Biomedical subjects

Hartmut Beug

Publications and source records attributed to Hartmut Beug.

At least 37 records · Page 2Linked to original sources

PubMed2005

Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth.

Colorectal carcinoma (CRC) is a major cause of morbidity and mortality in Western countries. It has so far been molecularly defined mainly by alterations of the Wnt pathway. We show here for the first time that aberrant activities of the signal transducer and activator of transcription STAT3 actively contribute to this malignancy and, thus, are a potential therapeutic target for CRC. Constitutive STAT3 activity was found to be abundant in dedifferentiated cancer cells and infiltrating lymphocytes of CRC samples, but not in non-neoplastic colon epithelium. Cell lines derived from malignant colorectal tumors lost persistent STAT3 activity in culture. However, implantation of colon carcinoma cells into nude mice resulted in restoration of STAT3 activity, suggesting a role of an extracellular stimulus within the tumor microenvironment as a trigger for STAT activation. STAT3 activity in CRC cells triggered through interleukin-6 or through a constitutively active STAT3 mutant promoted cancer cell multiplication, whereas STAT3 inhibition through a dominant-negative variant impaired IL-6-driven proliferation. Blockade of STAT3 activation in CRC-derived xenograft tumors slowed down their development, arguing for a contribution of STAT3 to colorectal tumor growth.

Animals↗

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

Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors.

Outgrowth, long-term self-renewal, and terminal maturation of human erythroid progenitors derived from umbilical cord blood in serum-free medium can be modulated by steroid hormones. Homogeneous erythroid cultures, as characterized by flow cytometry and dependence on a specific mixture of physiologic proliferation factors, were obtained within 8 days from a starting population of mature and immature mononuclear cells. Due to previous results in mouse and chicken erythroblasts, the proliferation-promoting effect of glucocorticoids was not unexpected. Surprisingly, however, androgen had a positive effect on the sustained expansion of human female but not male erythroid progenitors. Under optimal conditions, sustained proliferation of erythroid progenitors resulted in a more than 10(9)-fold expansion within 60 days. Terminal erythroid maturation was significantly improved by adding human serum and thyroid hormone (3,5,3'-triiodothyronine [T3]) to the differentiation medium. This resulted in highly synchronous differentiation of the cells toward enucleated erythrocytes within 6 days, accompanied by massive size decrease and hemoglobin accumulation to levels comparable to those in peripheral blood erythrocytes. Thus, obviously, different ligand-activated nuclear hormone receptors massively influence the decision between self-renewal and terminal maturation in the human erythroid compartment.

Androgens↗

PubMed2004

Evidence for a size-sensing mechanism in animal cells.

Continuously proliferating cells exactly double their mass during each cell cycle. Here we have addressed the controversial question of if and how cell size is sensed and regulated. We used erythroblasts that proliferate under the control of a constitutively active oncogene (v-ErbB) or under the control of physiological cytokines (stem cell factor, erythropoietin and v-ErbB inhibitor). The oncogene-driven cells proliferated 1.7 times faster and showed a 1.5-fold increase in cell volume. The two phenotypes could be converted into each other 24 h after altering growth factor signalling. The large cells had a higher rate of protein synthesis, together with a shortened G1 phase. Additional experiments with chicken erythroblasts and mouse fibroblasts, synchronized by centrifugal elutriation, provided further evidence that vertebrate cells can respond to cell size alterations (induced either through different growth factor signalling or DNA synthesis inhibitors) by compensatory shortening of the subsequent G1 phase. Taken together, these data suggest that an active size threshold mechanism exists in G1, which induces adjustment of cell-cycle length in the next cycle, thus ensuring maintenance of a proper balance between growth and proliferation rates in vertebrates.

Animals↗

PubMed2004

Translational control of putative protooncogene Nm23-M2 by cytokines via phosphoinositide 3-kinase signaling.

The expansion and differentiation of hematopoietic progenitors is regulated by cytokine and growth factor signaling. To examine how signal transduction controls the gene expression program required for progenitor expansion, we screened ATLAS filters with polysome-associated mRNA derived from erythroid progenitors stimulated with erythropoietin and/or stem cell factor. The putative proto-oncogene nucleoside diphosphate kinase B (ndpk-B or nm23-M2) was identified as an erythropoietin and stem cell factor target gene. Factor-induced expression of nm23-M2 was regulated specifically at the level of polysome association by a phosphoinositide 3-kinase-dependent mechanism. Identification of the transcription initiation site revealed that nm23-M2 mRNA starts with a terminal oligopyrimidine sequence, which is known to render mRNA translation dependent on mitogenic factors. Recently, the nm23-M2 locus was identified as a common leukemia retrovirus integration site, suggesting that it plays a role in leukemia development. The expression of Nm23 from a retroviral vector in the absence of its 5'-untranslated region caused constitutive polysome association of nm23-M2. Polysome-association and protein expression of endogenous nm23-M2 declined during differentiation of erythroid progenitors, suggesting a role for Nm23-M2 in progenitor expansion. Taken together, nm23-m2 exemplifies that cytokine-dependent control of translation initiation is an important mechanism of gene expression regulation.

