Stable expression of human cytochrome P450 2D6*10 in HepG2 cells.
OBJECTIVE
Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs. In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established.


METHODS
Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from human liver tissue and cloned into pGEM-T vector. cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 cells. Expression of mRNA was validated by RT-PCR. Enzyme activity of catalyzing dextromethorphan O-demethylation in postmitochondrial supernatant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC).


RESULTS
The cloned cDNA had 4 base differences, e.g. 100 C-T, 336 T-C, 408 C-G and 1 457 G-C, which resulted in P34S, and S486T amino acid substitutions, and two samesense mutations were 112 F and 136 V compared with that reported by Kimura et al (GenBank accession number: M33388). P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 allele. The relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31 +/- 0.19 nmol/min(-1)/mg(-1) S9 protein (n=3), but was undetectable in parental HepG2 cells.


CONCLUSIONS
cDNA of human CYP2D6*10 can be successfully cloned. A cell line, HepG2-CYP2D6*10, expressing CYP2D6*10 mRNA and having metabolic activity, has been established.
Zhuge
Jian
J
Department of Pathology and Pathophysiology, School of Medicine, Zhejiang University, Hangzhou 310031, Zhejiang Province, China.
Yu
Ying-Nian
YN
Wu
Xiao-Dan
XD
D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D008113:Liver Neoplasms; D010090:Oxidoreductases, O-Demethylating / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D010090:Oxidoreductases, O-Demethylating; C052861:dextromethorphan O-demethylase; D019389:Cytochrome P-450 CYP2D6