TY - JOUR A1 - Kim, Mia A1 - Grimmig, Tanja A1 - Grimm, Martin A1 - Lazariotou, Maria A1 - Meier, Eva A1 - Rosenwald, Andreas A1 - Tsaur, Igor A1 - Blaheta, Roman A1 - Heemann, Uwe A1 - Germer, Christoph-Thomas A1 - Waaga-Gasser, Ana Maria A1 - Gasser, Martin T1 - Expression of Foxp3 in Colorectal Cancer but Not in Treg Cells Correlates with Disease Progression in Patients with Colorectal Cancer JF - PLoS ONE N2 - Background Measles virus (MV) causes T cell suppression by interference with phosphatidylinositol-3-kinase (PI3K) activation. We previously found that this interference affected the activity of splice regulatory proteins and a T cell inhibitory protein isoform was produced from an alternatively spliced pre-mRNA. Hypothesis Differentially regulated and alternatively splice variant transcripts accumulating in response to PI3K abrogation in T cells potentially encode proteins involved in T cell silencing. Methods To test this hypothesis at the cellular level, we performed a Human Exon 1.0 ST Array on RNAs isolated from T cells stimulated only or stimulated after PI3K inhibition. We developed a simple algorithm based on a splicing index to detect genes that undergo alternative splicing (AS) or are differentially regulated (RG) upon T cell suppression. Results Applying our algorithm to the data, 9% of the genes were assigned as AS, while only 3% were attributed to RG. Though there are overlaps, AS and RG genes differed with regard to functional regulation, and were found to be enriched in different functional groups. AS genes targeted extracellular matrix (ECM)-receptor interaction and focal adhesion pathways, while RG genes were mainly enriched in cytokine-receptor interaction and Jak-STAT. When combined, AS/RG dependent alterations targeted pathways essential for T cell receptor signaling, cytoskeletal dynamics and cell cycle entry. Conclusions PI3K abrogation interferes with key T cell activation processes through both differential expression and alternative splicing, which together actively contribute to T cell suppression. KW - T cells KW - gene regulation KW - alternative splicing KW - measles virus KW - T cell receptors KW - reverse transcriptase-polymerase chain reaction KW - TCR signaling cascade KW - cell cycle and cell division Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130340 VL - 8 IS - 1 ER - TY - JOUR A1 - Liu, Dan A1 - Hu, Kai A1 - Niemann, Markus A1 - Herrmann, Sebastian A1 - Cikes, Maja A1 - Störk, Stefan A1 - Beer, Meinrad A1 - Gaudron, Philipp Daniel A1 - Morbach, Caroline A1 - Knop, Stefan A1 - Geissinger, Eva A1 - Ertl, Georg A1 - Bijnens, Bart A1 - Weidemann, Frank T1 - Impact of Regional Left Ventricular Function on Outcome for Patients with AL Amyloidosis JF - PLoS ONE N2 - Objectives The aim of this study was to explore the left ventricular (LV) deformation changes and the potential impact of deformation on outcome in patients with proven light-chain (AL) amyloidosis and LV hypertrophy. Background Cardiac involvement in AL amyloidosis patients is associated with poor outcome. Detecting regional cardiac function by advanced non-invasive techniques might be favorable for predicting outcome. Methods LV longitudinal, circumferential and radial peak systolic strains (Ssys) were assessed by speckle tracking imaging (STI) in 44 biopsy-proven systemic AL amyloidosis patients with LV hypertrophy (CA) and in 30 normal controls. Patients were divided into compensated (n = 18) and decompensated (n = 26) group based on clinical assessment and followed-up for a median period of 345 days. Results Ejection fraction (EF) was preserved while longitudinal Ssys (LSsys) was significantly reduced in both compensated and decompensated groups. Survival was significantly reduced in decompensated group (35% vs. compensated 78%, P = 0.001). LSsys were similar in apical segments and significantly reduced in basal segments between two patient groups. LSsys at mid-segments were significantly reduced in all LV walls of decompensated group. Patients were further divided into 4 subgroups according to the presence or absence of reduced LSsys in no (normal), only basal (mild), basal and mid (intermediate) and all segments of the septum (severe). This staging revealed continuously worse prognosis in proportion to increasing number of segments with reduced LSsys (mortality: normal 14%, mild 27%, intermediate 67%, and severe 64%). Mid-septum LSsys<11% suggested a 4.8-fold mortality risk than mid-septum LSsys≥11%. Multivariate regression analysis showed NYHA class and mid-septum LSsys were independent predictors for survival. Conclusions Reduced deformation at mid-septum is associated with worse prognosis in systemic amyloidosis patients