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Thomas Wirth

Publications and source records attributed to Thomas Wirth.

At least 37 records · Page 2Linked to original sources
PubMed2004

Epithelial-mesenchymal transition: NF-kappaB takes center stage.

Activation of the transcription factor NF-kappaB occurs in many human tumors, and studies have shown that NF-kappaB can promote cell proliferation and oncogenesis, possibly by protecting cells from apoptosis. Little is known, however, about whether NF-kappaB is involved in tumor progression including epithelial-mesenchymal transition (EMT), a central process governing both morphogenesis and carcinoma progression in multicellular organisms. In a combined in vitro/in vivo model of mammary carcinogenesis, NF-kappaB was essential both for the induction and maintenance of EMT and for in vivo metastasis. NF-kappaB and the signaling pathways that are involved in its activation should therefore receive attention as potential targets for the development of novel anti-metastatic cancer treatments.

Cell Differentiation↗
PubMed2004

Identification of novel Myc target genes with a potential role in lymphomagenesis.

The c-Myc transcription factor regulates a wide set of genes involved in processes such as proliferation, differentiation and apoptosis. Therefore, altered expression of Myc leads to deregulation of a large number of target genes and, as a consequence, to tumorigenesis. For understanding Myc-induced transformation, identification of these target genes is essential. In this study, we searched for Myc target genes involved in lymphomagenesis using different mouse T and B cell lymphoma cell lines transformed by a conditional Myc-allele. Target genes obtained by microarray experiments were further subjected to a kinetic analysis of mRNA expression upon Myc inactivation/reactivation, bioinformatic examination of Myc binding sites and chromatin immunoprecipitation. This approach allowed us to define targets whose activation is a direct consequence of Myc binding. Among the 38 novel Myc targets, we identified several genes implicated in the tumor development. These genes are not only relevant for mouse lymphomas because we observed their upregulation in human lymphomas as well. Our findings further the understanding of Myc-induced lymphomagenesis and help toward developing more efficient antitumor strategies.

Animals↗
PubMed2004

Reactivity, chemoselectivity, and diastereoselectivity of the oxyfunctionalization of chiral allylic alcohols and derivatives in microemulsions: comparison of the chemical oxidation by the hydrogen peroxide/sodium molybdate system with the photooxygenation.

The chiral allylic alcohols 1a-d and their acetate (1e) and silyl ether (1f) derivatives have been oxidized by the H2O2/MoO4(2)- system, a convenient and efficient chemical source of singlet oxygen. This chemical peroxidation (formation of the allylic hydroperoxides 2) has been conducted in various media, which include aqueous solutions, organic solvents, and microemulsions. The reactivity, chemoselectivity, and diastereoselectivity of this chemical oxidation are compared to those of the sensitized photooxygenation, with the emphasis on preparative applications in microemulsion media. While a similar threo diastereoselectivity is observed for both modes of peroxidation, the chemoselectivity differs significantly, since in the chemical oxidation with the H2O2/MoO4(2)- system the undesirable epoxidation by the intermediary peroxomolybdate competes efficiently with the desirable peroxidation by the in situ generated singlet oxygen. A proper choice of the type of microemulsion and the reaction conditions furnishes a high chemoselectivity (up to 97%) in favor of threo-diastereoselective (up to 92%) peroxidation.

Journal Article↗
PubMed2004

Overexpression of phospholipid-hydroperoxide glutathione peroxidase in human dermal fibroblasts abrogates UVA irradiation-induced expression of interstitial collagenase/matrix metalloproteinase-1 by suppression of phosphatidylcholine hydroperoxide-mediated NFkappaB activation and interleukin-6 release.

