Publications by authors named "Patrick Petzsch"

23 Publications

  • Page 1 of 1

The transcription factor reservoir and chromatin landscape in activated plasmacytoid dendritic cells.

BMC Genom Data 2021 Sep 20;22(1):37. Epub 2021 Sep 20.

Institute of Medical Microbiology and Hospital Hygiene, University of Düsseldorf, Düsseldorf, Germany.

Background: Transcription factors (TFs) control gene expression by direct binding to regulatory regions of target genes but also by impacting chromatin landscapes and modulating DNA accessibility for other TFs. In recent years several TFs have been defined that control cell fate decisions and effector functions in the immune system. Plasmacytoid dendritic cells (pDCs) are an immune cell type with the unique capacity to produce high amounts of type I interferons quickly in response to contact with viral components. Hereby, this cell type is involved in anti-infectious immune responses but also in the development of inflammatory and autoimmune diseases. To date, the global TF reservoir in pDCs early after activation remains to be fully characterized.

Results: To fill this gap, we have performed a comprehensive analysis in naïve versus TLR9-activated murine pDCs in a time course study covering early timepoints after stimulation (2 h, 6 h, 12 h) integrating gene expression (RNA-Seq) and chromatin landscape (ATAC-Seq) studies. To unravel the biological processes underlying the changes in TF expression on a global scale gene ontology (GO) analyses were performed. We found that 70% of all genes annotated as TFs in the mouse genome (1014 out of 1636) are expressed in pDCs for at least one stimulation time point and are covering a wide range of TF classes defined by their specific DNA binding mechanisms. GO analysis revealed involvement of TLR9-induced TFs in epigenetic modulation, NFκB and JAK-STAT signaling, and protein production in the endoplasmic reticulum. pDC activation predominantly "turned on" the chromatin regions associated with TF genes. Our in silico analyses pointed at the AP-1 family of TFs as less noticed but possibly important players in these cells after activation. AP-1 family members exhibit (1) increased gene expression, (2) enhanced chromatin accessibility in their promoter region, and (3) a TF DNA binding motif that is globally enriched in genomic regions that were found more accessible in pDCs after TLR9 activation.

Conclusions: In this study we define the complete set of TLR9-regulated TFs in pDCs. Further, this study identifies the AP-1 family of TFs as potentially important but so far less well characterized regulators of pDC function.
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http://dx.doi.org/10.1186/s12863-021-00991-2DOI Listing
September 2021

Microglia contributes to remyelination in cerebral but not spinal cord ischemia.

Glia 2021 Nov 14;69(11):2739-2751. Epub 2021 Aug 14.

Department of Neurology, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany.

Inflammation after injury of the central nervous system (CNS) is increasingly viewed as a therapeutic target. However, comparative studies in different CNS compartments are sparse. To date only few studies based on immunohistochemical data and all referring to mechanical injury have directly compared inflammation in different CNS compartments. These studies revealed that inflammation is more pronounced in spinal cord than in brain. Therefore, it is unclear whether concepts and treatments established in the cerebral cortex can be transferred to spinal cord lesions and vice versa or whether immunological treatments must be adapted to different CNS compartments. By use of transcriptomic and flow cytometry analysis of equally sized photothrombotically induced lesions in the cerebral cortex and the spinal cord, we could document an overall comparable inflammatory reaction and repair activity in brain and spinal cord between day 1 and day 7 after ischemia. However, remyelination was increased after cerebral versus spinal cord ischemia which is in line with increased remyelination in gray matter in previous analyses and was accompanied by microglia dominated inflammation opposed to monocytes/macrophages dominated inflammation after spinal cord ischemia. Interestingly remyelination could be reduced by microglia and not hematogenous macrophage depletion. Our results show that despite different cellular composition of the postischemic infiltrate the inflammatory response in cerebral cortex and spinal cord are comparable between day 1 and day 7. A striking difference was higher remyelination capacity in the cerebral cortex, which seems to be supported by microglia dominance.
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http://dx.doi.org/10.1002/glia.24068DOI Listing
November 2021

The signal transducer CD24 suppresses the germ cell program and promotes an ectodermal rather than mesodermal cell fate in embryonal carcinomas.

Mol Oncol 2021 Jul 22. Epub 2021 Jul 22.

Department of Urology, Urological Research Laboratory, Translational UroOncology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Germany.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.
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http://dx.doi.org/10.1002/1878-0261.13066DOI Listing
July 2021

Lymphotoxin-β-receptor (LTβR) signaling on hepatocytes is required for liver regeneration after partial hepatectomy.

