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Thomas Wirth

Publications and source records attributed to Thomas Wirth.

At least 55 records · Page 3Linked to original sources
PubMed2003

A telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer.

The catalytic component of human telomerase reverse transcriptase (hTERT) is not expressed in most primary somatic human cells, whereas the majority of cancer cells reactivate telomerase by transcriptional up-regulation of hTERT. Several studies demonstrated that the hTERT promoter can be used to restrict gene expression of E1-deleted replication defective adenoviral vectors to telomerase-positive cancer cells. In this study, a conditionally replicating adenovirus (hTERT-Ad) expressing E1A genes under control of a 255-bp hTERT-promoter was constructed. Additionally, an internal ribosomal entry site-enhanced green fluorescent protein cassette was inserted downstream of the E1B locus to monitor viral replication in vivo. Adenoviral replication of hTERT-Ad and enhancement of enhanced green fluorescent protein expression could be observed in all investigated telomerase-positive tumor cell lines. In contrast, hTERT-Ad infection of telomerase-negative primary human hepatocytes did not result in significant replication. The capability of hTERT-Ad to induce cytopathic effects in tumor cells was comparable with that of adenovirus wild type and significantly higher compared with ONYX-015, regardless of the p53 status of the tumor cells. Single application of low-dose hTERT-Ad to tumor xenografts led to significant inhibition of tumor growth, confirming the potential therapeutic value of conditionally replicative adenoviral vectors. These in vivo experiments also revealed that hTERT-Ad-mediated oncolysis was more efficient than ONYX-015 treatment. These results demonstrate that expression of E1A under transcriptional control of the hTERT promoter is sufficient for effective telomerase-dependent adenovirus replication as a promising perspective for the treatment of the majority of epithelial tumors.

Adenoviridae↗
PubMed2003

B cell-specific transgenic expression of Bcl2 rescues early B lymphopoiesis but not B cell responses in BOB.1/OBF.1-deficient mice.

Mice deficient for the transcriptional coactivator BOB.1/OBF.1 show several defects in B cell differentiation. Numbers of immature transitional B cells in the bone marrow are reduced and fewer B cells reach the periphery. Furthermore, germinal center B cells are absent and marginal zone (MZ) B lymphocytes are markedly reduced. Increased levels of B cell apoptosis in these mice prompted us to analyze expression and function of antiapoptotic proteins. Bcl2 expression is strongly reduced in BOB.1/OBF.1-deficient pre-B cells. When BOB.1/OBF.1-deficient mice were crossed with Bcl2-transgenic mice, B cell development in the bone marrow and numbers of B cells in peripheral lymphoid organs were normalized. However, neither germinal center B cells nor MZ B cells were rescued. Additionally, Bcl2 did not rescue the defects in signaling and affinity maturation found in BOB.1/OBF.1-deficient mice. Interestingly, Bcl2-transgenic mice by themselves show an MZ B cell defect. Virtually no functional MZ B cells were detected in these mice. In contrast, mice deficient for Bcl2 show a relative increase in MZ B cell numbers, indicating a previously undetected function of Bcl2 for this B cell compartment.

Animals↗
PubMed2003

Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4.

In this study we have identified members of the Toll-like receptor (TLR) family (namely, TLRs 4, 6, 8, and 9) as proteins to which the intracellular protein tyrosine kinase, Bruton's tyrosine kinase (Btk), binds. Detailed analysis of the interaction between Btk and TLR8 demonstrates that the presence of both Box 2 and 3 motifs in the Toll/interleukin-1 receptor domain was required for the interaction. Furthermore, co-immunoprecipitation experiments revealed that Btk can also interact with key proteins involved in TLR4 signal transduction, namely, MyD88, Mal (MyD88 adapter-like protein), and interleukin-1 receptor-associated kinase-1, but not TRAF-6. The ability of Btk to interact with TLR4 and Mal suggests a role for Btk in lipopolysaccharide (LPS) signal transduction. Stimulation of the human monocytic cell line THP-1 with LPS resulted in an increase in the level of tyrosine phosphorylation of Btk (indicative of activation). The autokinase activity of Btk was also stimulated after LPS stimulation. In addition, a dominant negative form of Btk inhibited TLR4-mediated activation of a nuclear factor kappaB (NFkappaB)-dependent reporter gene in HEK293 cells as well as LPS-induced activation of NFkappaB in the astrocytoma cell line U373 and the monocytic cell line RAW264.7. Further investigation revealed that the Btk-specific inhibitor, LFM-A13, inhibited the activation of NFkappaB by LPS in THP-1 cells. Our findings implicate Btk as a Toll/interleukin-1 receptor domain-binding protein that is important for NFkappaB activation by TLR4.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2003

