University of Frankfurt Medical School

Frankfurt am Main, Germany

University of Frankfurt Medical School

Frankfurt am Main, Germany

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Brendel C.,Paul Ehrlich Institute | Muller-Kuller U.,Paul Ehrlich Institute | Muller-Kuller U.,University of Frankfurt Medical School | Schultze-Strasser S.,Paul Ehrlich Institute | And 7 more authors.
Gene Therapy | Year: 2012

Protection against epigenetic silencing is a desirable feature of future gene therapy vectors, in particular for those applications in which transgene expression will not confer growth advantage to gene-transduced cells. The ubiquitous chromatin opening element (UCOE) consisting of the methylation-free CpG island encompassing the dual divergently transcribed promoters of the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) has been shown to shield constitutive active heterologous promoters from epigenetic modifications and chromosomal position effects. However, it is unclear if this element can be used to improve expression from tissue-specific enhancer/promoters, while maintaining tissue specificity in hematopoietic cells. Here, we evaluated the potential of the A2UCOE in combination with the myeloid-specific myeloid related protein 8 (MRP8) promoter to target transgene expression specifically to myeloid cells in vitro and in vivo from a self-inactivating lentiviral vector. The inclusion of the A2UCOE did not interfere with specific upregulation of MRP8 promoter activity during myeloid differentiation and mediated sustained and vector copy-dependent expression in myeloid cells. Notably, the A2UCOE did not protect the MRP8 promoter from methylation in the P19 embryonal carcinoma cell line, suggesting that this element maintains the inherent epigenetic state and transcriptional activity of cellular promoters in their native configuration. Thus, the A2UCOE could represent a useful protective genetic element in gene therapy vectors, ensuring physiological transcriptional regulation of tissue-specific promoters independent of the chromosomal integration site. © 2012 Macmillan Publishers Limited All rights reserved.


Schneider K.,Albert Ludwigs University of Freiburg | Weyerbrock A.,Albert Ludwigs University of Freiburg | Doostkam S.,Albert Ludwigs University of Freiburg | Plate K.,University of Frankfurt Medical School | Machein M.R.,Albert Ludwigs University of Freiburg
Journal of Neuro-Oncology | Year: 2015

Placenta growth factor (PlGF) is a member of vascular endothelial growth factor family which can promote cancer growth by various mechanisms. Placenta growth factor is upregulated in many neoplastic diseases and serum levels of PlGF are increased in cancer patients following anti-angiogenic therapy. However, its role in glioma growth is yet not fully elucidated. In this study we analyzed the expression of PlGF mRNA using real time PCR in human gliomas of different WHO grades. Placenta growth factor mRNA levels were highly variable and did not correlate with WHO grades, arguing against a significant role in glioma progression. The highest PlGF expression was observed in anaplastic astrocytomas whereas grade II astrocytomas and glioblastomas displayed lower levels of expression. Immunohistochemical analysis showed that PlGF was expressed by inflammatory and endothelial cells in addition to tumor cells. Placenta growth factor mRNA expression in 12 matched glioblastoma samples before and after therapy, including bevacizumab and cilengitide treatment was largely unaffected by the aforementioned treatment modalities. In vitro, the exposure of VEGFR-1 expressing glioma cells to bevacizumab did not increase the expression levels of PlGF mRNA. In summary, our results do not support the hypothesis that PlGF plays a major role in the resistance of gliomas after anti-angiogenic therapy. © 2014, Springer Science+Business Media New York.


