Peppelenbosch, M.P.

Fecal microbiota transfer may increase irritable bowel syndrome and inflammatory bowel diseases-associated bacteria (Article)

2013-04-01T00:00:00Z

Identification of lineage-uncommitted, long-lived, label-retaining cells in healthy human esophagus and stomach, and in metaplastic esophagus (Article)

2013-04-01T00:00:00Z

Background & Aims: The existence of slowly cycling, adult stem cells has been challenged by the identification of actively cycling cells. We investigated the existence of uncommitted, slowly cycling cells by tracking 5-iodo-2'-deoxyuridine (IdU) label-retaining cells (LRCs) in normal esophagus, Barrett's esophagus (BE), esophageal dysplasia, adenocarcinoma, and healthy stomach tissues from patients. Methods: Four patients (3 undergoing esophagectomy, 1 undergoing esophageal endoscopic mucosal resection for dysplasia and an esophagectomy for esophageal adenocarcinoma) received intravenous infusion of IdU (200 mg/m2 body surface area; maximum dose, 400 mg) over a 30-minute period; the IdU had a circulation half-life of 8 hours. Tissues were collected at 7, 11, 29, and 67 days after infusion, from regions of healthy esophagus, BE, dysplasia, adenocarcinoma, and healthy stomach; they were analyzed by in situ hybridization, flow cytometry, and immunohistochemical analyses. Results: No LRCs were found in dysplasias or adenocarcinomas, but there were significant numbers of LRCs in the base of glands from BE tissue, in the papillae of the basal layer of the esophageal squamous epithelium, and in the neck/isthmus region of healthy stomach. These cells cycled slowly because IdU was retained for at least 67 days and co-labeling with Ki-67 was infrequent. In glands from BE tissues, most cells did not express defensin-5, Muc-2, or chromogranin A, indicating that they were not lineage committed. Some cells labeled for endocrine markers and IdU at 67 days; these cells represented a small population (<0.1%) of epithelial cells at this time point. The epithelial turnover time of the healthy esophageal mucosa was approximately 11 days (twice that of the intestine). Conclusions: LRCs of human esophagus and stomach have many features of stem cells (long lived, slow cycling, uncommitted, and multipotent), and can be found in a recognized stem cell niche. Further analyses of these cells, in healthy and metaplastic epithelia, is required. © 2013 AGA Institute.

Hedgehog signalling as an antagonist of ageing and its associated diseases (Article)

2012-10-01T00:00:00Z

Hedgehog is an important morphogenic signal that directs pattern formation during embryogenesis, but its activity also remains present through adult life. It is now becoming increasingly clear that during the reproductive phase of life and beyond it continues to direct cell renewal (which is essential to combat the chronic environmental stress to which the body is constantly exposed) and counteracts vascular, osteolytic and sometimes oncological insults to the body. Conversely, down-regulation of hedgehog signalling is associated with ageing-related diseases such as type 2 diabetes, neurodegeneration, atherosclerosis and osteoporosis. Hence, in this essay we argue that hedgehog signalling is not only important at the start of life, but also constitutes an important anti-geriatric influence, and that enhanced understanding of its properties may contribute to developing rational strategies for healthy ageing and prevention of ageing-related diseases.

Knocking Down Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) Reverts Chemoresistance through Inactivation of Src and Bcr-Abl Proteins (Article)

2012-09-05T00:00:00Z

The development of multidrug resistance (MDR) limits the efficacy of continuous chemotherapeutic treatment in chronic myelogenous leukemia (CML). Low molecular weight protein tyrosine phosphatase (LMW-PTP) is up-regulated in several cancers and has been associated to poor prognosis. This prompted us to investigate the involvement of LMW-PTP in MDR. In this study, we investigated the role of LMW-PTP in a chemoresistant CML cell line, Lucena-1. Our results showed that LMW-PTP is highly expressed and 7-fold more active in Lucena-1 cells compared to K562 cells, the non-resistant cell line. Knocking down LMW-PTP in Lucena-1 cells reverted chemoresistance to vincristine and imatinib mesylate, followed by a decrease of Src and Bcr-Abl phosphorylation at the activating sites, inactivating both kinases. On the other hand, overexpression of LMW-PTP in K562 cells led to chemoresistance to vincristine. Our findings describe, for the first time, that LMW-PTP cooperates with MDR phenotype, at least in part, through maintaining Src and Bcr-Abl kinases in more active statuses. These findings suggest that inhibition of LMW-PTP may be a useful strategy for the development of therapies for multidrug resistant CML.