5' Untranslated Regions↗

PubMed2004

Control of erythropoiesis by erythropoietin and stem cell factor: a novel role for Bruton's tyrosine kinase.

Erythropoietin (Epo) and stem cell factor (SCF) are essential factors in the control of survival, expansion and differentiation of erythroid progenitors. Upon activation, their receptors, the EpoR and c-Kit, initiate multiple signalling pathways that control many cellular processes. To control erythropoiesis, the strength, duration and specificity of signalling must be tightly controlled. Negative feed-back regulation is extensively studied, but positive feed-forward control is relatively little studied. The cytoplasmic tyrosine kinase Bruton's tyrosine kinase (Btk) was found to be phosphorylated by Jak2 in response to Epo and appeared to be required for fast and efficient phosphorylation of Epo-induced targets including the EpoR itself and downstream targets such as PLCgamma and Stat5. Erythroid progenitors deficient in Btk fail to undergo renewal divisions and differentiate instead at low, physiologic concentrations of Epo and SCF. In addition, Btk is phosphorylated by SCF, which causes association of Btk with TRAIL-receptor1. In absence of Btk, erythroid progenitors are hypersensitive to TRAIL. Thus, Btk modulates signalling in erythroid progenitors to enhance expansion of erythroid progenitors. The complexity of signalling by the EpoR/c-Kit signalosome and its control by Btk is discussed with respect to normal and aberrant erythropoiesis.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2004

Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells.

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors, useful for both basic research and clinical applications. Besides their characterization in colony assays, protocols exist for the cultivation of lymphoid, myeloid, and erythroid cells. With the possible exception of mast cells, however, long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here, we describe for the first time an efficient yet easy strategy to generate mass cultures of pure, immature erythroid progenitors from mouse ES cells (ES-EPs), using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived, adult, definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions, ES-EPs differentiated into mature, enucleated erythrocytes. Importantly, ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition, the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells.

Aging↗

PubMed2004

Enhanced tenascin-C expression and matrix deposition during Ras/TGF-beta-induced progression of mammary tumor cells.

Overexpression of tenascin-C (TN-C) in breast carcinomas has been associated with a migratory or even invasive tumor cell phenotype. The mechanisms regulating expression and matrix deposition of TN-C in normal and cancerous breast tissues are, however, little understood. Here, we demonstrate that mouse mammary epithelial cells (EpH4) transformed by oncogenic Ha-Ras (EpRas) overexpress TN-C, which accumulates in the cytoplasm. When EpRas cells undergo epithelial-mesenchymal transition (EMT) in response to TGFbeta1, they secrete TN-C into the culture medium. In EpRas cells undergoing TGFbeta1-induced EMT in three-dimensional (3D)-collagen gel cultures, TN-C was deposited into an extracellular matrix (ECM) already containing fibronectin and perlecan. Under less physiological 2D plastic cultures, EpRas cells undergoing EMT failed to deposit TN-C into an (apparently incomplete) ECM. Ras-downstream signaling was dissected by pharmacological inhibitors and effector-specific Ras mutants (V12S35, V12C40), specifically inhibiting or activating ERK/MAPK or PI3K signaling, respectively. We showed that TN-C overexpression required a hyperactive ERK/MAPK-signaling pathway, while elevated PI3K signaling did not enhance TN-C expression. Similarly, tumors induced by cells exhibiting hyperactive ERK/MAPK signaling showed expression of TN-C in the tumor cells themselves, while only endothelial cells expressed TN-C in tumors caused by the V12C40 mutant (incapable of EMT in vivo). Taken together, our data indicate that hyperactive ERK/MAPK signaling causes enhanced expression of TN-C, while its secretion is induced by TGFbeta1 and both signals cooperate in TN-C matrix deposition. Importantly, both signals also cooperate to induce EMT in vitro and tumor progression/metastasis in vivo.

Animals↗

PubMed2004

beta-Catenin and TGFbeta signalling cooperate to maintain a mesenchymal phenotype after FosER-induced epithelial to mesenchymal transition.

