TY - GEN A1 - Herzog, Natalie A1 - Hansen, Max A1 - Miethbauer, Sebastian A1 - Schmidtke, Kai-Uwe A1 - Anderer, Ursula A1 - Lupp, Amelie A1 - Sperling, Sebastian A1 - Seehofer, Daniel A1 - Damm, Georg A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner T1 - Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures T2 - Cell Biology International N2 - 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. KW - cytochrome P450 enzyme Y1 - 2016 UR - http://onlinelibrary.wiley.com/doi/10.1002/cbin.10574/full U6 - https://doi.org/10.1002/cbin.10574 SN - 1095-8355 VL - 40 IS - 3 SP - 341 EP - 353 ER - TY - GEN A1 - Herzog, Natalie A1 - Hansen, Max A1 - Miethbauer, Sebastian A1 - Schmidtke, Kai-Uwe A1 - Anderer, Ursula A1 - Lupp, Amelie A1 - Sperling, Sebastian A1 - Seehofer, Daniel A1 - Damm, Georg A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner T1 - Primary like human hepatocytes genetically engineered to obtain proliferation competence display liver biotransformation activity in 2D and 3D culture systems T2 - Cell Biology International Y1 - 2016 SN - 1095-8355 SN - 1065-6995 VL - 40 IS - 3 SP - 341 EP - 353 ER - TY - GEN A1 - Steinbrecht, Susanne A1 - König, Rosalie A1 - Schmidtke, Kai-Uwe A1 - Herzog, Natalie A1 - Scheibner, Katrin A1 - Krüger-Genge, Anne A1 - Jung, Friedrich A1 - Kammerer, Sarah A1 - Küpper, Jan-Heiner T1 - Metabolic activity testing can underestimate acute drug cytotoxicity as revealed by HepG2 cell clones overexpressing cytochrome P450 2C19 and 3A4 T2 - Toxicology Y1 - 2018 U6 - https://doi.org/10.1016/j.tox.2018.11.008 SN - 0300-483X VL - 412 SP - 37 EP - 47 ER - TY - GEN A1 - Steinbrecht, Susanne A1 - Kiebist, Jan A1 - König, Rosalie A1 - Thiessen, Markus A1 - Schmidtke, Kai-Uwe A1 - Kammerer, Sarah A1 - Küpper, Jan-Heiner A1 - Scheibner, Katrin T1 - Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells T2 - AMB Express N2 - Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPAUPO). 4-OH-CPAUPO–induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPAUPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment. KW - Biocatalysis KW - Cyclophosphamide KW - Human drug metabolites KW - Peroxygenase KW - Toxicity Y1 - 2020 UR - https://amb-express.springeropen.com/articles/10.1186/s13568-020-01064-w U6 - https://doi.org/10.1186/s13568-020-01064-w SN - 2191-0855 VL - 10 ER - TY - PAT A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner A1 - Schmidtke, Kai-Uwe A1 - Miethbauer, Sebastian A1 - Herzog, Natalie T1 - Verfahren zur Herstellung von biogenen Stoffen T1 - Method for producing biogenic substances Y1 - 2020 UR - https://patents.google.com/patent/EP3068868B1/de ER - TY - PAT A1 - Scheibner, Katrin A1 - Kiebist, Jan A1 - Schmidtke, Kai-Uwe A1 - Küpper, Jan-Heiner T1 - Verfahren zur enzymatischen Herstellung von zytostatischen 4-Hydroxy-Oxazaphosphorinen“ Y1 - 2020 UR - https://www.b-tu.de/enzymtechnologie/publikationen/patente ER - TY - PAT A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner A1 - Jung, Friedrich T1 - In vitro Arzneimittel aus Prodrugs und deren Verwendung Y1 - 2021 UR - https://www.b-tu.de/enzymtechnologie/publikationen/patente ER -