Gene Therapy
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August 2000, Volume 7, Number 15, Pages 1274-1283
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Viral Transfer Technology
Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes
N B Beck, J S Sidhu and C J Omiecinski

Department of Environmental Health, University of Washington, 4225 Roosevelt Way, NE, # 100, Seattle, WA 98105-6099, USA

Correspondence to: C J Omiecinski

Abstract

Baculovirus transfection strategies have proven successful at transferring foreign DNA into hepatoma cells and primary hepatocytes. When testing the utility of these methodologies in cultured hepatocytes, we discovered that the presence of baculovirus disrupts the phenobarbital (PB) gene induction process, a potent transcriptional activation event characteristic of highly differentiated hepatocytes, and repressed expression of the albumin gene. In concert with previous reports from our laboratory demonstrating that increased cAMP levels can completely repress the induction of specific cytochrome P450 (CYP) genes, cAMP concentrations and PKA activities were measured in the primary hepatocytes subsequent to baculovirus exposure. However, neither parameter was affected by the presence of the virus. To evaluate whether immune response modulation was triggered by baculovirus exposure, RNase protection assays were performed and demonstrated that baculovirus infection activates TNF-alpha, IL-1alpha and IL-1beta expression in the primary hepatocyte cultures. Immunocytochemical experiments indicated that the production of cytokines was likely due to the presence of small numbers of Kupffer cells present in the culture populations. Exogenously added TNF-alpha was also effective in repressing PB induction, consistent with other reports indicating that inflammatory cytokines are capable of suppressing expression of biotransformation enzyme systems. Comparative studies demonstrated the specificity of these effects since exposures of hepatocytes to adenoviral vectors did not result in down-regulation of hepatic gene responsiveness. These results indicate that baculovirus vectors enhance the expression of inflammatory cytokines in primary hepatocyte cultures, raising concerns as to whether these properties will compromise the use of baculovirus vectors for study of cytochrome P450 gene regulation, as well as for liver-directed gene therapy in humans. Gene Therapy (2000) 7, 1274-1283.

Keywords

baculovirus; transfection; hepatocyte; cytochrome P450; cytokines; TNF-alpha

Received 23 February 1999; accepted 21 April 2000
August 2000, Volume 7, Number 15, Pages 1274-1283
Table of contents    Previous  Abstract  Next   Full text  PDF