Biol Chem 2021 Aug 7;402(9):1147-1154. Epub 2021 Jun 7.

Institute of Medical Microbiology and Hospital Hygiene, Medical Faculty, Heinrich Heine University Düsseldorf, University Hospital Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany.

Lymphotoxin-β-receptor deficient (LTβR) and Tumor Necrosis Factor Receptor p55 deficient (TNFRp55) mice show defects in liver regeneration (LR) after partial hepatectomy (PHx) with significantly increased mortality. LTβR and TNFRp55 belong to the core members of the TNF/TNFR superfamily. Interestingly, combined failure of LTβR and TNFRp55 signaling after PHx leads to a complete defect in LR. Here, we first addressed the question which liver cell population crucially requires LTβR signaling for efficient LR. To this end, mice with a conditionally targeted LTβR allele (LTβR) were crossed to AlbuminCre and LysozymeMCre mouse lines to unravel the function of the LTβR on hepatocytes and monocytes/macrophages/Kupffer cells, respectively. Analysis of these mouse lines clearly reveals that LTβR is required on hepatocytes for efficient LR while no deficit in LR was found in LTβR × LysMCre mice. Second, the molecular basis for the cooperating role of LTβR and TNFRp55 signaling pathways in LR was investigated by transcriptome analysis of etanercept treated LTβR (LTβR/ET) mice. Bioinformatic analysis and subsequent verification by qRT-PCR identified novel target genes (Cyclin-L2, Fas-Binding factor 1, interferon-related developmental regulator 1, Leucyl-tRNA Synthetase 2, and galectin-4) that are upregulated by LTβR/TNFRp55 signaling after PHx and fail to be upregulated after PHx in LTβR/ET mice.
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http://dx.doi.org/10.1515/hsz-2021-0152DOI Listing
August 2021

Acute myeloid leukemia-induced functional inhibition of healthy CD34+ hematopoietic stem and progenitor cells.

Stem Cells 2021 Sep 20;39(9):1270-1284. Epub 2021 May 20.

Department of Hematology, Oncology and Clinical Immunology, Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany.

Acute myeloid leukemia (AML) is characterized by an expansion of leukemic cells and a simultaneous reduction of normal hematopoietic precursors in the bone marrow (BM) resulting in hematopoietic insufficiency, but the underlying mechanisms are poorly understood in humans. Assuming that leukemic cells functionally inhibit healthy CD34+ hematopoietic stem and progenitor cells (HSPC) via humoral factors, we exposed healthy BM-derived CD34+ HSPC to cell-free supernatants derived from AML cell lines as well as from 24 newly diagnosed AML patients. Exposure to AML-derived supernatants significantly inhibited proliferation, cell cycling, colony formation, and differentiation of healthy CD34+ HSPC. RNA sequencing of healthy CD34+ HSPC after exposure to leukemic conditions revealed a specific signature of genes related to proliferation, cell-cycle regulation, and differentiation, thereby reflecting their functional inhibition on a molecular level. Experiments with paired patient samples showed that these inhibitory effects are markedly related to the immunomagnetically enriched CD34+ leukemic cell population. Using PCR, ELISA, and RNA sequencing, we detected overexpression of TGFβ1 in leukemic cells on the transcriptional and protein level and, correspondingly, a molecular signature related to TGFβ1 signaling in healthy CD34+ HSPC. This inhibitory effect of TGFβ1 on healthy hematopoiesis was functionally corrobated and could be pharmacologically reverted by SD208, an inhibitor of TGFβ receptor 1 signaling. Overall, these data indicate that leukemic cells induce functional inhibition of healthy CD34+ HSPC, at least in part, through TGFβ1, suggesting that blockage of this pathway may improve hematopoiesis in AML.
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http://dx.doi.org/10.1002/stem.3387DOI Listing
September 2021

Mobile SAR signal N-hydroxypipecolic acid induces NPR1-dependent transcriptional reprogramming and immune priming.

Plant Physiol 2021 Apr 19. Epub 2021 Apr 19.

Institute for Molecular Ecophysiology of Plants, Department of Biology, Heinrich Heine University, Universitätsstraße 1, D-40225 Düsseldorf, Germany.