Several signalling pathways contribute to the regulation of epithelial to mesenchymal transition (EMT), either during developmentally regulated processes or in cancer progression and metastasis. Induction of EMT in fully polarized mouse mammary epithelial cells (EpH4) by an inducible c-fos estrogen receptor (FosER) oncoprotein involves loss of E-cadherin expression, nuclear translocation of beta-catenin, and autocrine production of TGFbeta. Reporter assays demonstrate that both beta-catenin/LEF-TCF- and TGFbeta-Smad-dependent signalling activities are upregulated, probably coregulating mesenchymal-specific gene expression during EMT. Stable expression of E-cadherin in mesenchymal FosER cells decreased beta-catenin activity and reduced cell proliferation. However, these cells still exhibited a defect in epithelial polarization and expressed E-cadherin/beta-catenin complexes in the entire plasma membrane. On the other hand, inhibition of TGFbeta-Smad signalling in mesenchymal FosER cells induced flat, cobblestone-like clusters of cells, which relocalized beta-catenin to the plasma membrane but still lacked detectable E-cadherin. Interestingly, inhibition of TGFbeta signalling in the E-cadherin-expressing mesenchymal FosER cells caused their reversion to a polarized epithelial phenotype, in which E-cadherin, beta-catenin, and ZO-1 were localized at their correct lateral plasma membrane domains. These results demonstrate that loss of E-cadherin can contribute to increased LEF/TCF-beta-catenin signalling, which in turn cooperates with autocrine TGFbeta signalling to maintain an undifferentiated mesenchymal phenotype.

Active Transport, Cell Nucleus↗

PubMed2004

Btk is required for an efficient response to erythropoietin and for SCF-controlled protection against TRAIL in erythroid progenitors.

Regulation of survival, expansion, and differentiation of erythroid progenitors requires the well-controlled activity of signaling pathways induced by erythropoietin (Epo) and stem cell factor (SCF). In addition to qualitative regulation of signaling pathways, quantitative control may be essential to control appropriate cell numbers in peripheral blood. We demonstrate that Bruton's tyrosine kinase (Btk) is able to associate with the Epo receptor (EpoR) and Jak2, and is a substrate of Jak2. Deficiency of Btk results in reduced and delayed phosphorylation of the EpoR, Jak2, and downstream signaling molecules such as Stat5 and PLCgamma1 as well as in decreased responsiveness to Epo. As a result, expansion of erythroid progenitors lacking Btk is impaired at limiting concentrations of Epo and SCF. In addition, we show that SCF induces Btk to interact with TNF-related apoptosis-inducing ligand (TRAIL)-receptor 1 and that lack of Btk results in increased sensitivity to TRAIL-induced apoptosis. Together, our results indicate that Btk is a novel, quantitative regulator of Epo/SCF-dependent expansion and survival in erythropoiesis.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2004

Glucocorticoid receptor function in hepatocytes is essential to promote postnatal body growth.

Mice carrying a hepatocyte-specific inactivation of the glucorticoid receptor (GR) gene show a dramatic reduction in body size. Growth hormone signaling mediated by the Stat5 transcription factors is impaired. We show that Stat5 proteins physically interact with GR and GR is present in vivo on Stat5-dependent IGF-I and ALS regulatory regions. Interestingly, mice with a DNA-binding-deficient GR but an unaltered ability to interact with STAT5 (GR(dim/dim)) have a normal body size and normal levels of Stat5-dependent mRNAs. These findings strongly support the model in which GR acts as a coactivator for Stat5-dependent transcription upon GH stimulation and reveal an essential role of hepatic GR in the control of body growth.

Animals↗

PubMed2004

FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1.

Erythropoiesis requires tight control of expansion, maturation, and survival of erythroid progenitors. Because activation of phosphatidylinositol-3-kinase (PI3K) is required for erythropoietin/stem cell factor-induced expansion of erythroid progenitors, we examined the role of the PI3K-controlled Forkhead box, class O (FoxO) subfamily of Forkhead transcription factors. FoxO3a expression and nuclear accumulation increased during erythroid differentiation, whereas untimely induction of FoxO3a activity accelerated differentiation of erythroid progenitors to erythrocytes. We identified B cell translocation gene 1 (BTG1)/antiproliferative protein 2 as a FoxO3a target gene in erythroid progenitors. Promoter studies indicated BTG1 as a direct target of FoxO3a. Expression of BTG1 in primary mouse bone marrow cells blocked the outgrowth of erythroid colonies, which required a domain of BTG1 that binds protein arginine methyl transferase 1. During erythroid differentiation, increased arginine methylation coincided with BTG1 expression. Concordantly, inhibition of methyl transferase activity blocked erythroid maturation without affecting expansion of progenitor cells. We propose FoxO3a-controlled expression of BTG1 and subsequent regulation of protein arginine methyl transferase activity as a novel mechanism controlling erythroid expansion and differentiation.

3T3 Cells↗

PubMed2004

Immortalized p19ARF null hepatocytes restore liver injury and generate hepatic progenitors after transplantation.

