Transduction of cytochrome P3-450 by retroviruses: constitutive expression of enzymatically active microsomal hemoprotein in animal cells.
Cell lines were established which produce replication-defective ecotropic and amphotropic host range recombinant retroviruses containing the cDNA for mouse cytochrome P3-450 as well as the bacterial Neo gene for G418 resistance. The G418-resistant clones derived from virus-infected cultures were analyzed for the expression, subcellular localization, and catalytic activities of the cytochrome P3-450. Southern blot analysis of the genomic DNAs indicates that the viral DNA was stably integrated into the cellular DNA. Western blot analysis of the proteins showed that the size of the constitutively expressed product was Mr 54,000, indistinguishable from the cytochrome P3-450 found in mouse liver microsomes. Spectral characterization of the P3-450 proteins indicates that the newly synthesized apoprotein incorporated heme and integrated into the microsomes. Enzymatic analysis of the cell homogenates in vitro and of the dividing cells in situ showed very high acetanilide hydroxylase activity and very low aryl hydrocarbon hydroxylase activity, a diagnostic feature of the cytochrome P3-450. The precise transmission of the recombinant retroviral sequences into the target cells and the exceptional fidelity of expression of the enzyme in cells will allow the analysis of an increasing number of cloned genes of cytochrome P-450s by defining the individual enzyme specificities, their physiological role in cells, and consequences of their functional expression, such as in toxicity, mutagenesis, and carcinogenesis.
Battula
N
N
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D006367:HeLa Cells; D006420:Hemeproteins / Q000235:genetics*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D014162:Transfection
D004274:DNA, Recombinant; D006420:Hemeproteins; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C023738:acetanilide hydroxylase