Altered regulation of Cyp1a-1 gene expression during cultivation of mouse hepatocytes in primary culture.
Alterations in Cyp1a-1 gene expression in adult C57BL/6 mouse hepatocytes were followed after transferring them to primary culture during the initial 5 days. Changing the medium to a fresh one was associated with considerable amounts of Cyp1a-1 gene mRNA with a peak at around 6 hr after the medium change, followed by a decrease to negligible levels 24 hr later. Treatment of hepatocytes with cycloheximide increased the medium change-associated mRNA expression, the levels being equivalent to those observed after treatment with 3.2-25.6 nM 3-methylcholanthrene plus cycloheximide. With increasing length of culture period, cycloheximide-aided enhancement of the medium change-associated mRNA transcription increased. Although the chemical alone did not induce Cyp1a-1 gene transcripts in hepatocytes at day 1 or 2 of cultivation, for which medium had been changed 24 hr previously, prominent induction of transcripts was evident at later periods, the levels being elevated in accordance with length of time in culture. To examine whether or not the mRNA transcribed under these culture conditions was translatable, the cells were treated with actinomycin D after washing out the cycloheximide, in order to inhibit degradation of the generated mRNA (Nemoto N and Sakurai J, Carcinogenesis 12: 2115-2121, 1991). After these procedures significant elevation of aryl hydrocarbon hydroxylase activity was observed in hepatocytes, the rise being well correlated with elevated levels of mRNA transcripts. The observations suggest that the Cyp1a-1 gene might be expressed at low levels during the initial phase of cultivation of mouse hepatocytes in primary culture. Whether this expression might be essential for mouse hepatocytes to adapt to culture conditions is unclear. The findings do suggest, however, that superinducibility of the gene expression after cycloheximide treatment might be a result of a regulatory mechanism operating after adaptation to culture.
Nemoto
N
N
Department of Experimental Pathology, Cancer Institute, Tokyo, Japan.
Sakurai
J
J
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis; D002478:Cells, Cultured / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D003609:Dactinomycin / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D008099:Liver / Q000201:enzymology; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D013997:Time Factors
D003609:Dactinomycin; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D003513:Cycloheximide; D001189:Aryl Hydrocarbon Hydroxylases