N-Hydroxypipecolic acid (NHP) accumulates in the plant foliage in response to a localized microbial attack and induces systemic acquired resistance (SAR) in distant leaf tissue. Previous studies indicated that pathogen inoculation of Arabidopsis (Arabidopsis thaliana) systemically activates SAR-related transcriptional reprogramming and a primed immune status in strict dependence of FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), which mediates the endogenous biosynthesis of NHP. Here, we show that elevations of NHP by exogenous treatment are sufficient to induce a SAR-reminiscent transcriptional response that mobilizes key components of immune surveillance and signal transduction. Exogenous NHP primes Arabidopsis wild-type and NHP-deficient fmo1 plants for a boosted induction of pathogen-triggered defenses, such as the biosynthesis of the stress hormone salicylic acid (SA), accumulation of the phytoalexin camalexin and branched-chain amino acids, as well as expression of defense-related genes. NHP also sensitizes the foliage systemically for enhanced SA-inducible gene expression. NHP-triggered SAR, transcriptional reprogramming and defense priming are fortified by SA accumulation and require the function of the transcriptional co-regulator NON-EXPRESSOR OF PR GENES1 (NPR1). Our results suggest that NPR1 transduces NHP-activated immune signaling modes with predominantly SA-dependent and minor SA-independent features. They further support the notion that NHP functions as a mobile immune regulator capable of moving independently of active SA signalling between leaves to systemically activate immune responses.
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http://dx.doi.org/10.1093/plphys/kiab166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260123PMC
April 2021

Lymphotoxin β Receptor: a Crucial Role in Innate and Adaptive Immune Responses against Toxoplasma gondii.

Infect Immun 2021 05 17;89(6). Epub 2021 May 17.

Institute of Medical Microbiology and Hospital Hygiene, Heinrich Heine University, Düsseldorf, Germany

The lymphotoxin β receptor (LTβR) plays an essential role in the initiation of immune responses to intracellular pathogens. In mice, the LTβR is crucial for surviving acute toxoplasmosis; however, until now, a functional analysis was largely incomplete. Here, we demonstrate that the LTβR is a key regulator required for the intricate balance of adaptive immune responses. -infected LTβR-deficient (LTβR) mice show globally altered interferon-γ (IFN-γ) regulation, reduced IFN-γ-controlled host effector molecule expression, impaired T cell functionality, and an absent anti-parasite-specific IgG response, resulting in a severe loss of immune control of the parasites. Reconstitution of LTβR mice with toxoplasma immune serum significantly prolongs survival following infection. Notably, analysis of RNA-seq data clearly indicates a specific effect of infection on the B cell response and isotype switching. This study uncovers the decisive role of the LTβR in cytokine regulation and adaptive immune responses to control .
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http://dx.doi.org/10.1128/IAI.00026-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316152PMC
May 2021

TBBPA targets converging key events of human oligodendrocyte development resulting in two novel AOPs

ALTEX 2021 23;38(2):215-234. Epub 2020 Oct 23.

IUF - Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany.

Myelinating oligodendrocytes (OLs) establish saltatory nerve conduction during white matter development. Thus, interference with oligodendrogenesis leads to an adverse outcome on brain performance in the child due to aberrant myelination. An intertwined network of hormonal, transcriptional and biosynthetic processes regulates OL development, thereby simultaneously creating various routes of interference for environmental toxicants. The flame retardant tetrabromobisphenol A (TBBPA) is debated as an endocrine disruptor, especially of the thyroid hormone (TH) system. We identified how TBBPA interferes with the establishment of a population of maturing OLs by two independent modes-of-action (MoA), dependent and independent of TH signaling. Combining the previously published oligodendrocyte maturation assay (NPC6) with large-scale transcriptomics, we describe TBBPA as a TH disruptor, impairing human OL maturation in vitro by dysregulation of oligodendrogenesis-associated genes (i.e., MBP, KLF9 and EGR1). Furthermore, TBBPA disrupts a gene expression network regulating cholesterol homeostasis, reducing OL numbers independently of TH signaling. These two MoA converge in a novel putative adverse outcome pathway (AOP) network on the key event (KE) hypomyelination. Comparative analyses of human and rat neural progenitor cells (NPCs) revealed that human oligodendrogenesis is more sensitive to endocrine disruption by TBBPA. Therefore, ethical, cost-efficient and species-overarching in vitro assays are needed for developmental neurotoxicity hazard assessment. By incorporation of large-scale transcriptomic analyses, we brought the NPC6 assay to a higher readiness level for future applications in a regulatory context. The combination of phenotypic and transcriptomic analyses helps to study MoA to eventually build AOPs for a better understanding of neurodevelopmental toxicity.
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http://dx.doi.org/10.14573/altex.2007201DOI Listing
October 2020

CDK4/6 inhibition presents as a therapeutic option for paediatric and adult germ cell tumours and induces cell cycle arrest and apoptosis via canonical and non-canonical mechanisms.

