Abstract
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.
Highlights
Cell lines were established which produce replica- zymes display overlapping substrate specificities, and a tion-defective ecotropic and amphotropic host range single cytochrome P-450 may metabolize several substrates recombinant retrovirusescontainingthe cDNA for and asingle substrate inturn may bemetabolized by different mouse cytochrome P3-450 aswell as the bacterialNeo cytochrome P-450s [3]
The purpose of this investigation was to develop a system in which the coding sequences of cytochromes P-450 were constitutively expressed in cultured cells as enzymatically active proteins in their natural microsomal environment reflecting physiological specificity.Here Idescribe the construction of recombinant retroviruses containing the coding sequence for cytochrome P3-450, capable of infecting most mammalian cells
Enzyme Assays-Acetanilide hydroxylase activity was determined by measuring the conversion of [14C]acetanilideto its hydroxylated derivatives [8].The substrate and its metabolites were separated by silica gel thin layer chromatography (TLC)
Summary
- From the Laboratorv of Molecular Carcinogenesis.National Cancer Institute, National Institutes of Health, Bethesda, Maryland20892. Cell lines were established which produce replica- zymes display overlapping substrate specificities, and a tion-defective ecotropic and amphotropic host range single cytochrome P-450 may metabolize several substrates recombinant retrovirusescontainingthe cDNA for and asingle substrate inturn may bemetabolized by different mouse cytochrome P3-450 aswell as the bacterialNeo cytochrome P-450s [3]. Available techniques do not permit gene for G418 resistance. The G418-resistant clones direct measurement of the activity of the individual forms, derived from virus-infected cultures waenraelyzed for but theyprovide a composite profile of all the enzymespresent the expression, subcellular localization, and catalytic activities of the cytochrome P3-450.Southern blot analysis of the genomic DNAs indicates that the viral
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