Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7

Abstract Primary human hepatocytes are necessary to evaluate cytotoxicity, drug metabolism, and drug–drug interactions for candidate compounds in early-phase drug discovery and development. However, these analyses are often hampered by limited resources and functional or genetic variation among lots...

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Autores principales:	Takafumi Ueyama, Saori Tsuji, Takemi Sugiyama, Masako Tada
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/586c1d68c8994fc387df6a32a7dc4378
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spelling	oai:doaj.org-article:586c1d68c8994fc387df6a32a7dc43782021-12-02T16:07:44ZFluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A710.1038/s41598-017-03146-52045-2322https://doaj.org/article/586c1d68c8994fc387df6a32a7dc43782017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03146-5https://doaj.org/toc/2045-2322Abstract Primary human hepatocytes are necessary to evaluate cytotoxicity, drug metabolism, and drug–drug interactions for candidate compounds in early-phase drug discovery and development. However, these analyses are often hampered by limited resources and functional or genetic variation among lots. HepaRG human hepatocellular carcinoma cells can differentiate into mature hepatocyte-like cells (HepLCs) that possess similar metabolic activity to human hepatocytes. We previously established transgenic HepaRG cells carrying a dual reporter that express red fluorescent protein (RFP) under the transcriptional regulation of CYP3A7 in the hepatoblast-like cell state and enhanced green fluorescent protein (EGFP) under the transcriptional regulation of CYP3A4 following HepLC differentiation. In this study, we successfully isolated a subclone of transgenic CYP3A4G/7R HepaRG cells with an improved HepLC differentiation potency. Midazolam metabolism by CYP3A4 in these HepLCs was comparable to that in wild-type HepLCs. The EGFP fluorescence intensity was greatly induced by rifampicin (RIF) treatment. There was a strong correlation between fluorometric and metabolic analyses. The fold change in EGFP-positive cells was comparable to those in the CYP3A4 mRNA level and luminescence of proluciferin metabolites. RIF treatment and cell proliferation increased the RFP-positive cell number. Thus, CYP3A4G/7R HepLCs provide a real-time, multiwell-based system to co-evaluate CYP3A4 induction and hepatic regeneration.Takafumi UeyamaSaori TsujiTakemi SugiyamaMasako TadaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-8 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Takafumi Ueyama Saori Tsuji Takemi Sugiyama Masako Tada Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7
description	Abstract Primary human hepatocytes are necessary to evaluate cytotoxicity, drug metabolism, and drug–drug interactions for candidate compounds in early-phase drug discovery and development. However, these analyses are often hampered by limited resources and functional or genetic variation among lots. HepaRG human hepatocellular carcinoma cells can differentiate into mature hepatocyte-like cells (HepLCs) that possess similar metabolic activity to human hepatocytes. We previously established transgenic HepaRG cells carrying a dual reporter that express red fluorescent protein (RFP) under the transcriptional regulation of CYP3A7 in the hepatoblast-like cell state and enhanced green fluorescent protein (EGFP) under the transcriptional regulation of CYP3A4 following HepLC differentiation. In this study, we successfully isolated a subclone of transgenic CYP3A4G/7R HepaRG cells with an improved HepLC differentiation potency. Midazolam metabolism by CYP3A4 in these HepLCs was comparable to that in wild-type HepLCs. The EGFP fluorescence intensity was greatly induced by rifampicin (RIF) treatment. There was a strong correlation between fluorometric and metabolic analyses. The fold change in EGFP-positive cells was comparable to those in the CYP3A4 mRNA level and luminescence of proluciferin metabolites. RIF treatment and cell proliferation increased the RFP-positive cell number. Thus, CYP3A4G/7R HepLCs provide a real-time, multiwell-based system to co-evaluate CYP3A4 induction and hepatic regeneration.
format	article
author	Takafumi Ueyama Saori Tsuji Takemi Sugiyama Masako Tada
author_facet	Takafumi Ueyama Saori Tsuji Takemi Sugiyama Masako Tada
author_sort	Takafumi Ueyama
title	Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7
title_short	Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7
title_full	Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7
title_fullStr	Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7
title_full_unstemmed	Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7
title_sort	fluorometric evaluation of cyp3a4 expression using improved transgenic heparg cells carrying a dual-colour reporter for cyp3a4 and cyp3a7
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/586c1d68c8994fc387df6a32a7dc4378
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Fluorometric evaluation of CYP3A4 expression using improved transgenic HepaRG cells carrying a dual-colour reporter for CYP3A4 and CYP3A7

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