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) exhibits high specific activity in reducing phosphatidylcholine hydroperoxides (PCOOHs) and thus may play a central role in protecting the skin against UV irradiation-triggered detrimental long term effects like cancer formation and premature skin aging. Here we addressed the role of PHGPx in the protection against UV irradiation-induced expression of matrix metalloproteinase-1 (MMP-1). For this purpose, we created human dermal fibroblast cell lines overexpressing human PHGPx. Overexpression led to a significant increase in PHGPx activity. In contrast to a maximal 4.5-fold induction of specific MMP-1 mRNA levels in vector-transfected cells at 24 h after UVA irradiation, no MMP-1 induction occurred at any studied time point after UVA treatment of PHGPx-overexpressing fibroblasts. As interleukin-6 (IL-6) was earlier shown to mediate the UVA induction of MMP-1, we studied whether PHGPx overexpression might interfere with the NFkappaB-mediated IL-6 induction and downstream signaling. Using transient transfections of IL-6 promoter constructs containing NFkappaB binding sites, we observed a high induction of the reporter gene luciferase in vector-transfected control cells and a significantly lower induction in PHGPx-overexpressing fibroblasts following UVA irradiation. Consistently both UVA irradiation and treatment of fibroblasts with PCOOHs led to phosphorylation and nuclear translocation of the p65 subunit, whereas cells overexpressing PHGPx exhibited impaired NFkappaB activation, p65 phosphorylation, and nuclear translocation. In line with this, the PHGPx-overexpressing fibroblasts showed a reduced constitutive and UVA irradiation-induced IL-6 release. After incubating PHGPx-overexpressing cells with PCOOHs a reduced induction of IL-6 was observed. This together with the suppression of UVA irradiation-induced IL-6 release in the presence of Trolox, a chain breaker of PCOOH-initiated lipid peroxidation, indicates that UVA irradiation-induced PCOOHs and subsequent lipid peroxides initiate the NFkappaB-mediated induction of IL-6, which mediates the induction of MMP-1. Our finding is particularly relevant in light of the already available small molecule mimetics of PHGPx.

Active Transport, Cell Nucleus↗
PubMed2004

Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression.

Immunoglobulin production is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) in spite of functional clonal rearrangements. The presence of "crippling" mutations in coding and regulatory regions, as well as down-regulation of B-cell-specific transcription factors, has been suggested as a potential reason for the lack of immunoglobulin (Ig) chain gene transcription. We have investigated the impact of epigenetic silencing in suppressing Ig heavy (H)-chain expression. Chromatin immunoprecipitation (ChIP) was used to analyze transcription factor binding to octamer motifs present in the IgH regulatory regions. Transcription factors were bound to these motifs in control cell lines, however, they were absent in the cHL-derived cell lines KMH2, L1236, and L428. Ectopic expression of octamer-binding transcription factor (Oct2) and/or B-cell Oct binding protein/Oct-binding factor (BOB.1/OBF.1) did not result in any measurable binding to these sites. Increased histone 3 Lysine 9 (H3-K9) methylation was observed in the promoter region of the IgH locus in L428 and L1236 cells. This is a typical feature of heterochromatic, transcriptionally silent regions. Treatment of cHL-derived cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) partially reactivated IgH transcription and affected chromatin modifications. Our results suggest an important role of epigenetic silencing in the inhibition of IgH transcription in HRS cells.

Antimetabolites, Antineoplastic↗
PubMed2004

Iodine electrophiles in stereoselective reactions: recent developments and synthetic applications.

The regio- and stereocontrolled functionalisation of carbon-carbon double bonds bears enormous potential in organic synthesis. This area has been extensively studied and reviewed as alkenes are amongst the most important starting materials for synthetic chemists, accessible in many varieties and in large quantities. We focus in this tutorial review only on recent developments using iodine electrophiles for the functionalisation of alkenes although transition-mediated reactions and functionalisations with chalcogen electrophiles also play an important role. New synthetic applications using this methodology showing scope and limitations of iodine-mediated processes also within the context of other electrophilic reactions are highlighted.

Iodine↗
PubMed2004

Reversible lymphomagenesis in conditionally c-MYC expressing mice.

It is well documented that deregulation of MYC leads to tumor development, yet many aspects of this process are only partially understood. We have established a transgenic mouse model in which c-MYC is conditionally expressed in lymphoid cells using the tetracycline-regulated system of gene regulation. Mice with continuously expressed transgenic c-MYC died of invasive T- or B-cell lymphomas within 4 months. Lymphomas developing in transgenic mice were c-MYC dependent since doxycycline treatment led to tumor regression. Using transplantation of established tumor cell lines labeled with GFP, we followed the fate of neoplastic cells in recipients upon MYC inactivation. This approach allowed us to elucidate both apoptosis and differentiation as mechanisms of tumor elimination. Comparative genomic hybridization (CGH) and FISH analyses were performed in order to analyze possible chromosomal aberrations induced by c-MYC. We observed that overexpression of c-MYC is sufficient to induce recurrent patterns of genomic instability. The main observation was a gain of genomic material that corresponded to chromosome 15 in several T-cell tumors, which could be identified as trisomy.