Caspase-mediated cleavage converts the tumor necrosis factor (TNF) receptor-associated factor (TRAF)-1 from a selective modulator of TNF receptor signaling to a general inhibitor of NF-kappaB activation.

The role of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-1 in NF-kappaB activation by various members of the TNF receptor family is not well understood, and conflicting data have been published. Here, we show that TRAF1 differentially affects TRAF2 recruitment and activation of NF-kappaB by members of the TNF receptor family. Interestingly, a naturally occurring caspase-derived cleavage product of TRAF1 solely comprising its TRAF domain (TRAF1-(164-416)) acted as a general inhibitor of NF-kappaB activation. In contrast, a corresponding fragment generated by cleavage of TRAF3 showed no effect in this regard. In accordance with these functional data, TRAF1, but not TRAF3, interacted with the IKK complex via its N-TRAF domain. Endogenous TRAF1 and the overexpressed TRAF domain of TRAF1 were found to be constitutively associated with the IKK complex, whereas endogenous receptor interacting protein was only transiently associated with the IKK complex upon TNF stimulation. Importantly, the caspase-generated TRAF1-fragment, but not TRAF1 itself inhibited IKK activation. Our results suggest that TRAF1 and TRAF1-(164-416) exert their regulatory effects on receptor-induced NF-kappaB activation not only by modulation of TRAF2 receptor interaction but especially TRAF1-(164-416) also by directly targeting the IKK complex.

Caspases↗
PubMed2003

The Yaa mutation promoting murine lupus causes defective development of marginal zone B cells.

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of an as yet unidentified mutant gene, Yaa (Y-linked autoimmune acceleration). In view of a possible role of marginal zone (MZ) B cells in murine SLE, we have explored whether the expression of the Yaa mutation affects the differentiation of MZ and follicular B cells, thereby implicating the acceleration of the disease. In this study, we show that both BXSB and C57BL/6 Yaa mice, including two different substrains of BXSB Yaa males that are protected from SLE, displayed an impaired development of MZ B cells early in life. Studies in bone marrow chimeras revealed that the loss of MZ B cells resulted from a defect intrinsic to B cells expressing the Yaa mutation. The lack of selective expansion of MZ B cells in diseased BXSB Yaa males strongly argues against a major role of MZ B cells in the generation of pathogenic autoantibodies in the BXSB model of SLE. Furthermore, a comparative analysis with mice deficient in CD22 or expressing an IgM anti-trinitrophenyl/DNA transgene suggests that the hyperreactive phenotype of Yaa B cells, as judged by a markedly increased spontaneous IgM secretion, is likely to contribute to the enhanced maturation toward follicular B cells and the block in the MZ B cell generation.

Animals↗
PubMed2002

A cellular model for Friedreich Ataxia reveals small-molecule glutathione peroxidase mimetics as novel treatment strategy.

Friedreich Ataxia (FRDA), the most prevalent of the inherited ataxias, is a multi-systemic disease with loss of sensory neurons and life-threatening hypertrophic cardiomyopathy as its most severe manifestations. Reduced levels of the mitochondrial protein frataxin lead to cell-damaging oxidative stress and consequently FRDA is considered as a model for more common neurodegenerative disorders in which reactive radicals and oxidative stress are involved. We have developed a cellular assay system that discriminates between fibroblasts from FRDA patients and unaffected donors on the basis of their sensitivity to pharmacological inhibition of de novo synthesis of glutathione. With this assay we observed that supplementation with selenium effectively improved the viability of FRDA fibroblasts, indicating that basal selenium concentrations are not sufficient to allow an adequate increase in the activity of certain detoxification enzymes (such as GPX). Furthermore, we characterized potential drug candidates and found that idebenone, a mitochondrially localized antioxidant that ameliorates cardiomyopathy in FRDA patients, as well as other lipophilic antioxidants protected FRDA cells from cell death. Our results also demonstrate for the first time that small-molecule GPX mimetics have potential as a novel treatment strategy for Friedreich Ataxia and presumably also for other neurodegenerative diseases with mitochondrial impairment.