Br J Cancer 2020 08 18;123(3):378-391. Epub 2020 May 18.

Department of Urology, Urological Research Lab, Translational UroOncology, University Hospital Düsseldorf, Düsseldorf, Germany.

Background: Germ cell tumours (GCTs) are the most common solid malignancies in young men. Although high cure rates can be achieved, metastases, resistance to cisplatin-based therapy and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In this study, we analysed the potential of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors palbociclib and ribociclib (PaRi) as molecular drugs to treat cisplatin-resistant and -sensitive paediatric and adult GCTs.

Methods: Ten GCT cell lines, including cisplatin-resistant subclones and non-malignant controls, were treated with PaRi and screened for changes in viability (triphenyl tetrazolium chloride (XTT) assay), apoptosis rates (flow cytometry, caspase assay), the cell cycle (flow cytometry), the transcriptome (RNA-sequencing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and on protein level (western blot). Expression profiling was performed on paediatric and adult GCT tissues (expression microarrays, qRT-PCR, immunohistochemistry, 'The Cancer Genome Atlas' database).

Results: We demonstrate that adult GCTs highly express CDK4, while paediatric GCTs strongly express CDK6 instead. Thus, both GCT types are potentially treatable by PaRi. GCTs presented as highly sensitive towards PaRi, which caused a decrease in viability, cell cycle arrest and apoptosis. Although GCTs mainly arrested in the G1/G0 phase, some embryonal carcinoma cell lines were able to bypass the G1/S checkpoint and progressed to the G2/M phase. We found that upregulation of CDK3 and downregulation of many mitosis regulation factors, like the HAUS genes, might be responsible for bypassing the G1/S checkpoint and termination of mitosis, respectively. We postulate that GCT cells do not tolerate these alterations in the cell cycle and eventually induce apoptosis.

Conclusion: Our study highlights PaRi as therapeutic options for cisplatin-resistant and -sensitive paediatric and adult GCTs.
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http://dx.doi.org/10.1038/s41416-020-0891-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403155PMC
August 2020

Knockdown of UTX/KDM6A Enriches Precursor Cell Populations in Urothelial Cell Cultures and Cell Lines.

Cancers (Basel) 2020 Apr 21;12(4). Epub 2020 Apr 21.

Department of Urology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany.

The histone demethylase UTX (gene: ) directs cell and tissue differentiation during development. Deleterious mutations in occur in many human cancers, most frequently in urothelial carcinoma. The consequences of these mutations are poorly understood; plausibly, they may disturb urothelial differentiation. We therefore investigated the effects of UTX siRNA-mediated knockdown in two in vitro models of urothelial differentiation; namely, primary cultures of urothelial epithelial cells treated with troglitazone and PD153035 and the immortalized urothelial cell line HBLAK treated with high calcium and serum. In both models, efficient UTX knockdown did not block morphological and biochemical differentiation. An apparent delay was due to a cytotoxic effect on the cell cultures before the initiation of differentiation, which induced apoptosis partly in a p53-dependent manner. As a consequence, slowly cycling, smaller, KRT14 precursor cells in the HBLAK cell line were enriched at the expense of more differentiated, larger, proliferating KRT14 cells. UTX knockdown induced apoptosis and enriched KRT14 cells in the BFTC-905 papillary urothelial carcinoma cell line as well. Our findings suggest an explanation for the frequent occurrence of mutations across all stages and molecular subtypes of urothelial carcinoma, whereby loss of UTX function does not primarily impede later stages of urothelial differentiation, but favors the expansion of precursor populations to provide a reservoir of potential tumor-initiating cells.
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http://dx.doi.org/10.3390/cancers12041023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226239PMC
April 2020

Characterization and application of electrically active neuronal networks established from human induced pluripotent stem cell-derived neural progenitor cells for neurotoxicity evaluation.

Stem Cell Res 2020 05 10;45:101761. Epub 2020 Mar 10.

