Establishment of a stable human cell line, HPL-A3, for use in reporter gene assays of cytochrome P450 3A inducers.
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 D(3))-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.
Department of Molecular Toxicology and Global Center of Excellence Program, School of Pharmaceutical Sciences, University of Shizuoka, Japan.
Masashi
M
Sekimoto
Shinsuke
S
Sano
Takuomi
T
Hosaka
Kiyomitsu
K
Nemoto
Masakuni
M
Degawa
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects*; D020536:Enzyme Activators / Q000494:pharmacology*; D017930:Genes, Reporter; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D000077297:Pregnane X Receptor; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol / Q000378:metabolism*; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D012293:Rifampin / Q000494:pharmacology; D014162:Transfection; D014807:Vitamin D / Q000031:analogs & derivatives / Q000494:pharmacology
D002121:Calcium Channel Blockers; D020536:Enzyme Activators; D000077297:Pregnane X Receptor; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D014807:Vitamin D; C097949:1,25-dihydroxyvitamin D; D008156:Luciferases; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D012293:Rifampin