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DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4)
Hilmar Quentmeier, Sonja Eberth, Julia Romani, Herbert A Weich, Margarete Zaborski, Hans G Drexler
BMC Cancer , 2012, DOI: 10.1186/1471-2407-12-19
Abstract: Real-time (RT) PCR analysis was performed to quantify KDR and FLT4 expression in some ninety leukemia/lymphoma cell lines, human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs). Western blot analyses and flow cytometric analyses confirmed results at the protein level. After bisulfite conversion of DNA we determined the methylation status of KDR and FLT4 by DNA sequencing and by methylation specific PCR (MSP). Western blot analyses were performed to examine the effect of VEGF-C on p42/44 MAPK activation.Expression of KDR and FLT4 was observed in cell lines from various leukemic entities, but not in lymphoma cell lines: 16% (10/62) of the leukemia cell lines expressed KDR, 42% (27/65) were FLT4 positive. None of thirty cell lines representing six lymphoma subtypes showed more than marginal expression of KDR or FLT4. Western blot analyses confirmed KDR and FLT4 protein expression in HDMECs, HUVECs and in cell lines with high VEGF-R mRNA levels. Mature VEGF-C induced p42/44 MAPK activation in the KDR- /FLT4+ cell line OCI-AML1 verifying the model character of this cell line for VEGF-C signal transduction studies. Bisulfite sequencing and MSP revealed that GpG islands in the promoter regions of KDR and FLT4 were unmethylated in HUVECs, HDMECs and KDR+ and FLT4+ cell lines, whereas methylated cell lines did not express these genes. In hypermethylated cell lines, KDR and FLT4 were re-inducible by treatment with the DNA demethylating agent 5-Aza-2'deoxycytidine, confirming epigenetic regulation of both genes.Our data show that VEGF-Rs KDR and FLT4 are silenced by DNA methylation. However, if the promoters are unmethylated, other factors (e.g. transactivation factors) determine the extent of KDR and FLT4 expression.Vascular endothelial growth factors (VEGFs) and their corresponding receptors (VEGF-Rs) are important regulators of angiogenesis and lymphangiogensis. VEGF-A binds VEGF-R1 (FLT1) and VEGF-R2 (KDR). Both tyrosine kinase
CD7 in acute myeloid leukemia: correlation with loss of wild-type CEBPA, consequence of epigenetic regulation
Sonja R?hrs, Michaela Scherr, Julia Romani, Margarete Zaborski, Hans G Drexler, Hilmar Quentmeier
Journal of Hematology & Oncology , 2010, DOI: 10.1186/1756-8722-3-15
Abstract: As already described for primary AML cells, the majority of AML cell lines tested were either C/EBPα+/CD7- or C/EBPα-/CD7+. However, the existence of isolated CD7+ cell lines expressing wild-type C/EBPα challenges the notion that C/EBPα acts as a unique repressor of CD7. Furthermore, ectopic expression of CEBPA did not reduce CD7 in CD7+ cells and knock-down of C/EBPα failed to induce CD7 in CD7- cells. In contrast, the DNA demethylating agent Aza-2'deoxycytidine triggered CD7 expression in CD7- AML and in T-cell lines suggesting epigenetic regulation of CD7. Bisulfite sequencing data confirmed that CpGs in the CD7 exon1 region are methylated in CD7- cell lines, and unmethylated in CD7+ cell lines.We confirmed an inverse correlation between the expression of wild-type CEBPA and of CD7 in AML cells. Our results contradict the hypothesis that C/EBPα acts as repressor for CD7, and instead show that epigenetic mechanisms are responsible for CD7 regulation, in AML cells as well as in T-cells, the typical CD7 expressing cell type.CCAAT/enhancer binding factor alpha (CEBPA), located on chromosome 19q13.1 encodes a transcription factor that is of importance for granulocytic differentiation [1]. C/EBPα is upregulated during myelomonocytic development and positively affects expression of granulocyte differentiation related genes such as the G-CSF receptor (GCSFR), myeloperoxidase and neutrophil elastase (ELA2) [2-4]. CEBPA mutations are found in 5 - 14% of acute myeloid leukemia (AML) cases [5]. C/EBPα mutant proteins block the effect of wild-type C/EBPα on target genes in a dominant-negative manner [6]. This might be the reason why patients with CEBPA mutations and those with a silenced CEBPA promoter are found in the same AML subclass according to gene expression profiling [7]. Also expression of the T-cell marker CD7 has been associated with CEBPA mutations and with CEBPA hypermethylation [7,8].CD7 is expressed in 30% of AML cases and CD7 positivity is linked with poor pro
