Maintenance of phenobarbital-inducible Cyp2b gene expression in C57BL/6 mouse hepatocytes in primary culture as spheroids.
Expression of the Cyp2b-9 and Cyp2b-10 genes was investigated in primary cultured adult C57BL/6NCrj mouse hepatocytes in monolayers or during the formation of spheroids (multicellular aggregates). Both the constitutive and phenobarbital-inducible expression of Cyp2b-9 and Cyp2b-10 mRNA decreased rapidly after transferring the hepatocytes to monolayer culture. The decrease was dependent on cell-density, and became more rapid in more densely seeded cells. However, in spheroid culture, the Cyp2b-9 and Cyp2b-10 mRNAs were induced by phenobarbital at high levels for at least 4 days either with continuous exposure from the start of cultivation or for 24 h before harvesting. The expression of both Cyp2b-9 and Cyp2b-10 species was confirmed by either reverse transcriptase-polymerase chain reaction or Western blotting. Although more Cyp2b-9 than Cyp2b-10 was expressed in the liver of control mice, the amounts of the latter became relatively overwhelming in untreated hepatocytes because of a faster decline of Cyp2b-9 species in culture. The level of mRNA induced by phenobarbital was concentration-dependent, the highest being at 2 or 4 mM, which was equivalent to in vivo treatment levels. There was more Cyp2b-10 species than those of Cyp2b-9 after exposure to phenobarbital both in vivo and in vitro. Phenobarbital also induced CYP1A2 mRNA, which was again peculiar to spheroid culture. Although the expression levels of both Cyp2b-9 and Cyp2b-10 species was very low in hepatocytes cultured without dexamethasone even in the presence of phenobarbital, the addition of 10(-7) or 10(-6) M dexamethasone caused an increase in the mRNA. When given concomitantly with phenobarbital, the expression was greatly enhanced. Nicotinamide or isonicotinamide similarly enhanced the expression of the two mRNA species, but the levels in monolayer cultures were still far lower than those in vivo. In contrast to the findings for mRNA expression, the protein levels in the presence of nicotinamide were similar to those in vivo under both monolayer and spheroid conditions. Since previous efforts to maintain expression of the CYP2B1 and CYP2B2 genes in primary cultured rat hepatocytes, orthologous to mouse Cyp2b-9 and Cyp2b-10 genes, required special cell attachment factors, our spheroid culture system, in which mouse hepatocytes are simply seeded onto noncoated dishes, has advantages for mechanistic studies.
Nemoto
N
N
Department of Experimental Pathology, Cancer Institute, Tokyo, Japan.
Sakurai
J
J
Funae
Y
Y
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009536:Niacinamide / Q000494:pharmacology; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D010634:Phenobarbital / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis
D012333:RNA, Messenger; D009536:Niacinamide; C027681:isonicotinamide; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D010634:Phenobarbital