with LV hypertrophy. KW - regression analysis KW - ejection fraction KW - echocardiography KW - cardiac transplantation KW - deformation KW - amyloidosis KW - prognosis KW - stem cell transplantation Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130293 VL - 8 IS - 3 ER - TY - JOUR A1 - Rasche, Leo A1 - Duell, Johannes A1 - Morgner, Charlotte A1 - Chatterjee, Manik A1 - Hensel, Frank A1 - Rosenwald, Andreas A1 - Einsele, Hermann A1 - Topp, Max S. A1 - Brändlein, Stephanie T1 - The Natural Human IgM Antibody PAT-SM6 Induces Apoptosis in Primary Human Multiple Myeloma Cells by Targeting Heat Shock Protein GRP78 JF - PLoS ONE N2 - In contrast to other haematological malignancies, targeted immunotherapy has not entered standard treatment regimens for de novo or relapsed multiple myeloma (MM) yet. While a number of IgG-formatted monoclonal antibodies are currently being evaluated in clinical trials in MM, our study aimed to investigate whether the fully human IgM monoclonal antibody PAT-SM6 that targets a tumour-specific variant of the heat shock protein GRP78 might be an attractive candidate for future immunotherapeutic approaches. We here show that GRP78 is stably and consistently expressed on the surface on tumour cells from patients with de novo, but also relapsed MM and that binding of PAT-SM6 to MM cells can specifically exert cytotoxic effects on malignant plasma cells, whereas non-malignant cells are not targeted. We demonstrate that the induction of apoptosis and, to a lesser extent, complement dependent cytotoxicity is the main mode of action of PAT-SM6, whereas antibody dependent cellular cytotoxicity does not appear to contribute to the cytotoxic properties of this antibody. Given the favourable safety profile of PAT-SM6 in monkeys, but also in a recent phase I trial in patients with malignant melanoma, our results form the basis for a planned phase I study in patients with relapsed MM. KW - cytotoxicity KW - apoptosis KW - immunohistochemistry techniques KW - enzyme-linked immunoassays KW - multiple myeloma KW - cell staining KW - cell binding KW - complement system Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130125 VL - 8 IS - 5 ER - TY - JOUR A1 - Huang, Bei A1 - Belharazem, Djeda A1 - Li, Li A1 - Kneitz, Susanne A1 - Schnabel, Philipp A. A1 - Rieker, Ralf J. A1 - Körner, Daniel A1 - Nix, Wilfried A1 - Schalke, Berthold A1 - Müller-Hermelink, Hans Konrad A1 - Ott, German A1 - Rosenwald, Andreas A1 - Ströbel, Philipp A1 - Marx, Alexander T1 - Anti-apoptotic signature in thymic squamous cell carcinomas – functional relevance of anti-apoptotic BIRC3 expression in the thymic carcinoma cell line 1889c JF - Frontiers in Oncology N2 - The molecular pathogenesis of thymomas and thymic arcinomas (TCs) is poorly understood and results of adjuvant therapy are unsatisfactory in case of metastatic disease and tumor recurrence. For these clinical settings, novel therapeutic strategies are urgently needed. Recently, limited sequencing efforts revealed that a broad spectrum of genes that play key roles in various common cancers are rarely affected in thymomas and TCs, suggesting that other oncogenic principles might be important.This made us re-analyze historic expression data obtained in a spectrumof thymomas and thymic squamous cell carcinomas (TSCCs) with a custom-made cDNA microarray. By cluster analysis, different anti-apoptotic signatures were detected in type B3 thymoma and TSCC, including overexpression of BIRC3 in TSCCs. This was confirmed by qRT-PCR in the original and an independent validation set of tumors. In contrast to several other cancer cell lines, the BIRC3-positive TSCC cell line, 1889c showed spontaneous apoptosis after BIRC3 knock-down. Targeting apoptosis genes is worth testing as therapeutic principle in TSCC. KW - gene expression KW - MTCH2 KW - targeted KW - myasthenia gravis KW - apoptosis KW - thymus KW - thymoma KW - thymic carcinoma Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132214 VL - 3 IS - 316 ER - TY - JOUR A1 - Wolfahrt, Sonja A1 - Herman, Sandra A1 - Scholz, Claus-Jürgen A1 - Sauer, Georg A1 - Deissler, Helmut T1 - Identification of alternative transcripts of rat CD9 expressed by tumorigenic neural cell lines and in normal tissues JF - Genetics and Molecular Biology N2 - CD9 is the best-studied member of the tetraspanin family of transmembrane proteins. It is involved in various fundamental cellular processes and its altered expression is a characteristic of malignant cells of different origins. Despite numerous investigations confirming its fundamental role, the heterogeneity of CD9 or other tetraspanin proteins was considered only to be caused by posttranslational modification, rather than