Animals↗
PubMed2004

Myosin light chain 1 atrial isoform (MLC1A) is expressed in pre-B cells under control of the BOB.1/OBF.1 coactivator.

The BOB.1/OBF.1 protein is a B-cell-specific coactivator of the Oct1 and Oct2 transcription factors. It is involved in mediating the transcriptional activity of the Oct proteins. However, animals deficient for BOB.1/OBF.1 showed virtually normal expression of genes that contain octamer motifs in their regulatory regions. To identify new genes that are regulated by BOB.1/OBF.1, we took advantage of a previously described cell system. RNAs differentially expressed in a BOB.1/OBF.1-deficient pre-B cell line and a derivative of this cell line expressing a hormone dependent BOB.1/OBF.1-estrogen receptor (BobER) fusion protein were isolated. Using the cDNA representational difference analysis method we could identify myosin light chain 1 atrial (MLC1A) isoform as a gene regulated by BOB.1/OBF.1. MLC1A was so far unknown to be expressed in tissues other than muscle. Here we demonstrate that MLC1A is indeed expressed in mouse pre-B cells. Analysis of the expressed mRNA revealed an alternative 5' promoter element and an alternative splice product, which had not yet been described for the murine gene. Cotransfection experiments with reporter constructs driven by the MLC1A promoter suggest that the regulation by BOB.1/OBF.1 is indirect. Consistent with this conclusion is the observation that transcriptional induction of the endogenous MLC1A gene by BOB.1/OBF.1 requires de novo protein synthesis.

Animals↗
PubMed2004

Doxycycline regulation in a single retroviral vector by an autoregulatory loop facilitates controlled gene expression in liver cells.

The tetracycline system has limitations in liver cells, such as toxic effects and low controllability. We generated different retroviral vectors for controlled gene expression in liver cells, in which the regulatory elements were arranged in different patterns. Only the organization of the tetracycline system in an autoregulatory loop in the sense orientation results in high retroviral titres and in tight regulation of gene expression in highly differentiated hepatoma cells. Because of the toxicity of the transactivator tTA, it was impossible to establish doxycycline-dependent stable HepG2 cell lines. To avoid sequelching-related toxicity in liver cells, we replaced tTA with new non-toxic transactivators. By using tTA2, tTA3 and tTA4, we observed tight doxycycline-dependent gene expression in 23, 49 and 45% of the isolated clones. The tTA4 vector was used to transduce hepatocytes of mice in vivo. Tight doxycycline-controllable gene regulation was also observed in the liver of mice, confirming our hypothesis that retroviral vectors with autoregulatory loops of the tetracycline system facilitate inducible gene expression in the liver in vivo. Our new retroviral vector system allows rapid isolation of controllable clones in a very high yield and should make the tetracycline system more applicable to liver-derived cells and in liver gene therapy in vivo.

Animals↗
PubMed2004

Differential Emu enhancer activity and expression of BOB.1/OBF.1, Oct2, PU.1, and immunoglobulin in reactive B-cell populations, B-cell non-Hodgkin lymphomas, and Hodgkin lymphomas.

It has previously been demonstrated that in cultured and in situ tumour cells of classical Hodgkin lymphoma (cHL), the immunoglobulin (Ig) promoter is inactive and its transcription factors Oct2 and/or BOB.1/OBF.1 are down-regulated. In this study, the analysis of these transcription factors has been extended to a broad spectrum of B-cell malignancies and the findings have been related to the situation in normal B-cells of various differentiation stages and to the expression of Ig. Furthermore, an additional Ig transcription factor, PU.1, recently described to be absent from cHL, and a further regulatory element of the Ig gene, the intronic Emu enhancer, have been studied. BOB.1/OBF.1 and Oct2 were present in all B-cells expressing Ig, whereas PU.1 proved to be absent from late B-cell differentiation stages and from a subset of germinal centre B-cells. Interestingly, there were several normal (eg germinal centre centroblasts and monocytoid B-cells) and malignant B-cell populations (eg a proportion of diffuse large B-cell lymphomas, DLBCLs) that were Ig-negative, despite their BOB.1/OBF.1 and Oct2 expression. This study further shows that absence of PU.1 alone, as well as inactivation of the intronic Emu enhancer, is not sufficient to down-regulate Ig transcription. Taken together, the simultaneous absence of PU.1, Oct2, and/or BOB.1/OBF.1 is unique to Hodgkin and Reed-Sternberg (HRS) cells and cannot be detected in normal B-cell subsets or B-cell non-Hodgkin lymphomas (B-NHLs). This supports the concept that the down-regulation of Ig in cHL does not reflect a physiological situation, but a defect probably closely linked to the pathogenesis of cHL.

