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.

Regulation of Cytosolic Sulfotransferases in Models of Human Hepatocyte Development

“…It has been classified as a bipotent hepatic progenitor cell line that can be differentiated into both cholangiocyte-like and hepatocyte-like cells (Parent et al, 2004). Many studies have demonstrated that HepaRG-derived hepatocytelike cells exhibit properties of adult human hepatocytes, including the expression of xenobiotic-metabolizing enzymes, transporters, and the transcription factors that control hepatocellular gene expression (Aninat et al, 2006;Lubberstedt et al, 2011;Gerets et al, 2012;Hoekstra et al, 2013;Tsuji et al, 2014). Undifferentiated and differentiated HepaRG cells have distinct gene expression profiles that tend to reflect the patterns seen in fetal and adult human liver, respectively.…”

“…Undifferentiated and differentiated HepaRG cells have distinct gene expression profiles that tend to reflect the patterns seen in fetal and adult human liver, respectively. Genes that are primarily expressed in fetal hepatocytes, such as CYP3A7 and pyruvate kinase muscle isozyme, are more highly expressed in undifferentiated HepaRG cells, whereas genes that are predominantly expressed in adult hepatocytes, such as CYP3A4 and CYP2E1, are more highly expressed in differentiated HepaRG cells (Tsuji et al, 2014;Bucher et al, 2017). HepaRG cells are becoming established as a useful model for studying hepatocellular differentiation, xenobiotic metabolism and toxicity, and development of liver diseases (Sharanek et al, 2015;Nunn et al, 2016;Rodrigues et al, 2016;Sayyed et al, 2016;Xia et al, 2016).…”

“…Most studies using HepaRG cells have used differentiated cells as a model to complement the use of primary cultured human hepatocytes (Josse et al, 2008; Lubberstedt et al, 2011; Gerets et al, 2012;Klein et al, 2015). However, few studies have evaluated the changes in xenobiotic-metabolizing enzyme expression that occur as HepaRG cells pass through the stages of the differentiation process (Aninat et al, 2006;Hart et al, 2010;Ceelen et al, 2011;Tsuji et al, 2014;Bucher et al, 2017), and no studies have determined expression of the individual SULTs. We found that SULT1B1, SULT1C2, SULT1C3, SULT1C4, and SULT1E1 mRNA levels were highest in confluent HepaRG cells, whereas SULT2A1 RNA levels increased throughout the differentiation process.…”

A Cytoplasmic Form of Gaussia luciferase Provides a Highly Sensitive Test for Cytotoxicity

“…The expression units were placed between insulator sequences (INS) to prevent inappropriate inactivation by position effects. A loxP site was also placed into the vector, facilitating easy integration of a single copy vector into an acceptor loxP site previously placed in a specific chromosomal region of the host cell genome by Cre-mediated site-specific recombination [ 19 ]. The ORFs of G-Luc and C-Luc with KDEL were amplified using 3’ reverse primers containing the 12-base sequence encoding KDEL ( Table 1 ).…”

Rotavirus in Organ Transplantation: Drug-Virus-Host Interactions

“…This means that a decreased gastrointestinal transit time caused by rotavirus infection forces the metabolism of tacrolimus in lower intestinal tissue . Oxidation of 50–60% of clinical drugs is through CYP3A4 enzyme . CYP3A4 is highly expressed at the apex of the villus enterocytes adjacent to the microvillus border, whereas the crypt cells have null expression.…”