Targeting Hedgehog signaling and understanding refractory response to treatment with Hedgehog pathway inhibitors (Article)

2012-08-01T00:00:00Z

Hedgehog (Hh) signaling is a principal component of the morphogenetic code best known to direct pattern formation during embryogenesis. The Hh pathway remains active in adulthood however where it guides tissue regeneration and remodeling and Hh production in the niche plays an important role in maintaining stem cell compartment size. Deregulated Hh signaling activity is associated, depending on the context, with both cancer initiation and progression. Interestingly, the Hh pathway is remarkably druggable, raising hopes that inhibition of the pathway could support anticancer therapy. Indeed, a large body of preclinical data supports such an action, but promising clinical data are still limited to basal cell carcinoma (BSC) and medulloblastoma. Nevertheless cancer resistance against Hh targeting has already emerged as a major problem. Here we shall review the current situation with respect to targeting the Hh pathway in cancer in general and in chemotolerance in particular with a focus on the problems associated with the emergence of tumors resistant to treatment with inhibitors targeting the Hh receptor Smoothened (SMO).

Pancreatic cancer: Promise for personalised medicine? (Article)

2012-05-01T00:00:00Z

Pancreatic cancer has an infaust prognosis and is the fourth commonest cause of cancer related death in men. Design of rational treatment has been hampered by lack of insight into the pathogenesis of the disease. Recently more insight has been gained into a number of crucial aspects of pancreatic carcinogenesis, in particular the cell types that can give rise to oncological transformation in the pancreas, different modes of interaction between transformed pancreatic cells and the stroma that fosters further disease progression, the need of the pancreatic tumour cells to overcome the pressure of immune surveillance and the various changes in intercellular biochemistry that tumour cells employ to both sustain chemoresistance and metastasis. Although still largely incomplete, this new knowledge opens novel avenues on more successful treatment of the disease through personalised medicine.

Absence of ABCG2-mediated mucosal detoxification in patients with active inflammatory bowel disease is due to impeded protein folding (Article)

2012-01-01T00:00:00Z

Xenotoxic damage in inflammatory diseases, including IBD (inflammatory bowel disease), is compounded by reduced activity of the xenobiotic transporter ABCG2 (ATP-binding-cassette G2) during the inflammatory state. An association between the activation of the unfolded protein response pathway and inflammation prompted us to investigate the possibility that reduced ABCG2 activity is causally linked to this response. To this end, we correlated expression of ABCG2 and the unfolded protein response marker GRP78 (glucose-regulated protein of 78 kDa) in colon biopsies from healthy individuals (n=9) and patients with inactive (n=67) or active (n=55) IBD, ischaemic colitis (n=10) or infectious colitis (n=14). In addition, tissue specimens throughout the small bowel from healthy individuals (n=27) and from patients with inactive (n=9) or active (n=25) Crohn's disease were co-stained for ABCG2 and GRP78. In all biopsies from patients with active inflammation, irrespective of the underlying disease, an absolute negative correlation was observed between epithelial ABCG2 expression and GRP78 expression, suggesting that inflammation-dependent activation of the unfolded protein response is responsible for suppression of ABCG2 function. The link between the unfolded protein response and functional ABCG2 expression was further corroborated by live imaging of ABCG2-expressing cells, which showed that various inflammatory mediators, including nitric oxide, activate the unfolded protein response and concomitantly reduce plasma membrane localization as well as transport function of ABCG2. Thus a novel mechanism for explaining xenobiotic stress during inflammation emerges in which intestinal inflammation activates the unfolded protein response, in turn abrogating defences against xenobiotic challenge by impairing ABCG2 expression and function. © The Authors Journal compilation

