Nijmegen Center for Molecular Life science

Nijmegen, Netherlands

Nijmegen Center for Molecular Life science

Nijmegen, Netherlands
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Christianson H.C.,Lund University | Svensson K.J.,Lund University | Van Kuppevelt T.H.,Nijmegen Center for Molecular Life science | Li J.-P.,Uppsala University | And 2 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

Extracellular vesicle (EV)-mediated intercellular transfer of signaling proteins and nucleic acids has recently been implicated in the development of cancer and other pathological conditions; however, the mechanism of EV uptake and how this may be targeted remain as important questions. Here, we provide evidence that heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived EVs with exosome-like characteristics. Internalized exosomes colocalized with cell-surface HSPGs of the syndecan and glypican type, and exosome uptake was specifically inhibited by free HS chains, whereas closely related chondroitin sulfate had no effect. By using several cell mutants, we provide genetic evidence of a receptor function of HSPG in exosome uptake, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Further, enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous PG biosynthesis by xyloside significantly attenuated exosome uptake. We provide biochemical evidence that HSPGs are sorted to and associate with exosomes; however, exosomeassociated HSPGs appear to have no direct role in exosome internalization. On a functional level, exosome-induced ERK1/2 signaling activation was attenuated in PG-deficient mutant cells as well as in WT cells treated with xyloside. Importantly, exosome-mediated stimulation of cancer cell migration was significantly reduced in PG-deficient mutant cells, or by treatment of WT cells with heparin or xyloside. We conclude that cancer cell-derived exosomes use HSPGs for their internalization and functional activity, which significantly extends the emerging role of HSPGs as key receptors of macromolecular cargo.

Christianson H.C.,Skåne University Hospital | van Kuppevelt T.H.,Nijmegen Center for Molecular Life science | Belting M.,Skåne University Hospital
PLoS ONE | Year: 2012

Tumor development requires angiogenesis and anti-angiogenic therapies have been introduced in the treatment of cancer. In this context, heparan sulfate proteoglycans (HSPGs) emerge as interesting targets, owing to their function as co-receptors of major, pro-angiogenic factors. Accordingly, previous studies have suggested anti-tumor effects of heparin, i.e. over-sulfated HS, and various heparin mimetics; however, a significant drawback is their unspecific mechanism of action and potentially serious side-effects related to their anticoagulant properties. Here, we have explored the use of human ScFv anti-HS antibodies (αHS) as a more rational approach to target HSPG function in endothelial cells (ECs). αHS were initially selected for their recognition of HS epitopes localized preferentially to the vasculature of patient glioblastoma tumors, i.e. highly angiogenic brain tumors. Unexpectedly, we found that these αHS exhibited potent pro-angiogenic effects in primary human ECs. αHS were shown to stimulate EC differentiation, which was associated with increased EC tube formation and proliferation. Moreover, αHS supported EC survival under hypoxia and starvation, i.e. conditions typical of the tumor microenvironment. Importantly, αHS-mediated proliferation was efficiently counter-acted by heparin and was absent in HSPG-deficient mutant cells, confirming HS-specific effects. On a mechanistic level, binding of αHS to HSPGs of ECs as well as glioblastoma cells was found to trigger p38 MAPK-dependent signaling resulting in increased proliferation. We conclude that several αHS that recognize HS epitopes abundant in the tumor vasculature may elicit a pro-angiogenic response, which has implications for the development of antibody-based targeting of HSPGs in cancer. © 2012 Christianson et al.

Neri F.,Human Genetics Foundation HuGeF | Krepelova A.,Human Genetics Foundation HuGeF | Krepelova A.,University of Siena | Incarnato D.,Human Genetics Foundation HuGeF | And 8 more authors.
Cell | Year: 2013

Summary The de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methyltransferase that cooperates with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESCs), but its function in these cells is unknown. Through genome-wide analysis of Dnmt3L knockdown in ESCs, we found that Dnmt3L is a positive regulator of methylation at the gene bodies of housekeeping genes and, more surprisingly, is also a negative regulator of methylation at promoters of bivalent genes. Dnmt3L is required for the differentiation of ESCs into primordial germ cells (PGCs) through the activation of the homeotic gene Rhox5. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low methylation levels at the H3K27me3 regions. Thus, in ESCs, Dnmt3L counteracts the activity of de novo DNA methylases to maintain hypomethylation at promoters of bivalent developmental genes. © 2013 Elsevier Inc.

