title: Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.
abstract: Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.
authors.affiliation: Bio Frontier Project Promotion Section, Organization for Tottori Industrial Promotion, Yonago, Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Tsuji
authors.affiliation: Division of Molecular Genetics and Biofunction, Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Japan.
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Kawamura
authors.affiliation: Division of Molecular Genetics and Biofunction, Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Japan.
authors.forename: Musashi
authors.initials: M
authors.lastname: Kubiura
authors.affiliation: Division of Molecular Genetics and Biofunction, Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Hayashi
authors.affiliation: Division of Laboratory Animal Science, Research Center for Bioscience and Technology, Tottori University, Yonago, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ohbayashi
authors.affiliation: Division of Molecular Genetics and Biofunction, Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Japan; Chromosome Engineering Research Center, Tottori University, Yonago, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.affiliation: Biopredic International, Rennes, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Chesné
authors.affiliation: Chromosome Engineering Research Center, Tottori University, Yonago, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.affiliation: Chromosome Engineering Research Center, Tottori University, Yonago, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Tada
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System; D005434:Flow Cytometry; D005453:Fluorescence; D022781:Hepatocytes; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008856:Microscopy, Fluorescence / Q000379:methods
chemical_list: D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A
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