3-Methylcholanthrene-mediated induction of cytochrome P4501A2 in human hepatoma HepG2 cells as quantified by the reverse transcription-polymerase chain reaction.
The feasibility of using HepG2 human hepatoma cells to study the regulation of the expression of the cytochrome P4501A2 gene (CYP1A2) was examined. The reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that HepG2 cells constitutively express CYP1A2 and are able to respond to 3-methylcholanthrene (3MC) by an induction of CYP1A2 mRNA. In these studies, selected sequences from intron-exon junctions were used as mRNA-specific primers for both CYP1A2 and the beta-actin gene. The level of induction was quantitated based on two parameters within the exponential phase of the amplification: the difference in the number of cycles that yields the same level of amplification and the efficiency of the PCR reaction. Using this method, it was estimated that the CYP1A2 steady-state mRNA level increased to a maximum of 12-fold at 24 h after exposure of the cells to 3MC. Both a reduction in basal expression as well as an increased accumulation of CYP1A2 mRNA appeared responsible for the overall induction at 24 and 48 h. These results suggested that the HepG2 cell line would be appropriate for studying the regulation of CYP1A2 expression.
Chung
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Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835.
Bresnick
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E
D001483:Base Sequence; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D008748:Methylcholanthrene / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D012194:RNA-Directed DNA Polymerase