3′-UTR polymorphism in the human CYP2A6 gene affects mRNA stability and enzyme expression

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Abstract

Cytochrome P450 2A6 (CYP2A6) is the major nicotine C-oxidase in human and participates in the metabolism of drugs and precarcinogens. The CYP2A6 gene is highly polymorphic and more than 22 different alleles have been described. We here focused on the polymorphism in the 3′-UTR region, in particular the common CYP2A6*1B allele, carrying an unequal crossover element from the pseudogene CYP2A7. Analysis of CYP2A6 expression in a human liver bank (n = 46) revealed that the protein level and catalytic activity using coumarin as a substrate were all higher, following a linear gene–dose relationship, in livers carrying one or two copies of CYP2A6*1B, as compared to other CYP2A6 allelic variants. Different variants of the CYP2A6 3′-UTR were cloned into a modified pGL3 plasmid downstream of the luciferase reporter gene. The plasmids, having the proximal promoter of CYP2A6 gene, were transfected into HeLa cells or injected into the tail veins of male CD1 mice. In both systems, the 3′-UTR CYP2A6*1B constructs caused higher reporter gene activity and the CYP2A7 3′-UTR construct lower activity, compared to the CYP2A6*1 3′-UTR constructs. Two SNPs differentiating the 3′-UTR between CYP2A7 and CYP2A6*1B were found to be of importance for the expression in both systems. Analysis of reporter enzyme degradation in HeLa cells showed that luciferase-3′-UTR-CYP2A6*1A had a half-life of approximately 4.9 h as compared to 6.3 h for luciferase-3′-UTR-CYP2A6*1B. In conclusion, we identified polymorphic motifs in the CYP2A6 3′-UTR of importance for CYP2A6 mRNA stabilization and enzyme expression. Such polymorphism has been described to influence the in vivo rate of nicotine elimination and possibly the cigarette consumption and risk of smoking induced lung cancer.

Section snippets

Material and methods

Subjects. Genomic DNAs and total mRNAs were extracted from 46 human liver samples obtained from Swedish kidney donors as described before [17]. Liver microsomes were prepared from 29 samples among the Swedish donors. 102 Turkish genomic DNA samples were collected from healthy individuals for pharmacogenetic studies at the Department of Biochemistry, Faculty of Pharmacy, University of Istanbul [9]. The Ethics Committee at the Karolinska Institutet approved the use of the human samples for the

Interindividual variation in hepatic CYP2A6 expression and activity

The protein level and the catalytic activity of CYP2A6 were determined in human liver samples and compared to previous data on the mRNA level in the same livers [18]. The association between the gene expression and catalytic activity of CYP2A6 and the influence of the CYP2A6*1B allele are shown in Fig. 2. The mRNA level, protein level as well as the catalytic activity of CYP2A6 measured by the formation of 7-hydroxycoumarin from coumarin were all in accordance with each other. The CYP2A6

Discussion

The results obtained indicate that the 3′-UTR plays an important role in regulation of CYP2A6 gene expression. This observation was supported by analysis of CYP2A6 genotype and phenotype in different liver samples as well as by results from both in vivo and in vitro transfection experiments using reporter constructs containing variant CYP2A6 3′-UTRs.

The CYP2A6*1B allele carries a gene conversion with the CYP2A7 gene in the 3′-UTR [3], [21]. Several studies on healthy volunteers indicated that

Acknowledgments

This study was supported by grants from The Swedish Cancer Foundation, The Swedish Research Council and by NIH (NIGMS 1-R01 GM60548).

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    Abbreviations: 3′-UTR, 3′-untranslated region; SNP, single nucleotide polymorphism; NNK, methylnitrosamino-1-(3-pyridyl)-1-butanone; NDEA, N-nitrosodiethylamine; ActD, actinomycin D.

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