B-Lymphocytes↗
PubMed2004

Tumor-specific adenoviral gene therapy: transcriptional repression of gene expression by utilizing p53-signal transduction pathways.

Adenoviral gene expression that is repressed by p53 in nontransformed cells could provide a tumor-specific gene therapy approach for a large subset of tumors. Adenoviral infection in vivo induces stabilization of p53, which can be utilized for a strategy that includes p53-dependent expression of a transcriptional repressor and a target promoter,which is highly susceptible for transcriptional repression. Therefore, we constructed different versions of CMV-promoters (CMVgal) with binding sites for GAL4-DBD and investigated 11 GAL4-DBD fusion proteins to elucidate the most effective repressor domain to silence CMVgal activity. The transcriptional repressor GAL4-KRAB-A under control of a p53-dependent promoter facilitates strong CMVgal-mediated gene expression specifically in p53 mutant cells by a double-recombinant adenoviral vector (Ad-RGCdR). GAL4-KRAB-A mediates strong transcriptional repression of Ad-RGCdR in p53 wild-type cells, which could be further enhanced by preactivation of p53-signalling following low-dose chemotherapy prior to adenoviral infection. By utilizing p53 signalling involved in chemotherapy and adenoviral infection, more than 99% of Ad-RGCdR gene expression could be repressed in p53 wild-type cells. Controlled gene expression from CMVgal promoters by transcriptional repression utilizing functional p53 signalling thus provides a very effective tool for tumor-specific adenoviral gene therapy.

Adenoviridae↗
PubMed2004

Protein transduction domains fused to virus receptors improve cellular virus uptake and enhance oncolysis by tumor-specific replicating vectors.

Expression of cellular receptors determines viral tropism and limits gene delivery by viral vectors. Protein transduction domains (PTDs) have been shown to deliver proteins, antisense oligonucleotides, liposomes, or plasmid DNA into cells. In our study, we investigated the role of several PTD motifs in adenoviral infection. When physiologically expressed, a PTD from human immunodeficiency virus transactivator of transcription (Tat) did not improve adenoviral infection. We therefore fused PTDs to the ectodomain of the coxsackievirus-adenovirus receptor (CAR(ex)) to attach PTDs to adenoviral fiber knobs. CAR(ex)-Tat and CAR(ex)-VP22 allowed efficient adenoviral infection in nonpermissive cells and significantly improved viral uptake rates in permissive cells. Dose-dependent competition of CAR(ex)-PTD-mediated infection using CAR(ex) and inhibition experiments with heparin showed that binding of CAR(ex)-PTD to both adenoviral fiber and cellular glycosaminoglycans is essential for the improvement of infection. CAR(ex)-PTD-treated adenoviruses retained their properties after density gradient ultracentrifugation, indicating stable binding of CAR(ex)-PTD to adenoviral particles. Consequently, the mechanism of CAR(ex)-PTD-mediated infection involves coating of the viral fiber knobs by CAR(ex)-PTD, rather than placement of CAR(ex) domains on cell surfaces. Expression of CAR(ex)-PTDs led to enhanced lysis of permissive and nonpermissive tumor cells by replicating adenoviruses, indicating that CAR(ex)-PTDs are valuable tools to improve the efficacy of oncolytic therapy. Together, our study shows that CAR(ex)-PTDs facilitate gene transfer in nonpermissive cells and improve viral uptake at reduced titers and infection times. The data suggest that PTDs fused to virus binding receptors may be a valuable tool to overcome natural tropism of vectors and could be of great interest for gene therapeutic approaches.

Adenoviridae↗
PubMed2004

NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression.