IUF - Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany; Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany. Electronic address:

Neurotoxicity is mediated by a variety of modes-of-actions leading to disturbance of neuronal function. In order to screen larger numbers of compounds for their neurotoxic potential, in vitro functional neuronal networks (NN) might be helpful tools. We established and characterized human NN (hNN) from hiPSC-derived neural progenitor cells by comparing hNN formation with two different differentiation media: in presence (CINDA) and absence (neural differentiation medium (NDM)) of maturation-supporting factors. As a NN control we included differentiating rat NN (rNN) in the study. Gene/protein expression and electrical activity from in vitro developing NN were assessed at multiple time points. Transcriptomes of 5, 14 and 28 days in vitro CINDA-grown hNN were compared to gene expression profiles of in vivo human developing brains. Molecular expression analyses as well as measures of electrical activity indicate that NN mature into neurons of different subtypes and astrocytes over time. In contrast to rNN, hNN are less electrically active within the same period of differentiation time, yet hNN grown in CINDA medium develop higher firing rates than hNN without supplements. Challenge of NN with neuronal receptor stimulators and inhibitors demonstrate presence of inhibitory, GABAergic neurons, whereas glutamatergic responses are limited. hiPSC-derived GABAergic hNN grown in CINDA medium might be a useful tool as part of an in vitro battery for assessing neurotoxicity.
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http://dx.doi.org/10.1016/j.scr.2020.101761DOI Listing
May 2020

AHR Signaling Dampens Inflammatory Signature in Neonatal Skin γδ T Cells.

Int J Mol Sci 2020 Mar 24;21(6). Epub 2020 Mar 24.

IUF-Leibniz Research Institute for Environmental Medicine, Auf'm Hennekamp 50, D-40225 Düsseldorf, Germany.

Background Aryl hydrocarbon receptor (AHR)-deficient mice do not support the expansion of dendritic epidermal T cells (DETC), a resident immune cell population in the murine epidermis, which immigrates from the fetal thymus to the skin around birth. Material and Methods In order to identify the gene expression changes underlying the DETC disappearance in AHR-deficient mice, we analyzed microarray RNA-profiles of DETC, sorted from the skin of two-week-old AHR-deficient mice and their heterozygous littermates. In vitro studies were done for verification, and IL-10, AHR repressor (AHRR), and c-Kit deficient mice analyzed for DETC frequency. Results We identified 434 annotated differentially expressed genes. Gene set enrichment analysis demonstrated that the expression of genes related to proliferation, ion homeostasis and morphology differed between the two mouse genotypes. Importantly, with 1767 pathways the cluster-group "inflammation" contained the majority of AHR-dependently regulated pathways. The most abundant cluster of differentially expressed genes was "inflammation." DETC of AHR-deficient mice were inflammatory active and had altered calcium and F-actin levels. Extending the study to the AHRR, an enigmatic modulator of AHR-activity, we found approximately 50% less DETC in AHRR-deficient mice than in wild-type-littermates. Conclusion AHR-signaling in DETC dampens their inflammatory default potential and supports their homeostasis in the skin.
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http://dx.doi.org/10.3390/ijms21062249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139545PMC
March 2020

Combination of Decitabine and Entinostat Synergistically Inhibits Urothelial Bladder Cancer Cells via Activation of FoxO1.

Cancers (Basel) 2020 Feb 3;12(2). Epub 2020 Feb 3.

Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Duesseldorf, 40225 Duesseldorf, Germany.

Occurrence of cisplatin-resistance in bladder cancer is frequent and results in disease progression. Thus, novel therapeutic approaches are a high medical need for patients suffering from chemotherapy failure. The purpose of this study was to test the combination of the DNA methyltransferase inhibitor decitabine (DAC) with the histone deacetylase inhibitor entinostat (ENT) in bladder cancer cells with different platinum sensitivities: J82, cisplatin-resistant J82CisR, and RT-112. Intermittent treatment of J82 cells with cisplatin resulted in the six-fold more cisplatin-resistant cell line J82CisR. Combinations of DAC and/or ENT plus cisplatin could not reverse chemoresistance. However, the combination of DAC and ENT acted cytotoxic in a highly synergistic manner as shown by Chou-Talalay analysis via induction of apoptosis and cell cycle arrest. Importantly, this effect was cancer cell-selective as no synergism was found for the combination in the non-cancerous urothelial cell line HBLAK. Expression analysis indicated that epigenetic treatment led to up-regulation of forkhead box class O1 (FoxO1) and further activated proapoptotic Bim and the cell cycle regulator p21 and reduced expression of survivin in J82CisR. In conclusion, the combination of DAC and ENT is highly synergistic and has a promising potential for therapy of bladder cancer, particularly in cases with platinum resistance.
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http://dx.doi.org/10.3390/cancers12020337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073167PMC
February 2020

Targeted misexpression of NAC052, acting in H3K4 demethylation, alters leaf morphological and anatomical traits in Arabidopsis thaliana.