Tiacci E.,University of Duisburg - Essen | Tiacci E.,University of Perugia | Doring C.,University of Frankfurt Medical School | Doring C.,Goethe University Frankfurt | And 9 more authors.
Blood | Year: 2012

The pathogenesis of classical Hodgkin lymphoma (cHL), the most common lymphoma in the young, is still enigmatic, largely because its Hodgkin and Reed-Sternberg (HRS) tumor cells are rare in the involved lymph node and therefore difficult to analyze. Here, by overcoming this technical challenge and performing, for the first time, a genome-wide transcriptional analysis of microdissected HRS cells compared with other B-cell lymphomas, cHL lines, and normal B-cell subsets, we show that they differ extensively from the usually studied cHL cell lines, that the lost B-cell identity of cHLs is not linked to the acquisition of a plasma cell-like gene expression program, and that Epstein-Barr virus infection of HRS cells has a minor transcriptional influence on the established cHL clone. Moreover, although cHL appears a distinct lymphoma entity overall, HRS cells of its histologic subtypes diverged in their similarity to other related lymphomas. Unexpectedly, we identified 2 molecular subgroups of cHL associated with differential strengths of the transcription factor activity of the NOTCH1, MYC, and IRF4 proto-oncogenes. Finally, HRS cells display deregulated expression of several genes potentially highly relevant to lymphoma pathogenesis, including silencing of the apoptosis-inducer BIK and of INPP5D, an inhibitor of the PI3K-driven oncogenic pathway. © 2012 by The American Society of Hematology.


Hahn M.,Ludwig Maximilians University of Munich | Hahn M.,Adolf Butenandt Institute | Dambacher S.,Ludwig Maximilians University of Munich | Dambacher S.,Adolf Butenandt Institute | And 26 more authors.
Genes and Development | Year: 2013

Cohesin plays an important role in chromatid cohesion and has additional functions in higher-order chromatin organization and in transcriptional regulation. The binding of cohesin to euchromatic regions is largely mediated by CTCF or the mediator complex. However, it is currently unknown how cohesin is recruited to pericentric heterochromatin in mammalian cells. Here we define the histone methyltransferase Suv4-20h2 as a major structural constituent of heterochromatin that mediates chromatin compaction and cohesin recruitment. Suv4- 20h2 stably associates with pericentric heterochromatin through synergistic interactions with multiple heterochromatin protein 1 (HP1) molecules, resulting in compaction of heterochromatic regions. Suv4-20h mutant cells display an overall reduced chromatin compaction and an altered chromocenter organization in interphase referred to as "chromocenter scattering." We found that Suv4-20h-deficient cells display chromosome segregation defects during mitosis that coincide with reduced sister chromatid cohesion. Notably, cohesin subunits interact with Suv4-20h2 both in vitro and in vivo. This interaction is necessary for cohesin binding to heterochromatin, as Suv4- 20h mutant cells display substantially reduced cohesin levels at pericentric heterochromatin. This defect is most prominent in G0-phase cells, where cohesin is virtually lost from heterochromatin, suggesting that Suv4-20h2 is involved in the initial loading or maintenance of cohesion subunits. In summary, our data provide the first compelling evidence that Suv4-20h2 plays essential roles in regulating nuclear architecture and ensuring proper chromosome segregation. © 2013 by Cold Spring Harbor Laboratory Press.


Markoutsa S.,Goethe University Frankfurt | Surun D.,University of Frankfurt Medical School | Karas M.,Goethe University Frankfurt | Hofmann B.,Goethe University Frankfurt | And 2 more authors.
FEBS Journal | Year: 2014

The enzyme 5-lipoxygenase (5-LO) catalyzes the first reactions in the biosynthesis of leukotrienes, powerful lipid mediators that are involved in several physiological and pathological processes. 5-LO activity is tightly regulated by several factors, including post translational modifications (PTMs). Phosphorylations of 5-LO by the kinases extracellular signal-regulated kinase 2 (Erk2), mitogen-activated protein kinase activated protein kinase 2 (MK2) and protein kinase A (PKA) have been described to regulate 5-LO activity. Furthermore, 5-LO phosphorylation is considered a determinant of drug candidate potency. However, no evidence on a molecular level, as can be provided by MS, has as yet been presented for these PTMs. Here, we employ a workflow including different proteolytic cleavages and phosphopeptide enrichment for detection of 5-LO phosphorylation by MALDI-MS. Proof for the known phosphorylation sites of MK2 (Ser271) and PKA (Ser523) was provided by MS after in vitro phosphorylation, but not for the postulated Erk2 site (Ser663). Detection limits have been determined for all three sites. Moreover, we identified novel tyrosine kinase target sites within 5-LO using in silico and in vitro methods. Tyr42, Tyr53 and either Tyr94 or Tyr445 were phosphorylated by the Src kinases Fgr, hematopoietic cell kinase (HCK) and Yes. To analyze the phosphorylation state in the cellular context, we created stably 5-LO-transduced Mono Mac 6 cells. Here, we only detected phospho-Ser271 by MS, whereas immunoblot analysis indicated tyrosine phosphorylation, phospho-Ser271 and phospho-Ser663. Unexpectedly, phospho-Ser271 occurred independent of cell stimulation. Taken together, we describe a method for the molecular analysis of 5-LO phosphorylation, provide insights regarding the occurrence of known phosphorylation sites partly in contrast to earlier studies and present first evidence on novel phosphosites. © 2014 FEBS.