BCR-ABL1-independent PI3Kinase activation causing imatinib-resistance
Hilmar Quentmeier, Sonja Eberth, Julia Romani, Margarete Zaborski, Hans G Drexler
Journal of Hematology & Oncology , 2011, DOI: 10.1186/1756-8722-4-6
Abstract: Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and all showed resistance to second generation TKI, nilotinib or dasatinib. STAT5, ERK1/2 and the ribosomal S6 protein (RPS6) are BCR-ABL1 downstream effectors, and all three proteins are dephosphorylated by imatinib in sensitive cell lines. TKI-resistant phosphorylation of RPS6, but responsiveness as regards JAK/STAT5 and ERK1/2 signalling were characteristic for resistant cell lines. PI3K pathway inhibitors effected dephosphorylation of RPS6 in imatinib-resistant cell lines suggesting that an oncogene other than BCR-ABL1 might be responsible for activation of the PI3K/AKT1/mTOR pathway, which would explain the TKI resistance of these cells. We show that the TKI-resistant cell line KCL-22 carries a PI3Kα E545G mutation, a site critical for the constitutive activation of the PI3K/AKT1 pathway. Apoptosis in TKI-resistant cells could be induced by inhibition of AKT1, but not of mTOR.We introduce five Philadelphia-chromosome positive cell lines as TKI-resistance models. None of these cell lines carries mutations in the kinase domain of BCR-ABL1 or other molecular aberrations previously indicted in the context of imatinib-resistance. These cell lines are unique as they dephosphorylate ERK1/2 and STAT5 after treatment with imatinib, while PI3K/AKT1/mTOR activity remains unaffected. Inhibition of AKT1 leads to apoptosis in the imatinib-resistant cell lines. In conclusion, Ph+ cell lines show a form of imatinib-resistance attributable to constitutive activation of the PI3K/AKT1 pathway. Mutations in PIK3CA, as observed in cell line KCL-22, or PI3K activating oncogenes may undelie TKI-resistance in these cell lines.Expression of the Philadelphia chromosome (Ph), resulting from fusion of the non-receptor tyrosine kinase ABL1 on chromosome 9 with BCR on chromosome 21
SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines
Hilmar Quentmeier, Bj?rn Schneider, Sonja R?hrs, Julia Romani, Margarete Zaborski, Roderick AF MacLeod, Hans G Drexler
Journal of Hematology & Oncology , 2009, DOI: 10.1186/1756-8722-2-3
Abstract: Of 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET(TAF-Iβ)-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF-I isoforms revealed that the cell lines also expressed TAF-Iα-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH) and array-based copy number analysis were both consistent with del(9)(q34.11q34.13) as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein.Cell lines LOUCY and MEGAL express the recently described SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.Leukemia subtypes are often associated with specific recurrent chromosome translocations. Translocations may function by constitutively activating proto-oncogenes or they may create new oncogenes by fusing two formerly independent genes. The SET-NUP214 (TAF-1/CAN) gene fusion has previously been described as result of a chromosomal translocation t(9;9)(q34;q34) in a case of acute undifferentiated leukemia [1]. The fusion gene appears to inhibit differentiation, while secondary chromosomal aberrations are necessary to induce tumorigenesis [2,3]. Recent studies have shown that the SET-NUP214 fusion can also result from a recurrent deletion, del(9)(q34.11q34.13) in patients with T-cell acute lymphoblastic leukemia (T-ALL) [4]. It has also been reported in a singl
Inhibition of PI3K/mTOR Overcomes Nilotinib Resistance in BCR-ABL1 Positive Leukemia Cells through Translational Down-Regulation of MDM2
Jie Ding, Julia Romani, Margarete Zaborski, Roderick A. F. MacLeod, Stefan Nagel, Hans G. Drexler, Hilmar Quentmeier
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0083510
Abstract: Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.
