative supercoiling. We can use binding of a Z-DNA specific antibody at low concentrations in metabolically active permeabilized nuclei to detect naturally occurring Z-DNA formation. Previously we identified three sequence elements in the human c-myc gene that adopt the Z-DNA conformation in the transcribed gene. The three elements are found far upstream (Z1), close to the main transcription start site (Z2) and in the first intron (Z3). Here we measure the persistence of Z-DNA at these three sites under the influence of various metabolic inhibitors. This provides some insight into the varying levels of negative supercoiling. alpha-Amanitin, an inhibitor of transcription, reduced the persistence of Z-DNA in all three elements. Aphidicolin, an inhibitor of replication, increased the persistence of Z-DNA in one element without significantly influencing the other two elements. When camptothecin an inhibitor of topoisomerase I was added in the presence of alpha-amanitin, the persistence of Z-DNA was extended in all three elements. However, in the presence of aphidicolin no effect of camptothecin on Z-DNA formation was observed.

Amanitins↗

PubMed1997

Efficient preparation of single-stranded DNA for in vitro selection.

In vitro selection of aptamers requires the reliable enzymatic preparation of large amounts of (+) single-stranded DNA molecules. This can be achieved by selective enzymatic digest of 5'-phosphorylated (-) strands from PCR products, a method already widely used in sequencing of PCR products. Here we present an adaptation of this method to prepare large pools of single-stranded DNA molecules for in vitro selection.

DNA, Single-Stranded↗

PubMed1996

Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression.

Glucose is the preferred carbon source for most eukaryotic cells and has profound effects on many cellular functions. How cells sense glucose and transduce a signal into the cell is a fundamental, unanswered question. Here we describe evidence that two unusual glucose transporters in the yeast Saccharomyces cerevisiae serve as glucose sensors that generate an intracellular glucose signal. The Snf3p high-affinity glucose transporter appears to function as a low glucose sensor, since it is required for induction of expression of several hexose transporter (HXT) genes, encoding glucose transporters, by low levels of glucose. We have identified another apparent glucose transporter, Rgt2p, that is strikingly similar to Snf3p and is required for maximal induction of gene expression in response to high levels of glucose. This suggests that Rgt2p is a high glucose-sensing counterpart to Snf3p. We identified a dominant mutation in RGT2 that causes constitutive expression of several HXT genes, even in the absence of the inducer glucose. This same mutation introduced into SNF3 also causes glucose-independent expression of HXT genes. Thus, the Rgt2p and Snf3p glucose transporters appear to act as glucose receptors that generate an intracellular glucose signal, suggesting that glucose signaling in yeast is a receptor-mediated process.

Amino Acid Sequence↗

PubMed1996

Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation.

The intron of the corticotropin-releasing hormone (corticoliberin; CRH) gene contains a sequence of over 100 bp of alternating purine/pyrimidine residues. We have used binding of a Z-DNA-specific antibody in metabolically active, permeabilized nuclei to study the formation of Z-DNA in this sequence at various levels of transcription. In the NPLC human primary liver carcinoma cell line, activation of cAMP-dependent pathways increased the level of transcription, while adding glucocorticoids inhibited transcription of the CRH gene. These cells respond in a manner similar to hypothalamic cells. Z-DNA formation in this sequence was detected at the basal level of transcription, as well as after stimulation with forskolin. Inhibition of transcription by dexamethasone abolished Z-DNA formation. Z-DNA formation in the WC gene (c-myc) was affected differently in the same experiment. Thus, changes in Z-DNA formation in the CRH gene are gene specific and are linked to the transcription of the gene.

Base Sequence↗

PubMed1996

Analysis of a 26,756 bp segment from the left arm of yeast chromosome IV.

The nucleotide sequence of a 26.7 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome IV is presented. An analysis of this segment revealed 11 open reading frames (ORFs) longer than 300 bp and one split gene. These ORFs include the genes encoding the large subunit of RNA polymerase II, the biotin apo-protein ligase, an ADP-ribosylation factor (ARF 2), the 'L35'-ribosomal protein, a rho GDP dissociation factor, and the sequence encoding the protein phosphatase 2A. Further sequence analysis revealed a short ORF encoding the ribosomal protein YL41B, an intron in a 5' untranslated region and an extended homology with another cosmid (X83276) located on the same chromosome. The potential biological relevance of these findings is discussed.

ADP-Ribosylation Factors↗