| Summary: | Most hepatoma cell lines lack proper expression and induction of CYP3A4 enzyme, which limits their use for predicting drug metabolism and toxicity. Nuclear receptor pregnane X receptor (PXR) has been well recognized for its critical role in regulating expression of CYP3A4 gene. However, its physiological activity of binding to the particular site of promoter is significantly weakened in hepatic cell lines. To address this problem, we created "chimeric PXR" constructs by appending a strong activation domain (AD) from p53 subunit to either N- or C- termini of the human PXR (hPXR), that is, hPXR-p53 and p53-hPXR. C3A, a hepatoma cell line, was used as the cell model to test the regulation effect of chimeric hPXR over wild type (WT) hPXR on CYP3A4 expression at gene, protein, and metabolism levels, respectively. Compared with C3A cells transiently transfected with WT hPXR, the activity of CYP3A4.XREM.luc reporter gene in C3A cells transfected with hPXR-p53 or p53-hPXR increased 5- and 9-fold respectively, and the levels of CYP3A4 mRNA expression increased 3.5- and 2.6-fold, respectively. C3A cells stably transfected with hPXR-p53-AD exhibited an improved expression of CYP3A4 at both gene (2-fold) and protein (1.5-fold) levels compared to WT C3A cells. Testosterone, a CYP3A4-specific substrate, was used for detecting the metabolism activity of CYP3A4. No testosterone metabolite could be detected in microsomes from WT C3A cells and WT C3A cells-based array, while the formation of 6β-hydroxytestosterone metabolite in the transfected cells was 714 and 55 pmol/mg protein/min, respectively. In addition, all the above expression levels in the transfected cell models could be further induced with additional treatment of Rifampicin, a specific inducer for CYP3A4. In conclusion, our study established a proof-of-principle example that genetic modification with chimeric hPXR-p53-AD could improve CYP3A4 metabolism ability in hepatic cell line.
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