Lindau D.,Nijmegen Center for Molecular Life science | Gielen P.,Nijmegen Center for Molecular Life science | Kroesen M.,Nijmegen Center for Molecular Life science | Wesseling P.,Radboud University Nijmegen | And 2 more authors.
Immunology | Year: 2013

Myeloid-derived suppressor cells (MDSC) and regulatory T (Treg) cells are major components of the immune suppressive tumour microenvironment (TME). Both cell types expand systematically in preclinical tumour models and promote T-cell dysfunction that in turn favours tumour progression. Clinical reports show a positive correlation between elevated levels of both suppressors and tumour burden. Recent studies further revealed that MDSCs can modulate the de novo development and induction of Treg cells. The overlapping target cell population of Treg cells and MDSCs is indicative for the importance and flexibility of immune suppression under pathological conditions. It also suggests the existence of common pathways that can be used for clinical interventions aiming to manipulate the TME. Elimination or reprogramming of the immune suppressive TME is one of the major current challenges in immunotherapy of cancer. Interestingly, recent findings suggest that natural killer T (NKT) cells can acquire the ability to convert immunosuppressive MDSCs into immunity-promoting antigen-presenting cells. Here we will review the cross-talk between MDSCs and other immune cells, focusing on Treg cells and NKT cells. We will consider its impact on basic and applied cancer research and discuss how targeting MDSCs may pave the way for future immunocombination therapies. © 2012 Nijmegen Centre for Molecular Life Sciences Immunology © 2012 Blackwell Publishing Ltd.

Jacobs J.F.M.,Radboud University Nijmegen | Jacobs J.F.M.,Nijmegen Center for Molecular Life science | Van Der Molen R.G.,Radboud University Nijmegen | Keren D.F.,University of Michigan
American Journal of Clinical Pathology | Year: 2014

Objectives: IgG4-related disease (IgG4-RD) is an increasingly recognized syndrome of unknown etiology that can affect a wide variety of organs. The commonly shared features include tumor-like swelling of the involved organs, a lymphoplasmacytic infiltrate enriched with polyclonal IgG4-positive plasma cells, variable degree of fibrosis, and elevated serum concentrations of polyclonal IgG4. Methods: In a qualitative retrospective study, the electrophoretic characteristics of serum from patients with increased polyclonal IgG4 were studied to see if a reproducible pattern could be identified. Results: We demonstrate that a characteristic focal band bridging the β and γ fraction by serum protein electrophoresis may be a first serologic indication for IgG4-RD. We further demonstrate that significant κ:λ skewing can occur in the polyclonal IgG4 fraction. Conclusions: The focal band detected by electrophoresis in sera from patients with IgG4-RD can be confirmed as polyclonal by immunofixation or immunosubtraction. Because these bands may be predominately of one light chain isotype, they could be misinterpreted as monoclonal gammopathies. © American Society for Clinical Pathology.

Glasson S.S.,Pfizer | Chambers M.G.,Eli Lilly and Company | Van Den Berg W.B.,Nijmegen Center for Molecular Life science | Little C.B.,University of Sydney
Osteoarthritis and Cartilage | Year: 2010

Aim: To describe a histologic scoring system for murine osteoarthritis (OA) that can be applied universally to instability, enzymatic, transgenic and spontaneous OA models. Methods: Scientists with expertise in assessing murine OA histopathology reviewed the merits and drawbacks of methods described in the literature. A semi-quantitative scoring system that could reasonably be employed in any basic cartilage histology laboratory was proposed. This scoring system was applied to a set of 10 images of the medial tibial plateau and femoral condyle to yield 20 scores. These images were scored twice by four experienced scorers (CL, SG, MC, TA), with a minimum time interval of 1 week between scores to obtain intra-observer variability. An additional three novice scorers (CR, CL and MM) with no previous experience evaluated the images to determine the ease of use and reproducibility across laboratories. Results: The semi-quantitative scoring system was relatively easy to apply for both experienced and novice scorers and the results had low inter- and intra-scorer variability. The variation in scores across both the experienced and novice scorers was low for both tibia and femur, with the tibia always having greater consistency. Conclusions: The semi-quantitative scoring system recommended here is simple to apply and required no specialized equipment. Scoring of the tibial plateaus was highly reproducible and more consistent than that of the femur due to the much thinner femoral cartilage. This scoring system may be a useful tool for both new and experienced scorers to sensitively evaluate models and OA mechanisms, and also provide a common paradigm for comparative evaluation across the many groups performing these analyses. © 2010 Osteoarthritis Research Society International.

