Establishment of a Stable Human Cell Line, HPL-A3, for Use in Reporter Gene Assays of Cytochrome P450 3A Inducers
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概要
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We have established a stable human cell line, termed HPL-A3, by co-transfection of a human pregnane X receptor (hPXR) expression vector and a reporter plasmid (p3A4-hPXRE-Luc) containing a luciferase gene and a promoter/enhancer region of the human cytochrome P450 3A4 (CYP3A4) gene into a human hepatoma-derived cell line, HepG2. We then examined the usefulness of HPL-A3 for a chemically activated luciferase expression (CALUX) assay of human CYP3A inducers. The induction of CALUX in HPL-A3 by hPXR activators, including rifampicin, occurred in time- and concentration-dependent fashions, whereas no such induction was observed using rat/mouse PXR activators, such as pregnenolone-16α-carbonitrile and dexamethasone. The hPXR activator-mediated induction of CYP3As, especially CYP3A4, was observed at levels of both mRNA and enzyme activity. Furthermore, there were positive correlations between chemical-mediated inductions of CALUX and CYP3A4 mRNA levels. In addition, the induction of CALUX by dihydropyridine calcium channel blockers, which are known to act as CYP3A inducers in rats, was observed in HPL-A3 cells. Interestingly, expression levels of not only hPXR but also of vitamin D receptor (VDR), a transcription factor that positively regulates CYP3A subfamily genes, were significantly increased in HPL-A3 cells compared with those in the parental cell line, HepG2. Consequently, VDR ligand (1,25-dihydroxyvitamin D3)-mediated inductions of CALUX and CYP3A4 mRNA were observed in the cells. These findings verified the usefulness of HPL-A3 for the screening of CYP3A inducers, which can activate the hPXR and/or hVDR.
著者
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NEMOTO Kiyomitsu
Department of Molecular Toxicology and COE Program in the 21st Century, School of Pharmaceutical Sci
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SEKIMOTO Masashi
Department of Molecular Toxicology and COE Program in the 21st Century, School of Pharmaceutical Sci
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Degawa Masakuni
Department Of Hygienic Chemistry Faculty Of Pharmaceutical Sciences Tohoku University
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Sekimoto Masashi
Department of Molecular Toxicology and Global Center of Excellence Program, School of Pharmaceutical Sciences, University of Shizuoka
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Hosaka Takuomi
Department of Molecular Toxicology and Global Center of Excellence Program, School of Pharmaceutical Sciences, University of Shizuoka
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Sano Shinsuke
Department of Molecular Toxicology and Global Center of Excellence Program, School of Pharmaceutical Sciences, University of Shizuoka
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