alternative splicing. Here we describe the first identification of CD9 transcript variants expressed by cell lines derived from fetal rat brain cells. Variant mRNA-B lacks a potential translation initiation codon in the alternative exon 1 and seems to be characteristic of the tumorigenic BT cell lines. In contrast, variant mRNA-C can be translated from a functional initiation codon located in its extended exon 2, and substantial amounts of this form detected in various tissues suggest a contribution to CD9 functions. From the alternative sequence of variant C, a different membrane topology ( 5 transmembrane domains) and a deviating spectrum of functions can be expected. KW - tetraspanin KW - CD9 KW - antigen KW - cancer KW - noncoding RNAs KW - nervous system KW - poor prognosis KW - tetraspanin protein KW - transcript KW - splice variant KW - membrane topology Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131801 VL - 36 IS - 2 ER - TY - JOUR A1 - Dmochewitz, Lydia A1 - Förtsch, Christina A1 - Zwerger, Christian A1 - Vaeth, Martin A1 - Felder, Edward A1 - Huber-Lang, Markus A1 - Barth, Holger T1 - A Recombinant Fusion Toxin Based on Enzymatic Inactive C3bot1 Selectively Targets Macrophages JF - PLoS ONE N2 - Background: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. Methodology/Principal Findings: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (~50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages. Conclusions/Significance: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases. KW - C3 KW - mammalian cells KW - clostridium botulinum KW - ADP-ribosylation toxins KW - botulinum C2 toxin KW - membrane translocation KW - bacterial toxins KW - actin filaments KW - cellular uptake KW - ribosyltransferase Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131189 VL - 8 IS - 1 ER - TY - JOUR A1 - Leich, E. A1 - Weißbach, S. A1 - Klein, H.-U. A1 - Grieb, T. A1 - Pischimarov, J. A1 - Stühmer, T. A1 - Chatterjee, M. A1 - Steinbrunn, T. A1 - Langer, C. A1 - Eilers, M. A1 - Knop, S. A1 - Einsele, H. A1 - Bargou, R. A1 - Rosenwald, A. T1 - Multiple myeloma is affected by multiple and heterogeneous somatic mutations in adhesion- and receptor tyrosine kinase signaling molecules JF - Blood Cancer Journal N2 - Multiple myeloma (MM) is a largely incurable plasma cell malignancy with a poorly understood and heterogeneous clinical course. To identify potential, functionally relevant somatic mutations in MM, we performed whole-exome sequencing of five primary MM, corresponding germline DNA and six MM cell lines, and developed a bioinformatics strategy that also integrated published mutational data of 38 MM patients. Our analysis confirms that identical, recurrent mutations of single genes are infrequent in MM, but highlights that mutations cluster in important cellular pathways. Specifically, we show enrichment of mutations in adhesion molecules of MM cells, emphasizing the important role for the interaction of the MM cells with their microenvironment. We describe an increased rate of mutations in receptor tyrosine kinases (RTKs) and associated signaling effectors, for example, in EGFR, ERBB3, KRAS and MAP2K2, pointing to a role of aberrant RTK signaling in the development or progression of MM. The diversity of mutations affecting different nodes of a particular signaling network appears to be an intrinsic feature of individual MM samples, and the elucidation of intra- as well as interindividual redundancy in mutations that affect survival pathways will help to better tailor targeted therapeutic strategies to the specific needs of the MM patient. KW - multiple myeloma KW - somatic mutations KW - whole-exome sequencing KW - adhesion KW - receptor tyrosine kinases Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128663 VL - 3 IS - e102 ER - TY - JOUR A1 - Schlereth, Katharina A1 - Heyl, Charlotte A1 - Krampitz, Anna-Maria A1 - Mernberger, Marco A1 - Finkernagel, Florian A1 - Scharfe, Maren A1 - Jarek, Michael A1 - Leich, Ellen A1 - Rosenwald, Andreas A1 - Stiewe, Thorsten T1 - Characterization of the p53 Cistrome - DNA Binding Cooperativity Dissects p53's Tumor Suppressor Functions JF - PLOS Genetics N2 - p53 protects us from cancer by transcriptionally regulating tumor suppressive programs designed to either prevent the development or clonal expansion of malignant cells. How p53 selects target genes in the genome in a context-and tissue-specific manner remains largely obscure. There is growing evidence that the ability of p53 to bind DNA in a cooperative manner prominently influences target gene selection with activation of the apoptosis program being completely dependent on DNA binding cooperativity. Here, we used ChIP-seq to