Martens J.H.A.,Nijmegen Center for Molecular Life science | Brinkman A.B.,Nijmegen Center for Molecular Life science | Simmer F.,Nijmegen Center for Molecular Life science | Francoijs K.-J.,Nijmegen Center for Molecular Life science | And 4 more authors.
Cancer Cell | Year: 2010

Many different molecular mechanisms have been associated with PML-RARα-dependent transformation of hematopoietic progenitors. Here, we identified high confidence PML-RARα binding sites in an acute promyelocytic leukemia (APL) cell line and in two APL primary blasts. We found colocalization of PML-RARα with RXR to the vast majority of these binding regions. Genome-wide epigenetic studies revealed that treatment with pharmacological doses of all-trans retinoic acid induces changes in H3 acetylation, but not H3K27me3, H3K9me3, or DNA methylation at the PML-RARα/RXR binding sites or at nearby target genes. Our results suggest that PML-RARα/RXR functions as a local chromatin modulator and that specific recruitment of histone deacetylase activities to genes important for hematopoietic differentiation, RAR signaling, and epigenetic control is crucial to its transforming potential. © 2010 Elsevier Inc. All rights reserved.

Kroesen M.,Nijmegen Center for Molecular Life science
Immunotherapy | Year: 2012

Neuroblastoma (NBL) is an aggressive malignancy of the sympathetic nervous system. Advanced-stage NBLs prove fatal in approximately 50% of patients within 5 years. Therefore, new treatment modalities are urgently needed. Immunotherapy is a treatment modality that can be combined with established forms of treatment. Administration of monoclonal antibodies or dendritic cell-based therapies alone can lead to favorable clinical outcomes in individual cancer patients; for example patients with melanoma, lymphoma and NBL. However, clinical benefit is still limited to a minority of patients, and further improvements are clearly needed. In this article, we review the most commonly used approaches to treat patients with NBL and highlight the prerequisites and opportunities of cell-based immunotherapy, involving both innate and adaptive immune-effector cells. Furthermore, we discuss the potential of the combined application of immunotherapy and novel tumor-targeted therapies for the treatment of both cancer in general and NBL in particular.

Arif M.A.,Nijmegen Center for Molecular Life science | Frank W.,Ludwig Maximilians University of Munich | Khraiwesh B.,King Abdullah University of Science and Technology
International Journal of Molecular Sciences | Year: 2013

RNA interference (RNAi) is a mechanism that regulates genes by either transcriptional (TGS) or posttranscriptional gene silencing (PTGS), required for genome maintenance and proper development of an organism. Small non-coding RNAs are the key players in RNAi and have been intensively studied in eukaryotes. In plants, several classes of small RNAs with specific sizes and dedicated functions have evolved. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs are synthesized from a short hairpin structure while siRNAs are derived from long double-stranded RNAs (dsRNA). Both miRNA and siRNAs control the expression of cognate target RNAs by binding to reverse complementary sequences mediating cleavage or translational inhibition of the target RNA. They also act on the DNA and cause epigenetic changes such as DNA methylation and histone modifications. In the last years, the analysis of plant RNAi pathways was extended to the bryophyte Physcomitrella patens, a non-flowering, non-vascular ancient land plant that diverged from the lineage of seed plants approximately 450 million years ago. Based on a number of characteristic features and its phylogenetic key position in land plant evolution P. patens emerged as a plant model species to address basic as well as applied topics in plant biology. Here we summarize the current knowledge on the role of RNAi in P. patens that shows functional overlap with RNAi pathways from seed plants, and also unique features specific to this species. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

Hoeijmakers W.A.,Nijmegen Center for Molecular Life science
Methods in molecular biology (Clifton, N.J.) | Year: 2013

Transcriptome analysis by next-generation sequencing (RNA-seq) allows investigation of a transcriptome at unsurpassed resolution. One major benefit is that RNA-seq is independent of a priori knowledge on the sequence under investigation, thereby also allowing analysis of poorly characterized Plasmodium species. Here we provide a detailed protocol for RNA isolation and fragmentation, ribosomal RNA depletion, and cDNA synthesis that enables the preparation of a sequencing library from 1 to 2 μg of total RNA. Although we focus our discussion on the quantitative measurement of gene expression, this protocol is suited for many applications of RNA-seq and allows analysis of most RNA species.

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