Heidler J.,University of Frankfurt Medical School | Strecker V.,Goethe University Frankfurt | Csintalan F.,Goethe University Frankfurt | Bleier L.,Goethe University Frankfurt | Wittig I.,Goethe University Frankfurt
Methods in Molecular Biology | Year: 2013

Blue native electrophoresis (BNE) is a long established method for the analysis of native protein complexes. Applications of BNE range from investigating subunit composition, stoichiometry, and assembly of single protein complexes to profiling of whole complexomes. BNE is an indispensible tool to diagnostically analyze cells and tissues from patients with mitochondrial disorders or model organisms. Since functional proteomic studies often require quantification of protein complexes, we describe here different quantification methods subsequent to protein complex separation by BNE. © 2013 Springer Science+Business Media, LLC.


Wempe F.,University of Frankfurt Medical School | De-Zolt S.,University of Frankfurt Medical School | Koli K.,University of Frankfurt Medical School | Bangsow T.,University of Frankfurt Medical School | And 6 more authors.
Disease models & mechanisms | Year: 2010

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Cigarette smoking has been identified as one of the major risk factors and several predisposing genetic factors have been implicated in the pathogenesis of COPD, including a single nucleotide polymorphism (SNP) in the latent transforming growth factor (TGF)-beta binding protein 4 (Ltbp4)-encoding gene. Consistent with this finding, mice with a null mutation of the short splice variant of Ltbp4 (Ltbp4S) develop pulmonary emphysema that is reminiscent of COPD. Here, we report that the mutational inactivation of the antioxidant protein sestrin 2 (sesn2) partially rescues the emphysema phenotype of Ltbp4S mice and is associated with activation of the TGF-beta and mammalian target of rapamycin (mTOR) signal transduction pathways. The results suggest that sesn2 could be clinically relevant to patients with COPD who might benefit from antagonists of sestrin function.


Kippenberger S.,University of Frankfurt Medical School | Hofmann M.,University of Frankfurt Medical School | Zoller N.,University of Frankfurt Medical School | Thaci D.,University of Frankfurt Medical School | And 3 more authors.
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2010

In normal epithelial cells hemidesmosomes mediate stable adhesion to the underlying basement membrane. In carcinoma cells a functional and spatial dissociation of the hemidesmosomal complex is observed stimulating the hypothesis that the β4 integrin may trigger essential signalling cascades determining cell fate. In the present study we dissected the signalling pathways giving rise to PKB/Akt and ERK1/2 activation in response to β4 ligation by 3E1. It was found that the activation of PKB/Akt is sensitive towards alterations of the keratin filament as demonstrated by using KEB-7 cells that carry a keratin mutation typical for epidermolysis bullosa simplex. Similar results were achieved by chemically induced keratin aggregations. Of note, the signalling to ERK1/2 was not affected. ERK1/2 activation utilizes an EGF-R transactivation mechanism as shown by dominant-negative expression experiments and also by treatment with a specific inhibitor (AG1478). Downstream from the EGF-R the activation of ERK1/2 takes the prototypical signalling cascade via Shc, Ras and Raf-1 as demonstrated by dominant-negative expression experiments. Taken together our data define a new model of β4-dependent PKB/Akt and ERK1/2 activation demonstrating the keratin filament as a structure necessary in signal transmission. © 2010 Elsevier B.V.