comprehensively profile the cistrome of p53 mutants with reduced or increased cooperativity. The analysis highlighted a particular relevance of cooperativity for extending the p53 cistrome to non-canonical binding sequences characterized by deletions, spacer insertions and base mismatches. Furthermore, it revealed a striking functional separation of the cistrome on the basis of cooperativity; with low cooperativity genes being significantly enriched for cell cycle and high cooperativity genes for apoptotic functions. Importantly, expression of high but not low cooperativity genes was correlated with superior survival in breast cancer patients. Interestingly, in contrast to most p53-activated genes, p53-repressed genes did not commonly contain p53 binding elements. Nevertheless, both the degree of gene activation and repression were cooperativity-dependent, suggesting that p53-mediated gene repression is largely indirect and mediated by cooperativity-dependently transactivated gene products such as CDKN1A, E2F7 and non-coding RNAs. Since both activation of apoptosis genes with non-canonical response elements and repression of pro-survival genes are crucial for p53's apoptotic activity, the cistrome analysis comprehensively explains why p53-induced apoptosis, but not cell cycle arrest, strongly depends on the intermolecular cooperation of p53 molecules as a possible safeguard mechanism protecting from accidental cell killing. KW - cell-cycle arrest KW - gene expression KW - breast cancer KW - human genome KW - transcriptional repression KW - consensus DNA KW - in-vivo KW - apoptosis KW - network KW - damage Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127579 SN - 1553-7404 VL - 9 IS - 8 ER - TY - JOUR A1 - Mueller, Kerstin A1 - Quandt, Jasmin A1 - Marienfeld, Ralf B. A1 - Weihrich, Petra A1 - Fiedler, Katja A1 - Claussnitzer, Melina A1 - Laumen, Helmut A1 - Vaeth, Martin A1 - Berberich-Siebelt, Frederike A1 - Serfling, Edgar A1 - Wirth, Thomas A1 - Brunner, Cornelia T1 - Octamer-dependent transcription in T cells is mediated by NFAT and \(NF-\kappa B\) JF - Nucleic Acids Research N2 - The transcriptional co-activator BOB.1/OBF.1 was originally identified in B cells and is constitutively expressed throughout B cell development. BOB.1/OBF.1 associates with the transcription factors Oct1 and Oct2, thereby enhancing octamer-dependent transcription. In contrast, in T cells, BOB.1/OBF.1 expression is inducible by treatment of cells with PMA/Ionomycin or by antigen receptor engagement, indicating a marked difference in the regulation of BOB.1/OBF.1 expression in B versus T cells. The molecular mechanisms underlying the differential expression of BOB.1/OBF.1 in T and B cells remain largely unknown. Therefore, the present study focuses on mechanisms controlling the transcriptional regulation of BOB.1/OBF.1 and Oct2 in T cells. We show that both calcineurin- and \(NF-\kappa B\)-inhibitors efficiently attenuate the expression of BOB.1/OBF.1 and Oct2 in T cells. In silico analyses of the BOB.1/OBF.1 promoter revealed the presence of previously unappreciated combined NFAT/\(NF-\kappa B\) sites. An array of genetic and biochemical analyses illustrates the involvement of the \(Ca^{2+}\)/calmodulin-dependent phosphatase calcineurin as well as NFAT and \(NF-\kappa B\) transcription factors in the transcriptional regulation of octamer-dependent transcription in T cells. Conclusively, impaired expression of BOB.1/OBF.1 and Oct2 and therefore a hampered octamer-dependent transcription may participate in T cell-mediated immunodeficiency caused by the deletion of NFAT or \(NF-\kappa B\) transcription factors. KW - germinal center formation KW - OBF-1 OCA-B KW - coactivator OBF-1 KW - gene expression KW - functional characterization KW - immunoglobulin promoters KW - OCT-1-deficient mice KW - embryonic lethality KW - endothelial cells KW - murine homolog Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123280 SN - 1362-4962 VL - 41 IS - 4 ER - TY - JOUR A1 - Timofeev, Oleg A1 - Schlereth, Katharina A1 - Wanzel, Michael A1 - Braun, Attila A1 - Nieswandt, Bernhard A1 - Pagenstecher, Axel A1 - Rosenwald, Andreas A1 - Elsässer, Hans-Peter A1 - Stiewe, Thorsten T1 - p53 DNA Binding Cooperativity Is Essential for Apoptosis and Tumor Suppression In Vivo JF - Cell Reports N2 - Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53(E177R) mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer. KW - mutant p53 KW - senescence KW - mice KW - tumorigenesis KW - restoration KW - damage responses KW - antioxidant function KW - p53-inducible regulator KW - p53-dependent apoptosis KW - cell-cycle arrest Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122168 VL - 3 ER -