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Search Results: 1 - 10 of 197105 matches for " Hans G Drexler "
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Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin
Cord C. Uphoff,Sabine-A. Denkmann,Hans G. Drexler
Journal of Biomedicine and Biotechnology , 2012, DOI: 10.1155/2012/267678
Abstract: A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day
Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II, and SMRV in Human and Other Primate Cell Lines
Cord C. Uphoff,Sabine A. Denkmann,Klaus G. Steube,Hans G. Drexler
Journal of Biomedicine and Biotechnology , 2010, DOI: 10.1155/2010/904767
Abstract: The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections.
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
Methylation of miR-34a, miR-34b/c, miR-124-1 and miR-203 in Ph-negative myeloproliferative neoplasms
Chor Sang Chim, Thomas S Wan, Kwan Yeung Wong, Tsz Kin Fung, Hans G Drexler, Kit Fai Wong
Journal of Translational Medicine , 2011, DOI: 10.1186/1479-5876-9-197
Abstract: We studied DNA methylation of these miRs in Philadelphia-negative (Ph-ve) myeloproliferative neoplasms (MPNs). Methylation-specific PCR (MSP), verified by direct sequencing of the methylated MSP products, was performed in cell lines, normal controls and diagnostic marrow samples of patients with MPNs.Methylation of these miRs was absent in the normal controls. miR-34b/c were homozygously methylated in HEL cells but heterozygously in MEG-01. In HEL cells, homozygous miR-34b/c methylation was associated with miR silencing, and 5-aza-2'-deoxycytidine treatment led to re-expression of both miR-34b and miR-34c, consistent with that both miRs are under the regulation of the same promoter CpG island. miR-34a was heterozygously methylated in MEG-01 and K-562. miR-203 was completely unmethylated in K-562 and SET-2 but no MSP amplification was found in both HEL and MEG-01, suggestive of miR deletion. In primary samples, four each had miR-34b/c and -203 methylation, in which two had concomitant methylation of miR-34b/c and -203. miR-34a was methylated in one patient and none had methylation of miR-124-1. Seven patients (15.6%) had methylation of at least one of the four miRs. miR methylation did not correlate with clinical parameters, disease complications or JAK2 V617F mutation.This is the first report of miR hypermethylation in MPNs. miR-203 hypermethylation is not specific to Ph+ve leukemias but also present in Ph-ve MPNs. miR-34b/c methylation was associated with reversible miR silencing. There was no correlation of miR methylation with clinical demographic data or outcome.Philadelphia-negative (Ph-ve) myeloproliferative neoplasm (MPN) is a stem cell disease with proliferation of myeloid lineage, leading to the development of distinct clinical entities including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) [1-3]. JAK2 V617F mutation, resulting in constitutive activation of JAK-STAT signaling, occurs in about half of the patients wi
Polycomb repressor complex 2 regulates HOXA9 and HOXA10, activating ID2 in NK/T-cell lines
Stefan Nagel, Letizia Venturini, Victor E Marquez, Corinna Meyer, Maren Kaufmann, Michaela Scherr, Roderick AF MacLeod, Hans G Drexler
Molecular Cancer , 2010, DOI: 10.1186/1476-4598-9-151
Abstract: This analysis showed high expression levels of HOXA9, HOXA10 and ID2 in NK-cell lines in addition to T-cell line LOUCY, suggesting leukemic deregulation therein. Overexpression experiments, chromatin immuno-precipitation and promoter analysis demonstrated that HOXA9 and HOXA10 directly activated expression of ID2. Concomitantly elevated expression levels of HOXA9 and HOXA10 together with ID2 in cell lines containing MLL translocations confirmed this form of regulation in both ALL and acute myeloid leukemia. Overexpression of HOXA9, HOXA10 or ID2 resulted in repressed expression of apoptosis factor BIM. Furthermore, profiling data of genes coding for chromatin regulators of homeobox genes, including components of polycomb repressor complex 2 (PRC2), indicated lacking expression of EZH2 in LOUCY and exclusive expression of HOP in