Primary hepatocytes are blocked in mitotic activity and well-defined culture conditions only allow the limited expansion of these cells. Various genetic modifications have therefore been employed to establish immortalized hepatic cell lines, but, unfortunately, proper hepatocyte cultures conducting a faithful hepatic gene expression program and lacking malignancy are hardly available. Here we report the immortalization of primary hepatocytes isolated from p19(ARF) null mice using the rationale that loss of p19(ARF) lowers growth-suppressive functions of p53 and bypasses cellular senescence without losing genetic stability. The established hepatocytes, called MIM, express liver-specific markers, show a nontumorigenic phenotype, and competence to undergo Fas-mediated apoptosis. Intrasplenic transplantation of GFP-expressing parental MIM cells into Fas-injured livers of SCID mice revealed liver-reconstituting activity. In the regenerated liver, MIM cells localized in small-sized clusters and showed presence in structures comparable to canals of Hering, the site of oval cells. Transplantation of MIM-Bcl-X(L) cells, which are protected against apoptosis, and successive Fas-induced liver damage, enhanced donor-derived liver repopulation by showing differentiation into cholangiocytes and cells expressing markers characteristic of both fetal hepatocytes and oval cells. In conclusion, these data indicate that long-term cultivated p19(ARF) null hepatocytes are capable of generating hepatic progenitor cells during liver restoration, and thus represent a highly valuable tool to study the differentiation repertoire of hepatocytes.

Animals↗

PubMed2004

Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis.

Carcinomas arising from epithelial cells represent the most prevalent malignancies in humans, and metastasis is the major cause for the death of carcinoma patients. The breakdown of epithelial cell homeostasis leading to aggressive cancer progression has been correlated with the loss of epithelial characteristics and the acquisition of a migratory phenotype. This phenomenon, referred to as epithelial to mesenchymal transition (EMT), is considered as a crucial event in late stage tumorigenesis. Here we summarize the multitude of EMT models derived from different tissues, and review the diversity of molecular mechanisms contributing to the plasticity of epithelial cells. In particular, the synergism between activation of Ras, provided by the aberrant stimulation of receptor tyrosine kinases, and transforming growth factor (TGF)-beta signaling plays a pivotal role in inducing EMT of various epithelial cell types. Cytokines such as TGF-beta and extracellular matrix molecules are thought to fundamentally contribute to the microenvironmental interaction between stromal and malignant cells, and provide the basis for a broad repertoire of epithelial differentiation. Investigations of EMT tumor models, which represent in vitro correlates to local invasion and metastasis in vivo, facilitate the identification of diagnostic markers for a more accurate and faithful clinical and pathological assessment of epithelial tumors. In addition, the analysis of molecular mechanisms involved in EMT might yield novel therapeutic targets for the specific treatment of aggressive carcinomas.

Animals↗

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↗

PubMed2004

The Btk inhibitor LFM-A13 is a potent inhibitor of Jak2 kinase activity.

LFM-A13, or alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide, was shown to inhibit Bruton's tyrosine kinase (Btk). Here we show that LFM-A13 efficiently inhibits erythropoietin (Epo)-induced phosphorylation of the erythropoietin receptor, Janus kinase 2 (Jak2) and downstream signalling molecules. However, the tyrosine kinase activity of immunoprecipitated or in vitro translated Btk and Jak2 was equally inhibited by LFM-A13 in in vitro kinase assays. Finally, Epo-induced signal transduction was also inhibited in cells lacking Btk. Taken together, we conclude that LFM-A13 is a potent inhibitor of Jak2 and cannot be used as a specific tyrosine kinase inhibitor to study the role of Btk in Jak2-dependent cytokine signalling.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2003

The MEK-1/ERKs signalling pathway is differentially involved in the self-renewal of early and late avian erythroid progenitor cells.

Making decisions between self-renewal and differentiation is a central ability of stem cells. Elucidation of molecular networks governing this decision is therefore of prime importance. A model of choice to explore this question is represented by chicken erythroid progenitors, in which self-renewal versus differentiation as well as progenitor maturation are regulated by external factor combinations. We used this system to study whether similar or different signalling pathways were involved in the self-renewal of early, immature or more mature erythroid progenitors. We show that a transforming growth factor (TGF)-alpha-activated Ras/MEK-1/ERK1/2 pathway is strictly required for immature self-renewing cells but becomes fully dispensable when those cells are induced to differentiate. Consequently, pharmacological inhibition of this pathway led to spontaneous differentiation, only dependent on the presence of survival signals. Conversely, ectopic expression of a constitutive form of MEK-1 stimulates renewal and arrests differentiation process. Finally, we demonstrate that the ERK/MAPK signalling pathway is required in early but not in late primary erythroid progenitors, which can be turned into each other by different growth factor combinations specifically driving their renewal. To the best of our knowledge, this is the first description of a central role of ERK/MAPK signalling in regulating progenitor plasticity in the same cell type under different environmental conditions.

Animals↗

PubMed2003

Expression profiling of epithelial plasticity in tumor progression.

Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and trans-differentiation to mesenchymal-like cells with invasive behavior.

Animals↗