@article{HuangBelharazemLietal.2013, author = {Huang, Bei and Belharazem, Djeda and Li, Li and Kneitz, Susanne and Schnabel, Philipp A. and Rieker, Ralf J. and K{\"o}rner, Daniel and Nix, Wilfried and Schalke, Berthold and M{\"u}ller-Hermelink, Hans Konrad and Ott, German and Rosenwald, Andreas and Str{\"o}bel, Philipp and Marx, Alexander}, title = {Anti-apoptotic signature in thymic squamous cell carcinomas - functional relevance of anti-apoptotic BIRC3 expression in the thymic carcinoma cell line 1889c}, series = {Frontiers in Oncology}, volume = {3}, journal = {Frontiers in Oncology}, number = {316}, doi = {10.3389/fonc.2013.00316}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132214}, year = {2013}, abstract = {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.}, language = {en} } @article{SchlerethHeylKrampitzetal.2013, author = {Schlereth, Katharina and Heyl, Charlotte and Krampitz, Anna-Maria and Mernberger, Marco and Finkernagel, Florian and Scharfe, Maren and Jarek, Michael and Leich, Ellen and Rosenwald, Andreas and Stiewe, Thorsten}, title = {Characterization of the p53 Cistrome - DNA Binding Cooperativity Dissects p53's Tumor Suppressor Functions}, series = {PLOS Genetics}, volume = {9}, journal = {PLOS Genetics}, number = {8}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1003726}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127579}, pages = {e1003726}, year = {2013}, abstract = {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.}, language = {en} } @article{MuellerQuandtMarienfeldetal.2013, author = {Mueller, Kerstin and Quandt, Jasmin and Marienfeld, Ralf B. and Weihrich, Petra and Fiedler, Katja and Claussnitzer, Melina and Laumen, Helmut and Vaeth, Martin and Berberich-Siebelt, Frederike and Serfling, Edgar and Wirth, Thomas and Brunner, Cornelia}, title = {Octamer-dependent transcription in T cells is mediated by NFAT and \(NF-\kappa B\)}, series = {Nucleic Acids Research}, volume = {41}, journal = {Nucleic Acids Research}, number = {4}, issn = {1362-4962}, doi = {10.1093/nar/gks1349}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123280}, pages = {2138-2154}, year = {2013}, abstract = {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.}, language = {en} } @article{AukemaKreuzKohleretal.2014, author = {Aukema, Sietse M. and Kreuz, Markus and Kohler, Christian W. and Rosolowski, Maciej and Hasenclever, Dirk and Hummel, Michael and K{\"u}ppers, Ralf and Lenze, Diddo and Ott, German and Pott, Christiane and Richter, Julia and Rosenwald, Andreas and Szczepanowski, Monika and Schwaenen, Carsten and Stein, Harald and Trautmann, Heiko and Wessendorf, Swen and Tr{\"u}mper, Lorenz and Loeffler, Markus and Spang, Rainer and Kluin, Philip M. and Klapper, Wolfram and Siebert, Reiner}, title = {Biological characterization of adult MYC-translocation-positive mature B-cell lymphomas other than molecular Burkitt lymphoma}, series = {Haematologica}, volume = {99}, journal = {Haematologica}, number = {4}, issn = {1592-8721}, doi = {10.3324/haematol.2013.091827}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116882}, pages = {726-735}, year = {2014}, abstract = {Chromosomal translocations affecting the MYC oncogene are the biological hallmark of Burkitt lymphomas but also occur in a subset of other mature B-cell lymphomas. If accompanied by a chromosomal break targeting the BCL2 and/or BCL6 oncogene these MYC translocation-positive (MYC+) lymphomas are called double-hit lymphomas, otherwise the term single-hit lymphomas is applied. In order to characterize the biological features of these MYC+ lymphomas other than Burkitt lymphoma we explored, after exclusion of molecular Burkitt lymphoma as defined by gene expression profiling, the molecular, pathological and clinical aspects of 80 MYC-translocation-positive lymphomas (31 single-hit, 46 double-hit and 3 MYC+-lymphomas with unknown BCL6 