Wang Jue, Pitarque Mariá, Ingelman-Sundberg Magnus
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden.
Biochem Biophys Res Commun. 2006 Feb 10;340(2):491-7. doi: 10.1016/j.bbrc.2005.12.035. Epub 2005 Dec 19.
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 CYP2A61B 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 CYP2A61B, 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 CYP2A61B constructs caused higher reporter gene activity and the CYP2A7 3'-UTR construct lower activity, compared to the CYP2A61 3'-UTR constructs. Two SNPs differentiating the 3'-UTR between CYP2A7 and CYP2A61B 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-CYP2A61A had a half-life of approximately 4.9h as compared to 6.3h 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.
细胞色素P450 2A6（CYP2A6）是人体内主要的尼古丁C-氧化酶，参与药物和前致癌物的代谢。CYP2A6基因具有高度多态性，已发现22种以上不同的等位基因。我们在此重点研究3'-非翻译区（3'-UTR）的多态性，特别是常见的CYP2A61B等位基因，它携带来自假基因CYP2A7的不等交换元件。对一个人类肝脏库（n = 46）中CYP2A6表达的分析表明，与其他CYP2A6等位基因变体相比，携带一个或两个CYP2A61B拷贝的肝脏中，以香豆素为底物时的蛋白质水平和催化活性均更高，且呈线性基因剂量关系。将CYP2A6 3'-UTR的不同变体克隆到荧光素酶报告基因下游的改良pGL3质粒中。这些含有CYP2A6基因近端启动子的质粒被转染到HeLa细胞中，或注射到雄性CD1小鼠的尾静脉中。在这两种系统中，与CYP2A61 3'-UTR构建体相比，3'-UTR CYP2A61B构建体导致更高的报告基因活性，而CYP2A7 3'-UTR构建体导致较低的活性。发现区分CYP2A7和CYP2A61B之间3'-UTR的两个单核苷酸多态性（SNP）对两种系统中的表达都很重要。对HeLa细胞中报告酶降解的分析表明，荧光素酶-3'-UTR-CYP2A61A的半衰期约为4.9小时，而荧光素酶-3'-UTR-CYP2A6*1B的半衰期为6.3小时。总之，我们在CYP2A6的3'-UTR中鉴定出对CYP2A6 mRNA稳定性和酶表达很重要的多态性基序。这种多态性已被描述为会影响体内尼古丁消除率，并可能影响香烟消费量以及吸烟诱发肺癌的风险。
