Quantitative analysis of constitutive and inducible CYPs mRNA expression in the HepG2 cell line using reverse transcription-competitive PCR.
Drug interactions which affect drug metabolism are of clinical importance. It is, however, difficult to estimate drug interactions in human from results obtained in animal experiments. In our previous study, we demonstrated that a combination of the HepG2 cell line and semiquantitative reverse transcription-PCR (RT-PCR) could be used to evaluate the degree of CYP3A mRNA induction by various drugs. Using an RT-competitive PCR (RT-cPCR) with beta-actin as the standard in this study, the constitutive and rifampicin (RFP)-induced expression of CYP3A4, CYP2C9, CYP2E1, and CYP1A2 mRNA in the HepG2 cells could be quantitatively and reproducibly determined. 120 h-treatment of HepG2 cells with 50 micromol/l RFP induced maximally 8.4- and 6.0-fold the expression of CYP3A4 and CYP2C9 mRNA, respectively, in comparison with untreated cells. On the other hand, mRNA level in CYP2E1 and CYP1A2 was not significantly changed by 50 micromol/l RFP after 24 to 120 h. To our knowledge, we report for the first time quantitative profiles of CYPs mRNA in HepG2 cells. This study demonstrates the efficiency of a combination of HepG2 cells and RT-cPCR in the evaluation of CYPs mRNA-induction by drugs.
Sumida
A
A
Clinical Evaluation of Medicines and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Fukuen
S
S
Yamamoto
I
I
Matsuda
H
H
Naohara
M
M
Azuma
J
J
D001189:Aryl Hydrocarbon Hydroxylases; D006528:Carcinoma, Hepatocellular; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics*; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D006899:Mixed Function Oxygenases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; C510163:CYP3A4 protein, human