J Exp Bot 2020 02;71(4):1434-1448

Institute of Plant Molecular and Developmental Biology, Heinrich-Heine-University, Duesseldorf, Germany.

In an effort to identify genetic regulators for the cell ontogeny around the veins in Arabidopsis thaliana leaves, an activation-tagged mutant line with altered leaf morphology and altered bundle sheath anatomy was characterized. This mutant had a small rosette area with wrinkled leaves and chlorotic leaf edges, as well as enhanced chloroplast numbers in the (pre-)bundle sheath tissue. It had a bundle-specific promoter from the gene GLYCINE DECARBOXYLASE SUBUNIT-T from the C4 species Flaveria trinervia (GLDTFt promoter) inserted in the coding region of the transcriptional repressor NAC052, functioning in H3K4 demethylation, in front of an alternative start codon in-frame with the natural start codon. Reconstruction of the mutation event of our activation-tagged line by creating a line expressing an N-terminally truncated sequence of NAC052 under control of the GLDTFt promoter confirmed the involvement of NAC052 in leaf development. Our study not only reveals leaf anatomic and transcriptomic effects of an N-terminally truncated NAC052 under control of the GLDTFt promoter, but also identifies NAC052 as a novel genetic regulator of leaf development.
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http://dx.doi.org/10.1093/jxb/erz509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031063PMC
February 2020

Distinctive mutational spectrum and karyotype disruption in long-term cisplatin-treated urothelial carcinoma cell lines.

Sci Rep 2019 10 9;9(1):14476. Epub 2019 Oct 9.

Department of Urology, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

The DNA-damaging compound cisplatin is broadly employed for cancer chemotherapy. The mutagenic effects of cisplatin on cancer cell genomes are poorly studied and might even contribute to drug resistance. We have therefore analyzed mutations and chromosomal alterations in four cisplatin-resistant bladder cancer cell lines (LTTs) by whole-exome-sequencing and array-CGH. 720-7479 genes in the LTTs contained point mutations, with a characteristic mutational signature. Only 53 genes were mutated in all LTTs, including the presumed cisplatin exporter ATP7B. Chromosomal alterations were characterized by segmented deletions and gains leading to severely altered karyotypes. The few chromosomal changes shared among LTTs included gains involving the anti-apoptotic BCL2L1 gene and losses involving the NRF2 regulator KEAP1. Overall, the extent of genomic changes paralleled cisplatin treatment concentrations. In conclusion, bladder cancer cell lines selected for cisplatin-resistance contain abundant and characteristic drug-induced genomic changes. Cisplatin treatment may therefore generate novel tumor genomes during patient treatment.
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http://dx.doi.org/10.1038/s41598-019-50891-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785536PMC
October 2019

Novel technique for the simultaneous isolation of cardiac fibroblasts and epicardial stromal cells from the infarcted murine heart.

Cardiovasc Res 2020 04;116(5):1047-1058

Institut für Molekulare Kardiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany.

Aims: Myocardial infarction (MI) leads to activation of cardiac fibroblasts (aCFs) and at the same time induces the formation of epicardium-derived cells at the heart surface. To discriminate between the two cell populations, we elaborated a fast and efficient protocol for the simultaneous isolation and characterization of aCFs and epicardial stromal cells (EpiSCs) from the infarcted mouse heart.

Methods And Results: For the isolation of aCFs and EpiSCs, infarcted hearts (50 min ischaemia/reperfusion) were digested by perfusion with a collagenase-containing medium for only 8 min, while EpiSCs were enzymatically removed from the outside by applying mild shear forces via a motor driven device. Cardiac fibroblasts (CFs) isolated from unstressed hearts served as control. Viability of isolated cells was >90%. Purity of EpiSCs was confirmed by immunofluorescence staining and qPCR of various mesenchymal markers including Wilms-tumor-protein-1. Microarray analysis of CFs, aCFs, and EpiSCs on day 5 post-MI revealed a unique gene expression pattern in the EpiSC fraction, which was enriched for epithelial markers and epithelial to mesenchymal transition-related genes. Compared to aCFs, 336 significantly altered gene entities were identified in the EpiSC fraction. qPCR analysis showed high expression of Serpinb2, Cxcl13, Adora2b, and Il10 in EpiSCs relative to CFs and aCFs. Furthermore, microarray data identified Ddah1 and Cemip to be highly up-regulated in aCFs compared to CFs. Immunostaining of the infarcted heart revealed a unique distribution of Dermokine, Aquaporin-1, Cytokeratin, Lipocalin2, and Periostin within the epicardial cell layer.