Patent
University Of Frankfurt Medical School | Date: 2010-11-24

The present invention relates to a method of producing a cell comprising a conditionally active transgene in its genome, the method comprising (a) introducing into the cell a targeting vector, wherein the targeting vector comprises (i) a 5 recombinase recognition site specifically recognised by a first recombinase, wherein the first recombinase is endogenously present in the cell or wherein the first recombinase or a nucleic acid molecule encoding said first recombinase in expressible form is introduced into the cell; followed by (ii) a 5 recombinase recognition site specifically recognised by a second recombinase, wherein the second recombinase is not endogenously present or is not active in the cell; followed by (iii) a selection cassette comprising a positively selectable marker gene; followed by (iv) a 3 recombinase recognition site specifically recognised by a third recombinase, wherein the third recombinase is not endogenously present or is not active in the cell; followed by (v) the transgene; followed by (vi) a 3 recombinase recognition site specifically recognised by a fourth recombinase, wherein the fourth recombinase is endogenously present in the cell or wherein the fourth recombinase or a nucleic acid molecule encoding said fourth recombinase in expressible form is introduced into the cell; wherein the genome of the cell comprises a 5 recombinase recognition site and a 3 recombinase recognition site that are identical to the recombinase recognition sites of (i) and (vi), and wherein said recombinase recognition sites comprised in the genome of the cell are located 3 of an endogenous cellular promoter such that introduction of the targeting vector into the genome by site specific recombination results in the promoter being operatively linked to the selectable marker gene; and (b) culturing the cell in the presence of a selection medium specific for the selectable marker encoded by the selectable marker gene of (iii). The present invention further relates to a method of producing a conditional transgenic non-human mammalian animal as well as to a conditional transgenic non-human mammalian animal obtainable by said method. The present invention also relates to a transgenic TDP-43 mouse, comprising a transgenic cassette in intron 1 of the mouse Tardbp gene.


Patent
University Of Frankfurt Medical School | Date: 2011-06-01

The present invention relates to a method of producing a cell comprising a conditionally active transgene in its genome, the method comprising (a) introducing into the cell a targeting vector, wherein the targeting vector comprises (i) a 5 recombinase recognition site specifically recognised by a first recombinase, wherein the first recombinase is endogenously present in the cell or wherein the first recombinase or a nucleic acid molecule encoding said first recombinase in expressible form is introduced into the cell; followed by (ii) a 5 recombinase recognition site specifically recognised by a second recombinase, wherein the second recombinase is not endogenously present or is not active in the cell; followed by (iii) a selection cassette comprising a positively selectable marker gene; followed by (iv) a 3 recombinase recognition site specifically recognised by a third recombinase, wherein the third recombinase is not endogenously present or is not active in the cell; followed by (v) the transgene; followed by (vi) a 3 recombinase recognition site specifically recognised by a fourth recombinase, wherein the fourth recombinase is endogenously present in the cell or wherein the fourth recombinase or a nucleic acid molecule encoding said fourth recombinase in expressible form is introduced into the cell; wherein the genome of the cell comprises a 5 recombinase recognition site and a 3 recombinase recognition site that are identical to the recombinase recognition sites of (i) and (vi), and wherein said recombinase recognition sites comprised in the genome of the cell are located 3 of an endogenous cellular promoter such that introduction of the targeting vector into the genome by site specific recombination results in the promoter being operatively linked to the selectable marker gene; and (b) culturing the cell in the presence of a selection medium specific for the selectable marker encoded by the selectable marker gene of (iii). The present invention further relates to a method of producing a conditional transgenic non-human mammalian animal as well as to a conditional transgenic non-human mammalian animal obtainable by said method. The present invention also relates to a transgenic TDP-43 mouse, comprising a transgenic cassette in intron 1 of the mouse Tardbp gene.

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