status). Comparison of single-hit and double-hit lymphomas revealed no difference in MYC partner (IG/non-IG), genomic complexity, MYC expression or gene expression profile. Double-hit lymphomas more frequently showed a germinal center B-cell-like gene expression profile and had higher IGH and MYC mutation frequencies. Gene expression profiling revealed 130 differentially expressed genes between BCL6(+)/MYC+ and BCL2(+)/MYC+ double-hit lymphomas. BCL2(+)/MYC+ double-hit lymphomas more frequently showed a germinal center B-like gene expression profile. Analysis of all lymphomas according to MYC partner (IG/non-IG) revealed no substantial differences. In this series of lymphomas, in which immunochemotherapy was administered in only a minority of cases, single-hit and double-hit lymphomas had a similar poor outcome in contrast to the outcome of molecular Burkitt lymphoma and lymphomas without the MYC break. Our data suggest that, after excluding molecular Burkitt lymphoma and pediatric cases, MYC+ lymphomas are biologically quite homogeneous with single-hit and double-hit lymphomas as well as IG-MYC and non-IG-MYC+ lymphomas sharing various molecular characteristics.}, language = {en} } @article{SanderdeJongRosenwaldetal.2014, author = {Sander, Brigitta and de Jong, Daphne and Rosenwald, Andreas and Xie, Wanling and Balagu{\´e}, Olga and Calaminici, Maria and Carreras, Joaquim and Gaulard, Philippe and Gribben, John and Hagenbeek, Anton and Kersten, Marie Jos{\´e} and Molina, Thierry Jo and Lee, Abigail and Montes-Moreno, Santiago and Ott, German and Raemaekers, John and Salles, Gilles and Sehn, Laurie and Thorns, Christoph and Wahlin, Bjorn E. and Gascoyne, Randy D. and Weller, Edie}, title = {The reliability of immunohistochemical analysis of the tumor microenvironment in follicular lymphoma: a validation study from the Lunenburg Lymphoma Biomarker Consortium}, series = {Haematologica}, volume = {99}, journal = {Haematologica}, number = {4}, issn = {1592-8721}, doi = {10.3324/haematol.2013.095257}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116875}, pages = {715-725}, year = {2014}, abstract = {The cellular microenvironment in follicular lymphoma is of biological and clinical importance. Studies on the clinical significance of non-malignant cell populations have generated conflicting results, which may partly be influenced by poor reproducibility in immunohistochemical marker quantification. In this study, the reproducibility of manual scoring and automated microscopy based on a tissue microarray of 25 follicular lymphomas as compared to flow cytometry is evaluated. The agreement between manual scoring and flow cytometry was moderate for CD3, low for CD4, and moderate to high for CD8, with some laboratories scoring closer to the flow cytometry results. Agreement in manual quantification across the 7 laboratories was low to moderate for CD3, CD4, CD8 and FOXP3 frequencies, moderate for CD21, low for MIB1 and CD68, and high for CD10. Manual scoring of the architectural distribution resulted in moderate agreement for CD3, CD4 and CD8, and low agreement for FOXP3 and CD68. Comparing manual scoring to automated microscopy demonstrated that manual scoring increased the variability in the low and high frequency interval with some laboratories showing a better agreement with automated scores. Manual scoring reliably identified rare architectural patterns of T-cell infiltrates. Automated microscopy analyses for T-cell markers by two different instruments were highly reproducible and provided acceptable agreement with flow cytometry. These validation results provide explanations for the heterogeneous findings on the prognostic value of the microenvironment in follicular lymphoma. We recommend a more objective measurement, such as computer-assisted scoring, in future studies of the prognostic impact of microenvironment in follicular lymphoma patients.