Genetically modified lactococcus lactis for delivery of human interleukin-10 to dendritic cells (Article)

2012-01-01T00:00:00Z

Interleukin-10 (IL-10) plays an indispensable role in mucosal tolerance by programming dendritic cells (DCs) to induce suppressor Th-cells. We have tested the modulating effect of L. lactis secreting human IL-10 (L.lacti s IL-10) on DC function in vitro. Monocyte-derived DC incubated with L.lacti s IL-10 induced effector Th-cells that markedly suppressed the proliferation of allogenic Th-cells as compared to L. lactis. This suppressive effect was only seen when DC showed increased CD83 and CD86 expression. Furthermore, enhanced production of IL-10 was measured in both L.lacti s IL-10 -derived DC and Th-cells compared to L. lactis-derived DC and Th-cells. Neutralizing IL-10 during DC-Th-cell interaction and coculturing L.lacti s IL-10 -derived suppressor Th-cells with allogenic Th-cells in a transwell system prevented the induction of suppressor Th-cells. Only 130pg/mL of bacterial-derived IL-10 and 40 times more exogenously added recombinant human IL-10 were needed during DC priming for the generation of suppressor Th-cells. The spatially restricted delivery of IL-10 by food-grade bacteria is a promising strategy to induce suppressor Th-cells in vivo and to treat inflammatory diseases.

A phospholipidomic analysis of all defined human plasma lipoproteins (Article)

2011-12-01T00:00:00Z

Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are performed. LC-ESI/MS, LC-ESI-MS/MS and High Performance Thin Layer Chromatography (HPTLC) analysis of different lipoprotein fractions collected from pooled plasma revealed the presence of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyeline (SM) only on lipoproteins and phosphatidylcholine (PC), Lyso-PC on both lipoproteins and plasma lipoprotein free fraction (PLFF). Cardiolipin, phosphatidylglycerol (PG) and Phosphatidylserine (PS) were observed neither in the lipoprotein fractions nor in PLFF. All three approaches led to the same results regarding phospholipids occurrence in plasma lipoproteins and PLFF. A high abundancy of PE and SM was observed in VLDL and LDL fractions respectively. This study provides for the first time the knowledge about the phospholipid composition of all defined plasma lipoproteins.

Statins augment the chemosensitivity of colorectal cancer cells inducing epigenetic reprogramming and reducing colorectal cancer cell 'stemness' via the bone morphogenetic protein pathway (Article)

2011-11-01T00:00:00Z

Background: Promoter hypermethylation is an important and potentially reversible mechanism of tumour suppressor gene silencing in cancer. Compounds that demethylate tumour suppressor genes and induce differentiation of cancer cells, but do not have toxic side effects, would represent an exciting option in cancer therapy. Statins are cholesterol-lowering drugs with an excellent safety profile and associated with a reduced incidence of various cancers including colorectal cancer (CRC). The authors have previously shown that statins act by activating tumour suppressive bone morphogenetic protein (BMP) signalling in CRC, increasing expression of BMP2. BMP2 is silenced by hypermethylation in gastric cancer. Aim: To investigate whether BMP2 is methylated in CRC, whether statins can reverse this, and what implications this has for the use of statins in CRC. Methods: Methylation-specific PCR, bisulphite sequencing, immunoblotting, reverse transcription PCR, quantitative PCR, fluorescence-activated cell sorting analysis, an in vitro DNA methyltransferase (DNMT) assay, and cell viability studies were performed on CRC cells. The effect of statins was confirmed in a xenograft mouse model. Results: BMP2 is silenced by promoter hypermethylation in cell lines with the hypermethylator phenotype and in primary tumours. Treatment with lovastatin downregulates DNMT activity, leading to BMP2 promoter demethylation and to upregulation of expression of BMP2 as well as other genes methylated in CRC. Statins alter gene expression, indicating a shift from a stem-like state to a more differentiated state, thereby sensitising cells to the effects of 5-fluorouracil. In a xenograft mouse model, simvastatin treatment induces BMP2 expression, leading to differentiation and reduced proliferation of CRC cells. Conclusions: Statins act as DNMT inhibitors, demethylating the BMP2 promoter, activating BMP signalling, inducing differentiation of CRC cells, and reducing 'stemness'. This study indicates that statins may be able to be used as differentiating agents in combined or adjuvant therapy in CRC with the CpG island methylator phenotype.

