Ciaramella, Giuseppe; (1995) Development of adult hepatocyte culture systems capable of expressing genes coding for the major proteins responsible for foreign compound metabolism. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cytochromes P450 play a central role in the synthesis of endogenous compounds, such as steroids and fatty acids, and in the metabolism of many foreign compounds, including dietary components, therapeutic drugs and carcinogens. The expression of several cytochromes P450 is increased considerably upon exposure to certain xenobiotics. For example, treatment with the antiepileptic drug phenobarbital causes a dramatic increase in the abundance in rat liver of mRNAs encoding two members of the CYP2B subfamily, namely CYP2B1 and CYP2B2. Until recently, however, the phenobarbital response could not be reproduced in culture and therefore studies of the regulation of the expression of CYP2B1/2 have been hampered by the absence of a suitable cell culture system. We have developed culture conditions for both primary rat hepatocytes and a highly differentiated rat hepatoma cell line, FAZA 967, that support the induction of CYP2B gene expression by phenobarbital. Using the RNase protection method we found that the induction of CYP2B1/2 mRNAs is comparable in magnitude to that observed in vivo. We found also that in these cell systems, as is the case in rat liver, CYP2B1/2 gene expression is induced by the barbiturate-antagonist picrotoxin, at levels comparable to those observed using phenobarbital as the inducer. The expression and induction by phenobarbital of other drug-metabolising enzymes, namely P450 reductase, FMOl, GST 1-1/2-2, 3-3/4-4, and GST7-7, has also been monitored using immunoblotting, and found to reflect the in vivo situation. To demonstrate the possibility of using the primary hepatocyte system in promoter mapping studies, we have developed a highly efficient protocol for the transfection of DNA into these cells, based on lipofection. We demonstrated that in this system the immediate early promoter of human cytomegalovirus can promote the expression in large amounts of catalytically active ?-galactosidase and luciferase enzymes. In initial attempts aimed at defining regions of the CYP2B2 gene promoter important for its regulation, we found that a DNA fragment located between -368 and -4 of the 5'-flanking region of a CYP2B2 gene was not sufficint to drive the expression of the luciferase gene. Finally, we have attempted to establish a conditionally immortalised hepatocyte cell system by isolating hepatocytes from the livers of transgenic mice, harbouring a mutant, temperature sensitive form, of the SV40 virus large T antigen, whose expression is under the regulation of a promoter that is responsive to y-interferon. These cells could be kept in culture for longer than a month, could be passaged and secreted albumin.

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