RETROVIRUS-MEDIATED STABLE EXPRESSION OF HUMAN CYP2A6 IN MAMMALIAN-CELLS

To study the pharmacological and toxicological significance of the hum an cytochrome P450 isoform CYP2A6, we expressed it in mammalian cells by retrovirus-mediated gene transfer. The LXSN vector and PA317 packag ing cells were used to create amphotropic recombinant retroviruses con taining CYPZA6 cDNA. NIH 3T3 and HeLa cells were infected with these r etroviruses and cell clones expressing CYP2A6 were selected. The integ ration of the CYP2A6 construct was verified by PCR analysis and northe rn blot analysis showed that a 5 kb mRNA containing the CYP2A6 was pre sent in the cells. The integrated cDNA directed the expression of cata lytically active CYP2A6 enzyme which has remained stable over numerous cell passages. No oxidation of several other P450 substrates was dete cted. The promutagen aflatoxin B1 was metabolized to intermediates bin ding to the host cell genomic DNA by the 3T3 2A6 cells. These cell lin es are thus well suited for the study of the catalytic profile and the biological consequences of promutagen activation by the human CYP2A6 isoform.