Gene therapy against hepatocellular carcinoma using p48 gene

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Double-stranded RNA-dependent protein kinase (PKR) is a key factor involved in interferon (IFN)-induced antiviral actions. Since p48, together with signal transducers and activators of transcription 1 and 2 (STAT1 and STAT2), is an indispensable mediator in IFN-α signaling pathways, we investigated the effect of p48 gene transduction on PKR expression and its activity in HuH-7 human hepatoma cells.HuH-7 cells were infected or transfected with p48 gene expression adenoviral vector or plasmid vector, respectively, and incubated with or without IFN-α, then PKR expression and phosphorylation of α-subunit of eukaryotic protein synthesis initiation factor-2 (eIF2 α) in the cells were examined. In addition, PKR activity inhibiting protein translation was determined by the decrease of chloramphenicol acetyltransferase (CAT) gene translation or α-fetoprotein secretion.p48 overexpression itself could not stimulate PKR expression. However, p48 overexpression in combination with interferon-α treatment caused a marked increase in PKR expression and augmented the phosphorylation of eIF2 α, by which the transfected CAT gene translation, as well as the endogenous α-fetoprotein synthesis, was blocked without affecting their mRNA levels.These results suggest that p48 gene transduction may provide a strategy to enhance the IFN-mediated PKR expression and its activity in hepatocytes.