Retrovirus-mediated stable expression of human CYP2A6 in mammalian cells.
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.
Salonpää
P
P
Department of Pharmacology and Toxicology, University of Oulu, Finland.
Hakkola
J
J
Pasanen
M
M
Pelkonen
O
O
Vähäkangas
K
K
Battula
N
N
Nouso
K
K
Raunio
H
H
D016475:3T3 Cells; D016604:Aflatoxin B1 / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D065637:Cytochrome P-450 CYP2A6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*
D065607:Cytochrome P-450 Enzyme Inhibitors; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6