Mapping of interactions between human macrophages and Staphylococcus aureus reveals an involvement of MAP kinase signaling in the host defense (Article)

2011-09-02T00:00:00Z

Staphylococcus aureus is a dangerous opportunistic human pathogen that causes serious invasive diseases when it reaches the bloodstream. Recent studies have shown that S. aureus is highly resistant to killing by professional phagocytes and that such cells even provide a favorable environment for intracellular survival of S. aureus. Importantly, the reciprocal interactions between phagocytes and S. aureus have remained largely elusive. Here we have employed kinase profiling to define the nature and time resolution of the human THP-1 macrophage response toward S. aureus and proteomics to identify the response of S. aureus toward macrophages. The results of these studies reveal major macrophage signaling pathways triggered by S. aureus and proteomic signatures of the responses of S. aureus to macrophages. We also identify human proteins bound to S. aureus that have potential roles in bacterial killing and internalization. Most noticeably, our observations challenge the classical concept that macrophage responses are mainly mediated through Toll-like receptor 2 and NF-kB signaling and highlight the important role of the stress-activated MAP kinase signaling in orchestrating the host defense.

Major remodelling of the murine stem cell kinome following differentiation in the hematopoietic compartment (Article)

2011-08-05T00:00:00Z

The changes in signal transduction associated with the acquisition of specific cell fates remain poorly understood. We performed massive parallel assessment of kinase signatures of the radiations of the hematopoietic system, including long-term repopulating hematopoietic stem cells (LT-HSC), short-term repopulating HSC (ST-HSC), immature natural killer (iNK) cells, NK cells, B cells, T cells, and myeloid cells. The LT-HSC kinome is characterized by noncanonical Wnt, Ca2+and classical protein kinase C (PKC)-driven signaling, which is lost upon the transition to ST-HSC, whose kinome signature prominently features receptor tyrosine kinase (RTK) activation of the Ras/MAPK signaling cassette. Further differentiation to iNK maintains signaling through this cassette but simultaneously leads to activation of a PI3K/PKB/Rac signaling, which becomes the dominant trait in the kinase signature following full differentiation toward NK cells. Differentiation along the myeloid and B cell lineages is accompanied by hyperactivation of both the Ras/MAPK and PI3K/PKB/Rac signaling cassette. T cells, however, deactivate signaling and only display residual G protein-coupled pathways. Thus, differentiation along the hematopoietic lineage is associated with major remodelling of cellular kinase signature.

Impeded protein folding and function in active inflammatory bowel disease (Article)

2011-08-01T00:00:00Z

The intestinal tract is covered by a total of 300 square metres of IECs (intestinal epithelial cells) that covers the entire intestinal mucosa. For protection against luminal xenobiotics, pathogens and commensal microbes, these IECs are equipped with membrane-bound transporters as well as the ability to secrete specific protective proteins. In patients with active IBD (inflammatory bowel disease), the expression of these proteins, e.g. ABC (ATP-binding cassette) transporters such as ABCG2 (ABC transporter G2) and defensins, is decreased, thereby limiting the protection against various luminal threats. Correct ER (endoplasmic reticulum)-dependent protein folding is essential for the localization and function of secreted and membrane-bound proteins. Inflammatory triggers, such as cytokines and nitric oxide, can impede protein folding, which causes the accumulation of unfolded proteins inside the ER. As a result, the unfolded protein response is activated which can lead to a cellular process named ER stress. The protein folding impairment affects the function and localization of several proteins, including those involved in protection against xenobiotics. In the present review, we discuss the possible inflammatory pathways affecting protein folding and eventually leading to IEC malfunction in patients with active IBD. ©The Authors Journal compilation

