@misc{HerzogHansenMiethbaueretal., author = {Herzog, Natalie and Hansen, Max and Miethbauer, Sebastian and Schmidtke, Kai-Uwe and Anderer, Ursula and Lupp, Amelie and Sperling, Sebastian and Seehofer, Daniel and Damm, Georg and Scheibner, Katrin and K{\"u}pper, Jan-Heiner}, title = {Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures}, series = {Cell Biology International}, volume = {40}, journal = {Cell Biology International}, number = {3}, issn = {1095-8355}, doi = {10.1002/cbin.10574}, pages = {341 -- 353}, abstract = {Primary human hepatocytes are in great demand during drug development and in hepatology. However, both scarcity of tissue supply and donor variability of primary cells create a need for the development of alternative hepatocyte systems. By using a lentivirus vector system to transfer coding sequences of Upcyte® proliferation genes, we generated non-transformed stable hepatocyte cultures from human liver tissue samples. Here, we show data on newly generated proliferation-competent HepaFH3 cells investigated as conventional two-dimensional monolayer and as organotypical three-dimensional (3D) spheroid culture. In monolayer culture, HepaFH3 cells show typical cobblestone-like hepatocyte morphology and anchorage-dependent growth for at least 20 passages. Immunofluorescence staining revealed that characteristic hepatocyte marker proteins cytokeratin 8, human serum albumin, and cytochrome P450 (CYP) 3A4 were expressed. Quantitative real-time PCR analyses showed that expression levels of analyzed phase I CYP enzymes were at similar levels compared to those of cultured primary human hepatocytes and considerably higher than in the liver carcinoma cell line HepG2. Additionally, transcripts for phase II liver enzymes and transporter proteins OATP-C, MRP2, Oct1, and BSEP were present in HepaFH3. The cells produced urea and converted model compounds such as testosterone, diclofenac, and 7-OH-coumarin into phases I and II metabolites. Interestingly, phases I and II enzymes were expressed at about the same levels in convenient monolayer cultures and complex 3D spheroids. In conclusion, HepaFH3 cells and related primary-like hepatocyte lines seem to be promising tools for in vitro research of liver functions and as test system in drug development and toxicology analysis.}, language = {en} } @misc{KadeHerzogSchmidtkeetal., author = {Kade, S. and Herzog, Natalie and Schmidtke, Kai-Uwe and K{\"u}pper, Jan-Heiner}, title = {Chronic ethanol treatment depletes glutathione regeneration capacity in hepatoma cell line HepG2}, series = {Journal of Cellular Biotechnology}, volume = {1}, journal = {Journal of Cellular Biotechnology}, number = {2}, issn = {2352-3697}, doi = {10.3233/JCB-15019}, pages = {183 -- 190}, language = {en} } @misc{HerzogHansenMiethbaueretal., author = {Herzog, Natalie and Hansen, Max and Miethbauer, Sebastian and Schmidtke, Kai-Uwe and Anderer, Ursula and Lupp, Amelie and Sperling, Sebastian and Seehofer, Daniel and Damm, Georg and Scheibner, Katrin and K{\"u}pper, Jan-Heiner}, title = {Primary like human hepatocytes genetically engineered to obtain proliferation competence display liver biotransformation activity in 2D and 3D culture systems}, series = {Cell Biology International}, volume = {40}, journal = {Cell Biology International}, number = {3}, issn = {1095-8355}, pages = {341 -- 353}, language = {en} } @misc{HerzogKatzenbergerMartinetal., author = {Herzog, Natalie and Katzenberger, Nadine and Martin, Frank and Schmidtke, Kai-Uwe and K{\"u}pper, Jan-Heiner}, title = {Generation of cytochrome P450 3A4-overexpressing HepG2 cell clones for standardization of hepatocellular testosterone 6β-hydroxylation activity}, series = {Journal of Cellular Biotechnology}, volume = {1}, journal = {Journal of Cellular Biotechnology}, number = {1}, issn = {2352-3697}, doi = {10.3233/JCB-15002}, pages = {15 -- 26}, language = {en} } @misc{KuenzelRauschSchaefferetal., author = {K{\"u}nzel, Stephan R. and Rausch, Johanna S. E. and Sch{\"a}ffer, Charlotte and Hoffmann, Maximilian and K{\"u}nzel, Karolina and Klapproth, Erik and Kant, Theresa and Herzog, Natalie and K{\"u}pper, Jan-Heiner and Lorenz, Kristina and Dudek, Svenja and Emig, Ramona and Ravens, Ursula and Rog-Zielinska, Eva A. and Peyronnet, R{\´e}mi and El-Armouche, Ali}, title = {Modeling atrial fibrosis in vitro - Generation and characterization of a novel human atrial fibroblast cell line}, series = {FEBS Open Bio}, volume = {10}, journal = {FEBS Open Bio}, number = {7}, issn = {2211-5463}, doi = {10.1002/2211-5463.12896}, pages = {1210 -- 1218}, abstract = {Atrial fibrillation (AF) is regularly accompanied by cardiac fibrosis and concomitant heart failure. Due to the heterogeneous nature and complexity of fibrosis, the knowledge about the underlying mechanisms is limited, which prevents effective pharmacotherapy. A deeper understanding of cardiac fibroblasts is essential to meet this need. We previously described phenotypic and functional differences between atrial fibroblasts from patients in sinus rhythm and with AF. Herein, we established and characterized a novel human atrial fibroblast line, which displays typical fibroblast morphology and function comparable to primary cells but with improved proliferation capacity and low spontaneous myofibroblast differentiation. These traits make our model suitable for the study of fibrosis mechanisms and for drug screening aimed at developing effective antifibrotic pharmacotherapy.