Benzoquinones↗
PubMed2002

Rocaglamide derivatives are potent inhibitors of NF-kappa B activation in T-cells.

Crude extracts from different Aglaia species are used as anti-inflammatory remedies in the traditional medicine of several countries from Southeast Asia. Because NF-kappaB transcription factors represent key regulators of genes involved in immune and inflammatory responses, we supposed that the anti-inflammatory effects of Aglaia extracts are mediated by the inhibition of NF-kappaB activity. Purified compounds of Aglaia species, namely 1H-cyclopenta[b]benzofuran lignans of the rocaglamide type as well as one aglain congener were tested for their ability to inhibit NF-kappaB activity. We show that a group of rocaglamides represent highly potent and specific inhibitors of tumor necrosis factor-alpha (TNFalpha) and phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB-dependent reporter gene activity in Jurkat T cells with IC(50) values in the nanomolar range. Some derivatives are less effective, and others are completely inactive. Rocaglamides are able to suppress the PMA-induced expression of NF-kappaB target genes and sensitize leukemic T cells to apoptosis induced by TNFalpha, cisplatin, and gamma-irradiation. The suppression of NF-kappaB activation correlated with the inhibition of induced IkappaB(alpha) degradation and IkappaB(alpha) kinase activation. The level of interference was determined and found to be localized upstream of the IkappaB kinase complex but downstream of the TNF receptor-associated protein 2. Our data suggest that rocaglamide derivatives could serve as lead structures in the development of anti-inflammatory and tumoricidal drugs.

Animals↗
PubMed2002

Lack of correlation between NF-kappaB activation and induction of programmed cell death in PC12 pheochromocytoma cells treated with 6-hydroxydopamine or the cannabinoid receptor 1-agonist CP55,940.

NF-kappaB is a transcriptional regulator that plays a key role in immunity, inflammation and programmed cell death. We generated a PC12 cell line termed PC12kappaBluc that contains an integrated NF-kappaB-responsive reporter gene to directly measure NF-kappaB activity. The "classical" activators of NF-kappaB, phorbol 12-O-tetradecanoate-13-acetate and tumor necrosis factor alpha, strongly induced NF-kappaB activity in PC12kappaBluc cells. Activation of NF-kappaB could be attenuated by preincubating the cells with the cAMP analogue dbcAMP or via expression of the superrepressor IkappaBalphaS32A/S36A. PC12kappaBluc cells were subjected to several apoptotic paradigms, including treatment with 6-hydroxydopamine, H2O2, K2Cr2O7, MnCl2, C2-ceramide or the cannabinoid receptor-1 agonist CP55,940. A simultaneous measurement of the NF-kappaB activity revealed that only administration of 6-hydroxydopamine or CP55,940 increased NF-kappaB activity. Using pharmacological and genetic strategies to attenuate NF-kappaB transcriptional activity, we demonstrate that the elevation of NF-kappaB activity by 6-hydroxydopamine and CP55,940 is not an integral part of the apoptotic signaling cascade in PC12 cells.

Adrenergic Agents↗
PubMed2002

Neuronal cell death induced by antidepressants: lack of correlation with Egr-1, NF-kappa B and extracellular signal-regulated protein kinase activation.