}, language = {en} } @phdthesis{Hock2013, author = {Hock, Matthias}, title = {Analyse der NFATc1-Genexpression durch eGFP-BAC-Reporterm{\"a}use}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-80596}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {In Lymphozyten wird nach Antigenaktivierung die Expression des Nfatc1-Gens durch Aktivierung des P1-Promoters stark induziert. Dagegen ist die, durch den Promoter P2 vermittelte Expression ebenso wie die der anderen NFAT Faktoren c2 und c3 konstitutiv. Die Akkumulation der dabei gebildeten Isoform NFATc1/αA ist sowohl f{\"u}r Effektorfunktionen wie die Zytokinproduktion sowie die Proliferation und das {\"U}berleben der aktivierten Zellen wichtig (Chuvpilo et al., 2002). Um die Expression des Nfatc1-Gens auf Einzelzellebene messen zu k{\"o}nnen, wurden BAC (bacterial artificial chromosom) transgene Mauslinien generiert, die einen 210kb großen Bereich des Nfatc1-Gens der Maus enthalten. In diesen Lokus wurde ein eGFP-Reportergen innerhalb des allen Isoformen gemeinsamen, dritten Exons integriert. In dieser Arbeit wird durch semiquantitative RT-PCR-Experimente von Gesamt-Milzzellen und TLymphozyten gezeigt, dass in den B6/NFATc1-eGFP-BAC-Reporterm{\"a}usen die Expression der eGFP-cDNA analog zum endogenen Nfatc1-Lokus der Kontrolle der beiden Promotoren P1 und P2 unterliegt. In Western Blot Experimenten wird in diesen Zellen mittels eines NFATc1α-spezifischen Antik{\"o}rpers eine induzierbare und CsA-sensitive α-GFP-Isoform - vergleichbar mit der endogenen NFATc1α-Isoform - nachgewiesen. Gleichzeitig zeigen NFATc1-Antik{\"o}rper das konstitutiv exprimierte GFPβ-Protein. Die Korrelation der Expression von NFATc1 und GFP auf mRNA- und Proteinebene machen in B6/NFATc1-eGFP-BAC-Reporterm{\"a}usen das GFP-Protein somit zu einem sensitiven und spezifischen Marker der NFATc1-Aktivit{\"a}t. In FACS-Analysen gibt der Anstieg der GFP-Fluoreszenzintensit{\"a}t bei Stimulation von Gesamt- Milzzellen bzw. T-Lymphozyten um bis auf das Dreifache die Induktion von NFATc1 wider. Unter dem Einfluss von CsA verbleibt die GFPFluoreszenzintensit{\"a}t auf dem Niveau unstimulierter Zellen. Die GFPFluoreszenz korreliert dar{\"u}ber hinaus bei Prim{\"a}rstimulation mit der Expression des IL-2-Gens, dessen Promotor mit 5 NFAT-Bindestellen den Prototyp eines NFATc1-Targets darstellt (Serfling et al., 1989). Die Analyse der Koexpression von NFATc1 und GFP mittels Fluoreszenzmikroskopie zeigt in allen stimulierten, GFP-positiven CD4+-Lymphozyten die nukle{\"a}re Lokalisation von 75 NFATc1, vor allem von NFATc1α. Die Analyse des GFP-Ph{\"a}notyps in alloreaktiven T-Zellen zeigt bei Abstoßungsreaktionen in vitro („Mixed Lymphocyte Reactions") eine selektive Zunahme der Fluoreszenz dieser Zellen um bis auf das Vierfache, was die Rolle von NFATc1 f{\"u}r die Effektorfunktion aktivierter T-Lymphozyten verdeutlicht. GFP und das endogene NFATc1 werden bei Stimulation konventioneller T-Zellen (Tcons, CD4+CD25-FoxP3-) stark exprimiert, w{\"a}hrend nat{\"u}rliche regulatorische T-Zellen (nTregs, CD4+CD25+FoxP3+) konstant geringe NFATc1- und GFP-Konzentrationen zeigen. In induzierten regulatorischen T-Lymphozyten (iTregs) supprimiert TGF- β konzentrationsabh{\"a}ngig die GFP-Fluoreszenz bis auf das Niveau unstimulierter Lymphozyten. W{\"a}hrend in nTregs die Suppression des Nfatc1- Gens im wesentlichen durch FoxP3 erfolgt (Torgerson et al., 2009), scheint dies in iTregs vor allem {\"u}ber den TGF-β Signalweg vermittelt zu werden. Die Analyse der GFP-Expression in den verschiedenen Stadien der TZellentwicklung zeigt weiterhin deutliche Unterschiede in der Aktivit{\"a}t des Nfatc1-Gens. Dies wird durch die starke Aktivit{\"a}t des BAC-Genlokus in CD4- CD8- DN Thymozyten, welche eine sechsfach h{\"o}here GFP-Expression aufweisen als CD4+CD8+ DP Zellen, deutlich.}, subject = {NFATc1}, language = {de} }