Vascular endothelial growth factor receptor 2 (VEGFR-2) signalling activity in paediatric pilocytic astrocytoma is restricted to tumour endothelial cells (Article)

2011-08-01T00:00:00Z

Aims: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed. Methods: VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels. Results: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3 was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively. Conclusions: Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours. © 2011 The Authors. Neuropathology and Applied Neurobiology

Widespread deregulation of phosphorylation-based signaling pathways in multiple myeloma cells:Opportunities for therapeutic intervention (Article)

2011-07-01T00:00:00Z

Multiple myeloma (MM) is a neoplasm of plasma cell origin that is largely confined to the bone marrow (BM). Chromosomal translocations and other genetic events are known to contribute to deregulation of signaling pathways that lead to transformation of plasma cells and progression to malignancy. However, the tumor stroma may also provide trophic support and enhance resistance to therapy. Phosphorylation of proteins on tyrosine, serine and threonine residues plays a pivotal role in cell growth and survival. Therefore, knowing the status of phosphorylation-based signaling pathways in cells may provide key insights into how cell growth and survival is promoted in tumor cells. To provide a more comprehensive molecular analysis of signaling disruptions in MM, we conducted a kinome profile comparison of normal plasma cells and MM plasma cells as well as their surrounding cells from normal BM and diseased BM. Integrated pathway analysis of the profiles obtained reveals deregulation of multiple signaling pathways in MM cells but also in surrounding bone marrow blood cells compared to their normal counterparts. The deregulated kinase activities identified herein, which include the mTOR (mammalian target of rapamycin)/p70S6K and ERK1/2 (extracellular signal-regulated kinases 1 and 2) pathways, are potential novel molecular targets in this lethal disease.

The long and winding road to rational treatment of cancer associated with LKB1/AMPK/TSC/mTORC1 signaling (Article)

2011-05-19T00:00:00Z

The liver kinase B1 (LKB1)/adenosine mono-phosphate-activated protein kinase (AMPK)/tuberous sclerosis complex (TSC)/mammalian target of rapamycin (mTOR) complex (mTORC1) cassette constitutes a canonical signaling pathway that integrates information on the metabolic and nutrient status and translates this into regulation of cell growth. Alterations in this pathway are associated with a wide variety of cancers and hereditary hamartoma syndromes, diseases in which hyperactivation of mTORC1 has been described. Specific mTORC1 inhibitors have been developed for clinical use, and these drugs have been anticipated to provide efficient treatment for these diseases. In the present review, we provide an overview of the metabolic LKB1/AMPK/TSC/mTORC1 pathway, describe how its aberrant signaling associates with cancer development, and indicate the difficulties encountered when biochemical data are extrapolated to provide avenues for rational treatment of disease when targeting this signaling pathway. A careful examination of preclinical and clinical studies performed with rapamycin or derivatives thereof shows that although results are encouraging, we are only half way in the long and winding road to design rationale treatment targeted at the LKB1/AMPK/TSC/mTORC1 pathway. Inherited cancer syndromes associated with this pathway such as the Peutz-Jeghers syndrome and TSC, provide perfect models to study the relationship between genetics and disease phenotype, and to delineate the complexities that underlie translation of biochemical and genetical information to clinical management, and thus provide important clues for devising novel rational medicine for cancerous diseases in general.

High intra-uterine exposure to infliximab following maternal anti-TNF treatment during pregnancy (Article)

