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  • Viral Transfer Technology
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Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes

Gene Therapy volume 7, pages 1274–1283 (2000)Cite this article

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-α, IL-1α and IL-1β 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-α 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.

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Acknowledgements

We would like to acknowledge the generous support of Drs Stella Thompson, Yi-Min Zheng, and Allan Rettie for their help with baculovirus preparation and plaque assay determinations and to Dr Sean Boyle for his adenovirus expertise. We thank Dr Terrance Kavanagh for expert assistance with scanning laser cytometry and data analysis. We are grateful to Dr Richard Boyce for assistance with baculovirus protocols, to Dr Jonathan Reichner for the donation of the KU-1 antibody, to Dr Mark Kay for donation of adenovirus vectors, and to Dr Fred Farin, Julia H Tracy and Fei Liu for their excellent technical assistance. This research was supported by a grant from the National Institute of General Medical Sciences (GM32281) and a National Institute of Environmental Health Sciences Center Grant (ES07033). NBB was supported by an NIEHS training grant (ES07032). CJO is a Burroughs Wellcome Fund Toxicology Scholar.

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

    NB Beck, JS Sidhu & CJ Omiecinski

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Beck, N., Sidhu, J. & Omiecinski, C. Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes. Gene Ther 7, 1274–1283 (2000). https://doi.org/10.1038/sj.gt.3301246

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