Gene therapy against hepatocellular carcinoma using p48 gene

双链RNA依赖蛋白激酶(PKR)是干扰素诱导的抗病毒作用的关键因子。鉴于P48与信号转导和转录激活因子1和2(STAT1和STAT2)一起是干扰素-α信号通路中不可缺少的介导者，我们研究了P48基因转导对HH-7人肝癌细胞PKR表达及其活性的影响。将P48基因表达的腺病毒载体或质粒载体分别感染或转染HU-7细胞，并与干扰素-α共同孵育，检测PKR在细胞中的表达和真核合成蛋白合成起始因子2(EIF2)α亚单位α的磷酸化。此外，抑制蛋白质翻译的活性取决于氯霉素乙酰转移酶(CAT)基因翻译或α-甲胎蛋白分泌的减少。p48过表达本身不能刺激PKR的表达。然而，联合干扰素-α治疗后，p48基因过表达可显著增加pKR的表达，增强eIF2mRNA的磷酸化，从而阻断CAT基因的翻译和内源性α的合成，而不影响其α的表达水平。

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.