2011-05-01T00:00:00Z

Aliment Pharmacol Ther 2011; 33: 1053-1058 Summary Background Typically, inflammatory bowel disease (IBD) patients are in their reproductive years, raising questions about safely using antitumour necrosis factor antibodies like infliximab (IFX) during pregnancy. IgG antibodies naturally cross the placenta, especially during the last trimester. To prevent foetal intra-uterine exposure, stopping IFX treatment at gestational week 30 is recommended. However, whether this limits intra-uterine and early postnatal IFX exposure is unestablished. Aim To determine the intra-uterine exposure to IFX following maternal treatment with IFX. Methods Four pregnant IBD patients intentionally continued IFX during pregnancy. IFX levels were assessed in newborns' cord blood and the mothers' peripheral blood at delivery. The children's development during the first 3-6 months, infections, vaccine reactions and antibody responses to vaccinations against Haemophilus influenzae type b and Pneumococcus were assessed. Results The patients stopped IFX therapy at gestational week 21, 26, 26 and 30, respectively. In three infants, therapeutic IFX levels were present in cord blood at levels of 5.5-13.7 Îg/mL and were two- to three-fold higher than in the peripheral blood of their mothers. During the 3- to 6-month follow-up, the children developed normally without signs of infections or allergic reactions, and had normal antibody titres after routine childhood vaccinations. Conclusion The use of IFX until gestational week 30 leads to foetal intra-uterine exposure to IFX at levels that exceed those in the mothers' peripheral blood. Although no short-term complications were detected, the high IFX levels observed in newborns raise concerns about unknown effects of IFX on the developing immune system.

Van anus tot kanis (Inaugural Lecture)

2011-04-29T00:00:00Z

Rede, In verkorte vorm uitgesproken ter gelegenheid van het aanvaarden van het ambt van bijzonder hoogleraar met als leeropdracht Experimentele Gastroenterologie aan het Erasmus MC, faculteit van de Erasmus Universiteit Rotterdam op 29 april 2011

Kinase activity profiling of pneumococcal pneumonia (Article)

2011-04-11T00:00:00Z

Background: Pneumonia represents a major health burden. Previous work demonstrated that although the induction of inflammation is important for adequate host defense against pneumonia, an inability to regulate the host's inflammatory response within the lung later during infection can be detrimental. Intracellular signaling pathways commonly rely on activation of kinases, and kinases play an essential role in the regulation of the inflammatory response of immune cells. Methodology/Principal Findings: Pneumonia was induced in mice via intranasal instillation of Streptococcus (S.) pneumoniae. Kinomics peptide arrays, exhibiting 1024 specific consensus sequences for protein kinases, were used to produce a systems biology analysis of cellular kinase activity during the course of pneumonia. Several differences in kinase activity revealed by the arrays were validated in lung homogenates of individual mice using western blot. We identified cascades of activated kinases showing that chemotoxic stress and a T helper 1 response were induced during the course of pneumococcal pneumonia. In addition, our data point to a reduction in WNT activity in lungs of S. pneumoniae infected mice. Moreover, this study demonstrated a reduction in overall CDK activity implying alterations in cell cycle biology. Conclusions/Significance: This study utilizes systems biology to provide insight into the signaling events occurring during lung infection with the common cause of community acquired pneumonia, and may assist in identifying novel therapeutic targets in the treatment of bacterial pneumonia.

IDH1 R132H decreases proliferation of glioma cell lines in vitro and in vivo (Article)

2011-03-01T00:00:00Z

Objective: A high percentage of grade II and III gliomas have mutations in the gene encoding isocitrate dehydrogenase (IDH1). This mutation is always a heterozygous point mutation that affects the amino acid arginine at position 132 and results in loss of its native enzymatic activity and gain of alternative enzymatic activity (producing D-2-hydroxyglutarate). The objective of this study was to investigate the cellular effects of R132H mutations in IDH1. Methods: Functional consequences of IDH1R132Hmutations were examined among others using fluorescence-activated cell sorting, kinome and expression arrays, biochemical assays, and intracranial injections on 3 different (glioma) cell lines with stable overexpression of IDH1R132H. Results: IDH1R132Hoverexpression in established glioma cell lines in vitro resulted in a marked decrease in proliferation, decreased Akt phosphorylation, altered morphology, and a more contact-dependent cell migration. The reduced proliferation is related to accumulation of D-2-hydroxyglutarate that is produced by IDH1R132H. Mice injected with IDH1R132HU87 cells have prolonged survival compared to mice injected with IDH1wtor green fluorescent protein-expressing U87 cells. Interpretation: Our results demonstrate that IDH1R132Hdominantly reduces aggressiveness of established glioma cell lines in vitro and in vivo. In addition, the IDH1R132H-IDH1wtheterodimer has higher enzymatic activity than the IDH1R132H-IDH1R132Hhomodimer. Our observations in model systems of glioma might lead to a better understanding of the biology of IDH1 mutant gliomas, which are typically low grade and often slow growing. Copyright

Essential role for the d-asb11 cul5 box domain for proper notch signaling and neural cell fate decisions in Vivo (Article)

2010-12-03T00:00:00Z

ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11Cul). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11Cul fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11Cul mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.