Conclusions: We describe the simultaneous isolation of viable, purified fractions of aCFs and EpiSCs from the infarcted mouse heart. In this study, several differentially expressed markers for aCFs and EpiSCs were identified, underlining the importance of cell separation to study heterogeneity of stromal cells in the healing process after MI.
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http://dx.doi.org/10.1093/cvr/cvz193DOI Listing
April 2020

HDAC5 Expression in Urothelial Carcinoma Cell Lines Inhibits Long-Term Proliferation but Can Promote Epithelial-to-Mesenchymal Transition.

Int J Mol Sci 2019 Apr 30;20(9). Epub 2019 Apr 30.

Department of Urology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany.

Class I histone deacetylases (HDACs) generally promote cell proliferation and tumorigenesis, whereas class IIA HDACs like HDAC4 and HDAC5 may promote or impede cancer development in a tissue-dependent manner. In urothelial carcinoma (UC), HDAC5 is often downregulated. Accordingly, HDAC5 was weakly expressed in UC cell lines suggesting a possible tumor-suppressive function. We therefore characterized the effects of stable HDAC5 expression in four UC cell lines (RT112, VM-Cub-1, SW1710 and UM-UC-3) with different phenotypes reflecting the heterogeneity of UC, by assessing proliferation, clonogenicity and migration ability. Further, we detailed changes in the proteome and transcriptome by immunoblotting, mass spectrometry and RNA sequencing analysis. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line (VM-Cub-1) induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were confirmed by comprehensive proteomics and transcriptomics analyses. In contrast to HDAC5, overexpression of HDAC4 exerted only weak effects on cell proliferation and phenotypes. We conclude that overexpression of HDAC5 may generally decrease proliferation in UC, but, intriguingly, may induce EMT on its own in certain circumstances.
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http://dx.doi.org/10.3390/ijms20092135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539474PMC
April 2019

Contingencies of UTX/KDM6A Action in Urothelial Carcinoma.

Cancers (Basel) 2019 Apr 4;11(4). Epub 2019 Apr 4.

Department of Urology, Medical Faculty, Heinrich Heine University, 40225 Düsseldorf, Germany.

The histone demethylase Ubiquitously Transcribed Tetratricopeptide Repeat Protein X-Linked (UTX/KDM6A) demethylates H3K27me2/3 at genes and enhancers and is often inactivated by mutations in urothelial carcinoma (UC). The consequences of its inactivation are however poorly understood. We have investigated the consequences of moderate UTX overexpression across a range of UC cell lines with or without mutations in or its interaction partners and in a normal control cell line. Effects on cell proliferation, especially long-term, varied dramatically between the cell lines, ranging from deleterious to beneficial. Similarly, effects on global gene expression determined by RNA-Seq were variable with few overlapping up- or downregulated genes between the cell lines. Our data indicate that UTX does not act in a uniform fashion in UC. Rather, its effect depends on several contingencies including, prominently, the status of KMT2C and KMT2D which interact with UTX in the COMPASS complex. In particular, we provide evidence that these factors determine the amount of nuclear UTX.
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http://dx.doi.org/10.3390/cancers11040481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520694PMC
April 2019

GLI3 repressor but not GLI3 activator is essential for mouse eye patterning and morphogenesis.

Dev Biol 2019 06 3;450(2):141-154. Epub 2019 Apr 3.

Institute for Animal Developmental and Molecular Biology, Heinrich Heine University, 40225 Düsseldorf, Germany. Electronic address:

Since 1967, it is known that the loss of GLI3 causes very severe defects in murine eye development. GLI3 is able to act as a transcriptional activator (GLI3-A) or as a transcriptional repressor (GLI3-R). Soon after the discovery of these GLI3 isoforms, the question arose which of the different isoforms is involved in eye formation - GLI3-A, GLI3-R or even both. For several years, this question remained elusive. By analysing the eye morphogenesis of Gli3 mouse embryos that lack GLI3-A and GLI3-R and of Gli3 mouse embryos in which only GLI3-A is missing, we revealed that GLI3-A is dispensable in vertebrate eye formation. Remarkably, our study shows that GLI3-R is sufficient for the creation of morphologically normal eyes although the molecular setup deviates substantially from normality. In depth-investigations elucidated that GLI3-R controls numerous key players in eye development and governs lens and retina development at least partially via regulating WNT/β-CATENIN signalling.
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http://dx.doi.org/10.1016/j.ydbio.2019.02.018DOI Listing
June 2019

IGF1 Treatment Improves Cardiac Remodeling after Infarction by Targeting Myeloid Cells.