}, language = {en} } @misc{ScheibnerKuepperSchmidtkeetal., author = {Scheibner, Katrin and K{\"u}pper, Jan-Heiner and Schmidtke, Kai-Uwe and Miethbauer, Sebastian and Herzog, Natalie}, title = {Verfahren zur Herstellung von biogenen Stoffen}, language = {de} } @misc{SteinbrechtKoenigSchmidtkeetal., author = {Steinbrecht, Susanne and K{\"o}nig, Rosalie and Schmidtke, Kai-Uwe and Herzog, Natalie and Scheibner, Katrin and Kr{\"u}ger-Genge, Anne and Jung, Friedrich and Kammerer, Sarah and K{\"u}pper, Jan-Heiner}, title = {Metabolic activity testing can underestimate acute drug cytotoxicity as revealed by HepG2 cell clones overexpressing cytochrome P450 2C19 and 3A4}, series = {Toxicology}, volume = {412}, journal = {Toxicology}, issn = {0300-483X}, doi = {10.1016/j.tox.2018.11.008}, pages = {37 -- 47}, language = {en} } @misc{SteinbrechtPfeiferHerzogetal., author = {Steinbrecht, Susanne and Pfeifer, Nadine and Herzog, Natalie and Katzenberger, Nadine and Schulz, Christian and Kammerer, Sarah and K{\"u}pper, Jan-Heiner}, title = {HepG2-1A2 C2 and C7: Lentivirus vector-mediated stable and functional overexpression of cytochrome P450 1A2 in human hepatoblastoma cells}, series = {Toxicology Letters}, volume = {319}, journal = {Toxicology Letters}, issn = {0378-4274}, doi = {10.1016/j.toxlet.2019.11.006}, pages = {155 -- 159}, abstract = {Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min-1 x mg-1 protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B1 (EC50<100 nM) when compared to parental HepG2 cells (EC50 ∼5 μM). Thus, newly established cell lines are an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP1A2.}, language = {en} } @misc{KuenzelHoffmannWeberetal., author = {K{\"u}nzel, Stephan R. and Hoffmann, Maximilian and Weber, Silvio and K{\"u}nzel, Karolina and K{\"a}mmerer, Susanne and G{\"u}nscht, Mario and Klapproth, Erik and Rausch, Johanna S. E. and Sadek, Mirna S. and Kolanowski, Tomasz and Meyer-Roxlau, Stefanie and Piorkowski, Christopher and Tugtekin, Sems M. and Rose-John, Stefan and Yin, Xiaoke and Mayr, Manuel and Kuhlmann, Jan Dominik and Wimberger, Pauline and Gr{\"u}tzmann, Konrad and Herzog, Natalie and K{\"u}pper, Jan-Heiner and O'Reilly, Molly and Kabir, S. Nashitha and Sommerfeld, Laura C. and Guan, Kaomei and Wielockx, Ben and Fabritz, Larissa and Nattel, Stanley and Ravens, Ursula and Dobrev, Dobromir and Wagner, Michael and El-Armouche, Ali}, title = {Diminished PLK2 Induces Cardiac Fibrosis and Promotes Atrial Fibrillation}, series = {Circulation Research}, volume = {129}, journal = {Circulation Research}, number = {8}, issn = {1524-4571}, doi = {10.1161/CIRCRESAHA.121.319425}, pages = {804 -- 820}, abstract = {Rationale: Fibrosis promotes the maintenance of atrial fibrillation (AF), making it resistant to therapy. Improved understanding of the molecular mechanisms leading to atrial fibrosis will open new pathways toward effective antifibrotic therapies. Objective: This study aims to decipher the mechanistic interplay between PLK2 (polo-like kinase 2) and the profibrotic cytokine OPN (osteopontin) in the pathogenesis of atrial fibrosis and AF. Methods and Results: Atrial PLK2 mRNA expression was 10-fold higher in human fibroblasts than in cardiomyocytes. Compared with sinus rhythm, right atrial appendages and isolated right atrial fibroblasts from patients with AF showed downregulation of PLK2 mRNA and protein, along with increased PLK2 promotor methylation. Genetic deletion as well as pharmacological inhibition of PLK2 induced profibrotic phenotype conversion in cardiac fibroblasts and led to a striking de novo secretion of OPN. Accordingly, PLK2-deficient (PLK2 knockout) mice showed cardiac fibrosis and were prone to experimentally induced AF. In line with these findings, OPN plasma levels were significantly higher only in patients with AF with atrial low-voltage zones (surrogates of fibrosis) compared with sinus rhythm controls. Mechanistically, we identified ERK1/2 as the relevant downstream mediator of PLK2 leading to increased OPN expression. Finally, oral treatment with the clinically available drug mesalazine, known to inhibit ERK1/2, prevented cardiac OPN overexpression and reversed the pathological PLK2 knockout phenotype in PLK2 knockout mice. Conclusions: Abnormal PLK2/ERK1/2/OPN axis function critically contributes to AF-related atrial fibrosis, suggesting reinforcing PLK2 activity and/or OPN inhibition as innovative targets to prevent fibrosis progression in AF. Mesalazine derivatives may be used as lead compounds for the development of novel anti-AF agents targeting fibrosis.}, language = {en} } @misc{SchulzHerzogKubicketal., author = {Schulz, Christian and Herzog, Natalie and Kubick, Stefan and Jung, Friedrich and K{\"u}pper, Jan-Heiner}, title = {Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies}, series = {Cells}, volume = {12}, journal = {Cells}, number = {17}, issn = {2073-4409}, doi = {10.3390/cells12172140}, abstract = {In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising "humanised" in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine.}, language = {en} }