Hedgehog signaling maintains chemoresistance in myeloid leukemic cells (Article)

2010-12-02T00:00:00Z

The development of resistance against chemotherapy remains one of the major challenges in the clinical management of leukemia. There is still limited insight into the molecular mechanisms that maintain the chemotherapy-resistant phenotype, despite the obvious clinical relevance that such knowledge would have. In this study, we show that the chemotherapy-resistant phenotype of myeloid leukemia cells correlates with activation of the Hedgehog (Hh) pathway, whereas in chemosensitive cells, such activation is less pronounced. Importantly, the overexpression of Hh pathway components induces chemoprotection and inhibition of the pathway reverts chemoresistance of Lucena-1 cells, apparently by interfering with P-glycoprotein-dependent drug resistance. Our data thus identify the Hh pathway as an essential component of multidrug resistance (MDR) myeloid leukemia and suggest that targeting the Hh pathway might be an interesting therapeutic avenue for overcoming MDR resistance in myeloid leukemia.

Biological effects of propionic acid in humans; metabolism, potential applications and underlying mechanisms (Article)

2010-11-01T00:00:00Z

Undigested food is fermented in the colon by the microbiota and gives rise to various microbial metabolites. Short-chain fatty acids (SCFA), including acetic, propionic and butyric acid, are the principal metabolites produced. However, most of the literature focuses on butyrate and to a lesser extent on acetate; consequently, potential effects of propionic acid (PA) on physiology and pathology have long been underestimated. It has been demonstrated that PA lowers fatty acids content in liver and plasma, reduces food intake, exerts immunosuppressive actions and probably improves tissue insulin sensitivity. Thus increased production of PA by the microbiota might be considered beneficial in the context of prevention of obesity and diabetes type 2. The molecular mechanisms by which PA may exert this plethora of physiological effects are slowly being elucidated and include intestinal cyclooxygenase enzyme, the G-protein coupled receptors 41 and 43 and activation of the peroxisome proliferator-activated receptor γ, in turn inhibiting the sentinel transcription factor NF-κB and thus increasing the threshold for inflammatory responses in general. Taken together, PA emerges as a major mediator in the link between nutrition, gut microbiota and physiology.

Role of defective autophagia and the intestinal flora in Crohn disease (Article)

2010-10-01T00:00:00Z

The precise mechanisms underlying the development of Crohn disease (CD) remain controversial, but sufficient data have been collected to suggest that an uncontrolled immune response within the intestinal mucosa leads to inflammation in a genetically susceptible host. Although lack of mucosal regulatory T cells causes colitis in humans and experimental rodents, patients with CD have more rather than less regulatory activity in the intestine, apparently excluding defects in tolerance as the cause of CD. Genome-wide association studies have identified many gene variants that confer susceptibility and which seem associated to diminished functioning of especially innate immunity. In apparent agreement, CD patients are impaired with respect to innate immune responses and controlling bacterial flora in the intestine. Furthermore, severe genetic deficiencies in innate immunity, like e.g., lack of NADP oxidase activity or diminished function of the Wiskott Aldrich syndrome protein are associated with colitis in mice and men, and are often mistakenly diagnosed as CD. Thus we favor the view that the primary defect in CD is a lack in innate immunity, causing second tier immunological defenses to combat otherwise easily controlled bacterial breaches of the mucosal barrier.