The transcription factor NF-kappaB is activated in a range of human cancers and is thought to promote tumorigenesis, mainly due to its ability to protect transformed cells from apoptosis. To investigate the role of NF-kappaB in epithelial plasticity and metastasis, we utilized a well-characterized in vitro/in vivo model of mammary carcinogenesis that depends on the collaboration of the Ha-Ras oncoprotein and TGF-beta. We show here that the IKK-2/IkappaBalpha/NF-kappaB pathway is required for the induction and maintenance of epithelial-mesenchymal transition (EMT). Inhibition of NF-kappaB signaling prevented EMT in Ras-transformed epithelial cells, while activation of this pathway promoted the transition to a mesenchymal phenotype even in the absence of TGF-beta. Furthermore, inhibition of NF-kappaB activity in mesenchymal cells caused a reversal of EMT, suggesting that NF-kappaB is essential for both the induction and maintenance of EMT. In line with the importance of EMT for invasion, blocking of NF-kappaB activity abrogated the metastatic potential of mammary epithelial cells in a mouse model system. Collectively, these data provide evidence of an essential role for NF-kappaB during distinct steps of breast cancer progression and suggest that the cooperation of Ras- and TGF-beta-dependent signaling pathways in late-stage tumorigenesis depends critically on NF-kappaB activity.

Animals↗
PubMed2003

Direct iodination of alkanes.

[reaction: see text] A cheap and efficient iodination of hydrocarbons can be achieved by generating tert-butyl hypoiodite from iodine and sodium tert-butoxide. The alkane is reactant and solvent, and this metal-free process provides a clean solution for their direct iodination.

Journal Article↗
PubMed2003

Expression of the aldehyde dehydrogenase 2-like gene is controlled by BOB.1/OBF.1 in B lymphocytes.

BOB.1/OBF.1 is a lymphocyte-restricted transcriptional coactivator. It binds to the Oct1 and Oct2 transcription factors and increases their transactivation potential. Targeted gene disruption experiments revealed that BOB.1/OBF.1 is critical at different stages of B cell development. A large number of genes expressed in B cells contain octamer motifs in their regulatory regions. However, only few genes have been described so far whose expression is dependent on BOB.1/OBF.1. To understand the molecular basis of BOB.1/OBF.1 function in B cell development, we searched for BOB.1/OBF.1 target genes by expression profiling. We have identified genes both induced and repressed by BOB.1/OBF.1. Using different genetic systems, we demonstrate regulation of a selection of these genes. Identified targets included genes encoding Ahd2-like, AKR1C13, Rbp1, Sdh, Idh2, protocadherin gamma, alpha-catenin, Ptprs, Id3, and Creg. Classification of BOB.1/OBF.1 target genes by function suggests that they affect various aspects of B cell physiology such as cellular metabolism, cell adhesion, and differentiation. To better understand the mechanism of BOB.1/OBF.1 action, we cloned the promoter of the gene encoding Ahd2-like, the gene showing the strongest regulation by BOB.1/OBF.1. This promoter indeed contains a perfect octamer motif. Furthermore, the motif was recognized by the Oct transcription factors as well as BOB.1/OBF.1 in vitro and in vivo, as shown by electromobility shift and chromatin immunoprecipitation assays. Transient transfections confirm that this promoter is activated by BOB.1/OBF.1. Our observations suggest that by regulating genes in different functional pathways, BOB.1/OBF.1 has a widespread effect on B cell development and function.

Aldehyde Dehydrogenase↗
PubMed2003

Critical role of RelB serine 368 for dimerization and p100 stabilization.

In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory kappaB proteins. In most other cell types inhibitory kappaB proteins prevent nuclear translocation and activation of NFkappaB. We reasoned that this characteristic might be because of post-translational modifications of RelB. In Drosophila, signal-dependent phosphorylation of the Rel homologue Dorsal at serine 317 has been shown to be critical for nuclear import. The evolutionary conservation of this serine prompted us to analyze the function of the corresponding site in RelB. As a model system we used the murine S107 plasmacytoma cell line, which lacks endogenous RelB expression. Analysis of S107 cells expressing wild type RelB and serine 368 mutants reveals that serine 368 is not required for nuclear import but that it is critical for RelB dimerization with other members of the NFkappaB family. Similar effects were obtained when the conserved serine in RelA was mutated. We further demonstrate that expression of functional RelB, but not of serine 368 mutants, severely reduces p52 generation and strongly increases expression of the p52 precursor, p100. Wild type RelB, but not mutant RelB, prolonged p100 half-life. We therefore suggest an inhibitory effect of RelB on p100 processing, which is possibly regulated in a signal-dependent manner.

3T3 Cells↗