Epigenetic regulation of CD44 in Hodgkin and non-Hodgkin lymphoma
Sonja Eberth, Bj?rn Schneider, Andreas Rosenwald, Elena M Hartmann, Julia Romani, Margarete Zaborski, Reiner Siebert, Hans G Drexler, Hilmar Quentmeier
BMC Cancer , 2010, DOI: 10.1186/1471-2407-10-517
Abstract: We screened for TSG methylation using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in 40 lymphoma-derived cell lines representing anaplastic large cell lymphoma, Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma and mantle cell lymphoma (MCL) as well as in 50 primary lymphoma samples. The methylation status of differentially methylated CD44 was verified by methylation-specific PCR and bisulfite sequencing. Gene expression of CD44 and its reactivation by DNA demethylation was determined by quantitative real-time PCR and on the protein level by flow cytometry. Induction of apoptosis by anti-CD44 antibody was analyzed by annexin-V/PI staining and flow cytometry.On average 8 ± 2.8 of 24 TSG were methylated per lymphoma cell line and 2.4 ± 2 of 24 TSG in primary lymphomas, whereas 0/24 TSG were methylated in tonsils and blood mononuclear cells from healthy donors. Notably, we identified that CD44 was hypermethylated and transcriptionally silenced in all BL and most FL and DLBCL cell lines, but was usually unmethylated and expressed in MCL cell lines. Concordant results were obtained from primary lymphoma material: CD44 was not methylated in MCL patients (0/11) whereas CD44 was frequently hypermethylated in BL patients (18/29). In cell lines with CD44 hypermethylation, expression was re-inducible at mRNA and protein levels by treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine, confirming epigenetic regulation of CD44. CD44 ligation assays with a monoclonal anti-CD44 antibody showed that CD44 can mediate apoptosis in CD44+ lymphoma cells. CD44 hypermethylated, CD44- lymphoma cell lines were consistently resistant towards anti-CD44 induced apoptosis.Our data show that CD44 is epigenetically regulated in lymphoma and undergoes de novo methylation in distinct lymphoma subtypes like BL. Thus CD44 may be a promising new epigenetic marker for diagnosis and a potential therape
Ionic Liquids Applied to Improve the Dispersion of Coagent Particles in an Elastomer
Magdalena Maciejewska,Marian Zaborski
Journal of Composites , 2013, DOI: 10.1155/2013/286534
Abstract: The aim of this work was to study the activity of several ionic liquids (alkylimidazolium salts) that are used to improve the dispersion of coagent particles in peroxide-cross-linked hydrogenated acrylonitrile butadiene elastomer (HNBR). Hydrotalcite grafted with monoallyl maleate was applied as a coagent for the HNBR vulcanization. In this paper, we discuss the effect of the ionic liquids (alkylimidazolium salts) with respect to their anion (bromide, chloride, tetrafluoroborate, and hexafluorophosphate) and the length of alkyl chain in the cation (allyl-, ethyl-, butyl-, hexyl-, and octyl-) on the vulcanization kinetics of rubber compounds. The influence of ionic liquids on the cross-link density, the mechanical properties of the vulcanizates, and their resistance to weather ageing were also studied. Alkylimidazolium salts seem to improve the dispersion of the coagent particles and to be active in the cross-linking of HNBR with peroxide. The type of ionic liquid considerably influences the activity of the coagent particles toward the HNBR. The application of ionic liquids increases the cross-link density of the vulcanizates and improves their resistance to weather aging. 1. Introduction Ionic liquids (ILs) are generally defined as salts with melting points below 100°C [1, 2]. The definition of ILs clearly distinguishes them from other molten salts. Research on ILs has been one of the most rapidly growing fields in chemistry and industry in recent years. This rapid growth is mainly due to the many unique properties of ionic liquids. They are able to solvate a large variety of polar and nonpolar organic compounds and show potentially “environmentally friendly” characteristics (negligible vapor pressure, flammability) [3]. Their chemical and physical properties can be tuned for a wide range of potential applications by varying the cations and anions. The antielectrostatic properties of ILs have also been recognized [3]. In the past few years, ILs not only have been employed as solvents for various types of polymerization [4, 5] but also have been used to dissolve polymers [6, 7], to impart functionality and to create new polymer composites [8]. Applications of ILs as solvents for polymerization processes, as components of polymeric matrices (such as polymer gels), as templates for porous polymers, and as novel electrolytes for electrochemical polymerizations have been reviewed [9, 10]. However, the application of ILs to improve the dispersion of coagents based on hydrotalcite for elastomer cross-linking with peroxides has not yet been reported. Thus, this