Reversible phosphorylation in haematological malignancies: Potential role for protein tyrosine phosphatases in treatment? (Article)

2010-08-09T00:00:00Z

Most aspects of leukocyte physiology are under the control of reversible tyrosine phosphorylation. It is clear that excessive phosphorylation of signal transduction elements is a pivotal element of many different pathologies including haematological malignancies and accordingly, strategies that target such phosphorylation have clinically been proven highly successful for treatment of multiple types of leukemias and lymphomas. Cellular phosphorylation status is dependent on the resultant activity of kinases and phosphatases. The cell biology of the former is now well understood; for most cellular phosphoproteins we now know the kinases responsible for their phosphorylation and we understand the principles of their aberrant activity in disease. With respect to phosphatases, however, our knowledge is much patchier. Although the sequences of whole genomes allow us to identify phosphatases using in silico methodology, whereas transcription profiling allows us to understand how phosphatase expression is regulated during disease, most functional questions as to substrate specificity, dynamic regulation of phosphatase activity and potential for therapeutic intervention are still to a large degree open. Nevertheless, recent studies have allowed us to make meaningful statements on the role of tyrosine phosphatase activity in the three major signaling pathways that are commonly affected in leukemias, i.e. the Ras-Raf-ERK1/2, the Jak-STAT and the PI3K-PKB-mTOR pathways. Lessons learned from these pathways may well be applicable elsewhere in leukocyte biology as well.

Anchoring skeletal muscle development and disease: The role of ankyrin repeat domain containing proteins in muscle physiology (Article)

2010-08-01T00:00:00Z

The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies.

Anchoring skeletal muscle development and disease: The role of ankyrin repeat domain containing proteins in muscle physiology (Article)

2010-08-01T00:00:00Z

Bone marrow stromal cell interaction reduces Syndecan-1 expression and induces kinomic changes in myeloma cells (Article)

2010-07-01T00:00:00Z

CD138 (Syndecan 1) is a heparan sulfate proteoglycan that concentrates heparan sulfate-binding growth factors on the surface of normal and malignant plasma cells (multiple myeloma, MMC). Recent studies have shown the presence of a CD138-negative fraction of MMC within myelomatous bone marrow (BM). We employed kinome array technology to characterize this fraction at a molecular level, using a myeloma cell line model. Compared to CD138-positive cells, CD138-negative MMC showed (i) a reduced activity of kinases involved in cell cycle progression, in agreement with a decreased labeling index and (ii) reduced Rho signaling to F-actin. Interestingly, CD138 mRNA and protein expression was reduced upon interaction of MM cells with stromal cell lines and primary mesenchymal cultures, which was accompanied by the acquisition of an increased Bcl6/Blimp1 ratio. Co-culture induced an increased activity of kinases involved in adhesion and a decreased S-phase transition in both CD138-positive and -negative fractions. In addition, CD138-negative MMC demonstrated an increased STAT3 and ERK1/2 activation compared to CD138+ MMC, in agreement with a lower sensitivity to compound exposure. The presence of a less mature, more resistant CD138-negative myeloma cell fraction within bone marrow microniches might contribute to high incidence of relapse of Myeloma patients.

Rupture of vulnerable atherosclerotic plaques: MicroRNAs conducting the orchestra? (Article)

2010-02-01T00:00:00Z

MicroRNAs (miRNAs) are tiny, endogenous nucleotides that bind to mRNA and induce translation repression within metazoan cells. Since their discovery in 1993 in Caenorhabditis elegans and the demonstration of miRNAs in Homo sapiens in 2000, research has been fruitful in deciphering the role of these nucleotides in development, tissue homeostasis, and pathologic processes. In humans, around 700 human miRNA nucleotides have been verified, which interfere with 30% of all genes. Recently, the role of miRNA in cardiovascular research gained attention and the involvement of miRNAs in several cardiovascular diseases has been identified. In this review, we focus on the role of miRNAs in atherosclerosis and in particular on the potential role of miRNAs in the development of vulnerable atherosclerotic plaques. The role of miRNA in the main characteristics of these plaques, inflammation, angiogenesis, and apoptosis will be discussed. Finally, the future perspectives and miRNA-based diagnostic and therapeutic potentials will be highlighted.