The role of ribonucleic acid and protein synthesis in microsomal aryl hydrocarbon hydroxylase induction in cell culture. The independence of transcription and translation.

Aryl hydrocarbon hydroxylase is a microsomal mixed function oxygenase induced by benz[a]anthracene and other polycyclic hydrocarbons in mammalian cell culture. Actinomycin D or Z-mercapto-1-(/3-4-pyridethyl)benzimidazole (inhibitors of RNA synthesis) or cycloheximide or puromycin (inhibitors of protein synthesis), added simultaneously with benz[a]anthracene, completely prevents aryl hydrocarbon hydroxylase induction. During treatment with inducer, the stimulation of hydroxylase activity becomes insensitive to inhibition by actinomycin D but continues to be sensitive to inhibition by cycloheximide. The phase of induction requiring RNA synthesis is independent of translation; in cells previously treated with benz[o]anthracene plus cycloheximide, the enzyme activity rises for 8 hours after the cells are placed in fresh control medium. Addition of actinomycin D to these previously treated cells does not inhibit the rise in hydroxylase activity, suggesting that RNA synthesis has occurred in the absence of translation. Furthermore, when actinomycin D is added to cells previously treated with benz[a]anthracene plus Z-mercapto-l-(fl-4-pyridethyl)benzimidazole, the increase in aryl hydrocarbon hydroxylase activity is prevented. Induction of increased hydroxylase activity seems to require at least two phases. The initial phase appears to involve synthesis of an induction-specific RNA, and this phase is cycloheximide-insensitive and hence translationindependent. The later phase of enzyme induction involves translation related to an induction-specific RNA, and this stage is insensitive to inhibition by actinomycin D and, thus, is transcription-independent. The increased enzyme activity may be due to an increase in the amount of either enzyme protein or other protein involved in enzyme activation.