Salonpää P, Hakkola J, Pasanen M, Pelkonen O, Vähäkangas K, Battula N, Nouso K, Raunio H
Department of Pharmacology and Toxicology, University of Oulu, Finland.
Eur J Pharmacol. 1993 Aug 2;248(2):95-102. doi: 10.1016/0926-6917(93)90030-t.
To study the pharmacological and toxicological significance of the human cytochrome P450 isoform CYP2A6, we expressed it in mammalian cells by retrovirus-mediated gene transfer. The LXSN vector and PA317 packaging cells were used to create amphotropic recombinant retroviruses containing CYP2A6 cDNA. NIH 3T3 and HeLa cells were infected with these retroviruses and cell clones expressing CYP2A6 were selected. The integration of the CYP2A6 construct was verified by PCR analysis and northern blot analysis showed that a 5 kb mRNA containing the CYP2A6 was present in the cells. The integrated cDNA directed the expression of catalytically active CYP2A6 enzyme which has remained stable over numerous cell passages. No oxidation of several other P450 substrates was detected. The promutagen aflatoxin B1 was metabolized to intermediates binding to the host cell genomic DNA by the 3T3 2A6 cells. These cell lines are thus well suited for the study of the catalytic profile and the biological consequences of promutagen activation by the human CYP2A6 isoform.
为了研究人类细胞色素P450同工酶CYP2A6的药理和毒理学意义，我们通过逆转录病毒介导的基因转移在哺乳动物细胞中表达了该酶。使用LXSN载体和PA317包装细胞来制备含有CYP2A6 cDNA的双嗜性重组逆转录病毒。用这些逆转录病毒感染NIH 3T3和HeLa细胞，并筛选出表达CYP2A6的细胞克隆。通过PCR分析验证了CYP2A6构建体的整合，Northern印迹分析表明细胞中存在一条含有CYP2A6的5 kb mRNA。整合的cDNA指导了具有催化活性的CYP2A6酶的表达，该酶在多次细胞传代过程中保持稳定。未检测到其他几种P450底物的氧化。前诱变剂黄曲霉毒素B1被3T3 2A6细胞代谢为与宿主细胞基因组DNA结合的中间体。因此，这些细胞系非常适合用于研究人类CYP2A6同工酶的催化特征和前诱变剂激活的生物学后果。