The tricyclic antidepressants (TCA) amitriptyline and desipramine and the serotonin reuptake inhibitor fluoxetine induce, at microM concentrations, cell death in HT22 immortalized hippocampal neurons and PC12 pheochromocytoma cells. Here, we show that these neurotoxic effects are accompanied by a selective activation of extracellular signal-regulated protein kinase (ERK), the biosynthesis of the transcription factor Egr-1 and an increase in the transcriptional activity of NF-kappa B. However, an impairment of both ERK activation and Egr-1 biosynthesis by the MAP kinase kinase-1 (MEK-1) inhibitor PD98059 did not block cell death. Moreover, stimulation of ERK phosphorylation and Egr-1 biosynthesis by sphingosine-1-phosphate did not induce cell death, indicating that stimulation of the ERK signaling pathway and Egr-1 biosynthesis are not required for neuronal cell death induced by antidepressants. Likewise, attenuation of antidepressant-induced NF-kappa B activity by elevation of the intracellular cAMP concentration or by retroviral driven expression of the non-degradable superrepressor I kappa B alpha S32A/S36A demonstrated that the elevation of NF-kappa B activity by amitriptyline, desipramine and fluoxetine is not an integral part of the apoptotic signaling cascade triggered by these compounds.

Amitriptyline↗
PubMed2002

Loss of PU.1 expression is associated with defective immunoglobulin transcription in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease.

Immunoglobulin transcription is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin disease (cHD). We recently demonstrated that defective immunoglobulin promoter transcription correlates with the down-regulation of the B-cell transcription factors Oct2 and BOB.1/OBF.1. These results prompted us to investigate whether immunoglobulin enhancer activity is also impaired in HRS cells and whether as yet unidentified factors could be necessary for immunoglobulin enhancer activity in HRS cells of cHD. Here we analyzed 30 cases of cHD for expression of the Ets family member PU.1 that is known to collaborate with multiple transcription factors and to regulate expression of immunoglobulin genes. We show that PU.1 is not expressed in primary and cultured HRS cells. Reintroduction of PU.1 and Oct2 in cultured HRS cells restored the activity of cotransduced immunoglobulin enhancer constructs. Our study identifies PU.1 deficiency as a recurrent defect in HRS cells that might contribute to their impairment of immunoglobulin transcription.

DNA-Binding Proteins↗
PubMed2002

New and efficient chiral selenium electrophiles.

New chiral diselenides were prepared in a few steps from readily available starting materials. The selenium electrophiles generated from these diselenides were used for the efficient stereoselective inter- and intramolecular functionalization of alkenes. The substitution pattern influences the stereoselectivities and protection of the hydroxy moiety in the chiral side chain led to increased selectivities and yields in the selenenylation reactions. An additional substituent in the second ortho position was advantageous as well. Addition products with up to 96% de were obtained. The influence of the nucleophile on the outcome of selenenylations of alkenes was studied to some extent as well.

Journal Article↗
PubMed2002

Reagent-controlled stereoselective iodolactonizations.

[reaction: see text] Iodocyclizations are important transformations and among stereocontrolled iodocyclizations mostly substrate-controlled versions using a chiral auxiliary have been successfully investigated. This work reports on stereoselective reagent-controlled iodolactonizations applying a new method using a combination of ICl and a primary amine leading to the highest selectivities known so far.

Journal Article↗
PubMed2002

Reduction of marginal zone B cells in CD22-deficient mice.

CD22 is a B cell-specific member of the immunoglobulin superfamily and binds to sialic acid. CD22 inhibits B cell receptor signaling. Mice deficient for CD22 show a largely normal B cell development. Here, we have performed a detailed analysis of the splenic B cell population and found that the subset of marginal zone (MZ) B cells was selectively reduced in CD22-deficient mice. CD22-deficient mice showed a lack of TNP-ficoll capturing cells in the MZ and a reduced response to TNP-ficoll, particularly when the antigen was applied intravenously. CD22-deficient B cells showed both enhanced motility as well as enhanced chemotaxis to certain chemokines. The altered chemokine responsiveness or the higher signaling capacity of CD22-deficient B cells may lead to the compromised MZ B cell compartment, as both processes have previously been shown to affect MZ composition.

Animals↗
PubMed2002

CD22 regulates early B cell development in BOB.1/OBF.1-deficient mice.

