Dell, Helen J.; (1997) The regulation of cytochrome P450 2B gene expression by the anti-epileptic drug phenobarbital. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Expression of the Cytochrome P450 CYP2B2 gene is increased in rat liver following exposure to the anti-epileptic drug phenobarbital. The proximal 5' flanking sequence of this gene was studied to identify cis-acting DNA elements and trans-acting proteins which are involved in this induction mechanism. C/EBPα and C/EBPβ bind within the region from -34 to -86bp of the CYP2B2 promoter. C/EBPβ is involved in complexes which increase in abundance in response to phenobarbital treatment of the animal. Neither C/EBPα or C/EBPβ increase in amount following phenobarbital treatment. However, formation of complexes containing C/EBPα and/or C/EBPβ is inhibited by alkaline phosphatase treatment of nuclear protein extracts. Competition gel retardations suggest that a GC box binding protein and AP1 are also involved in the complexes that bind to the -34 to -86bp region of the CYP2B2 gene. The study of the mechanism of phenobarbital induction of cytochromes P450 2B1 and 2B2 has suffered from the lack of an hepatocyte culture system or cell line capable of maintaining their expression and induction. Dr. G. Ciaramella set up a primary hepatocyte culture system that was capable of maintaining CYP2B1/2 mRNAs and their induction by phenobarbital for 4 days. This system was characterised further and its suitability for use in studying the phenobarbital induction mechanism was demonstrated. Treatment of these cells with a cAMP analogue, a protein phosphatase inhibitor and a cAMP-dependent protein kinase activator inhibited the phenobarbital response at the protein level. The primary hepatocytes were transfected with constructs containing sections of the CYP2B2 5' flanking sequence attached to a luciferase reporter gene. Expression from the construct containing the -2915bp to +29bp region of CYP2B2 increased slightly in response to phenobarbital treatment of the cells.

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