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

Baculovirus transfection strategies have proven successful at transferring foreign DNA into hepatoma cells and primary hepatocytes. When testing the u tility of these methodologies in cultured hepatocytes, we discovered that t he presence of baculovirus disrupts the phenobarbital (PB) gene induction p rocess, a potent transcriptional activation event characteristic of highly differentiated hepatocytes, and repressed expression of the albumin gene. I n concert with previous reports from our laboratory demonstrating that incr eased cAMP levels can completely repress the induction of specific cytochro me P450 (CYP) genes, cAMP concentrations and PKA activities were measured i n the primary hepatocytes subsequent to baculovirus exposure. However, neit her parameter was affected by the presence of the virus. To evaluate whethe r immune response modulation was triggered by baculovirus exposure, RNase p rotection assays were performed and demonstrated that baculovirus infection activates TNF-alpha, IL-1 alpha: and IL-1 beta expression in the primary h epatocyte cultures. Immunocytochemical experiments indicated that the produ ction of cytokines was likely due to the presence of small numbers of Kupff er cells present in the culture populations. Exogenously added TNF-alpha wa s also effective in repressing PB induction, consistent with other reports indicating that inflammatory cytokines are capable of suppressing expressio n 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. T hese results indicate that baculovirus vectors enhance the expression of in flammatory cytokines in primary hepatocyte cultures, raising concerns as to whether these properties will compromise the use of baculovirus Vectors fo r study of cytochrome P450 gene regulation, as well as for liver-directed g ene therapy in humans.