O pesquisador frente à avalia??o na pós-gradua??o: em pauta novos modos de subjetiva??o
Axt, Margarete;
Psicologia & Sociedade , 2004, DOI: 10.1590/S0102-71822004000100006
Abstract: thinking the current capes model of post-graduation evaluation, in brazil, brings into a reflexive visibility some components of the mechanism that ended producing this subjectvation device of the researcher, since its creation in the seventies, through the process of globalization of the economy and expansion of telecommunications until the creation of the world trade organization in the nineties, followed by a stimulation of competition and individualization strategies. from the lines that come to us with several effects, configuring a complex context of brazilian post-graduation, i bring three issues to be discussed - indicators of the researcher's productivity; quality indicators defined by the qualis of their areas; and the ressuscitated fee for cnpq board -, as i am able to trace at this moment, trying to give them an outline of the scene in course, in the researcher's perspective. historically produced, these scenes, in perspective, may suggest an increasing process of shattering to the researcher in different levels: (a) inside the post-graduation program, by the continuous requirement to reach the expected quality rates; (b) in the researcher's own knowledge area, specially when working in two post-graduation programs, needing to attend to two qualis; (c) inside research groups, by asymmetries that are formed in the relations due to differentiated treatment towards cnpq scholarship holders (national council of scientific and technological development). if evaluation is a necessary condition to increase research and post-graduation education excellence, it is not, however, the only condition: it is needed to create conditions to possibilities in micropolitics instances to propose, based on collective evaluations, intervention strategies that work linking and producing new connections, in the countercurrent of the homogeneous, individualizing thinking and excluding competition. such movements, when acquiring strength and power, may provoke the emergence, in m
Pensando a educa o musical imaginativamente: uma filosofia da educa o musical por Estelle Ruth Jorgensen Thinking music education imaginatively: a philosophy of music education by Estelle Ruth Jorgensen
Margarete Arroyo
Per Musi , 2013,
Mundo da vida e pesquisa em educa o: ressonancias, implica es, replica es = World of life and researches in education: resonances, implications, responses
Axt, Margarete
Letras de Hoje , 2011,
Abstract: é propósito aqui criar algumas possibilidades de inflex o que permitam pensar em quest es postas pela via da pesquisa-forma o enquanto plano de experimenta o no ambito da própria escola. Esta modalidade investigativa afirma seu aspecto pro-ativo no sentido de que: ao mesmo tempo em que o pesquisador desenvolve seu trabalho, ele n o pode se descuidar da forma o propriamente dita, tendo em vista um cuidado ético de respeito ao outro, nesta rela o. Trata-se, também, de considerar a rela o academia-comunidade segundo um vínculo de indissociabilidade e de simultaneidade entre as dimens es universitárias pesquisa-extens o (forma o). N o mais pesquisa antecedendo e guiando extens o, mas pesquisa e extens o (forma o) fazendo-se juntas no mesmo contexto problemático em que se encontram em contato. Esta perspectiva oportuniza desenhar um plano, ao mesmo tempo de implica o-vivencia o do mundo da vida tal como se desenrola na escola, e de experimenta o-experiencia o de algumas alian as entre este mundo da vida na escola e o mundo teórico da academia, n o opondo um ao outro, antes compondo-os no tra ado de uma filosofia primeira, assim como proposta pelo filósofo russo Mikhail Bakhtin.
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