Establishment of a Human Hepatoma Cell Line HepG2-A10 for a Reporter Gene Assay of Arylhydrocarbon Receptor Activators

Abstract

To establish a human hepatic cell line for a convenient reporter gene assay of arylhydrocarbon receptor (AhR) activators, the chimera plasmid containing xenobiotic responsible element (XRE), minimal SV40 promoter, and luciferase reporter gene and the expression vector pRC/CMV containing a neomycin-resistant gene were co-transfected into a human hepatoma cell line, HepG2. Then, antibiotic (G418)-resistant HepG2 cells were selected and cloned. A cell clone, HepG2-A10, showed the highest responsibility to 3-methylcholanthrene (MC)-mediated induction of luciferase among the clones obtained. Expression levels of luciferase activity in HepG2-A10 cells were increased in a dose-and time-dependent manner by treatment with either MC, an AhR-ligand type activator, or omeprazole (OME), a non-AhR ligand type activator. In addition, expression levels of cytochrome P4501A subfamily genes (CYP1A1 and CYP1A2) were also increased in a dose-and time-dependent manner by treatment with MC. The presentˆndings demonstrate that a newly established human HepG2-A10 is a useful cell line for a convenient reporter gene assay of AhR activators.

Establishment of a Stable Human Cell Line, HPL-A3, for Use in Reporter Gene Assays of Cytochrome P450 3A Inducers

“…17,18) Clone HPL-A3 showed the greatest ability to induce chemical-mediated luciferase activity among the clones examined. Therefore, HPL-A3 was used for further experiments.…”

“…Gene Expression of CYP3A4 and Its Positive Transcriptional Regulators Constitutive gene expression levels of the positive transcriptional nuclear receptors (PXR, VDR, and CAR) of the CYP3A4 gene and of their partner protein, retinoid X receptor α (RXRα), were determined by reverse transcription (RT)-PCR method 17,18) with the appropriate primer sets. [19][20][21][22][23] The primer sets and amplification protocol used are shown in Table 1.…”

Augmentation of 3‐methylcholanthrene‐induced bioactivation in the human hepatoma cell line HepG2 by the calcium channel blocker nicardipine

“…To determine a mechanism for the Nicmediated induction of the CYP1 mRNAs, the ability of Nic to activate AhR was examined using a HepG2-A10 cell line, which was established from HepG2 cells for screening of AhR activators including AhR ligands. (19) The results indicated that Nic weakly activated AhR (inducing luciferase), suggesting that Nic-mediated induction likely occurs only partially through activation of AhR.…”

“…A human hepatoma cell line, HepG2, was obtained from the ATCC (Manassas, VA, USA), and HepG2‐A10 cells were previously established from HepG2 cells for use in a convenient reporter gene assay of AhR activators in our laboratory. ( 19 ) The HepG2 and HepG2‐A10 cells were maintained in DMEM supplemented with 5% FBS, 4 mM l ‐glutamine and 60 μg/mL kanamycin sulfate at 37°C in a 5% CO 2 humidified incubator.…”