BOB.1/OBF.1 (also called OCA-B), a B lymphocyte-specific transcriptional coactivator, is recruited to octamer-containing promoters by interacting with the Oct-1 or Oct-2 proteins. BOB.1/OBF.1-deficient mice show impaired secondary immunoglobulin isotype secretion and complete absence of germinal centers. Furthermore, numbers of splenic B cells are reduced due to a developmental block at the transitional B cell stage in the bone marrow. We found that surface expression of CD22 is selectively increased on B lineage cells in the bone marrow of BOB.1/OBF.1-deficient mice. CD22 is known as a negative regulator of B cell receptor signaling. We therefore investigated whether defects in B cell development in the BOB.1/OBF.1-deficient mice might be due to CD22 up-regulation. Mice were generated lacking both genes. In BOB.1/OBF.1xCD22 double-deficient mice, numbers of transitional B cells in the bone marrow were normal. Consequently, double-deficient mice also had normal B to T cell ratios in the spleen. We show that BOB.1/OBF.1(-/-) B cells were incapable to induce BCR-triggered Ca(2+) mobilization. This Ca(2+)-signalling defect was restored in BOB.1/OBF.1xCD22 double-deficient B cells. Nevertheless, double-deficient animals were unable to mount humoral immune responses and to form germinal centers. Finally, we demonstrate that CD22(-/-) splenic B cells proliferate independently of BOB.1/OBF.1 upon stimulation with LPS. These studies suggest that the B cell differentiation defect observed in BOB.1/OBF.1(-/-) mice is BCR-signal dependent. However, the impairment in germinal center formation is caused by a different mechanism.

Animals↗
PubMed2002

Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth.

It has been suggested that carcinoembryonic antigen (CEA) enhances metastatic seeding of colon cancer cells due to its homo- and heterophilic binding properties. Our recent finding that endogenous CEA protects colon cancer cells against apoptosis suggests a more complex role of CEA in cancer progression. In this study we compared the in vitro effects of endogenous CEA on tumor cell aggregation and cell cycle regulation of human HT29 colon cancer cells with the corresponding in vivo effects, i.e. tumor cell seeding and formation of metastatic lesions. Stable expression of CEA targeted ribozymes (Rz) under control of a tet-off promoter system allowed regulation of CEA levels on the mRNA and protein level by 50%. Downregulation of CEA levels inhibited tumor cell aggregation by 70%. In accordance with previous studies, reduction of CEA levels increased in vitro the apoptotic rate and reduced colony formation by 30% to 50%. To determine the in vivo effect of CEA-dependent aggregate formation and its growth regulating role under apoptotic stress, HT29 cells with high and low CEA levels, respectively, were injected into nude mice. Immunostaining of lung microsections revealed similar numbers of tumor cells one hour after injection. 24 h later virtually all cells were removed from the lung in both groups. However, after 6 weeks all doxycycline treated mice (Rz off = CEA high) showed 14.5 +/- 4.6 metastatic lung lesions/mouse while 0.2 +/- 0.2 lesions/mouse appeared in the untreated group (Rz on = CEA low) (P < 0.001). Our study demonstrates a multifunctional role of CEA and indicates a prometastatic role of CEA independent of its adhesive function possibly due to its anti-apoptotic function.

Animals↗
PubMed2002

BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment.

Marginal-zone (MZ) B cells represent a first line of defense against particulate blood-borne antigens. Together with the B1 cells, they are responsible for the early response against type II T-independent antigens. The molecular pathways controlling the development of MZ B cells are only poorly understood. We found that these cells are virtually absent in mice deficient in the BOB.1/OBF.1 coactivator. Loss of these B cells was demonstrated by the lack of cells showing the appropriate cell surface phenotype but also by histological analyses and tri-nitro-phenol-Ficoll capturing. The lack of these cells is a B-cell-intrinsic defect, as shown by bone marrow complementation experiments. We also show that the expression of BOB.1/OBF.1 in peripheral B cells is required for the development of MZ B lymphocytes. Our analysis of BOB.1/OBF.1-deficient splenic B cells reveals alterations in cell motility, tumor necrosis factor receptor expression, and B-cell receptor (BCR) signaling. These changes could contribute to the loss of MZ B lymphocytes by altering the maturation of the cells. Interestingly, development of and BCR signaling in B1 B cells are completely normal in BOB.1/OBF.1 mutant mice.

Animals↗