Saturation of transgene protein synthesis from mRNA in cells producing a large number of transgene mRNA.

Experimental results have suggested that transgene expression can be saturated when large amounts of plasmid vectors are delivered into cells. To investigate this saturation kinetic behavior, cells were transfected with monitoring and competing plasmids using cationic liposomes. Even although an identical amount of a monitoring plasmid expressing firefly luciferase (FL) was used for transfection, transgene expression from the plasmid was greatly affected by the level of transgene expression from competing plasmids expressing renilla luciferase (RL). Similar results were obtained by exchanging the monitoring and competing plasmids. The competing plasmid-dependent reduction in transgene expression from the monitoring plasmid was also observed in mouse liver after hydrodynamic injection of plasmids. On the other hand, the mRNA and protein expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an endogenous gene, in the liver hardly changed even when transgene expression process is saturated. The expression of FL from a monitoring plasmid was significantly restored by siRNA-mediated degradation of RL mRNA that was expressed from a competing plasmid. These results suggest that the efficiency of protein synthesis from plasmid vectors is reduced when a large amount of mRNA is transcribed with no significant changes in endogenous gene expression.

Positive correlation between the generation of reactive oxygen species and activation/reactivation of transgene expression after hydrodynamic injections into mice.

PURPOSE: Hydrodynamic injection has been shown to reactivate silenced transgene expression in mouse liver. In this study, the roles of inflammatory cytokines and reactive oxygen species (ROS) in the reactivation were examined. METHODS: Production of inflammatory cytokines and ROS by hydrodynamic injection of saline was examined in mice that had received a hydrodynamic injection of a plasmid expressing Gaussia luciferase. The level of reporter gene expression was used as an indicator of the reactivation. The involvement of cytokines and ROS was examined by depleting Kupffer cells or by pre-administration of antioxidants, respectively. RESULTS: A hydrodynamic injection of saline induced a significant production of interleukin (IL)-6. Depleting Kupffer cells using clodronate liposomes markedly reduced the IL-6 production but had no significant effect on the transgene expression. On the other hand, an injection of catalase or N-acetylcysteine significantly inhibited the hydrodynamic injection-induced reactivation of silenced transgene expression. The silenced expression was also reactivated by carbon tetrachloride, an inducer of oxidative stress in the liver, in a dose-dependent manner, and this reactivation was significantly inhibited by catalase. CONCLUSIONS: These findings show a positive correlation between the generation of ROS and the reactivation of silenced transgene expression after hydrodynamic injections.

Gene silencing of beta-catenin in melanoma cells retards their growth but promotes the formation of pulmonary metastasis in mice.

Altered expression of beta-catenin, a key component of the Wnt signaling pathway, is involved in a variety of cancers because increased levels of beta-catenin protein are frequently associated with enhanced cellular proliferation. Although our previous study demonstrated that gene silencing of beta-catenin in melanoma B16-BL6 cells by plasmid DNA (pDNA) expressing short-hairpin RNA targeting the gene (pshbeta-catenin) markedly suppressed their growth in vivo, gene silencing of beta-catenin could promote tumor metastasis by the rearranging cell adhesion complex. In this study, we investigated how silencing of beta-catenin affects metastatic aspects of melanoma cells. Transfection of B16-BL6 cells with pshbeta-catenin significantly reduced the amount of cadherin protein, a cell adhesion molecule binding to beta-catenin, with little change in its mRNA level. Cadherin-derived fragments were detected in culture media of B16-BL6 cells transfected with pshbeta-catenin, suggesting that cadherin is shed from the cell surface when the expression of beta-catenin is reduced. The mobility of B16-BL6 cells transfected with pshbeta-catenin was greater than that of cells transfected with any of the control pDNAs. B16-BL6 cells stably transfected with pshbeta-catenin (B16/pshbeta-catenin) formed less or an equal number of tumor nodules in the lung than cells stably transfected with other plasmids when injected into mice via the tail vein. However, when subcutaneously inoculated, B16/pshbeta-catenin cells formed more nodules in the lung than the other stably transfected cells. These results raise concerns about the gene silencing of beta-catenin for inhibiting tumor growth, because it promotes tumor metastasis by reducing the amount of cadherin in tumor cells.