NK-cell lines. Subsequent treatment of T-cell lines JURKAT and LOUCY with DZNep, an inhibitor of EZH2/PRC2, resulted in elevated and unchanged HOXA9/10 expression levels, respectively. Moreover, siRNA-mediated knockdown of EZH2 in JURKAT enhanced HOXA10 expression, confirming HOXA10-repression by EZH2. Additionally, profiling data and overexpression analysis indicated that reduced expression of E2F cofactor TFDP1 contributed to the lack of EZH2 in LOUCY. Forced expression of HOP in JURKAT cells resulted in reduced HOXA10 and ID2 expression levels, suggesting enhancement of PRC2 repression.Our results show that major differentiation factors of the NK-cell lineage, including HOXA9, HOXA10 and ID2, were (de)regulated via PRC2 which therefore contributes to T-cell leukemogenesis.Adult lymphopoiesis starts with progenitor cells which originate from CD34+ hematopoietic stem cells (HSC) in the bone marrow. While the development of natural killer (NK)- cells completes primarily in the bone marrow, T-cells finalize their differentiation in the thymus [1-3]. Nevertheless, the facts that NK-cell differentiation also occurs in the thymus and early thymoc
Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications
Stefan Nagel, Stefan Ehrentraut, Jürgen Tomasch, Hilmar Quentmeier, Corinna Meyer, Maren Kaufmann, Hans G. Drexler, Roderick A. F. MacLeod
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0061447
Abstract: Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies.
Metal Ion Concentrations in Body Fluids after Implantation of Hip Replacements with Metal-on-Metal Bearing – Systematic Review of Clinical and Epidemiological Studies
Albrecht Hartmann, Franziska Hannemann, J?rg Lützner, Andreas Seidler, Hans Drexler, Klaus-Peter Günther, Jochen Schmitt
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0070359
Abstract: Introduction The use of metal-on-metal (MoM) total hip arthroplasty (THA) increased in the last decades. A release of metal products (i.e. particles, ions, metallo-organic compounds) in these implants may cause local and/or systemic adverse reactions. Metal ion concentrations in body fluids are surrogate measures of metal exposure. Objective To systematically summarize and critically appraise published studies concerning metal ion concentrations after MoM THA. Methods Systematic review of clinical trials (RCTs) and epidemiological studies with assessment of metal ion levels (cobalt, chromium, titanium, nickel, molybdenum) in body fluids after implantation of metalliferous hip replacements. Systematic search in PubMed and Embase in January 2012 supplemented by hand search. Standardized abstraction of pre- and postoperative metal ion concentrations stratified by type of bearing (primary explanatory factor), patient characteristics as well as study quality characteristics (secondary explanatory factors). Results Overall, 104 studies (11 RCTs, 93 epidemiological studies) totaling 9.957 patients with measurement of metal ions in body fluids were identified and analyzed. Consistently, median metal ion concentrations were persistently elevated after implantation of MoM-bearings in all investigated mediums (whole blood, serum, plasma, erythrocytes, urine) irrespective of patient characteristics and study characteristics. In several studies very high serum cobalt concentrations above 50 μg/L were measured (detection limit typically 0.3 μg/L). Highest metal ion concentrations were observed after treatment with stemmed large-head MoM-implants and hip resurfacing arthroplasty. Discussion Due to the risk of local and systemic accumulation of metallic products after treatment with MoM-bearing, risk and benefits should be carefully balanced preoperatively. The authors support a proposed ?time out“ for stemmed large-head MoM-THA and recommend a restricted indication for hip resurfacing arthroplasty. Patients with implanted MoM-bearing should receive regular and standardized monitoring of metal ion concentrations. Further research is indicated especially with regard to potential systemic reactions due to accumulation of metal products.
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