Mol Ther 2019 01 1;27(1):46-58. Epub 2018 Nov 1.

Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany. Electronic address:

Insulin-like growth factor 1 (IGF1) is an anabolic hormone that controls the growth and metabolism of many cell types. However, IGF1 also mediates cardio-protective effects after acute myocardial infarction (AMI), but the underlying mechanisms and cellular targets are not fully understood. Here we demonstrate that short-term IGF1 treatment for 3 days after AMI improved cardiac function after 1 and 4 weeks. Regional wall motion was improved in ischemic segments, scar size was reduced, and capillary density increased in the infarcted area and the border zone. Unexpectedly, inducible inactivation of the IGF1 receptor (IGF1R) in cardiomyocytes did not attenuate the protective effect of IGF1. Sequential cardiac transcriptomic analysis indicated an altered myeloid cell response in the acute phase after AMI, and, notably, myeloid-cell Igf1r mice lost the protective IGF1 function after I/R. In addition, IGF1 induced an M2-like anti-inflammatory phenotype in bone marrow-derived macrophages and enhanced the number of anti-inflammatory macrophages in heart tissue on day 3 after AMI in vivo. In summary, modulation of the acute inflammatory phase after AMI by IGF1 represents an effective mechanism to preserve cardiac function after I/R.
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http://dx.doi.org/10.1016/j.ymthe.2018.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319026PMC
January 2019

Genome Sequence of the Acidophilic Sulfate-Reducing Peptococcaceae Strain CEB3.

Genome Announc 2015 Aug 6;3(4). Epub 2015 Aug 6.

Institute of Biological Sciences, TU Bergakademie Freiberg, Freiberg, Germany

We report the draft genome of the Peptococcaceae strain CEB3 that originated from an acidic (pH 2.5) stream draining an abandoned copper mine. Strain CEB3 is one of the very few reported acidophilic sulfate-reducing isolates. The 5.04-Mb draft genome harbors 5,069 predicted protein-encoding and 66 RNA genes.
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http://dx.doi.org/10.1128/genomeA.00886-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541261PMC
August 2015

Genome Sequence of the Moderately Acidophilic Sulfate-Reducing Firmicute Desulfosporosinus acididurans (Strain M1T).

Genome Announc 2015 Aug 6;3(4). Epub 2015 Aug 6.

Institute of Biological Sciences, TU Bergakademie Freiberg, Freiberg, Germany

Microbial dissimilatory sulfate reduction is commonplace in many anaerobic environments, though few acidophilic bacteria are known to mediate this process. We report the 4.64-Mb draft genome of the type strain of the moderate acidophile Desulfosporosinus acididurans, which was isolated from acidic sediment in a river draining the Soufrière volcano, Montserrat.
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http://dx.doi.org/10.1128/genomeA.00881-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541271PMC
August 2015

A newly identified fatty alcohol oxidase gene is mainly responsible for the oxidation of long-chain ω-hydroxy fatty acids in Yarrowia lipolytica.

FEMS Yeast Res 2014 Sep 2;14(6):858-72. Epub 2014 Jul 2.

Institute of Microbiology, Dresden University of Technology, Dresden, Germany.

Nine potential (fatty) alcohol dehydrogenase genes and one alcohol oxidase gene were identified in Yarrowia lipolytica by comparative sequence analysis. All relevant genes were deleted in Y. lipolytica H222ΔP which is lacking β-oxidation. Resulting transformants were tested for their ability to accumulate ω-hydroxy fatty acids and dicarboxylic acids in the culture medium. The deletion of eight alcohol dehydrogenase genes (FADH, ADH1-7), which may be involved in ω-oxidation, led only to a slightly increased accumulation of ω-hydroxy fatty acids, whereas the deletion of the fatty alcohol oxidase gene (FAO1), which has not been described yet in Y. lipolytica, exhibited a considerably higher effect. The combined deletion of the eight (fatty) alcohol dehydrogenase genes and the alcohol oxidase gene further reduced the formation of dicarboxylic acids. These results indicate that both (fatty) alcohol dehydrogenases and an alcohol oxidase are involved in ω-oxidation of long-chain fatty acids whereby latter plays the major role. This insight marks the first step toward the biotechnological production of long-chain ω-hydroxy fatty acids with the help of the nonconventional yeast Y. lipolytica. The overexpression of FAO1 can be further used to improve existing strains for the production of dicarboxylic acids.
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http://dx.doi.org/10.1111/1567-1364.12176DOI Listing
September 2014
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